Biomarker Consortium

GI Cancer

Colorectal Cancer

DNA mismatch repair/microsatellite instability.

DNA mismatch repair (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 one (or more) MMR genes can result from either somatic or germline mutations, as well as from epigenetic silencing.

 Lynch syndrome arises from inherited (germline) mutations in four MMR genes (MLH1, MSH2, MSH6, and PMS2) and stands as the predominant genetically linked predisposition to colorectal cancer (CRC), accounting for approximately 2% to 4% of all CRC cases.

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

 Microsatellite instability (MSI) refers to a hyper-mutable phenotype that results when MMR genes are inactive or faulty.MSI status is generally categorized into three types: high microsatellite instability (MSI-H), low microsatellite instability (MSI-L) and microsatellite stability (MSS). 

The National Comprehensive Cancer Network guidelines recommended universal MMR and MSI testing in all newly diagnosed patients with CRC.

 Several techniques are used to test for the presence of MMR deficiency (dMMR) in CRC, including immunohistochemical (IHC) or polymerase chain reaction- (PCR-) based assays as well as NGS based testing. IHC assays test for the protein expression of 4 MMR genes known to be mutated in Lynch Syndrome.Under normal physiological conditions all 4 MMR proteins are expressed.However, the loss of expression of one or more of these suggests the presence of a MMR gene mutation and warrants further testing, including screening for the presence of BRAF V600E mutations. 

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

 Based findings from the CheckMate-142 trial, nivolumab received accelerated approval as a single agent in this setting; approval was later extended to nivolumab in combination with ipilimumab in 2018.

 Pembrolizumab received approval as a first-line agent for the treatment of dMMR/MSI-H metastatic CRC in 2020 based on safety and efficacy demonstrated in the KEYNOTE-177 trial.

Subsequently, in 2021, dostarlimabreceived accelerated approval for dMMR recurrent or advanced solid tumors with no satisfactory alternative treatment options based on findings from GARNET.

Learn more about Nivolumab and Ipilimumab >

Modrich P, Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. AnnuRev Biochem. 1996;65:101-33.

Marti TM, Kunz C, Fleck O. DNA mismatch repairand mutation avoidance pathways. J Cell Physiol. 2002;191:28-41. 

Bellacosa A. Functional interactions and signaling properties of mammalian DNA mismatch repair proteins. Cell Death Differ. 2001;8:1076-92.

Peters AC, Young LC, Maeda T, et al. Mammalian DNAmismatch repair protects cells from UVB-induced DNAdamage by facilitating apoptosis and p53 activation. DNARepair (Amst) 2003;2:427-35.

Modrich P, Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem 1996; 65:101-33.

Herman JG, Umar A, Polyak K, et al. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl AcadSciUSA1998;95:6870-5.

Hampel H, Frankel WL, Martin E, et al. Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol 2008;26:5783-5788.

Lynch HT, de la Chapelle A. Hereditary colorectal cancer. N Engl J Med 2003;348:919-932.

Aaltonen LA, Salovaara R, Kristo P, et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screeningfor the disease. N Engl J Med 1998;338:1481-1487.

Hampel H, Frankel WL, Martin E, et al. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med 2005;352:1851-1860

Bonneville R, Krook MA, Chen HZ, et al. Detection of microsatellite instability biomarkers via next-generation sequencing. Methods Mol Biol.2020;2055:119–32.

Li K, Luo H, Huang L, Luo H, Zhu X. Microsatellite instability: a review of what the oncologist should know. Cancer Cell Int.2020;20:16. doi: 10.1186/s12935-019-1091-8.

National Comprehensive Cancer Network. Clinical Practice Guidelines in Oncology. Colon Cancer, v1.2024. Accessed April 10, 2024. 

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

National Comprehensive Cancer Network. Clinical Practice Guidelines in Oncology. Rectal Cancer, v1.2024. Accessed April 10, 2024.

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

KEYTRUDA (pembrolizumab) [package insert].Whitehouse Station, NJ, USA: Merck & Co., Inc.; 12/2018.

OPDIVO (Nivolumab) [package insert].Princeton, NJ, USA: Bristol-Myers Squibb Company; 3/2024.

YERVOY (Ipilimumab) [package insert].Princeton, NJ, USA: Bristol-Myers Squibb Company; 2/2023.

JEMPERLI (Dostarlimab) [package insert].Durham, NC, USA: GlaxoSmithKline; 3/2024.

FDA.gov. FDA grants nivolumab accelerated approval for MSI-H or dMMR colorectal cancer. Updated August 1, 2017. Accessed April 10, 2024.

FDA.gov. FDA grants accelerated approval to ipilimumab for MSI-H or dMMRmetastatic colorectal cancer. Updated July11, 2018. Accessed April 10, 2024.

FDA.gov. FDA approves pembrolizumab for first-line treatment of MSI-H/dMMR colorectal cancer. Updated June 30, 2020. Accessed April 10, 2024.

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

MSI-H/dMMR

Neurotrophic-tropomyosin receptor tyrosine kinase (NTRK)

The neurotrophic-tropomyosin receptor tyrosine kinase 1 (NTRK1), NTRK2, and NTKR3 genes encode for proteins TRKA, TRKB, and TRKC, respectively.

 NTRK proteins localize to cell membranes, and their endogenous ligands are neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, and neurotrophin-5.

 NTRK gene activation activates downstream signaling pathways RAS-RAF-MEK-ERK, phospholipase C, and phosphatidylinositol 3 kinase (PI3K).

The NTRK proteins are involved in the growth and proliferation of neural cells in particular, and gene fusions can induce tumorigenesis in neural and non-neural cell cancers.1 When oncogenic, NTRK gene fusions are drivers for tumorigenesis and lead to constitutively active downstream signaling of various growth pathways.1,4 

The rarity of NTRK gene fusions in CRC makes it challenging to elucidate clinicopathological characteristics. In a 2022 study, researchers analyzed a cohort of 16 patients with NTRK+ colorectal cancer (CRC) and found that of the three genes, NTRK1 rearrangements were the most common, and TPM3 was the most common fusion partner in introns 7-11.5 NTRK+ CRC variants were more likely to have microsatellite instability.

NTRK gene fusions are prevalent in approximately 1% of solid tumors but are even more rare in colorectal cancer (0.35-0.7%).

 Testing should be included as part of a broader molecular profiling but is included in current guidelines because targeted TRK inhibitors are available and approved to treat CRC solid tumors.

 Guidelines endorse using multiple testing methodologies, depending on the accessibility of assays and available testing material.

 Feasible testing methods include immunohistochemistry (ICH), fluorescence in situ hybridization (FISH), DNA-based next-generation sequencing, and RNA-based NGS.

 The ideal approach appears to be RNA-based NGS because of the higher rates of false positives and lack of evidence of functional transcription present in other methods.

There are currently two treatment options for unresectable or metastatic solid tumors with NTRK gene fusions: larotrectinib and entrectinib.

 Larotrectinib and entrectinib are tyrosine kinase inhibitors specifically targeting TRKA, TRKB, and TRKC, anaplastic lymphoma kinase (ALK), and the proto-oncogene tyrosine-protein kinase 1 (ROS1).

 These targeted therapies are only appropriate for a small subset of CRC patients but offer an additional option with an attractive safety profile and an overall response rate of 57-79%.

1. 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 Therapeutics. JCO Precision Oncology. 2018;(2):1-20. doi:10.1200/PO.18.00183

2. Manea CA, Badiu DC, Ploscaru IC, et al. A review of NTRK fusions in cancer. Annals of Medicine & Surgery. 2022;79. doi:10.1016/j.amsu.2022.103893

3. Solomon JP, Hechtman JF. Detection of NTRK Fusions: Merits and Limitations of Current Diagnostic Platforms. Cancer Research. 2019;79(13):3163-3168. doi:10.1158/0008-5472.CAN-19-0372

4. National Comprehensive Cancer Network. NCCN guidelines version 1.2024: Colon Cancer. https://www.nccn.org/professionals/physician_gls/pdf/colon.pdf

5. Wang H, Li Z, Ou Q, et al. NTRK fusion positive colorectal cancer is a unique subset of CRC with high TMB and microsatellite instability. Cancer Medicine. 2022;11(13):2541-2549. doi:10.1002/cam4.4561

6. Cocco E, Benhamida J, Middha S, et al. Colorectal Carcinomas Containing Hypermethylated MLH1 Promoter and Wild-Type BRAF/KRAS Are Enriched for Targetable Kinase Fusions. Cancer Research. 2019;79(6):1047-1053. doi:10.1158/0008-5472.CAN-18-3126

7. Vitraki (Larotrectinib) [Package Insert]. Loxo Oncology; 2018.

8. Rozlytrek (Entrectinib) [Package Insert]. Genentech; 2019.

9. FDA approves larotrectinib for solid tumors with NTRK gene fusions | FDA. Accessed March 18, 2024. https://www.fda.gov/drugs/fda-approves-larotrectinib-solid-tumors-ntrk-gene-fusions

10. Drilon A, Laetsch TW, Kummar S, et al. Efficacy of Larotrectinib in TRK Fusion–Positive Cancers in Adults and Children. N Engl J Med. 2018;378(8):731-739. doi:10.1056/NEJMoa1714448

11. FDA approves entrectinib for NTRK solid tumors and ROS-1 NSCLC | FDA. Accessed March 19, 2024. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-entrectinib-ntrk-solid-tumors-and-ros-1-nsclc

12. Demetri GD, Paz-Ares L, Farago AF, et al. Efficacy and safety of entrectinib in patients with NTRK fusion-positive (NTRK-fp) Tumors: Pooled analysis of STARTRK-2, STARTRK-1 and ALKA-372-001. Annals of Oncology. 2018;29:viii713. doi:10.1093/annonc/mdy424.017

NTRK fusions

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

The RAS-RAF-MEK-ERK pathway is an important protein kinase cascade that regulates cell differentiation, proliferation, and survival in response to hormones, cytokines, and growth factors.

 There are 3 functional RAF proteins in humans, including V-Raf murine sarcoma viral oncogene homolog B (BRAF).

 BRAF is a serine/threonine-specific protein kinase with 3 conserved domains: CR1, CR2, and CR3.

 The wild-type BRAF protein forms a strong hydrophobic interaction in residues 596-600, which keeps it in its inactive state. BRAF becomes activated once the activation loop is phosphorylated, destabilizing the hydrophobic site and enabling downstream kinase activity.

 mutation is an acquired gene mutation more common in older patients (>60 years), proximal tumor location, locally advanced characteristics, and poor cell differentiation.

 V600E mutation is associated with worse survival after recurrence in patients with stage III colon cancer and is associated with a shorter median progression-free and median overall survival than patients with wild-type 

 can result in constitutive activation of the 

 mutations are more common in cancer types that also harbor 

 mutations, including colorectal cancer, borderline ovarian cancers, and malignant melanoma.

 This association occurs because activation of the RAS-RAF-MEK-ERK pathway can be achieved by cancer cells through mutation at various points along the pathway.

 gain synergistic effects when combined with other agents that target kinases in the same pathway.

Patients with metastatic colorectal cancer (mCRC) should have their tumor genotyped with a next-generation sequencing (NGS) panel in a CLIA-certified laboratory that is qualified to perform high-complexity molecular pathology testing. BRAF V600E may also be tested with immunohistochemistry (IHC).

 Testing may be performed on either the primary tumor or the metastases.

A single drug regimen is FDA-approved for treating adult patients with mCRC with a BRAF V600E mutation detected by an FDA-approved test after prior therapy: encorafenib in combination with cetuximab.

 NCCN guidelines version 1.2024 also recommend encorafenib in combination with panitumumab.

 Encorafenib is a kinase inhibitor of BRAF V600E, wild-type BRAF, and CRAF.

 Cetuximab is an epidermal growth factor receptor (EGFR) inhibitor.

Clancy C, Burke JP, Kalady MF, Coffey JC. BRAF mutation is associated with distinct clinicopathological characteristics in colorectal cancer: a systematic review and meta-analysis. 

. 2013;15(12):e711-e718. doi:10.1111/codi.12427

Wan PTC, Garnett MJ, Roe SM, et al. 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

Cutler RE, Stephens RM, Saracino MR, Morrison DK. Autoregulation of the Raf-1 serine/threonine kinase. 

. 1998;95(16):9214-9219. doi:10.1073/pnas.95.16.9214

Sinicrope FA, Shi Q, Allegra CJ, et al. Association of DNA mismatch repair and mutations in BRAF and KRAS with survival after recurrence in stage III colon cancers. 

. 2017;3(4):472-480. doi:10.1001/jamaoncol.2016.5469

Tol J, Nagtegaal ID, Punt CJ. BRAF mutation in metastatic colorectal cancer. 

. 2009;361(1):98-99. doi:10.1056/nejmc0904160

Davies H, Bignell GR, Cox C, et al. Mutations of the 

. 2002;417(6892):949-954. doi:10.1038/nature00766

NCCN. Clinical Practice Guidelines in Oncology. Colon cancer, version 1.2024. https://www.nccn.org/professionals/physician_gls/pdf/colon.pdf

Tabernero J, Grothey A, Van Cutsem E, et al. Encorafenib plus cetuximab as a new standard of care for previously treated BRAF V600E-mutant metastatic colorectal cancer: Updated survival results and subgroup analyses from the BEACON study. 

2021;39(4):273-284. doi:10.1200/JCO.20.02088

FDA approves encorafenib in combination with cetuximab for metastatic colorectal cancer with a BRAF V600E mutation. US FDA. Updated April 9, 2020. Accessed May 21, 2024. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-encorafenib-combination-cetuximab-metastatic-colorectal-cancer-braf-v600e-mutation. 

Erbitux (cetuximab). Package insert. Eli Lilly and Company; 2021. https://uspl.lilly.com/erbitux/erbitux.html#pi

BRAF V600E

Human epidermal growth factor receptor2 (HER2); erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERB-B2) 

The HER2 oncogene, located on chromosome 17q12, encodes a transmembrane tyrosine kinase receptor that is part of the epidermal growth factor receptor family.

 HER2 initiates downstream signaling pathways related to cell proliferation by forming homodimers or heterodimers with other EGFR family members.

 Amplification or overexpression of HER2 results in the increased activation of mitogenic signaling, leading to uncontrolled cell proliferation and tumorigenesis. In colorectal cancer (CRC) HER2 amplification is more frequently observed in distal (rectal) and left-sided tumors, while being less common in proximal and right-sided CRC.

 HER2-amplified CRC is also associated with a greater number of metastatic sites, particularly lung and brain metastases. While the prognostic significance of HER2 amplification in CRC remains unclear, it may serve as a predictive marker for resistance to anti-EGFR therapies.

 HER2 amplification or overexpression occurs in approximately 3-5% of metastatic CRC, with a higher reported prevalence in RAS/BRAF wild type tumors.

The National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in colon and rectal cancer, recommend testing for HER2 amplification in patients with metastatic CRC and no KRAS/NRAS or BRAF mutations.

 Testing for HER2 overexpression is generally performed via immunohistochemistry (IHC) assays, with positivity defined as 3+ staining in greater than 50% of tumor cells. Reflex testing with fluorescence in situ hybridization (FISH) is recommended in patients with IHC 2+ to identify HER2 amplification, with positivity defined as a HER2:CEP17 ratio greater than or equal to 2 in more than 50% of tumor cell. Next generation sequencing (NGS) may also be used to identify HER2 amplification.

The FDA has approved two targeted therapies for HER2+ metastatic CRC.

 The first of these, tucatinib, is a HER2-targeted tyrosine-kinase inhibitor. Tucatinib received accelerated approval in combination with the anti-HER2 monoclonal antibody, trastuzumab, for RAS wild-type HER2+ unresectable or metastatic CRC that has progressed on chemotherapy with fluoropyrimidine, oxaliplatin, and irinotecan.

 This approval was based on findings from the phase 2 MOUNTAINEER study.Trastuzumab deruxtecan (T-DXd) is a HER2-directed antibody drug conjugate (ADC) consisting of a humanized anti-HER2 IgG1 antibody linked to the topoisomerase I inhibitor payload, deruxtecan, via a cleavable tetrapeptide linker.

 Upon binding of HER2 on tumor cells, T-DXd undergoes internalization and intracellular linker cleavage, thereby releasing the DXd payload and subsequently causing DNA damage and apoptotic cell death. T-DXd was granted accelerated approval for previously treated unresectable or metastatic HER2+ (IHC 3+) solid tumors with no satisfactory alternative treatment options, marking it as the first tumor-agnostic approved ADC. Accelerated approval was based on findings from three clinical trials: DESTINY-PanTumor02 (NCT04482309), DESTINY-Lung01 (NCT03505710), and DESTINY-CRC02 (NCT04744831).

Learn more about Fam-Trastuzumab Deruxtecan nxki (T-DXd) >

Rubin I and Yarden, Y. The basic biology of HER2. Ann Oncol. 2001;12(supp1):S3-S8.

Schechter AL, Stern DF, Vaidyanathan L, et al. The neu oncogene: an erb-B-related gene encoding a 185,000-Mr tumour antigen. Nature. 1984;312(5994):513‐516.

Ahcene Djaballah S, Daniel F, Milani A, Ricagno G, Lonardi S. HER2 in colorectal cancer: the long and winding road from negative predictive factor to positive actionable target. Am Soc Clin Oncol Educ Book.2022;42:1-14. doi: 10.1200/EDBK_351354.

Sartore-Bianchi A, Lonardi S, Martino C, et al. Pertuzumab and trastuzumab emtansine in patients with HER2-amplified metastatic colorectal cancer: the phase II HERACLES-B trial. ESMO Open. 2020;5:e000911.

Siena S, Di Bartolomeo M, Raghav K, et al; DESTINY-CRC01 investigators. Trastuzumab deruxtecan (DS-8201) in patients with HER2-expressing metastatic colorectal cancer (DESTINY-CRC01): a multicentre, open-label, phase 2 trial. Lancet Oncol. 2021;22:779-789.

Strickler JH, Ng K, Cercek A, et al. MOUNTAINEER: open-label, phase II study of tucatinib combined with trastuzumab for HER2-positive metastatic colorectal cancer (SGNTUC-017, trial in progress). J Clin Oncol. 2021;39:3s (suppl; abstract TPS153).

Sartore-Bianchi A, Amatu A, Porcu L, et al. HER2 positivity predicts unresponsiveness to EGFR-targeted treatment in metastatic colorectal cancer. Oncologist. 2019;24:1395-1402.

Raghav K, Loree JM, Morris JS, et al. Validation of HER2 amplification as a predictive biomarker for anti-epidermal growth factor receptor antibody therapy in metastatic colorectal cancer. JCO Precis Oncol.2019;3:1-13. doi: 10.1200/PO.18.00226.

National Comprehensive Cancer Network. Clinical Practice Guidelines in colon cancer, version 1.2024. Accessed April 22, 2024. 

National Comprehensive Cancer Network. Clinical Practice Guidelines in rectal cancer, version 1.2024. Accessed April 22, 2024. 

TUKYSA (tucatinib) [package insert].Bothell, WA, USA: Seagen Inc., 01/2023.

ENHERTU (fam-trastuzumab deruxtecan-nxki) [package insert].Basking Ridge, NJ, USA: Daiichi Sankyo, Inc., 02/2024.

FDA.gov. FDA grant accelerated approval to tucatinib with trastuzumab for colorectal cancer.Updated January 19, 2023. Accessed April 22, 2024. 

https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grants-accelerated-approval-tucatinib-trastuzumab-colorectal-cancer

FDA.gov. FDA grant accelerated approval to fam-trastuzumab deruxtecan-nxki for unresectable or metastatic HER2-positive solid tumors. Updated April 5, 2024. Accessed April 22, 2024. 

https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grants-accelerated-approval-fam-trastuzumab-deruxtecan-nxki-unresectable-or-metastatic-her2

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