Genetic analysis in the clinical management of biliary tract cancer

Abstract Biliary tract cancer (BTC) is clinically and pathologically heterogeneous and responds inadequately to treatment. A small section of patients develop resectable disease, although the relapse rates are high; the benefits of adjuvant capecitabine chemotherapy for BTC are now understood, and gemcitabine‐based combination chemotherapy is the first line of therapeutic strategy for BTC; however, alternative therapy for BTC is not known. Genomic profiling can provide detailed information regarding the carcinogenesis, identification, and therapy for BTC. Currently, confirmed restorative targets for BTC are lacking. In this review, we aimed to analyze the preclinical and clinical implications of a spectrum of genomic alterations associated with new potentially remedial targets. We focused on eight draggable genes for BTC, which were described as having evidence of therapeutic impact (evidence level 2A‐3B) based on the clinical practice guidance for next‐generation sequencing in cancer diagnosis and treatment; these include ERBB2, NTRK1, RNF43, CDK6, CDKN2B, FGFR2, IDH1, and IDH2. Moreover, some of the BTC present microsatellite instability, hypermutation, and germline variants, which we also reviewed. Finally, we discussed the therapeutic options based on the next‐generation sequencing findings in BTC. Studies have demonstrated that BTC includes subgroups with individually distinct driver mutations, most of which will be targeted with new treatment plans.


| INTRODUC TI ON
Biliary tract cancers (BTCs) include intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and gallbladder carcinoma. BTC occurs in the epithelial cellular lining of the bile duct and may appear at specific anatomical regions (intrahepatic, extrahepatic, and gallbladder). Although BTC is predominant in East and South Asian countries and in certain regions of South America, the worldwide prevalence of BTC is increasing rapidly. 1,2 Studies show that BTC is not a single distinct disorder, but consists of several diseases with specific demographics, molecular features, and treatment options.
Intrahepatic cholangiocarcinoma is the second-most typical major hepatic malignancy, with an increasing global incidence, especially in the European hemisphere; this is perhaps because of the elevated percentage of overweight individuals and incidence of hepatitis C infection. [3][4][5][6][7][8][9] In Asia, intrahepatic cholangiocarcinoma is principally associated with parasitic infections. [3][4][5][6][7][8][9] The majority of extrahepatic cholangiocarcinoma occurs within the hepatic duct bifurcation; in 30% of cases, the disease develops within the distal common bile duct and occurs with pain-free jaundice. 7 Gallbladder carcinoma is undoubtedly an unusual disease linked to cholecystitis, cholelithiasis, and obesity. 7 Although surgical resection is the most accepted treatment method for treating these tumors, the tumors are inoperable for a significant proportion of patients. 7,[10][11][12] For patients with unresectable advanced/recurrent BTC, gemcitabine-based combination chemotherapy is the first line of therapeutic strategy, such as gemcitabine plus cisplatin, 13,14 gemcitabine plus S-1, 15 and gemcitabine/cisplatin/S-1 combination chemotherapy. 16,17 The efficacy of gemcitabine-based combination chemotherapy on survival is encouraging; the median overall survival following these therapies has been reported to range from 11.2 to 16.2 months. [13][14][15][16][17] On the other hand, no scientific molecular markers for earlier medical diagnosis have been identified, and effective specific molecular therapies are unavailable; consequently, the 5-year survival rate is extremely minimal (10% for cholangiocarcinoma and <5% for gallbladder carcinoma). 4,9,18 Hence, understanding the molecular features of this disease might assist in developing targeted therapeutics. 19,20 Patients with tumors developing in the vicinity of bile ducts present with biliary obstruction because of regional infiltration in the biliary tract. A small section of patients can probably be identi-

| G ENE TI C ALTER ATI ON S IN B ILIARY TR AC T C AN CER
Advancements in molecular biology have resulted in the recognition of numerous gene irregularities. Gene panel testing and the efficient use of genomic mutation analysis via next-generation sequencing (NGS) or related approaches competent at synchronized recognition of multiple genomic mutations are utilized for elucidating cancer-associated genomic mutation(s) in specific individuals and for designing the most suitable customized treatment. An average test panel addresses gene history, which is considered to be beneficial for forecasting responses to medication and prognosis, resulting in conclusive medical diagnosis. These panels can concurrently display a large number of transcripts and offer an array of information that uncovers genomic variations, which include gene mutations, deletions, insertions, gene fusions, and duplications. In addition, the extent to which information is integrated in gene panel tests is controlled by the innovations in diagnostic and treatment strategies. 21 In 2017, Valle et al 22

| H UMAN EPIDERMAL G ROW TH FAC TOR RECEP TOR (H ER) FAMILY AND ERB B2 AMPLIFI C ATI ON
Evidence indicates that HER2 might be used as a novel restorative target in patients with BTCs. 23 The frequency of HER2 amplification or overexpression is noted in roughly 4%-28.6% of GBC, 19,23-28 4%-11% of extrahepatic cholangiocarcinoma, [26][27][28] and 0.6%-5% of intrahepatic cholangiocarcinoma. [26][27][28] Previous reports show that the principal site of occurrence of HER2-positive BTC varies; the ratio of HER2 3+ in immunohistochemistry was at its maximum in GBCs, followed by that in extrahepatic cholangiocarcinoma. 27,29 In addition, recent genomic research has shown how mutational information is BTC -specific, as indicated by their principal sites of occurrence or etiological factors. 19,[30][31][32] Studies show that BTC pathogenesis might be unique for these variables, and therefore, various therapeutic approaches might be required, which will depend on patient-specific medical details, as well as the outcomes of inherited or molecular profiling. 23 Studies (using immunohistochemistry) have not been able to illustrate the prognostic effect of HER2 overexpression in patients with BTC because of comparatively smaller sample size and heterogeneity of individual attributes. 23,33,34 Some other studies have indicated that HER2 overexpression is associated with poor prognosis of patients without any targeted therapies, and that these patients might benefit from targeting the HER2 signaling pathway. 35,36 Trastuzumab is a monoclonal antibody targeting HER2. Certain in BTC who were treated with HER2-targeted therapy (trastuzumab and pertuzumab); two patients showed partial response, while the other three patients had stable disease beyond 120 days. 40 In addition, the SUMMIT trial using the pan-HER kinase inhibitor neratinib included nine patients with HER2-mutated BTC; the objective response rate at week 8 was 22.2% and clinical benefit (stable disease or partial response lasting at least 24 weeks) rate was 33.3%. 41 (Table 1).

| T YROS INE RECEP TOR K INA S E (TRK ) FUS I ON G ENE
Based on scientific evidence, the entire response rate of larotrectinib in TRK fusion-positive tumor types was nearly 80% (95% CI, 67-90), irrespective of the tumor type. 42 Studies show that specific mutations can be created to cope with the acquired mutations in the kinase domain; for instance, LOXO-195 is presently being assessed in adults and children in a phase I-II study. 42 Larotrectinib-related adverse events that resulted in dose reductions were unusual in this study; in a study of 55 patients with TRK fusion-positive cancer, therapy was not halted for any of the patients due to drug-related unwanted effects. 42 Larotrectinib had noticeable and durable anti-tumor action in patients with TRK fusion-positive cancers, irrespective of the chronological age of the affected person or tumor variety. Long-lasting responses were noticed irrespective of patient age, tumor tissue, and the position of fusion. 42 Long-term management with larotrectinib is possible for patients with minimal side-effects. 42 Nevertheless, another study regarding extended follow-up of a larger patient cohort indicated TA B L E 1 Draggable genes with therapeutic impact for biliary tract cancer partly cited from the clinical practice guidance for nextgeneration sequencing in cancer diagnosis and treatment (Edition 1.0) 21 that experience may offer additional comprehension of the safety profile of this agent.

| FIB ROB L A S T G ROW TH FAC TOR RECEP TOR (FG FR) FUS I ON G ENE
In a recent study on whole exome and transcriptome sequencing,

| ISOCITR ATE DEHYDROG ENA S E (IDH) ALTER ATI ON S
Mutations in the genes encoding isocitrate dehydrogenase (IDH1 and IDH2) are observed more regularly in noninfectious cholangiocarcinomas. 52 IDH1 and IDH2 mutations were also identified (19%) in the Johns Hopkins group. 52 These mutations were grouped in formerly recognized hot spots (codons 132 and 172) and were related to poor prognosis. 53,54 These variations in analysis could be because of differences in sample size and in the basic features of these two studies. 53

| MI CROSATELLITE IN S TAB ILIT Y-HI G H (MS I -H) & HYPERMUTATI ON
Mutational load has been shown to be elevated in tumors that can be effectively eradicated using immunotherapies, for instance, in melanoma and lung cancer. 56 For example, therapy using checkpoint inhibitors in tumors with mismatch repair deficiency was shown to be effective in a phase II study, attaining approximately 40% of the target results. 57 Mutational load is high in BTCs. 57

| G ERMLINE VARIANTS IN B ILIARY TR AC T C AN CER
The genetic attributes of BTC are not completely understood, and its molecular profiles are heterogeneous. Large sample sizes are required for extensive evaluation of the molecular basis of BTC. Individuals with germline mutations in breast cancer gene 2 (BRCA2) are at high risk of BTC as well as of pancreatic cancer. 64 In a recent study involving 412 BTC samples from Japanese and Italian populations, 32 frequently mutated genes, including a novel deletion of MUC17 at 7q22.1, were recognized, some of which adversely affected clinical prognosis. 65 The other significantly and commonly mutated genes included TP53, KRAS, SMAD4, NF1, ARID1A, PBRM1, and ATR, some of which negatively affected patient prognosis. 65 Notably, they observed that at least 11% of BTC cases had deleterious germline mutations in cancer-predisposing genes. 65 Zou et al 55  A study reported several changes in FGFR2, such as three-gene fusions, while another report identified FGFR2 fusions in primary hepatic cholangiocarcinoma. 49 In particular, the most common variations were within ARID1A (36%), IDH1/2 (36%), and TP53 (36%), in addition to the amplification of MCL1 (21%). 49 Nearly 66% of patients within this study harbored genomic changes, which might be associated with targeted treatments, and, therefore, therapy options can be possibly customdesigned for individual patients. 49 In In addition, a repeated gene fusion of ETV6 and NTRK3 (ETV6-NTRK3) has been defined in congenital fibrosarcoma. 66

| K R A S -B R AF-MEK-ERK PATHWAY
As with several types of cancer, the RAS-RAF-MEK-ERK signal transduction pathway is often dysregulated in cholangiocarcinoma. 70 Epidermal growth factor (EGF) and platelet-derived growth

| CON CLUS IONS
The treatment options for patients with advanced BTC are improving; owing to intercontinental cooperation toward understanding and treating BTC, the latter can no longer be considered "rare diseases." Studies have demonstrated that BTC includes subgroups with individually distinct driver mutations, most of which can be targeted with new treatment plans. Systemic treatment plans, such as targeted therapies and immunotherapy for BTC, is improving rapidly.
In addition, development of numerous pathway-targeted therapies, together with modulation of the immune environment, provides assurance to patients with these disorders. For decreasing the incidence of BTC, robust specialized medical advancement, along with basic and translation analysis, is required. Identification of inherited driver mutations and translational research are required for providing a distinct opinion regarding the past, present, and future of BTCs.

D I SCLOS U R E
Funding: Authors declare no financial support for this article.