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Two cases of intrahepatic cholangiocellular carcinoma with high insertion-deletion ratios that achieved a complete response following chemotherapy combined with PD-1 blockade

Two cases of intrahepatic cholangiocellular carcinoma with high insertion-deletion ratios that... Background: Insertion–deletion mutations (indels) may generate more tumour-specific neoantigens with high affinity to major histocompatibility complex class I. A high indel ratio is also related to a good response to programmed death-1 (PD-1) checkpoint blockade in melanoma and renal cell carcinoma. However, the correlation between a high indel ratio and the immunotherapy response in intrahepatic cholangiocarcinoma (ICC) is unknown. Case presentation: Two patients with relapsed ICC at stage IIIb were treated with PD-1 blockade combined with chemotherapy. After 7 and 4 months of chemotherapy and PD-1 blockade (3 and 15 cycles, and 5 and 6 cycles, respectively), magnetic resonance imaging and positron emission tomography with computed tomography imaging showed that both patients achieved a complete response (CR), which has lasted up to nearly 16 and 13 months to date, respectively. Whole-exome sequencing and immunohistochemistry analysis showed that both patients had cancers with microsatellite stability (MSS) and mismatch repair (MMR) proficiency, weak PD-L1 expression, and a tumour mutation burden (TMB) of 2.95 and 7.09 mutations/Mb, respectively. Patient 2 had mutations of TP53 and PTEN that are known to confer sensitivity to immunotherapy, and the immunotherapy-resistant mutation JAK2, whereas patient 1 had no known immunotherapy response-related mutations. However, the indel ratios of the two patients (48 and 66.87%) were higher than the median of 12.77% determined in a study of 71 ICC patients. Moreover, comparison to six additional ICC patients who showed a partial response, stable disease, or progressive disease after PD-1 blockade treatment alone or in combination with chemotherapy demonstrated no difference in PD-L1 expression, TMB, MSI, and MMR status from those of the two CR patients, whereas the indel frequency was significantly higher in the CR patients. Conclusions: These two cases suggest that indels might be a new predictor of PD-1 blockade response for ICC patients beside PD-L1 expression, TMB, MSI, and dMMR, warranting further clinical investigation. Keywords: Indel, Intrahepatic cholangiocarcinoma, Combination immunotherapy, PD-1 blockade, Whole-exome sequencing * Correspondence: lsc620213@126.com Department of Hepatobiliary Surgery, the First Medical Center, Chinese PLA General Hospital, Medical School of Chinese PLA, 28 Fuxing Road, Beijing 100853, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 2 of 8 Background showed durable responses with a 40% objective response Intrahepatic cholangiocarcinoma (ICC) is an aggressive rate for ICC [12]. However, these studies did not deter- malignancy with a poor prognosis. After curative resec- mine the molecular characteristics of the ICC patients that tion, the 5-year survival rate and median survival time of showed a clinical benefit from the treatment. Further- ICC patients is 30% and 28 months, respectively [1], and more, the combination of pembrolizumab and chemother- the recurrence rate has been reported to be in the range apy has also shown a good response in a case report of a of 40–80% [2]. Other than surgical resection, standard single ICC patient. This ICC patient had a high tumour treatment options for ICC include liver transplantation, mutation burden (TMB) of 19.3 mutations/Mb, but with a gemcitabine-based chemotherapeutic regimen, and microsatellite instability (MSI) and MMR proficiency loco-regional therapies such as transarterial chemoem- (pMMR) [13]. Thus, identifying the subsets of ICC pa- bolization and conformal external-beam radiation ther- tients that are most likely to benefit from PD1/PD-L1 apy; however, given the poor outcome, more effective blockade alone and in combination remains a challenge treatments are urgently needed. and hindrance to effective personalized medicine. Similar to virus-associated cancers, viral hepatitis and Insertion–deletion mutations (indels) cause frameshift Clonorchis sinensis infection are known risk factors for mutations that not only alter the amino acid compos- ICC [3]. These infections often cause immune exhaustion, ition of the proteins but may also lead to early termin- which is mediated through the programmed cell death ation of protein synthesis. Indels and single-nucleotide 1-ligand 1 (PD-L1)/programmed cell death 1 (PD-1) path- variants (SNVs) together determine the TMB. Notably, way, similar to the immunosuppressive mechanism of indels could produce more than three times the neoanti- cancer [4]. Indeed, several studies have shown that PD1/ gens with high affinity to major histocompatibility com- PD-L1 blockade can be effective in the treatment of plex class I (MHC-I) (IC50 < 50 nM), and nine times the virus-related cancers [5]. Moreover, a study including 27 mutation-specific neoantigens compared with SNVs. This patients with ICC showed that 100, 30, and 41% of the high neoantigen load induced by indels was associated cases had infiltrated lymphocytes, positive PD-L1 expres- with HLA I presentation, CD8 T cell activation, and sion, and positive human leukocyte antigen class I antigen increased cytolytic activity compared with the high (HLA I) expression, respectively [6]. In another study, 39 SNV neoantigen group [14]. Consequently, the indel of 54 patients with ICC were found to be positive for count has been significantly associated with a response PD-L1 expression within the tumour front using immuno- to a checkpoint inhibitor across three separate melan- histochemistry (IHC), and the overall survival of these oma cohorts and in patients with advanced renal cell patients was reduced by approximately 60% compared carcinoma [14, 15]. However, there are limited data on with that of patients without PD-L1 expression [7]. Simi- the ability of indels to predict a response to PD-1/ larly, 260 patients with biliary tract cancer (BTC) that had PD-L1 blockers alone and in combination. Here, we re- a relatively poor prognosis showed higher PD-L1 expres- port two patients with ICC with high indel ratios who sion [8]. Collectively, these studies provide a biological ra- were successfully treated with PD-1 blockers plus tionale for the treatment of ICC patients with PD1/PD-L1 chemotherapy, both of whom showed weak PD-L1 ex- blockade. pression and a microsatellite stable (MSS) status, and More importantly, clinical trials have demonstrated a without dMMR. Moreover, one of the patients had a benefit of PD-1/PD-L1 blockers for patients with ICC. relatively low TMB. Comparison of the features of these The PD-L1 positive BTC cohort of the KEYNOTE-028 patients to others reported in the literature, as well as basket trial showed that four of 24 patients who were to six additional cases of ICC that received PD-L1 ther- PD-L1 positive showed a partial response (PR), and four apywithout aCR, points to apotential roleof indels as patients had stable disease (SD) [9]. Another basket trial a key factor determining the response to therapy, war- including four patients with DNA mismatch repair defi- ranting further consideration. ciency (dMMR) cholangiocarcinoma demonstrated that one patient had a complete response (CR) and the other Case presentation A (Patient 1) three patients had SD after PD-1 blockade therapy [10]. Patient 1 (Fig. 1 and Table 1) is a 50-year-old male with Further, three recent studies reported encouraging clin- moderately differentiated ICC staged at IIIb. He was ical outcomes. In a Phase II study on patients with admitted to the hospital in January 2016 due to upper advanced refractory BTC treated with nivolumab, 17% of abdominal pain. He had a history of hepatitis B for 10 the 29 patients achieved a PR, 38% showed SD, and years, and his Child-Pugh class was A. Magnetic reson- there was an overall 55% disease control rate (DCR). No ance imaging (MRI) revealed a mass in the left outer grade IV or V toxicities were reported [11]. Asian BTC lobe, which grew outward and invaded the diaphragm. patients that received M7824 after chemotherapy failure, The tumour marker carcinoembryonic antigen was ele- which targets PD-1 and transforming growth factor-β, vated at 10.14 μg/L. He underwent left hemihepatectomy Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 3 of 8 Fig. 1 Imaging of patient 1 over the course of therapy. a showed timeline of the clinical course. Representative micrographs of PD-L1 expression + + (b) and percentage of CD8 T cells (c) within tumor (original magnification ×400). The positive rate of PD-L1 and CD8 T cells were < 5% and 10%, respectively. MRI and PET-CT imaging (d) showed the lesion around the margin and enlarged lymph nodes disappeared after treatment with pembrolizumab in combination with tegafur on September 14, 2017. The CR was sustained up to 9 January 2019 and hepatoduodenal ligament skeletonization on February determine the presence of SNVs, indels, the TMB, 16, 2016. The tumour was 11 cm × 9 cm × 6 cm, and no copy number variations (CNVs), MSI status, and lymph node metastases were found. Intraoperative radio- dMMR by bioinformatics methods. The TMB was de- therapy was performed on the liver section using 9-mV termined to be 2.95 mutations/Mb, and a total of 25 photon beams with a single dose of 12 Gy, which could non-synonymous mutations (NSMs) were detected in eliminate the residual tumour due to invasion of the dia- the whole genome, including 12 indels and 13 SNVs. phragm and the venous root of the liver. The tumour was This patient harboured only one clinically actionable found to be positive for cytokeratin 18 (CK18) and was mutation in FGF4, which amplified to reveal a copy negative for Arg-1, hepatocyte, glypican-3 (GPC-3), and number of 3.64. No SNVs were detected in MLH1, CK7 in IHC analysis. The tumour proportion score (TPS) MSH2, MSH6,and PMS2 (Additional file 1), suggest- of the PD-L1 expression level was < 5% determined using ing pMMR, and the MSI was 0.01%. monoclonal mouse anti-human PD-L1 clone (22C3) anti- The left hepatic lobe lesions and retroperitoneal left body by allred criteria, and the frequency of infiltrating lymph nodes were treated with Cyberknife (52 Gy/4 F for CD8 T cells was 10%. 4 days) on February 4, 2017 and February 9, 2017, Liver resection margin recurrence and abdominal respectively. Tegafur and pembrolizumab were initiated lymph node metastasis were detected using MRI and 9 days apart. Tegafur chemotherapy was started at 40 mg positron emission tomography-computed tomography twice a day every 3 weeks but was withdrawn due to (PET-CT) after 11 months. MRI showed a marginal le- development of thrombocytopenia and leukopenia after sion of 4 × 1.5 cm in the left lobe of the liver, along with three cycles. Pembrolizumab was administered at 150 mg an enlarged hepatic hilar (1.6 × 1.5 cm) and retroperiton- every 3 weeks for 15 cycles for about 1 year. On June 6, eal lymph nodes (5.2 × 3 cm and 2.8 × 2.6 cm). PET-CT 2017, the abdominal MRI showed that most of the lesions scans also revealed abnormal hypermetabolic lesions in in the margin, and all the lesions in the hilar and retro- these locations. peritoneal lymph nodes had disappeared. It was specu- Whole-exome sequencing (WES) was applied to the lated that the residual lesions at the margin might tissue resected after surgery, and the data were used to represent a surgical reaction. The patient was deemed to Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 4 of 8 Table 1 Baseline, mutation characteristics, treatment, and outcomes Patient Reference 12 3 4 5 6 7 8 Baseline characteristics Age (years) 50 67 60 51 65 59 55 60 Sex male male female female female male male male Surgery Yes Yes Yes No Yes Yes No Yes Stage IIIb IIIb IIIb IIIb IIIb IIIb IV IIIb Lymph node metastasis + – ++ + ++ + Virus infection HBV NHVI NHVI NHVI NHVI NHVI HCV HBV Tumour PD-L1 < 5% < 5% < 1% unknown > 90% > 90% unknown < 5% expression level (%) Percentage of CD8 T cells 10% 10% 10% unknown 20% 20% unknown 30% Mutation characteristics TMB (mutations/Mb) 2.95 7.09 5.62 4.18 8.47 34.84 5.38 6.01 7.4 (median) [21] TMB (NSM) 25 163 114 66 209 1088 106 127 47 (median) [22] Number of SNVs 13 54 85 52 173 771 82 105 41 (median) [22] Number of indels 12 109 29 14 36 317 24 22 6 (median) [22] indels ratio (%) 48.00% 66.87% 25.44% 21.21% 17.22% 29.16% 22.64% 17.32% 12.77% (median) [22] MSI-H/MSS MSS MSS MSS MSS MSS MSI-H MSS MSS 3.2% MSI-H [20] dMMR/pMMR pMMR pMMR pMMR pMMR pMMR dMMR pMMR pMMR 2% dMMR [10] Percentage of CNVs (%) 3.1 4.2 9.29 2.66 4.75 0.78 3.67 5.92 Mutations conferring NA TP53/PTEN KRAS NA BRAF/GITR MLH1/GITR TP53 TP53 sensitivity to immunotherapy Mutations conferring JAK2 B2M chromosome 11q13 resistance to immunotherapy Treatment Therapeutic regimen (cycles) tegafur (3) + tegafur (5, but irregular) nivolumab (4) + nivolumab nivolumab nivolumab (8) + nivolumab (8) + pembrolizumab (6) pembrolizumab + pembrolizumab (6) cisplatin (1) + (15) (8) cisplatin (1) + lenvatinib (8 months (15) + cyberknife (2) gemcitabine (1) gemcitabine (1) continuously) Outcome Type of response CR CR PD SD PD PR PD PR PFS (months) 16 13 Unknown 7 5 3 Unknown 10 The whole exons of tumour tissues removed during surgery and matched white blood cells were sequenced by the Illumina NovaSeq 6000 Sequencing System with average sequence coverage of 200X. We calculated SNVs, indels, and copy number variations (CNVs) by the mutect2 algorithm, and CNVnator software, respectively. MSI was identified using msisensor, and dMMR was identified by analysing SNVs and indels in MLH1, MSH2, MSH6, and PMS2 Abbreviations: CNG copy number gain, CNL copy number loss, NHVI no hepatitis virus infection Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 5 of 8 show a CR on September 14, 2017 and was still in remis- ARID2 are included in the COSMIC top 20 mutated genes sion at the last follow-up of January 10, 2019 based on (Additional file 1). The TMB was 7.09 mutations/Mb, in- PET-CT analysis. No side effects of pembrolizumab were cluding 109 indels (66.87%) and 54 SNVs. The tumour ex- observed. hibitedpMMR andMSI (0.01%). He wasstarted on a regimen of tegafur and pembrolizumab in late August. Un- Case presentation B (Patient 2) fortunately, he experienced the common adverse event to Patient 2 (Fig. 2 and Table 1) is a 67-year-old male with tegafur of pruritus, determined to be of grade 2 according to no hepatitis virus infection. He underwent extended right the standard CTCAE5.0 criteria. After withdrawing tegafur, hemihepatectomy, left hepaticojejunostomy, perihepatic the pruritus disappeared, and the drug was thus switched to lymphadenectomy, and portal vein reconstruction on May an irregular administration schedule as of December 23, 16, 2017. The tumour measured 7.6 cm × 7 cm × 7 cm, with 2017 with an increase in the dose from 40 mg to 60 mg and nerve invasion accompanied by microvascular invasion. No to be taken twice a day until the beginning of February tumour was found in the liver margin and bile duct margin 2018. Pembrolizumab was administrated intravenously (150 after the surgery. Lymphatic metastasis was detected in mg every 3 weeks for six cycles). PET-CT scans revealed that groups 8 and 12A. IHC showed Arg-1 (−), CK18 (+), GPC-3 the enlarged lymph nodes had disappeared. This CR was (−), hepatocyte (−), Ki-67 positivity of 65%, and CK19 (+). achieved in less than 4 months, and the patient continued to Accordingly, he was diagnosed with iCCA stage IIIb. Similar be in remission for 13 months up to the last follow-up. to Patient 1, PD-L1 expression was detected on < 5% of the tumour cells, and the percentage of CD8 T cells was 10%. Discussion Lymph node metastasis in the hepatoportal area was de- This is the first report demonstrating two ICC patients tected using MRI and PET-CT on August 16, 2017. WES with high indel rates that achieved a CR after PD-1 revealed 163 NSMs, including clinically actionable alter- blockade combined with chemotherapy. We also analysed ations in PTEN and TP53. In addition, TP53, SMAD4,and the presence of genomic alterations known to be involved Fig. 2 a Timeline of the clinical course in patient 2. The positive rate of PD-L1 (b) and CD8 T cells (c) in patient 2 were < 5% and 10% evaluated by IHC, respectively (magnification ×400). Patient 2 had a complete metabolic response after treatment with pembrolizumab in combination with tegafur, with no residual hypermetabolic uptake on post-treatment imaging (d) Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 6 of 8 in the response and resistance to immunotherapy using Compared with the PR, SD, and PD groups of these six WES [16–18]. Patient 2 harboured three such mutations, additional patients, Patient 1 had the lowest TMB and Pa- including TP53 and PTEN inactivating mutants, which tient 2 had a TMB that was not significantly different from can induce a strong potential response, and JAK2 muta- that of the other patients. However, the indel rate of the tions, which have been associated with immune therapy CR groups was higher than that of the other groups, and resistance. However, Patient 1 did not have any related was significantly different from that reported previously mutations. Both patients had an MSI frequency of 0.01%, [22](P = 0.001 and P < 0.001, respectively). which is well within the spectrum of MSS tumours Immunotherapy has achieved great success, with 10– [19, 20], and also showed a pMMR molecular phenotype. 35% of patients showing a response to single immune The median TMB of patients with ICC varies among checkpoint blockade inhibitors [24]. Recently, more clin- studies. However, the TMB levels of Patients 1 and 2 ical effort has focused on combination immunotherapy were lower or equal to the median reported for 69 Chinese such as immune checkpoint inhibitors combined with ICC patients [21]. In addition, the TMB of Patient 1 was chemotherapy, radiotherapy, and/or targeted therapy. half the median TMB reported for 71 cholangiocarcinoma Chemotherapy can increase the immunogenicity of tumour patients, whereas that of Patient 2 was nearly four times cells mainly by regulating the antigenicity and adjuvanticity higher than the median [22]. Moreover, both patients of [24]. Chemotherapy drugs can destroy genes and trigger this study showed weak PD-L1 expression (< 5% of tumour new mutations, thereby increasing antigenicity, although cells), suggesting a weak PD-L1 status. However, IHC these new antigens appear to be expressed at lower levels showed that CD8 T cell infiltration was moderate at 10% in tumours [25]. Chemical agents have been developed that in both patients. A previous report showed that patients trigger immunogenic cell death, resulting in the release of with a higher density of CD8 T cells at the edge of tumour damage-associated molecular patterns to subsequently acti- invasion respond better to treatment [23]; thus, infiltrated vate inherent and adaptive immune responses. Chemother- CD8 T cells may be a prerequisite for effective immuno- apy also depletes regulatory T cells and myeloid-derived therapy. Overall, although these two patients did not show suppressor cells, which may result in the formation of typical characteristics of an immunological benefit such as further hot immune tumours [26]. a high TMB, high MSI, and dMMR, compared with the Trials of the combination of chemotherapy with an median indel rate of 12.77% for ICC patients [22], the indel immune checkpoint blocker (CIT) have been completed rates of these two patients were substantially higher at 48 for patients with non-small cell lung cancer (NSCLC), and 66.87%, respectively. Moreover, the CR lasted for 16 demonstrating that CIT was more effective than chemo- and 13 months, respectively, as of January 2019, consistent therapy alone. In two phase-3 studies (KEYNOTE-189 with previous reports that high indel counts are associated and KEYNOTE-407), pembrolizumab plus pemetrexed with a good response to immunotherapy in melanoma and and platinum-based or carboplatin and taxane chemother- renal cell carcinoma [14, 15]. apy both showed a significantly improved response rate For comparison, we also analysed the cases of an add- (47.6% vs. 18.9% and 58.4 vs. 35.0%) and progression-free itional six ICC patients that received PD-1 treatment, alone survival (PFS) (8.8 months vs. 4.9 months and 6.4 months or in combination, but did not show a CR (Table 1). Pa- vs. 4.8 months, respectively). Nivolumab and atezolizumab tients 4, 5, and 8 were treated with PD-1 blockade alone; combined with chemotherapy also showed positive results. patients 3 and 6 received a combination of PD-1 blockade In the PACIFIC trial, patients with locally advanced unre- and chemotherapy; and patient 7 was treated with a com- sectable NSCLC received chemoradiation followed by bination of PD-1 blockade and lenvatinib. Among the pa- durvalumab, which resulted in significant improvement in tients showing a PR, patient 6 had high-MSI status and median PFS and 18-month PFS rates compared chemora- dMMR, and also showed the highest TMB of 34.84 muta- diation followed by placebo [27]. Moreover, among 14 tions/Mb with high PD-L1 expression (> 90%). By contrast, patients with ICC who failed previous anticancer therapy patient 8 also showed a PR and did not share these charac- that received lenvatinib combined with pembrolizumab or teristics, demonstrating weak PD-L1 expression (< 5%). nivolumab, three patients achieved a PR and the DCR was Among the patients that showed progressive disease (PD), 92.9%. A total of 450 cancer genes and whole exons were patient 5 had high PD-L1 (> 90%) and an inactivated BRAF sequenced in seven patients, revealing four patients with a mutation, which is associated with sensitivity to high TMB greater than 12 mutations/Mb, and one of the immunotherapy. Patients 3 and 7 harboured both of four was MSI-H [28]. immunotherapy-sensitive and -resistant mutations, dem- In the KEYNOTE-189 and KEYNOTE-407 trials, pa- onstrating similar complexity in the mutation profile as de- tients with less than 1% PD-L1 expression also responded termined for Patient 2 (Additional file 1). However, these to CIT, and there was no association of PD-L1 expression mutations were only analysed at the DNA levels, and thus with a clinical benefit [27]. The expression of PD-L1 may transcriptome analysis is necessary for confirmation. be affected by both time fluctuations and intratumoural Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 7 of 8 heterogeneity, so that it is not always associated with Authors’ contributions MS, HW, YLuo, QX, and GW were responsible for WES and the whole-exome a better outcome. Moreover, one ICC patient with a data analysis. TW and XW were responsible for the collection and analysis of low TMB showed tumour shrinkage on a regimen of clinical data. YLi supervised the pathology interpretation of the patient tissue lenvatinib combined with PD-1 blockade [28]. A study samples. YC, XL, and XX took care of patients and communicated with patients. MS and BH were responsible for writing the manuscript. SL was on pancreatic cancer suggested that the quality of a responsible for analysis of data, data interpretation, and revision. All authors tumour antigen could better predict the survival of read and approved the final manuscript. patients after surgery but not the number of tumour Ethics approval and consent to participate antigens [29]. Our present cases also showed that a The two patients provided informed consent. high TMB, MSI-H, and PD-L1 expression cannot completely predict all patients who will benefit from Consent for publication N/A combination immunotherapy. Hence, it is still an un- met need to identify which patients may receive the Competing interests benefit from single or combined immunotherapy. The authors declare that they have no competing interests. Compared with immunotherapy alone, it is more diffi- cult to explore how the combination of conventional Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in therapy might promote immunotherapy and to screen published maps and institutional affiliations. patients that will receive clinical benefits, as biopsy sampling is required, which is not standard in routine Author details Department of Hepatobiliary Surgery, the First Medical Center, Chinese PLA clinical practice. In addition to PD-L1 expression, high General Hospital, Medical School of Chinese PLA, 28 Fuxing Road, Beijing MSI, dMMR, and high TMB which have been approved 2 100853, China. Radiotherapy Department, Chinese PLA General Hospital, by the Food and Drug Administration as biomarkers of Beijing 100853, China. GenomiCare Biotechnology (Shanghai) Co., Ltd., Shanghai 201210, China. immunotherapy [30], our results further suggest that the indel ratio may be associated with a response to PD-1 Received: 5 December 2018 Accepted: 22 April 2019 blockade for ICC patients. However, clinical studies with larger samples are required to validate this association References and understand the underlying mechanism. 1. Mavros MN, Economopoulos KP, Alexiou VG, Pawlik TM. Treatment and prognosis for patients with intrahepatic cholangiocarcinoma: systematic review and meta-analysis. JAMA Surg. 2014;149:565–74. Additional file 2. Wirth TC, Vogel A. Surveillance in cholangiocellular carcinoma. Best Pract Res Clin Gastroenterol. 2016;30:987. 3. Cardinale V, Bragazzi MC, Carpino G, Di Matteo S, Overi D, Nevi L, et al. Additional file 1: List of mutated genes in the eight ICC patients, and Intrahepatic cholangiocarcinoma: review and update. 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Two cases of intrahepatic cholangiocellular carcinoma with high insertion-deletion ratios that achieved a complete response following chemotherapy combined with PD-1 blockade

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Medicine & Public Health; Oncology; Immunology
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Abstract

Background: Insertion–deletion mutations (indels) may generate more tumour-specific neoantigens with high affinity to major histocompatibility complex class I. A high indel ratio is also related to a good response to programmed death-1 (PD-1) checkpoint blockade in melanoma and renal cell carcinoma. However, the correlation between a high indel ratio and the immunotherapy response in intrahepatic cholangiocarcinoma (ICC) is unknown. Case presentation: Two patients with relapsed ICC at stage IIIb were treated with PD-1 blockade combined with chemotherapy. After 7 and 4 months of chemotherapy and PD-1 blockade (3 and 15 cycles, and 5 and 6 cycles, respectively), magnetic resonance imaging and positron emission tomography with computed tomography imaging showed that both patients achieved a complete response (CR), which has lasted up to nearly 16 and 13 months to date, respectively. Whole-exome sequencing and immunohistochemistry analysis showed that both patients had cancers with microsatellite stability (MSS) and mismatch repair (MMR) proficiency, weak PD-L1 expression, and a tumour mutation burden (TMB) of 2.95 and 7.09 mutations/Mb, respectively. Patient 2 had mutations of TP53 and PTEN that are known to confer sensitivity to immunotherapy, and the immunotherapy-resistant mutation JAK2, whereas patient 1 had no known immunotherapy response-related mutations. However, the indel ratios of the two patients (48 and 66.87%) were higher than the median of 12.77% determined in a study of 71 ICC patients. Moreover, comparison to six additional ICC patients who showed a partial response, stable disease, or progressive disease after PD-1 blockade treatment alone or in combination with chemotherapy demonstrated no difference in PD-L1 expression, TMB, MSI, and MMR status from those of the two CR patients, whereas the indel frequency was significantly higher in the CR patients. Conclusions: These two cases suggest that indels might be a new predictor of PD-1 blockade response for ICC patients beside PD-L1 expression, TMB, MSI, and dMMR, warranting further clinical investigation. Keywords: Indel, Intrahepatic cholangiocarcinoma, Combination immunotherapy, PD-1 blockade, Whole-exome sequencing * Correspondence: lsc620213@126.com Department of Hepatobiliary Surgery, the First Medical Center, Chinese PLA General Hospital, Medical School of Chinese PLA, 28 Fuxing Road, Beijing 100853, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 2 of 8 Background showed durable responses with a 40% objective response Intrahepatic cholangiocarcinoma (ICC) is an aggressive rate for ICC [12]. However, these studies did not deter- malignancy with a poor prognosis. After curative resec- mine the molecular characteristics of the ICC patients that tion, the 5-year survival rate and median survival time of showed a clinical benefit from the treatment. Further- ICC patients is 30% and 28 months, respectively [1], and more, the combination of pembrolizumab and chemother- the recurrence rate has been reported to be in the range apy has also shown a good response in a case report of a of 40–80% [2]. Other than surgical resection, standard single ICC patient. This ICC patient had a high tumour treatment options for ICC include liver transplantation, mutation burden (TMB) of 19.3 mutations/Mb, but with a gemcitabine-based chemotherapeutic regimen, and microsatellite instability (MSI) and MMR proficiency loco-regional therapies such as transarterial chemoem- (pMMR) [13]. Thus, identifying the subsets of ICC pa- bolization and conformal external-beam radiation ther- tients that are most likely to benefit from PD1/PD-L1 apy; however, given the poor outcome, more effective blockade alone and in combination remains a challenge treatments are urgently needed. and hindrance to effective personalized medicine. Similar to virus-associated cancers, viral hepatitis and Insertion–deletion mutations (indels) cause frameshift Clonorchis sinensis infection are known risk factors for mutations that not only alter the amino acid compos- ICC [3]. These infections often cause immune exhaustion, ition of the proteins but may also lead to early termin- which is mediated through the programmed cell death ation of protein synthesis. Indels and single-nucleotide 1-ligand 1 (PD-L1)/programmed cell death 1 (PD-1) path- variants (SNVs) together determine the TMB. Notably, way, similar to the immunosuppressive mechanism of indels could produce more than three times the neoanti- cancer [4]. Indeed, several studies have shown that PD1/ gens with high affinity to major histocompatibility com- PD-L1 blockade can be effective in the treatment of plex class I (MHC-I) (IC50 < 50 nM), and nine times the virus-related cancers [5]. Moreover, a study including 27 mutation-specific neoantigens compared with SNVs. This patients with ICC showed that 100, 30, and 41% of the high neoantigen load induced by indels was associated cases had infiltrated lymphocytes, positive PD-L1 expres- with HLA I presentation, CD8 T cell activation, and sion, and positive human leukocyte antigen class I antigen increased cytolytic activity compared with the high (HLA I) expression, respectively [6]. In another study, 39 SNV neoantigen group [14]. Consequently, the indel of 54 patients with ICC were found to be positive for count has been significantly associated with a response PD-L1 expression within the tumour front using immuno- to a checkpoint inhibitor across three separate melan- histochemistry (IHC), and the overall survival of these oma cohorts and in patients with advanced renal cell patients was reduced by approximately 60% compared carcinoma [14, 15]. However, there are limited data on with that of patients without PD-L1 expression [7]. Simi- the ability of indels to predict a response to PD-1/ larly, 260 patients with biliary tract cancer (BTC) that had PD-L1 blockers alone and in combination. Here, we re- a relatively poor prognosis showed higher PD-L1 expres- port two patients with ICC with high indel ratios who sion [8]. Collectively, these studies provide a biological ra- were successfully treated with PD-1 blockers plus tionale for the treatment of ICC patients with PD1/PD-L1 chemotherapy, both of whom showed weak PD-L1 ex- blockade. pression and a microsatellite stable (MSS) status, and More importantly, clinical trials have demonstrated a without dMMR. Moreover, one of the patients had a benefit of PD-1/PD-L1 blockers for patients with ICC. relatively low TMB. Comparison of the features of these The PD-L1 positive BTC cohort of the KEYNOTE-028 patients to others reported in the literature, as well as basket trial showed that four of 24 patients who were to six additional cases of ICC that received PD-L1 ther- PD-L1 positive showed a partial response (PR), and four apywithout aCR, points to apotential roleof indels as patients had stable disease (SD) [9]. Another basket trial a key factor determining the response to therapy, war- including four patients with DNA mismatch repair defi- ranting further consideration. ciency (dMMR) cholangiocarcinoma demonstrated that one patient had a complete response (CR) and the other Case presentation A (Patient 1) three patients had SD after PD-1 blockade therapy [10]. Patient 1 (Fig. 1 and Table 1) is a 50-year-old male with Further, three recent studies reported encouraging clin- moderately differentiated ICC staged at IIIb. He was ical outcomes. In a Phase II study on patients with admitted to the hospital in January 2016 due to upper advanced refractory BTC treated with nivolumab, 17% of abdominal pain. He had a history of hepatitis B for 10 the 29 patients achieved a PR, 38% showed SD, and years, and his Child-Pugh class was A. Magnetic reson- there was an overall 55% disease control rate (DCR). No ance imaging (MRI) revealed a mass in the left outer grade IV or V toxicities were reported [11]. Asian BTC lobe, which grew outward and invaded the diaphragm. patients that received M7824 after chemotherapy failure, The tumour marker carcinoembryonic antigen was ele- which targets PD-1 and transforming growth factor-β, vated at 10.14 μg/L. He underwent left hemihepatectomy Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 3 of 8 Fig. 1 Imaging of patient 1 over the course of therapy. a showed timeline of the clinical course. Representative micrographs of PD-L1 expression + + (b) and percentage of CD8 T cells (c) within tumor (original magnification ×400). The positive rate of PD-L1 and CD8 T cells were < 5% and 10%, respectively. MRI and PET-CT imaging (d) showed the lesion around the margin and enlarged lymph nodes disappeared after treatment with pembrolizumab in combination with tegafur on September 14, 2017. The CR was sustained up to 9 January 2019 and hepatoduodenal ligament skeletonization on February determine the presence of SNVs, indels, the TMB, 16, 2016. The tumour was 11 cm × 9 cm × 6 cm, and no copy number variations (CNVs), MSI status, and lymph node metastases were found. Intraoperative radio- dMMR by bioinformatics methods. The TMB was de- therapy was performed on the liver section using 9-mV termined to be 2.95 mutations/Mb, and a total of 25 photon beams with a single dose of 12 Gy, which could non-synonymous mutations (NSMs) were detected in eliminate the residual tumour due to invasion of the dia- the whole genome, including 12 indels and 13 SNVs. phragm and the venous root of the liver. The tumour was This patient harboured only one clinically actionable found to be positive for cytokeratin 18 (CK18) and was mutation in FGF4, which amplified to reveal a copy negative for Arg-1, hepatocyte, glypican-3 (GPC-3), and number of 3.64. No SNVs were detected in MLH1, CK7 in IHC analysis. The tumour proportion score (TPS) MSH2, MSH6,and PMS2 (Additional file 1), suggest- of the PD-L1 expression level was < 5% determined using ing pMMR, and the MSI was 0.01%. monoclonal mouse anti-human PD-L1 clone (22C3) anti- The left hepatic lobe lesions and retroperitoneal left body by allred criteria, and the frequency of infiltrating lymph nodes were treated with Cyberknife (52 Gy/4 F for CD8 T cells was 10%. 4 days) on February 4, 2017 and February 9, 2017, Liver resection margin recurrence and abdominal respectively. Tegafur and pembrolizumab were initiated lymph node metastasis were detected using MRI and 9 days apart. Tegafur chemotherapy was started at 40 mg positron emission tomography-computed tomography twice a day every 3 weeks but was withdrawn due to (PET-CT) after 11 months. MRI showed a marginal le- development of thrombocytopenia and leukopenia after sion of 4 × 1.5 cm in the left lobe of the liver, along with three cycles. Pembrolizumab was administered at 150 mg an enlarged hepatic hilar (1.6 × 1.5 cm) and retroperiton- every 3 weeks for 15 cycles for about 1 year. On June 6, eal lymph nodes (5.2 × 3 cm and 2.8 × 2.6 cm). PET-CT 2017, the abdominal MRI showed that most of the lesions scans also revealed abnormal hypermetabolic lesions in in the margin, and all the lesions in the hilar and retro- these locations. peritoneal lymph nodes had disappeared. It was specu- Whole-exome sequencing (WES) was applied to the lated that the residual lesions at the margin might tissue resected after surgery, and the data were used to represent a surgical reaction. The patient was deemed to Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 4 of 8 Table 1 Baseline, mutation characteristics, treatment, and outcomes Patient Reference 12 3 4 5 6 7 8 Baseline characteristics Age (years) 50 67 60 51 65 59 55 60 Sex male male female female female male male male Surgery Yes Yes Yes No Yes Yes No Yes Stage IIIb IIIb IIIb IIIb IIIb IIIb IV IIIb Lymph node metastasis + – ++ + ++ + Virus infection HBV NHVI NHVI NHVI NHVI NHVI HCV HBV Tumour PD-L1 < 5% < 5% < 1% unknown > 90% > 90% unknown < 5% expression level (%) Percentage of CD8 T cells 10% 10% 10% unknown 20% 20% unknown 30% Mutation characteristics TMB (mutations/Mb) 2.95 7.09 5.62 4.18 8.47 34.84 5.38 6.01 7.4 (median) [21] TMB (NSM) 25 163 114 66 209 1088 106 127 47 (median) [22] Number of SNVs 13 54 85 52 173 771 82 105 41 (median) [22] Number of indels 12 109 29 14 36 317 24 22 6 (median) [22] indels ratio (%) 48.00% 66.87% 25.44% 21.21% 17.22% 29.16% 22.64% 17.32% 12.77% (median) [22] MSI-H/MSS MSS MSS MSS MSS MSS MSI-H MSS MSS 3.2% MSI-H [20] dMMR/pMMR pMMR pMMR pMMR pMMR pMMR dMMR pMMR pMMR 2% dMMR [10] Percentage of CNVs (%) 3.1 4.2 9.29 2.66 4.75 0.78 3.67 5.92 Mutations conferring NA TP53/PTEN KRAS NA BRAF/GITR MLH1/GITR TP53 TP53 sensitivity to immunotherapy Mutations conferring JAK2 B2M chromosome 11q13 resistance to immunotherapy Treatment Therapeutic regimen (cycles) tegafur (3) + tegafur (5, but irregular) nivolumab (4) + nivolumab nivolumab nivolumab (8) + nivolumab (8) + pembrolizumab (6) pembrolizumab + pembrolizumab (6) cisplatin (1) + (15) (8) cisplatin (1) + lenvatinib (8 months (15) + cyberknife (2) gemcitabine (1) gemcitabine (1) continuously) Outcome Type of response CR CR PD SD PD PR PD PR PFS (months) 16 13 Unknown 7 5 3 Unknown 10 The whole exons of tumour tissues removed during surgery and matched white blood cells were sequenced by the Illumina NovaSeq 6000 Sequencing System with average sequence coverage of 200X. We calculated SNVs, indels, and copy number variations (CNVs) by the mutect2 algorithm, and CNVnator software, respectively. MSI was identified using msisensor, and dMMR was identified by analysing SNVs and indels in MLH1, MSH2, MSH6, and PMS2 Abbreviations: CNG copy number gain, CNL copy number loss, NHVI no hepatitis virus infection Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 5 of 8 show a CR on September 14, 2017 and was still in remis- ARID2 are included in the COSMIC top 20 mutated genes sion at the last follow-up of January 10, 2019 based on (Additional file 1). The TMB was 7.09 mutations/Mb, in- PET-CT analysis. No side effects of pembrolizumab were cluding 109 indels (66.87%) and 54 SNVs. The tumour ex- observed. hibitedpMMR andMSI (0.01%). He wasstarted on a regimen of tegafur and pembrolizumab in late August. Un- Case presentation B (Patient 2) fortunately, he experienced the common adverse event to Patient 2 (Fig. 2 and Table 1) is a 67-year-old male with tegafur of pruritus, determined to be of grade 2 according to no hepatitis virus infection. He underwent extended right the standard CTCAE5.0 criteria. After withdrawing tegafur, hemihepatectomy, left hepaticojejunostomy, perihepatic the pruritus disappeared, and the drug was thus switched to lymphadenectomy, and portal vein reconstruction on May an irregular administration schedule as of December 23, 16, 2017. The tumour measured 7.6 cm × 7 cm × 7 cm, with 2017 with an increase in the dose from 40 mg to 60 mg and nerve invasion accompanied by microvascular invasion. No to be taken twice a day until the beginning of February tumour was found in the liver margin and bile duct margin 2018. Pembrolizumab was administrated intravenously (150 after the surgery. Lymphatic metastasis was detected in mg every 3 weeks for six cycles). PET-CT scans revealed that groups 8 and 12A. IHC showed Arg-1 (−), CK18 (+), GPC-3 the enlarged lymph nodes had disappeared. This CR was (−), hepatocyte (−), Ki-67 positivity of 65%, and CK19 (+). achieved in less than 4 months, and the patient continued to Accordingly, he was diagnosed with iCCA stage IIIb. Similar be in remission for 13 months up to the last follow-up. to Patient 1, PD-L1 expression was detected on < 5% of the tumour cells, and the percentage of CD8 T cells was 10%. Discussion Lymph node metastasis in the hepatoportal area was de- This is the first report demonstrating two ICC patients tected using MRI and PET-CT on August 16, 2017. WES with high indel rates that achieved a CR after PD-1 revealed 163 NSMs, including clinically actionable alter- blockade combined with chemotherapy. We also analysed ations in PTEN and TP53. In addition, TP53, SMAD4,and the presence of genomic alterations known to be involved Fig. 2 a Timeline of the clinical course in patient 2. The positive rate of PD-L1 (b) and CD8 T cells (c) in patient 2 were < 5% and 10% evaluated by IHC, respectively (magnification ×400). Patient 2 had a complete metabolic response after treatment with pembrolizumab in combination with tegafur, with no residual hypermetabolic uptake on post-treatment imaging (d) Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 6 of 8 in the response and resistance to immunotherapy using Compared with the PR, SD, and PD groups of these six WES [16–18]. Patient 2 harboured three such mutations, additional patients, Patient 1 had the lowest TMB and Pa- including TP53 and PTEN inactivating mutants, which tient 2 had a TMB that was not significantly different from can induce a strong potential response, and JAK2 muta- that of the other patients. However, the indel rate of the tions, which have been associated with immune therapy CR groups was higher than that of the other groups, and resistance. However, Patient 1 did not have any related was significantly different from that reported previously mutations. Both patients had an MSI frequency of 0.01%, [22](P = 0.001 and P < 0.001, respectively). which is well within the spectrum of MSS tumours Immunotherapy has achieved great success, with 10– [19, 20], and also showed a pMMR molecular phenotype. 35% of patients showing a response to single immune The median TMB of patients with ICC varies among checkpoint blockade inhibitors [24]. Recently, more clin- studies. However, the TMB levels of Patients 1 and 2 ical effort has focused on combination immunotherapy were lower or equal to the median reported for 69 Chinese such as immune checkpoint inhibitors combined with ICC patients [21]. In addition, the TMB of Patient 1 was chemotherapy, radiotherapy, and/or targeted therapy. half the median TMB reported for 71 cholangiocarcinoma Chemotherapy can increase the immunogenicity of tumour patients, whereas that of Patient 2 was nearly four times cells mainly by regulating the antigenicity and adjuvanticity higher than the median [22]. Moreover, both patients of [24]. Chemotherapy drugs can destroy genes and trigger this study showed weak PD-L1 expression (< 5% of tumour new mutations, thereby increasing antigenicity, although cells), suggesting a weak PD-L1 status. However, IHC these new antigens appear to be expressed at lower levels showed that CD8 T cell infiltration was moderate at 10% in tumours [25]. Chemical agents have been developed that in both patients. A previous report showed that patients trigger immunogenic cell death, resulting in the release of with a higher density of CD8 T cells at the edge of tumour damage-associated molecular patterns to subsequently acti- invasion respond better to treatment [23]; thus, infiltrated vate inherent and adaptive immune responses. Chemother- CD8 T cells may be a prerequisite for effective immuno- apy also depletes regulatory T cells and myeloid-derived therapy. Overall, although these two patients did not show suppressor cells, which may result in the formation of typical characteristics of an immunological benefit such as further hot immune tumours [26]. a high TMB, high MSI, and dMMR, compared with the Trials of the combination of chemotherapy with an median indel rate of 12.77% for ICC patients [22], the indel immune checkpoint blocker (CIT) have been completed rates of these two patients were substantially higher at 48 for patients with non-small cell lung cancer (NSCLC), and 66.87%, respectively. Moreover, the CR lasted for 16 demonstrating that CIT was more effective than chemo- and 13 months, respectively, as of January 2019, consistent therapy alone. In two phase-3 studies (KEYNOTE-189 with previous reports that high indel counts are associated and KEYNOTE-407), pembrolizumab plus pemetrexed with a good response to immunotherapy in melanoma and and platinum-based or carboplatin and taxane chemother- renal cell carcinoma [14, 15]. apy both showed a significantly improved response rate For comparison, we also analysed the cases of an add- (47.6% vs. 18.9% and 58.4 vs. 35.0%) and progression-free itional six ICC patients that received PD-1 treatment, alone survival (PFS) (8.8 months vs. 4.9 months and 6.4 months or in combination, but did not show a CR (Table 1). Pa- vs. 4.8 months, respectively). Nivolumab and atezolizumab tients 4, 5, and 8 were treated with PD-1 blockade alone; combined with chemotherapy also showed positive results. patients 3 and 6 received a combination of PD-1 blockade In the PACIFIC trial, patients with locally advanced unre- and chemotherapy; and patient 7 was treated with a com- sectable NSCLC received chemoradiation followed by bination of PD-1 blockade and lenvatinib. Among the pa- durvalumab, which resulted in significant improvement in tients showing a PR, patient 6 had high-MSI status and median PFS and 18-month PFS rates compared chemora- dMMR, and also showed the highest TMB of 34.84 muta- diation followed by placebo [27]. Moreover, among 14 tions/Mb with high PD-L1 expression (> 90%). By contrast, patients with ICC who failed previous anticancer therapy patient 8 also showed a PR and did not share these charac- that received lenvatinib combined with pembrolizumab or teristics, demonstrating weak PD-L1 expression (< 5%). nivolumab, three patients achieved a PR and the DCR was Among the patients that showed progressive disease (PD), 92.9%. A total of 450 cancer genes and whole exons were patient 5 had high PD-L1 (> 90%) and an inactivated BRAF sequenced in seven patients, revealing four patients with a mutation, which is associated with sensitivity to high TMB greater than 12 mutations/Mb, and one of the immunotherapy. Patients 3 and 7 harboured both of four was MSI-H [28]. immunotherapy-sensitive and -resistant mutations, dem- In the KEYNOTE-189 and KEYNOTE-407 trials, pa- onstrating similar complexity in the mutation profile as de- tients with less than 1% PD-L1 expression also responded termined for Patient 2 (Additional file 1). However, these to CIT, and there was no association of PD-L1 expression mutations were only analysed at the DNA levels, and thus with a clinical benefit [27]. The expression of PD-L1 may transcriptome analysis is necessary for confirmation. be affected by both time fluctuations and intratumoural Sui et al. Journal for ImmunoTherapy of Cancer (2019) 7:125 Page 7 of 8 heterogeneity, so that it is not always associated with Authors’ contributions MS, HW, YLuo, QX, and GW were responsible for WES and the whole-exome a better outcome. Moreover, one ICC patient with a data analysis. TW and XW were responsible for the collection and analysis of low TMB showed tumour shrinkage on a regimen of clinical data. YLi supervised the pathology interpretation of the patient tissue lenvatinib combined with PD-1 blockade [28]. A study samples. YC, XL, and XX took care of patients and communicated with patients. MS and BH were responsible for writing the manuscript. SL was on pancreatic cancer suggested that the quality of a responsible for analysis of data, data interpretation, and revision. All authors tumour antigen could better predict the survival of read and approved the final manuscript. patients after surgery but not the number of tumour Ethics approval and consent to participate antigens [29]. Our present cases also showed that a The two patients provided informed consent. high TMB, MSI-H, and PD-L1 expression cannot completely predict all patients who will benefit from Consent for publication N/A combination immunotherapy. Hence, it is still an un- met need to identify which patients may receive the Competing interests benefit from single or combined immunotherapy. The authors declare that they have no competing interests. Compared with immunotherapy alone, it is more diffi- cult to explore how the combination of conventional Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in therapy might promote immunotherapy and to screen published maps and institutional affiliations. patients that will receive clinical benefits, as biopsy sampling is required, which is not standard in routine Author details Department of Hepatobiliary Surgery, the First Medical Center, Chinese PLA clinical practice. In addition to PD-L1 expression, high General Hospital, Medical School of Chinese PLA, 28 Fuxing Road, Beijing MSI, dMMR, and high TMB which have been approved 2 100853, China. Radiotherapy Department, Chinese PLA General Hospital, by the Food and Drug Administration as biomarkers of Beijing 100853, China. GenomiCare Biotechnology (Shanghai) Co., Ltd., Shanghai 201210, China. immunotherapy [30], our results further suggest that the indel ratio may be associated with a response to PD-1 Received: 5 December 2018 Accepted: 22 April 2019 blockade for ICC patients. However, clinical studies with larger samples are required to validate this association References and understand the underlying mechanism. 1. Mavros MN, Economopoulos KP, Alexiou VG, Pawlik TM. Treatment and prognosis for patients with intrahepatic cholangiocarcinoma: systematic review and meta-analysis. JAMA Surg. 2014;149:565–74. Additional file 2. Wirth TC, Vogel A. Surveillance in cholangiocellular carcinoma. Best Pract Res Clin Gastroenterol. 2016;30:987. 3. Cardinale V, Bragazzi MC, Carpino G, Di Matteo S, Overi D, Nevi L, et al. Additional file 1: List of mutated genes in the eight ICC patients, and Intrahepatic cholangiocarcinoma: review and update. 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FDA: Food and Drug Administration; ICC: Intrahepatic cholangiocarcinoma; 7. Gani F, Nagarajan N, Kim Y, Zhu Q, Luan L, Bhaijjee F, et al. Program death 1 IHC: Immunohistochemistry; Indels: Insertion–deletion mutations; MHC- immune checkpoint and tumor microenvironment: implications for patients I: Major histocompatibility complex class I; MRI: Magnetic resonance imaging; with intrahepatic cholangiocarcinoma. Ann Surg Oncol. 2016;23:2610–7. MSI-H: High microsatellite instability; MSS: Microsatellite stable; NSCLC: Non- 8. Nakamura H, Arai Y, Totoki Y, Shirota T, Elzawahry A, Kato M, et al. Genomic small cell lung cancer; NSMs: Nonsynonymous mutations; PD-1: Programmed spectra of biliary tract cancer. Nat Genet. 2015;47:1003. cell death protein 1; PD-L1: Programmed cell death 1-ligand 1; PET– 9. 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Journal for ImmunoTherapy of Cancer – Springer Journals

Published: May 7, 2019

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