Molecular Testing in Metastatic Colorectal Cancer: Understanding How, When, and What to Profile

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“In line with the emergence of targeted therapies, molecular biomarker testing in metastatic colorectal cancer has evolved over the past decade,” noted Jeanne Tie, MD, MBChB, FRACP, who acknowledged there is confusion about the best ways to use molecular testing in the clinic. Dr. Tie, who is Associate Professor, University of Melbourne, Australia, and Lower GI Medical Oncology and Trials Lead at the Peter MacCallum Cancer Centre, explained the options to clinicians listening in to the ASCO20 Virtual Education Program.1

Jeanne Tie, MD, MBChB, FRAC

Jeanne Tie, MD, MBChB, FRAC

Because outcomes are improved when novel therapies are applied, all patients newly diagnosed with colorectal cancer should be tested for mutations in KRAS, NRAS, and BRAF V600E, as well as for microsatellite instability–high (MSI-H) status or mismatch repair deficiency (dMMR). In the refractory setting, clinicians should also look for HER2 amplifications and rare fusions such as NTRK, especially in patients with RAS/BRAF wild-type disease and in those with MSI-H tumors. These amplifications and fusions were found to be actionable in 2019, with promising results seen with trastuzumab (plus pertuzumab or lapatinib) and approval by the U.S. Food and Drug Administration for larotrectinib and entrectinib.

Despite these advances, a retrospective review of 23 United States–based oncology practices demonstrated that routine molecular testing remains suboptimal. In a study by Gutierrez et al, less than 60% of patients underwent testing for any of these markers—a rate that had not increased since 2013.2

Inadequate genotyping may be related to patient factors, such as poor performance status, which preclude any active treatment. An analysis of the Australian metastatic colorectal cancer (TRACC) registry included only patients fit enough for treatment.3 The investigators reported a steady increase in testing since 2009, ultimately 80%, reflecting “a definite learning curve among oncologists regarding genetic profiling,” Dr. Tie said.

Goals of Genomic Testing

There are three main considerations in deciding which test to do for a given patient: (1) clinical context (treatment-naive or treatment-refractory and tissue availability); (2) information that is needed for the treatment of the patient; and (3) cost and funding, which are important for choosing between single-gene testing and next-generation sequencing, noted Dr. Tie. For example, for a 33-year-old patient with de novo unresectable metastatic disease, useful information would be genomic changes that affect standard-of-care therapies, facilitate a prognosis, and help to determine hereditary risk. Testing would establish the potential benefit of standard treatment: for RAS wild-type tumors, an anti-EGFR agent; and for BRAF V600E–mutated tumors, a BRAF inhibitor/ EGFR inhibitor combination.

“A retrospective review of 23 United States–based oncology practices demonstrated that routine molecular testing remains suboptimal.”
— Jeanne Tie, MD, MBChB, FRACP

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Other information would also be derived, however. “Along with being a therapeutic target, the BRAF V600E mutation is a poor prognostic marker,” Dr. Tie said. “If you determine this by testing early, you may be encouraged to treat this patient more aggressively upfront, such as with FOLFOXIRI [leucovorin, fluorouracil, oxaliplatin, irinotecan], and use BRAF-targeted treatment in the second line.”

The status of BRAF and MSI is also a component in screening for Lynch syndrome, which has obvious implications for the patient’s family.

After disease progression with standard treatment, clinicians should look for rare targets that may be either actionable or render the patient eligible for a clinical trial. They include HER2 amplifications and NTRK fusions in RAS/BRAF wild-type tumors, KRAS G12C mutations, and class II/III BRAF mutations. Testing can also identify acquired-resistance factors—RAS/BRAF V600E, EGFR ECD and PIK3CA mutations, and cMET/HER2 amplifications—which may also present opportunities for clinical trial enrollment.

Differences Among Genomic Tests

Detection capabilities vary among the testing methods, noted Dr. Tie. Next-generation sequencing of DNA and RNA offers “one-stop shops” that can identify multiple genetic alterations simultaneously. This type of sequencing detects point mutations and single nucleotide variants, indels (insertions/deletions), copy number variations and amplifications, MSI status, and tumor mutational burden (TMB). Next-generation RNA sequencing detects fusions, splice variants, and gene expression.

“However, the simplest and least expensive testing is polymerase chain reaction (DNA) or reverse transcription–polymerase chain reaction (RT-PCR) RNA testing,” said Dr. Tie. Such testing detects point mutations and single nucleotide variants, MSI status, and NTRK fusions (with RT-PCR).

Fluorescence in situ hybridization is a DNA-based technique that detects chromosomal abnormalities—amplifications, deletions, and fusions (HER2 and NTRK). However, it can be labor-intensive and requires experienced pathology,” she noted. Immunohistochemistry measures protein expression at the cellular level and provides information on the status of MMR, HER2, and NTRK.

The commercial and academic oncogenic panels vary primarily in terms of the number of genes profiled, the inclusion of RNA analysis, the use of a matched normal sample, and TMB cutoff. Turn-around time has improved for all tests and is now comparable, at 2 to 3 weeks.

“Use of a sequential single-gene approach or next-generation sequencing is dependent on resources and funding,” Dr. Tie noted.

“Use of a sequential single-gene approach or next-generation sequencing is dependent on resources and funding.”
— Jeanne Tie, MD, MBChB, FRACP

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MMR/MSI and NTRK Fusion Testing

“Given the therapeutic and hereditary risk implications, it’s important to understand MSI or MMR testing in more detail,” Dr. Tie continued.

Immunohistochemistry detects the presence or absence of four MMR gene proteins (MLH1 loss, MSH2, MSH6, and PMS2 loss). Tumors are labeled as “deficient” when there is loss of their expression. “Its advantages are it’s inexpensive, fast, and widely available,” she added.

Molecular polymerase chain reaction–based analysis assesses the stability of microsatellite markers, which are short, repetitive DNA sequences that are a direct measure of genomic instability caused by the dysfunctional MMR system. It is usually performed only in expert pathology labs.

“The concordance between immunohistochemistry and polymerase chain reaction testing is generally high (96%), but it’s not perfect. So, the decision about which screening test to use primarily depends on availability, expertise, and resources,” indicated Dr. Tie.

Next-generation sequencing is an emerging technique to test for MSI. A study using the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay showed 99.4% accuracy in detecting MSI-H colorectal and endometrial tumors.4

Next-generation sequencing can also accurately detect other molecular changes such as NTRK fusions. “These fusions are extremely rare in colorectal cancer (< 1% prevalence), so detection is “challenging,” but they are “therapeutically important” and therefore worth testing for, according to Dr. Tie.

One study using the MSK-IMPACT panel to test 2,315 colorectal tumors found that “activating kinase fusions” (such as NTRK) occurred only in RAS/BRAF wild-type tumors.5 Enriching for this subset, testing revealed these alterations in 15% of patients; further enriching for MSI-H tumors with MLH1 hypermethylation, a 42% prevalence was found. “This is intriguing and opens up another treatment opportunity for MSI-H tumors after patients’ tumors fail to respond to therapy with an immune checkpoint inhibitor,” she said.

The European Society for Medical Oncology (ESMO) has proposed an algorithm for NTRK gene fusion testing.6 For tumors with a low prevalence of this alteration, such as colorectal tumors, ESMO recommends a front-line DNA- or RNA-based method. Alternatively, a “two-stop” approach can be considered in which immunohistochemistry is used first and any positive test is confirmed by next-generation sequencing.

Which Tissue Sample?

Another question raised by clinicians is whether to test on primary or metastatic tissue. High genomic concordance (> 90%) has been found between these tumor sites for KRAS, NRAS, BRAF, and MSI, suggesting “key driver mutations and MSI/MMR status do not change over the disease course…. Thus, mutation testing can be done on either,” according to Dr. Tie.

The same high concordance has been found between tumor tissue and plasma RAS/BRAF genotyping (ie, in circulating tumor DNA). “Plasma can be a source” of sampling, especially when tissue is difficult to obtain, she said.

“Repeat testing is not required for standard application, unless you are looking for acquired mutations to targeted therapy,” Dr. Tie added.

Barriers to Next-Generation Sequencing—or Any Testing

“Next-generation sequencing profiling can provide us with a large amount of genomic information, so why not do it on all our patients?” Dr. Tie questioned. “The most widespread argument against next-generation sequencing is its additional cost to payers and patients, with little clinical benefit to justify it because the chance of finding an actionable result, especially in colorectal cancer, is extremely low.” The cost of next-generation sequencing can exceed $4,000, vs $300 to $400 for a single-gene assay.

“The cost of next-generation sequencing can exceed $4,000, vs $300 to $400 for a single-gene assay.”
— Jeanne Tie, MD, MBChB, FRACP

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Another barrier to next-generation sequencing is oncologists’ lack of confidence in testing, according to a national survey conducted in 2017.7 Clinicians reported being most confident using single-gene testing and least confident with whole-genome or whole-exome sequencing to guide patient care. Their confidence varied by testing platform, patient volume, genomic training, and practice infrastructure.

Clinical utility, or the extent to which incorporating genetic testing into patient care improves outcome, “remains to be defined,” Dr. Tie added. Pooled data from more than 13,000 patients included in 12 genomically matched trials reported that for every 1,000 patients who underwent genomic profiling, testing revealed 400 targetable mutations. However, just 120 patients (12%) received matched therapy, and 8 to 30 patients (0.8%–3.0%) responded to the treatment.8

“We need to move the field forward. To do so, more imaginative and strategic designs of genomically matched trials will be required,” Dr. Tie concluded. 

DISCLOSURE: Dr. Tie has received honoraria from Amen, FivepHusion, Merck Serono, and Servier; has served as a consultant or advisor to AstraZeneca/MedImmune, Bristol Myers Squibb, and Merck Serono; and has been reimbursed for travel, accommodations, or other expenses by Merck Serono.


1. Tie J: How, when, and what to profile? ASCO20 Virtual Education Program.

2. Gutierrez ME, Price KS, Lanman RB, et al: Genomic profiling for KRAS, NRAS, BRAF, microsatellite instability, and mismatch repair deficiency among patients with metastatic colon cancer. JCO Precis Oncol. December 6, 2019 (early release online).

3. Field K, Wong HL, Shapiro J, et al: Developing a national database for metastatic colorectal cancer management: Perspectives and challenges. Intern Med J. 43:1224-1231, 2013.

4. Middha S, Zhang L, Nafa K, et al: Reliable pan-cancer microsatellite instability assessment by using targeted next-generation sequencing data. JCO Precis Oncol. October 3, 2017 (early release online).

5. 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 Res 79:1047-1053, 2019.

6. Marchiò C, Scaltriti M, Ladanyi M, et al: ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research. Ann Oncol 30:1417-1427, 2019.

7. de Moor JS, Gray SW, Mitchell SA, et al: Oncologist confidence in genomic testing and implications for using multimarker tumor panel tests in practice. JCO Precis Oncol. June 11, 2020 (early release online).

8. Tannock IF, Hickman JA: Molecular screening to select therapy for advanced cancer? Ann Oncol 30:661-663, 2019.