Historically, treatment for colorectal cancer (CRC) has been guided primarily by cancer stage, morphology, and family history. However, as technological developments over the last decade have reduced the turnaround time and cost of molecular methods, DNA testing of tumor tissue has rapidly become the standard of practice for personalizing treatment, particularly in the case of metastatic disease.
Predictive biomarkers help direct therapy decisions by providing information on differences in treatment response in biomarker-positive patients compared to biomarker-negative patients. Molecular analysis of tumor tissue also provides treatment-independent prognostic information on outcomes such as overall survival (OS) time or progression-free survival time.
In 2017, the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology established evidence-based guidelines for molecular biomarker testing of colorectal tumor tissue as an aid in directing treatment (3). The guideline committee reviewed 123 articles published from January 1, 2008, to February 12, 2015, and established 21 guidelines. Six of these centered on specific tumor tissue biomarkers (NRAS, KRAS, BRAF, PIK3CA, PTEN) and mismatch repair (MMR) testing that may determine etiology, stratify patients by prognosis, or measure treatment response. We expand on five of these guideline statements in this article.
A Molecular History of CRC
CRC is a heterogeneous disease resulting from the accumulation of genetic and epigenetic alterations. The etiology of CRC impacts treatment, prognosis, management, and surveillance frequency.
The overall 5-year CRC survival rate is approximately 65%, with the main prognostic factor for survival being cancer stage at diagnosis. The 5-year survival rate is approximately 90% for Stage I and declines to about 70% for Stage II, 58% for Stage III, and less than 15% for Stage IV, with mortality largely attributed to metastasis (1). At the time of diagnosis, 25% of CRC patients present with metastasis and nearly 50% of all patients with CRC will develop metastasis, with the liver being the most common site (2). Early intervention and successful resection of the primary tumor are often curative; however, early stage disease typically does not present with symptoms, highlighting the importance of CRC screening programs.
Colonoscopy remains the gold standard CRC screening method but suffers from low compliance due to the invasive nature of the test. Noninvasive stool-based screening tests such as fecal occult blood tests (FOBT) and fecal immunochemical tests likewise have less than ideal participation rates. The dietary restrictions patients need to adhere to before FOBT also limit its uptake, and this method generally has low overall sensitivity.
The serum-based tumor marker carcinoembryonic antigen (CEA) has low sensitivity and specificity for CRC, particularly in the early stages when resection would have the most impact. Consequently, CEA testing is only recommended for monitoring cancer recurrence, not detecting the disease.
Most CRC tumors are sporadic (70%–80%), and age remains the greatest risk factor. The genes most commonly mutated in CRC include APC (in about 80%–82% of cases), TP53 (48%–59%), KRAS (40%–45%), and PIK3CA (14%–18%); however, numerous other genes show mutations at significantly lower frequencies (4).
The pattern of mutations and epigenetic alterations of these genes influence how normal colon tissue progresses to CRC. The two most common pathways of tumor development are chromosomal instability (CIN) and microsatellite instability (MSI), responsible for 60%-75% and 10%–20% of CRC cases, respectively.
CIN is the most common pathway of CRC pathogenesis and also the cause of most sporadic CRC (5). The hypothesis is that as adenomatous tissue grows, it also accumulates genetic mutations or epigenetic changes to gene expression. Notably, only a small percentage of adenomatous polyps progress to CRC. Tumors arising in this pathway typically have aneuploidy and multiple somatic mutations. These can include loss of heterozygosity of APC and/or TP53 genes as well as activating mutations in KRAS and NRAS.
MSI drives the remaining 10%–20% of sporadic CRC and can be detected as alterations in the length of DNA microsatellite sequences that lead to a very high level of mutations. Functional defects in the DNA MMR system cause MSI-related tumors (5).
Biomarkers Used to Determine Etiology
Guideline Statement #2b BRAF p.V600 mutational analysis should be performed in deficient MMR tumors with loss of MLH1 to evaluate for Lynch Syndrome Risk. Presence of BRAF mutation strongly favors a sporadic pathogenesis. The absence of BRAF mutation does not exclude risk of Lynch syndrome. Strength of Recommendation: Recommendation
MSI occurs in a minority of sporadic CRC cases. In about 75% of these cases, MSI arises from epigenetic silencing via CpG methylation of the promoter for the MLH1 gene, one of the four MMR genes. Sporadic mutations in the other MMR genes (MSH2, MSH6, and PMS2) also occur, albeit more rarely. In addition to a sporadic pathogenesis, MSI arises from germline mutations to one of the four MMR genes or the EPCAM gene. These are the causative mutations of Lynch syndrome, the most common hereditary cause of CRC, accounting for between 2% and 4% of all CRC.
For tumors demonstrating a loss of MLH1, the current recommendation is to perform BRAF p.V600 mutational analysis of the tumor tissue. The BRAF p.V600 mutation is rarely associated with the germline mutations found in Lynch syndrome but occurs in approximately 75% of epigenetically silenced MLH1 in sporadic MSI tumors. As noted in the guideline statement, the presence of the BRAF mutation strongly suggests that the etiology of the disease is sporadic, rather than hereditary.
While the treatment modalities for sporadic and Lynch syndrome tumors may not differ, identifying patients with Lynch syndrome is important, as it is inherited in an autosomal dominant manner and increases the risk of endometrial, ovarian, gastric, and other cancers (6, 7).
Biomarkers for Predicting Treatment Response
Guideline Statement #1: Patients with colorectal carcinoma being considered for anti-EGFR therapy must receive RAS mutational testing. Mutational analysis should include KRAS and NRAS codons 12 and 13 of exon 2, 59 and 61 of exon 3, and 117 and 146 of exon 4 (“expanded” or “extended” RAS). Strength of Recommendation: Recommendation.
Patients with unresectable or partially resectable metastatic CRC may benefit from adding anti-epidermal growth factor receptor (EGFR)-targeted monoclonal antibody therapies (cetuximab and panitumumab) to their standard chemotherapy regimen. Early studies examining the effectiveness of anti-EGFR therapy demonstrated that this approach improved overall response and reduced risk of disease progression when compared to a standard chemotherapy regimen (2, 8).
While impressive, these initial studies examined the effect of anti-EGFR therapy in an unselected population and found that less than 20% of participants benefited (9). Subsequent studies demonstrated that patients with activating mutations in downstream effectors of EGFR such as KRAS and NRAS had significantly worse response rates (10-13). These activating mutations in KRAS and NRAS produce effectors that are independent of EGFR’s binding to its ligand, rendering the monoclonal antibody therapy ineffective. This applies to a relatively large population of CRC patients as approximately 40% have an activating KRAS mutation, and 7% have an activating NRAS mutation. Patients with wild-type KRAS and NRAS have a significantly improved overall response to anti-EGFR therapy with longer progression-free survival and a higher 5-year OS rate.
These studies also found that KRAS and NRAS mutation status does not influence patient OS for those only receiving supportive care, reinforcing RAS mutational status as a predictive rather than prognostic biomarker.
Guideline Statement #4: There is insufficient evidence to recommend BRAF c.1799 p.V600 mutational status as a predictive molecular biomarker for response of anti-EGFR inhibitors. Strength of Recommendation: No recommendation.
The BRAF activating mutation BRAF c.1799 p.V600 occurs in approximately 8%–12% of patients with stage IV CRC and about 14% of patients with stage II and III disease. This and the RAS mutations often are mutually exclusive (13). The low prevalence of BRAF mutations clouds their predictive value in patients with stage IV CRC, and testing for whether BRAF-mutant tumors are resistant to anti-EGFR antibody remains controversial.
Published studies have yielded varied and conflicting conclusions. Several have reported that patients with this mutation have a poorer response rate to chemotherapy in combination with cetuximab compared to patients with wild-type BRAF; however, a modest beneficial impact from adding anti-EGFR agents also has been reported (13-15).
Guideline Statement #2a BRAF p.V600 (BRAF c.1799 [p.V600]) mutational status should be performed in colorectal tissue in patients with colorectal carcinoma for prognostic stratification. Strength of Recommendation: Recommendation.
This recommendation is supported by numerous publications that have demonstrated that stage II-IV patients with BRAF p.V600 mutations have shorter progression-free survival and OS time (16). While there is currently insufficient evidence to determine whether patients with the BRAF p.V600 mutation benefit from anti-EGFR therapy, standard treatment regimens are likely insufficient for this population. There also are encouraging findings from clinical trials exploring the combination of standard anti-EGFR therapy with novel BRAF inhibitors.
Guideline Statement #3 Clinicians should order mismatch repair status testing in patients with CRCs for the identification of patients at high risk for Lynch syndrome and/or prognostic stratification. Strength of Recommendation: Recommendation.
In addition to identifying patients at risk for Lynch syndrome, MMR testing provides prognostic data for sporadic CRC. Patients with early stage MSI tumors have a better prognosis than those with microsatellite stable tumors. In a meta-analysis summarizing 20 studies that included 9,243 patients, patients with a high level of MSI had a longer OS time. Patients with MSI-positive tumors also had longer overall disease-free survival than patients without MSI-positive tumors (17).
In addition to the prognostic value of MSI status, emerging data indicates that patients with tumors positive for MMR defects may have a better response rate to immune checkpoint inhibitors such as pembrolizumab (18).
Other Potential Biomarkers
Guideline Statement #5 There is insufficient evidence to recommend PIK3CA mutational analysis of colorectal carcinoma tissue for therapy selection outside of a clinical trial. Strength of Recommendation: No recommendation.
Despite screening, some patients with wild-type RAS mutations still fail to respond to anti-EGFR monoclonal therapy. Activating mutations in KRAS and NRAS account for just 40% of anti-EGFR-resistant stage IV CRC patients, suggesting the possibility of other potential negative predictive biomarkers.
Several studies have evaluated PIK3CA as a negative predictive marker to anti-EGFR monoclonal antibodies. Approximately 40% of PIK3CA mutations co-occur with RAS mutations and nearly 50% of PIKC3A co-occur with BRAF mutations, making it difficult to elucidate the importance of PIKC3A as an independent predictive and prognostic marker (13). To understand these effects, more studies are needed with sufficiently large cohorts of patients with wild-type NRAS, wild-type BRAF, and mutant PIK3CA.
Some evidence also suggests that another gene, TP53, holds promise as a predictive biomarker to assess treatment response. TP53 is the most frequent somatic gene mutation in human cancer and is found in approximately half of all adenocarcinomas, making it an exciting potential target for personalized therapy (4, 21).
TP53 has been reported to predict the effect of adjuvant 5-fluorouracil therapy in patients with stage III (N1) CRC (19). In addition, mutant-TP53 patients with metastatic CRC who received neo-adjuvant chemotherapy had statistically significant poorer outcomes, with decreased 5-year OS rate (20). The role of TP53 as a prognostic biomarker is still being evaluated: The same study reported no difference in the rate of 5-year OS for mutant-TP53 and wild-type TP53 patients who did not receive neo-adjuvant therapy (20).
Unlike KRAS, which harbors several high-frequency mutations, numerous discrete TP53 mutations occur at relatively low frequencies. One study of 456 CRC patients detected more than 130 discrete mutations, with the two most prevalent accounting for just 14% of TP53-positive mutations and 7% of all CRCs tested (21). Future studies coupling discrete mutational status with treatment response and overall survival could be immensely valuable for developing exquisitely personalized therapies.
Available treatments for CRC continue to advance, and researchers rapidly are discovering new information about tumorigenesis pathways. These insights are now being translated into new potential treatments in clinical trials targeted to the unique features of each patient’s tumor composition, with the goal of improving OS of CRC patients. Continued research will hopefully lead to finely and precisely tailored treatments.
Zahra Shajani-Yi, PhD, NRCC, is an assistant professor at Vanderbilt University School of Medicine in the department of pathology, microbiology and immunology. She is also associate director of the clinical chemistry laboratory and medical director of esoteric chemistry at Vanderbilt University Medical Center in Nashville, Tennessee.+Email: email@example.com
Mark A. Cervinski, PhD, DABCC, FAACC, is an associate professor at the Geisel School of Medicine at Dartmouth in the department of pathology and laboratory medicine. He is also director of the clinical chemistry laboratory and point-of-care testing at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire.+Email: firstname.lastname@example.org
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