The Oncologist

Chen, Helen X.; Rubinstein, Larry V.; Shankar, Lalitha K.; Abrams, Jeffrey S.

2014 The Oncologist

doi: 10.1634/theoncologist.2014-0126pmid: 24755462

The Response Evaluation Criteria in Solid Tumors (RECIST) criteria, which categorize quantitative tumor size changes into complete response, partial response (PR), stable disease (SD), or progressive disease (PD) [1], provide standardized, objective measurements of tumor response to therapy and have been used extensively over the last decade for early to late drug development and regulatory approvals, as well as for treatment decisions in individual patient care. The RECIST criteria were generated based on data from cytotoxic chemotherapy trials; their limitations for the assessment of molecularly targeted agents have been increasingly recognized with the advance of new therapies. In particular, the cutoff of 30% change in the sum of longest diameters (ΔSLD) as the criterion of response has been criticized for not adequately capturing potentially effective therapies. As exemplified by antiangiogenic therapies, such as sorafenib in renal cell carcinoma (RCC) and hepatocellular carcinoma, drugs with very low rates of RECIST-defined responses (10% or less) can still succeed and confer significant clinical benefit. For that reason, many clinical trials also use SD as an additional indicator of therapeutic effect. However, inclusion of SD and progression-free survival (PFS) in the efficacy readout often requires randomized trials to distinguish the drug effect from the natural course of the tumor. For patients receiving therapy, the implication of SD is often uncertain, because the criterion encompasses a wide range of tumor size changes, from 29% reduction to 19% increase. Exploration and validation of optimal criteria of tumor burden changes or functional imaging parameters as markers of drug effect and/or clinical benefit have the promise to improve the efficiency of both development of new therapeutics and therapeutic management of individual patients. A study published in this issue of The Oncologist [2] represents one of the many retrospective analyses of the relation between tumor size changes and clinical outcomes in patients treated with vascular endothelial growth factor (VEGF) pathway-targeting agents, focusing on a specific patient care question: what degree of tumor size change early in the course of therapy may predict the clinical outcome in the patient and therefore provide guidance for decisions on further treatment? The analysis was based on 66 patients treated with 1 of the 6 different VEGF-pathway inhibitors, and thresholds of −30% (as in RECIST) or −10% ΔSLDs were tested for their ability to classify patients with good or poor outcomes. This analysis concluded that ≥10% reduction in SLD (responders) at the first scan was associated with significantly better outcomes, compared with that of nonresponders (those who did not achieve 10% SLD reduction). Time to treatment failure was 8.4 months versus 4.1 months, and overall survival was 35 months versus 15 months, both with p values < .01. In contrast, the RECIST threshold of −30% ΔSLDs at the first scan failed to predict patient outcome (TTF of 6.9 months versus 5.5 months). It further suggested that −10% ΔSLDs at first scan could be used for treatment decisions as to whether the anti-VEGF therapies should be continued, although the negative predictive value of “nonresponse,” by either the −10% or −30% cutoffs, was not discussed. As already recognized by the authors, there are multiple limitations of this series, including limited sample size and small numbers in each marker subgroup, heterogeneity of the VEGF-targeting therapies ranging from tyrosine-kinase inhibitors (TKIs) to monoclonal antibodies, and inconsistency in the timing of first scans (20–170 days from start of therapy) on which the cutoff optimization was based. Despite the limitations, this study joined a large body of independent retrospective studies that collectively demonstrated a significant correlation between tumor size changes at the first scan and the clinical outcome [3–5]. One of the largest series was reported by Thiam et al. [5] based on 334 patients with advanced RCC treated on the sunitinib arm in a phase III trial for sunitinib versus interferon-α. It tested a series of ΔSLD thresholds at the first scan at 6 weeks (−45%, −30%, −20%, −10%, 0%, +10%) for their correlations with PFS and found that ΔSLDs of −10% provided the optimal cutoff that distinguished the PFS outcomes (median PFS of 5.6 months versus 11 months). The −10% cutoff was also examined in a number of other retrospective studies in independent patient cohorts and was consistently found to be significantly associated with outcomes ([5–7] and this study). What is the clinical utility of this finding? Although ≥10% shrinkage is clearly associated with significantly better outcome, the practical concern for a given patient is the likelihood of benefiting from therapy if that threshold is not reached. As an inherent limitation of post-treatment biomarkers, it is not possible to distinguish between predictive and prognostic markers because all marker subgroups would have received the same therapy. PFS of 5 months was often selected as an arbitrary landmark to estimate presence or lack of therapeutic benefit from VEGF-pathway inhibitors in first-line metastatic RCC. In several studies, including Thiam et al. [5] and the current study, median PFS values in nonresponders by the −10% criterion were 5–6 months, indicating that although these patients did not do well on average, 50% of the patients were progression-free for longer than 5–6 months. Furthermore, in tumors in which the target pathways remain relevant through progression, continuation of therapy may be beneficial whichever PD criteria are used. Given that the treatment benefit in an individual patient cannot be ruled out with high certainty, the value of this early post-treatment marker would depend on the availability of better, less toxic treatment options. Is this new criterion of response (≥10% SLD reduction) better than RECIST in guiding treatment decisions? The answer depends on how RECIST is used. It is clear that objective response (≥30% SLD reduction) is not a good predictor of outcomes. However, treatment decisions in general practice are not based on response, but rather on progression. Patients not achieving PR, but in SD, by RECIST definition (ΔSLDs in the range of −29% to +19%) would continue therapy until PD. To demonstrate the potential advantage of the −10% cutoff over the current practice, outcome differences using the cutoffs of RECIST PR/SD versus −10% should be compared. However, such comparisons are not always feasible because the numbers of patients with PD on first scans are usually very small, at least for anti-VEGF therapies in RCC. On the other hand, the use of the SD category as a basis for continuing therapy is conceivably problematic, because the tumor size could have increased by up to 19%. Indeed, lack of tumor shrinkage (0% SLD reduction) or +10% SLD at the first scan was associated with an extremely poor outcome in patients treated with sunitinib, with a median PFS of 1.5 months [5]. Further studies with sufficient power would be required to confirm this finding. What is the general implication of the findings on the use of tumor imaging in drug development? This study, among many others, reinforced the notion that clinically viable targeted agents may have low response rates as defined by RECIST, but tumor shrinkage with smaller magnitudes is common. These studies further suggest that modification of the response criteria to capture and categorize minor responses may provide a more sensitive readout of the therapeutic effect and potentially improve the accuracy of early drug evaluation. On the other hand, finding the optimal cutoff can be challenging, because the threshold may differ for the same agents in different indications or for different agents in the same indication. For example, although studies on VEGFR TKIs in metastatic RRC identified 10% as the optimal cutoff, similar tests for everolimus in second-line metastatic RCC failed to identify any cutoffs (from −45% to +20%) that separate better and poorer outcomes [8]. Technical limitations, such as measurement variability, may also limit the choice of thresholds that can be adopted. Efforts are under way by the RECIST committee to expand the database to include large randomized clinical trials of targeted agents, as well as studies using molecular imaging with fluorodeoxyglucose-positron emission tomography (FDG-PET), with the plan to evaluate and update the guidelines and criteria to meet the needs of drug development and patient care in the era of novel therapies. In summary, the current study and others focusing on VEGF-pathway inhibitors in advanced RCC highlight the observation that tumor shrinkage below the threshold of RECIST can be associated with significant clinical benefit, and that flexibility in the size change categorization should be considered in future modification of the response criteria. However, whether −10% or other cutoffs would be optimal for treatment decisions in individual patients is uncertain and would require further, sufficiently powered, prospective studies. It should be noted that criteria for one agent or one disease setting may not be generalizable to others. As newer therapeutic agents become available in various clinical settings, it may be necessary to further modify existing tumor size-based response criteria, verify promising molecular and functional imaging methods, and investigate new technologies. Finally, before optimal criteria are defined for dichotomous characterization of tumor responses, it would be desirable to collect tumor size measurements as a continuous variable and evaluate the reporting of antitumor activity based on several cutoffs, rather than just one, for responses or progression. Disclosures The authors indicated no financial relationships. Editor's Note: See the related article, “10% Tumor Diameter Shrinkage on the First Follow-Up Computed Tomography Predicts Clinical Outcome in Patients With Advanced Renal Cell Carcinoma Treated With Angiogenesis Inhibitors: A Follow-Up Validation Study,” by Katherine M. Krajewski et al., on page 507 of this issue. References 1 Therasse P , Arbuck SG, Eisenhauer EA. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada . J Natl Cancer Inst . 2000 ; 92 : 205 – 216 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Krajewski KM , Franchetti Y, Nishino M. 10% tumor diameter shrinkage on the first follow-up computed tomography predicts clinical outcome in patients with advanced renal cell carcinoma treated with angiogenesis inhibitors: A follow-up validation study . The Oncologist . 2014 ; 19 : 507 – 514 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Van der Veldt AA , Haanen JB, Van den Eertwegh AJ. Targeted therapy for renal cell cancer: Current perspectives . Discov Med . 2010 ; 10 : 394 – 405 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 4 Krajewski KM , Guo M, Van den Abbeele AD. Comparison of four early posttherapy imaging changes (EPTIC; RECIST 1.0, tumor shrinkage, computed tomography tumor density, Choi criteria) in assessing outcome to vascular endothelial growth factor-targeted therapy in patients with advanced renal cell carcinoma . Eur Urol . 2011 ; 59 : 856 – 862 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Thiam R , Fournier LS, Trinquart L. Optimizing the size variation threshold for the CT evaluation of response in metastatic renal cell carcinoma treated with sunitinib . Ann Oncol . 2010 ; 21 : 936 – 941 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Abel EJ , Culp SH, Tannir NM. Early primary tumor size reduction is an independent predictor of improved overall survival in metastatic renal cell carcinoma patients treated with sunitinib . Eur Urol . 2011 ; 60 : 1273 – 1279 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Smith AD , Lieber ML, Shah SN. Assessing tumor response and detecting recurrence in metastatic renal cell carcinoma on targeted therapy: Importance of size and attenuation on contrast-enhanced CT . AJR Am J Roentgenol . 2010 ; 194 : 157 – 165 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Oudard S , Thiam R, Fournier LS. Optimisation of the tumour response threshold in patients treated with everolimus for metastatic renal cell carcinoma: Analysis of response and progression-free survival in the RECORD-1 study . Eur J Cancer . 2012 ; 48 : 1512 – 1518 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Disclosures of potential conflicts of interest may be found at the end of this article. © 2014 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Dizon, Don S.

2014 The Oncologist

doi: 10.1634/theoncologist.2014-0116pmid: 24721814

Open in new tabDownload slide Don S. Dizon Open in new tabDownload slide Don S. Dizon I still remember when I professed my intention to become an oncologist while in residency. Some of my colleagues were supportive, but others were openly disdainful: “What a terrible field—all death and dying, and all that false hope.” I remember the thought that went through my mind at that time, too. It was an Emily Dickinson poem: Hope is the thing with feathers That perches in the soul, And sings the tune—without the words, And never stops at all … Oncology is a field in continual evolution. Although once our best treatments involved hormone manipulations and mutilating surgery, we now are well on the path toward precision medicine. This is the hope of clinicians in our field—better treatments, less toxicity, more individualization—treatment that can cure this devastating disease and, short of that, turn it from life-threatening to chronic. Death is not a “given” in our clinics and inpatient units anymore. Most of our patients are not dying of cancer; they are living with cancer. Despite our hope and advancements in treatment, the reality is that not all of our patients will live through cancer, walk through treatment without side effects, or hear the word “remission” based on the latest imaging test. Some of our patients will die of their cancers. As often as we greet new patients and embrace patients we have known over a longer period of time, we will say (and have said) good-bye to many others. When a patient dies of cancer, hope can be replaced with questions. Could we have done something more? Should we have done something different? Would I make the same recommendations if I could counsel him (or her) all over again? Hope can transform into something altogether different, like the sun clouding over and its yellow yielding to graying skies. Some would call it guilt, but I think it is more than that. I think this quote attributed to the preacher Horace Bushnell was more correct when he said, “Guilt is the very nerve of sorrow.” I have come to realize that perhaps “guilt” is really sadness. In this issue of The Oncologist, three extraordinary experiences of patient care are presented, and each represents a strong narrative of that emotional and sensitive part of our jobs centering around the end of life and, ultimately, the death of a patient. They are shared as recollections of individuals, as in the cases of Drs. Halmos [1] and O'Reilly [2], and of the team experience recalled during Schwartz Rounds at Massachusetts General Hospital [3]. Each demonstrates that we are not immune to our patients’ end-of-life experiences. We are a part of that, and we should acknowledge how hard it can be. Dr. Halmos is at the movies with his kids when the experience is interrupted by news of a patient's sudden turn, ICU stay, and quick death [1]. The interruption is irreversible, and his attention is split now. On the one hand, he is a father with his children out at a family event; on the other, he is a doctor with responsibilities to his patient, recalling conversations had, treatments tried, and the stark realization that his patient is at “the end of the road.” Dr. O'Reilly shares his experience from a place two steps removed from that of Dr. Halmos [2]. He recalls the death of a patient and a letter received posthumously, written months before and meant to be read after the patient had died. The letter was one of thanks: “Thanks for trying, thanks for hoping, and thanks to your family for their sacrifice.” No reply is expected, no confirmation of receipt necessary. Indeed, this patient is now deceased, and as Dr. O'Reilly notes quite starkly, “Any inadequate reply that I could compose will never be read. It can't be.” Dr. Schapira et al. tackle the real-time experience of caring for a person who is not only at the end of the cancer journey but who is also a colleague [3]. Boundary issues are explored, including the challenges of being a patient at an institution where the patient was also a provider, the desires of family and the wishes of the patient, the relationships built on collegiality and now challenged by illness. It is a story of empathy, sorrow, and support—the inpatient team experience of dying and death. One might wonder what the consequences of these experiences are. There are no words of wisdom regarding the “appropriate” way to respond to these situations, and I do not think there will ever be a definitive response. Just as each patient's journey after cancer is individual, perhaps each practitioner's approach to end of life is also personal. It is my hope that these narratives stimulate the reader to ask himself or herself, “How would I respond?” For me, I have learned that I cannot go to the funerals of patients. It hurts too much, and I am not ashamed to admit that. Instead, I have made it a point to say good-bye to patients at the end of their lives, when I feel their time has come and it is likely that I will not see them again: “It has been the greatest honor to know you and to be with you during your cancer. You have taught me how to care, and I will not forget you.” Looking someone in their eyes, acknowledging the moment, and saying good-bye has provided for me the best coping mechanism—closure. These narratives in medicine reflect the human experience of oncology. They remind us that we are not alone in feeling guilt, pain, and even sorrow when our patients die. Indeed, the reader may see himself or herself in these stories or recall another personal experience. And perhaps that it is the bigger point: to emphasize the art of oncology, the humanism in medicine, and to remind us that we are not superhuman and we are not gods. Our patients’ end-of-life experiences are part of the journey that we, as their oncologists, embarked on with them at the beginning. Whether or not we are able to be present with each patient at the time of his or her death, it remains a powerful experience for those of us on the clinical side of the therapeutic relationship. Beyond biology and targeted therapy, we are equally as human as our patients. When it comes to the end-of-life journey, as clinicians, we must feel it, experience it, and acknowledge it. Acknowledgment Thanks to my dear friend Amy Fries for helping review and edit this commentary. Disclosures Don S. Dizon: UpToDate (E). (C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board EDITOR'S NOTE: See the articles referenced in this commentary on pages 545–549, 574–575, and 576 of this issue. References 1 Halmos B . The seventh door . The Oncologist . 2014 ; 19 : 574 – 575 . Google Scholar Crossref Search ADS PubMed WorldCat 2 O'Reilly S . The letter to which I couldn't reply . The Oncologist . 2014 ; 19 : 576 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Schapira L , Blaszkowsky LS, Cashavelly BJ. Caring for one of our own . The Oncologist . 2014 ; 19 : 545 – 549 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Disclosures of potential conflicts of interest may be found at the end of this article. © 2014 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Seidman, Andrew D.; Chan, Stephen; Wang, Jin; Zhu, Chao; Xu, Cong; Xu, Binghe

2014 The Oncologist

doi: 10.1634/theoncologist.2013-0428pmid: 24705980

Abstract Introduction. In two randomized phase III trials of patients with metastatic breast cancer (MBC), gemcitabine-docetaxel (GD) and capecitabine-docetaxel (CD) had similar efficacy, but distinct safety profiles. Methods. Data from two GD versus CD studies were pooled; overall survival (OS), progression-free survival (PFS), and overall response rate (ORR) were determined. Cox proportional hazards models identified prognostic factors associated with improved OS and PFS. Using a multivariate prognostic model incorporating identified adverse prognostic factors, we grouped MBC patients into low-, intermediate-, and high-risk categories. Hazard ratios (HRs) of GD over CD for OS and PFS were determined for subsets of patients. Results. Baseline demographics of the pooled population were mostly well balanced. In the pooled population, there were no significant differences between GD versus CD for OS (HR = 1.02; p = .824), PFS (HR = 1.15; p = .079), and ORR (p = .526). In the pooled crossover population, there were trends toward improved OS (HR = 0.82; p = .171) and PFS (HR = 0.93; p = .557) with GD. Several prognostic factors (including prior adjuvant taxane) for improved OS or PFS were identified; however, there were no significant interactions between treatment arms and prognostic factors for PFS or OS, except number of metastatic sites. In the prognostic model, median OS and PFS were numerically lower in the high-risk group versus the intermediate- and low-risk groups. Conclusion. This analysis confirms the lack of efficacy difference between GD and CD in the pooled population, crossover population, and almost all subpopulations. Several prognostic factors were associated with improved outcomes in the pooled population. 摘要 引言. 两项在转移性乳腺癌（MBC）患者中的随机III期试验显示，吉西他滨-多西他赛（GD）与卡培他滨-多西他赛（CD）的有效性相似，但安全性不同。 方法. 汇总来自比较GD与CD的两个试验的研究数据；分析总生存期（OS）、无疾病进展生存期（PFS）以及总缓解率（ORR）。使用Cox比例风险模型识别与OS和PFS改善相关的预后因素。将识别出的不良预后因素合并到多变量预后模型中，在此基础上将MBC患者分成低危、中危和高危组。对于亚组患者，分析在OS与PFS指标上GD优于CD的风险比（HR）。 结果. 纳入汇总分析的人群基线人口学特征总体上平衡。汇总人群中，GD与CD在OS（HR = 1.02；p = 0.824）、PFS（HR = 1.15；p = 0.079）、ORR（p = 0.526）上的差异无统计学意义。汇总的交叉换组人群中，GD获得的OS（HR = 0.82；p = 0.171）、PFS（HR = 0.93；p = 0.557）倾向于更佳。研究发现了有利于OS或PFS的几项预后因素（包括既往接受过紫杉烷类辅助治疗）；然而，治疗分组与OS或PFS的预后因素之间并无显著的交互关系，转移部位数量除外。在预后模型中，高危组中位OS和PFS在数值上低于中危组和低危组。 结论. 本次分析证实，GD与CD在汇总人群、交叉换组人群以及几乎所有亚组人群中有效性无差异。几项预后因素与汇总人群的良好转归相关。The Oncologist 2014;19:443-452 Gemcitabine, Docetaxel, Capecitabine, Metastatic breast cancer, Pooled analysis Implications for Practice: In two randomized phase III trials of metastatic breast cancer patients, gemcitabine-docetaxel and capecitabine-docetaxel had similar efficacy, but distinct safety profiles. This pooled analysis confirmed the lack of efficacy difference between gemcitabine-docetaxel and capecitabine-docetaxel in the pooled population, the pooled crossover population, and almost all examined subpopulations. This analysis also identified several prognostic factors (Eastern Cooperative Oncology Group performance status, estrogen receptor status, prior adjuvant taxane, and number of metastatic sites) that were associated with both improved overall survival and progression-free survival in the overall pooled population. The choice of regimen should be guided by the clinical characteristics and tolerance to toxicities of the individual patient while considering the approved indications of the drugs. Introduction Globally, female breast cancer accounted for 23% of total cancer cases and 14% of cancer deaths in 2008, making this the most frequently diagnosed cancer and the leading cause of cancer death among females [1]. Despite advances in treatment, the long-term prognosis for women with metastatic breast cancer (MBC) is poor [2, 3]. In these patients, systemic chemotherapy can prolong survival and improve quality of life [4, 5]. However, new and better treatment options are needed to improve outcomes. In addition, the best use of existing agents has yet to be determined. Several agents, including gemcitabine, docetaxel, and capecitabine, have single-agent activity in advanced breast cancer [6–8]. Relative to single-agent therapy, combinations can significantly improve time to progression (TTP) and response, with a small increase in overall survival (OS) [9]. However, it is unclear whether combinations are more effective than the same agents administered sequentially [10]. In addition, combination therapy is usually associated with increased toxicity [9, 10]. In order to improve outcomes and minimize toxicity, treatments have combined drugs with distinct mechanisms of action (and sometimes synergistic activity) and partially nonoverlapping toxicities. When combined with taxanes, both gemcitabine and capecitabine have superior efficacy relative to taxane monotherapy [11, 12]. Gemcitabine-paclitaxel was associated with improved OS, TTP, and response relative to paclitaxel monotherapy and had manageable toxicity [11]. This regimen is now indicated in the U.S. for treatment of patients with MBC who have relapsed following anthracycline-based adjuvant/neoadjuvant chemotherapy unless clinically contraindicated [13] and has similar indications in the European Union and China [14, 15]. Likewise, the combination of capecitabine and docetaxel (capecitabine-docetaxel) was associated with improved OS, TTP, and response relative to docetaxel monotherapy [12] and is approved for use in the United States, European Union, and China [16–18]. Because of the synergy of gemcitabine and docetaxel in vitro [19], the combination of gemcitabine and docetaxel (gemcitabine-docetaxel) was explored in patients with MBC. This doublet was tested in nonrandomized clinical trials and demonstrated activity and tolerability [20–28]. More recently, gemcitabine-docetaxel and capecitabine-docetaxel were compared in two randomized phase III trials of patients with MBC [29, 30]. One of these trials had a planned crossover to the alternate single agent [30]. In these phase III trials, gemcitabine-docetaxel had similar efficacy to capecitabine-docetaxel, but the toxicity profile of the regimens differed. Here, we performed a pooled analysis of these phase III trials to confirm the efficacy of gemcitabine-docetaxel versus capecitabine-docetaxel in MBC patients, and to identify subsets of patients who may derive the most benefit from each regimen. Materials and Methods Patients In total, 780 patients were enrolled in the two international randomized phase III trials. Both studies enrolled patients ≥18 years old with histologically or cytologically confirmed MBC [29, 30]. In the Chan et al. trial (00191438), patients had measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) [31] and a Karnofsky performance status ≥70 [29]. Treatment with one prior anthracycline regimen in the neoadjuvant/adjuvant or first-line metastatic setting was required. Prior taxane treatment was permitted in the neoadjuvant/adjuvant setting if completed 6 months before enrollment. In the Seidman et al. trial (00191152), patients had an Eastern Cooperative Oncology Group performance status (ECOG PS) ≤1 and measurable or nonmeasurable disease. Patients may have completed neoadjuvant or adjuvant taxane therapy ≥6 months before enrollment [30]. Prior anthracycline, hormone, or immunotherapy, and no more than one prior line of chemotherapy for MBC were allowed. Patients who received prior taxane therapy for MBC were excluded. In both trials, patients provided written informed consent according to local guidelines. The studies were conducted per the principles of Good Clinical Practice and the Declaration of Helsinki. Treatment Patients were randomized to receive either gemcitabine-docetaxel or capecitabine-docetaxel [29, 30]. In the Chan trial, patients assigned to gemcitabine-docetaxel received gemcitabine (1,000 mg/m2 30-minute i.v. infusion) on days 1 and 8 and docetaxel (75 mg/m2 60-minute i.v. infusion) on day 1 [29]. Patients assigned to capecitabine-docetaxel received oral capecitabine (1,250 mg/m2 twice daily) on days 1 through 14 and docetaxel (75 mg/m2 60-minute i.v. infusion) on day 1. The capecitabine dose was based on the label [16]. Cycles were repeated every 21 days until progressive disease or unacceptable toxicity. In the Seidman trial, patients assigned to gemcitabine-docetaxel received gemcitabine (1,000 mg/m2 30-minute i.v. infusion) on days 1 and 8 and docetaxel (75 mg/m2 60-minute i.v. infusion) on day 1 [30]. Patients assigned to capecitabine-docetaxel received oral capecitabine (1,000 mg/m2 twice daily) on days 1 through 14 and docetaxel (75 mg/m2 60-minute i.v. infusion) on day 1. Cycles were repeated every 21 days until disease progression. The capecitabine dose was reduced because of the high incidence of diarrhea and hand-foot syndrome that was observed in the earlier Chan trial [29]. Patients who progressed on induction gemcitabine-docetaxel or capecitabine-docetaxel received single-agent capecitabine or gemcitabine, respectively (using the induction doses and schedules) within 4 weeks of documented progressive disease. Dose reductions were described in the original reports [29, 30]. Efficacy Evaluations In the Chan trial, the primary endpoint was progression-free survival (PFS); secondary endpoints were OS, overall response rate (ORR), time-to-treatment failure, safety, and quality of life [29]. In the Seidman trial, the primary endpoint was TTP; secondary endpoints were ORR, OS, and safety. Time-to-treatment failure was added as a post hoc analysis [30]. Tumor responses were evaluated using RECIST 1.0 criteria [31] every third cycle. Confirmatory scans were performed at least 3 weeks after the first evidence of response [29, 30]. Statistical Analyses Patient-level data from two individual studies were pooled for analyses. OS was calculated from the date of randomization until death from any cause or censored at last known alive date. PFS was calculated from the date of randomization until first date of documented progression or death from any cause or censored last follow-up visit for patients who were still alive and progression-free. OS and PFS were estimated using the Kaplan-Meier product limit method [32]. Cox proportional hazards models [33] and log-rank tests, stratified by study, were used to calculate hazard ratios (HRs) and to compare survival curves of the two treatment arms for OS and PFS. Pooled ORR (defined as the proportion of patients with a best overall response of complete response [CR] or partial response [PR]) and disease control rate (DCR, defined as the proportion of patients with a best overall response of CR, PR, or stable disease) were also calculated in the two treatment arms and compared using the Cochran-Mantel-Haenszel test stratified by study. Potential prognostic factors were identified initially by searching available baseline variables that significantly influenced OS or PFS at a level of p < .05 in the univariate analyses, and then were included in the multivariate analyses using stepwise Cox proportional hazards modeling for OS or PFS. Factors with p values <.05 in the multivariate analyses were considered statistically significant and prognostic. All p values were two-sided and were not adjusted for multiplicity. Caution should be used when interpreting these p values. The crossover population consisted of patients who received induction gemcitabine-docetaxel and then, upon progression, crossed over to capecitabine, and patients who received induction capecitabine-docetaxel and then, upon progression, crossed over to gemcitabine. Induction PFS was estimated for all crossover patients from the time of randomization to the date of first progressive disease or death from any cause, whichever occurred first. Results Patient Demographics Table 1 shows the baseline demographics for the pooled population. From the Chan trial, 305 patients (153 gemcitabine-docetaxel; 152 capecitabine-docetaxel) were randomized [29]; from the Seidman trial, 475 patients (239 gemcitabine-docetaxel induction phase; 236 capecitabine-docetaxel induction phase) were randomized [30]. A minority of patients received prior chemotherapy for MBC (20.9% gemcitabine-docetaxel; 19.1% capecitabine-docetaxel). The arms were well balanced, with the possible exceptions of crossover status and progesterone receptor status. HER2 status was not available in the Seidman trial [30] and prior use of trastuzumab was unknown in both trials. Table 1 Baseline demographics of pooled population Open in new tab Table 1 Baseline demographics of pooled population Open in new tab Efficacy Pooled Efficacy of Gemcitabine-Docetaxel Versus Capecitabine-Docetaxel In the pooled population, OS for patients randomized to gemcitabine-docetaxel versus capecitabine-docetaxel was not statistically different (stratified log-rank p = .824, HR = 1.02, 95% CI, 0.86–1.20; median 21.5 months vs. 22.0 months) (Fig. 1A). In the pooled population, PFS for patients randomized to gemcitabine-docetaxel versus capecitabine-docetaxel was not statistically different (stratified log-rank p = .079, HR = 1.15, 95% CI, 0.98–1.35; median 8.5 months vs. 8.5 months) (Fig. 1B). Figure 1 Open in new tabDownload slide Kaplan-Meier curves of the pooled population. (A): Overall survival. (B): Progression-free survival. Abbreviations: CD, capecitabine-docetaxel; CI, confidence interval; GD, gemcitabine-docetaxel; HR, hazard ratio. Figure 1 Open in new tabDownload slide Kaplan-Meier curves of the pooled population. (A): Overall survival. (B): Progression-free survival. Abbreviations: CD, capecitabine-docetaxel; CI, confidence interval; GD, gemcitabine-docetaxel; HR, hazard ratio. In the pooled population, the ORR was 32.1% (95% CI, 27.5–37.0) for gemcitabine-docetaxel and 34.3% (95% CI, 29.6–39.2) for capecitabine-docetaxel (Cochran-Mantel-Haenszel p = .526). The DCR (CR + PR + stable disease) was 56.6% (95% CI, 51.6–61.6) for gemcitabine-docetaxel and 57.5 (95% CI, 52.4–62.4) for capecitabine-docetaxel (Cochran-Mantel-Haenszel p = .781). Pooled Efficacy of Crossover Population In the pooled crossover population, although there was a trend favoring gemcitabine-docetaxel, the difference in OS among patients initially receiving gemcitabine-docetaxel versus capecitabine-docetaxel was not statistically significant (unstratified log-rank p = .171, HR = 0.82, 95% CI, 0.62–1.09; median 25.5 months vs. 23.5 months) (Fig. 2A). Likewise, there was a trend toward improved PFS of the induction phase with gemcitabine-docetaxel, but the difference in PFS in the pooled crossover population receiving gemcitabine-docetaxel versus capecitabine-docetaxel was not statistically significant (unstratified log-rank p = .557, HR = 0.93, 95% CI, 0.73–1.19; 8.3 months vs. 6.5 months) (Fig. 2B). Figure 2 Open in new tabDownload slide Kaplan-Meier curves of the crossover subpopulation within the pooled population. (A): Overall survival. (B): Progression-free survival. Abbreviations: CD-G, capecitabine-docetaxel crossed over to gemcitabine; CI, confidence interval; GD-C, gemcitabine-docetaxel crossed over to capecitabine; HR, hazard ratio. Figure 2 Open in new tabDownload slide Kaplan-Meier curves of the crossover subpopulation within the pooled population. (A): Overall survival. (B): Progression-free survival. Abbreviations: CD-G, capecitabine-docetaxel crossed over to gemcitabine; CI, confidence interval; GD-C, gemcitabine-docetaxel crossed over to capecitabine; HR, hazard ratio. Prognostic Factors Using a univariate Cox proportional hazards model, we found that several potential prognostic factors were associated with improved OS or PFS at a significance level of p < .05 (Table 2). For OS, these were race, ECOG PS, estrogen receptor status, progesterone receptor status, prior surgery, prior radiotherapy, prior adjuvant taxane, and number of metastatic sites; for PFS, these were ECOG PS, estrogen receptor status, progesterone receptor status, prior adjuvant taxane, time since diagnosis, and number of metastatic sites. These factors were chosen for further multivariate analysis using the stepwise multivariate Cox proportional hazards modeling. As a result, race, ECOG PS, estrogen receptor status, prior radiotherapy, prior adjuvant taxane, and number of metastatic sites were significant at p < .05 for OS, and ECOG PS, estrogen receptor status, prior adjuvant taxane, and number of metastatic sites were significant at p < .05 for PFS (Table 3). Table 2 Prognostic factors for pooled overall survival and progression-free survival (univariate analysis) Open in new tab Table 2 Prognostic factors for pooled overall survival and progression-free survival (univariate analysis) Open in new tab Table 3 Prognostic factors for pooled overall survival and progression-free survival (multivariate analysis) Open in new tab Table 3 Prognostic factors for pooled overall survival and progression-free survival (multivariate analysis) Open in new tab A multivariate prognostic model was constructed by incorporating all identified adverse prognostic factors. The prognostic factors were grouped according to the criteria shown in supplemental online Table 1. For OS, the low-risk group (n = 121) had none or one negative prognostic factor, the intermediate-risk group (n = 500) had two or three negative prognostic factors, and the high-risk group (n = 159) had four to six negative prognostic factors; for PFS, the low-risk group (n = 121) had no negative prognostic factors, the intermediate-risk group (n = 558) had one or two negative prognostic factors, and the high-risk group (n = 101) had three or four negative prognostic factors. The median OS was 11.3 (95% CI, 9.5–14.1) months in the high-risk group, 22.9 (95% CI, 20.3–24.9) months in the intermediate-risk group, and 36.5 (95% CI, 27.5–44.5) months in the low-risk group (Fig. 3A). The median PFS was 4.4 (95% CI, 3.5–5.5) months in the high-risk group, 8.6 (95% CI, 7.8–9.2) months in the intermediate-risk group, and 11.2 (95% CI, 9.3–13.4) months in the low-risk group (Fig. 3B). In all risk groups, the p value was <.001 using a stratified log-rank test. Figure 3 Open in new tabDownload slide Kaplan-Meier curves of the risk groups. (A): Overall survival. (B): Progression-free survival. The stratified log-rank test for both overall survival and progression-free survival risk groups was p < .001. Abbreviations: CI, confidence interval; Int, intermediate. Figure 3 Open in new tabDownload slide Kaplan-Meier curves of the risk groups. (A): Overall survival. (B): Progression-free survival. The stratified log-rank test for both overall survival and progression-free survival risk groups was p < .001. Abbreviations: CI, confidence interval; Int, intermediate. Subgroup Analyses for Prognostic Factors Figure 4A shows the subgroup analysis for OS. Although there were trends toward a more favorable OS with capecitabine-docetaxel in some subgroups (nonwhite race, ECOG PS >0, estrogen receptor negative, prior radiotherapy, prior taxane usage, one or two metastatic sites, and high-risk group), no interaction tests were statistically significant at the p = .05 level (p values not shown), suggesting that there is no heterogeneity of treatment effects across the levels of the prognostic factors and two treatment arms. Also, in most subgroups, there were trends toward a more favorable PFS with capecitabine-docetaxel (Fig. 4B). However, with the exception of a possible interaction between metastatic sites and treatment arms (favored capecitabine-docetaxel in patients with one or two vs. more than two metastatic sites; p = .026), interaction tests for other prognostic factors were not statistically significant (p values not shown). Figure 4 Open in new tabDownload slide Forest plots. Subgroups were identified using a multivariate Cox model. Squares indicate point estimates; horizontal lines indicate 95% CIs. (A): Overall survival. (B): Progression-free survival. HR >1 favors CD; HR <1 favors GD. For PFS, Wald p = .026 for treatment arm and number of metastatic sites. Abbreviations: CD, capecitabine-docetaxel; CI, confidence interval; ECOG PS, Eastern Cooperative Oncology Group performance status; ERS, estrogen receptor status; GD, gemcitabine-docetaxel; HR, hazard ratio; OS, overall survival; PFS, progression-free survival. Figure 4 Open in new tabDownload slide Forest plots. Subgroups were identified using a multivariate Cox model. Squares indicate point estimates; horizontal lines indicate 95% CIs. (A): Overall survival. (B): Progression-free survival. HR >1 favors CD; HR <1 favors GD. For PFS, Wald p = .026 for treatment arm and number of metastatic sites. Abbreviations: CD, capecitabine-docetaxel; CI, confidence interval; ECOG PS, Eastern Cooperative Oncology Group performance status; ERS, estrogen receptor status; GD, gemcitabine-docetaxel; HR, hazard ratio; OS, overall survival; PFS, progression-free survival. Discussion Despite improvements in outcomes for patients with MBC, the optimal use of existing chemotherapeutic agents continues to be debated. Gemcitabine and capecitabine in combination with taxanes are routinely used in first-line MBC patients who received prior anthracyclines, but the optimal use of these combinations is unknown. This pooled analysis of two international phase III trials that compared gemcitabine-docetaxel with capecitabine-docetaxel [29, 30] was performed to provide guidance for the best use of these agents. Results obtained with the pooled population confirm the lack of efficacy difference between gemcitabine-docetaxel and capecitabine-docetaxel that had been previously reported for the individual phase III trials [29, 30]. In the pooled population, there were no between-arms differences in OS (HR = 1.02), PFS (HR = 1.15), or ORR (p = .526). The median OS (21.5 months) and median PFS (8.5 months) reported here for the pooled gemcitabine-docetaxel arm are at least equivalent to those obtained for the gemcitabine-paclitaxel combination in the registration trial (OS = 18.6 months; TTP = 6.14 months), whereas the ORR for gemcitabine-docetaxel in the pooled population (32.1%) seems lower than that in the registration trial (41.4%) [11]. Here, the capecitabine-docetaxel combination (median OS = 22.0 months; median PFS = 8.5 months) outperformed itself relative to the registration trial (median OS = 14.5 months; median TTP = 6.1 months) [12]. However, patients in the capecitabine registration trial [12] received more prior treatments than patients in the pooled population [29, 30]. Because there was no efficacy difference between gemcitabine-docetaxel and capecitabine-docetaxel in the pooled population, it was of interest to identify subsets of patients that may benefit the most from these regimens. Our data also show that although there were trends toward improved OS and PFS with gemcitabine-docetaxel in the pooled crossover population, the differences between gemcitabine-docetaxel and capecitabine-docetaxel were not statistically significant. A possible explanation is that capecitabine was better tolerated as a single agent than when combined with docetaxel. This is supported by the toxicity profiles of the Chan and Seidman trials; both trials had high rates of toxicity-related discontinuations in the capecitabine-docetaxel arms compared with the gemcitabine-docetaxel arms (Seidman = 28.4% vs. 18.0%; Chan = 27% vs. 13% in the induction phases) [29, 30]. However, this was a post hoc subset analysis, and the usual caveats regarding subset analyses should be noted [34]. It should also be noted that in the crossover population, data for secondary progression (e.g., from crossover to further progression) were not collected in the Chan trial [29], so the induction PFS in the crossover population should be viewed with caution. Given the limitations of this subset analysis, the trends toward improved OS and PFS with gemcitabine-docetaxel suggest that gemcitabine-docetaxel followed by capecitabine might be a preferred sequence option for certain MBC patients and may warrant further evaluation. However, it should be noted that gemcitabine in combination with docetaxel is currently not approved in the U.S., European Union, or China for the treatment of MBC. Although several prognostic factors were associated with improved outcomes in the overall pooled population, there were no interactions in OS or PFS between gemcitabine-docetaxel and capecitabine-docetaxel and any of the tested prognostic factors, with the possible exception of longer PFS in the capecitabine-docetaxel arm in the subgroup of patients with one or two sites of metastases. This suggests that there is no evidence of benefiting more in either regimen within each level of prognostic factors. Using risk factors identified in a multivariate prognostic model, we categorized patients into low-, intermediate-, and high-risk groups. The high-risk group had worse outcomes (OS and PFS) than the other risk groups. However, no statistically significant interaction was found between treatment arms and risk groups. These data show that prior adjuvant taxane was a prognostic factor for both OS and PFS. However, it should be noted that in the Seidman trial [30], a large proportion of patients had an unknown adjuvant taxane status (67.8% gemcitabine-docetaxel; 69.5% capecitabine-docetaxel). For the purpose of the pooled analysis, patients with an unknown taxane status were considered to have received no prior adjuvant taxane, but it is possible that this assumption was incorrect for some patients. Another potential issue with this analysis is that, in the Chan trial, enrolled patients were required to have received one prior anthracycline regimen [29], whereas the Seidman trial did not have this requirement, so a significant proportion of patients had not been exposed to anthracyclines (approximately 42%) [30]. Finally, it should be noted that patients with prior exposure to adjuvant taxanes may acquire drug resistance to taxanes. Thus, non–cross-resistant regimens should be evaluated in those patients toward optimization. Finally, it should be noted that, with the exception of capecitabine, the drug doses and schedules were identical in the parent trials. The Chan trial used the approved capecitabine dose (1,250 mg/m2), whereas the Seidman trial used a reduced dose (1,000 mg/m2) [29, 30]. When used as a component of capecitabine-docetaxel or as monotherapy, the capecitabine dose can be reduced without compromising TTP or OS [35]. Therefore, the efficacy results should not have been affected by using pooled data from the previously described trials [29, 30]. Because the reduced capecitabine dose is associated with a decreased incidence of treatment-related adverse events, particularly hand-foot syndrome, diarrhea, and stomatitis [35], it would not have been appropriate to perform a safety analysis on the pooled population. Nonetheless, based on the parent trials, it is known that gemcitabine-docetaxel and capecitabine-docetaxel have different toxicity profiles; capecitabine-docetaxel is generally associated with higher incidences of grade 3-4 gastrointestinal toxicity, hand-foot syndrome, and mucositis, and gemcitabine-docetaxel is generally associated with more grade 3-4 fatigue, elevated liver enzymes, neutropenia, leukopenia, and thrombocytopenia [29, 30]. Despite these differences, toxicity-related discontinuations in the capecitabine-docetaxel arm (28.4%) were significantly greater (p = .009) than in the gemcitabine-docetaxel arm (18.0%) in the Seidman trial [30], which is consistent with toxicity-related discontinuations observed in the Chan trial (capecitabine-docetaxel = 27%; gemcitabine-docetaxel = 13%) [29]. Conclusion Results from this analysis confirm the lack of efficacy difference between gemcitabine-docetaxel and capecitabine-docetaxel in the pooled population. In addition, there are no efficacy differences between regimens in the crossover population, as well as in almost all examined subpopulations. Several prognostic factors, such as ECOG PS, estrogen receptor status, prior adjuvant taxane, and number of metastatic sites, were associated with both improved OS and PFS in the overall pooled population. The choice of regimen should be guided by the clinical characteristics and tolerance to toxicities of the individual patient. Acknowledgments We thank Tan Ding and Weishan Shi for assistance in statistical computation. Medical writing support was provided by Lori Kornberg (Inventiv Health Clinical). Writing support (funded by Eli Lilly and Company) included drafting of the manuscript, preparing references, and assembling figures/tables. Andrew D. Seidman and Stephen Chan contributed to this manuscript equally as co-first authors. Author Contributions Conception/design: Binghe Xu, Andrew D. Seidman, Stephen Chan, Jin Wang, Cong Xu Provision of study material or patients: Binghe Xu, Stephen Chan, Cong Xu Collection and/or assembly of data: Binghe Xu, Andrew D. Seidman, Stephen Chan Data analysis and interpretation: Binghe Xu, Andrew D. Seidman, Stephen Chan, Jin Wang, Chao Zhu, Cong Xu Manuscript writing: Andrew D. Seidman, Stephen Chan, Chao Zhu Final approval of manuscript: Binghe Xu, Andrew D. Seidman, Stephen Chan, Jin Wang, Chao Zhu, Cong Xu Disclosures Jin Wang: Eli Lilly (E); Chao Zhu: Eli Lilly (E); Cong Xu: Eli Lilly (E). The other authors indicated no financial relationships. (C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board References 1 Jemal A , Bray F, Center MM. Global cancer statistics . CA Cancer J Clin . 2011 ; 61 : 69 – 90 . 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Dose-adjusting capecitabine minimizes adverse effects while maintaining efficacy: A retrospective review of capecitabine for metastatic breast cancer . Clin Breast Cancer . 2011 ; 11 : 349 – 356 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Disclosures of potential conflicts of interest may be found at the end of this article. © 2014 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Vasan, Neil; Yelensky, Roman; Wang, Kai; Moulder, Stacy; Dzimitrowicz, Hannah; Avritscher, Rony; Wang, Baliang; Wu, Yun; Cronin, Maureen T.; Palmer, Gary; Symmans, W. Fraser; Miller, Vincent A.; Stephens, Philip; Pusztai, Lajos

2014 The Oncologist

doi: 10.1634/theoncologist.2013-0377pmid: 24710307

Abstract Background. The aim of this study was to assess the frequency of potentially actionable genomic alterations in breast cancer that could be targeted with approved agents or investigational drugs in clinical trials using a next-generation sequencing-based genomic profiling assay performed in a Clinical Laboratory Improvement Amendments-certified and College of American Pathologists-accredited commercial laboratory. Methods. Fifty-one breast cancers were analyzed, including primary tumor biopsies of 33 stage I–II and 18 stage IV cancers (13 soft tissue, 3 liver, and 2 bone metastases). We assessed 3,230 exons in 182 cancer-related genes and 37 introns in 14 genes often rearranged in cancer for base substitutions, indels, copy number alterations, and gene fusions. The average median sequencing depth was 1,154×. Results. We observed 158 genomic alterations in 55 genes in 48 of 51 (94%) tumors (mean 3.1, range 0–9). The average number of potentially therapeutically relevant alterations was similar in primary (1.6, range 0–4) and in heavily pretreated metastatic cancers (2.0, range 0–4) (p = .24). The most common actionable alterations were in PIK3CA (n = 9, phosphatidylinositol 3-kinase [PI3K]/mammalian target of rapamycin [mTOR] inhibitors), NF1 (n = 7, PI3K/mTOR/mitogen-activated protein kinase inhibitors), v-akt murine thymoma viral oncogene homolog 1-3 (n = 7, PI3K/mTOR/AKT inhibitors), BRCA1/2 (n = 6, poly[ADP-ribose] polymerase inhibitors), and CCND1,2 and CCNE (n = 8)/cycline dependent kinase (CDK)6 (n = 1) (CDK4/6 inhibitors), KIT (n = 1, imatinib/sunitinib), ALK (n = 1, crizotinib), FGFR1,2 (n = 5, fibroblast growth factor receptor inhibitors), and EGFR (n = 2, epidermal growth factor receptor inhibitors). Our sequencing assay also correctly identified all six cases with HER2 (ERBB2) amplification by fluorescence in situ hybridization when tumor content was adequate. In addition, two known activating HER2 mutations were identified, both in unamplified cases. Conclusion. Overall, 84% of cancers harbored at least one genomic alteration linked to potential treatment options. Systematic evaluation of the predictive value of these genomic alterations is critically important for further progress in this field. 摘要 背景 本研究的目的是，在获得临床实验室改进修正案认证及美国病理学家学会认可的商业实验室中，使用一种新一代测序型基因组分析技术对乳腺癌进行测序分析，以确定癌灶内那些可作为已批准药物或临床试验性药物治疗靶标的基因组突变的发生频率。 方法 我们共分析了 51 例癌灶，包括 33 例 I-II 期原发性肿瘤活检组织和 18 例 IV 期癌灶活检组织（13 例为软组织灶，3 例为肝脏病灶，2 例为骨转移灶）。我们对 182 个癌症相关基因的 3230 个外显子和 14 个在癌症中常发生重排之基因的 37 个内含子进行了测序，以查找其中的碱基替代、插入缺失、拷贝数突变和基因融合。平均中位测序深度为 1154×。 结果 我们在 51 个瘤灶的 48 个 (94%) 内的 55 个基因中发现了 158 个基因组突变（平均 3.1，范围为 0-9）。原发性癌灶和进行过多重预先治疗的转移癌灶中可作为潜在治疗靶标的平均突变数大体相似（前者为 1.6，范围为 0-4；后者为 2.0，范围为 0-4）(p = 0.24)。最常见的可靶定突变为 PIK3CA （n =9，磷脂酰肌醇 3-激酶 [PI3K]/雷帕霉素的哺乳类动物靶标 [mTOR] 抑制剂）、NF1 （n =7，PI3K/mTOR/丝裂原活化蛋白激酶抑制剂）、v-akt 鼠科胸腺瘤病毒致癌基因同源体 1-3（n =7，PI3K/mTOR/AKT 抑制剂）、 BRCA1/2 （n =6，聚[ADP-核糖]聚合酶抑制剂）、CCND 1,2 和 CCNE (n =8)/细胞周期素依赖性抑制剂 (CDK)6 (n = 1)（CDK4/6 抑制剂）、KIT （n =1，伊马替尼/舒尼替尼）、ALK （n =1，克唑替尼）、FGFR1,2 （n =5，成纤维细胞生长因子受体抑制剂）以及 EGFR （n =2，表皮生长因子受体抑制剂）。在肿瘤量充足的情况下，我们的测序分析还通过荧光原位杂交法，准确地鉴定出了所有六例 HER2 (ERBB2) 扩增突变。此外，我们还检测出了两例已知的活化 HER2 突变，均出现在非扩增病例中。 结论 总体而言，84% 的癌症存在至少一种可作为潜在治疗靶标的基因组突变。对这些基因组突变的预测价值进行系统性评估，对推动这一领域的进步具有重要意义。The Oncologist 2014;19:453–458 Next-generation sequencing, Precision medicine, Molecularly targeted therapy, Predictive markers Implications for Practice: The technical ability to perform molecular profiling in the clinic is broadly available; it is now critically important to focus on assessing the clinical utility of molecular profiling as a patient selection tool. Introduction An increasing number of molecularly targeted drugs are available in the clinic as approved drugs (Table 1) or in the context of clinical trials (http://www.clinicaltrials.gov). These drugs target specific molecular abnormalities, including mutated protein kinases and amplified or rearranged genes. Cancers that carry these abnormalities often, but not always, respond to the corresponding targeted therapies. For example, the HER2 gene-amplified breast cancers benefit from HER2-targeted therapies [1]. Chronic myeloid leukemia with the BCR-ABL translocation responds to inhibitors of the BCR-ABL kinase [2]. Lung cancers with activating mutations of EGFR can benefit from epidermal growth factor receptor (EGFR) inhibitors [3], and lung cancers that carry an activating rearrangement of the ALK kinase often respond to anaplastic lymphoma kinase (ALK) inhibitors [4]. BRAF-mutant melanoma may respond to v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors [5], and activating mutations in c-KIT or PDGFR render gastrointestinal stromal tumors sensitive to KIT inhibitors [6]. The repertoire of genomic abnormalities and their incidence differ across different histologic types of cancer, but most abnormalities are not unique to any particular cancer type [7]. Although the same genomic abnormality may play a more important driver role in one type of cancer compared with another, there is also evidence to support that different types of cancers could respond to the same biologically targeted agent if they harbor the sensitizing genomic abnormality. For example, HER2-targeted therapies are effective in gastric and esophageal adenocarcinomas that have HER2 gene amplification [8]. The BRAF inhibitor vemurafenib has shown promising results in patients with BRAF mutant metastatic papillary thyroid cancer and malignant histocytosis [9]. The purpose of the current study is to survey the potentially targetable genomic abnormalities in primary and metastatic breast cancers using a standardized, commercially available next-generation sequencing (NGS)-based genomic profiling assay on routine clinical tissue samples. Table 1 U.S. Food and Drug Administration-approved molecularly targeted drugs for cancer Open in new tab Table 1 U.S. Food and Drug Administration-approved molecularly targeted drugs for cancer Open in new tab Several studies have examined the mutational landscape of breast cancer using whole genome or partial genome sequencing [10–12]. Many of the initial whole genome and whole exome sequencing studies included very few patients and had limited sensitivity because of low sequencing depth. Other studies included a larger number of patients but restricted the analysis to a modest number of known oncogenic mutations [13–15]. The most comprehensive genomic analysis of breast cancer was recently reported by the Cancer Genome Atlas Network (TCGA) [16]. Whole exome sequencing was performed on 507 breast cancers with modest sequencing depth (30% of target sequences had coverage <20-fold). Low to moderate coverage limits the sensitivity to detect genomic events that may be restricted to relatively small tumor cell subpopulations. In aggregate, the above studies have established TP53 and PI3KCA as the most frequently mutated genes in breast cancer and also revealed a large number of low-frequency potentially druggable genomic anomalies in variable proportions of breast cancers. These observations raise the possibility that subsets of breast cancers may be candidates for targeted therapies aimed at rare genomic abnormalities. In this study, we performed full sequencing of 3,230 exons in 182 cancer-related genes and 37 introns in 14 genes that are frequently rearranged in cancers. As a result of the targeted sequencing method and the high, uniform coverage (>99% of exons covered ≥100×), our detection sensitivity for minor allele variants is significantly higher than those achieved in the TCGA. The analysis was performed using a standard operating procedure in a CLIA-certified (Clinical Laboratory Improvement Amendments, http://www.cms.gov/clia), CAP-accredited (College of American Pathologists) commercial molecular diagnostic laboratory; therefore, the results can be directly incorporated into clinical trials as patient selection criteria or used for medical decision making. Materials and Methods Patients and Samples Fifty-one breast cancers were analyzed, including primary tumor biopsies in 1 stage I, 17 stage II, and 15 stage III primary breast cancers and 18 total stage IV metastasis biopsies of 13 soft tissue, 3 liver, and 2 bone tumors. The mean age was 52 years (range 28–86 years), 58% ER+, 20% HER2+, 31% triple negative. Fine-needle aspirations (FNA) of the primary cancers were obtained prospectively at the time of diagnosis before any therapy in the context of a prospective biomarker discovery protocol at MD Anderson Cancer Center. Metastatic cancer biopsies, also obtained through FNA, were collected in the context of a prospective biomarker validation trial [17]. Patients with metastatic breast cancers had received at least two prior lines of therapy for metastatic cancer and were exposed to an average of seven different drugs (range 5–17), including adjuvant therapy before the biopsies were obtained. Both tissue collection studies were approved by the Institutional Review Committee of the University of Texas MD Anderson Cancer Center, and all patients signed informed consent. Analysis was performed on the already collected tissue samples, and the results were not included in the medical records or used for treatment decision making. Next-Generation Sequencing (NGS)-Based Genomic Profiling Two FNA aspirates were pooled into a single vial for subsequent molecular analysis, and NGS-based targeted sequencing was performed by Foundation Medicine. Genomic DNA was extracted using the Maxwell 16 FFPE Plus LEV DNA Purification kit (Promega, Madison, WI, http://www.promega.com) and quantified using a PicoGreen fluorescence assay (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Fifty to 200 ng of DNA was sheared to 100–400 base pair fragments by sonication, followed by end-repair, dA-addition, and ligation of indexed, Illumina (San Diego, CA, http://www.illumina.com) sequencing adaptors. Sequencing libraries were hybridization captured using a pool of >24,000 individually synthesized 5′-biotinylated DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, http://www.idtdna.com/Home/Home.aspx) that were designed to target 182 cancer-related genes and 37 additional introns in 14 genes often rearranged in cancer, corresponding to 1.14 million total base pairs (supplemental online Table 1). DNA sequencing is performed using the HiSeq-2000 instrument (Illumina) with 49 × 49 paired-end reads. Point mutations (base substitutions), short insertions/deletions (indels), focal amplifications, homozygous deletions, and chromosomal rearrangements were analyzed using Bayesian algorithms, local assembly, comparison with process-matched normal controls, and analysis of chimeric pairs, respectively. The method was optimized to detect >5% mutant allele frequency (MAF) of base substitutions and >10% MAF of indels with >99% accuracy. The validated accuracy of copy number alterations was >95%. Methodological details and analytical validity of the assay were reported previously [18]. The results were annotated and interpreted through dbSNP, COSMIC, and medical literature to assemble the final report of actionable genomic alterations. We did not analyze matched normal specimens; however, all reported mutations have been identified in previously published cancer-sequencing studies and were therefore considered somatic events. An alteration was categorized as potentially “actionable” if linked to an approved therapy in breast cancer or another solid tumor, or if it mapped to a pathway targetable with an approved drug or a drug in clinical trials. Results All biopsies yielded the 50 ng of DNA required for analysis. The average median sequencing depth was 1,154-fold with >99% of nucleotides covered ≥100-fold. The time from DNA processing to final report required 7–14 days (Fig. 1). We observed 158 genomic alterations in 48 of 51 (94%) tumors (mean 3.1, range 0–9) in 55 genes. In total, 30 of 33 (91%) primary and 18 of 18 (100%) metastatic breast cancers had at least one genomic alteration. The 158 genomic alterations observed included 45 base substitutions (15%), 24 short insertions/deletions (28%), 63 focal amplifications (40%), 18 homozygous deletions (11%), and 8 rearrangements (5%) (Fig. 2). Overall, 84% of samples harbored at least one actionable alteration (Fig. 3). These actionable targets included the following: PIK3CA (n = 9, 18%), HER2 (n = 9, 18%), NF1 (n = 7, 14%), MCL1 (n = 6, 12%), and PTEN (n = 5, 10%). Twenty-five other genes with actionable alterations were identified, occurring at lower frequencies (n = 4 or fewer, <10%). Figure 1 Open in new tabDownload slide Schematic illustrating tumor sample preparation, sequencing library preparation, the analysis pipeline, and clinical reporting. Reprinted with modification from Nature Biotech 31:1023-1031, 2013. Figure 1 Open in new tabDownload slide Schematic illustrating tumor sample preparation, sequencing library preparation, the analysis pipeline, and clinical reporting. Reprinted with modification from Nature Biotech 31:1023-1031, 2013. Figure 2 Open in new tabDownload slide Pie chart of the alteration classes in the 51 breast cancers sequenced in this study. Figure 2 Open in new tabDownload slide Pie chart of the alteration classes in the 51 breast cancers sequenced in this study. Figure 3 Open in new tabDownload slide Distribution of alterations observed in this study. For abbreviations and information on these genes, please see http://www.genecards.org. Abbreviations: FGFR, fibroblast growth factor receptor; MEK, mitogen-activated protein kinase kinase; PARP, poly(ADP-ribose) polymerase; PI3, phosphatidylinositol 3; PI3K, phosphatidylinositol 3-kinase. Figure 3 Open in new tabDownload slide Distribution of alterations observed in this study. For abbreviations and information on these genes, please see http://www.genecards.org. Abbreviations: FGFR, fibroblast growth factor receptor; MEK, mitogen-activated protein kinase kinase; PARP, poly(ADP-ribose) polymerase; PI3, phosphatidylinositol 3; PI3K, phosphatidylinositol 3-kinase. The specific alterations that were observed in the most frequently actionable targets included the following: PIK3CA C420R, E542K, E545A, E545K, H1047R (H1047R occurred in four cases); HER2 amplification and HER2 S310F and L755S mutations; NF1 truncation; MCL1 amplification; PTEN truncation; and PTEN S170I mutation. Sequencing-based HER2 copy number assessment correctly identified HER2 gene amplification in all six cases that were HER2-positive by routine clinical testing and had adequate tumor cellularity for NGS analysis. Additional mutations in other well-known oncogenes were observed, including AKT1 E17K (three cases); ALK V757M; DNMT3A R882C; ESR1 Y537C, Y537N, and D538G; KIT K642E; and KRAS G12D. Numerous other alterations with no available therapeutic implications were also observed in each cancer, including MYC amplification (n = 8, 16%) and TP53 alteration (n = 32, 63%: 31 mutations and 1 homozygous deletion), which were the most frequent alterations observed. Thirty-nine (76%) tumors exhibited multiple alterations (Fig. 4). Only nine (18%) tumors exhibited single genetic alterations, and three (6%) tumors exhibited no detectable alterations. The average number of alterations was 2.9 in primary tumors and 3.4 in heavily pretreated metastatic cancers (p = .31). The average number of actionable alterations was 1.6 in primary (range 0–4) and 2.0 (range 0–4) in heavily pretreated metastatic cancers (p = .24). Figure 4 Open in new tabDownload slide Comprehensive annotation of genomic alterations of 51 breast cancers. Figure 4 Open in new tabDownload slide Comprehensive annotation of genomic alterations of 51 breast cancers. Discussion This study, along with numerous other reports, demonstrates that comprehensive sequencing of potentially druggable genes can be performed on small, routine, diagnostic needle biopsies in a clinically relevant time frame. The incorporation of NGS to profile cancer biopsies for therapeutic targets has become a reality in the clinic. Our results also indicate that a large fraction of breast cancers contains genomic abnormalities that may render them susceptible to approved or investigational therapies. The distribution and frequency of the most frequent genomic alterations observed in this study are similar to the findings reported by the TCGA breast cancer project including TP53 and PI3KCA mutations and HER2 amplification [16]. Importantly, sequencing-based assessment of HER2 gene copy number correctly identified six of the six clinically HER2-amplified cases when assay requirements were met. We also identified functional mutations in the HER2 gene (S310F, L755S) in two cases in the absence of amplification. Both of these mutations have previously been reported in the literature; S310F is an activating mutation [19], and L755S is a mediator of resistance to lapatinib [20]. We also detected numerous, less frequent but directly targetable alterations in KIT (K642E, an activating mutation commonly seen in gastrointestinal stromal tumors [21] and in acral and mucosal melanomas [22]), ALK (V757M, mutation in the extracellular domain that was previously observed in colorectal cancer [10]; ALK translocations cause non-small cell lung cancer, and mutations cause neuroblastoma [23]), and AKT1 mutation (E17K, a constitutively activating mutation that was also seen in colorectal, endometrial, lung, and ovarian cancers and acute leukemias [24]). We observed amplifications of the EGFR, FGFR1, FGFR2, AKT2, and AKT3 genes, all of which can be directly targeted by approved or investigational drugs. A second collectively large group includes alterations that are not direct drug targets but represents biological pathways that could be targeted by drugs. These include deletion and truncation of neurofibromin/NF1 (Y1625fs*5, A2646fs*14, Q1399*, W1559*, and c.1393-1G>T splice site mutation), a negative regulator of the RAS signal transduction pathway, and a mutation in KRAS (G12D, a mutation that confers resistance to anti-EGFR therapy in colorectal cancer). Amplification of MCL1 gene that encodes BCL2-like protein inhibits apoptosis and amplifications in cyclins E and D, and MYC, as well as homozygous loss of PTEN, BRCA2, and truncation of BRCA1 (S1253fs*10, Q1756fs*74, K1759fs*70) and BRCA2 (V1610fs*4, L2092fs*7). It is also important to recognize that almost all cancers harbored multiple abnormalities. The average number of targetable alterations per sample was surprisingly similar, 1.6 in primary versus 2.0 in heavily treated metastatic breast cancers. The multiplicity of anomalies suggests that ultimately combinatorial therapies may be required for optimal efficacy. Studying the functional interaction between the multiple somatic alterations that tumors acquire and the equally large number of functional germline polymorphisms that we all carry represents a very important and fertile ground for research. However, it is also important to remember that, despite the multiplicity of functional genomic alterations, targeting single alterations can provide clinical benefit (e.g., antiestrogens, HER2-targeted therapies, etc.), which justifies clinical testing of targeted therapies. The increasing clinical availability of NGS-based profiling assays and the results that they generate raise several important clinical questions. The most important one is how to act on the results. Currently, all U.S. Food and Drug Administration (FDA)-approved therapies that target a specific molecular abnormality in breast cancer are HER2-targeted agents (trastuzumab, lapatinib, pertuzumab, and ado-trastuzumab-emtansine). However, there are 26 other FDA-approved drugs (Table 1) that target specific somatic molecular abnormalities seen in cancer, most of which are encountered at low frequencies in breast cancer. It is attractive to consider the use of these targeted agents in tumor types in which they have not been approved to evaluate the predictive utility of target profiling [25, 26]. The safety profiles of already approved agents are already well established, and the key challenge is to assess clinical benefit. Many academic institutions pursue “molecular triaging” or “basket” studies that involve performing molecular target profiling and using the results to steer patients to clinical trials that test targeted therapies [27]. The U.S. National Cancer Institute recently announced plans for the MATCH trial that aims to provide access to therapies that target the specific mutations found in a cancer [28]. Pharmaceutical companies are also considering providing a portfolio of their approved drugs and some investigational agents for molecular triaging studies that encompass multiple cancer types; however, no such clinical trial is open yet. The fragmentation of the patient population into very small, molecularly defined subsets will be a challenge during the implementation of these trials. It is hard to envisage efficiently running separate studies for BRAF mutant, HER2 mutant, AKT mutant, ALK mutant, cKIT mutant, and FGFR-amplified breast cancer subsets. More importantly, only a minority of cancer patients receive their care through large academic centers and have access to these studies; overall, only 5%–10% of U.S. cancer patients participate in clinical trials [29]. One could also consider other innovative ways to harness the broad scientific interest in this field and the motivation of patients with incurable diseases to broaden their treatment options. Establishment of a nationwide registry of molecularly targeted therapies of rare genomic abnormalities could rapidly move the field forward. The registry would collect basic clinical and molecular information and simple, but informative, outcome data such as duration of therapy (i.e., a composite endpoint of tolerability and cancer control). Such registry could even be linked to coverage for treatment and maintained by the Center for Medicaid and Medicare Services or other third-party payers. Drug activity could be assessed and made public at predefined milestones, for example, after accruing the first 100 patients nationwide with a particular molecular anomaly. The registry would not replace rigorous clinical trials but could identify clinical scenarios in which further trials are needed. Conclusion In summary, NGS-based genomic profiling of DNA from breast cancer needle biopsies to assess potential therapeutic targets is readily available. Target profiling showed a high frequency of genomic alterations linked to potential treatment option with approved or investigational drugs. These alterations include multiple different types of abnormalities, including gene amplification and deletions, frame shifts, small insertions and deletions (indels), single-nucleotide substitutions, and gene rearrangements with fusion genes. These results raise a broad spectrum of distinct clinically testable therapeutic hypotheses for individual patients. Testing the clinical efficacy of these treatment options represents a major challenge for the traditional clinical trial system as a result of the varied and individually small patient subsets. Innovative approaches to provide access to potentially effective drugs and to capture systematically the outcome of therapy are needed to move the field forward and to provide benefit to the greatest number of patients without long delays. Author Contributions Conception/Design: Roman Yelensky, Kai Wang, Stacy Moulder, W. Fraser Symmans, Philip Stephens, Yun Wu, Baliang Wang, Rony Avritscher, Lajos Pusztai, Vincent Miller Provision of study material or patients: Kai Wang, Stacy Moulder, W. Fraser Symmans, Philip Stephens, Yun Wu, Baliang Wang, Rony Avritscher, Lajos Pusztai Collection and/or assembly of data: Neil Vasan, Gary Palmer, Lajos Pusztai Data analysis and interpretation: Neil Vasan, Roman Yelensky, Hannah Dzimitrowicz, Gary Palmer, Lajos Pusztai, Maureen Cronin Manuscript writing: Neil Vasan, Roman Yelensky, Lajos Pusztai Final approval of manuscript: Gary Palmer, Lajos Pusztai, Maureen Cronin, Vincent Miller Disclosures Lajos Pusztai: Foundation Medicine (RF); Roman Yelensky: Foundation Medicine (E, IP, OI); Kai Wang: Foundation Medicine (E, OI); Gary Palmer: Foundation Medicine (E, OI); Philip Stephens: Foundation Medicine (E, OI); Maureen Cronin: Celgene (E); Celgene, Foundation Medicine (OI); Vincent Miller: Foundation Medicine (E, OI). The other authors indicated no financial relationships. (C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board References 1 Hudis CA . Trastuzumab—mechanism of action and use in clinical practice . N Engl J Med . 2007 ; 357 : 39 – 51 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Capdeville R , Buchdunger E, Zimmermann J. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug . Nat Rev Drug Discov . 2002 ; 1 : 493 – 502 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Lynch TJ , Bell DW, Sordella R. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib . N Engl J Med . 2004 ; 350 : 2129 – 2139 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Gandhi L , Jänne PA. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Disclosures of potential conflicts of interest may be found at the end of this article. © 2014 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Schneider, Jeffrey G.; Farhadfar, Nosha; Sivapiragasam, Abirami; Geller, Matthew; Islam, Shahidul; Selbs, Elena

2014 The Oncologist

doi: 10.1634/theoncologist.2013-0311pmid: 24705979

Abstract Introduction. Excision repair cross-complementation group 1 (ERCC1) expression by non-small cell lung cancer (NSCLC) has been reported to predict resistance to platinum-based therapies. On this basis, several commercial laboratories have offered ERCC1 testing to facilitate clinical decision making, but the reliability of such assays has recently been called into question. Methods. First, three large commercial laboratories were queried for their cumulative ERCC1 test results in NSCLC patients to compare their independent rates of ERCC1 expression. Second, identical tumor blocks from individual NSCLC patients underwent round-robin analysis to evaluate interlaboratory concordance for ERCC1 expression. Third, a retrospective review of medical records from NSCLC patients identified those who were both highly responsive and resistant to platinum-based chemotherapies. Tumor blocks from these patients were then used in a gold standard analysis to determine individual laboratory sensitivity and specificity for ERCC1 results. Results. Significant differences were observed in independent laboratory ERRC1 expression rates (Clarient 70% vs. Genzyme 60% vs. Third Laboratory 44%, p < .0001 for all two-way comparisons). Only 4 of 18 tumors examined in round-robin analysis were fully concordant (κ ≤ 0.222 for all two-way comparisons). In preselected platinum responsive and resistant specimens, none of these three commercially marketed laboratory assays achieved a specificity of greater than 50%. Conclusion. The results of commercial laboratory testing for ERCC1 are inconsistent and unreliable. Better validation and postmarketing surveillance should be mandated before tumor biomarker assays are allowed to enter the clinical arena. ERCC1, Tumor predictive biomarker, Platinum sensitivity, Lung cancer Implications for Practice: Prior reports have suggested that clinical benefit from platinum-based chemotherapy may be predicted by determining tumor expression levels of the excision repair cross-complementation group 1 (ERCC1) enzyme. On this basis, ERCC1 testing has been recognized in consensus guidelines and offered by commercial laboratories. In this study, we compared ERCC1 expression levels on identical tumor specimens, as determined by three different commercial laboratories. We also evaluated each laboratory's ERCC1 assay for its ability to correctly predict platinum resistance or sensitivity in tumor specimens that were preselected on the basis of clinically observed platinum responsiveness. ERCC1 testing was found to be both highly discordant and uniformly unreliable at all three laboratories. We conclude that these ERCC1 assays should not be used in routine clinical practice or recommended in current practice guidelines. Our experience also demonstrates how postmarketing surveillance may help to ensure the technical reliability and clinical utility of predictive tumor biomarker assays. Introduction Platinum-based chemotherapies represent the first-line metastatic treatment of choice [1, 2] and the only effective adjuvant option for most patients with non-small cell lung cancer (NSCLC) [3–5]. Even so, these therapies have modest benefits that must be weighed against significant treatment-related side effects [6]. Predictive markers are much needed to allow selective treatment of the subpopulation of NSCLC patients who are most likely to benefit from platinum therapies while sparing the remainder from treatment-associated morbidity. Excision repair cross-complementation group 1 (ERCC1) is one of the critical proteins involved in the process whereby cells normally repair platinated DNA and circumvent treatment-induced cytotoxicity [7–11]. Consequently, the upregulation of ERCC1 by tumors represents a plausible mechanism for their resistance to platinum-based chemotherapies [11–13]. Numerous prior reports have described an association between the tumor expression of ERCC1 and lack of clinical benefits following platinum-based chemotherapy [14–20]. In the adjuvant setting, Olaussen et al. reported that early-stage NSCLC patients whose tumors were “positive” for high levels of ERCC1 protein expression derived no benefit from platinum-based therapies (hazard ratio for death, 1.14, p = .40), whereas ERCC1 “negative” patients realized important platinum attributable benefit (hazard ratio for death, 0.65, p = .002) [18]. In the metastatic setting, Cobo et al. prospectively randomized NSCLC patients to standard platinum-based chemotherapy (control group) versus ERCC1-directed therapy (experimental group). Patients in the experimental group, who received platinum therapy only if their tumors demonstrated low ERCC1 mRNA expression and nonplatinum chemotherapy otherwise, experienced superior response rates (51% vs. 39%, p = .02) [15]. Based on such reports suggesting clinical utility of ERCC1 testing as well as its recognition in current National Comprehensive Cancer Network guidelines [21], several commercial laboratories have offered ERCC1 testing to assist medical oncologists and their patients when deciding whether to administer platinum-based chemotherapy regimens. However, ERCC1 testing methodology and criteria for high and low results are nonstandardized and may vary considerably between laboratories. In addition, technical problems with ERCC1 assays have become apparent and prior results suggesting clinical utility have not been reproducible [22]. This study was performed to evaluate ERCC1 testing offered by three large commercial laboratories. Methods Participating Commercial Laboratories Clarient, Inc. (Aliso, CA, http://www.clarientinc.com), Genzyme Corporation (Cambridge, MA, http://www.genzyme.com), and “Third Laboratory” (requesting anonymity after learning of study results) agreed to participate in the conduct of this study and confirmed the accuracy of this report. Each of these laboratories offered ERCC1 testing to facilitate NSCLC patient management, and all three assays had been used by physicians at Winthrop-University Hospital (Mineola, NY) prior to the initiation of this study. All three laboratories agreed to perform ERCC1 testing without charge when requested specifically for the purposes of this study. The design and conduct of this protocol were approved by the Winthrop-University Hospital Institutional Review Board as well as by each of the participating commercial laboratories. Study Design ERCC1 testing was performed on tumor specimens from three separate cohorts of NSCLC patients. First, we compared the prevalence of “high” versus “low” ERCC1 expression in NSCLC as reported by each independent laboratory. Second, we chose 18 patients treated at Winthrop-University Hospital whose tumors had already been tested for ERRC1 expression at Clarient and arranged for retesting of their same tumor specimens at Genzyme and Third Laboratory. Finally, we retrospectively reviewed the charts of more than 300 NSCLC patients treated at Winthrop-University Hospital to identify 12 who were “platinum responders” and another 12 who were “platinum nonresponders” to platinum-based chemotherapy administered as first-line treatment for metastatic disease. Platinum responders were required to have achieved a partial response, whereas nonresponders were required to have had progressive disease while receiving systemic platinum-based chemotherapy using either World Health Organization (WHO) [23] or Response Evaluation Criteria in Solid Tumors (RECIST) [24] criteria. In addition, charts were reviewed to confirm the impression of clinical benefit in responders and worsening in nonresponders. Slides were cut from a single pretreatment tumor block for each of these 24 tumors, divided equally, and sent to each of the three commercial laboratories for their independent ERCC1 testing. Specimens were deidentified and coded so that no laboratory knew tumor response status or any other tumor- or patient-related characteristics before reporting its ERCC1 assay results. ERCC1 Testing Methodologies Each of the three laboratories performed ERCC1 testing according to the same protocol and standards as incorporated in its own commercially offered test. Clarient and Genzyme determined ERCC1 protein expression by immunohistochemical (IHC) analysis using the same antibody reagent, designated 8F1, and protocol as reported by Olaussen et al. [18, 25] The proportion (expressed as a percentage) and the intensity (0, 1+, 2+, or 3+) of IHC-positive staining tumor nuclei were determined in identical fashion by both Clarient and Genzyme. However, each laboratory had developed its own criteria for reporting a final test result as “positive” or “negative” for ERCC1 expression. For Genzyme, a positive result required 3+ staining in at least 10% of nuclei. A positive result for Clarient required 2+ or 3+ staining in at least 50% of examined tumor nuclei. Importantly, after recognizing a change in the performance of newer 8F1 antibody lots, Clarient chose to modify its commercial assay by retitering the 8F1 antibody from 1:200 to 1:8000. These modified assay conditions took effect commercially in November 2009 and were applied to only the third cohort of patients in this study. Third Laboratory determined ERCC1 mRNA gene expression by quantitative real-time polymerase chain reaction (RT-PCR) assay according to a proprietary procedure [26a] as previously reported by Lord et al. [26b] ERCC1 RNA levels were reported as the ratio of ERCC1 gene transcripts to β-actin reference gene transcripts. Ratios above 1:7 were reported as “positive” for ERCC1 expression and lower ratios were reported as “negative.” We do recognize that because of posttranscriptional and posttranslational regulatory mechanisms, the accurate measurement of ERCC1 mRNA and protein expression for ERCC1 need not be concordant. However, all of these ERCC1 assays ultimately report a clinical result intended to reflect platinum sensitivity or resistance. We chose to compare all three laboratory results on the basis of this clinical predictive outcome measure. Statistical Analysis For cohort 1, involving separate patient populations tested at each of the three participating laboratories, descriptive statistics were analyzed by proportion. A two-sample test of proportions was used to compare these independent ERCC1 expression rates. For cohort 2, Cochran's Q test was used to determine concordance for ERCC1 results in identical patients tested at all three laboratories [27]. McNemer's test of paired proportions was applied to determine discordance between laboratories. After applying Bonferroni correction for multiple comparisons, none of our results changed from being statistically significant to nonsignificant using p < .05 as a measure of statistical significance. κ statistics were calculated to evaluate interlaboratory concordance. For cohort 3, clinically determined chemotherapy response provided a “gold standard” against which each laboratory's results were compared using McNemer's test. Because we were rigorous in our definition of platinum-sensitive and -resistant tumor specimens, our cohort 3 sample size was limited and no a priori power analysis was performed. As such, this gold standard analysis should be considered exploratory. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated using exact binomial proportions [28]. Calculations were performed using SAS 9.3 (SAS Institute, Cary, NC, http://www/sas/com) and Stata/SE 10.0 for Windows (StataCorp, College Station, TX, http://www.stata.com). Results Aggregate ERCC1 Expression at Three Different Laboratories Considerable variation in the prevalence of ERCC1 expression was noted at Clarient (70%, n = 1,083), Genzyme (60%, n = 1,070), and Third Laboratory (44%, n = 2,320). These differences in aggregate ERCC1 expression rates, as shown in Table 1, were highly significant (p < .0001 for all two-way comparisons) and motivated us to compare ERCC1 expression results that these three laboratories found on identical tumor specimens. Table 1 Commercial laboratory testing of ERCC1 in non-small cell lung cancer Open in new tab Table 1 Commercial laboratory testing of ERCC1 in non-small cell lung cancer Open in new tab ERCC1 Expression in Identical Tumor Blocks at Three Different Laboratories Tumors from 18 NSCLC patients were tested for ERCC1 expression at all three commercial laboratories. Results are delineated in Table 1 and interlaboratory concordance is depicted in Figure 1. As shown, all three laboratories reported the same “positive” or “negative” result in only 4 of these 18 specimens. Clarient and Genzyme, the two laboratories using identical IHC testing reagents and methodology, reported concordant results in 11 of the 18 tumors (61%) tested. All 7 of the discordant cases were reported as positive by Clarient and negative by Genzyme. The results reported by these two laboratories were shown to correlate poorly (κ: 0.222; 95% CI: 0.017–0.55) and the difference between ERCC1 expression reported by Clarient (89%) and Genzyme (50%) on identical tumor blocks was highly significant (p = .015). Discordance could not be attributed to the different intensity/percentage staining criteria used by these two laboratories in determining ERCC1 expression. This was determined by repeating comparisons after applying Clarient criteria to Genzyme specimens and alternatively after applying Genzyme criteria to Clarient specimens. Third Laboratory's ERCC1 results were concordant with those of Genzyme in only 39% (κ: –0.222; 95% CI: −0.646–0.202) and with Clarient in only 44% (κ: 0.117; 95% CI: −0.053–0.289) of cases. Figure 1 Open in new tabDownload slide Laboratory concordance for excision repair cross-complementation group 1 (ERCC1) expression results. Venn diagram with overlap indicating concordant ERCC1 results. As depicted, all three laboratories were concordant for “positive” or “negative” ERCC1 result in only 4 of 18 identical tumor blocks examined in round-robin analysis. Figure 1 Open in new tabDownload slide Laboratory concordance for excision repair cross-complementation group 1 (ERCC1) expression results. Venn diagram with overlap indicating concordant ERCC1 results. As depicted, all three laboratories were concordant for “positive” or “negative” ERCC1 result in only 4 of 18 identical tumor blocks examined in round-robin analysis. ERCC1 Expression in Selected Platinum Responders and Nonresponders Tested at Three Laboratories Specimens from 2 of the 12 selected platinum nonresponders and 1 of the 12 responders were deemed inadequate for ERCC1 determination by all three laboratories. Patient and tumor characteristics for each of the remaining 11 responders and 10 nonresponders are shown in Table 2. Responders and nonresponders were similar in age, gender, and the carboplatin-doublet chemotherapy regimens they received. Histology was adenocarcinoma in all patients, except for 2 nonresponders with squamous cell carcinoma. Per selection criteria for this study, all responders had partial response defined by WHO (n = 2), RECIST (n = 2), or both (n = 7) criteria, whereas all nonresponders had progression of disease defined by both WHO and RECIST criteria, except for 1 patient who had just 15% increase in maximum tumor diameter (RECIST criteria), but 42% increase in product of bidimensional target lesion measurements (WHO criteria) [24, 29]. Not surprisingly, responders received more cycles of carboplatin than nonresponders (median, 5 vs. 2.5 cycles, p = .003) and lived longer (median 25 vs. 9 months, p = .03). Only 3 nonresponders survived longer than 9 months, and all achieved major objective treatment response to second-line nonplatinum therapies. Table 2 Characteristics of platinum responders and nonresponders Open in new tab Table 2 Characteristics of platinum responders and nonresponders Open in new tab The ability of commercial laboratory ERCC1 assays to correctly identify our platinum-responsive and -nonresponsive patients is shown in Figure 2 and Table 3. Results were reported by Clarient for 21 tumors and by Genzyme for 20, whereas Third Laboratory was able to provide results for only 8, reporting “insufficient material” in the remaining 13 cases. This was somewhat unexpected because one of the perceived advantages of Third Laboratory's RT-PCR assay was that it might provide an enhanced ability to derive results from smaller specimens not amenable to IHC testing. Figure 2 Open in new tabDownload slide Accuracy of excision repair cross-complementation group 1 results in predefined platinum responders and nonresponders. Figure 2 Open in new tabDownload slide Accuracy of excision repair cross-complementation group 1 results in predefined platinum responders and nonresponders. Table 3 Sensitivity, specificity, and positive and negative predictive values for excision repair cross-complementation group 1 predicting platinum resistance at three commercial laboratories Open in new tab Table 3 Sensitivity, specificity, and positive and negative predictive values for excision repair cross-complementation group 1 predicting platinum resistance at three commercial laboratories Open in new tab Initially, we were also surprised to find that Clarient, the laboratory that had previously been observed to report the highest rate of ERCC1 expression (Table 1), only reported one high ERCC1 expression result in this entire cohort of patients. Only after we queried Clarient about these unexpected results did we learn that its commercial assay had been deliberately modified to decrease the prevalence of high ERCC1 results, with that change having been implemented and applied between the second and third cohorts of this study (as above). Consequently, Clarient's assay reported low ERCC1 results that correctly predicted response in all 11 platinum responders, but also incorrectly predicted platinum response in 9 of 10 nonresponders. Genzyme reported low ERCC1 results in 8 of 11 responders, but also in 5 of 9 nonresponders. Third Laboratory was unable to perform its RT-PCR-based mRNA assay on the majority of specimens, reporting low ERCC1 results in 3 of 4 responders and 2 of 4 nonresponders. Disappointingly, none of the three commercial assays reported a significantly higher frequency of low ERCC1 results in responders as compared with nonresponders in this selected cohort. The sensitivity, specificity, positive predictive value, and negative predictive value for each laboratory are shown in Table 3. Discussion Having entered an era in which cancer treatments are increasingly predicated on the results of tumor biomarker assays, the reliability of such assays is of the utmost importance. Our data demonstrate that ERCC1 testing, as performed in each of these three laboratories, is highly unreliable. Significant interlaboratory discrepancies in ERCC1 expression rates were confirmed by retesting specimens from the same tumor blocks at all three laboratories. Given that the test is reported as a simple dichotomous result (positive or negative), the degree of discordance that we observed between laboratories was both disappointing and alarming. Our results demonstrated that in 78% of cases, an ERCC1 result reported by one of these three laboratories would have been different if the test had been performed at one of the other two laboratories. In other words, if ERCC1 testing were used to decide whether to administer or withhold platinum-based chemotherapy, then that decision would most often be determined by which laboratory the test was sent to rather than any true difference in the biology of the tumor being tested. In the case of Clarient, the treatment decision would have likely been different if the test had been sent before or after their ERCC1 assay had been modified. None of the ERCC1 assays that we studied could reliably distinguish between patients carefully selected as platinum responders and nonresponders. Because all patients were treated with platinum-doublet therapies, it is conceivable that responders who were determined to have high ERCC1 expression may have been truly resistant to the platinum therapy, but sensitive to the other coadministered cytotoxic agent. Nonresponder patients were, however, selected on the basis of being highly resistant to all administered agents in the platinum regimen, making their classification as “platinum nonresponders” more straightforward. Nevertheless, even in these refractory patients, none of the three commercially available assays correctly predicted refractory disease (high ERCC1) in more than half of tested specimens. In other words, none of the assays that we evaluated could predict platinum resistance on the basis of ERCC1 expression with a specificity of greater than 50%. Recently, Friboulet et al. reported that none of the 16 currently available 8F1 antibody lots could reliably identify the ERCC1 isoform responsible for nucleotide excision repair and platinum resistance [22]. ERCC1 expression measured by these newer lots of 8F1 did not provide predictive utility in a newly studied cohort of patients receiving adjuvant cisplatin-based chemotherapy or in a reassessment of the original International Adjuvant Lung Trial cohort in which 8F1 had been previously validated. Importantly, when ERCC1 expression was measured with currently available 8F1 antibody lots, results were discordant with those previously observed with the original, and no longer available, 8F1 antibody. These authors suggest that the inadequacy of ERCC1 assays may reflect a change in the performance of currently available 8F1 antibody, but problems with ERCC1 measurement by 8F1 have been apparent since at least 2007 [30–32]. Prior reports have raised several other potential explanations as to why ERCC1 testing might not provide a reliable means of selecting patients for platinum or nonplatinum therapies. First, the very notion that measuring the expression of a single protein could adequately assess the complex process of DNA repair has been questioned [7, 32]. Second, low expression of ERCC1 has been shown to be associated with other genomic alterations, including those that affect sensitivity to nonplatinum chemotherapies [33, 34]. Third, others have previously noted that thresholds for ERCC1 “expression” had been arbitrarily assigned and inadequately validated [35]. Fourth, several prior studies had been unable to confirm the predictive utility of ERCC1 expression in platinum-treated lung cancer patients [31, 36]. Fifth, the measurement of ERCC1 protein by IHC and mRNA by RT-PCR has shown inconsistent correlation [37]. Sixth, the 8F1 antibody, most frequently used to measure ERCC1 protein levels, is unable to differentiate between normal and ERCC1-deficient cell lines, possibly due to excessive background cytoplasmic staining by 8F1 [30, 38, 39]. Based on such considerations, two consensus panels have appropriately recommended against the use of ERCC1 testing in routine clinical practice [2, 40]. Even the authors of studies previously reporting possible clinical utility of ERCC1 testing have responded to stated concerns by declaring that the test is still “not applicable for standard use in the everyday clinic” [41]. Yet, current National Comprehensive Cancer Network guidelines state that “Multiple translational investigations have provided evidence for the predictive use of ERCC-1 levels to assess the efficacy of platinum-based chemotherapies in NSCLC” [21] and the test remains commercially available. Conclusion This report provides direct evidence that currently marketed ERCC1 assays are unreliable. Initially perceived discrepancies between individual laboratory rates of ERCC1 expression suggested that some of these results were suspect. Discordance in laboratory round-robin analysis demonstrated that most were inconsistent. Sensitivity and specificity analysis among defined platinum responders and nonresponders confirmed that all were inadequate. We conclude that commercial laboratories should not offer ERCC1 testing until assays are better standardized and results are more thoroughly validated. Two important lessons may be gleaned from the oncology community's recent experience with ERCC1 testing. First, the process by which tumor biomarkers are validated and allowed to enter routine clinical practice is currently inadequate. Guideline committees, thought leaders, and clinicians should insist on more robust and consistent data before adopting tools that may alter patient care. Second, even after such tumor biomarker assays are incorporated into clinical practice, postmarketing scrutiny remains essential. This report suggests that such scrutiny may be achieved by simply demanding consistency of same-specimen results as well as by more ambitious efforts to develop gold standard specimens to ensure reliability of laboratory results. Acknowledgments This study was not supported by any external funding. The three commercial laboratories participating in this study performed ERCC1 assays at no charge when specimens were submitted specifically for the conduct of this study. Those three companies had no role in the analysis of data or in the writing of this manuscript. All three companies reviewed a final draft of this manuscript and confirmed that all technical aspects of their performance in this study have been accurately reported. We acknowledge and appreciate the cooperation of each of the three commercial laboratories participating in this study. Each laboratory participated with the knowledge that results might prove unfavorable to the marketing of its ERCC1 assays, but also with the understanding that such postmarketing surveillance might provide valuable insights for clinicians involved in the management of non-small cell lung cancer patients. Author Contributions Conception/design: Jeffrey G. Schneider, Nosha Farhadfar, Abirami Sivapiragasam, Matthew Geller, Elena Selbs Provision of study material or patients: Jeffrey G. Schneider, Nosha Farhadfar, Abirami Sivapiragasam, Matthew Geller, Elena Selbs Collection and/or assembly of data: Jeffrey G. Schneider, Nosha Farhadfar, Abirami Sivapiragasam, Matthew Geller Data analysis and interpretation: Jeffrey G. Schneider, Nosha Farhadfar, Abirami Sivapiragasam, Shahidul Islam Manuscript writing: Jeffrey G. Schneider, Nosha Farhadfar, Abirami Sivapiragasam, Shahidul Islam, Elena Selbs Final approval of manuscript: Jeffrey G. Schneider, Nosha Farhadfar, Abirami Sivapiragasam, Matthew Geller, Shahidul Islam, Elena Selbs Disclosures The authors indicated no financial relationships. For Further Reading: Jared M. Weiss, Thomas E. Stinchcombe. Second-Line Therapy for Advanced NSCLC. The Oncologist 2013;18:947–953. Implications for Practice: The landscape of first-line treatment of non-small cell lung cancer has generated challenges for clinical decisions in second-line therapy. For the patient treated with standard chemotherapy in the first line who has a treatable molecular change, this change should be targeted. More specifically, the patient with an epidermal growth factor receptor (EGFR) mutation should be treated with an EGFR tyrosine kinase inhibitor, and the patient with EML4/ALK rearrangement should be treated with crizotinib. However, these agents are increasingly being used in the first line, and most patients do not have these molecular changes. This leaves the clinician with many challenging questions regarding second-line therapy. How should the patient without treatable mutations be treated? Which clinical trials are most promising? How should the patient treated with a targeted agent in the first line be treated in the second line? This review addresses these issues, exploring the key existing data available to help guide informed clinical decisions. References 1 Schiller JH , Harrington D, Belani CP. Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer . N Engl J Med . 2002 ; 346 : 92 – 98 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Azzoli CG , Baker S Jr, Temin S. American Society of Clinical Oncology Clinical Practice Guideline update on chemotherapy for stage IV non-small-cell lung cancer . J Clin Oncol . 2009 ; 27 : 6251 – 6266 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Arriagada R , Bergman B, Dunant A. 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Roth, Joshua A.; Billings, Paul; Ramsey, Scott D.; Dumanois, Robert; Carlson, Josh J.

2014 The Oncologist

doi: 10.1634/theoncologist.2013-0357pmid: 24710309

Abstract Purpose. Life Technologies has developed a 14-gene molecular assay that provides information about the risk of death in early stage non-squamous non-small cell lung cancer patients after surgery. The assay can be used to identify patients at highest risk of mortality, informing subsequent treatments. The objective of this study was to evaluate the cost-effectiveness of this novel assay. Patients and Methods. We developed a Markov model to estimate life expectancy, quality-adjusted life years (QALYs), and costs for testing versus standard care. Risk-group classification was based on assay-validation studies, and chemotherapy uptake was based on pre- and post-testing recommendations from a study of 58 physicians. We evaluated three chemotherapy-benefit scenarios: moderately predictive (base case), nonpredictive (i.e., the same benefit for each risk group), and strongly predictive. We calculated the incremental cost-effectiveness ratio (ICER) and performed one-way and probabilistic sensitivity analyses. Results. In the base case, testing and standard-care strategies resulted in 6.81 and 6.66 life years, 3.76 and 3.68 QALYs, and $122,400 and $118,800 in costs, respectively. The ICER was $23,200 per QALY (stage I: $29,200 per QALY; stage II: $12,200 per QALY). The ICER ranged from “dominant” to $92,100 per QALY in the strongly predictive and nonpredictive scenarios. The model was most sensitive to the proportion of high-risk patients receiving chemotherapy and the high-risk hazard ratio. The 14-gene risk score assay strategy was cost-effective in 68% of simulations. Conclusion. Our results suggest that the 14-gene risk score assay may be a cost-effective alternative to standard guideline-based adjuvant chemotherapy decision making in early stage non-small cell lung cancer. Gene-expression profiling, Non-small cell lung cancer, Cost effectiveness Implications for Practice: Gene-expression profiles, like the 14-gene risk score assay, provide prognostic information that may improve adjuvant chemotherapy decision making in early stage non-small cell lung cancer relative to standard stage-based approaches. We evaluated the clinical and economic impacts of the 14-gene risk score assay and report a testing strategy that is likely to result in additional quality-adjusted survival at an additional cost that is generally considered to be cost-effective in the United States. Our findings can be used to inform decisions about clinical implementation and payer considerations. Introduction Non-small cell lung cancer (NSCLC) is among the most commonly diagnosed malignancies in the U.S., with approximately 192,000 incident cases in 2012 [1]. Cases diagnosed in the early stages (i.e., stage I or II) have better survival prognosis relative to those diagnosed at more advanced stages, yet overall 5-year survival remains poor at 50% [2–4]. In stage I and II NSCLC, standard care typically begins with surgery to remove the malignant tissue [5, 6]. After surgery, patients and clinicians are faced with a decision about whether to use adjuvant chemotherapy to attempt to reduce the risk of disease recurrence. This decision is typically informed by a variety of clinical and pathologic factors, with disease stage playing a prominent role [5, 6]. These prognostic factors are used to risk stratify patients so that the balance of benefits and risks of adjuvant chemotherapy can be weighed [7]. Large clinical trials have demonstrated that adjuvant chemotherapy does not provide a survival benefit on average in stage I NSCLC but shows moderate survival benefit in stage II NSCLC [8]. Accordingly, clinical guidelines from the American Society of Clinical Oncology and National Comprehensive Cancer Network generally recommend that stage I patients not receive adjuvant chemotherapy and that stage II patients receive chemotherapy [5, 6, 8]. However, even among stage I patients, the 5-year risk of recurrence remains high at 24%. In addition, adjuvant chemotherapy is often considered for high-risk subgroups, such as those with poorly differentiated tumors, vascular invasion, wedge resection, tumors >4 cm, visceral pleural involvement, and/or incomplete lymph node sampling [7, 9]. The limited success of the traditional TNM staging system for predicting outcomes after primary therapy has led to efforts to identify biomarkers of disease risk and treatment response [10–14]. One example is a novel 14-gene risk score (RS) assay (Pervenio Lung RS; Life Technologies, Rockville, MD, http://www.lifetech.com) developed by Kratz and colleagues [12]. In a study of this assay, patients with completely resected non-squamous non-small cell lung tumors were shown to have a continuum of overall survival prognosis based on the assay's risk score classification. Stage I patients classified as low, intermediate, and high risk had median overall survival times of 113 months, 91 months, and 59 months, respectively [12]. Stage II patients classified as low, intermediate, and high risk had median overall survival times of 62 months, 49 months, and 33 months, respectively [12]. Although currently established as a prognostic test, the 14-gene RS may also provide information in early stage NSCLC to improve targeting of adjuvant chemotherapy, as has been seen with similar assays in early stage breast cancer [15]. Prospective research is under way to further investigate the ability of the 14-gene RS to inform adjuvant chemotherapy treatment decisions (i.e., as a predictive test), but endpoints will not be reached for several years [16]. In the interim, thousands of patients with early stage NSCLC, in consultation with their physicians, will face adjuvant chemotherapy treatment decisions [10, 17]. In this study, we inform these decisions by using simulation modeling to evaluate the potential health outcomes and cost-effectiveness of the 14-gene RS relative to standard guideline-based care. Our findings could be used to inform decisions about clinical implementation and payer considerations. Materials and Methods Overview We developed an integrated decision tree and Markov state-transition model to simulate health outcomes for a cohort of 67-year-old patients with completely resected stage I/II non-squamous NSCLC from the time of adjuvant chemotherapy decision making until death (Fig. 1) [12]. The model compares health outcomes for the cohort in two clinical strategies: a 14-gene RS strategy and a standard guideline-based strategy. In the 14-gene RS strategy, patients undergo gene-expression profiling and are classified as having low-, intermediate-, or high-risk overall survival prognosis, and subsequent treatments are affected by patients’ risk status (i.e., high-risk patients are recommended to receive adjuvant chemotherapy). In the standard-care strategy, adjuvant chemotherapy decisions are informed solely by standard clinical-pathological factors (e.g., stage, tumor size, histology). The cohort is then tracked for disease recurrence and mortality over a lifetime horizon. Model outcomes include life years, quality-adjusted life years (QALYs), and direct medical expenditure (referred to as “costs”). Our analysis took a payer perspective, and cost and QALY outcomes were discounted at 3% per year. Figure 1 Open in new tabDownload slide Simplified decision model (A) and Markov model schematics (B). Abbreviations: NSCLC, non-small cell lung cancer; RS, risk score. Figure 1 Open in new tabDownload slide Simplified decision model (A) and Markov model schematics (B). Abbreviations: NSCLC, non-small cell lung cancer; RS, risk score. The decision model was implemented in Microsoft Excel (Microsoft Inc., Redmond, WA, http://www.microsoft.com). Model Structure The model was developed as a Markov cohort, tracking long-term outcomes in 1-month cycles for each patient cohort strata defined by stage (I or II), 14-gene RS risk group (low, intermediate, or high), and adjuvant chemotherapy use or nonuse. The model consists of five health states following diagnosis and primary treatment (nodule resection): observation, chemotherapy, postchemotherapy, recurrence, and death (Fig. 1). In addition, we tracked adverse event rates related to chemotherapy use (anemia, neutropenia, febrile neutropenia, fatigue, anorexia, and nausea or vomiting) [18]. We calculated outcomes for the 14-gene RS and standard-care strategies using weighted averages of outcomes from stage and risk subgroups. Model Inputs Model inputs were derived from the 14-gene RS validation studies; a physician decision-making survey; the U.S. Surveillance, Epidemiology, and End Results (SEER) database; published literature; and government sources [4, 11, 19–22]. All model parameters and data sources are provided in Table 1. Mean input values and uncertainty ranges were derived directly from source studies when possible. Table 1 Model input values, distributions, and data sources Open in new tab Table 1 Model input values, distributions, and data sources Open in new tab Risk-Group Distribution Inputs The proportions of patients with early stage NSCLC classified by the 14-gene RS as low, intermediate, and high risk were derived from the test-validation results reported by Kratz and colleagues [12]. Specifically, we based the risk-group distribution of stage I patients on that of 433 patients in the Kaiser Permanente validation cohort (28% low, 20% intermediate, 52% high) and the risk-group distribution of stage II patients on that of 222 patients in the China Clinical Trials Consortium cohort (16% low, 14% intermediate, 70% high) [12]. Chemotherapy Use and Effectiveness Inputs Chemotherapy utilization rates in the 14-gene RS and usual-care strategies were based on the findings of a physician survey that evaluated pre- and post-testing adjuvant chemotherapy recommendations [23]. Updated results from that study tracked 58 physicians in community-practice settings who provided adjuvant chemotherapy recommendations for 120 patients based on clinical-pathological factors and then made another recommendation after receiving the 14-gene RS risk-group classification. We assumed that the proportion of patients receiving chemotherapy in the standard-care strategy was equal to that recommended to receive chemotherapy before 14-gene RS testing (i.e., 14.3% of stage I and 68.8% of stage II) and that the proportion of patients receiving chemotherapy in the 14-gene RS strategy was equal to that recommended to receive chemotherapy after testing (i.e., 3.8% of low-risk, 26.3% of intermediate-risk, and 70.6% of high-risk patients among stage I and 1.0% of low-risk, 57.1% of intermediate risk, and 95.2% of high-risk patients among stage II). Our standard-care chemotherapy-utilization rates were consistent with prior reports in the peer-reviewed literature [24]. Patients that received chemotherapy were assumed to get the cisplatin and vinorelbine regimen used in the International Adjuvant Lung Cancer Trial (IALT) and recommended by National Comprehensive Cancer Network and American Society of Clinical Oncology guidelines [5, 7, 25]. We modeled the impact of adjuvant chemotherapy through its effect on the rate of disease recurrence (local and distant). To estimate the effects of chemotherapy in the 14-gene RS risk groups, we evaluated health outcomes across three plausible scenarios representing the likely range of outcomes in clinical practice. In our base case, we used stage I and II distant recurrence-free survival hazard ratios reported in the Lung Adjuvant Cisplatin Evaluation (LACE) meta-analysis conducted by Douillard and colleagues and distributed the overall recurrence rate benefit in each stage to the risk groups based on baseline recurrence risk (Fig. 2) [8]. Specifically, we assumed that chemotherapy-benefit scales, from least benefit to greatest benefit, within its uncertainty range (i.e., 95% confidence interval for the hazard ratio), according to underlying prognosis for the stage I and II cohort (Fig. 2). This assumption is supported by evidence in the peer-reviewed literature demonstrating increased benefit from adjuvant chemotherapy in patient subgroups with increased baseline risk of disease recurrence and death [8]. We also evaluated a scenario with a nonpredictive chemotherapy effect (i.e., equal chemotherapy benefit across all risk groups), in which the 14-gene RS strategy improved health outcomes solely through its impact on treatment decisions (with more patients receiving chemotherapy because of classification as high-risk). These patients then were assumed to receive the average stage-specific chemotherapy benefit. Last, we evaluated a strongly predictive chemotherapy scenario based on the findings of an analogous molecular marker study conducted by Zhu and colleagues with patients from the JBR.10 trial (a phase III randomized controlled trial that evaluated cisplatin and vinorelbine versus observation alone in stage I/II NSCLC) [25, 26]. The chemotherapy effects from this study are not directly applicable, given differing gene panels and risk-group cutoffs, but nonetheless, it can be used to represent the upper end of the spectrum of predictive impact that could be achieved. Collectively, these scenarios represent the plausible range of chemotherapy impacts in clinical practice and facilitate evaluation of the plausible range of cost-effectiveness of the 14-gene RS assay strategy. Figure 2 Open in new tabDownload slide HR distributions for stage I patients (A) and stage II patients (B) by risk group. To derive mean HRs by risk group, we assumed that the HRs had a log-normal distribution, generated the cumulative HR distribution, scaled the distribution with the increasing risk of death in the underlying population as calculated by the 14-gene risk score assay, used the risk category cutoffs to segment the population, and calculated the mean HR by risk group. Abbreviation: HR, hazard ratio. Figure 2 Open in new tabDownload slide HR distributions for stage I patients (A) and stage II patients (B) by risk group. To derive mean HRs by risk group, we assumed that the HRs had a log-normal distribution, generated the cumulative HR distribution, scaled the distribution with the increasing risk of death in the underlying population as calculated by the 14-gene risk score assay, used the risk category cutoffs to segment the population, and calculated the mean HR by risk group. Abbreviation: HR, hazard ratio. Adjuvant Chemotherapy Adverse Event Rates and Resource Utilization We considered grade 3/4 chemotherapy adverse events occurring in ≥5% of patients in the JBR.10 trial (Table 1) [27]. Neutropenia was assumed to require two office visits, treatment with 500 mg of clindamycin three times daily for 1 month, and 500 mg of amoxicillin three times daily for 1 month. We assumed that 70% of febrile neutropenia cases required inpatient treatment, and the remaining 30% was treated in an outpatient setting with two office visits and 2 g of cefepime three times daily for 2 days. Anemia was assumed to be treated with one vial of erythropoietin injected per week until disease recurrence. Fatigue, anorexia, nausea, and vomiting were assumed to require two office visits. Recurrence Inputs We estimated lung cancer recurrence using the relationship between lung cancer-specific mortality and recurrence (local and distant), as reported in the IALT study [20]. Specifically, we applied the observed IALT ratio of recurrence to lung cancer-specific mortality to the 14-gene RS validation cohort to obtain recurrence rates for the initial 5 years of follow-up. In years 5–10 of follow-up, we applied recurrence rates derived from a study of long-term outcomes in early stage NSCLC from Maeda and colleagues [28]. We assumed that no recurrences occurred beyond 10 years of follow-up [28]. Mortality Inputs We implemented separate overall mortality rates by disease stage and risk-group classification, in accordance with the findings of the 14-gene RS validation study [12]. Overall mortality rates were divided into lung cancer-specific mortality and other-cause mortality by subtracting other-cause mortality from overall mortality. Other-cause mortality rates were derived from life tables for former smokers reported as part of the National Cancer Institute CISNET project [29]. Cost Inputs We utilized adjuvant chemotherapy, adverse event treatment, and procedure costs based on the 2013 U.S. Centers for Medicare and Medicaid Services reimbursement schedule (Table 1). The cost of the 14-gene RS assay ($3,995) was provided by Life Technologies. Model Validation and Calibration We calibrated our model in each stage and risk-group strata by fixing postrecurrence mortality (in accordance with current evidence) and adjusting the annual recurrence rate to align simulated 5-year overall survival with overall survival rates from the 14-gene RS clinical studies [12, 30]. We validated our long-term survival outcomes by comparing mean overall survival with mean survival in similar patients in the JBR.10 clinical trial [31]. We also calculated 5-year overall survival hazard ratios for stage I and II patients receiving chemotherapy (vs. observation only) and evaluated whether they were within the stage-specific 95% confidence intervals reported in the LACE meta-analysis [8]. Model Outcomes We used our model to calculate overall life expectancy, quality-adjusted life years (QALYs), and direct medical expenditures for the 14-gene RS and standard-care strategies. The QALY is a standard metric from comparative effectiveness research that incorporates a quality of life “utility score” adjustment applied to life expectancy [32–34]. A utility score of 0 represents the value for death, and 1 represents the value for “full” health. Thus, 10 years of life at a utility of 0.5 is equivalent to 5 years of life with full health [33]. The QALY allows consideration of morbidity and mortality in a single measure, allows for comparability between studies, and is considered the gold standard metric in cost-effectiveness studies [35, 36]. These outcomes enable calculation of the incremental cost-effectiveness ratio (ICER), the ratio of the difference in costs between strategies and the difference in effects (e.g., QALYs) between strategies. We also conducted similar analyses stratified by disease stage (I or II) to evaluate whether there were differential health outcome impacts and ICERs. Sensitivity Analyses We evaluated outcome uncertainty using one-way and probabilistic sensitivity analyses. In our one-way sensitivity analysis, we propagated low- and high-input-value estimates through the model and obtained the resulting range of incremental QALYs and costs for each individual input. We present our one-way sensitivity analysis results in tornado diagrams displaying the 10 most influential model inputs. We also conducted a probabilistic sensitivity analysis using Monte Carlo simulation [37–39]. This approach involved specifying the distribution of model inputs, simultaneously sampling parameter sets from the distributions, and propagating the values through the model framework to calculate the joint distribution of model outcomes [37, 38]. We used the probabilistic sensitivity analysis results to calculate 95% credible intervals (95% CI) around model outcomes, and we display these results in the form of cost-effectiveness acceptability curves. Willingness-to-Pay (per QALY) Threshold We evaluated the cost-effectiveness of the 14-gene RS at willingness-to-pay thresholds ranging from $50,000 to $200,000 per QALY [40–43]. This range reflects the implied willingness to pay for cancer treatments in the U.S. and is consistent with values used in prior analyses [40, 44, 45]. Results Base Case Results In our base case analysis, the 14-gene RS strategy and the standard-care strategy respectively resulted in 54% and 32% of patients receiving adjuvant chemotherapy, in 6.81 and 6.66 life years, in 3.76 and 3.68 QALYs, and in lifetime costs of $118,000 and $116,200 (Table 2). Accordingly, the 14-gene RS strategy resulted in greater life expectancy and QALYs compared with the standard-care strategy at a greater overall cost. The resulting incremental cost-effectiveness ratio for the combined analysis of stage I and II patients was $23,200 per QALY gained. Table 2 Health outcomes by scenario Open in new tab Table 2 Health outcomes by scenario Open in new tab Stage-Stratified Results When we restricted our analysis to stage I patients, the 14-gene RS strategy and the standard-care strategy respectively resulted in 43% and 14% of patients receiving adjuvant chemotherapy, in 7.56 and 7.41 life years, in 4.18 and 4.11 QALYs, and in lifetime costs of $98,200 and $96,100 (Table 2). The resulting incremental cost-effectiveness ratio for only stage I patients was $29,200 per QALY gained. When we restricted our analysis to stage II patients, the 14-gene RS strategy and the standard-care strategy respectively resulted in 75% and 69% of patients receiving adjuvant chemotherapy, in 5.22 and 5.07 life years, in 2.86 and 2.78 QALYs, and in lifetime costs of $159,900 and $158,800 (Table 2). The resulting incremental cost-effectiveness ratio for only stage II patients was $12,200 per QALY gained. Calibration and Validation Results In our validation analysis comparing mean expected survival, the results estimated by the model were well aligned with those from the JBR.10 trial in patients undergoing observation only (5.97 years vs. 5.65 years; 95% CI: 4.98–6.32 years) and those receiving adjuvant chemotherapy (6.61 years vs. 7.00 years; 95% CI: 6.27–7.73 years) [31]. In addition, our simulated stage I and II 5-year overall survival hazard ratios were aligned with those reported in the LACE database meta-analysis (stage I overall survival, HR: 1.10 vs. 1.01; 95% CI: 0.78–1.30; stage II overall survival HR: 0.88 vs. 0.74; 95% CI: 0.60–0.91) [8]. Sensitivity Analysis Results Our one-way sensitivity analysis demonstrated that the base case QALY and cost results were most sensitive to the proportion of high-risk patients receiving chemotherapy, the high-risk recurrence hazard ratios, and the proportion of stage II patients receiving chemotherapy in the standard-care strategy (Fig. 3). Lifetime costs and the ICERs were modestly sensitive to the cost of the 14-gene RS. Figure 3 Open in new tabDownload slide One-way sensitivity analysis tornado diagrams for incremental quality-adjusted life years (A) and lifetime costs (B). Abbreviation: QALYs, quality-adjusted life years. Figure 3 Open in new tabDownload slide One-way sensitivity analysis tornado diagrams for incremental quality-adjusted life years (A) and lifetime costs (B). Abbreviation: QALYs, quality-adjusted life years. Our probabilistic sensitivity analysis results demonstrate that the 14-gene RS strategy is likely to be cost effective at willingness-to-pay thresholds greater than $40,000 per QALY (Fig. 4). Figure 4 Open in new tabDownload slide Cost-effectiveness acceptability curves for the base case predictive chemotherapy benefit, nonpredictive chemotherapy benefit, and strongly predictive chemotherapy benefit scenarios. Abbreviation: QALY, quality-adjusted life year. Figure 4 Open in new tabDownload slide Cost-effectiveness acceptability curves for the base case predictive chemotherapy benefit, nonpredictive chemotherapy benefit, and strongly predictive chemotherapy benefit scenarios. Abbreviation: QALY, quality-adjusted life year. Alternative Chemotherapy-Benefit Scenario Results In our scenario analysis evaluating a strongly predictive chemotherapy benefit by risk group, the 14-gene RS strategy and the standard-care strategy respectively resulted in 7.57 and 6.83 life years, in 4.18 and 3.78 QALYs, and in $98,300 and $111,546 of cost (Table 2). The 14-gene RS testing was a dominant strategy because it led to decreased lifetime cost of care and increased QALYs. In our scenario analysis evaluating a nonpredictive chemotherapy-benefit scenario by risk group, the 14-gene RS assay strategy and the standard-care strategy respectively resulted in 6.73 and 6.65 life years, in 3.72 and 3.68 QALYs, and in $119,900 and $116,300 of cost (Table 2). The resulting incremental cost-effectiveness ratio was $92,100 per QALY gained. The results of these alternative scenarios are shown in Table 2. Discussion Newly diagnosed early stage NSCLC patients continue to have suboptimal outcomes compared with many other types of cancer, as demonstrated by their poor disease-free and overall survival [46]. There are two primary mechanisms through which these health outcomes can be improved: the development of more effective and less toxic treatment regimens and the use of existing treatments to optimize risk-benefit tradeoffs for individual patients. The 14-gene RS is a prime example of the latter, providing information about mortality risk and potentially enabling physicians to limit the use of standard cytotoxic agents to those patients who stand to gain the greatest treatment benefit. We created a decision-analytic model to systematically evaluate the plausible range of clinical and economic impacts of the 14-gene RS strategy and to assess the potential cost-effectiveness of a 14-gene RS testing strategy relative to a standard-care strategy informed by clinical-pathological factors. We found that the 14-gene RS strategy has the potential to improve clinical outcomes and to be cost-effective under a wide variety of plausible assumptions. These projected benefits could potentially become more pronounced with the continued development of more effective treatments for early stage NSCLC patients. Despite the growing number of prognostic assays in early stage NSCLC, little has been published in the peer-reviewed literature about their potential cost-effectiveness. Consequently, this is the first peer-reviewed publication evaluating the potential clinical and economic outcomes of a practical prognostic assay in early stage NSCLC. Our study highlights several key drivers of the cost-effectiveness in this setting. Most important, assays must be able to classify patients into subgroups with clinically meaningful differences in disease prognosis and/or treatment effectiveness. In the case of the 14-gene RS assay, testing can identify subgroups of stage I disease with relatively poor overall survival prognosis and in which adjuvant chemotherapy may be beneficial and subgroups of stage II disease with relatively favorable prognosis and in which adjuvant chemotherapy may not be beneficial. Both represent a departure from current standard care, and it is through this mechanism that value can be realized in the form of improved survival, health-related quality of life, and/or reduced treatment costs. Beyond the direct value created as a function of the magnitude of the clinical benefits or cost savings, the total value created by a testing strategy is also substantially dictated by the degree to which patients and physicians are willing to follow its recommended treatment pathway. For this reason, post-testing patient and physician chemotherapy preferences are also important determinants of cost-effectiveness. Finally, because of the relatively moderate cost of testing and adjuvant chemotherapy (with standard platinum-doublet regimens) compared with the high cost of postrecurrence care, a key driver of cost-effectiveness is the impact of testing on recurrence rates. This study has several key limitations that should be noted. First and foremost, there is no direct evidence of a predictive effect for chemotherapy by 14-gene RS status. Clinical trials are currently under way to address this evidence gap; however, given the current understanding of the relationship between baseline prognosis and chemotherapy benefit, it is plausible that there is some differential chemotherapy impact by prognostic status. We addressed this issue by evaluating a scenario with current best evidence about the range of chemotherapy impact within and by stage and a scenario based on an analogous biomarker study using a retrospective trial-based analysis. In these scenarios, the ICER ranged from dominant to $92,100 per QALY, demonstrating that the 14-gene RS is expected to be a cost-effective intervention for a wide range of chemotherapy impacts in the U.S. The published data on the 14-gene RS assay does not include sufficient information about recurrence rates to derive direct estimates of disease-free survival by risk status. In the absence of such data, we estimated the recurrence rates for each risk group and stage combination using the observed relationship between lung cancer-specific mortality and recurrence (local and distant), as reported in the International Adjuvant Lung Cancer Trial [20]. Actual recurrence rates may differ from these estimates, but there is a strong correlation between disease recurrence and lung cancer-specific survival, as demonstrated in prior studies in this disease setting [8]. An additional limitation is that the data that were used to estimate the change in physician treatment selection after use of the 14-gene RS was obtained from a study that included known users of the assay [23]. These early adopters may be different from other physicians in ways that would affect our study results. Specifically, if fewer physicians changed their recommendations, the results would be attenuated. However, this is the best available evidence as to how the assay is used in current practice and its likely impact on actual treatment decisions because these data reflect the actual pre- and post-testing adjuvant chemotherapy recommendations made in clinical practice as opposed to hypothesized use or idealized guidelines-based use. Future studies will further inform this specific aspect of clinical utility (i.e., the impact of physician behavior). Last, it should be noted that cost impacts and efficiencies beyond those considered in this analysis might arise when the 14-gene RS is applied in clinical settings, and health economic impact should be reassessed accordingly. Conclusion The results of our analysis suggest that at implied willingness-to-pay levels in the U.S., the 14-gene RS assay is a cost-effective alternative to a standard guideline-based adjuvant chemotherapy decision-making strategy in early stage NSCLC. However, the predictive ability of the assay has great influence on cost-effectiveness, with a predictive assay being highly cost-effective and a nonpredictive assay being only marginally cost-effective. Future studies should address this question of differential chemotherapy benefit by risk group and should further examine post-testing chemotherapy preferences in community-practice settings. Acknowledgments We thank David Jablons, Michael Mann, Girish Putcha, and Janna Sipes for their thoughtful feedback and editing. This study was supported by funding from Life Technologies Corporation. Author Contributions Conception/Design: Joshua A. Roth, Paul Billings, Robert Dumanois, Scott D. Ramsey, Josh J. Carlson Provision of study material or patients: Joshua A. Roth, Paul Billings, Robert Dumanois, Josh J. Carlson Collection and/or assembly of data: Joshua A. Roth, Josh J. Carlson Data analysis and interpretation: Joshua A. Roth, Paul Billings, Robert Dumanois, Scott D. Ramsey, Josh J. Carlson Manuscript writing: Joshua A. Roth, Paul Billings, Robert Dumanois, Scott D. Ramsey, Josh J. Carlson Final approval of manuscript: Joshua A. Roth, Paul Billings, Robert Dumanois, Scott D. Ramsey, Josh J. Carlson Disclosures Josh J. Carlson: Life Technologies Corporation (C/A); Scott D. Ramsey: Life Technologies Corporation (C/A); Joshua A. Roth: Life Technologies Corporation (C/A); Robert Dumanois: Life Technologies (E); Paul Billings: Life Technologies (E, OI). (C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board References 1 Previous version: SEER cancer statistics review, 1975-2009 (vintage 2009 populations) . Available at http://seer.cancer.gov/csr/1975_2009_pops09/. Updated August 20, 2012. Accessed December 4, 2013. 2 El-Sherif A , Gooding WE, Santos R. Outcomes of sublobar resection versus lobectomy for stage I non-small cell lung cancer: A 13-year analysis . Ann Thorac Surg . 2006 ; 82 : 408 – 415; discussion 415–416 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Henschke CI , Yankelevitz DF, Libby DM. Survival of patients with stage I lung cancer detected on CT screening . N Engl J Med . 2006 ; 355 : 1763 – 1771 . 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Accessed December 4, 2013 Author notes Disclosures of potential conflicts of interest may be found at the end of this article. © 2014 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Dadu, Ramona; Waguespack, Steven G.; Sherman, Steven I.; Hu, Mimi I.; Busaidy, Naifa L.; Jimenez, Camilo; Habra, Mohammed A.; Ying, Anita K.; Bassett, Roland L.; Cabanillas, Maria E.

2014 The Oncologist

doi: 10.1634/theoncologist.2013-0409pmid: 24733667

Abstract Background. Sorafenib has proven efficacy in advanced differentiated thyroid cancer (DTC), but many patients must reduce the dose or discontinue treatment because of toxicity. The tolerability and efficacy of lower starting doses of sorafenib for DTC remain largely unstudied. Methods. We retrospectively examined overall survival, time to treatment failure, time to progression, discontinuation rates, and dose-reduction and interruption rates in patients with metastatic DTC treated with first-line sorafenib outside of a clinical trial. Two patient groups were compared; group 1 received the standard starting dose of 800 mg/day, and group 2 received any dose lower than 800 mg/day. Results. We included 75 adult patients, with 51 in group 1 and 24 in group 2. Mean age at diagnosis was 54 years, and 56% were male. The most common histologies included 43% papillary thyroid cancer of the conventional type, 15% papillary thyroid cancer of the follicular variant, and 15% Hürthle cell carcinoma. Time to treatment failure was 10 months (95% confidence interval [CI]: 5.6–14.3) in group 1 and 8 months (95% CI: 3.4–12.5) in group 2 (p = .56). Median overall survival was 56 months (95% CI: 30.6–81.3) in group 1 and 30 months (95% CI: 16.1–43.8) in group 2 (p = .08). Rates of discontinuation due to disease progression were 79% in group 1 and 91% in group 2, and 21% in group 1 and 9% in group 2 (p = .304) stopped treatment because of toxicity. Dose-reduction rates were 59% and 43% (p = .29), and interruption rates were 65% and 67% (p = .908) in group 1 and group 2, respectively. Conclusion. Efficacy and tolerability of sorafenib in treatment-naïve DTC patients does not appear to be negatively influenced by lower starting daily doses. 摘要 背景 索拉非尼对晚期分化型甲状腺癌 (DTC) 的疗效已获证实，但因其有毒副反应，很多患者不得不减量服用或中止用药。 关于索拉非尼在更低的起始剂量下使用时对 DTC 的疗效和耐受性，目前仍研究甚少。 方法 我们对一些在临床试验之外使用索拉非尼作为一线药物治疗转移性 DTC 的患者进行了一个回顾性分析，评估了他们的总生存期、至治疗失败的时间、至病情进展的时间、中止治疗率、减剂量和中断用药率。 我们共比较了两个患者组；组 1 接受的是 800 mg/天的标准起始剂量，组 2 接受的是低于 800 mg/天的剂量。 结果 我们共纳入了 75 名成人患者，其中组 1 为 51 名，组 2 为 24 名。他们的平均确诊年龄为 54 岁，男性占 56%。 最常见的组织类型包括普通型甲状腺乳头状癌 (占 43%) 、滤泡变异型甲状腺乳头状癌 (占 15%) 和 Hürthle 细胞癌 (占 15%) 。 组 1 的至治疗失败的时间为 10 个月 (95% 置信区间 [CI]： 5.6-14.3) ，组 2 为 8 个月 (95% CI: 3.4–12.5) (p = 0.56)。 组 1 的中位总生存期为 56 个月 (95% CI: 30.6–81.3)，组 2 为 30 个月 (95% CI: 16.1–43.8) (p = 0.08)。 因疾病进展而导致的中止治疗率在组 1 中为 79%，在组 2 中为 91%；因毒性作用而导致的停止治疗率在组 1 中为 21%，在组 2 中为 9% (p = 0.304)。 组 1 和组 2 的减剂量率分别为 59% 和 43% (p = 0.29)，用药中断率分别为 65% 和 67% (p = 0.908)。 结论 下调索拉非尼的日起始剂量对其在未获治疗的 DTC 患者中的疗效和耐受性似乎并不会造成负面影响。The Oncologist 2014;19:477–482 Time to failure, Overall survival, Dose reduction, Drug interruptions, Adverse events Implications for Practice: Sorafenib (800 mg daily) is the first oral tyrosine kinase inhibitor approved for differentiated thyroid cancer (DTC). Many patients must reduce the dose or discontinue treatment owing to toxicity. In clinical practice, outside the regimented scenario of a clinical trial, physicians sometimes choose to administer reduced doses in patients with multiple comorbidities. Our data suggest that treatment with sorafenib, when administered by experienced specialists outside of a clinical trial setting, resulted in similar efficacy and tolerability as previously reported in clinical trials. But, most importantly, we showed that the efficacy and tolerability of sorafenib in treatment-naïve DTC patients does not appear to be negatively influenced by lower starting daily doses. Introduction The incidence of thyroid cancer is rising annually. In 2013, an estimated 60,220 cases will be diagnosed, and despite standard treatment, 1,850 patients are expected to die of thyroid cancer [1]. Until recently, no efficacious, systemic therapies have been approved by the U.S. Food and Drug Administration for the treatment of differentiated thyroid cancer (DTC) refractory to radioactive iodine (RAI). However, the National Comprehensive Cancer Network and American Thyroid Association guidelines recommend tyrosine kinase inhibitors (TKIs) for progressive or symptomatic DTC refractory to RAI [2, 3]. Sorafenib is the TKI most studied and, therefore, the most frequently used as first-line treatment for progressive RAI-refractory DTC. It is an orally active multi-TKI with multiple targets, including vascular endothelial growth factor receptor 2 (VEGFR2), VEGFR3, BRAF, RET, and c-KIT. Sorafenib is currently approved in the U.S. and Europe for the treatment of advanced renal cell carcinoma and unresectable hepatocellular carcinoma. In November 2013, sorafenib became the first approved targeted agent for treatment of advanced DTC. The recommended dose is 800 mg daily (400 mg twice daily) in solid tumors, but no thyroid cancer patients were included in the phase I dose-finding trials [4–8]. In all phase II trials of sorafenib in patients with advanced RAI-refractory DTC, the total daily starting dose was 800 mg, but dose reductions were needed in up to 79% of patients, and the drug was discontinued owing to toxicity in up to 25% [9–13]. Because of the encouraging efficacy observed in phase II trials, a phase III placebo-controlled trial was conducted in patients with DTC who had not been treated previously with TKIs [14]. Results showed a progression-free survival advantage of 5 months in the sorafenib group. Unfortunately, dose reduction was needed in 64% of patients, and drug interruption was necessary in 66% of patients. Furthermore, the rate of drug discontinuation because of toxicity was 19%. It was recently reported that sorafenib therapy may have a mild but detectable impact on health-related quality of life [15]. Patients in the sorafenib arm had lower scores at first assessment (1 month after starting treatment) compared with placebo patients, possibly related to side effects of treatment. When mild side effects occur, they are managed conservatively, but dose reduction by one dose level (400 mg daily) and drug hold is needed when they are more severe. Proactive management is important because it may help decrease symptom severity and allow patients to maintain the benefit of therapy. It is unclear why tolerability of sorafenib in patients with thyroid cancer is so poor. In a very small study of nine patients with DTC receiving a total daily dose of 400 mg of sorafenib (200 mg twice daily), the efficacy was comparable to that reported by other trials, but none of the patients discontinued treatment because of treatment-related adverse events and no dose adjustments for toxicity were required, suggesting that sorafenib starting dose may have an impact on tolerability of sorafenib [16]. In clinical practice, outside of the regimented scenario of a clinical trial, physicians often face challenging situations when full-dose sorafenib is perceived as unsafe. These situations may include poor performance status, renal or hepatic dysfunction, high risk of bleeding or fistula formation in a previously irradiated area, and extremes of age. Consequently, we reviewed our clinical practice with sorafenib in patients who were not treated in the context of a clinical trial. Because more than half of the patients who started treatment with an 800-mg daily dose in a clinical trial required dose reductions, we sought to compare the efficacy and tolerability of the standard 800-mg starting dose with reduced starting doses in patients with advanced RAI-refractory DTC. Methods Study Population We retrospectively reviewed the records of adult patients (aged >18 years) with advanced metastatic DTC treated with sorafenib as first-line therapy outside of a clinical trial from January 2005 to July 2013 at the University of Texas MD Anderson Cancer Center. We collected the following demographic and clinicopathologic information for our analysis: age, sex, ethnicity, type of thyroid cancer, TNM stage at diagnosis, cumulative RAI activity, sites of metastases, time between diagnosis and start of treatment with sorafenib, starting dose of sorafenib, reported treatment interruptions or dose reductions and their causes, and date of death or last follow-up. We excluded patients who received an unknown starting dose of sorafenib. Patients were divided into two groups on the basis of sorafenib total starting daily dose; group 1 received a starting dose of 800 mg/day (400 mg twice daily), and group 2 received any starting dose lower than 800 mg/day. The reasons for choosing a reduced dose of sorafenib were not evaluable in all patients, but most were related to an increased risk of bleeding (history of ischemic colitis, Crohn's disease, decompensated cirrhosis with esophageal varices, atrial fibrillation during anticoagulation treatment with warfarin, hemoptysis, or history of treated brain metastases). Other comorbidities included renal failure on hemodialysis and poor performance status. In six patients, the treating physician decided to start with a lower dose with plans for upward titration if the drug was tolerated. This was achieved in four patients. Objectives We compared overall survival (OS) duration, time to treatment failure (TTF), time to progression (TTP), rates of sorafenib discontinuation because of progression or toxicity, and rates of dose reductions and drug interruption between the two groups. Definitions TTF was calculated as the time from the start of treatment with sorafenib to discontinuation because of disease progression or unacceptable toxicity. TTP was defined as the time from the start of treatment to discontinuation because of progression, as decided by the treating physician. Patients who were still receiving sorafenib at the time of our analysis were censored at the time of last follow-up. OS was calculated as the time from the start of treatment with sorafenib to the date of death from any cause. Patients who had not died at the time of analysis were censored at the time they were last known to be alive. Statistical Analysis Descriptive statistics were used to summarize patient characteristics by group. Comparisons were made between groups to identify any significant imbalances in patient characteristics. An independent samples t test was used to compare continuous variables, and a chi-square test was used to compare categorical variables. TTF, TTP, and OS for each group were illustrated using Kaplan-Meier plots, and a log-rank test was used to compare TTF, TTP, and OS distributions between groups. A p value <.05 was considered statistically significant. SPSS version 17 (IBM Corp., Armonk, NY, http://www-01.ibm.com/software/analytics/spss/) was used for statistical analysis. Results Study Population Seventy-five adult patients with metastatic DTC were treated with first-line sorafenib. Group 1 (800-mg/day starting dose) included 51 patients, and group 2 (<800-mg/day starting dose) included 24 patients. In group 2, only one patient received a 200-mg/day starting dose of sorafenib; the rest received 400 mg/day. Dose escalation to 800 mg daily was subsequently achieved in four patients who initially started with a lower dose. Patient Characteristics Baseline characteristics of all patients and comparisons between groups are shown in Table 1. The mean age at diagnosis was 54 years, and 42 patients (56%) were male. Most common types of DTC included the conventional type of papillary thyroid cancer (32 patients; 43%), followed by Hürthle cell carcinoma (11 patients; 15%) and follicular-variant papillary thyroid cancer (11 patients; 15%). Fifty-four patients (72%) had advanced tumors (T3 or T4), and 24 patients (32%) showed evidence of distant metastases at diagnosis. All patients had evidence of distant metastases at the time of sorafenib initiation, with the two most common sites being lung in 71 patients (95%) and bone in 24 patients (32%). Table 1 Baseline demographic and clinicopathologic characteristics of all patients in the study population and comparisons between groups 1 and 2 (group 1: starting dose of 800 mg/day of sorafenib; group 2: starting dose <800 mg/day of sorafenib) Open in new tab Table 1 Baseline demographic and clinicopathologic characteristics of all patients in the study population and comparisons between groups 1 and 2 (group 1: starting dose of 800 mg/day of sorafenib; group 2: starting dose <800 mg/day of sorafenib) Open in new tab Efficacy Time to Treatment Failure Seventy-one patients were included in the TTF analysis; four patients (all part of group 1) were excluded because the date of sorafenib discontinuation was unavailable. The median TTF for all patients was 9 months (95% confidence interval [CI]: 5.4–12.5). Median TTF was 10 months (95% CI: 5.6–14.3) in group 1 and 8 months (95% CI: 3.4–12.5) in group 2 (p = .56) (Fig. 1). TTF did not appear to be influenced by drug hold or dose reduction. Figure 1 Open in new tabDownload slide Time to treatment failure in group 1 (800-mg/day starting dose of sorafenib) compared with group 2 (<800-mg/day starting dose of sorafenib). ∗Four patients were excluded from group 1 because of an unknown date of sorafenib discontinuation. Abbreviations: CI, confidence interval; TTF, time to treatment failure. Figure 1 Open in new tabDownload slide Time to treatment failure in group 1 (800-mg/day starting dose of sorafenib) compared with group 2 (<800-mg/day starting dose of sorafenib). ∗Four patients were excluded from group 1 because of an unknown date of sorafenib discontinuation. Abbreviations: CI, confidence interval; TTF, time to treatment failure. Time to Progression Sixty-one patients were included in TTP analysis (39 patients in group 1 and 22 patients in group 2); 14 patients were excluded because they discontinued sorafenib because of toxicity or because the date of sorafenib discontinuation was unavailable. The median TTP for all patients was 10 months (95% CI: 5.1–14.8). Median TTP was 11 months in group 1 (95% CI: 6.1–15.8) and 8 months (95% CI: 5.8–10.1) in group 2 (p = .354). Overall Survival No patients were excluded for the OS analysis. The median OS duration of all patients was 39 months (95% CI: 17.9–60.0). Median OS was 56 months (95% CI: 30.6–81.3) in group 1 and 30 months (95% CI: 16.1–43.8) in group 2 (p = .08) (Fig. 2). The median follow-up time was 25 months in group 1 and 27.5 months in group 2. Figure 2 Open in new tabDownload slide Overall survival in group 1 (800-mg/day starting dose of sorafenib) compared with group 2 (<800-mg/day starting dose of sorafenib). Abbreviations: CI, confidence interval; OS, overall survival. Figure 2 Open in new tabDownload slide Overall survival in group 1 (800-mg/day starting dose of sorafenib) compared with group 2 (<800-mg/day starting dose of sorafenib). Abbreviations: CI, confidence interval; OS, overall survival. Tolerability Profile Reason for Sorafenib Discontinuation Forty-two of 51 patients discontinued sorafenib in group 1, and 22 of 24 patients discontinued sorafenib in group 2. Nine patients in group 1 and two patients in group 2 are still receiving sorafenib at the time of this analysis. Sorafenib was discontinued because of disease progression in 33 patients (79%) in group 1 and in 20 patients (91%) in group 2 and because of toxicity in 9 patients (21%) in group 1 and 2 patients (9%) in group 2 (p = .304) (Table 2). Toxic effects leading to sorafenib discontinuation included weight loss, anorexia, rash, squamous cell skin carcinoma, mucositis, diverticulitis, hand-foot syndrome, and altered mental status. Table 2 Tolerability of sorafenib in group 1 (800-mg/day starting dose) compared with group 2 (<800-mg/day starting dose) Open in new tab Table 2 Tolerability of sorafenib in group 1 (800-mg/day starting dose) compared with group 2 (<800-mg/day starting dose) Open in new tab Dose Reductions and Drug Interruptions Dose reductions and drug interruptions occurred in 46 of 51 patients in group 1 and 21 of 24 patients in group 2. Dose reduction was required in 27 patients (59%) in group 1 and 9 patients (43%) in group 2 (p = .29). Drug interruptions were necessary in 30 patients (65%) in group 1 and 14 patients (67%) in group 2 (p = .908) (Table 2). Discussion Sorafenib has shown efficacy in patients with advanced RAI-refractory DTC and, therefore, was recently approved for this disease. In phase II and III trials of sorafenib in patients with DTC, the starting daily dose was 800 mg. This was based on previous experience from phase I studies and from phase III studies in renal and hepatocellular carcinoma. Unfortunately, many patients with DTC receiving sorafenib 800 mg as starting daily dose require dose reduction or drug interruption; therefore, the maximum tolerated dose resulting in optimal efficacy should be considered. The only study to evaluate the effect of reduced starting doses was a very small prospective study of nine patients with DTC receiving a total daily dose of 400 mg [16]. Results of this study showed that the efficacy of the 400-mg/day dose was similar to that reported in the other trials, but none of these patients discontinued treatment because of treatment-related adverse events and no dose adjustments for toxicity were required. Our study included a large number of patients with treatment-naïve DTC who were treated with sorafenib outside a clinical trial, and we compared outcomes of patients who started with a daily dose of 800 mg with those who started at a lower dose. Our results for group 1 (800 mg/day) were very similar to those of the phase III clinical trial of sorafenib for patients with DTC receiving the same sorafenib starting dose [14]. Efficacy as measured by median TTP was 11 months for those patients receiving 800 mg/day in our study. Although not directly comparable, TTP in our study population is similar to the median progression-free survival rate of 10.8 months in the sorafenib group reported in the phase III clinical trial. An important finding of our study was that the median TTP was not statistically different between the two groups (11 months and 8 months), suggesting that efficacy may not be influenced by the sorafenib starting daily dose. In clinical practice, outside of the regimented scenario of a clinical trial, physicians often face challenging situations when full-dose sorafenib is perceived as unsafe because of comorbidities or relative contraindications to therapy. In this cohort of patients, treatment with a lower starting dose of sorafenib may still offer clinical benefit, as suggested by a median TTP of 8 months. Surprisingly, the lower starting dose of sorafenib was associated with similar high rates of discontinuation, dose reduction, and drug interruption when compared with the standard 800 mg dose. Because of a clinical benefit and similar toxicity profile, a lower dose of sorafenib may be started in challenging cases when full-dose sorafenib is perceived as unsafe. If tolerable, the dose may be subsequently increased. The discontinuation, drug-hold, and dose-reduction rates reported in the phase III trial in DTC (19%, 64%, and 66%, respectively) are very similar to the results we found in our study. Interestingly, in phase III trials of sorafenib in patients with renal and hepatocellular carcinoma, the discontinuation, dose-reduction, and drug-interruption rates (10%, 13%, and 21%, respectively, in patients with renal cell carcinoma and 11%, 26%, and 44%, respectively, in patients with hepatocellular carcinoma) were much lower than those observed in patients with thyroid cancer [17, 18]. It is unclear why patients with thyroid cancer have a different tolerability profile than patients with other cancers. One possible explanation is that patients with thyroid cancer continue treatment with sorafenib for a longer duration than patients with other cancers, and this increases the cumulative dose regardless of the starting dose. The median treatment durations noted for patients in phase III studies for DTC, renal cell carcinoma, and hepatocellular carcinoma were 46 weeks, 23 weeks, and 21 weeks, respectively [14, 17, 18]. Moreover, there are inherent differences in patients with DTC, including the presence of postoperative hypothyroidism, prior treatment with RAI, and the possibility of comorbidities such as hypoparathyroidism. A standardized approach related to prescribing commercially available tyrosine kinase inhibitors outside of a clinical trial for patients with advanced thyroid cancer was recently published [19]. This represents an effort to improve patient safety and monitoring and to prolong duration of therapy as long as it remains effective. In our study, the median OS duration for all DTC patients receiving first-line sorafenib was 39 months; the median OS was 56 months in group 1 (800-mg/day starting dose) compared with 30 months in group 2 (<800-mg/day starting dose). However, the difference was not statistically significant, possibly because of small sample size. Differences in median OS may be related to the dose effect itself, but this needs further study to be determined. Another hypothesis for the difference in OS is that the group of patients receiving a lower starting dose was selected because of worse performance status or medical comorbidities (as described under Methods). Our study contains several limitations. The retrospective nature of the study resulted in missing information regarding duration of treatment with various sorafenib starting daily doses before dose escalation and de-escalation, and this may have resulted in inhomogeneity between the two groups. There is also lack of information regarding patient performance status, evidence of disease progression before sorafenib initiation, radiographic assessment for evaluation of response rate and progression-free survival, and the type and grade of adverse events noted. Analysis of the effects of these variables on tolerability and outcomes is beyond the scope of this study. Conclusion Our data suggest that treatment with sorafenib, when administered by experienced specialists outside of a clinical trial setting, resulted in efficacy and tolerability similar to that previously reported in clinical trials. In clinical practice, physicians sometimes choose to administer reduced starting daily doses in patients with multiple comorbidities. Our results indicate that the efficacy of sorafenib in treatment-naïve patients with DTC does not appear to be negatively influenced by starting the treatment at a reduced daily dose. Interestingly, we did not find that reduced starting doses led to better tolerability; however, a prospective clinical trial randomizing patients with advanced DTC to receive different sorafenib starting doses would best address this question. Future studies analyzing the influence of dose intensity and total cumulative dose on outcomes and tolerability are also warranted. Acknowledgments This research was supported by the NIH and the National Cancer Institute under Grant P30CA016672. We thank Erica Goodoff for helping with grammatical assistance and stylistic suggestions. Author Contributions Conception/Design: Ramona Dadu, Maria E. Cabanillas Provision of study material or patients: Ramona Dadu, Steven G. Waguespack, Steven I. Sherman, Mimi I. Hu, Naifa L. Busaidy, Camilo Jimenez, Mohammed A. Habra, Anita K. Ying, Maria E. Cabanillas Collection and/or assembly of data: Ramona Dadu, Maria E. Cabanillas Data analysis and interpretation: Ramona Dadu, Steven G. Waguespack, Steven I. Sherman, Mimi I. Hu, Naifa L. Busaidy, Camilo Jimenez, Mohammed A. Habra, Anita K. Ying, Roland L. Bassett, Maria E. Cabanillas Manuscript writing: Ramona Dadu, Steven G. Waguespack, Steven I. Sherman, Mimi I. Hu, Naifa L. Busaidy, Camilo Jimenez, Mohammed A. Habra, Anita K. Ying, Maria E. Cabanillas Final approval of manuscript: Ramona Dadu, Steven G. Waguespack, Steven I. Sherman, Mimi I. Hu, Naifa L. Busaidy, Camilo Jimenez, Mohammed A. Habra, Anita K. Ying, Maria E. Cabanillas Disclosures Steven I. Sherman: Bayer (C/A); Naifa L. Busaidy: Bayer-Onyx (C/A); Bayer (RF). The other authors indicated no financial relationships. (C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board For Further Reading: Taofeek K. Owonikoko, Rajasree P. Chowdry, Zhengjia Chen et al. Clinical Efficacy of Targeted Biologic Agents as Second-Line Therapy of Advanced Thyroid Cancer. The Oncologist 2013:18;1262–1269. Implications for Practice: Significant benefit can be achieved in patients with iodine-refractory thyroid cancer treated with targeted agents in the first-line setting. It is currently unknown whether additional benefit would be obtained with the use of different biologic agents to treat patients after failing first-line therapy. This study reports the authors' experience using biologic agents as second-line treatment for advanced thyroid cancer and shows that patients derived additional benefit, albeit modest, in comparison to the front-line treatment. These findings are relevant for the clinical management of patients and for future studies of second-line targeted therapy of thyroid cancer. References 1 Siegel R , Naishadham D, Jemal A. Cancer statistics, 2012 . CA Cancer J Clin . 2012 ; 62 : 10 – 29 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Cooper DS , Doherty GM, Haugen BR. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer . Thyroid . 2009 ; 19 : 1167 – 1214 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Tuttle RM , Ball DW, Byrd D. Thyroid carcinoma . J Natl Compr Canc Netw . 2010 ; 8 : 1228 – 1274 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Strumberg D , Richly H, Hilger RA. Phase I clinical and pharmacokinetic study of the Novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors . J Clin Oncol . 2005 ; 23 : 965 – 972 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Moore M , Hirte HW, Siu L. Phase I study to determine the safety and pharmacokinetics of the novel Raf kinase and VEGFR inhibitor BAY 43-9006, administered for 28 days on/7 days off in patients with advanced, refractory solid tumors . Ann Oncol . 2005 ; 16 : 1688 – 1694 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Clark JW , Eder JP, Ryan D. Safety and pharmacokinetics of the dual action Raf kinase and vascular endothelial growth factor receptor inhibitor, BAY 43-9006, in patients with advanced, refractory solid tumors . Clin Cancer Res . 2005 ; 11 : 5472 – 5480 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Strumberg D , Clark JW, Awada A. Safety, pharmacokinetics, and preliminary antitumor activity of sorafenib: A review of four phase I trials in patients with advanced refractory solid tumors . The Oncologist . 2007 ; 12 : 426 – 437 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Awada A , Hendlisz A, Gil T. Phase I safety and pharmacokinetics of BAY 43-9006 administered for 21 days on/7 days off in patients with advanced, refractory solid tumours . Br J Cancer . 2005 ; 92 : 1855 – 1861 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Kloos RT , Ringel MD, Knopp MV. Phase II trial of sorafenib in metastatic thyroid cancer . J Clin Oncol . 2009 ; 27 : 1675 – 1684 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Hoftijzer H , Heemstra KA, Morreau H. Beneficial effects of sorafenib on tumor progression, but not on radioiodine uptake, in patients with differentiated thyroid carcinoma . Eur J Endocrinol . 2009 ; 161 : 923 – 931 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Gupta-Abramson V , Troxel AB, Nellore A. Phase II trial of sorafenib in advanced thyroid cancer . J Clin Oncol . 2008 ; 26 : 4714 – 4719 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Ahmed M , Barbachano Y, Riddell A. Analysis of the efficacy and toxicity of sorafenib in thyroid cancer: A phase II study in a UK based population . Eur J Endocrinol . 2011 ; 165 : 315 – 322 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Schneider TC , Abdulrahman RM, Corssmit EP. Long-term analysis of the efficacy and tolerability of sorafenib in advanced radio-iodine refractory differentiated thyroid carcinoma: Final results of a phase II trial . Eur J Endocrinol . 2012 ; 167 : 643 – 650 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Brose M , Nutting C, Jarzab B. Sorafenib in locally advanced or metastatic patients with radioactive iodine-refractory differentiated thyroid cancer: The phase III DECISION trial . J Clin Oncol . 2013 ; 39 ( suppl ): 4a . Google Scholar OpenURL Placeholder Text WorldCat 15 Schlumberger M , Jarzab B, Elisei R et al. Phase III randomized, double-blinded, placebo-controlled trial in locally advanced or metastatic patients with radioactive iodine (RAI)-refractory differentiated thyroid cancer (DTC) - exploratory analyses of patient-reported outcomes . Abstract 100 presented at: Annual Meeting of the American Thyroid Association ; October 16–20, 2013 ; San Juan, Puerto Rico . 16 Chen L , Shen Y, Luo Q. Response to sorafenib at a low dose in patients with radioiodine-refractory pulmonary metastases from papillary thyroid carcinoma . Thyroid . 2011 ; 21 : 119 – 124 . Google Scholar Crossref Search ADS PubMed WorldCat 17 Llovet JM , Ricci S, Mazzaferro V. Sorafenib in advanced hepatocellular carcinoma . N Engl J Med . 2008 ; 359 : 378 – 390 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Escudier B , Eisen T, Stadler WM. Sorafenib in advanced clear-cell renal-cell carcinoma . N Engl J Med . 2007 ; 356 : 125 – 134 . Google Scholar Crossref Search ADS PubMed WorldCat 19 Carhill AA , Cabanillas ME, Jimenez C. The noninvestigational use of tyrosine kinase inhibitors in thyroid cancer: Establishing a standard for patient safety and monitoring . J Clin Endocrinol Metab . 2013 ; 98 : 31 – 42 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Disclosures of potential conflicts of interest may be found at the end of this article. © 2014 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Kentwell, Jessica; Gundara, Justin S.; Sidhu, Stan B.

2014 The Oncologist

doi: 10.1634/theoncologist.2013-0458pmid: 24718512

Abstract Only recently has it been uncovered that the mammalian transcriptome includes a large number of noncoding RNAs (ncRNAs) that play a variety of important regulatory roles in gene expression and other biological processes. Among numerous kinds of ncRNAs, short noncoding RNAs, such as microRNAs, have been extensively investigated with regard to their biogenesis, function, and importance in carcinogenesis. Long noncoding RNAs (lncRNAs) have only recently been implicated in playing a key regulatory role in cancer biology. The deregulation of ncRNAs has been demonstrated to have important roles in the regulation and progression of cancer development. In this review, we describe the roles of both short noncoding RNAs (including microRNAs, small nuclear RNAs, and piwi-interacting RNAs) and lncRNAs in carcinogenesis and outline the possible underlying genetic mechanisms, with particular emphasis on clinical applications. The focus of our review includes studies from the literature on ncRNAs in traditional endocrine-related cancers, including thyroid, parathyroid, adrenal gland, and gastrointestinal neuroendocrine malignancies. The current and potential future applications of ncRNAs in clinical cancer research is also discussed, with emphasis on diagnosis and future treatment. Long noncoding RNA, Small untranslated RNA, Endocrine gland neoplasms, Tumor suppressor genes, Oncogenes Implications for Practice: Our knowledge of noncoding RNA (ncRNA) has boomed since the turn of the century, and progressive research in this field now aims to take advantage of our improved understanding of these key regulators. Present preclinical evidence in relation to endocrine malignancy has great potential to transform future management of a unique group of diseases that have failed to respond to conventional treatment. Epigenetic ncRNA regulation is clearly of great importance and should be harnessed in the future as a means of not only enhancing our understanding of endocrine malignancy but also, ultimately, of translating current knowledge into therapeutic success. Introduction The discovery that more than 95% of human transcriptional output is noncoding RNA (ncRNA) [1] raised questions about the traditional opinion in molecular biology that RNA is only a simple intermediary between DNA and protein. An ncRNA is, by definition, an RNA that does not transcribe for a protein, and there is presently an increasing number of well-characterized ncRNAs that are of evolving scientific and clinical interest [2]. It is now evident that ncRNAs play important functional and structural roles in both health and disease [3, 4]. Progressive research in this field aims to take advantage of our improved understanding of these key regulators to ultimately translate this knowledge into improved diagnostic, prognostic, and therapeutic options in oncology. This review summarizes the evidence to date and demonstrates the burgeoning potential of ncRNA-driven scientific and clinical research [5] Our knowledge of ncRNAs has boomed since the turn of the century, and a myriad of ncRNAs have now been characterized and can be broadly classified based on size [6]. Small noncoding RNAs (sncRNAs) are typically 18–200 nucleotides in length compared with long ncRNAs (lncRNAs), which can range from 200 nucleotides to more than 100 kilobase pairs. More specifically, sncRNAs are of vital importance to fundamental biological processes and, therefore, can contribute significantly to certain pathophysiological states. The most well-known families of sncRNAs are microRNAs (miRNAs) and small interfering RNAs, both of which have established roles in RNA interference [1, 7, 8]. Other sncRNA families include small nucleolar RNAs (snoRNAs), small nuclear RNAs, and piwi-interacting RNAs. Detailed descriptions of sncRNA biogenesis and function have been described previously [8–10]. For the purposes of this review, microRNAs will be the primary focus; however, additional ncRNAs of note will be discussed briefly to highlight their growing potential and our current understanding of the roles they play in cancer. Small Noncoding RNA MicroRNAs miRNAs are endogenous, single-stranded, short RNA sequences (∼22 nucleotides) that regulate gene expression at the post-transcriptional level by base pairing with target mRNA sequences. miRNA-mediated gene silencing is generally accomplished by imperfect base pairing of 5′ regions of miRNAs with the target mRNA sequence, leading to translational repression and/or mRNA degradation [8, 11–13]. Thousands of human miRNAs have now been characterized. They are known to play important roles in a wide variety of processes including growth, differentiation, immune reactions, and adaptation to physiological stress [1, 3, 7, 8, 10, 14, 15]. Given that a single miRNA can target hundreds of mRNAs and a single mRNA can be targeted by multiple miRNAs [16], they are attractive therapeutic targets in disease, particularly cancer [16, 17]. Of additional importance is the fact that miRNAs are stable in the circulation and, therefore, may be used as serum biomarkers [18, 19]. From a translational viewpoint, patterns of differential miRNA expression have been shown to be of diagnostic and prognostic utility in a multitude of cancers and are now on the cusp of clinical application (Tables 1–3). Table 1 MicroRNAs in thyroid malignancies Open in new tab Table 1 MicroRNAs in thyroid malignancies Open in new tab Table 2 MicroRNAs in adrenocortical carcinomas and pheochromocytoma Open in new tab Table 2 MicroRNAs in adrenocortical carcinomas and pheochromocytoma Open in new tab Table 3 MicroRNAs in gastrointestinal neuroendocrine malignancies Open in new tab Table 3 MicroRNAs in gastrointestinal neuroendocrine malignancies Open in new tab Small Nucleolar RNA and Piwi-Interacting RNA Small nucleolar RNAs (snoRNAs) and small nuclear RNAs are best known as guide molecules for site-specific methylation and pseudouridylation of other RNAs [8]. As their name implies, they are typically localized to the nucleolus or nucleus, respectively, where they play important roles in the modification and processing of ribosomal RNA. Certain snoRNAs have been shown to influence mRNA splicing and may even serve as miRNA precursors [8]. With regard to human disease, deletion of the snoRNA cluster SNORD116 C/D box is a paternally inherited deletion evident in Prader-Willi syndrome [19]. In addition, various snoRNAs are differentially expressed in non-small cell lung cancer [20], peripheral T-cell lymphoma [21], and prostate cancer [22]. Other studies have shown that a homozygous deletion of the snoRNA U50 is associated with prostate cancer development [23] and undergoes frequent deletion and transcriptional downregulation in breast cancer [24]. Of additional interest is the well-known small nuclear RNA 7SK (also known as RN7SK), which regulates transcription by inhibiting the activity of CDK9/cyclin T1 complexes [25]. HMGA1 has been identified as a novel RN7SK interaction gene and is often overexpressed in human malignancies, including thyroid cancer [26]. Intriguingly, RN7SK has also been shown to regulate expression of LARP7, leading to a novel syndrome of facial dysmorphism, dwarfism, and intellectual disability [27]. Although the precise mechanism by which this group of small RNAs contributes carcinogenesis is still unknown, it is likely that such disease associations reflect underlying mechanistic importance. Long Noncoding RNAs Long noncoding RNAs can be defined as RNA molecules greater than 200 nt in length [28] and can be divided into five subclasses depending on their genomic location (intronic or intergenic). They are generally involved in regulating genetic expression at various levels, including chromatin modification, transcription, and post-transcriptional processing, such as mRNA splicing and translation [12]. Given such intrinsic involvement, it is no surprise that they are now known to be of importance in a number of biological processes and pathological disease states [1, 10, 29]. Altered expression in cancer has recently been revealed [30, 31]. One defined mechanism of lncRNA action involves interaction with chromatin remodeling complexes, which are of known importance in carcinogenesis. The lncRNA ANRIL (antisense noncoding RNA in the INK4 locus), for example, has been shown to be altered in up to 30%–40% of human tumors [6]. This may relate to associations with three tumor-suppressor genes that are often deleted in this context [32]. Another example of a chromatin-modifying lncRNA is HOTAIR (HOX antisense intergenic RNA) [7]. Gupta et al. [33] found that HOTAIR expression is significantly upregulated in both primary and metastatic breast cancer (up to 2,000-fold compared with normal breast tissue); with regard to outcome associations, they also showed that expression positively correlated with metastasis and worse survival outcomes. HOTAIR has since been shown to promote metastasis by heightening the invasiveness of breast cancer cells by altering the expression of polycomb repressive complex 2, which reprograms the global chromatin state of tumor cells [34]. The advent of high-throughput gene-sequencing technologies has led to the recent discovery of a variety of novel lncRNAs. LSTINCT5 is an intergenic lncRNA identified by Silva et al. [35] that is overexpressed in breast and ovarian cancer cell lines. Functional importance was demonstrated through knockdown experiments that resulted in decreased proliferation in both cell lines [35]. Similarly, Prensner et al. [36] identified an lncRNA, later named PCAT1, that was selectively upregulated only in prostate cancer. Similar to HOTAIR, PCAT1 functions predominantly as a transcriptional repressor by facilitating transregulation of genes preferentially involved in mitosis and cell division, including known tumor suppressor genes such as BRCA2 [28]. Another novel lncRNA, prostate cancer noncoding RNA 1 (PCNCR1), was identified in a “gene desert” on chromosome 8q24.2 and is associated with susceptibility to prostate cancer [37]. The lncRNA differential display code 3 (DD3) is also highly overexpressed in prostate cancer, yet little is known about the role DD3 may play in prostate cancer progression [38]. DD3 has been developed into a highly specific, nucleic acid amplification-based marker of prostate cancer that demonstrated higher specificity than serum prostate-specific antigen [39, 40]. The rapid timeline of novel lncRNA discoveries suggests that their clinical utility in medicinal applications is only beginning [6]. Of additional interest is steroid receptor RNA activator (SRA), which was the first lncRNA shown to function as a gene regulator. SRA is overexpressed in breast, uterine, and ovarian tumors and increases cell proliferation in certain hormone-dependent breast cancers [41, 42] and prostate cancers [43]. BC200 (also known as BCYRN1) is also greatly upregulated in ovarian cancer [44] and is also significantly overexpressed in high-grade invasive breast tumors [45]. LncRNAs also play a role in post-transcriptional gene regulation and control of cellular growth [46]. The lncRNA growth arrest-specific 5 (GAS5), which also encodes some snoRNAs, functions by sensitizing to apoptosis, a function that has been illustrated in prostate cancer cell lines [47]. The functional impact of GAS5 has also been explored in breast cancer, in which GAS5 is relatively underexpressed and, as such, maintains tumor growth potential [48]. The significant role that ncRNAs play in oncology, although still evolving, is a considerable topic. In order to enhance the clarity of this review, endocrine malignancies will be the theme of specific interest. This includes cancers of traditional endocrine origin such as thyroid carcinoma, parathyroid carcinoma, adrenocortical carcinoma, pheochromocytoma, and gastrointestinal neuroendocrine tumors. Reviews of ncRNAs as they relate to other endocrine organs, including breast [34], ovary [49], and prostate [22], can be found elsewhere. Noncoding RNAs in Endocrine Malignancies Surgery is the curative treatment of choice for endocrine malignancies and may also be a vital therapeutic modality for locoregional disease control and an effective palliative option. Beyond surgery, endocrine cancers have generally suffered from a lack of tailored chemotherapeutic, hormonal, or biologic therapy options. This is typified by, for instance, medullary thyroid cancer, a disease in which outcomes have not improved for more than 30 years [50, 51]. This is particularly the case in sporadic forms of the disease, and although tyrosine kinase inhibitors have been a revolution for many neuroendocrine diseases, they have failed, as of yet, to demonstrate a survival advantage for medullary thyroid cancer patients. Newer therapies are required, and our ever-expanding knowledge base regarding ncRNAs may represent an opportunity to improve treatment outcomes. Noncoding RNAs and Thyroid Cancer Thyroid cancer is the most common endocrine malignancy [52] and the fifth most common cancer in women [53]. The incidence of thyroid cancer has increased continuously over the last three decades [53], highlighting the need to maintain a progressive treatment paradigm. A variety of thyroid carcinoma phenotypes exist, the most common being differentiated thyroid carcinomas (DTC) that include papillary thyroid carcinoma (PTC) and follicular subtypes. The overall prognosis for DTC is excellent, with 10-year survival greater than 90% [54]. However, conventional treatment options such as surgery and radiotherapy are not effective after metastasis, after which survival declines rapidly [54]. In contrast, anaplastic thyroid carcinoma and medullary thyroid carcinoma (MTC) are rare endocrine malignancies with poorer prognosis than DTC. Anaplastic thyroid carcinoma is often rapidly fatal and has median survival of less than 6 months [54]. Although MTC maintains a 10-year survival rate greater than 70% following appropriate surgery at diagnosis, metastases are common, and efficacious therapeutic options beyond surgery are still limited [54]. Several gene mutations have been shown to be of importance in thyroid cancer, and many of them (e.g., RET, RAS) involve the oncogenic mitogen-activated protein kinase pathway [52, 55]. Beyond this, however, epigenetic regulators (e.g., ncRNAs) are also being keenly investigated for clinical application. Although there are already established circulating biomarkers in many endocrine diseases, they are not always reliable. Circulating thyroglobulin, for example, is used as a biomarker to measure residual disease in PTC but is unreliable in 25% of cases [56]. Alternatively, novel biomarkers based on ncRNAs are on the horizon. Lee et al. [57] demonstrated that miR-222, miR-221, miR-146b, and miR-21 were lower in the serum of PTC patients after thyroidectomy (compared with controls), suggesting that elevated levels of these serum miRNAs strongly correlate with the presence of PTC. Two miRNAs in particular, miR-222 and miR-146b, also correlated with the presence of extrathyroidal extension prior to thyroidectomy. These results suggest that miRNA expression in serum not only correlates with the presence of PTC but also can predict for disease aggressiveness. Lee et al. demonstrated that miR-222, miR-221, miR-146b, and miR-21 were lower in the serum of PTC patients after thyroidectomy (compared with controls), suggesting that elevated levels of these serum miRNAs strongly correlate with the presence of PTC. Two miRNAs in particular, miR-222 and miR-146b, also correlated with the presence of extrathyroidal extension prior to thyroidectomy. These results suggest that miRNA expression in serum not only correlates with the presence of PTC but also can predict for disease aggressiveness. MTC is also a disease in which differential miRNA expression has proven to be of utility. The first study investigating the miRNA profile of MTC was performed by Nikiforova et al. [58]. This involved analysis of 42 thyroid cancers and an additional 62 fine-needle aspiration (FNA) samples that were subjected to microarray studies probing for 158 different miRNAs. When compared with normal thyroid tissue, 10 miRNAs were found to be significantly upregulated, and four (miR-323, miR-370, miR-129, and miR-137) were upregulated on the order of >100-fold change. Although the paper by Nikiforova et al. provided evidence of a distinct miRNA profile for MTC, no clinical data were presented; therefore, any clinical relevance is unknown. Following this work, the miR-200 family was implicated by Santarpia et al. in unpublished studies [59]. Microarray analysis of primary tumors (number not available) and corresponding metastatic tumor specimens yielded 16 miRNAs that were found to be differentially expressed between primary and metastatic tumor tissue. Further bioinformatic analyses identified purported gene targets known to be important for cell adhesion and migration. Subsequent miR-200 transfection of two human MTC cell lines (TT and MZ-CRC-1) induced a reduction in cell adhesion and an increase in cell detachment in vitro and characteristics thought to be consistent with a tendency to metastasize and more aggressive clinical behavior [59]. More recent work has been undertaken to define the miRNA profile of MTC at the Kolling Institute of Medical Research. Following array studies, validation work confirmed that miR-375 and miR-183 were significantly overexpressed in sporadic MTC (SMTC) versus hereditary MTC (HMTC) cases of disease. MiR-183 expression was also shown to be significantly associated with high-risk RET mutation status in addition to a tendency toward development of residual disease, lateral lymph node metastases, and mortality [60]. The most recent miRNA studies of MTC were published in 2012 by Mian et al. [61]. These studies examined clinical specimens of 34 cases of SMTC, 6 cases of HMTC, and an additional 2 cases of C-cell hyperplasia, with the aim of correlating miRNA expression with RET gene mutation status and outcome. In summary, overexpression of a number of miRNAs was defined in MTC (SMTC and HMTC were grouped together) and C-cell hyperplasia specimens (miR-21, miR-127, miR-154, miR-224, miR-323, miR-370, miR-9*, miR-183, and miR-375). More specifically, lower levels of miR-127 were observed in cases of SMTC with somatic RET mutations, as compared with wild-type RET. With regard to clinical outcome, miR-224 expression also correlated with an improved prognosis. Mian et al. concluded that miRNAs are significantly dysregulated in MTC and suggested that this may be a fundamental event in the pathogenesis of C-cell carcinogenesis [61]. The studies of Nikiforova et al. [58] also demonstrated that miRNA analysis of FNA tissue not only is possible but also improves the accuracy of this diagnostic test in assessment of the thyroid nodule. When at least one miRNA was overexpressed more than twofold, FNA test sensitivity improved to 100%, specificity was 94% and accuracy 95% [58]. With regard to potential biopsy of metastatic disease, it has also been shown that miRNA profiles in lymph node metastases are similar specifically to the primary tumor in thyroid cancer [62]. A number of lncRNAs have also been associated with thyroid carcinomas, including the lncRNAs AK023948 [63] and NAMA [64], which are both downregulated in PTC. AK023948 represents a possible candidate gene for PTC susceptibility [63], and NAMA was found to be a downstream target gene of the mitogen-activated protein kinase pathway and is associated with cell-growth arrest [64]. Papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3A) is another lncRNA found to be significantly underexpressed in PTC, with restoration of PTCSC3A in PTC cell lines inhibiting cell growth [65]. The significance of miRNAs in thyroid cancer is now also becoming a reality for additional lncRNAs candidate markers. Noncoding RNAs and Parathyroid Cancer Parathyroid carcinoma is a rare cause of primary hyperparathyroidism and may lead to intractable, potentially life-threatening hypercalcemia [54]. The rare nature of parathyroid carcinoma has meant that there are few studies examining the role that ncRNAs play in the pathogenesis of this disease. Current opportunities to exploit ncRNAs do exist, particularly with regard to diagnosis, which is frequently difficult, given the absence of definitive histological features and the requirement for local tissue invasion, by which time curative resection is not possible [54]. Aberrant parathyroid carcinoma miRNA expression has been identified in a small number of studies [66–68], suggesting that there may be a miRNA profile of diagnostic and prognostic potential. Corbetta et al. [66], for example, have shown that when compared with normal glands, malignant parathyroid tissue overexpresses miR-222 and miR-503. In addition, miR-296 and miR-139 were reportedly underexpressed in malignant tissue. Interestingly, miR-139 is located at a fragile chromosomal region often lost in multiple endocrine neoplasia type 1, implying that this miRNA may harbor significance in multigland endocrinopathy [66]. Many overexpressed miRNAs in parathyroid carcinoma are located at the C19MC genomic cluster. The loss of promoter methylation at the C19MC cluster is associated with high calcium levels and metastatic disease, suggesting an oncogenic role for these overexpressed miRNAs [68]. The functional role of what appears to be newly identified differential expression is beginning to be unveiled. Although current ncRNA research into parathyroid carcinoma is presently limited, it is a disease that would appear to be ideally suited to this realm of scientific exploration. The need for improved diagnostic criteria in particular should be an adequate stimulus for ongoing research interest. Noncoding RNA in Adrenocortical Carcinoma and Pheochromocytoma Adrenocortical carcinoma (ACC) is an aggressive tumor of the adrenal gland, associated with frequent metastasis and poor survival [69]. Patients with ACC have suffered from diagnostic criteria that, arguably, are still lacking [70], leading to delays in diagnosis and difficulty committing to appropriate therapy. Present treatment regimens are poorly tolerated, and tailored therapy has yet to become a reality. Clearly, ncRNAs may fill a significant biomarker, diagnostic, and therapeutic void for this disease. Soon et al. investigated whether differential miRNA expression could identify potential prognostic markers and therapeutic targets in ACC using a microarray analysis [71]. MiR-483-5p was found to be overexpressed in ACC compared with both adenoma and normal tissue. Cancers also demonstrated underexpression of miR-335 and miR-195. Clinical data correlation also yielded biomarker potential, with ACC underexpression of miR-195 and overexpression of miR-483-5p predicting poorer disease-specific prognosis. These results have been further validated more recently by Chabre et al., who confirmed miR-483-5p overexpression and miR-195 underexpression in ACC. In addition, differential expression of these miRNAs also correlated with a more aggressive phenotype of disease, associated with larger tumor size and poorer prognosis [44]. Significantly, the authors were also able to quantify the miRNA expression in patient serum, and levels of both miR-195 and miR-483-5p were shown to decrease significantly following resection of the primary ACC. This suggested that serum miRNA expression may be associated with a specific tumor burden, which has implications for the use of circulating miRNAs as biomarkers of disease. In contrast to the previous two studies, the works of Ozata et al. [69] failed to demonstrate a significant association between miR-483-5p or miR-195 and clinical outcome in their ACC cohort. This may be indicative of a heterogeneous patient population and may also relate to the current reliability of ncRNA technology that is not presently commercially available on a large scale. More work is required to ensure that ncRNA results fulfill the requirements of a clinically useful test, namely, to be valid, reliable, and reproducible. Table 3 summarizes the ncRNA research to date in ACCs and pheochromocytomas. Pheochromocytomas are rare, catecholamine-producing endocrine tumors of chromaffin cell origin that originate in the adrenal medulla [54, 72, 73]. They can also occur in extra-adrenal sites such as the chest and the pelvis [74]. They are unique tumors because they often occur within the context of hereditary endocrine syndromes and genetic mutations, such as multiple endocrine neoplasia type 2 and von Hippel-Lindau syndrome. Currently, there are no reliable histomorphological features to distinguish between benign and malignant pheochromocytoma [72], and a definitive diagnosis of malignancy relies on tumor metastases at sites where chromaffin tissue is normally absent [74, 75]. As in the previously discussed diagnostic conundrums of parathyroid carcinoma and ACC, ncRNAs may represent an alternative diagnostic tool with which to clarify a specific diagnosis and may have far-reaching clinical consequences. Currently, there are no reliable histomorphological features to distinguish between benign and malignant pheochromocytoma, and a definitive diagnosis of malignancy relies on tumor metastases at sites where chromaffin tissue is normally absent. As in the previously discussed diagnostic conundrums of parathyroid carcinoma and ACC, ncRNAs may represent an alternative diagnostic tool with which to clarify a specific diagnosis and may have far-reaching clinical consequences. Meyer-Rochow et al. were among the first groups to explore miRNA expression in malignant pheochromocytoma by using a microarray expression analysis [76]. Overexpression of miR-483-5p was identified in malignant tumors (compared with benign and normal adrenal tissue), and this was also shown to positively correlate with IGF2 overexpression. In addition, miR-15a and miR-16 were found to be underexpressed. The functional roles of miR-15a and miR-16 were further investigated in vitro, and it was determined they induced cell cycle arrest and reduced cellular proliferation [76]. From a translational viewpoint, the authors also examined the clinical utility of differential miRNA expression and reported that high IGF2 mRNA and low miR-15a expression could distinguish malignant from benign tumors with 80% sensitivity and 100% specificity [76]. These findings articulate how existing diagnostic schemas may benefit from the addition of ncRNA expression profiles. These results have been supported by the confirmatory studies of Patterson et al., who also identified overexpression of miR-483-5p in malignant pheochromocytomas [72], in addition to underexpression of miR-183 and miR-101. Such differential miRNA expression was also validated in serum samples. Beyond these findings, Tombol et al. [73] demonstrated that elevated expression of miR-885-5p and miR-1225-3p is seen in multiple endocrine neoplasia type 2-related and sporadic, recurring pheochromocytomas, respectively [73]. Current genetic markers for endocrine malignancies such as pheochromocytoma are of great utility but are not infallible. As discussed, ncRNA research may represent an opportunity to improve diagnostic and prognostic accuracy by identifying subtle epigenetic differences between tumors. This may also reveal the possibility of tailored therapy for diseases like endocrine malignancies that largely fail to respond to conventional chemotherapeutic regimens. Noncoding RNAs and Neuroendocrine Tumors There are few studies in the literature relating to ncRNAs and (non-MTC) neuroendocrine malignancies. Neuroendocrine tumors (NETs) most often occur in the intestines, pancreas, and lung and can be classified as functional NETs (hormone secreting) or nonfunctional (non-hormone secreting) [77]. Pancreatic endocrine tumors (PETs) are rare cancers that account for ∼1%–2% of all pancreatic malignancies, and 90% of them occur sporadically [77]. The only study in the literature to our knowledge investigating ncRNAs and PETs [78] found differential microRNA expression between PETs and normal pancreatic tissue, as well as being able to distinguish between pancreatic tumors of an islet cell origin from those of an acinar cell origin. Overexpression of miR-21 was also highly correlated with the presence of liver metastasis [78]. Two studies have investigated ncRNAs and small intestinal NETs, and only one study exists presently with a focus on lung NETs. miRNA-196a was shown to be overexpressed in small intestinal NETs in both studies [79, 80]. Overexpression was validated in malignant samples, and expression was differential between locally advanced and metastatic NET cell lines. Such differential expression presumably occurred through the proliferative effect of miR-196a on the PI3K/AKT/mTOR pathway and synthesis of HOXB/C genes [80]. Similarly, Lee et al. found miR-21 and miR-155 to be overexpressed in high-grade, invasive pulmonary NETs compared with carcinoid pulmonary NETs [81]. The oncogenic effect of miR-155 in these NETs was proposed to be due to the miR-155 effect on transforming growth factor-β to induce cell migration, invasion, and epithelial-mesenchymal transition [81]. Although basic scientific exploration of the role that ncRNAs play in NETs continues, there is little data available that is of translational significance. As with the other endocrine malignancies discussed, NETs are a disease that requires a dedicated research effort to forge new management paradigms, possibly based on ncRNA discovery [82]. Conclusion This review highlights the burgeoning potential of noncoding RNA as a diagnostic, prognostic, and therapeutic tool in cancer research. There is no question that both small and long ncRNAs have vital biological roles that influence a myriad of different pathways that are implicated in the oncogenesis and metastasis of cancer. Present preclinical evidence in relation to endocrine malignancy has great potential to transform future management of a unique group of diseases that have failed to respond to conventional management. Epigenetic ncRNA regulation is of great importance and should be harnessed in the future as a means of not only enhancing our understanding of endocrine malignancy but also, ultimately, of translating current knowledge into therapeutic success. Author Contributions Manuscript writing: Jessica Kentwell, Stan B. Sidhu, Justin Gundara Final approval of manuscript: Stan B. Sidhu, Justin Gundara Disclosures The authors indicated no financial relationships. 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Srivastava, Geetika; Renfro, Lindsay A.; Behrens, Robert J.; Lopatin, Margarita; Chao, Calvin; Soori, Gamini S.; Dakhil, Shaker R.; Mowat, Rex B.; Kuebler, J. Philip; Kim, George; Mazurczak, Miroslaw; Lee, Mark; Alberts, Steven R.

2014 The Oncologist

doi: 10.1634/theoncologist.2013-0401pmid: 24710310

Abstract Purpose. The Oncotype DX colon cancer assay is a clinically validated predictor of recurrence risk in stage II colon cancer patients. This prospective study evaluated the impact of recurrence score (RS) results on physician recommendations regarding adjuvant chemotherapy in T3, mismatch repair-proficient (MMR-P) stage II colon cancer patients. Patients and Methods. Stage IIA colon cancer patients were enrolled in 17 centers. Patient tumor specimens were assessed by the RS test (quantitative reverse transcription-polymerase chain reaction) and mismatch repair (immunohistochemistry). For each patient, the physician's recommended postoperative treatment plan of observation, fluoropyrimidine monotherapy, or combination therapy with oxaliplatin was recorded before and after the RS and mismatch repair results were provided. Results. Of 221 enrolled patients, 141 patients had T3 MMR-P tumors and were eligible for the primary analysis. Treatment recommendations changed for 63 (45%; 95% confidence interval: 36%–53%) of these 141 T3 MMR-P patients, with intensity decreasing for 47 (33%) and increasing for 16 (11%). Recommendations for chemotherapy decreased from 73 patients (52%) to 42 (30%), following review of RS results by physician and patient. Increased treatment intensity was more often observed at higher RS values, and decreased intensity was observed at lower values (p = .011). Conclusion. Compared with traditional clinicopathological assessment, incorporation of the RS result into clinical decision making was associated with treatment recommendation changes for 45% of T3 MMR-P stage II colon cancer patients in this prospective multicenter study. Use of the RS assay may lead to overall reduction in adjuvant chemotherapy use in this subgroup of stage II colon cancer patients. 摘要 目的 Oncotype DX 结肠癌测定是一项已通过临床验证的 II 期结肠癌复发风险预测工具。本前瞻性研究评估了复发评分 (RS) 对 T3 期错配修复精巧 (MMR-P) II 期结肠癌患者的辅助化疗治疗建议的影响。 患者与方法 我们在 17 个研究中心进行了 IIA 期结肠癌患者的招募。使用 RS 检测法（定量逆转录聚合酶链反应）和错配修复（免疫组织化学）对患者肿瘤样本进行了评估。我们记录了医生在获得 RS 和错配修复结果前及获得结果后为每一位患者建议的术后治疗方案，包括观察、氟嘧啶单药治疗，或氟嘧啶与奥沙利铂联合治疗。 结果 在 221 名入组患者中，有 141 名存在 T3 MMR-P 肿瘤并有资格被纳入主要分析。在这 141 名 T3 MMR-P 患者中，有 63 名的治疗建议发生了改变（45%；95% 置信区间： 36%–53%），其中 47 (33%) 名的治疗强度被下调，16 (11%) 名的治疗强度被上调。在医生和患者重审 RS 结果后，建议对其施用化疗的患者从 73 名 (52%) 下降至 42 名 (30%)。高 RS 值通常伴随治疗强度的增加，而低 RS 值通常伴随治疗强度的下降 (p = 0.011)。 结论 与传统临床病理评估相比，将 RS 结果纳入临床决策过程中导致了本前瞻性多中心研究中有 45% 的 T3 MMR-P II 期结肠癌患者的治疗建议发生了更改。RS 测定法的使用可能会从整体上降低这一II 期结肠癌患者亚组的辅助化疗使用率。The Oncologist 2014;19:492–497 Colon cancer, Chemotherapy, Adjuvant, Risk assessment, Decision analysis Implications for Practice: The use of adjuvant therapy for resected stage II colon cancer remains of uncertain benefit. Patients with high-risk features appear to gain benefit, whereas patients with a deficiency in mismatch repair enzymes have a low risk of recurrence and should not receive adjuvant chemotherapy. For all other stage II patients, defining recurrence risk has been challenging. Using the Oncotype DX colon cancer assay with a recurrence score in this patient group, we observed a change in the initial treatment recommendation in 45% of patients, indicating that the assay can provide meaningful information with regard to adjuvant therapy decisions. Introduction For patients with resected colon cancer, individualized approaches to adjuvant therapy decision making offer the promise of an improved, patient-specific balance between toxicity and treatment benefit. With the advent of new tools for risk assessment to individualize these decisions, an important consideration will be the level and the quality of evidence supporting not only the clinical validity of the tools but also whether these tools affect treatment decisions in practice in a clinically meaningful way. Although adjuvant therapy for patients with resected stage III colon cancer is routinely recommended, decision making regarding adjuvant treatment in stage II patients has remained a challenge. Overall, 75%–80% of patients with stage II colon cancers are cured with surgery alone, and adjuvant 5-fluorouracil (5-FU) chemotherapy has been shown to provide a relatively small absolute benefit (3%–4% absolute risk reduction for overall survival in the QUASAR trial and in meta-analysis of multiple trials) [1–4]. The use of oxaliplatin, together with 5-FU and leucovorin, has been advocated for high-risk stage II patients but remains of uncertain benefit in stage II patients lacking high-risk features [1]. The National Comprehensive Cancer Network recommends consideration of either observation or a fluoropyrimidine but not oxaliplatin for low-risk patients [5]. As such, it is critical to balance the side effects of chemotherapy with the expected benefit of treatment for these patients. The challenge has been in reliably identifying stage II patients, who lack traditional pathological high-risk features but who may still have an increased risk of recurrence and for whom adjuvant therapy should be considered, and low-risk patients, who can more appropriately be observed. Until recently, the risk of recurrence for stage II colon cancer patients following curative surgery was largely determined by traditional clinicopathological factors, including bowel obstruction or perforation, number of lymph nodes assessed, T stage, lymphovascular invasion, perineural invasion, and tumor grade [5, 6]. With the exception of T stage and nodal assessment, these markers have been significantly limited by lack of standardization, variable reproducibility, and inconsistent results in the literature [7, 8]. These limitations have prompted the development of biomarkers to better discriminate recurrence risk for stage II patients and thus to enable more informed treatment decisions. Two markers that have been well validated in prospectively designed studies are mismatch repair (MMR) status of the tumor tissue, in which MMR deficiency has been associated with lower recurrence risk [9], and the 12-gene recurrence score (RS; Oncotype DX colon cancer assay; Genomic Health, Inc., Redwood City, CA, http://www.oncotypedx.com). The Oncotype DX assay determines recurrence risk using a gene expression profile derived from 1,851 patients with stage II and III colon cancers enrolled in three National Surgical Adjuvant Breast and Bowel Project (NSABP) studies and an observational cohort from the Cleveland Clinic [10]. In these studies, 761 candidate genes identified from the colon cancer literature were evaluated to produce a final set of 12 genes (7 recurrence genes and 5 reference genes) and an algorithm that yields an RS result scaled from 0 to 100. In the prospectively designed QUASAR validation study [4], the RS was found to be significantly associated with risk of recurrence (hazard ratio per interquartile range: 1.38; 95% confidence interval [CI]: 1.11–1.74; p = .004) in stage II colon cancer patients treated with surgery alone. The prespecified low-risk group (RS values <30) had a 12% risk of recurrence, and the high-risk group (RS values ≥41) had a 22% risk of recurrence. The RS result remained a significant predictor of recurrence-free interval (RFI) in multivariable analyses controlling for T stage, MMR, number of nodes examined, lymphovascular invasion, and grade. In addition to the RS result, T stage and MMR were also found to be strong predictors of recurrence (both p < .001). Evaluation of patients randomized in QUASAR to observation versus adjuvant 5-FU revealed that proportional benefit of 5-FU was similar across the range of RS results such that patients with high-RS disease derived larger absolute treatment benefits than those with low-RS disease. The greatest value of the RS result resides in the ∼70% of patients for whom T stage and MMR status are not informative, that is, in patients with T3 stage and MMR-proficient (MMR-P) tumors. These results have now been confirmed in two independent, prospectively designed studies, including a study of stage II colon cancer patients from the Cancer and Leukemia Group B (CALGB) 9581 trial and a study of patients with stage II and III colon cancer from the NSABP C-07 trial [11]. To further the understanding of how physicians and patients use individualized, quantitative recurrence-risk information in clinical practice, we conducted a prospective multicenter study to evaluate how the clinically validated recurrence score assay affects adjuvant treatment decision making for patients with T3 MMR-P stage II colon cancer. Methods Patients Consecutive patients with resected stage II colon cancer were identified at the participating institutions for participation in this study. Based on annual estimates, sites seeing at least 60 new colon cancer cases (inclusive of all stages but exclusive of rectal cancers) or roughly 20 stage II cases per year were eligible to participate. Patients eligible to participate were required to have completely resected stage II colon cancer with T3 tumors. Other inclusion criteria included age ≥18 years, the ability to give consent and answer written questions in English, and candidacy for treatment with adjuvant chemotherapy. Major exclusion criteria included rectal tumors, T4 stage, perforations or obstructions, synchronous tumors, or contraindications for adjuvant chemotherapy according to a physician's judgment. A signed, written informed consent was obtained from all patients prior to their participation. This study was approved by the Mayo Clinic institutional review board and by the institutional review boards of the participating institutions within the Mayo Clinic Cancer Research Consortium. Unrestricted funding was provided by Genomic Health, Inc. Physician and Patient Assessments For consented patients, information was collected on patient demographics, performance status, comorbidities, and other clinical and pathological tumor characteristics. At the time of patient registration, the medical oncologist independently completed a baseline pre-Oncotype DX questionnaire, recording the planned treatments as observation, fluoropyrimidine monotherapy (5-FU [infusional or bolus] or capecitabine), or combination chemotherapy with oxaliplatin. Treatment recommendations were captured in the context of MMR status, if known, or with hypothetical MMR results. The patient's tumor specimen was then submitted for testing at Genomic Health with the commercially available MMR immunohistochemistry assay and the Oncotype DX colon cancer assay. After results of the Oncotype DX colon cancer assay became known and were thoroughly discussed with the patient, the medical oncologist completed a post-Oncotype DX questionnaire (supplemental online Appendix). Statistical Methods The primary objective was assessment of the impact of the Oncotype DX colon cancer assay (the RS result) on treatment recommendations by medical oncologists regarding adjuvant chemotherapy for T3 MMR-P stage II colon cancer patients. Changes in treatment recommendation included changes from chemotherapy to observation, changes from observation to chemotherapy, or changes in the chemotherapy regimen to exclude or include oxaliplatin. Changes in intensity of treatment recommendations from baseline to follow-up were defined as follows: changes from observation to any chemotherapy or from fluoropyrimidine monotherapy to combination chemotherapy with oxaliplatin constituted increased treatment intensity, whereas changes from combination chemotherapy with oxaliplatin to fluoropyrimidine monotherapy or from any chemotherapy to observation constituted decreased treatment intensity. Further exploratory analyses were performed to evaluate other clinical and pathological factors potentially related to changes in treatment plan. A target sample size of 170 patients with T3 MMR-P tumors was based on the desire for a 95% two-sided confidence interval of 12% for an anticipated overall treatment decision change rate of 20%. Because patients with MMR-P and MMR-deficient (MMR-D) tumors were eligible for the study and approximately 15% of subjects were anticipated to be MMR-D, the study targeted accrual of approximately 220 patients. The primary analysis was conducted in patients with resected T3 MMR-P stage II colon cancer; secondary analyses included both MMR-P and MMR-D patients. For the primary endpoint, the proportion of T3 MMR-P patients for whom the physicians’ treatment recommendation changed from baseline to follow-up was calculated along with the 95% confidence interval. The distribution of treatment recommendations at baseline and follow-up were separately examined for MMR-P and MMR-D patients. Changes in treatment recommendations were evaluated according to Oncotype DX recurrence score risk groups (as previously defined in QUASAR) [4] in MMR-P and MMR-D patients separately. Ordinal logistic regression was used in overall (MMR-P and MMR-D) and MMR-P-only populations to model association between the continuous RS and change in recommended treatment intensity, defined as “decreased intensity,” “increased intensity,” or “no change.” The appropriateness of the proportional odds assumption was confirmed. Potential nonlinearity of the effect of RS on the log-odds of treatment-recommendation change was examined via restricted cubic splines but was not detected. In additional secondary analyses for each population, prior knowledge of MMR status at baseline and MMR status (in the overall population only) were further examined for association with change in treatment recommendation using chi-square tests. An ordinal logistic regression model including RS and adjustment for knowledge of MMR at baseline was fitted for the primary evaluable (MMR-P) population. All analyses were carried out using SAS version 9.2 (SAS Institute, Inc., Cary, NC, http://www.sas.com). Results From May 21, 2010, through May 15, 2012, 221 patients were enrolled at 17 participating centers, with 2 patients deemed ineligible per protocol. Thirty-two patients were not evaluable, mostly because of incomplete data or loss to follow-up. The final evaluable population included 141 patients with T3 MMR-P tumors (the primary analysis population) and 46 patients with T3 MMR-D tumors. In the overall protocol-eligible population, patients had a median age of 66 years, were predominantly white, and had an Eastern Cooperative Oncology Group performance status of 0. Distribution of tumor characteristics (e.g., tumor grade, lymphovascular invasion) were generally as expected, including a high number of examined nodes (median: 18) reflecting contemporary standards for staging. More than 90% of patients had 12 or more lymph nodes assessed. A somewhat higher proportion of MMR-D tumors (46 of 187, 25%) was observed in this study compared with other large trials (14% in QUASAR, 21% in CALGB 9581) [4, 12]. Patient and tumor characteristics of all protocol-eligible patients (n = 219) and the subset of MMR-P patients evaluable for the primary endpoint (n = 141) were similar (Table 1). Table 1 Patient and tumor characteristics Open in new tab Table 1 Patient and tumor characteristics Open in new tab In the primary analysis, treatment recommendations changed for 45% (95% CI: 36%–53%) of T3 MMR-P patients. At baseline, 48% of T3 MMR-P patients were recommended to receive observation, 24% to receive fluoropyrimidine monotherapy, and 28% to receive combination chemotherapy with oxaliplatin. At postassay follow-up, most changes in treatment recommendation led to decreases in intensity of therapy (i.e., changes to observation or to remove oxaliplatin from the adjuvant therapy) (Table 2). Recurrence score values ranged from 2 to 57, with a median of 25. Most RS values (71%) were in the low-risk group, as defined in the QUASAR validation study (i.e., <30). The rate of change was fairly consistent across RS groups (46.0%, 39.4%, and 42.8% for low-, intermediate-, and high-RS groups, respectively) (Table 3). Among patients with changed treatment recommendations, as expected, the highest rate of recommendations for decreased treatment intensity (39.0%) occurred in the low-RS group. For the seven patients in the high-RS group, three patients (42.8%) had changes in treatment recommendation, all to increased treatment intensity. Among evaluable MMR-D patients (not shown), most treatment recommendations at both baseline and follow-up were for observation, with only three changes in each direction (increasing and decreasing intensity). Table 2 Pre- and postassay treatment recommendations, mismatch repair-proficiency patients Open in new tab Table 2 Pre- and postassay treatment recommendations, mismatch repair-proficiency patients Open in new tab Table 3 Treatment recommendation change by RS groups, mismatch repair-proficiency patients Open in new tab Table 3 Treatment recommendation change by RS groups, mismatch repair-proficiency patients Open in new tab In ordinal logistic regression, the RS result was significantly associated with changes in treatment recommendation in both the overall evaluable (MMR-P and MMR-D) population (p = .001) and the primary MMR-P population (p = .011). Lower RS values were associated with changes in the direction of lower intensity, and higher RS values were associated with changes to increased treatment intensity (Table 4). MMR status was significantly associated with change in recommendation (p < .001), with physicians of MMR-P patients significantly more likely to change treatment recommendations than those of MMR-D patients (45% vs. 15% change in recommendations for MMR-P vs. MMR-D patients, respectively) (Table 5). Knowledge of MMR status at baseline was not significantly associated with a change in recommendation in this population (p = .109). In the primary evaluable population (MMR-P), however, knowledge of MMR at baseline was moderately associated with treatment recommendation change (p = .040), with change more likely to occur among patients with unknown MMR status at baseline. Of note, in a multivariable ordinal regression analysis, RS remained a significant predictor of change in treatment intensity (p = .010) after adjusting for knowledge of MMR at baseline. Table 4 Association of recurrence score with change in treatment recommendation intensity, relevant to the median score (median score 25) Open in new tab Table 4 Association of recurrence score with change in treatment recommendation intensity, relevant to the median score (median score 25) Open in new tab Table 5 Association of MMR with change in treatment recommendation intensity Open in new tab Table 5 Association of MMR with change in treatment recommendation intensity Open in new tab Discussion The use of molecular markers of risk recurrence is becoming increasingly important in oncology to guide the use of expensive and often toxic therapies. For resected stage II colon cancer, traditional pathological or clinical findings (i.e., bowel obstruction or perforation, inadequate number of lymph nodes assessed, T4 stage, lymphovascular invasion, perineural invasion, high tumor grade) have been used to define a group of patients felt to be at high risk of recurrence and therefore more likely to benefit from chemotherapy. For the remaining group of patients with resected stage II colon cancer, no method of risk stratification existed until the recent introduction of molecular-based risk-scoring systems. In this prospective multicenter study, information provided by the recurrence score assay resulted in treatment recommendation changes for 45% of patients with T3 MMR-P (not high risk) stage II colon cancer. Changes in treatment recommendations were associated with RS results in the expected direction. The majority of changes in treatment recommendations led to decreases in the intensity of therapy. Given these findings, this study demonstrates the real-world clinical utility of the Oncotype DX colon cancer assay in standard-risk stage II colon cancer patients with T3 MMR-P tumors, a clinical setting in which conventional clinical and pathological measures are not informative. The high rate of treatment decision change observed in this study is, in part, likely to be a reflection of limited physician confidence in the conventional paradigm of qualitative risk assessment, based on conventional clinicopathological factors such as T stage, number of nodes examined, tumor grade, lymphovascular invasion, and bowel perforation or obstruction. Although T4 stage and MMR deficiency are now well established as predictors of recurrence risk in stage II colon cancer [9], relatively few stage II patients have either T4 or MMR-D tumors, and for the majority of patients who have T3 MMR-P tumors, clinicopathological factors have only limited utility in discriminating recurrence risk [6, 13]. The recurrence score assay has now been shown to provide more accurate, quantitative recurrence-risk information for these patients, and strong clinical validation data across three large, independent, prospectively designed studies (QUASAR, CALGB 9581, and NSABP C-07) [1, 11, 12] have demonstrated the ability of this standardized, quantitative assay to predict recurrence risk in stage II colon cancer beyond conventional clinicopathological factors. The availability of quantitative recurrence-risk information provided by the recurrence score assay, as contrasted with qualitative risk assessment with conventional factors, is likely to have been an important driver of changes in treatment recommendations in this study. Similar findings have been reported in multiple studies of node-negative, hormone receptor-positive breast cancer patients provided with quantitative recurrence-risk information with the Oncotype DX breast cancer assay, after which treatment decisions were changed in more than one third of cases [14, 15]. Some limitations of this study should be acknowledged. Compared with the validation studies, a higher proportion of patients were observed in the low-risk RS group (71%) and a lower percentage of patients were observed in the high-RS group (5%), potentially reflecting some degree of patient selection by the enrolling physicians and/or differences in patient populations observed in contemporary clinical practice compared with clinical trials. In addition, the study enrolled a larger proportion of patients with MMR-D tumors (∼25%) than the expected rate of 15% [9]. Consequently, the primary analysis population was limited to 141 T3 MMR-P patients, below the anticipated sample size of 170 patients. This is the first large, prospective, multicenter study to evaluate the impact of a gene expression-based recurrence-risk assessment tool on stage II colon cancer adjuvant treatment decision making in real-world clinical practice. An important strength of this study is its prospective design, conducted at 17 centers within an experienced community-based research network of medical oncology practices and led by an academic center (Mayo Clinic) with expertise in colon cancer. More than 90% of patients in the study had at least 12 lymph nodes examined, which is indicative of the quality of surgery and pathology within these centers and the representativeness of the stage II patient population studied. The study was designed to mirror conventional patient workflow and thus was able to document treatment decision making in real time as medical oncologists advised their patients with and without the results of the recurrence score assay. The high level of association observed in this study between RS results and changes in the intensity of recommended treatment demonstrates the value of the assay for clinical decision making across a wide variety of clinical practice settings. Conclusion Prospective evaluation of new clinical tools for treatment decision making will be critically important for assessing and validating their value and utility in real-world practice. The findings of this prospective multicenter study demonstrate that the quantitative recurrence-risk information provided by the recurrence score test is meaningful and important to practicing physicians and can guide treatment planning for T3 MMR-P stage II colon cancer patients in clinical practice. Moving forward, it will also be important to evaluate how the RS results affect the decision-making process for patients, including assessment of patient confidence in the treatment decision and the corresponding effects on patient quality of life. In this regard, a health economic analysis, performed as a substudy in this trial and reported separately, demonstrates that treatment decisions based on incorporation of the recurrence score assay, as observed in this study, would be expected to result in both significant increases in patient quality-adjusted life-years as well as cost savings for the health care system [16]. Incorporation of the RS into adjuvant treatment planning in stage II colon cancer patients with T3 MMR-P tumors informs clinical decision making for a large proportion of patients, with attendant expected positive impacts on patient care and health care resource utilization. Author Contributions Conception/Design: Geetika Srivastava, Lindsay A. Renfro, Margarita Lopatin, Calvin Chao, Mark Lee, Steven R. Alberts Provision of study material or patients: Robert J. Behrens, Gamini S. Soori, Shaker R. Dakhil, Rex B. Mowat, J. Philip Kuebler, George Kim, Miroslaw Mazurczak, Steven R. Alberts Collection and/or assembly of data: Lindsay A. Renfro, Robert J. Behrens, Shaker R. Dakhil, Rex B. Mowat, J. Philip Kuebler, George Kim, Miroslaw Mazurczak, Steven R. Alberts Data analysis and interpretation: Geetika Srivastava, Lindsay A. Renfro, Margarita Lopatin, Calvin Chao, Mark Lee, Steven R. Alberts Manuscript writing: Geetika Srivastava, Robert J. Behrens, Margarita Lopatin, Calvin Chao, J. Philip Kuebler, Mark Lee, Steven R. Alberts Final approval of manuscript: Geetika Srivastava, Lindsay A. Renfro, Robert J. Behrens, Margarita Lopatin, Calvin Chao, Gamini S. Soori, Shaker R. Dakhil, Rex B. Mowat, J. Philip Kuebler, George Kim, Miroslaw Mazurczak, Mark Lee, Steven R. Alberts Disclosures Mark Lee: Genomic Health, Inc. (E, OI); Calvin Chao: Genomic Health, Inc. (E, OI); Margarita Lopatin: Genomic Health, Inc. (E, OI). The other authors indicated no financial relationships. (C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board References 1 André T , Boni C, Navarro M. Improved overall survival with oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial . J Clin Oncol . 2009 ; 27 : 3109 – 3116 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Gill S , Loprinzi CL, Sargent DJ. Pooled analysis of fluorouracil-based adjuvant therapy for stage II and III colon cancer: Who benefits and by how much? . J Clin Oncol . 2004 ; 22 : 1797 – 1806 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Sargent D , Sobrero A, Grothey A. 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Google Scholar OpenURL Placeholder Text WorldCat Author notes Disclosures of potential conflicts of interest may be found at the end of this article. © 2014 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Riggs, Stephen B.; Burgess, Earl F.; Gaston, Kris E.; Merwarth, Caroline A.; Raghavan, Derek

2014 The Oncologist

doi: 10.1634/theoncologist.2013-0379pmid: 24718515

Abstract Postchemotherapy surgery for advanced testicular cancer has evolved over the last couple of decades. Patients with nonseminomatous germ cell tumors and residual retroperitoneal mass ≥1 cm should undergo postchemotherapy retroperitoneal lymph node dissection (RPLND). For seminoma, RPLND is considered in those patients with masses ≥3 cm that are also positron emission tomography positive. Masses that occur outside of the retroperitoneum should be completely resected with the possible exception of bilateral lung masses when resection of the first mass shows necrosis. The role of surgery in patients with extragonadal germ cell tumors is most vital in those with primary mediastinal nonseminomatous germ cell tumors. Importantly, patient selection, surgical planning, and consideration of referral to centers with this expertise are important to optimize success. 摘要 晚期睾丸癌的化疗后手术治疗在过去几十年经历了诸多演变。非精原细胞性生殖细胞瘤及腹膜后残留肿块 ≥1 cm 的患者，应接受化疗后腹膜后淋巴结清扫术 (RPLND)。对于精原细胞瘤而言，肿块 ≥3 cm 且正电子发射断层扫描呈阳性的患者可考虑施行 RPLND。发生在腹膜后腔以外的肿块应彻底切除，但首个切除肿块显示坏死的双侧肺肿块可能要除外。对于性腺外生殖细胞瘤而言，手术切除对那些存在原发性纵膈非精原细胞性生殖细胞瘤的患者具有最为重要的作用。值得注意的是，患者的选择、手术的方案以及考虑转院到具有此专门经验的医院对提高成功率具有重要意义。The Oncologist 2014;19:498–506 Retroperitoneal lymph node dissection, Testicular cancer, Postchemotherapy retroperitoneal lymph node dissection, Seminoma, Nonseminoma, Germ cell tumor Implications for Practice: Patients with advanced testicular cancer will often be considered for surgical consolidation following chemotherapy. This review article focuses on the evaluation and role of surgery in treatment of these complex patients. It underscores the selection of patients, vital role of surgery, as well as providing guidance in the use of surveillance as opposed to surgical extirpation. Introduction The role of surgery following primary chemotherapy for testicular cancer has evolved over the past 3 to 4 decades. Since the 1970s, there has been exploration of salvage surgery for those who do not achieve complete response to chemotherapy and even to consolidate successful chemotherapy by removing occult residual foci of germ cell malignancy or teratoma. Furthermore, it has become apparent that germ cell tumor can have transformation (either as a side effect of treatment or due to benign evolution) to mature teratoma that is inherently chemotherapy-resistant [1]. Our senior author and colleagues reported from the Royal Prince Alfred Hospital group its experience from 1977 to 1980 involving 21 patients (mix of stage II and III) who failed to achieve a clinical or radiological response (CR), of whom 14 had retroperitoneal node dissections, 5 had biopsy only, and 2 had thoracotomies for residual disease outside the retroperitoneum [2]. Several important observations were evident from this early series: first, the surgery was complex, requiring an average of 7 hours and at times requiring resection of adjacent organs. Second, those patients with tumor markers elevated at diagnosis and after primary chemotherapy either relapsed or had viable carcinoma at the time of surgery. Next, those patients with positive prechemotherapy tumor markers who subsequently normalized had a low probability of either relapse or viable carcinoma following surgery. In addition, multiple residual sites (e.g., lung, supraclavicular node, and retroperitoneum) did not necessarily have concordant pathology (i.e., one site could have necrosis and the other site viable germ cell tumor or teratoma) [2]. Other early series confirmed the prognostic importance of a good response to chemotherapy, marker status after induction chemotherapy, and tissue type in resected specimen. Finally, those with residual viable disease have the potential for adverse outcomes and should be considered for further chemotherapy [3–5]. It is our intent in this article to update the oncologist regarding the role of surgery after chemotherapy for advanced germ cell tumors. Imaging After Chemotherapy In nonseminomatous germ cell tumors (NSGCTs), the imaging of choice is usually computed tomography (CT), with fludeoxyglucose (FDG) positron emission tomography (PET) reserved for residual masses in seminomatous patients only. One complicating factor surrounding the use of FDG PET for evaluation in patients with NSGCTs is due to the possibility that any residual mass could contain viable tumor, teratoma, or fibrosis/necrosis/inflammation/fluid. Therefore, a positive PET scan in this population could represent viable germ cell or benign inflammation; fibrosis/necrosis and mature teratoma do not have affinity for the radiolabeled tracer unless they contain pockets of tissue fluid that allow passive absorption and retention of tracer. However, after chemotherapy for seminomatous tumors, there is only binary choice for the histology of the remaining mass: viable germ cell cancer or fibrosis, of which the latter appears to be much less “PET-active” [6]. The use of FDG PET in those patients with pure seminoma and especially masses >3 cm appears to have utility. De Santis et al. published a multicenter study involving 51 patients in whom 19 had residual masses >3 cm with a median follow-up of 34 months [7]. The positive predictive value and negative predictive value of FDG PET, irrespective of tumor size, were 100% and 96%, respectively. The two false negatives were seen in tumors ≤3 cm in size. Size >3 cm or ≤3 cm was confirmed to be a predictor of residual disease, as it has been in other series, with a cancer rate of 37% and 8%, respectively [7–10]. This series was followed up 1 year later and reported an additional false negative (3 of 11) in tumors <3 cm but maintained its perfect positive predictive value for a final sensitivity and specificity of 80% and 100%, respectively [11]. Therefore, all postchemotherapy masses >3 cm in size in patients with primary seminoma do not necessarily need treatment. A positive PET scan strongly suggests tumor (requiring additional therapy); therefore, we recommend the use of PET CT in this setting. Finally, it is worth mentioning that the timing of FDG PET after chemotherapy should be between 4 and 12 weeks, but no earlier because of the potential for inflammation-based false-positive results [7]. Retroperitoneal Lymph Node Dissection Retroperitoneal lymph node dissection (RPLND) (Fig. 1) was first performed in the 1950s using knowledge from lymphatic drainage studies derived many years earlier. The primary landing zones were further elucidated and determined to be predictable in subsequent studies [12–14]. In general, as evidenced from early studies involving RPLND in patients without prior treatment, right-sided tumors metastasize to the interaortocaval lymph nodes first (just below the left renal vein), followed by the precaval and paracaval lymph nodes. Left-sided testicular tumors metastasize to the para- and preaortic areas. Contralateral involvement is more frequent in right-sided tumors as well as in bulky retroperitoneal disease [12, 15]. The involvement of suprahilar zones is infrequent in patients with minimal to moderate retroperitoneal disease (old staging B1; current staging IIA), but this incidence increases with increasing retroperitoneal volume (old staging B1, B2; current staging IIB, IIC). Furthermore, ipsilateral or contralateral disease below the bifurcation of the internal iliac vessels seems to be present only in the setting of more bulky retroperitoneal adenopathy (stage IIB, IIC) [12]. Enthusiasm for a modified template in primary treatment for stages I and IIA was reinforced in subsequent years with reaffirmation of the predictable drainage patterns in testicular cancer. In addition, the hypothesis that the drainage follows that of the testicular veins (on either side) to a so-called “lymphatic epicenter” was refuted [14]. Figure 1 Open in new tabDownload slide Retroperitoneal lymph node dissection. (A): Aorta. (B): Vena cava. (C): Left renal vein. Figure 1 Open in new tabDownload slide Retroperitoneal lymph node dissection. (A): Aorta. (B): Vena cava. (C): Left renal vein. Ultimately, an attempt to achieve sympathetic preservation and antegrade ejaculation has resulted in greater use of either modified templates or nerve-sparing RPLND. The former is accomplished by staying above the inferior mesenteric arteries (based on the distribution of nodal disease referred to above), whereas a prospective nerve-sparing RPLND involves identification and preservation of sympathetic nerves from the T12–L3 thoracolumbar spinal cord in addition to the hypogastric plexus [15]. Nerve-sparing templates may even be applicable to postchemotherapy RPLNDs, whereas modified templates have, historically, been advocated for primary surgery [16], but its use in the postchemotherapy setting is evolving. Long-term follow-up studies that prove the absence of late relapse will be needed before such modified templates can be viewed as standard of care, and it seems unlikely that the hypothesis would ever be tested in a randomized trial. Indications for Postchemotherapy RPLND in Seminoma Germ Cell Tumors Postchemotherapy residual masses in patients with advanced seminoma warrant a different approach to that given to residual masses after chemotherapy in patients with nonseminoma. In general, the chance for malignancy is low if the tumor size is <3 cm and, therefore, observation is warranted. However, it increases to approximately 30% for residual masses ≥3 cm [9, 10]. Patients with pure seminoma and residual tumors >3 cm after chemotherapy should be submitted for PET CT as described above. If the PET scan is positive, then biopsy or surgery is indicated. Of importance, seminoma appears to remain sensitive to radiation after chemotherapy and, therefore, this approach, in addition to surgery, should be considered [9]. There appears to be an increased rate of perioperative morbidity after RPLND for seminoma. For reasons that are not clear, the residual masses after advanced seminoma treated with cisplatin-based chemotherapy are associated with much more extensive fibrosis and, thus, constitute a much more complex surgical challenge. Albeit retrospective, one study evaluating outcomes in patients submitted for RPLND with seminoma suggested a higher rate (38%) of additional operative procedures (e.g., nephrectomy, inferior vena cava resection, arterial grafting, or bowel resection) as compared with those undergoing the same procedure for nonseminoma (26.8%). As expected, the rate of postoperative complications was also greater in the seminoma group (24.7% vs. 20.3%) [17]. There appears to be an increased rate of perioperative morbidity after RPLND for seminoma. For reasons that are not clear, the residual masses after advanced seminoma treated with cisplatin-based chemotherapy are associated with much more extensive fibrosis and, thus, constitute a much more complex surgical challenge. Indications for Postchemotherapy RPLND in Nonseminoma Germ Cell Tumors Debate continues regarding which patients should be submitted for PC RPLND, most specifically around subcentimeter residual masses. Two divergent views are centered on the uncertainty and unpredictability regarding the biologic behavior of residual microscopic teratoma as well as the possibility of viable cancer. Furthermore, there remains variation among institutions as to what constitutes a radiographically normal retroperitoneum. The proponents of RPLND (Table 1) cite several observations, including a Norwegian study demonstrating 33% (29 of 87) of tumors ≤2 cm as having viable germ cell or teratoma, of which 55% (16 of 29) were ≤1 cm [18]. Furthermore, Carver et al., from Memorial Sloan-Kettering Cancer Institute, reported on 532 patients who underwent RPLND after chemotherapy: 154 patients had residual tumors ≤1 cm, of which 28% had either teratoma and/or viable germ cell (the majority were teratoma) [19]. On multivariate analysis, teratoma within the primary specimen as well as relative change in nodal size predicted teratoma within the retroperitoneum [19]. Table 1 Postchemotherapy pathology for patients with residual masses ≤2 cm and ≤1 cm Open in new tab Table 1 Postchemotherapy pathology for patients with residual masses ≤2 cm and ≤1 cm Open in new tab In contrast, the group from Indiana University (Table 2) has advocated surveillance of subcentimeter residual masses based on review of their experience. In 2009, Ehrlich et al. reported on 141 patients determined to have a CR after chemotherapy, defined as normalization of tumor markers as well as radiographic disease <1 cm [20]. Median follow-up was 15.5 years, and 12 (9%) patients experienced relapse, with four (3%) deaths attributed to germ cell tumor (GCT). Only six sites of relapse were in the retroperitoneum, suggesting that one half of the patients with relapse had the potential to benefit from PC RPLND. International Germ Cell Consensus Classification (IGCCC) predicted outcome with good-risk disease enjoying a 99% cause-specific survival versus 73% if disease risk was intermediate to poor. However, only 32 (29%) patients of the entire cohort were classified as intermediate to poor risk [20, 21]. Another retrospective study (Table 2) in support of surveillance was reported in 2008, evaluating 276 patients from both the British Columbia Cancer Agency and the Oregon Testis Cancer Program [22]. Like the series from Indiana University, 161 underwent surveillance for CR (same criteria as above); however, the median follow-up was much shorter (only 40 months), lending uncertainty to the potential for further late relapses. Ten patients (6%) relapsed at a median of 52 months, all of whom were salvaged and continuously in remission after a median follow-up of 64 months. Interestingly, all but two of the relapses were considered to have pristine postchemotherapy scans (i.e., no radiographic residual disease), and all but one of the relapses were in the retroperitoneum. Salvage therapy consisted of nine RPLNDs (one after additional chemotherapy) and one received only systemic chemotherapy. These two surveillance studies illustrate that the most likely recurrence site for patients who do not undergo immediate postchemotherapy RPLND is the retroperitoneum [23]. Table 2 Outcome of surveillance for patients with complete response (≤1 retroperitoneal mass; normalized tumor markers) after first-line chemotherapy Open in new tab Table 2 Outcome of surveillance for patients with complete response (≤1 retroperitoneal mass; normalized tumor markers) after first-line chemotherapy Open in new tab The German Testicular Group analyzed the pathological outcomes of patients undergoing postchemotherapy RPLND for any size tumor [24]. Size correlated with potential findings: those with tumors <1 cm had a 9.4% and 21% chance of harboring either viable cancer or mature teratoma, respectively. These findings elevated to 21% and 25% for postchemotherapy tumors 1–1.5 cm and, ultimately, to 36% and 42% for those tumors greater >1.5 cm in size [21, 24]. The usual histologic findings after PC RPLND have been reported as 40%–50% fibrosis/necrosis, 35%–40% teratoma, and approximately 10% viable germ cell tumor; of importance, a higher proportion of viable cancer was noted in the early series, prior to the implementation of predictive algorithms that matched aggression of chemotherapy to anticipated prognosis. Therefore, based on the considerations above, one's ability to predict (to some degree) the histology of a postchemotherapy mass is based on the size of the mass after primary chemotherapy [21, 23, 24]. Several groups have evaluated predictive models in an attempt to determine retroperitoneal pathology [25–29]; unfortunately, all had variable success. It appears that patients who achieve a complete radiographic response (normalization of tumor markers and radiographic disease ≤1 cm) after primary chemotherapy usually do not require postchemotherapy surgery [23]. Of course, these patients must be followed carefully as some will require salvage RPLND and consideration of further chemotherapy depending on the histologic results. Of note, we strongly advocate review of images by a highly experienced surgeon, oncologist, and radiologist to make this determination. Any residual tumor ≥1 cm in size should be removed because of the increasing probability of either viable tumor or teratoma [18, 21, 23, 24]. Finally, historical data have suggested that the morbidity was as much as twofold greater (20.7%) in patients undergoing postchemotherapy RPLND as compared with primary RPLND [15, 30]. Often this was secondary to pulmonary toxicity (adult respiratory distress syndrome or prolonged ventilation) from bulky retroperitoneal disease and bleomycin-induced pulmonary toxicity. However, current literature suggests that the morbidity after postchemotherapy RPLND is more comparable to that seen after primary RPLND with the exception of greater blood loss and operative time in addition to a reduced chance to maintain antegrade ejaculations [31]. This shift is most likely due to surgical experience, collaboration with other surgical disciplines (i.e., a qualified and experienced vascular surgeon is vital), and improved chemotherapy that has resulted in smaller residual volumes of tumor. All of the above underscores the need to have this surgery performed at centers with considerable expertise as well as appropriate ancillary services. Chemotherapy After Postchemotherapy RPLND Current treatment paradigms predominantly involve second-line chemotherapy after the finding of viable GCT following postchemotherapy surgery (including RPLND and metastasectomy). Fizazi et al., in a retrospective analysis, evaluated the outcomes of 238 patients, all of whom had normal tumor markers before resection and residual viable NSGCT (so-called “surgical complete response”) removed following initial induction chemotherapy [32]. Seventy percent of their cohort received various postsurgery (second-line) chemotherapy regimens. Patients were stratified to one of three groups based on three identified risk factors: complete resection, <10% of viable malignant cells, and good-risk IGCCC. Those with all three factors were considered good risk, those without one of the risk factors were considered intermediate risk, and those without two or three were considered poor risk. Patients in the favorable group had a 100% overall survival (OS) rate at 5 years irrespective of postoperative chemotherapy. After adjustment for tumor volume, risk status, and status of resection, postoperative chemotherapy was associated with a significantly better progression-free survival (PFS) (p < .001) but not overall survival [32]. A follow-up validation study was performed in 2008 across 12 institutions [33]. Ninety percent of patients underwent first-line chemotherapy with cisplatin. Median follow-up was 65 months and, similar to the first study, 5-year PFS for the entire cohort was 65%, with 5-year OS being 72%. The indices of complete resection, <10% viable germ cell and IGCCC risk status, were together highly predictive of both PFS (p = .0008) and OS (p = .003). However, as with the first study, there was no evidence of a survival benefit with postsurgery chemotherapy; thus, the index above does prognosticate but does not predict response to treatment [33]. This does bring into question the need for second-line chemotherapy in all patients with viable GCT after surgery in the postchemotherapy setting, especially for those patients who have undergone complete resection. However, the two studies above were retrospective, analyzing a cohort among multiple institutions and over many years, thus weakening the interpretation of the data. We currently consider observation versus several cycles of adjuvant cisplatin-based chemotherapy for those patients with residual viable GCT who have received induction chemotherapy only [34], with the duration of chemotherapy predicated on apparent response and toxicity; however, we favor the concept that this would be a fertile area for a multicenter collaborative trial to identify a truly optimal approach. Postchemotherapy Surgery for Sites Outside the Retroperitoneum The concordance between retroperitoneal masses and masses outside the retroperitoneum is incomplete, with outcomes dictated by the ability to resect all residual masses in addition to tumor marker status and whether viable GCT remains. In fact, the histologic discordance between sites is reported to be between 25% and 47% [35, 36]. Interestingly, the concordance between patients with bilateral residual lung masses who have pure necrosis in the first lung appears very good, with 19 of 20 (95%) also having this finding in the second lung [37]. Therefore, careful consideration to observation regarding a contralateral lung mass after the finding of necrosis in the first can be given; however, this does not hold true for mediastinal masses. Our view is that, in general, all masses should be considered for simultaneous resection if technically feasible. The use of surgery for either relapse or in the primary setting in patients with metastatic germ cell tumor to the brain remains a topic of debate. Prospective trials are lacking and almost certainly will not be conducted as this constitutes less than 10% of patients with advanced germ cell tumors and less than 1% of all germ cell tumor patients [38]. National Comprehensive Cancer Network guidelines recommend radiation for patients with brain metastasis following chemotherapy, with surgery reserved for consideration in those in whom it appears feasible (solitary metastasis) [39]. However, the evidence basis for this recommendation is not particularly strong. In the presence of significant elements of choriocarcinoma (predicted by the histology of the primary tumor or of metastases at other sites or very high circulating human chorionic gonadotropin), we believe that initial surgical resection of isolated brain metastases may be safer (if feasible) to avoid life-threatening intracranial hemorrhage after chemotherapy or radiotherapy. Bone metastasis is also rare, constituting less than 1% of metastasis at the time of primary diagnosis or relapse [40]. However, it has been shown in patients specifically with poor-risk disease to constitute up to 9% [41]. In this study, Oechsle et al. retrospectively reviewed 40 patients with bone metastases from a cohort of 434 patients with poor-risk disease [41]. All patients underwent primary high-dose cisplatin-based chemotherapy with peripheral blood stem cell reinfusion. Four patients (10%) underwent surgical consolidation, all of whom had the finding of necrosis at the time of surgery [41]. As with disease of the brain, the ultimate timing and role of surgery for bone metastasis remain uncertain. Extragonadal Germ Cell Tumors Extragonadal germ cell tumors represent less than 5% of all adult germ cell malignancies. Most of these are located in the anterior mediastinum followed by the retroperitoneum and very rarely in the pineal gland or presacral area [42]. Those containing seminoma are considered good or intermediate IGCCC risk and are recommended to undergo chemotherapy, usually with very good outcomes irrespective of location. Unfortunately, primary mediastinal nonseminomatous germ cell tumors (PMNSGCTs) are considered IGCCC poor risk and carry only a 40%–50% rate of survival after combination treatment with cisplatin-based chemotherapy and surgery [42, 43]. This is inferior to those occurring in the retroperitoneum, which are considered IGCCC good or intermediate classification depending on tumor marker status. Surgery plays a vital role in the management of PMNSGCTs as there is a high rate of viable tumor at the time of resection after chemotherapy [44, 45]. In general, we advocate that patients with PMNSGCTs are treated initially with VP-16, etopside or vinblastine plus ifosfamide and cisplatin, or another poor-risk cisplatin-based regimen (to avoid bleomycin and its potential pulmonary toxicity) followed by thoracotomy and resection (residual mass is usually present). It is important that this complex surgery is undertaken by an experienced thoracic surgeon who has done this type of surgery, assuming the disease is deemed potentially resectable [45]. Because of the lack of effective salvage therapy, resection should be considered even in the face of elevated tumor markers as well as at the time of any recurrence [42, 45]. Surgery After Salvage or Second-Line Chemotherapy It should be mentioned that the paradigm for aggressive surgical resection after salvage or second-line chemotherapy is to some degree different. In general, these are patients who progressed after first-line chemotherapy or who remain with unresectable disease. Residual masses after second-line chemotherapy have been associated with a much higher chance of residual GCT [10, 35], and surgery is often considered even in the face of elevated tumor marker status because of the paucity of effective, alternative options. However, Eggener et al., in a review of 71 patients after multiple chemotherapy regimens in 2007 (90% received second-line chemotherapy only), suggested that there was declining incidence of viable GCT in the retroperitoneal residual mass (ultimately paralleling that seen after primary chemotherapy) [46]. Overall, the rate of viable GCT was 28%; however, when analyzing the subset of patients who received taxane therapy, this rate dropped from 42% to 14%. Finally, with this reduction came higher rates of fibrosis (63% vs. 39%) with a relatively stable distribution of teratoma (31% vs. 33%). Of note, the rate of viable GCT or teratoma if located outside of the retroperitoneum was 31%. The 10-year disease-specific survival (DSS) was 70%, but was most favorable for those with a finding of only fibrosis (87%), as compared with teratoma (47%) or viable GCT (47%). On multivariate analysis, tumor size ≥5 cm as well as the presence of GCT predicted DSS [46]. All of the above supports the vital role of postchemotherapy surgery after second-line chemotherapy while at the same time suggesting that second-line taxane (paclitaxel, ifosfamide, and cisplatin or paclitaxel and ifosfamide followed by carboplatin and etoposide plus peripheral blood stem) therapy has improved the outcome and shifted histopathological distribution similar to that seen after first-line platinum-based therapy. Template Controversy regarding the anatomical extent of RPLND after chemotherapy continues. Patients in the 1970s and 1980s often had high-volume residual disease necessitating full bilateral retroperitoneal dissection including suprahilar dissection. However, current chemotherapy regimens tend to leave remaining disease burden very low and restricted to the primary landing zones with contralateral crossover currently less likely [21, 47]. Some have advocated use of intraoperative frozen sections to guide extent of surgery. Herr et al. evaluated 62 patients, of whom 37 underwent limited lymph node dissection based on the finding of necrosis on frozen section [48]. The remaining patients underwent bilateral full template dissection of the retroperitoneum after frozen section revealed viable germ cell or teratoma. Fourteen patients had relapse, of whom only 1 had germ cell or teratoma in the retroperitoneum after limited dissection. Interestingly, there were six surgical complications, five of which occurred after bilateral dissection [48]. Others have suggested that a template RPLND is appropriate in those patients presenting with low-volume retroperitoneum disease (<5cm, stage IIA or IIB). Beck et al. evaluated 100 patients submitted for modified template RPLND (for postchemotherapy residual disease) after primary chemotherapy, of which there were only four recurrences in the retroperitoneum after 32 months of follow-up [47]. Interestingly, the locations of all recurrences were deemed to also be outside the boundaries of a full bilateral dissection. In this highly select population (98% had good-risk disease and 94% were stage IIA or B), a modified template did not appear to affect outcome [47]. Another 152 patients with postchemotherapy residual masses were retrospectively reviewed by Heidenreich et al. from two tertiary referral centers, of whom 98 underwent modified template resection if the mass was located in the primary landing zone and <5 cm [49]. Mean length of surgery was significantly longer (90 minutes) in the full bilateral group as compared with the modified group. Antegrade ejaculation was preserved in 85% of patients undergoing modified template (average mass size of 4.5 cm) but only 25% in those undergoing full bilateral dissection (average mass size 11 cm). For the entire analyzed cohort, there were nine recurrences (three after modified dissection [3%] and six after full template dissection [12%]), with only one recurrence occurring within the boundary of a modified template dissection and all others occurring outside the boundaries of a full template dissection. Interestingly, all recurrences within the modified group were outside of the retroperitoneum; however, three in the full bilateral group were suprahilar in location [49]. Obviously, there is some selection bias with the average size for patients undergoing modified template being 4.5 cm versus 11 cm for full bilateral dissection. Also, 30% and 15% were IGCCC poor-risk classification for the full and modified groups, respectively [49]. What we take away from this, in contrast to earlier series [50], is that patients with postchemotherapy residual masses need to be carefully considered for a surgical plan. Also, with the advancement of systemic therapy, the current tumor burden of residual disease does not parallel that seen 20 years ago and, thus, the primary landing zones are more likely to be the only site of disease. Therefore, in patients with stage IIB or lower with a postchemotherapy residual mass within the primary known landing site, the surgeon can consider template dissection. High-volume tumors (i.e., stage IIC) and those with either pre- or postchemotherapy masses outside of the predicted lymphatic drainage should be submitted for full bilateral template as well as consideration for suprahilar and iliac dissection. Ultimately, however, there is little substitute for surgeon experience and judgment; therefore, we find this to be paramount in deciding the extent of dissection. It should also be emphasized that this type of surgery should not be undertaken solo by a surgeon who is inexperienced in the nuances of postchemotherapy germ cell cancer surgery. Laparoscopy The use of laparoscopy in postchemotherapy RPLND appears technically feasible but uncertain with regards to its long-term outcome [51, 52]. Most of the current experience is in the primary setting as opposed to after chemotherapy. In general, the total lymph nodes removed in series evaluating laparoscopic RPLND have been considerably less than those reported for open series [53–55]. Understanding the utmost importance of “controlling the retroperitoneum,” we have not advocated laparoscopy in the postchemotherapy setting. Currently, the gold standard for postchemotherapy RPLND remains an open surgical approach. “Redo” RPLND As mentioned above, it is of utmost importance to achieve complete resection at the time of initial surgery. Hendry et al. reported on 442 patients undergoing RPLND for radiographic masses ≥1 cm, of whom 112 received their surgery in a salvage fashion [56]. The salvage group consisted of a referred population who had recurrent disease after observation of a known para-aortic mass. Also, they were submitted for reinduction chemotherapy prior to surgery. Complete resection was accomplished in 87% of the primary group versus 72% of the salvage group, and lack of complete resection was elucidated on multivariate analysis as a predictor for OS. The need for concurrent nephrectomy was statistically twice as high in the salvage group, with an operative mortality 1.8% as compared with 0.9% in the primary group. Finally, overall survival was improved by an absolute difference of 33% in the primary (89%) as compared with the salvage (56%) group [56]. Incomplete control of the retroperitoneum after primary RPLND has been shown by others to be associated with an increased complication rate as compared with primary RPLND [57]. This at times requires coordination from additional surgical subspecialties (e.g., vascular surgery). McKernion et al. evaluated 34 patients who underwent primary PC RPLND who subsequently were submitted for reoperation [58]. Teratoma was the most common histologic finding at PC RPLND and at the time of reoperation. Notably, at a median follow-up of 29.5 months, the disease-specific survival rate for patients undergoing a second RPLND following PC RPLND was 56% [58]. Sonneveld et al. evaluated 51 patients with residual teratoma after postchemotherapy RPLND, of whom 15.7% (8) were deemed by the surgeon to be “incomplete” resections. Of these 8 patients, 4 relapsed with disease in the retroperitoneum, emphasizing the importance of complete resection [59]. Another more recent study evaluated the outcomes of 18 patients undergoing repeat RPLND, of whom 3 (16.7%) were deemed “out-of-field” recurrence, leaving the remaining 83% as “in-field” recurrences. Most recurrences were located in the interaortocaval, para-aortic, and suprahilar locations, with 10 patients (63%) having residual teratoma or GCT. Adjunctive procedures such as thoracotomy or vessel resection and grafting were required 55% of the time, and the overall postoperative complication rate was 38.8% [57]. This increased rate of perioperative complications as well as location of recurrence have been supported by others [58, 60]. The location of the recurrences may be related to incomplete surgery with respect to the renoaortic junction and the need to control (and dissect) all major tributaries before addressing any retroperitoneal mass. Of note, patients with teratoma after primary or postchemotherapy RPLND often have teratoma on repeat RPLND (as evidenced by 12 of 15 patients in one series [58]). Our own experience and review of the above literature give us some insight into how to approach those patients presenting with residual masses in the retroperitoneum after primary RPLND who are also marker-negative. First, patients with the appearance of teratoma on imaging (cystic masses) would be best served with upfront repeat surgery. Teratoma only on initial RPLND (whether it be primary or after chemotherapy) often predicts teratoma in the residual retroperitoneum mass, and these patients also should be submitted for repeat surgery. It is reasonable to use percutaneous biopsy to attempt to identify patients who may have only necrosis/fibrosis. This would be most appropriate in those patients with negative tumor markers, no teratomatous elements at the time of initial RPLND, and no radiographic features suggesting teratoma. It is quite evident that salvage chemotherapy cannot salvage inadequate surgical resection, and complete surgical resection of any residual masses is imperative, as this may be the only prognostic factor that we can actually control. Moreover, it is obvious that these complex surgeries need to be planned and coordinated appropriately to optimize surgical outcomes and success. It is quite evident that salvage chemotherapy cannot salvage inadequate surgical resection, and complete surgical resection of any residual masses is imperative, as this may be the only prognostic factor that we can actually control. Moreover, it is obvious that these complex surgeries need to be planned and coordinated appropriately to optimize surgical outcomes and success. “Desperation” Postchemotherapy RPLND The use of “desperation” PC RPLND has been coined to refer to the use of surgery in patients with increased serum tumor markers after chemotherapy [21, 61]. At the time of desperation surgery, the incidence of viable GCT ranges from 40% to 81%, considerably higher than that seen after first-line chemotherapy and normalized tumor markers [61]. Beck et al. reported on 114 patients with metastatic germ cell tumor and elevated tumor markers after first- or second-line chemotherapy [62]. Retroperitoneal pathology was GCT in 53.5% (28% after first-line chemotherapy; 75.8% after second-line chemotherapy), teratoma in 34.2%, and fibrosis in 12.3%. There was a 54% 5-year overall survival for the entire cohort. Predictors of adverse outcomes in the induction group were retroperitoneal histology (finding of cancer was least favorable), whereas increasing beta-human chorionic gonadotropin, elevated alpha-feto protein (continuous variable), redo RPLND, and GCT histology predicted adverse outcome in the salvage chemotherapy group [62]. Literature review on the concept of desperation RPLND suggests that results are more favorable among those with stable or declining tumor markers as opposed to elevated ones. Also, in general, 50% of patients with elevated tumor markers at the time of surgery will have mature teratoma or fibrosis/necrosis, with a long-term disease-free interval in one third of patients with viable GCT [61–63]. It should be underscored that the use of RPLND in the face of rising tumor markers and the appearance of resectable disease constitutes an infrequent clinical condition. General guidelines for considering surgery include declining or plateau serum tumor markers after chemotherapy, slowly increasing tumor markers after an initial complete response to chemotherapy (primary or secondary), resectable disease (one to two sites), increasing markers, and apparently resectable disease after all systemic options have been used [62]. Conclusion Improvement in systemic therapy has, to a large degree, now relegated surgery for advanced GCTs to the postchemotherapy setting. Its use within the armamentarium for treatment of advanced GCTs remains vital. We have learned much over the past several decades. Masses outside the retroperitoneum should be completely resected with the possible exception of bilateral residual lung masses when resection of the first mass shows necrosis. For seminoma, postchemotherapy RPLND or biopsy and radiation should be considered for masses >3 cm that are also PET-positive. For NSCGT, postchemotherapy RPLND should be performed for tumors ≥1 cm and normal markers, those with plateauing tumor markers, and those with residual in-field masses and negative markers after prior RPLND. Additional indications include normal markers or plateauing markers after salvage chemotherapy, and in the desperation setting if the tumor appears resectable. It remains vitally important to “control the retroperitoneum” by resection of all disease the first time, thus avoiding the potential disturbing consequences associated with redo RPLND. Although our understanding regarding its use has evolved, it remains a complex procedure with the potential for significant complications; therefore, it is paramount to consider referral to facilities with this expertise. Author Contributions Conception/design: Stephen B. Riggs, Earl F. Burgess, Kris E. Gaston, Derek Raghavan Provision of study material or patients: Stephen B. Riggs Collection and/or assembly of data: Stephen B. Riggs, Caroline A. Merwarth Manuscript writing: Stephen B. Riggs, Derek Raghavan Final approval of manuscript: Stephen B. Riggs, Earl F. Burgess, Kris E. Gaston, Caroline A. Merwarth, Derek Raghavan Disclosures Derek Raghavan: Sanofi Aventis (C/A). The other authors indicated no financial relationships. (C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board Editor's Note: For further reading on germ cell tumors, watch for the forthcoming commentary by Sara J. Stoneham et al., “Adolescents and Young Adults with a “Rare” Cancer: Getting Past Semantics to Optimal Care for Patients with Germ Cell Tumors.” Germ cell tumors are the third most common cancer diagnosis in adolescent and young adult (AYA) patients aged 15–24. Many cancers that arise in AYA patients, including germ cell tumors, are defined as “rare” because they are relatively infrequent during early childhood and older adulthood. 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