Molecular pathology and biomarkers – implications for systemic therapy
Increasingly, biomarker expression is driving therapeutic decision-making in medicine. Obtaining tissue to confirm the diagnosis of suspected colorectal cancer is fundamental prior to commencement of systemic therapy.
Overview of evidence (non-systematic literature review)[edit source]
No systematic reviews were undertaken for this topic. Practice points were based on selected reviews, primary studies, and other clinical practice guidelines (see Guideline development process).
RAS mutation testing[edit source]
Among patients with metastatic colorectal cancer, RAS mutation status permits clinicians to identify individuals who might benefit from strategies targeting the epidermal growth factor receptor (EGFR). Anti-EGFR monoclonal antibodies (cetuximab and panitumumab) should only be prescribed for patients whose tumours are RAS wild-type. As yet, there are no accepted biologic or molecular markers of responsiveness to bevacizumab or to conventional cytotoxic chemotherapy agents, although these are active areas of research.
Tumour overexpression of several genes involved in the EGFR signalling pathway and downstream events might identify patients who are most likely to respond to anti-EGFR agents. It is now well established that activating mutations in KRAS, which result in constitutive activation of the RAS-RAF-ERK pathway, result in resistance to anti-EGFR therapy. Activating mutations in KRAS are detected in approximately 40% of metastatic colorectal cancers.
In metastatic colorectal cancer, KRAS mutations are mainly found in exon 2 (codons 12, 13). Retrospective analyses of pivotal clinical trials for the anti-EGFR monoclonal antibodies, cetuximab and panitumumab, have shown that patients with metastatic colorectal cancer whose tumours contain activating mutations in KRAS exon 2 (codons 12, 13) do not derive a benefit from EGFR monoclonal antibody therapy. Furthermore, evidence from the PRIME study with panitumumab, from the CRYSTAL study with cetuximab, and from other studies of EGFR monoclonal antibody therapies, has shown that mutations other than those in KRAS exon 2 – i.e. exons 3 and 4 of KRAS and exons 2, 3 and 4 of NRAS (extended RAS analysis) – also predict a lack of response to EGFR-targeting monoclonal antibodies and that these therapies may, in fact, have a detrimental effect in patients with RAS-mutant disease, specifically when combined with an oxaliplatin-based cytotoxic backbone.
These findings were supported by results from the phase II PEAK study, in which patients with KRAS and NRAS exon 2, 3 and 4 wild-type metastatic colorectal cancer treated with the combination of leucovorin calcium (folinic acid), 5-fluorouracil (5FU) and oxaliplatin (FOLFOX) regimen ‘6’ (FOLFOX6) plus panitumumab showed longer progression-free survival than those treated with FOLFOX6 plus bevacizumab, and a trend towards improved overall survival.
Next-generation sequencing techniques to identify additional RAS-activating mutations were used to analyses tumour samples previously tested for KRAS exon 2 mutations from patients previously enrolled in the phase III trial of panitumumab in chemorefractory metastatic colorectal cancer. When treated with panitumumab, patients with RAS wild-type tumours achieved response rates with of 15%, compared with 1% among those with RAS-mutant tumours.
Similar findings have been reported with cetuximab in patients with RAS wild-type tumours (according to extended RAS analysis). The addition of cetuximab to FOLFOX regimen ‘4’ (FOLFOX4) or to the combination of folinic acid, 5FU and irinotecan hydrochloride (FOLFIRI) was associated with improved treatment outcomes across all efficacy end points.
The importance of extended RAS testing was accentuated in the phase III FIRE-3 trial, in which patients with previously untreated metastatic colorectal cancer with RAS wild-type tumours receiving FOLFIRI and cetuximab showed an improvement in overall survival, compared with patients with RAS mutation receiving the same regimen (median 33.1 versus 28.7 months).
The weight of evidence indicates that anti-EGFR monoclonal antibody therapy should be restricted to those patients whose tumours lack mutations after extended RAS testing.
Harbouring a RAS mutation is therefore a negative predictive marker of treatment outcome in patients with metastatic colorectal cancer who receive anti-EGFR therapies. Extended RAS testing is therefore required for all patients who are candidates for anti-EGFR therapy. To allow for the development of a strategic management plan, patients with metastatic colorectal cancer should have their tumour tested for RAS mutations at the time of diagnosis of their metastatic disease.
RAS testing should be carried out on all patients at the time of diagnosis of metastatic colorectal cancer.
RAS mutational status is a negative predictive biomarker for therapeutic choices involving EGFR antibody therapies in metastatic colorectal cancer.
Cetuximab and panitumumab should only be considered for the treatment of patients with RAS wild-type metastatic colorectal cancer.
BRAF mutation testing[edit source]
BRAF is a component of the RAS-RAF-MAPK signalling pathway. Activating mutations, which are mutually exclusive with KRAS mutations, are found in approximately 5–10% of metastatic colorectal cancers.
BRAF mutations (most of which are V600E mutations) have consistently been associated with poor prognosis overall and as such their presence is considered to be a negative prognostic marker in metastatic colorectal cancer patients. An Australian retrospective analysis of patients with metastatic colorectal cancer demonstrated that two-thirds of BRAF-mutant patients’ primary lesions were located on the right side of the colon and associated with an increased incidence of peritoneal and distant lymph node metastases, but fewer pulmonary metastases. This study also reported a median survival of 10.4 months among patients with BRAF-mutant tumours, compared with 34.7 months for patients with BRAF wild-type tumours.
Moreover, BRAF mutations also appear to have predictive value, according to accumulating data. Evidence increasingly suggests that response to EGFR-targeted agents is less likely in patients whose tumours harbor BRAF mutations (particularly the BRAF V600E mutation).
At least two meta-analyses have addressed the efficacy of EGFR antibody therapies in patients with RAS wild-type/BRAF mutated tumours. Although neither analysis found a survival advantage for the addition of EGFR antibody therapy, they reached somewhat different conclusions:
- The first meta-analysis included 10 randomised controlled trials (RCTs) comparing cetuximab or panitumumab alone or plus chemotherapy with standard therapy or best supportive care (one phase II and nine phase III trials). Six trials were conducted in the first-line treatment setting, two for second-line therapy and two in patients with chemorefractory disease. Among patients with RAS wild-type/BRAF-mutant tumours, compared with control regimens, the addition of an anti-EGFR monoclonal antibody did not significantly improve progression-free survival (hazard ratio [HR] 0.88, 95% confidence interval [CI] 0.67 to 1.14), overall survival (HR 0.91, 95% CI 0.62 to 1.34), or objective response rate (relative risk [RR] 1.31, 95% CI 0.83 to 2.08).
- The second meta-analysis included eight RTCs; four conducted in the first-line setting, three in the second-line setting, and one in patients with chemorefractory disease. Among patients with RAS wild-type/BRAF mutant metastatic colorectal cancer, there was no significant overall survival benefit for the addition of anti-EGFR therapies (HR 0.97, 95% CI 0.67 to 1.41). In contrast, overall survival was significantly greater in patients with RAS wild-type BRAF wild-type tumours (HR 0.81; 95% CI 0.7 to 0.95). When comparing the overall survival benefit between BRAF mutant and BRAF wild-type tumours, the test for interaction was not statistically significant. The authors concluded that the observed differences in the effect of anti-EGFR therapies on overall survival according to BRAF mutation status could have been due to chance, and that the evidence was insufficient to state that BRAF-mutant tumours attain a different treatment benefit from anti-EGFR agents compared to individuals with BRAF wild-type tumours.
Results from TRIBE study has shown promising outcomes for patients with BRAF-mutated tumours treated with aggressive systemic therapy consisting of leucovorin calcium (folinic acid), 5FU, oxaliplatin and irinotecan hydrochloride (FOLFOXIRI) plus bevacizumab. In this trial, patients with metastatic colorectal cancer who received FOLFOXIRI plus bevacizumab showed 2.5 months longer progression-free survival than those who were treated with FOLFIRI. However, the overall survival results remained disappointing for patients with BRAF-mutated tumours, compared with those with BRAF wild-type tumours (19.0 months versus 41.7 months).
Clinical trials are currently underway to test targeted therapies in BRAF-mutated metastatic colorectal cancer, akin to the development of therapies for BRAF-mutated metastatic melanoma. Early results are promising but have generally been less favourable than the melanoma trials. Early studies evaluating single-agent BRAF inhibitor therapy or combination BRAF/mitogen-activated protein kinase (MEK) inhibition has yielded disappointing results.
EGFR activation has been implicated in the pathogenesis of BRAF mutant colorectal cancer. Therefore, the combination of BRAF/MEK inhibition and anti-EGFR therapy has recently been evaluated in a trial comparing (i) dabrafenib plus panitumumab, (ii) trametinib plus panitumumab, and (iii) the combination of dabrafenib, trametinib and panitumumab. In the dabrafenib/panitumumab treatment arm, the objective response rate was 10%, and 80% of patients achieved stable disease. With trametinib/panitumumab no patients attained objective response but 53% showed stable disease. However, combined BRAF/MEK inhibition with panitumumab yielded an 18% objective response rate and 67% of patients showed stable disease.
Somatic BRAF V600E mutations have been associated with sporadic cases of DNA mismatch repair deficiency showing microsatellite instability stability (MSI) phenotype. On the contrary, BRAF V600E mutation is not associated with the MSI phenotype due to a germline mutation in mismatch repair (Lynch Syndrome). BRAF V600E mutations have been proposed as a means of excluding Lynch syndrome. Subsets of patients with BRAF mutations in codons 594 and 596 have been shown to have microsatellite stability and significantly longer survival times, compared with those who have BRAF V600E disease.
The BRAF mutation status should ideally be performed at the time of diagnosis of metastatic colorectal cancer, as this represents a distinct biologic subtype.
The presence of a BRAF mutation in metastatic colorectal cancer is considered a poor prognostic marker.
BRAF mutation status in combination with testing for DNA mismatch repair deficiency can assist in the identification of a germline versus somatic cause of DNA mismatch repair deficiency.
The preponderance of the available evidence is that response to EGFR-targeted agents is less likely in patients whose tumours harbour a BRAF mutation.
Metastatic colorectal cancer patients with a BRAF mutation should be considered for a clinical trial where available or triplet chemotherapy if suitable.
Microsatellite instability (MSI) testing[edit source]
Approximately 10% of colorectal carcinomas demonstrate MSI. Distinct from the majority of colorectal cancers with chromosomal instability, tumours with MSI retain intact chromosomal numbers but contain microsatellite repeats due to deficiency in DNA mismatch repair which are thought to contribute to the early steps of tumorigenesis in colorectal cancer. While emerging clinical data have highlighted improved prognosis of tumours with MSI in early colorectal cancer, potentially circumventing the need for adjuvant chemotherapy, the implications of MSI in metastatic colorectal cancer remain uncertain.
A retrospective analysis in patients with metastatic colorectal cancer observed that MSI phenotype was associated with younger age (median 67 years), higher risk of poor differentiation (58%), and a higher risk of stage IV disease at presentation 45%. BRAF V600E mutations were present in 30% of patients with MSI.
Most studies have shown MSI not to be relevant as a predictive marker for various chemotherapeutic agents. However in a pooled analysis of four phase III studies (CAIRO, CAIRO2, COIN and FOCUS), BRAF mutations have been shown to be more frequent in patients with tumours exhibiting MSI than in those with microsatellite-stable tumours. Furthermore, in this analysis, progression-free survival and overall survival were significantly worse for patients with tumours with MSI, compared with those with microsatellite-stable tumours (HR, 1.33; 95% CI 1.12 to 1.57 and HR 1.35; 95% CI 1.13 to 1.61, respectively), and for patients with BRAF-mutant tumours when compared with those with BRAF wild-type tumours (HR 1.34; 95% CI 1.17 to 1.54 and HR 1.91; 95% CI 1.66–2.19, respectively).
Emerging data have shown DNA mismatch repair status to predict the clinical benefit of immune checkpoint blockade with pembrolizumab in patients with metastatic colorectal cancer. A phase II study evaluating pembrolizumab in patients with colorectal cancer reported immune-related objective response rates and immune-related 6-month progression-free survival rates of 40% (4 out of 10 patients) and 78% (7 out of 9 patients), respectively, for patients with DNA mismatch repair deficiency tumours, and 0% and 11% for those with DNA mismatch repair-proficient tumours. The study reported excellent rates of median progress-free survival and overall survival (maturity not reached) in the cohort with DNA mismatch repair deficiency tumours versus 2.2 and 5.0 months, respectively, in the cohort with DNA mismatch repair-proficient tumours.
CheckMate-142 is a phase II study evaluating the role of nivolumab, alone or in combination with ipilimumab, in heavily pre-treated MSI-high colorectal cancer. This study also had a cohort of non-MSI patients. In preliminary results, the objective response rate in the nivolumab-alone arm was 27%, compared with 15% in the combination treatment arm. Stable disease was reported in 24% in the nivolumab arm and 65% in the combination treatment arm. Median overall survival was more than 16 months in the nivolumab arm and has not been reached in the combination arm.
While these data provide proof of principle as to the potential for benefit from immunotherapy in metastatic colorectal cancer, it is premature to conclude, based upon these small studies, that immune checkpoint inhibitors represent a standard treatment for metastatic DNA mismatch repair-deficient colorectal cancer. Confirmation in larger data sets is needed, as is further exploration of the data from these trials, to understand why there was a complete lack of response in microsatellite-stable tumours, which represents the vast majority of patients with metastatic colorectal cancer.
MSI testing in the metastatic setting can be useful to help identify patients who require referral for further genetic testing and counselling.
BRAF V600 mutational analysis should be done in conjunction with MSI testing for prognostic stratification.
MSI testing may be a predictive marker for the use of immune checkpoint inhibitors in the treatment of patients with metastatic colorectal cancer.
Emerging biomarkers[edit source]
There is a growing list of additional biomarkers that may impact on responses to agents we have available for the treatment of metastatic colorectal cancer. At the present time, emerging biomarkers are not recommended for routine patient management outside of clinical trial settings.
In particular, there is a growing list of biomarkers beyond RAS mutations that may influence responses to EGFR targeted therapies. These include HER2, MET and KRAS gene amplification, ligands such as transforming growth factor-α (TGF-α), amphiregulin and epiregulin, EGFR mutations and alterations/mutations in HER3, PI3KCAand PTEN.
It seems likely in the future that a comprehensive biomarker analysis will be required to identify the subgroup of patients with metastatic colorectal cancer who will truly benefit from treatment with an anti-EGFR agent.
Although metastatic colorectal cancer is primarily considered to be a genetic disease characterised by the sequential accumulation of genetic of genetic mutations, evidence now suggests that epigenetic alterations add further complexity to pathogenesis, aetiology and prognosis of subgroups of the disease.
Emerging biomarkers are not recommended for routine patient management outside of the clinical trial setting.
Left-sided versus right-sided tumours[edit source]
Evidence is emerging to support the premise that left-sided and right-sided colon tumours have clinically significant differences. They differ with respect to biology, pathology and epidemiology, and previous data suggest a mortality difference between left- and right-sided colon tumours. Patients with right-sided colon tumour tend to have more poorly differentiated, higher incidence of mutant KRAS, mutated PIK3CA and mutant BRAF tumours, fewer liver and lung metastases, and shorter interval between diagnosis and study entry.
Two recent meta-analyses have provided data on the prognostic and predictive value of primary tumour location in patients with RAS wild-type metastatic colorectal cancer:
- The first meta-analysis included five first line randomised controlled studies and performed two separate analyses. One evaluated the predictive relevance of primary tumour location for anti-EGFR therapy combined with standard chemotherapy compared with chemotherapy alone (CRYSTAL and PRIME studies), and the other evaluated the impact of primary tumour location on therapy with either anti-EGFR plus chemotherapy or anti-VEGFR combined with chemotherapy (CALGB/SWOG 80405, FIRE-3 and PEAK studies). In addition, 14 first line studies were evaluated assessing prognostic impact of primary tumour location.
- The second meta-analysis included the same 5 first line randomised controlled studies (CRYSTAL, PRIME, PEAK, FIRE-3 and CALGB 80405) and one second line study (20050181) and performed a pooled analysis of all 6 trials.
The primary tumour was located in the right colon in 27% of patients in the first meta-analysis and 23.9% in the second. Both meta-analyses showed that right sided colon cancer was associated with a poorer prognosis. Overall survival for right sided colon cancer remained below 20 months in many studies, and in both the first and second line setting patients with right sided colon cancer had a worse prognosis regardless of treatment type received. However, this was numerically less pronounced and not statistically significant in those patients receiving chemotherapy and bevacizumab in the CALGB trial.
In terms of predictive role of primary tumour location, both meta-analyses showed a significant benefit from the addition of anti-EGFR therapy in patients with left sided colon cancer. In the meta-analysis by Holch et al a significant benefit was seen with the addition of anti-EGFR therapy to chemotherapy compared with chemo-therapy alone for both overall survival (HR 0.69; 95% CI 0.58-0.83, p<0.0001), progression free survival (HR 0.65; 95% CI 0.44-0.88, p=0.008) and response rate. When comparing chemotherapy plus anti-EGFR to chemotherapy plus anti-VEGF therapy, left sided colon cancer was associated with improved outcomes in those who received anti-EGFR therapy. This benefit was significant for overall survival (HR 0.71; 95% CI 0.58-0.85, p=0.0003), and response rate (HR 1.49; 95% CI 1.16-1.9, p=0.002) but not for progression free survival (HR 0.86; 95% CI0.73-1.02, p=0.084).
These findings were confirmed in the meta-analysis by Arnold et al which performed a pooled analysis of all six included studies comparing chemotherapy plus anti-EGFR to chemotherapy +/- bevacizumab and observed a significant benefit of chemotherapy plus anti-EGFR in left sided colon cancers for both overall survival (HR 0.75; 95% CI 0.67-0.84, p<0.001) and progression free survival (HR 0.78; 95% CI 0.70-0.87, p<0.001).
In contrast, the Holch et al meta-analysis did not show any significant benefit to the addition of an anti-EGFR to chemotherapy in right sided colon cancer, however the sample size was small (right sided n=172). When comparing chemotherapy plus anti-EGFR to chemotherapy plus anti-VEGF there was a significant improvement in progression free survival for those who received anti-VEGF (HR 1.53; 95% CI 1.16-2.01, p=0.003), and a trend to improved overall survival which was not statistically significant (HR 1.3; 95% CI 0.97-1.74, p=0.081). There was no significant difference between the two treatment in terms of response rate but numerically favoured anti-EGFR (HR1.2; 95% CI 0.77-1.87, p=0.432).
These findings were confirmed in the Arnold meta-analysis. In a pooled analysis of all 6 trials comparing chemotherapy plus anti-EGFR with chemotherapy +/- anti-VEGF there was no benefit seen from anti-EGFR in right sided colon cancer for overall survival (HR 1.12; 95% CI 0.87-1.45, p=0.381), or progression free survival (1.12; 95% CI 0.87-1.44, p=0.365). There was a non-significant trend to improved response rate with anti-EGFR (HR 1.47; 95% CI 0.94-2.29, p=0.089). In analysis of the individual studies by side, there was limited if any benefit seen with the addition of anti-EGFR therapy in right sided colon cancer in any study. Only the CRYSTAL (n=84) and second-line 20050181 (n=70) numerically favoured the addition of anti-EGFR therapy but this was not statistically significant and included only small numbers of patients. As in the first meta-analysis there was a significant improvement in outcome for patients with right sided tumours receiving chemotherapy and anti-VEGF compared with anti-EGFR plus chemotherapy in the CALGB 80405 study (progression free survival 10.2 months vs 7.5 months, p=0.007, n=149) and a non-significant trend to improved outcome in FIRE-3 (n=88) and PEAK (n=36).
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The location of the primary tumour is a strong prognostic factor. Patients with left sided primary tumours have a favourable outcome compared with those with right sided tumours regardless of treatment type received.
Left sided colorectal cancer should be considered for initial doublet chemotherapy and anti-EGFR therapy where appropriate. Alternate options remain appropriate based on patient preference and comorbidity.
Sequential use of all available therapies should continue to be utilised in patients with colorectal cancer regardless of the side of the primary tumour, provided it is appropriate for the individual patient.
Future trials for colon cancer should stratify patients by 'sidedness,' to better understand this issue.
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