Recent improvements in our understanding of the biology of colorectal cancer have led to the identification of several important prognostic and predictive markers of disease-associated risk and treatment response for the individual patient. Proper utilization of these biomarkers can enable physicians to tailor therapeutic strategies to maximize the likelihood of response and minimize treatment toxicity. In the management of colorectal cancer, tremendous progress has been made in the development of strategies for immune checkpoint inhibition; in refinement of agents and approaches used in targeted therapy; and in techniques for molecular subtyping of tumor samples that have identified patient subgroups with clinically relevant cellular differences potentially affecting clinical management and treatment outcome. In this article, we discuss several of the commonly tested markers in colorectal cancer—including microsatellite instability, RAS/RAF, DPD, HER2, UTG1A1, TS, and Immunoscore—and highlight their prevalence, prognostic and predictive value, and current role in the overall treatment paradigm.
Colorectal cancer is the fourth most common cancer diagnosed in the United States and the second leading cause of cancer-related death. Approximately 140,000 cases are diagnosed annually in the United States, and 1 in 5 patients have metastatic disease at the time of diagnosis. Advances in management of colorectal cancer, especially of metastatic disease, have increased the median overall survival (OS) time from 12 months to more than 30 months, due in large part to gains over the last few years in the development of targeted therapy and molecular testing. Historically, prognostic and predictive markers in colorectal cancer have relied on clinical and pathologic features only. For example, over the last few decades, lymph node positivity and the presence of metastasis were the only prognostic/predictive markers in colorectal cancer.
In contrast, the postgenomic era has been marked by the evolution of tumor molecular profiling, with the discovery of multiple new oncologic markers for use not only with targeted therapy but also with standard cytotoxic therapy. The information gained from tumor testing allows us to begin to move away from the currently employed trial-and-error methods and towards selecting the right patient for the right treatment. In this review, we discuss several of the most commonly tested markers in colorectal cancer and explore the rapidly changing landscape into which oncologists must integrate an ever-increasing array of molecular information. We highlight the new treatment opportunities that molecular testing is beginning to provide, and describe the ways in which clinicians can optimize the use of newly available molecular-based tools in the face of the rising cost of cancer care.
We also discuss the current standard of care for colorectal cancer, which includes testing for the RAS gene and microsatellite instability (MSI)/microsatellite stability (MSS) in tumor cells. We describe other recommended genetic evaluations, including testing for mutations of BRAF, UGT1A1, and DPD, and for amplification of HER2. Recent studies of testing for expression of the TS gene and TS protein, often considered to be confined to the research setting, are also evaluated. Finally, we highlight biomarkers for which there is emerging evidence of clinical relevance in colorectal cancer but currently no widespread adoption into clinical practice, including Immunoscore and consensus molecular subtypes.
MSI/Mismatch Repair (MMR) Status
MMR status should be tested in all patients with colorectal cancer, independent of the initial stage of disease. Microsatellites are short, repetitive nucleotide sequences of variable length that are distributed throughout genomic DNA, and MMR proteins are DNA repair enzymes that correct insertion/deletion loops, as well as base pair mismatches that occur during DNA replication. Approximately 20% of colorectal cancer tumors have MSI, a condition of genetic hypermutability characterized by truncation or expansion of microsatellites caused directly by impaired MMR enzyme activity. Dysfunctional MMR activity may result from a germline mutation in one of the four major MMR genes: MLH1, MSH2, MSH6, and PMS2. Germline MMR gene mutation is observed in 15% to 20% of patients with colorectal cancer; a mutation in any of the four MMR genes represents a defining clinical feature of Lynch syndrome, which occurs in 3% to 5% of cases. Disruption of MMR activity also may occur sporadically (as seen in 80% of colorectal cancer patients) as a consequence of methylation of CpG islands (regions with at least 200 base pairs in which the frequency of the cytosine-guanine sequence is higher than in other regions, with “p” simply indicating that “C” and “G” are connected by a phosphodiester bond) in MMR promoter sequences (most commonly in MLH1).[5,6] A subset of MMR-deficient (MMRD) tumors appears to be sporadically associated with the CpG island methylator phenotype (CIMP). The presence of CIMP-mediated MSI tumors is inversely correlated to that of tumors with the chromosomal instability pathway (CIN) phenotype, suggesting little overlap between the molecular mechanisms underlying CIMP vs CIN; also, CIMP-positive cases tend to have activating mutations in BRAF.[3,4,7]
MMR status can be evaluated by MSI testing, which is typically performed using polymerase chain reaction (PCR) to amplify a standard panel of microsatellite sequences in both tumor and normal tissue from the same patient. A positive result, MSI-high (MSI-H), is defined as expansion or contraction of at least 40% of microsatellite sequences in the tumor compared with normal DNA; a status of MSI-low (MSI-L), or MSS, is considered negative. Alternatively, tumors can be evaluated for expression of four major MMR proteins (MLH1, MSH2, MSH6, and PMS2) using immunohistochemistry (IHC) testing, in which the loss of nuclear expression, or MMRD status, is closely correlated with having an MSI-H status. IHC results of MMRD and/or MSI-H typically warrant PCR-based testing for germline mutations, to determine whether the patient has Lynch syndrome. The sensitivity of IHC and PCR testing methods is similar; however, MMR testing using IHC is more practical and less expensive. Algorithms are evolving to include testing for somatic BRAF mutations and MLH1 promoter hypermethylation, the presence of which effectively precludes the need for testing tumor samples for germline mutations associated with Lynch syndrome.
Most studies indicate that MSI-H status in colorectal cancer is prognostic of a better clinical outcome compared with MSI-L/MSS disease. In one study, colorectal cancer–specific mortality hazard ratios (HRs) for MSI-H vs MSI-L/MSS were 0.48 (95% CI, 0.27–0.87; P = .02) and 0.25 (95% CI, 0.12–0.52; P < .001) for BRAF-mutated and wild-type disease, respectively. Other studies suggest that the favorable prognosis of MSI-H may be limited to patients without BRAF mutations. In the adjuvant setting, MSI-H status is a predictive marker for lack of response to fluorouracil (5-FU)-based chemotherapy compared with MSS disease. In fact, in one study of 570 patients, there was a statistical trend towards worse disease-free survival and OS in patients with MSI-H disease treated with adjuvant 5-FU-based chemotherapy compared with patients who did not receive 5-FU. Consequently, the presence of MSI-H is often used to identify patients with stage II disease who should not be offered adjuvant chemotherapy. It is unknown whether or not patients with MSI-H stage II disease and BRAF mutations benefit from adjuvant chemotherapy. The role of MSI status in decision making regarding chemotherapy for stage III colorectal cancer is less clear.
MSI-H does not appear to have the same predictive value for chemotherapy response in the metastatic setting. In one study, the HR for response was 0.82 (95% CI, 0.65–1.03; P = .09), reflecting a trend towards a better treatment outcome than that of patients with MSI-L/MSS tumors but failing to reach statistical significance. However, recent data suggest that patients with MSI-H metastatic disease respond preferentially to immune checkpoint inhibition with the programmed death 1 (PD-1) inhibitor pembrolizumab. In a recent phase II study of 41 heavily pretreated patients with progressive metastatic carcinoma, the objective response rate (ORR) based on immune-related response criteria was 40% (4 of 10 patients) and the progression-free survival (PFS) rate was 78% (7 of 9 patients) in the patients with MSI-H colorectal cancer. In the patients with MMR-proficient tumors (ie, MSS tumors, MSI-L tumors, and tumors with intact MMR proteins), the corresponding response rates were 0% (0 of 8 patients) and 11% (2 of 18 patients), respectively.[12,13] Notably, the microenvironment in MMRD tumors (those with MSI; ie, large numbers of genomic mutations, particularly in areas of repetitive DNA sequences) has been found to be highly immunogenic. In addition to a microenvironment characterized by dense infiltration with immune cells and T helper type 1–associated cytokines,[14,15] recent studies have shown that MMRD tumors strongly express PD-1, programmed death ligand 1 (PD-L1), cytotoxic T-lymphocyte–associated antigen 4, the LAG3 protein, and indoleamine 2,3-dioxygenase; these findings suggest that the immune-active microenvironment of MMRD tumors is counterbalanced by immune inhibitory signals resisting tumor cell apoptosis, and that these signals represent targets for immune checkpoint inhibitor therapy.[14,15] Larger, prospective studies are needed in order to confirm this clinical observation. Additional studies are also needed to clarify the role of immune checkpoint inhibition in both untreated metastatic disease and in high-risk stage II and III colorectal cancer with MSI-H.
The greater susceptibility to immune checkpoint inhibition in tumors with MMRD has been hypothesized to stem from the high numbers of mutation-associated tumor neoantigens arising from the many somatic mutations occurring in tumors with MSI. In fact, Le et al found that MMRD tumors carried a mean of 1,782 somatic mutations per tumor and an average of 578 potential mutation-associated neoantigens, whereas tumor samples from patients with MMR-proficient colorectal cancers had a mean of 73 mutations per tumor and an average of 21 mutation-associated neoantigens. Recognition of mutation-associated neoantigens is an important component of the endogenous antitumor immune response; this has been supported by research showing that higher mutational load in patients with bladder cancer, melanoma, and lung cancer was correlated to a better response to treatment with various immune checkpoint inhibitors.[14,16-18] Notably, a study from Memorial Sloan Kettering Cancer Center showed that using next-generation sequencing panels to evaluate mutational load in colorectal cancer provided a reliable surrogate for MSI testing to identify patients who could potentially respond to treatment with immune checkpoint inhibitors. Indeed, high-throughput sequencing technologies could also be used to identify oncogenic drivers and therapeutic targets in tumor tissue, as well as in the evaluation of the immunogenicity of the tumor microenvironment.
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