Biomarker Consortium

GI Cancer

Biliary Tract Cancer

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF)

 gene belongs to the RAS-RAF-MEK-ERK signal transduction pathway, also called the MAPK pathway, and is involved in cell growth, differentiation, and proliferation.

 Proteins within the MAPK pathway are predominantly serine/threonine kinases that propagate the transduction pathway or activate downstream signal proteins for metabolism, mitosis, motility, and apoptosis.

 The 3 RAF proteins in humans are ARAF, BRAF, and CRAF; ARAF and CRAF require additional phosphorylation for full activity; subsequently, BRAF is the most active in humans.

 mutation is V600E, which flips the protein into a constitutively active conformation.

The mutated V600E protein stimulates ERK signaling in vivo.5 Other mutations cause reduced kinase activity, which activates wild-type CRAF and results in the same outcome of increased ERK signaling and tumorigenesis.

 V600E mutation status does not appear to correlate to survivability in biliary tract cancer (BTC).

 However, the power of studies to date has been low, so this conclusion may have limited applicability in light of future research.

 mutations appear exclusive to cholangiocarcinomas (CCAs), indicating that testing may provide nonspecific diagnostic support.

 Most intrahepatic CCAs are detected after the tumor is unresectable.

 mutations provides an additional treatment option prior to metastasis.

Experts recommend BRAF testing for patients with unresectable or metastatic gallbladder cancer, intrahepatic CCA, and extrahepatic CCA.1 Common testing modalities are next-generation sequencing (NGS) or polymerase chain reaction (PCR) testing of tumor tissue.1 NGS can also be used on cell-free DNA to detect 

BTCs are rare; they account for 3% of gastrointestinal cancers in adults, and 

 mutations account for approximately 1% to 5% of these.

 Overall prognosis is poor, with a 5-year survival of 5% to15%.

The first and currently only FDA-approved treatment for subsequent-line therapy for 

mutated BTC is dabrafenib with trametinib.

Dabrafenib is an inhibitor of mutated BRAF proteins, which are constitutively active; they stimulate tumor cell proliferation via the MAPK pathway.

 Trametinib is a reversible inhibitor of mitogen-activated protein kinase kinase 1 (MEK1) and MEK2.3 MEK proteins are upstream regulators of the MAPK pathway.

 Tumor cells that initially respond to dabrafenib can develop resistance via the MEK protein, providing the rationale for combination therapy.

Learn more about Dabrafenib and Trametinib >

NCCN. Clinical Practice Guidelines in Oncology. Biliary tract cancer, version 1.2024. Accessed March 9, 2024. 

https://www.nccn.org/professionals/physician_gls/pdf/btc.pdf

Tafinlar. Prescribing information. Novartis; 2024. Accessed March 11, 2024. 

https://www.novartis.com/us-en/sites/novartis_us/files/tafinlar.pdf

Mekinist. Prescribing information. Novartis; 2024. Accessed March 11, 2024. 

https://www.novartis.com/us-en/sites/novartis_us/files/mekinist.pdf

Winstead E. FDA approves dabrafenib–trametinib for BRAF-positive cancers. National Cancer Institute. July 21, 2022. Accessed March 9, 2024. 

https://www.cancer.gov/news-events/cancer-currents-blog/2022/fda-dabrafenib-trametinib-braf-solid-tumors

Wan PTC, Garnett MJ, Roe SM, et al; Cancer Genome Project. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. 

 2004;116(6):855-867. doi:10.1016/s0092-8674(04)00215-6

Cargnello M, Roux PP. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. 

 2011;75(1):50-83. doi:10.1128/MMBR.00031-10

Goeppert B, Frauenschuh L, Renner M, et al. BRAF V600E-specific immunohistochemistry reveals low mutation rates in biliary tract cancer and restriction to intrahepatic cholangiocarcinoma. 

 2014;27(7):1028-1034. doi:10.1038/modpathol.2013.206

Lamarca A, Barriuso J, McNamara MG, Valle JW. Molecular targeted therapies: Ready for “prime time” in biliary tract cancer. 

 2020;73(1):170-185. doi:10.1016/j.jhep.2020.03.007

BRAF V600E

IDH1

Neurotrophic tropomyosin receptor kinase (NTRK)

 genes encode for tropomyosin receptor kinases (TRKs) that attach to cell membranes and are involved in normal nervous system development.

Under normal circumstances, neurotrophins activate TRK receptors, which lead to downstream ERK signaling, cellular proliferation, and growth.

This receptor family has 3 known members: TRKA, TRKB, and TRKC.2 Their respective endogenous ligands are nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3, 4, and 5.

Other pathways implicated in broad NTRK signaling include mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/AKT.

 gene fusions are a driver gene for tumorigenesis.

Oncogenesis occurs via hybrid gene fusions that result in the rearrangement of NTRK sequences to attach to constitutively activated genes.

 gene fuses with an N-terminal target, which results in an overexpression of the kinase domain of kinase function by the chimeric protein.

 gene fusions in solid tumors is approximately 1% but is more prevalent in certain rare tumors.

 They are present in less than 1% of BTC, and the rarity of these oncogenes limits our ability to form precise incidence estimates.

 gene fusions are found in multiple unresectable or metastatic biliary tract cancers including gallbladder malignancies, intrahepatic cholangiocarcinomas (CCAs), and extrahepatic CCAs.

 Testing for these gene fusions is recommended for these 3 types of biliary tract cancer (BTC).

 fusions, National Comprehensive Cancer Network guidelines recommend multigene next-generation sequencing (NGS), preferably using a transcriptome-based approach.

While immunohistochemistry, fluorescence in situ hybridization (FISH), and reverse transcriptase–chain polymerase reaction have been used to detect 

 fusions, these methods are not mentioned in the guidelines for BTC.

Two drugs have been approved by the FDA for the treatment of 

 gene fusion–positive, unresectable or metastatic solid tumors including BTC.

 Larotrectinib was approved in 2018, and entrectinib was approved in 2019.

 Entrectinib is an inhibitor of TRKA, TRKB, and TRK3; larotrectinib is a selective pan-TRK inhibitor.

 Both have shown specificity to patients with 

 gene fusions with little to no activity when fusions are absent.

 Both TRK inhibitors have a favorable safety profile with a 57% to 75% response rate in pretreated, NTRK-positive tumors.

Farha N, Dima D, Ullah F, Kamath S. Precision oncology targets in biliary tract cancer. cancers. 2023;15(7):2105. doi:10.3390/cancers15072105

Manea CA, Badiu DC, Ploscaru IC, et al. A review of NTRK fusions in cancer. 

 2022;79:103893. doi:10.1016/j.amsu.2022.103893

Okamura R, Boichard A, Kato S, Sicklick JK, Bazhenova L, Kurzrock R. Analysis of NTRK alterations in pan-cancer adult and pediatric malignancies: Implications for NTRK-targeted therapeutic

Lange A, Lo HW. Inhibiting TRK proteins in clinical cancer therapy. 

. 2018;10(4):105. doi:10.3390/cancers10040105

Demols A, Rocq L, Perez-Casanova L, et al. A two-step diagnostic approach for NTRK gene fusion detection in biliary tract and pancreatic adenocarcinomas. 

 2023;28(7):e520-e525. doi:10.1093/oncolo/oyad075

NCCN. Clinical Guidelines in Oncology. Pancreatic adenocarcinoma, version 1.2024. Accessed June 11, 2024. 

https://www.nccn.org/professionals/physician_gls/pdf/pancreatic.pdf

Rozlytrek. Prescribing information. Genentech; 2024. Accessed June 11 ,2024. 

https://www.gene.com/download/pdf/rozlytrek_prescribing.pdf

Vitrakvi. Prescribing information. Bayer HealthCare Pharmaceuticals; 2023. Accessed June 11, 2024. 

https://labeling.bayerhealthcare.com/html/products/pi/vitrakvi_PI.pdf

Hong DS, DuBois SG, Kummar S, et al. Larotrectinib in patients with TRK fusion-positive solid tumours: a pooled analysis of three phase 1/2 clinical trials. 

. 2020;21(4):531-540. doi:10.1016/S1470-2045(19)30856-3

NTRK fusions

DNA mismatch repair/microsatellite instability

DNA MMR is involved in preserving genomic fidelity by repairing DNA after mismatching errors after replication or recombination.

 MMR is also involved in cell cycle regulation and apoptosis in response to DNA damage.

 Four genes regulate MMR mechanisms, and the dimer proteins they form are responsible for mismatch recognition, insertion-deletion loops, base excision, and other functions. Biallelic inactivation of 1 or more MMR genes can result from either somatic or germline mutations as well as from epigenetic silencing.

Germline mutations in MMR genes are frequently associated with Lynch syndrome, which is known to increase the risk of several types of cancers.

Microsatellites are short tandem nucleotide repeats (1-6 nucleotides) scattered throughout the genome and prone to high rates of mutation.

 MSI refers to a hypermutable phenotype that results when MMR genes are inactive or faulty. MSI status is generally categorized into 3 types: high MSI (MSI-H), low MSI (MSI-L), and microsatellite stability (MSS). The incidence of advanced biliary tract cancers (BTCs) with MMR-deficiency (dMMR)/MSI-H is approximately 1% to 3%.

The National Comprehensive Cancer Network guidelines recommended MMR and MSI testing in patients with unresectable or metastatic BTCs including intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and gallbladder cancer.

 Several techniques are used to test for the presence of dMMR in colorectal cancer, including immunohistochemistry (IHC) or polymerase chain reaction (PCR)–based assays as well as next-generation sequencing (NGS) testing. IHC assays assess expression of MMR proteins, PCR evaluates tumor DNA for microsatellite repeats, and NGS identifies inactivating mutations in MMR genes. MSI-H status correlates with the loss expression in 2 of 4 MMR proteins and abnormal microsatellites in 2 or more regions.

The FDA has approved several immunotherapy agents for the treatment of patients with dMMR/MSI-H solid tumors that act by inhibiting the programmed death ligand 1 (PD-L1) receptor found on T cells, thereby restoring the antitumor immune response.

 In 2023, pembrolizumab received approval for the treatment of adult and pediatric patients who have unresectable or metastatic MSI-H or dMMR solid tumors as determined by an FDA-approved test, who have progressed following prior treatment, and who have no satisfactory alternative treatment options.

 Subsequently, based on KEYNOTE-966 study (NCT04003636) findings, pembrolizumab was approved as a first-line therapy option to be used in combination with gemcitabine and cisplatin for locally advanced unresectable or metastatic BTC.

 Dostarlimab has also received accelerated approval for dMMR recurrent or advanced solid tumors with no satisfactory alternative treatment options based on findings from the GARNET trial (NCT02715284).

Modrich P, Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. 

. 1996; 65:101-33. doi:10.1146/annurev.bi.65.070196.000533

Marti TM, Kunz C, Fleck O. DNA mismatch repair and mutation avoidance pathways. 

Bellacosa A. Functional interactions and signaling properties of mammalian DNA mismatch repair proteins. 

. 2001; 8:1076-1092. doi:10.1038/sj.cdd.4400948

Peters AC, Young LC, Maeda T, Tron VA, Andrew SE. Mammalian DNA mismatch repair protects cells from UVB-induced DNA damage by facilitating apoptosis and p53 activation. 

 2003; 2:427-435. doi:10.1016/s1568-7864(03)00003-x

 1996; 65:101-133. doi:10.1146/annurev.bi.65.070196.000533

Herman JG, Umar A, Polyak K, et al. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. 

 1998;95:6870-6875. doi:10.1073/pnas.95.12.6870

Hampel H, Frankel WL, Martin E, et al. Feasibility of screening for Lynch syndrome among patients with colorectal cancer. 

 2008;26:5783-5788. doi:10.1200/JCO.2008.17.5950

Lynch HT, de la Chapelle A. Hereditary colorectal cancer. 

 2003;348:919-932. doi:10.1056/NEJMra012242

Aaltonen LA, Salovaara R, Kristo P, et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. 

 1998;338:1481-1487. doi:10.1056/NEJM199805213382101

Hampel H, Frankel WL, Martin E, et al. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). 

 2005;352:1851-1860. doi:10.1056/NEJMoa043146

Bonneville R, Krook MA, Chen HZ, et al. Detection of microsatellite instability biomarkers via next-generation sequencing. 

 2020;2055:11-32. doi:10.1007/978-1-4939-9773-2_5

Li K, Luo H, Huang L, Luo H, Zhu X. Microsatellite instability: a review of what the oncologist should know. 

 2020;20:16. doi:10.1186/s12935-019-1091-8

NCCN. Clinical Practice Guidelines in Oncology. Biliary tract cancers, version 1.2024. Accessed April 10, 2024. 

Keytruda. Prescribing information. Merck & Co; 12018. Accessed April 10, 2024. 

https://www.merck.com/product/usa/pi_circulars/k/keytruda/keytruda_pi.pdf

Jemperli. Prescribing information. GlaxoSmithKline; 2024. Accessed April 10, 2024. 

https://gskpro.com/content/dam/global/hcpportal/en_US/Prescribing_Information/Jemperli/pdf/JEMPERLI-PI-MG.PDF

FDA converts to full approval indication for KETRUDA (pembrolizumab) for certain adult and pediatric patients with advanced microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors. Press release. Merck. March 29, 2023. Accessed April 10, 2024. 

https://www.merck.com/news/fda-converts-to-full-approval-indication-for-keytruda-pembrolizumab-for-certain-adult-and-pediatric-patients-with-advanced-microsatellite-instability-high-msi-h-or-mismatch-repair-deficient/

FDA approves pembrolizumab with chemotherapy for biliary tract cancer. FDA. November 1, 2023. Accessed April 10, 2024. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-pembrolizumab-chemotherapy-biliary-tract-cancer

FDA grants accelerated approval to dostarlimab-gxly for dMMR advanced solid tumors. FDA. Updated July 18, 2023. Accessed April 10, 2024. 

https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grants-accelerated-approval-dostarlimab-gxly-dmmr-advanced-solid-tumors

MSI-H/dMMR

 gene was first discovered and characterized in 1985 on chromosome 10.

 encodes a transmembrane tyrosine kinase with multiple active extracellular sites including a cysteine-rich domain and cadherin-like domain 1 (CLD1), CLD2, CLD3, and CLD4.

 Under normal circumstances, RET protein is involved in the development of the kidneys and enteric nervous system as well as the homeostasis of neural, neuroendocrine, and hematopoietic cells.

Four glial cell line–derived neurotrophic factor (GDNF) family ligands (GFL) are endogenous ligands for RET; these include GDNF, persephin, artemin, and neurturin.

 Rather than bind directly to RET proteins, GFLs first bind to GDNF family receptor-α (GFRα) coreceptors, and this complex mediates RET homodimerization.

 The activated complex autophosphorylates and activates signal transduction pathways MAPK, RAS, ERK, PI3K, AKT, phospholipase C-γ, and JAK-STAT.

 gene fusions are found most frequently in thyroid cancers and non–small cell lung cancer while the incidence in BTC is relatively low (1%-2%) among cancers overall and even lower among patients with BTC (< 1%).

Oncogenic RET proteins are involved in tumor cell proliferation, invasion, differentiation, and migration, and oncogenesis occurs via 2 primary mechanisms.

 The first mechanism is via constitutive activation of kinase activity, and the second is chromosomal rearrangements that produce a chimeric protein fusion of the 

The National Comprehensive Cancer Network (NCCN) guidelines recommend testing for 

gene fusions for all patients with unresectable or metastatic gallbladder cancer, intrahepatic cholangiocarcinoma (CCA), and extrahepatic CCA.

The preferred test is next-generation sequencing (NGS), because traditional testing methods have limited utility for detecting 

Currently, the only FDA-approved treatment for adult patients with locally advanced or metastatic BTC tumors with a 

 Selpercatinib is a selective RET kinase inhibitor that inactivates wild-type RET and various mutated RET isoforms.9,10 Selpercatinib is well tolerated with a permanent discontinuation rate of 8% and a dose interruption rate of 64%.

Pralsetinib is a selective RET tyrosine kinase inhibitor mentioned within NCCN guidelines for BTC but does not currently have approval for treating tumor-agnostic solid tumors including BTCs.

 Pralsetinib is recommended under certain circumstances as first-line or subsequent-line systemic therapy.

Zhao L, Wang N, Zhang D, Jia Y, Kong F. A comprehensive overview of the relationship between RET gene and tumor occurrence. 

 2023;13:1090757. doi:10.3389/fonc.2023.1090757

Belli C, Penault-Llorca F, Ladanyi M, et al. ESMO recommendations on the standard methods to detect RET fusions and mutations in daily practice and clinical research. 

 2021;32(3):337-350. doi:10.1016/j.annonc.2020.11.021

Ibanez CF. Structure and physiology of the RET receptor tyrosine kinase. 

. 2013;5(2):a009134-a009134. doi:10.1101/cshperspect.a009134

Drilon A, Hu ZI, Lai GGY, Tan DSW. Targeting RET-driven cancers: Lessons from evolving preclinical and clinical landscapes. 

 2018;15(3):151-167. doi:10.1038/nrclinonc.2017.175

Carlomagno F. Thyroid cancer: Role of RET and beyond. 

Kato S, Subbiah V, Marchlik E, Elkin SK, Carter JL, Kurzrock R. RET aberrations in diverse cancers: next-generation sequencing of 4,871 patients. 

 2017;23(8):1988-1997. doi:10.1158/1078-0432.CCR-16-1679

NCCN. Clinical Practice Guidelines in Oncology. Pancreatic adenocarcinoma, version 1.2024. Accessed June 12, 2024. 

Shi M, Wang W, Zhang J, et al. Identification of RET fusions in a Chinese multicancer retrospective analysis by next-generation sequencing. 

 2022;113(1):308-318. doi:10.1111/cas.15181

Retevmo. Prescribing information. Eli Lilly and Company; 2024. Accessed June 12, 2024. 

https://uspl.lilly.com/retevmo/retevmo.html#pi

FDA D.I.S.C.O. Burst Edition: FDA approvals of Retevmo (selpercatinib) for adult patients with locally advanced or metastatic RET fusion-positive solid tumors, and Retevmo (selpercatinib) for adult patients with locally advanced or metastatic RET fusion-positive non-small cell lung cancer. FDA. November 7, 2022. Accessed April 1, 2024. 

https://www.fda.gov/drugs/resources-information-approved-drugs/fda-disco-burst-edition-fda-approvals-retevmo-selpercatinib-adult-patients-locally-advanced-or

Subbiah V, Hu MIN, Gainor JF, et al. Clinical activity of the RET inhibitor pralsetinib (BLU-667) in patients with RET fusion–positive solid tumors. 

 2021;39(suppl 3):467. doi:10.1200/JCO.2021.39.3_suppl.467

Fibroblast growth factor receptor 2 (FGFR2)

The FGFR family of tyrosine kinase receptors includes FGFR1, FGFR2, FGFR3, and FGFR4.

Upon growth factor binding, FGFRs dimerize and activate intracellular pathways crucial for cell proliferation, survival, and angiogenesis.

 gene situated on chromosome 10 has over 25 isoforms derived from alternative splicing.

 In cholangiocarcinomas (CCAs), more than 150 

 mutations, and they often occur along with 

 aberrations are found in 7.1% of solid tumors, with 

fusions occur in 5% to 7% of all CCAs and in 10% to 20% of intrahepatic CCAs (iCCAs), and they are influenced by geographical and etiological factors.

 Some studies suggest a favorable prognosis associated with 

 fusions, with longer overall survival (OS) and progression-free survival (PFS) noted in patients harboring these alterations and particularly in younger individuals.

The National Comprehensive Cancer Network guidelines recommend comprehensive molecular profiling for patients with unresectable or metastatic biliary tract cancers (BTCs).

fusions or rearrangements is recommended in patients with unresectable or metastatic intrahepatic CCAs and extrahepatic CCAs due to the high associated incidence, but it may also be considered for unresectable or metastatic gallbladder cancer. Next-generation sequencing (NGS) assays and break apart fluorescence in situ hybridization (FISH) assays are commonly used to identify patients with 

 Some fusion breakpoints can be detected using cell free DNA assays, but associated sensitivity is lower than for tumor tissue testing. Therefore, comprehensive NGS-based tissue testing remains preferred to optimize the identification of targetable aberrations.

The FDA has approved 2 targeted therapies for BTCs with 

 Both of these agents are small-molecule kinase inhibitors of FGFR1, FGFR2, FGFR3, and FGFR44. Pemigatinib received accelerated approval in 2020 for use in patients with unresectable, locally advanced or metastatic CCA with 

fusions or rearrangements based on findings from the FIGHT-202 study (NCT02924376).

 The FoundationOne CDx assay was also approved as a companion diagnostic for pemigatinib. Subsequently, in 2022, futibatinib received accelerated approval for previously treated, unresectable, locally advanced or metastatic, intrahepatic CCA harboring 

 fusions or rearrangements based on findings from the TAS-120-101 trial (NCT02052778).

 Another FGFR inhibitor, infigratinib, received accelerated approval in 2021; it was withdrawn from the US market in 2023.

Angerilli V, Fornaro L, Pepe F, et al. FGFR2 testing in cholangiocarcinoma: translating molecular studies into clinical practice. 

 2023;115(2):71-82. doi:10.32074/1591-951X-859

Katoh M. Fibroblast growth factor receptors as treatment targets in clinical oncology. 

 2019;16(2):105-122. doi:10.1038/s41571-018-0115-y

Neumann O, Burn TC, Allgäuer M, et al. Genomic architecture of FGFR2 fusions in cholangiocarcinoma and its implication for molecular testing. 

 2022;127(8):1540-1549. doi:10.1038/s41416-022-01908-1

Arai Y, Totoki Y, Hosoda F, et al. Fibroblast growth factor receptor 2 tyrosine kinase fusions define a unique molecular subtype of cholangiocarcinoma. 

 2014;59(4):1427-34. doi:10.1002/hep.26890

Wu YM, Su F, Kalyana-Sundaram S, et al. Identification of targetable FGFR gene fusions in diverse cancers. 

 2013;3(6):636-647. doi:10.1158/2159-8290.CD-13-0050

Chen L, Zhang Y, Yin L, et al. Fibroblast growth factor receptor fusions in cancer: opportunities and challenges. 

 2021;40(1):345. doi:10.1186/s13046-021-02156-6

Jain A, Borad MJ, Kelley RK, et al. Cholangiocarcinoma with 

 genetic aberrations: a unique clinical phenotype. 

Helsten T, Elkin S, Arthur E, Tomson BN, Carter J, Kurzrock R. The FGFR landscape in cancer: analysis of 4,853 tumors by next-generation sequencing. 

. 2016;22(1):259-267. doi:10.1158/1078-0432.CCR-14-3212

Graham RP, Barr Fritcher EG, Pestova E, et al. Fibroblast growth factor receptor 2 translocations in intrahepatic cholangiocarcinoma. 

. 2014;45(8):1630-1638. doi:10.1016/j.humpath.2014.03.014

Rizzato M, Brignola S, Munari G, et al. Prognostic impact of FGFR2/3 alterations in patients with biliary tract cancers receiving systemic chemotherapy: the BITCOIN study. 

Biliary Tract Cancer

Biliary Tract Cancer

Biomarkers