Randomized controlled phase III trial of adjuvant chemoimmunotherapy with activated cytotoxic T cells and dendritic cells from regional lymph nodes of patients with lung cancer

Randomized controlled phase III trial of adjuvant chemoimmunotherapy with activated cytotoxic T... Randomized controlled trial of adjuvant chemoimmunotherapy for lung cancer indicated a significant advantage in patients receiving immunotherapy. Herein we report the final results and immunological analysis with a median follow-up of 59.6 months. Patients with post-surgical lung cancer were randomly designated to receive either chemoimmunotherapy (group A, immunotherapy arm) or chemotherapy (group B, control arm). The immunotherapy comprised the adoptive transfer of autologous activated killer T cells and dendritic cells (AKT–DC). The 2- and 5-year overall survival (OS) rates were 96.0 and 69.4% in group A and 64.7 and 45.1% in group B, respectively. Multivariate analysis results revealed that the hazard ratio was 0.439. The 2- and 5-year recurrence-free survival rates were 70.0 and 57.9% in group A and 43.1 and 31.4% in group B, respectively. Subgroup analysis for the OS between treatment groups indicated that younger patients (≤ 55 years: HR 0.098), males (HR 0.474), patients with adenocarcinoma (HR 0.479), patients with stage III cancer (HR 0.399), and those who did not receive preoperative chemotherapy (HR 0.483) had lower HRs than those in the other groups. Immunological analysis + + of cell surface markers in regional lymph nodes of subjects receiving immunotherapy indicated that the CD8 /CD4 T-cell ratio was elevated in survivors. Patients with non-small-cell lung cancer benefited from adoptive cellular immunotherapy as an adjuvant to surgery. Patients with stage III cancer, those with adenocarcinoma, and those not receiving preoperative chemotherapy were good candidates. Lastly, cytotoxic T cells were important for a favorable chemoimmunotherapy outcome. Keywords Adjuvant therapy · Lung cancer · Regional lymph nodes · Cellular immunotherapy · Cytotoxic T cells AbbreviationsCI Confidence interval AKT–DC Activated killer T cells and dendritic cellsHR Hazard ratio CDTC Circulating and disseminating tumor cellsOS Overall survival RCT Randomized controlled trial RFS Recurrence-free survival Parts of this paper were presented at the 16th World Conference on Lung Cancer, WCLC, of the International Association for the study of Lung Cancer, IASLC, on September 6–9, 2015 in Denver, Introduction (Co, USA) [J Thorac Oncol 10:S 179 oral 04.01 Abstract] and at the European Society for Medical Oncology (ESMO 2017) Congress, Madrid Spain, on September 8–12, 2017 [ESMO 2017 Progress in diagnostic procedures and surgical technology 1144 Oral presentation]. has considerably improved the prognosis of lung cancer sur- gery [1, 2]. In advanced cases, patient outcomes remain poor * Hideki Kimura h.kimura1028@seikeikai-cmc.jp despite progress in adjuvant chemotherapy [3, 4] and molec- ular-targeted therapy [5]. Patients with stage IIIB and IV Department of Thoracic Surgery, Saiseikai-Narashino lung cancer and malignant pleural effusion, micrometastasis Hospital, Izumi-cho 1-1-1, Narashino, Chiba 275-8580, to mediastinal lymph nodes, or intrapulmonary metastasis Japan 2 are often identified after thoracotomy, shortly recur after Department of Thoracic Surgery, Chiba Cancer Center, surgery, and die early. Previously, we recruited advanced Chiba, Japan 3 lung cancer patients with poor prognoses who had under- Department of General Thoracic Surgery, Graduate School gone surgery for improving prognosis by immunotherapy of Medicine, Chiba University, Chiba, Japan Vol.:(0123456789) 1 3 Cancer Immunology, Immunotherapy in combination with adjuvant chemotherapy or molecular- Preparation of activated killer T cells and dendritic targeted therapy [6]. We present the results with a median cells from regional lymph nodes follow-up of 59.6  months and the associated statistical immunological analyses. The procedure involved in the preparation of AKT–DC has been previously described [6]. One to two grams of tumor- draining lymph nodes (TDLN) from intrapulmonary to medi- Patients and methods astinal lymph nodes with no metastasis was transferred to a sterile Petri dish and aseptically minced into 1-mm tissue The patients and methods are described in our previous fragments. The tissue preparation was then suspended in 50 ml report [6]. Alyse (ALyS505N: Cell Science and Technology Institute, Inc., Sendai, Japan) serum-free lymphocyte medium contain- Study design and inclusion criteria ing 400 IU/ml human recombinant interleukin 2, transferred to a 75-cm culture flask, and incubated at 37 °C in air contain- Patients with post-surgical NSCLC were randomly assigned ing 5% CO . When the TDLN started to release AKT–DC, to receive either adjuvant chemoimmunotherapy (group A, the tissues and cells were transferred to culture bags. The immunotherapy arm) or adjuvant chemotherapy (group B, AKT–DC were separated from the TDLN tissue by filtering control arm). Immunotherapy consisted of the adoptive through a nylon mesh and were then transferred to another set transfer of activated cytotoxic killer T cells and dendritic of bag, split 2–3 times, and harvested. Cells were suspended cells (AKT–DC) derived from the regional lymph nodes of in the cryoprotective agent CP-1 (Kyokuto Pharm. Co., Tokyo, patients with lung cancer. The patient inclusion criteria of Japan) with 4% human albumin and stored at 5–10 × 10 cells/ this study were as follows: post-surgical patients, < 76 years; bag (freeze bag F-100A: NIPRO Osaka, Japan) in − 80 °C Eastern Cooperative Oncology Group performance status until used. AKT–DC were intravenously infused 1 week after (PS), 0 or 1; adequate bone marrow, liver, and renal func- each course of chemotherapy and were then continued once tion; histology, primary NSCLC, including combined-type a month for the first 6 months after resection and then every small-cell carcinoma; pathological stage, IB with a tumor 2 months until 2 years after surgery. size > 5 cm or with severe vessel invasion and II–IV (TNM staging system version 6). Patients with clinical stage I and Immunological analysis II cancer received surgery and were pathologically stratified as group a: stage IB, group b: stage II, group c: stage IIIA, We selected patients who died within 3 years of recurrence and group d: stage IIIB, IV. Patients with clinical stage IIIA (n = 7) and compared the cell surface markers with that of cancer (single station N2 or T3N1) received two courses of other patients (n = 42) who were alive at 3 years in group A. induction chemotherapy and were stratified as group e: stage Mononuclear cells obtained from regional lymph nodes of the IIIA and group f: stage IIIB, IV diagnosed after thoracotomy patients after surgery were stained with immunofluorescence (pathological stages). Patients with stages IIIB or IV cancer and analyzed using flow cytometer before and 1–2 months and malignant pleural effusion, micrometastasis to medias- after the initiation of in vitro culture in IL2 when the cells tinal lymph nodes, or intrapulmonary metastasis identified actively proliferated. after thoracotomy were also included. Patients who under- went non-curative resection were included, but those with Flow cytometer exploratory thoracotomies or macroscopic residual tumors were excluded from this study. Cells were labeled with human monoclonal anti-CD8, HLA- DR, CD80, and CD4 antibodies conjugated with fluorescein Chemotherapy isothiocyanate (FITC; Becton, Dickinson and Co., NJ, USA) and anti-CD3, B7H1 (PD-L1), CD83, and CD25 antibodies We used platinum doublet regimens belonging to third-gen- conjugated with phycoerythrin (PE) and counted using a flow eration drugs as induction and adjuvant chemotherapy. Both cytometer (Cytomics-FC500; Beckman Coulter, CA, USA). groups received four courses of adjuvant chemotherapy after 7-Amino-actinomycin D (7AAD) was added to exclude non- surgery (groups a, b, c, and d). Patients with clinical stage viable cells. IIIA cancer (groups e and f) received two courses of induc- tion and adjuvant chemotherapy. After the confirmation of Statistical analysis recurrence, chemotherapy was resumed and EGFR-muta- tion-positive patients received EGFR-TKI. Immunotherapy The population for analysis was defined as randomly was continued or resumed with the patient’s consent in com- assigned patients eligible before treatment. Overall survival bination with chemotherapy. 1 3 Cancer Immunology, Immunotherapy (OS) was defined as the time from random assignment to One patient each from groups A and B was excluded from death from any cause. Recurrence-free survival (RFS) was the study due to a study violation or leukemic conversion of defined as the time from randomization to confirmation of AKT–DC after randomization. recurrence by the trial cancer board. Survival curves were estimated using the Kaplan–Meier method, and survival Overall survival rates with 95% confidence intervals (CIs) were calculated. The survival rates between the treatment arms were com- The difference in OS rates between the treatment arms was pared using the log-rank test, and hazard ratios (HR) were noted to be statistically significant (log-rank test, P = 0.0005) calculated using the Cox proportional hazards model with and in agreement with our initial findings from 4 years ago and without the following covariates: age, sex, histology, [6] (Fig. 2). The 2-, 5-, and 7-year OS rates were 96.0% (95% stage, and preoperative chemotherapy. The significance level CI 84.9–99.0), 69.4% (54.4–80.3), and 55.1 (34.3–71.7) of the two-tailed statistical test was 0.05. Statistical analyses in group A and 64.7% (50.0–76.1), 45.1% (31.2–58.0), were performed using the Translational Research Informat- and 38.1% (24.6–51.4) in group B, respectively. The HRs ics Center (TRI: TRILC1304) and by the Foundation for were 0.451 (95% CI 0.253–0.807) by univariate and 0.439 Biomedical Research and Innovation using SAS (version (0.239–0.807) by multivariate analysis using treatment, age, 9.3; SAS Institute, Cary, NC, USA). Interim analysis was sex, histology, stage, and preoperative chemotherapy. scheduled for 5 years after the initiation of the study regard- less of the number of enrolled patients. Recurrence‑free survival The 2-, 5-, and 7-year RFS rates were 70.0% (95% CI Results 55.3–80.7), 57.9% (43.1–70.2), and 47.5% (29.2–63.8) in group A and 43.1% (29.4–56.1), 31.4% (19.3–44.2), Consort diagram and 28.5% (16.7–41.5) in group B, respectively (Fig.  3). These differences in the RFS rates between the two treat- As shown in Fig.  1, 453 of 556 patients who underwent ment groups were also noted to be statistically significant surgery for NSCLC between April 2007 and July 2012 were (log-rank test, P = 0.0044). The HRs were 0.473 (95% CI excluded, and the remaining 103 patients were selected for 0.280–0.801) by univariate and 0.473 (0.275–0.812) by mul- randomization. Of the ineligible patients, 79 were excluded tivariate analysis in favor of group A. due to age (> 76 years), 303 were ineligible due to early- stage tumors. Among 62 patients with stage IIIB and IV OS using Cox proportional hazards model cancer, a sufficient number of AKT–DC (> 7 × 10 ) needed for subgroup analyses and treatment interactions for a course of treatment could not be obtained because of immunosuppression in 35 cases (56.5%), and these patients The HRs by subgroup analysis of OS between treatment were excluded from the study. Nine patients were excluded groups that were significantly lower than 1.0 in favor of due to hepatitis viral infections or due to refusal to provide an informed consent for immunotherapy. Lung Cancer Surgery Ineligible Eligible 103 Stratification Randomaization Ineligible 1 51 Ineligible 1 Group A GroupB (51) (50) Fig. 2 Overall survival (OS). OS was defined as the time from ran- Fig. 1 Consort diagram. Of the 556 patients who underwent surgery dom assignment to death from any cause and was estimated using the between April 2007 and July 2012, 103 were selected for randomiza- Kaplan–Meier method tion 1 3 Cancer Immunology, Immunotherapy conducted between 1986 and 1992 using LAK cells was reported in the journal Cancer [7]. The results of a phase II study conducted between 1998 and 2004 using AKT–DC obtained from the regional lymph nodes of patients with primary lung cancer predicted a promising outcome for a phase III study using this approach [8, 9]. The results of this phase III study clearly demonstrate that adoptive cel- lular immunotherapy benefits patients with lung cancer as an adjuvant to surgery. Subgroup analysis of OS, comparing the immuno- therapy arms using Cox models, indicated that younger patients, male patients, and patients with adenocarcinoma or stage III tumors are good candidates for immunother- apy. The prognosis for stages I and II is better than that for stage III in patients with NSCLC; however, chemoimmu- Fig. 3 Recurrence-free survival (RFS). RFS was defined as the time notherapy improved prognosis for stage III more efficiently from randomization to confirmation of recurrence by the trial cancer than it did for stage I and II; this finding was observed board. RFS was estimated using the Kaplan–Meier method in both male and female patients. While the prognosis was better for females than for males, it was significantly the immunotherapy arm (Table 1) were as follows: 0.098 improved by immunotherapy in males. Stage IIIB and IV (0.011–0.856), age ≤ 55; 0.474 (0.248–0.907), males; 0.479 tumors have evolved mechanisms for escaping immune (0.239–0.959), adenocarcinoma; 0.399 (0.194–0.822), stage response in the tumor microenvironment [10–13]. The III tumors; and 0.483 (0.245–0.951), those without preop- immune system is either inefficient or tolerates the growth erative chemotherapy. of tumors in stages IIIB or IV, which likely leads to inef- fective chemoimmunotherapy outcomes in those patients. RFS using Cox proportional hazards model Lung cancer patients with stage I and II tumors are good for subgroup analyses and treatment interactions candidates for surgery; however, surgery is not indicated in stage IIIA cases due to poor prognosis. If the prognosis As shown in Table 2, the HRs (95% CIs) of RFS by subgroup of advanced NSCLC can be improved by cell-mediated analysis significantly lower than 1.0 in favor of the immuno- immunotherapy, surgery may be added to the current treat- therapy arm were as follows: 0.058 (0.007–0.520), age ≤ 55; ment modalities for patients with stage IIIA cancer. 0.216 (0.059–0.791), females; 0.495 (0.269–0.909), adeno- The assessment of histological types in this RCT carcinoma; 0.446 (0.235–0.845), stage III tumors; and 0.507 showed that the HR of OS for adenocarcinoma was lower (0.271–0.948), patients without preoperative chemotherapy than that for squamous cell carcinoma. Patients with ade- (Table 2). nocarcinoma were demonstrated to benefit from immuno - therapy. It is speculated that metastatic tumors resulting Cell surface markers and survival from circulating and disseminating tumor cells (CDTC), the primary residual adenocarcinoma constituents in these + + The CD8 /CD4 T-cell ratio analyzed 1–2 months after the patients [14, 15], cannot escape immune surveillance and initiation of in vitro culture was higher in the survivors than are eventually eliminated by cell-mediated immunotherapy in the deceased (p = 0.013: Fig. 4). Additional analyses using [10, 16, 17]. Conversely, the residual pattern of squamous cell surface markers for determining the positive percentages cell carcinoma includes residual edges of the resected pri- + + + + + + of CD8, CD4, CD80, CD83 , HLA-DR, B7H1 , and mary tumor margin, but not CDTC. These residual tumors, T-reg (CD4 + CD25+) cells before and after in vitro culture like the original tumors, are capable of escaping immune failed to show significant relationships with survival. surveillance blocking immune response. We excluded cases with macroscopically residual tumors; however, microscopically residual tumors, such as those with posi- Discussion tive bronchial or chest wall margins, were included in the present trial. Squamous cell carcinoma invades the This study is a series of adjuvant immunotherapy tri- surrounding tissues that remain after resection, induces als in patients with post-surgical lung cancer extending immunosuppression, and blocks immune responses, and over 20 years, starting with Lymphokine activated killer may also prevent effective cell-mediated immunotherapy. (LAK) cells and continuing with AKT–DC. The first RCT 1 3 Cancer Immunology, Immunotherapy Table 1 OS using Cox models for subgroup analyses and treatment interactions Median OS (years) Variable Subgroup A B Forest plot Hazard ratio P* 55 or less (n = 0.098 (0.011, NR 3.96 0.1403 14) 0.856) 0.475 (0.158, Age 56-65 (n = 37) NR NR 1.426) 66 or more (n = 0.606 (0.290, 5.55 2.56 50) 1.267) 0.474 (0.248, Male (n = 75) NR 2.79 0.6538 0.907) Sex 0.399 (0.102, Female (n = 26) NR 5.68 1.557) 0.479 (0.239, Adeno (n = 73) NR 5.36 0.5404 0.959) Squamous (n = 0.623 (0.160, Histology NR 2.19 17) 2.435) 0.157 (0.017, others (n = 11) NR 1.74 1.419) 0.641 (0.212, I, II (n = 29) NR 6.49 0.4759 1.936) 0.399 (0.194, Stage III (n = 65) NR 2.78 0.822) IV (n =( 7) NR 7.29 0.356 0.032, 4.011) A,B,C,D (n = 0.483 (0.245, 6.63 3.58 0.8461 74) 0.951) Preoperative chemotherapy 0.409 (0.128, E,F (n = 27) NR 3.96 1.308) NR not reached to 50% *P value for treatment interaction Platinum doublet regimens belonging to the third-generation drugs are used for an induction and adjuvant chemotherapy. Both groups received four courses of adjuvant chemotherapy after surgery (group a, b, c, and d). Stage IIIA patients (group e and f) received two courses of induction chemotherapy before surgery Patients who did not receive preoperative chemotherapy responses may be abrogated by preoperative chemotherapy. had lower HRs and benefited from immunotherapy, whereas Cytotoxic anticancer drugs may negatively affect immune patients who received preoperative chemotherapy did not responses in regional lymph nodes, dampening the effect of significantly benefit from immunotherapy. Specific immune immunotherapy. 1 3 Cancer Immunology, Immunotherapy Table 2 RFS using Cox models for subgroup analyses and treatment interactions Median RFS Variable Subgroup (years) Forest plot Hazard Ratio p* A B 55 or less (n = 14) NR 1.36 0.058 (0.007, 0.520) 0.1799 Age 56-65 (n = 37) NR 2.04 0.419 (0.154, 1.134) 66 or more (n = 50) 2.55 1.38 0.649 (0.326, 1.293) Male (n = 75) 5.65 1.24 0.576 (0.320, 1.038) 0.1973 Sex Female (n = 26) NR 1.43 0.216 (0.059, 0.791) Adeno (n = 73) 5.92 1.38 0.495 (0.269, 0.909) 0.5805 Histology Squamous (n = 17) NR 1.40 0.635 (0.163, 2.471) others (n = 11) NR 0.96 0.253 (0.046, 1.406) I, II (n = 29) NR 6.49 0.610 (0.202, 1.842) 0.6170 Stage III (n = 65) 5.92 1.36 0.446 (0.235, 0.845) IV (n = 7) NR 2.06 0.437 (0.072, 2.651) A, B, C, D (n = 74) 5.92 1.73 0.507 (0.271, 0.948) 0.8244 Preoperative E, F (n = 27) NR 1.36 0.415 (0.154, 1.117) chemotherapy NR not reached to 50% *P value for treatment interaction Immunological analysis using cell surface markers of cytotoxic killer T cells seems to be more significant than that + + cultured lymphocytes indicated that the CD8 /CD4 T-cell of indirect support from helper T cells in eliminating CDTC. ratio was elevated in survivors. Analysis using other cell Most cancer recurrences result from CDTC, which are surface markers of lymphocytes before and after in vitro clinically undetectable at the time of resection of a primary culture failed to show any significant correlation with sur - carcinoma [14, 15]. The immune response against CDTC vival. These results indicated that C D8 cytotoxic T cells released from primary tumors is distinct from that against were more effective than CD4 helper T cells in the adju- original tumors regarding immunosuppression, which is vant therapy circumstances in this study. The effect of direct induced by several immune escape mechanisms. Original 1 3 Cancer Immunology, Immunotherapy for patients with lung cancer, there are certain limitations of this study. This study was carried out with a relatively small group of patients (only 103 patients) and at a single institution and only in Japan. Also, this was not a blinded study and the included patients were heterogeneous popu- lation. A large-scale, double-blind, randomized, multi- institutional trial is essential for ascertaining the efficacy of the presently described adjuvant cellular immunotherapy procedure and its clinical application. Successful dissemi- nation of skills is required for the culture of regional lymph nodes to successors and this requires experience, time, and financial resources. Close cooperation and collaboration to extend the study protocol nationwide will be immensely Fig. 4 Cell surface markers and survival. The relationship between beneficial to patients with lung cancer awaiting this cellular cell surface markers and survival was examined, which showed that immunotherapy. + + the CD8 /CD4 ratio was elevated in survivors Acknowledgements The authors are indebted to Mr. CWP Reynolds, associated with Tokyo Medical University, for his editing of this manu- tumors are not susceptible to cell-mediated immunother- script for its English language content. The authors would like to thank apy due to several immune escape mechanisms, whereas Enago (http://www.enago .jp) for the English language review. CDTC may get eliminated by cell-mediated immunotherapy because they can evade neither immune surveillance nor Author contributions Conception and design: HK, TN, TI. Collection and assembly of data: YM, AI. Data analysis and interpretation: HK, immune response. TI. Manuscript writing: HK. Final approval of manuscript: all authors. The target of immunotherapy in this trial was not the pri- mary lesion, but the undetectable tumor cells remaining after Funding No relevant funding. the resection of primary carcinoma of the lung. The phenotypic diversity of disseminated cells resulting Compliance with ethical standards from intra-tumor heterogeneity [18, 19] gives rise to clones that are resistant to chemotherapy and prevents tumor cell Conflict of interest The authors declare that they have no conflict of eradication by chemotherapy. The heterogeneity of tumor interest. cells enables them to escape even from molecular-targeted Ethical approval and ethical standards The trial protocol was reviewed therapy [20, 21]. The regional lymph nodes of patients with and approved by the ethics committee of Chiba Cancer Center and lung cancer are the organ where the first adaptive immune the University Hospital Medical Information Network in Japan response against cancer develops [22, 23]. Dendritic cells (UMIN:CCCI). Patient enrollment and random assignment were per- formed by Internet Data and Information Center for Medical Research at the tumor site internalize antigens, migrate to lymph (INDICE 000007525), which is supported by a Japanese government nodes, and induce naive T cells to become antigen-specific body. This study was conducted in accordance with the ethical princi- cytotoxic T cells [24]. They act as messengers between ples of the Declaration of Helsinki and the International Conference the innate and adaptive immune response. From initiation on Harmonization of Good Clinical Practice guidelines. to progression of cancer, tumor cells give rise to various Informed consent Informed consent was obtained from all individual antigens, which are recognized by dendritic cells. Regional participants included in the study. lymph nodes represent one of the frontline defense mecha- nism against cancer to cope with heterogeneous cancer cells. Open Access This article is distributed under the terms of the Crea- Cell-mediated immunotherapy derived from regional lymph tive Commons Attribution 4.0 International License (http://creat iveco nodes as a source of dendritic cells and cytotoxic T cells mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- may finally eradicate heterogeneous tumor cell clones that tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the disseminate throughout the body, carrying a wide variety of Creative Commons license, and indicate if changes were made. antigens before immunosuppression develops in microme- tastases. An important point to consider is whether the suf- ficient number of AKT–DC can be obtained from the patient for this cell-mediated immunotherapy, as we noticed that References AKT–DC could not be obtained in the required quantities 1. 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Randomized controlled phase III trial of adjuvant chemoimmunotherapy with activated cytotoxic T cells and dendritic cells from regional lymph nodes of patients with lung cancer

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Medicine & Public Health; Oncology; Immunology; Cancer Research
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Abstract

Randomized controlled trial of adjuvant chemoimmunotherapy for lung cancer indicated a significant advantage in patients receiving immunotherapy. Herein we report the final results and immunological analysis with a median follow-up of 59.6 months. Patients with post-surgical lung cancer were randomly designated to receive either chemoimmunotherapy (group A, immunotherapy arm) or chemotherapy (group B, control arm). The immunotherapy comprised the adoptive transfer of autologous activated killer T cells and dendritic cells (AKT–DC). The 2- and 5-year overall survival (OS) rates were 96.0 and 69.4% in group A and 64.7 and 45.1% in group B, respectively. Multivariate analysis results revealed that the hazard ratio was 0.439. The 2- and 5-year recurrence-free survival rates were 70.0 and 57.9% in group A and 43.1 and 31.4% in group B, respectively. Subgroup analysis for the OS between treatment groups indicated that younger patients (≤ 55 years: HR 0.098), males (HR 0.474), patients with adenocarcinoma (HR 0.479), patients with stage III cancer (HR 0.399), and those who did not receive preoperative chemotherapy (HR 0.483) had lower HRs than those in the other groups. Immunological analysis + + of cell surface markers in regional lymph nodes of subjects receiving immunotherapy indicated that the CD8 /CD4 T-cell ratio was elevated in survivors. Patients with non-small-cell lung cancer benefited from adoptive cellular immunotherapy as an adjuvant to surgery. Patients with stage III cancer, those with adenocarcinoma, and those not receiving preoperative chemotherapy were good candidates. Lastly, cytotoxic T cells were important for a favorable chemoimmunotherapy outcome. Keywords Adjuvant therapy · Lung cancer · Regional lymph nodes · Cellular immunotherapy · Cytotoxic T cells AbbreviationsCI Confidence interval AKT–DC Activated killer T cells and dendritic cellsHR Hazard ratio CDTC Circulating and disseminating tumor cellsOS Overall survival RCT Randomized controlled trial RFS Recurrence-free survival Parts of this paper were presented at the 16th World Conference on Lung Cancer, WCLC, of the International Association for the study of Lung Cancer, IASLC, on September 6–9, 2015 in Denver, Introduction (Co, USA) [J Thorac Oncol 10:S 179 oral 04.01 Abstract] and at the European Society for Medical Oncology (ESMO 2017) Congress, Madrid Spain, on September 8–12, 2017 [ESMO 2017 Progress in diagnostic procedures and surgical technology 1144 Oral presentation]. has considerably improved the prognosis of lung cancer sur- gery [1, 2]. In advanced cases, patient outcomes remain poor * Hideki Kimura h.kimura1028@seikeikai-cmc.jp despite progress in adjuvant chemotherapy [3, 4] and molec- ular-targeted therapy [5]. Patients with stage IIIB and IV Department of Thoracic Surgery, Saiseikai-Narashino lung cancer and malignant pleural effusion, micrometastasis Hospital, Izumi-cho 1-1-1, Narashino, Chiba 275-8580, to mediastinal lymph nodes, or intrapulmonary metastasis Japan 2 are often identified after thoracotomy, shortly recur after Department of Thoracic Surgery, Chiba Cancer Center, surgery, and die early. Previously, we recruited advanced Chiba, Japan 3 lung cancer patients with poor prognoses who had under- Department of General Thoracic Surgery, Graduate School gone surgery for improving prognosis by immunotherapy of Medicine, Chiba University, Chiba, Japan Vol.:(0123456789) 1 3 Cancer Immunology, Immunotherapy in combination with adjuvant chemotherapy or molecular- Preparation of activated killer T cells and dendritic targeted therapy [6]. We present the results with a median cells from regional lymph nodes follow-up of 59.6  months and the associated statistical immunological analyses. The procedure involved in the preparation of AKT–DC has been previously described [6]. One to two grams of tumor- draining lymph nodes (TDLN) from intrapulmonary to medi- Patients and methods astinal lymph nodes with no metastasis was transferred to a sterile Petri dish and aseptically minced into 1-mm tissue The patients and methods are described in our previous fragments. The tissue preparation was then suspended in 50 ml report [6]. Alyse (ALyS505N: Cell Science and Technology Institute, Inc., Sendai, Japan) serum-free lymphocyte medium contain- Study design and inclusion criteria ing 400 IU/ml human recombinant interleukin 2, transferred to a 75-cm culture flask, and incubated at 37 °C in air contain- Patients with post-surgical NSCLC were randomly assigned ing 5% CO . When the TDLN started to release AKT–DC, to receive either adjuvant chemoimmunotherapy (group A, the tissues and cells were transferred to culture bags. The immunotherapy arm) or adjuvant chemotherapy (group B, AKT–DC were separated from the TDLN tissue by filtering control arm). Immunotherapy consisted of the adoptive through a nylon mesh and were then transferred to another set transfer of activated cytotoxic killer T cells and dendritic of bag, split 2–3 times, and harvested. Cells were suspended cells (AKT–DC) derived from the regional lymph nodes of in the cryoprotective agent CP-1 (Kyokuto Pharm. Co., Tokyo, patients with lung cancer. The patient inclusion criteria of Japan) with 4% human albumin and stored at 5–10 × 10 cells/ this study were as follows: post-surgical patients, < 76 years; bag (freeze bag F-100A: NIPRO Osaka, Japan) in − 80 °C Eastern Cooperative Oncology Group performance status until used. AKT–DC were intravenously infused 1 week after (PS), 0 or 1; adequate bone marrow, liver, and renal func- each course of chemotherapy and were then continued once tion; histology, primary NSCLC, including combined-type a month for the first 6 months after resection and then every small-cell carcinoma; pathological stage, IB with a tumor 2 months until 2 years after surgery. size > 5 cm or with severe vessel invasion and II–IV (TNM staging system version 6). Patients with clinical stage I and Immunological analysis II cancer received surgery and were pathologically stratified as group a: stage IB, group b: stage II, group c: stage IIIA, We selected patients who died within 3 years of recurrence and group d: stage IIIB, IV. Patients with clinical stage IIIA (n = 7) and compared the cell surface markers with that of cancer (single station N2 or T3N1) received two courses of other patients (n = 42) who were alive at 3 years in group A. induction chemotherapy and were stratified as group e: stage Mononuclear cells obtained from regional lymph nodes of the IIIA and group f: stage IIIB, IV diagnosed after thoracotomy patients after surgery were stained with immunofluorescence (pathological stages). Patients with stages IIIB or IV cancer and analyzed using flow cytometer before and 1–2 months and malignant pleural effusion, micrometastasis to medias- after the initiation of in vitro culture in IL2 when the cells tinal lymph nodes, or intrapulmonary metastasis identified actively proliferated. after thoracotomy were also included. Patients who under- went non-curative resection were included, but those with Flow cytometer exploratory thoracotomies or macroscopic residual tumors were excluded from this study. Cells were labeled with human monoclonal anti-CD8, HLA- DR, CD80, and CD4 antibodies conjugated with fluorescein Chemotherapy isothiocyanate (FITC; Becton, Dickinson and Co., NJ, USA) and anti-CD3, B7H1 (PD-L1), CD83, and CD25 antibodies We used platinum doublet regimens belonging to third-gen- conjugated with phycoerythrin (PE) and counted using a flow eration drugs as induction and adjuvant chemotherapy. Both cytometer (Cytomics-FC500; Beckman Coulter, CA, USA). groups received four courses of adjuvant chemotherapy after 7-Amino-actinomycin D (7AAD) was added to exclude non- surgery (groups a, b, c, and d). Patients with clinical stage viable cells. IIIA cancer (groups e and f) received two courses of induc- tion and adjuvant chemotherapy. After the confirmation of Statistical analysis recurrence, chemotherapy was resumed and EGFR-muta- tion-positive patients received EGFR-TKI. Immunotherapy The population for analysis was defined as randomly was continued or resumed with the patient’s consent in com- assigned patients eligible before treatment. Overall survival bination with chemotherapy. 1 3 Cancer Immunology, Immunotherapy (OS) was defined as the time from random assignment to One patient each from groups A and B was excluded from death from any cause. Recurrence-free survival (RFS) was the study due to a study violation or leukemic conversion of defined as the time from randomization to confirmation of AKT–DC after randomization. recurrence by the trial cancer board. Survival curves were estimated using the Kaplan–Meier method, and survival Overall survival rates with 95% confidence intervals (CIs) were calculated. The survival rates between the treatment arms were com- The difference in OS rates between the treatment arms was pared using the log-rank test, and hazard ratios (HR) were noted to be statistically significant (log-rank test, P = 0.0005) calculated using the Cox proportional hazards model with and in agreement with our initial findings from 4 years ago and without the following covariates: age, sex, histology, [6] (Fig. 2). The 2-, 5-, and 7-year OS rates were 96.0% (95% stage, and preoperative chemotherapy. The significance level CI 84.9–99.0), 69.4% (54.4–80.3), and 55.1 (34.3–71.7) of the two-tailed statistical test was 0.05. Statistical analyses in group A and 64.7% (50.0–76.1), 45.1% (31.2–58.0), were performed using the Translational Research Informat- and 38.1% (24.6–51.4) in group B, respectively. The HRs ics Center (TRI: TRILC1304) and by the Foundation for were 0.451 (95% CI 0.253–0.807) by univariate and 0.439 Biomedical Research and Innovation using SAS (version (0.239–0.807) by multivariate analysis using treatment, age, 9.3; SAS Institute, Cary, NC, USA). Interim analysis was sex, histology, stage, and preoperative chemotherapy. scheduled for 5 years after the initiation of the study regard- less of the number of enrolled patients. Recurrence‑free survival The 2-, 5-, and 7-year RFS rates were 70.0% (95% CI Results 55.3–80.7), 57.9% (43.1–70.2), and 47.5% (29.2–63.8) in group A and 43.1% (29.4–56.1), 31.4% (19.3–44.2), Consort diagram and 28.5% (16.7–41.5) in group B, respectively (Fig.  3). These differences in the RFS rates between the two treat- As shown in Fig.  1, 453 of 556 patients who underwent ment groups were also noted to be statistically significant surgery for NSCLC between April 2007 and July 2012 were (log-rank test, P = 0.0044). The HRs were 0.473 (95% CI excluded, and the remaining 103 patients were selected for 0.280–0.801) by univariate and 0.473 (0.275–0.812) by mul- randomization. Of the ineligible patients, 79 were excluded tivariate analysis in favor of group A. due to age (> 76 years), 303 were ineligible due to early- stage tumors. Among 62 patients with stage IIIB and IV OS using Cox proportional hazards model cancer, a sufficient number of AKT–DC (> 7 × 10 ) needed for subgroup analyses and treatment interactions for a course of treatment could not be obtained because of immunosuppression in 35 cases (56.5%), and these patients The HRs by subgroup analysis of OS between treatment were excluded from the study. Nine patients were excluded groups that were significantly lower than 1.0 in favor of due to hepatitis viral infections or due to refusal to provide an informed consent for immunotherapy. Lung Cancer Surgery Ineligible Eligible 103 Stratification Randomaization Ineligible 1 51 Ineligible 1 Group A GroupB (51) (50) Fig. 2 Overall survival (OS). OS was defined as the time from ran- Fig. 1 Consort diagram. Of the 556 patients who underwent surgery dom assignment to death from any cause and was estimated using the between April 2007 and July 2012, 103 were selected for randomiza- Kaplan–Meier method tion 1 3 Cancer Immunology, Immunotherapy conducted between 1986 and 1992 using LAK cells was reported in the journal Cancer [7]. The results of a phase II study conducted between 1998 and 2004 using AKT–DC obtained from the regional lymph nodes of patients with primary lung cancer predicted a promising outcome for a phase III study using this approach [8, 9]. The results of this phase III study clearly demonstrate that adoptive cel- lular immunotherapy benefits patients with lung cancer as an adjuvant to surgery. Subgroup analysis of OS, comparing the immuno- therapy arms using Cox models, indicated that younger patients, male patients, and patients with adenocarcinoma or stage III tumors are good candidates for immunother- apy. The prognosis for stages I and II is better than that for stage III in patients with NSCLC; however, chemoimmu- Fig. 3 Recurrence-free survival (RFS). RFS was defined as the time notherapy improved prognosis for stage III more efficiently from randomization to confirmation of recurrence by the trial cancer than it did for stage I and II; this finding was observed board. RFS was estimated using the Kaplan–Meier method in both male and female patients. While the prognosis was better for females than for males, it was significantly the immunotherapy arm (Table 1) were as follows: 0.098 improved by immunotherapy in males. Stage IIIB and IV (0.011–0.856), age ≤ 55; 0.474 (0.248–0.907), males; 0.479 tumors have evolved mechanisms for escaping immune (0.239–0.959), adenocarcinoma; 0.399 (0.194–0.822), stage response in the tumor microenvironment [10–13]. The III tumors; and 0.483 (0.245–0.951), those without preop- immune system is either inefficient or tolerates the growth erative chemotherapy. of tumors in stages IIIB or IV, which likely leads to inef- fective chemoimmunotherapy outcomes in those patients. RFS using Cox proportional hazards model Lung cancer patients with stage I and II tumors are good for subgroup analyses and treatment interactions candidates for surgery; however, surgery is not indicated in stage IIIA cases due to poor prognosis. If the prognosis As shown in Table 2, the HRs (95% CIs) of RFS by subgroup of advanced NSCLC can be improved by cell-mediated analysis significantly lower than 1.0 in favor of the immuno- immunotherapy, surgery may be added to the current treat- therapy arm were as follows: 0.058 (0.007–0.520), age ≤ 55; ment modalities for patients with stage IIIA cancer. 0.216 (0.059–0.791), females; 0.495 (0.269–0.909), adeno- The assessment of histological types in this RCT carcinoma; 0.446 (0.235–0.845), stage III tumors; and 0.507 showed that the HR of OS for adenocarcinoma was lower (0.271–0.948), patients without preoperative chemotherapy than that for squamous cell carcinoma. Patients with ade- (Table 2). nocarcinoma were demonstrated to benefit from immuno - therapy. It is speculated that metastatic tumors resulting Cell surface markers and survival from circulating and disseminating tumor cells (CDTC), the primary residual adenocarcinoma constituents in these + + The CD8 /CD4 T-cell ratio analyzed 1–2 months after the patients [14, 15], cannot escape immune surveillance and initiation of in vitro culture was higher in the survivors than are eventually eliminated by cell-mediated immunotherapy in the deceased (p = 0.013: Fig. 4). Additional analyses using [10, 16, 17]. Conversely, the residual pattern of squamous cell surface markers for determining the positive percentages cell carcinoma includes residual edges of the resected pri- + + + + + + of CD8, CD4, CD80, CD83 , HLA-DR, B7H1 , and mary tumor margin, but not CDTC. These residual tumors, T-reg (CD4 + CD25+) cells before and after in vitro culture like the original tumors, are capable of escaping immune failed to show significant relationships with survival. surveillance blocking immune response. We excluded cases with macroscopically residual tumors; however, microscopically residual tumors, such as those with posi- Discussion tive bronchial or chest wall margins, were included in the present trial. Squamous cell carcinoma invades the This study is a series of adjuvant immunotherapy tri- surrounding tissues that remain after resection, induces als in patients with post-surgical lung cancer extending immunosuppression, and blocks immune responses, and over 20 years, starting with Lymphokine activated killer may also prevent effective cell-mediated immunotherapy. (LAK) cells and continuing with AKT–DC. The first RCT 1 3 Cancer Immunology, Immunotherapy Table 1 OS using Cox models for subgroup analyses and treatment interactions Median OS (years) Variable Subgroup A B Forest plot Hazard ratio P* 55 or less (n = 0.098 (0.011, NR 3.96 0.1403 14) 0.856) 0.475 (0.158, Age 56-65 (n = 37) NR NR 1.426) 66 or more (n = 0.606 (0.290, 5.55 2.56 50) 1.267) 0.474 (0.248, Male (n = 75) NR 2.79 0.6538 0.907) Sex 0.399 (0.102, Female (n = 26) NR 5.68 1.557) 0.479 (0.239, Adeno (n = 73) NR 5.36 0.5404 0.959) Squamous (n = 0.623 (0.160, Histology NR 2.19 17) 2.435) 0.157 (0.017, others (n = 11) NR 1.74 1.419) 0.641 (0.212, I, II (n = 29) NR 6.49 0.4759 1.936) 0.399 (0.194, Stage III (n = 65) NR 2.78 0.822) IV (n =( 7) NR 7.29 0.356 0.032, 4.011) A,B,C,D (n = 0.483 (0.245, 6.63 3.58 0.8461 74) 0.951) Preoperative chemotherapy 0.409 (0.128, E,F (n = 27) NR 3.96 1.308) NR not reached to 50% *P value for treatment interaction Platinum doublet regimens belonging to the third-generation drugs are used for an induction and adjuvant chemotherapy. Both groups received four courses of adjuvant chemotherapy after surgery (group a, b, c, and d). Stage IIIA patients (group e and f) received two courses of induction chemotherapy before surgery Patients who did not receive preoperative chemotherapy responses may be abrogated by preoperative chemotherapy. had lower HRs and benefited from immunotherapy, whereas Cytotoxic anticancer drugs may negatively affect immune patients who received preoperative chemotherapy did not responses in regional lymph nodes, dampening the effect of significantly benefit from immunotherapy. Specific immune immunotherapy. 1 3 Cancer Immunology, Immunotherapy Table 2 RFS using Cox models for subgroup analyses and treatment interactions Median RFS Variable Subgroup (years) Forest plot Hazard Ratio p* A B 55 or less (n = 14) NR 1.36 0.058 (0.007, 0.520) 0.1799 Age 56-65 (n = 37) NR 2.04 0.419 (0.154, 1.134) 66 or more (n = 50) 2.55 1.38 0.649 (0.326, 1.293) Male (n = 75) 5.65 1.24 0.576 (0.320, 1.038) 0.1973 Sex Female (n = 26) NR 1.43 0.216 (0.059, 0.791) Adeno (n = 73) 5.92 1.38 0.495 (0.269, 0.909) 0.5805 Histology Squamous (n = 17) NR 1.40 0.635 (0.163, 2.471) others (n = 11) NR 0.96 0.253 (0.046, 1.406) I, II (n = 29) NR 6.49 0.610 (0.202, 1.842) 0.6170 Stage III (n = 65) 5.92 1.36 0.446 (0.235, 0.845) IV (n = 7) NR 2.06 0.437 (0.072, 2.651) A, B, C, D (n = 74) 5.92 1.73 0.507 (0.271, 0.948) 0.8244 Preoperative E, F (n = 27) NR 1.36 0.415 (0.154, 1.117) chemotherapy NR not reached to 50% *P value for treatment interaction Immunological analysis using cell surface markers of cytotoxic killer T cells seems to be more significant than that + + cultured lymphocytes indicated that the CD8 /CD4 T-cell of indirect support from helper T cells in eliminating CDTC. ratio was elevated in survivors. Analysis using other cell Most cancer recurrences result from CDTC, which are surface markers of lymphocytes before and after in vitro clinically undetectable at the time of resection of a primary culture failed to show any significant correlation with sur - carcinoma [14, 15]. The immune response against CDTC vival. These results indicated that C D8 cytotoxic T cells released from primary tumors is distinct from that against were more effective than CD4 helper T cells in the adju- original tumors regarding immunosuppression, which is vant therapy circumstances in this study. The effect of direct induced by several immune escape mechanisms. Original 1 3 Cancer Immunology, Immunotherapy for patients with lung cancer, there are certain limitations of this study. This study was carried out with a relatively small group of patients (only 103 patients) and at a single institution and only in Japan. Also, this was not a blinded study and the included patients were heterogeneous popu- lation. A large-scale, double-blind, randomized, multi- institutional trial is essential for ascertaining the efficacy of the presently described adjuvant cellular immunotherapy procedure and its clinical application. Successful dissemi- nation of skills is required for the culture of regional lymph nodes to successors and this requires experience, time, and financial resources. Close cooperation and collaboration to extend the study protocol nationwide will be immensely Fig. 4 Cell surface markers and survival. The relationship between beneficial to patients with lung cancer awaiting this cellular cell surface markers and survival was examined, which showed that immunotherapy. + + the CD8 /CD4 ratio was elevated in survivors Acknowledgements The authors are indebted to Mr. CWP Reynolds, associated with Tokyo Medical University, for his editing of this manu- tumors are not susceptible to cell-mediated immunother- script for its English language content. The authors would like to thank apy due to several immune escape mechanisms, whereas Enago (http://www.enago .jp) for the English language review. CDTC may get eliminated by cell-mediated immunotherapy because they can evade neither immune surveillance nor Author contributions Conception and design: HK, TN, TI. Collection and assembly of data: YM, AI. Data analysis and interpretation: HK, immune response. TI. Manuscript writing: HK. Final approval of manuscript: all authors. The target of immunotherapy in this trial was not the pri- mary lesion, but the undetectable tumor cells remaining after Funding No relevant funding. the resection of primary carcinoma of the lung. The phenotypic diversity of disseminated cells resulting Compliance with ethical standards from intra-tumor heterogeneity [18, 19] gives rise to clones that are resistant to chemotherapy and prevents tumor cell Conflict of interest The authors declare that they have no conflict of eradication by chemotherapy. The heterogeneity of tumor interest. cells enables them to escape even from molecular-targeted Ethical approval and ethical standards The trial protocol was reviewed therapy [20, 21]. The regional lymph nodes of patients with and approved by the ethics committee of Chiba Cancer Center and lung cancer are the organ where the first adaptive immune the University Hospital Medical Information Network in Japan response against cancer develops [22, 23]. Dendritic cells (UMIN:CCCI). Patient enrollment and random assignment were per- formed by Internet Data and Information Center for Medical Research at the tumor site internalize antigens, migrate to lymph (INDICE 000007525), which is supported by a Japanese government nodes, and induce naive T cells to become antigen-specific body. This study was conducted in accordance with the ethical princi- cytotoxic T cells [24]. They act as messengers between ples of the Declaration of Helsinki and the International Conference the innate and adaptive immune response. From initiation on Harmonization of Good Clinical Practice guidelines. to progression of cancer, tumor cells give rise to various Informed consent Informed consent was obtained from all individual antigens, which are recognized by dendritic cells. Regional participants included in the study. lymph nodes represent one of the frontline defense mecha- nism against cancer to cope with heterogeneous cancer cells. Open Access This article is distributed under the terms of the Crea- Cell-mediated immunotherapy derived from regional lymph tive Commons Attribution 4.0 International License (http://creat iveco nodes as a source of dendritic cells and cytotoxic T cells mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- may finally eradicate heterogeneous tumor cell clones that tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the disseminate throughout the body, carrying a wide variety of Creative Commons license, and indicate if changes were made. antigens before immunosuppression develops in microme- tastases. An important point to consider is whether the suf- ficient number of AKT–DC can be obtained from the patient for this cell-mediated immunotherapy, as we noticed that References AKT–DC could not be obtained in the required quantities 1. 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Cancer Immunology, ImmunotherapySpringer Journals

Published: May 31, 2018

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