Tumor Biomarker Testing for Metastatic Colorectal Cancer: a Canadian Consensus Practice Guideline

The systemic therapy management of metastatic colorectal cancer (mCRC) has evolved from primarily cytotoxic chemotherapies to now include targeted agents given alone or in combination with chemotherapy, and immune checkpoint inhibitors. A better understanding of the pathogenesis and molecular drivers of colorectal cancer not only aided the development of novel targeted therapies but led to the discovery of tumor mutations which act as predictive biomarkers for therapeutic response. Mutational status of the KRAS gene became the first genomic biomarker to be established as part of standard of care molecular testing, where KRAS mutations within exons 2, 3, and 4 predict a lack of response to anti- epidermal growth factor receptor therapies. Since then, several other biomarkers have become relevant to inform mCRC treatment; however, there are no published Canadian guidelines which reflect the current standards for biomarker testing. This guideline was developed by a pan-Canadian advisory group to provide contemporary, evidence-based recommendations on the minimum acceptable standards for biomarker testing in mCRC, and to describe additional biomarkers for consideration.

inclusion of targeted agents in a patient's treatment regimen. Furthermore, there is now an established role for immunotherapy checkpoint inhibitors (pembrolizumab, nivolumab, and ipilimumab) in biomarker-defined populations of mCRC.
Clinical trials in mCRC continue to take a biomarker-driven approach, with many new predictive biomarkers linked to pre-existing and novel therapies on the cusp of being clinically relevant. With no national guidelines reflecting current biomarker requirements in mCRC, this guideline was developed by a pan-Canadian advisory group to provide contemporary, evidence-based recommendations on the minimum acceptable standards for tumor biomarker testing in mCRC, and to describe emerging biomarkers for consideration.

Guideline development
A pan-Canadian advisory group of medical oncologists and pathologists specializing in CRC was formed to develop the practice guideline. Consensus was reached on guideline methods and recommendation statements through two virtual meetings. Grading strength of recommendations was based on the GRADE system. 7 (Table 1) The guideline development and literature search were focused on answering the following questions: 1. What tumor biomarkers are important to inform treatment selection in mCRC? 2. What tumor biomarkers have emerging actionability in mCRC? 3. What are the optimal methods for performing tumor biomarker testing in mCRC? 4. When should tumor biomarker testing be performed?
The literature search was conducted in two steps. First, international guidelines on biomarker testing and treatment for CRC were identified through an internet search of international health organizations. Since the last guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology (ASCO) was published in February 2017 and included a systematic literature review at a publication cut-off date of February 2015, 8 references from this publication were used to support guideline statements. The second step involved a literature search in MEDLINE using the OvidSP database, with publication cut-off dates between 1 February 2015 and 1 February 2022. Literature search included the terms 'colorectal neoplasms', 'molecular targeted therapy' or 'antineoplastic agents', and 'biomarkers'. The search was filtered to include practice guidelines, consensus documents, systematic reviews, meta-analyses, randomized controlled trials, comparative studies, reviews, and evaluation studies. In addition to journal articles, the search identified meeting abstracts from ASCO, ASCO-Gastrointestinal Cancers Symposium, and European Society for Medical Oncology. Reference lists from identified publications were also scanned for additional relevant reports.

Minimum biomarker testing standards in mCRC
This section states the minimum biomarker testing required across all Canadian jurisdictions for patients with CRC prior to initial treatment in the metastatic setting ( Figure 1). Recommendations for assessment of these biomarkers are based on adequate evidence demonstrating clinical actionability, meaning the status of the biomarker is needed to inform likely response, benefit, and/or access to Health Canada-approved therapies ( Table 2).

Extended RAS testing (including KRAS and NRAS)
Analysis of KRAS and NRAS mutation status is well-established as standard of care, with all international guidelines reviewed in the literature search recommending mutation testing for these genes (Table 3). These recommendations are based on the predictive value of KRAS and NRAS mutation status for the efficacy of cetuximab and panitumumab in patients with mCRC.
In the initial analyses of two phase III, randomized controlled trials, cetuximab or panitumumab in combination with best supportive care (BSC) demonstrated significantly prolonged progression-free survival (PFS) compared with BSC alone in unselected patients with relapsed mCRC. 14,15 However, data reported from subsequent clinical studies of anti-EGFR monoclonal antibodies, including retrospective analyses of the aforementioned trials, demonstrated that benefit from these novel therapies was limited to RAS wild-type mCRC 5,[16][17][18][19][20][21][22][23][24][25][26] (Table 4). These findings      Missense mutations in KRAS and NRAS genes have been reported in approximately 50 and 5% of advanced CRCs, respectively, with the majority of mutations occurring in codons 12 and 13 within exon 2 of KRAS. 40 Because of this high mutational frequency, most trial analyses initially evaluated efficacy outcomes based only on KRAS codon 12 and 13 mutation status. However, an exploratory analysis of the PRIME trial showed that missense mutations in exons 3 and 4 of KRAS and exons 2, 3, and 4 of NRAS occurred in a combined 17% of patients and were also indicators of inferior PFS and OS in patients with mCRC receiving panitumumab plus FOLFOX. 26 Other  Table 4).
Location of primary tumor has also been shown to impact prognosis and response to anti-EGFR therapy, with retrospective analyses from the Intergroup 80405, CRYSTAL, FIRE-3, PEAK, PRIME, and PARADIGM trials showing that patients with left-sided tumors, but not those with right-sided tumors, benefited from the addition of anti-EGFR therapy to their treatment (Yoshino, and , 2021 (Table 3).
BRAF mutation status is additionally recommended to select patients for treatment with BRAF inhibitors. Although BRAF inhibitor monotherapy is effective in patients with melanoma and BRAF V600E mutations, it has produced low response rates in BRAF V600E-mutated CRCs. 60 Evidence from preclinical studies suggest that this lack of response is caused by feedback reactivation of EGFR and subsequent initiation of downstream signaling. 61 (Table 3) The frequency of dMMR/MSI and its significance in the management of CRC varies by disease stage. It occurs in approximately 20, 12, and 5% of patients with stage II, III, and IV CRC, respectively. 68 In stage II-III CRC, dMMR/ MSI-H strongly correlates with an improved prognosis compared with MMR proficient/microsatellite stable (pMMR/MSS) tumors and is a predictor for lack of benefit from fluoropyrimidine monotherapy in stage II patients. 69,70 Conversely, dMMR/MSI-H appears to be associated with worse prognosis in patients with mCRC. [71][72][73][74][75] This finding may be related to the enrichment of BRAF V600 mutations in patients with sporadic dMMR/MSI mCRC. 74,76 International guidelines have acknowledged the emerging value of MMR and MSI testing to predict response to immune checkpoint inhibitors. In early phase clinical trials, the anti-programmed death-1 (PD-1) receptor antibody, pembrolizumab, showed activity in patients with dMMR/ MSI-H mCRC, with ORRs between 33 and 53%. 77 The anti-PD-1 antibody nivolumab also has conditional approval from Health Canada, in combination with the anti-cytotoxic T-lymphocyte-associated antigen 4 agent ipilimumab, for patients with dMMR/MSI-H mCRC after prior fluoropyrimidine-based therapy in combination with oxaliplatin or irinotecan. This was based on results from the multi-cohort, phase II CHECKMATE 142 study, where patients treated with nivolumab and ipilimumab achieved an ORR of 55% and a disease control rate for ⩾12 weeks of 80%. 82 In another cohort of patients with previously untreated mCRC, nivolumab plus ipilimumab, achieved an ORR and disease control rate of 69 and 84%, respectively. At a median follow-up of 29.0 months, median PFS and OS were not reached. 83

Extended biomarker testing options
In addition to the minimum required biomarkers for testing in mCRC, the panel has agreed that the following biomarkers could be considered during later lines of therapy. These actionable biomarkers are required either to access current Health Canada-approved therapies or to confirm eligibility for ongoing clinical trials. Testing for these biomarkers may be considered earlier in the metastatic setting if a patient is not a good candidate for traditional chemotherapy, and they may be incorporated into initial testing when multigene next-generation sequencing (NGS) panels are used. It is important to acknowledge that publicly funded access to biomarker-linked therapies may vary across jurisdictions, which should be discussed with the patient.

NTRK testing
Neurotrophic tyrosine receptor kinase (NTRK) genes encode a family of transmembrane-receptor proteins, called tropomyosin receptor kinases (TRKs), which are involved in neural development. 84 Translocations in NTRK1, NTRK2, and NTRK3 genes (encoding TRKA, TRKB, and TRKC proteins) have gained enormous interest since the first gene fusion was detected in 1982, in a colorectal adenocarcinoma cell line. 85 Since then, over 80 different gene fusion partners have been identified across many tumor types. 84 These fusions typically involve the portion of an NTRK gene, which encodes for the tyrosine kinase domain joined with portions of genes that encode for dimerization motifs. 84 In this way, TRK proteins become constitutively activated and contribute to cancer pathogenesis through aberrant signaling of the MAPK and PI3K pathways.
NTRK gene fusions are now clinically actionable in any cancer type based on results from clinical trials investigating the TRK inhibitors larotrectinib and entrectinib. A pooled analysis of three trials evaluating larotrectinib monotherapy in 153 adult and pediatric patients with refractory cancers of various tumor histologies demonstrated an ORR of 79% and CR rate of 16%. 86 Responses were durable, leading to a median PFS of 28.3 months. Entrectinib, which targets TRK proteins, as well as c-ROS oncogene1 (ROS) and anaplastic lymphoma kinase (ALK), was studied in the STARTRK-1, STARTRK-2, and ALKA372-001 trials. A pooled analysis of these trials, including 54 adult patients with refractory malignancies, demonstrated an ORR of 57%, CR rate of 7%, and median PFS of 11.2 months. 87 Although subgroups of patients with CRC in these trials were small, response rates appeared lower than in the overall populations, with four of eight patients (50%) responding to larotrectinib and one of four patients (25%) responding to entrectinib. Additional studies are needed to better understand potential resistance mechanisms and whether patients with CRC benefit less from TRK inhibitors compared to patients with other tumor types. 88 Several methods can be used to detect NTRK gene fusions, including immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction, and NGS. There are also multiple assays available using each method, with different advantages and limitations for each. The optimal assay for testing NTRK gene fusions should thus be decided at each institution based on the testing parameters and outputs. 84,89,90 The ongoing CANTRK Ring study, which aims to harmonize and standardize Canadian molecular pathology laboratory approaches to NTRK testing, will also provide insight on optimal testing methods. 91 Given the low incidence of NTRK gene fusions in CRC (approximately 0.2%), 92 94 Testing for NTRK fusions prior to first-line treatment may also be considered in select patients who are not good candidates for cytotoxic chemotherapy.

HER2 testing
The ERBB2 gene (herein referred to as HER2) encodes for the ErbB2 (HER2) protein, which is part of a family of receptor tyrosine kinases, including EGFR, ErbB3, and ErbB4. Heterodimerization of any two ErbB family proteins initiates the activation of MAPK, PI3K, Protein Kinase C, and Stress Activated Protein Kinase pathways. 99 Around 2-5% of CRCs harbor HER2 gene amplifications, [100][101][102] and their occurrence is enriched in RAS and BRAF wildtype CRCs. 103 HER2 amplifications do not appear to be correlated with worse survival in CRC 104 ; however, evidence from small, retrospective studies show that HER2 amplifications are correlated with poorer response to anti-EGFR therapies. [105][106][107][108] This supports the value of HER2 amplification testing to inform treatment with anti-EGFR therapies.
While therapies targeting HER2 have become standard of care for the treatment of breast and gastroesophageal cancers with HER2 overexpression/gene amplifications, similar therapies are emerging for treating this subpopulation of patients with mCRC. The phase II HERACLES trial evaluated trastuzumab (an anti-HER2 antibody) and lapatinib (a small molecule inhibitor of HER2 and EGFR) in 35 patients with HER2positive refractory mCRC, as determined by IHC and FISH. 109 In the 32 patients evaluable for response, this dual HER2-targeted treatment produced an ORR of 28%, a CR rate of 3% (one patient), and 41% had stable disease. Median PFS was 4.7 months (95% CI: 3.7-6.1), and median OS was 10.0 months (95% CI: 7.9-15.8). Of note, central nervous system (CNS) metastasis occurred in 19% of patients, a high frequency, which mirrors disease progression outcomes with HER2-targeted therapies in breast and gastric cancers. 110 Therefore, evidence of HER2 amplification in mCRC should prompt vigilance in monitoring for CNS metastases, and presence of CNS metastases in CRC patients should prompt clinicians to consider testing for HER2 amplification regardless of therapy line. 111,112 Clinical trials evaluating other combinations of HER-targeted therapies in patients with HER2amplified mCRC are ongoing, with early analyses demonstrating response rates between 25 and 55% (Table 5). Notably, the phase II DESTINY-CRC01 evaluated trastuzumab deruxtecan, an anti-HER2 antibody-drug conjugate, in 78 patients with previously treated, RAS-wild-type, HER2-expressing mCRC. 113 Results reported for three cohorts based on HER2 expression level showed a 45% ORR for patients in cohort A [IHC 3+ or IHC 2+ and in situ hybridization (ISH) positive] and no confirmed response in either cohorts B or C (IHC 2+ and ISH negative or IHC 1+). In a subgroup analysis of cohort A, higher response rates were observed among patients with higher HER2 expression (ORR for IHC 3+ versus IHC 2+: 57.5 versus 7.7%). 118 The NCCN guidelines for CRC recommend testing for HER2 amplifications for patients with mCRC unless RAS/BRAF mutations have already been confirmed as HER2 amplification is rare in this subgroup of patients. 102,105 Several technologies can be used to test for HER2 amplifications, although the optimal testing method is unclear. Many clinical trials in mCRC have followed the methods described in the HERACLES study, which define HER2 positivity as tumors with 3+ HER2 score in >50% of cells by IHC or with 2+ HER2 score and a HER2:CEP17 ratio >2 in >50% of cells by FISH. 102,113,114 These are similar to the criteria for determining HER2 status in breast and gastroesophageal cancers except that the latter guidelines have a lower threshold for percentage of cells requiring positive staining (>10%). 119  TherapeuTic advances in Medical Oncology Volume 14 16 journals.sagepub.com/home/tam HER2 detection by NGS, in addition to detection by IHC and/or FISH. 116,117 Testing for HER2 variations may be ideally evaluated within a multigene NGS panel; however, not all panels allow for detection of copy number variations and further clinical validation would be required.

Tumor mutational burden testing
Tumor mutational burden (TMB) is a measure of the rate of somatic mutations occurring across all coding regions in a tumor genome. High TMB (TMB-H) leads to the production of tumor neoantigens, which increase the likelihood of stimulating an anti-tumor immune response. TMB has been assessed as a biomarker to predict response to immune checkpoint inhibitors. Since TMB is a continuous variable, thresholds for defining TMB-H vary among studies. In the phase II KEYNOTE-158 study, patients with a variety of solid tumors that were TMB-H, defined as 10 mutations/megabase (Mb) using the FoundationOne NGS assay, achieved an ORR of 29% with pembrolizumab treatment, compared to an ORR of 6% in the non-TMB-H cohort. 121 Notably, patients with mCRC were not included as a cohort in this study. Based on these results, the U.S. Food and Drug Administration granted accelerated approval to pembrolizumab for the treatment of unresectable or metastatic solid tumors with TMB-H (⩾10 mutations/Mb), using the FoundationOne companion diagnostic assay. However, pembrolizumab has not been approved by Health Canada for this indication.
The frequency of TMB-H in CRC is approximately 3% and is strongly correlated with MSI-H status. 122 In a study evaluating over 6000 CRC cases, 99.7% of MSI-H tumors were found to also have a TMB of ⩾12 mutations/Mb, whereas only 3% of pMMR/MSS cases were TMB-H. 122 The ability of TMB-H to predict response to pembrolizumab in MSS mCRC remains unclear. The Targeted Agent and Profiling Utilization Registry (TAPUR) study assessed the efficacy of pembrolizumab in 27 patients with refractory MSS mCRC and TMB-H at a cut-off of ⩾9 mutations/Mb. 123 This study found an ORR of only 11% and PFS of 9.3 weeks in patients with refractory mCRC receiving pembrolizumab monotherapy. Another study based in Japan found that 8 of 24 patients with pMMR/MSS CRC responded to a combination of regorafenib and nivolumab; however, no relationship between TMB-H and response was detected. 124 In a study by the Canadian Cancer Trials Group, which randomized 180 patients with refractory mCRC to treatment with durvalumab and tremelimumab or BSC, patients with plasma TMB ⩾ 28 mutations/Mb had a greater OS benefit (HR 0.34; 90% CI: 0.18-0.63; p = 0.004) compared to the overall population (HR 0.72; 90% CI: 0.54-0.97; p = 0.07). 125 However, in this same trial, the use of tissue TMB as a biomarker did not identify a group of patients with improved outcome following durvalumab and tremelimumab, and a cut point of 10 mutations/Mb did not result in improved outcomes (HR 0.54, 90% CI: 0.27-1.08, p = 0.14). 126 This suggests that optimization and validation of different TMB thresholds for different tumor types may be needed.

Other emerging predictive genomic alterations
Within the set of genes that are recommended to be assessed in mCRC, including KRAS/NRAS and BRAF, different types of genomic alterations that occur at a lower frequency are emerging as potential predictive biomarkers that require further validation. This includes RAS gene amplifications, which occur in 1-2% of patients with CRC and may be enriched in patients with a history of inflammatory bowel disease. 40,127,128 Non-V600E BRAF missense mutations occur in up to 2% of mCRC cases and continue to be investigated as predictors of anti-EGFR therapy response. 129 Some studies have reported different BRAF mutations having different impacts on response to anti-EGFR therapy, with one retrospective study showing reduced response in cases with mutations in codons 597 and 601 of BRAF compared to cases with mutations in codons 594 and 596. 130 Another study did not observe responses to anti-EGFR therapies in any atypical BRAF-mutated patients with CRC; however, stable disease was achieved in 6 of 11 patients (50%). 131 Genomic alterations in ERRB family genes other than HER2 amplifications may also be predictors of response to anti-EGFR therapies but require validation. These include missense mutations or insertion/ deletions with HER2 and amplifications in ERRB3/HER3 or ERBB1/EGFR genes. 132,133 Missense mutations within the HER2 gene occur in approximately 3% of CRCs. 101 Thus far, patients with mCRC harboring tumor HER2 mutations have not responded to single-agent HER2 small molecule inhibitors in clinical trials 134 ; however, this may be due to the varying sensitivities of different HER2 mutations to anti-HER2 monotherapy. 135  Mutations in the PIK3CA gene occur in 10-20% of patients with CRC and are commonly found in exon 9 (within the helical domain) and exon 20 (within the kinase domain). Given the role of PI3K in signal transduction downstream of EGFR, PIK3CA mutations have also been considered a contributor to the lack of response to anti-EGFR therapy observed in some RAS wild-type patients. 138 Studies have reported conflicting results on the value of PIK3CA as a predictive biomarker for response to EGFR inhibitors, with some studies concluding that PIK3CA is an independent predictor of lack of response to anti-EGFR therapy, and others not reporting a correlation. [139][140][141][142][143][144] This inconsistency may be due to differences in the frequency of PIK3CA mutations observed and their co-occurrence with KRAS mutations. A large retrospective analysis of 743 patients with mCRC revealed a negative correlation between PIK3CA mutation in exon 20 and response and survival following cetuximab treatment, which was not observed in patients with PIK3CA exon 9 mutations. 139 However, since exon 20 mutations were only present in 3% of patients, further validation is needed to recommend routine use of PIK3CA testing in clinical decision-making.
Targeting PIK3CA-mutated tumors with agents inhibiting the PI3K/AKT/mTOR pathway is also being explored in mCRC. Therapeutic response to PI3K inhibitors in PIK3CA-mutated mCRC has been variable thus far, which may be partly explained by the intricacy of the PI3K signaling network, which intertwines with several other compensatory pathways, leaving opportunities for resistance. [145][146][147][148] Thus, combination regimens including PI3K pathway inhibitors are underway (NCT04753203, NCT04495621, NCT02861 300, and NCT03711058). In addition, absence or presence of co-occurring genetic alterations may impact the efficacy of PI3K inhibitors in PIK3CA-mutated mCRC. For example, several reports of patients with PIK3CA mutated solid tumors who achieved a partial response or prolonged stable disease following PI3K inhibitor therapy have reported co-occurring mutations in ARID1. 148,149 Dysfunction in DNA damage response by mutations in the exonuclease domains of polymerase epsilon (POLE) and polymerase delta 1 (POLD1) leads to a hypermutated molecular phenotype and is thus also being explored as an independent marker for response to immune checkpoint inhibitors. 150 A large study analyzing the mutation profile of 47,721 solid tumors found that mutations in POLE and POLD1 were found in 7% of CRCs. 151 In the overall population, 26% of patients with POLE and POLD1 mutations were also MSI-H and mutated cases had a significantly higher TMB compared to wild-type cases. This study also reported an independent association between POLE/POLD1 mutations and benefit from immune checkpoint inhibitors. Several clinical trials are underway, which plan to investigate the role of POLE/POLD1 mutations on response to immune checkpoint inhibitors in mCRC (NCT031507061, NCT03435107, NCT03 461952, and NCT03767075).

Biomarker testing methodologies and reporting
Testing methods and specimens Many DNA-, RNA-, and protein-based assays are appropriate methods for evaluating the recommended mCRC biomarkers, if they are validated and performed by an accredited laboratory that follows quality guidelines, such as those set by the College of American Pathologists. 152 Biomarker analysis in mCRC is increasingly being performed with multi-gene NGS panels across Canadian academic centers. 153 This is likely due to the decreasing costs of NGS, and the many advantages to using multiplex testing in cancers with a rapidly evolving biomarker landscape, such as CRC. 154 Using NGS, many genes and multiple classes of genomic alterations can be assessed simultaneously with greater sensitivity than other genomic testing approaches. 155 In tumor sites where there are more than five actionable genomic biomarkers, NGS can be cost-and time-efficient, tissue-sparing, and can streamline the ordering and reporting of results for clinicians compared to sequential gene testing. 156,157 Given the increasing number of relevant biomarkers for mCRC, Formalin-fixed, paraffin-embedded (FFPE) tissue is the preferred specimen for testing given that it is the most common tissue preservation method used in surgical pathology practice. 8 Biomarker analysis using cytology specimens or different fixation protocols would require adequate validation. Either primary, metastatic, or recurrent tissue is an acceptable specimen for molecular biomarker evaluation, as several clinical studies have recorded concordance rates of over 90% for RAS and BRAF mutation status between primary and metastatic specimens. 158 As the storage time for FFPE blocks increase, DNA/RNA quality and antigenicity can decrease, impacting the success of downstream molecular analyses. DNA fragmentation and cytosine to uracil deamination commonly occur after formalinfixation and have been shown to increase with longer storage times, leading to a decrease in amplifiable DNA templates and G > A and C > T transitions, respectively. [159][160][161][162] One study reported significant degradation of DNA extracted from the same FFPE blocks of surgically resected carcinomas of the lung, colon, and urothelial tract after 4-6 years of storage. 162 This resulted in delayed target amplification of KRAS exon 2 with quantitative PCR, as well as a decrease in library yield and an increase in the number of single-nucleotide variants detected using NGS. The impact of increased FFPE tissue storage time on loss of antigenicity in the context of IHC assays is also well-documented, although the impact of storage time varies between antibodies used. 163,164 Thus, the panel recommends that a new biopsy may be considered if an FFPE tissue block older than 5 years is the only available sample for testing. As biomarker analysis can still be successful using samples from older archival blocks, despite decreased DNA quality, it is also reasonable to attempt biomarker testing first and consider repeat biopsy if biomarker testing is unsuccessful or quality controls are suboptimal.

Turnaround time and reporting
A rapid turnaround from sample acquisition to the reporting of biomarker results is necessary for preventing delayed treatment initiation. Metaanalyses covering studies across many tumor sites, including CRC, have reported an increased risk of death with every 4-week delay in initiation of curative treatment. 165,166 Studies evaluating the impact of treatment delay in mCRC are less clear and may be confounded by the poorer prognostic profile of patients receiving accelerated treatment. 167 A large retrospective study using data from the Taiwan Cancer Registry showed that an increase in the diagnosis to treatment interval for patients with mCRC, from less than 30 days to 31 to 150 days, resulted in a 37% increase in risk of death (HR 1.37, 95% CI: 1.28-1.47), when adjusted for other factors found to influence increase risk of death, including male gender, age >75 years, Charlson Comorbidity Index ⩾7, other catastrophic illnesses, lack of multidisciplinary team involvement, and treatment in a low volume center. 168 There is also evidence to support the improved outcomes for patients with mCRC when biomarker-driven treatment is initiated in the firstline setting. In the KEYNOTE-177 trial evaluating mCRC patients with dMMR/MSI-H tumors, not only was the median PFS significantly longer for patients receiving pembrolizumab versus chemotherapy plus bevacizumab or cetuximab, but also PFS after next line of treatment (PFS2) was prolonged [median not reached versus 23.5 months (HR 0.63; 95% CI: 0.45-0.88)]. 169 Thus, testing workflow and procedures should be optimized to ensure that molecular biomarker testing results be reported to the oncologist by the time of the first consultation. Guidelines from international pathology associations and Canadian consensus publications recommend a maximum of 10 working days from sample receipt by the testing laboratory to generation of a summary report, with the report being sent to the referring oncologist within 24 h. 6,8,[170][171][172] For samples requiring sendout to a reference lab, the suggested turnaround time from specimen acquisition to arrival in the reference lab is three working days. 172 Hospital systems should perform internal quality assurance assessments to evaluate whether turnaround time benchmarks are met. In cases where benchmarks are not met, strategies to improve turnaround time should be considered, which may include reflexive testing for all new CRC diagnoses, adjustments to workflow, and/or implementation of rapid biomarker testing methods. 8,173 Reporting of biomarker testing results should conform to existing guidelines (American College of Medical Genetics, College of American Pathologists). 174 used, including details of which genomic alterations can be detected and the limitations of the test, is important as biomarker standards evolve over time. For example, the current recommendations for extended RAS mutation testing only include the analysis of missense mutations within exons 2, 3, and 4; however, emerging evidence on the utility of testing for RAS gene amplifications may result in its widespread adoption, and it would therefore be important to report. In addition, with the increased use of comprehensive genomic profiling by NGS, several genomic alterations with varying clinical significance may be detected. Thus, it will be important to report the likely pathogenicity of the identified variant as well as an interpretation section describing the therapeutic or prognostic implications of the results. The panel also recommends that in cases where the minimum required biomarkers for CRC are tested within a larger multi-gene panel, that genomic alterations identified outside the required genes be reported to the oncologist. This practice may be beneficial for diagnosis, staging, clinical research purposes, determining patient eligibility for clinical trials, and allowing patients compassionate access to therapies.

Summary and future directions
Targeted therapies have increased the actionability of tumor molecular biomarkers in mCRC, particularly in earlier lines of treatment, and have brought the importance of timely molecular testing to the forefront. At minimum, the current biomarkers that must be evaluated to meet standard of care include mutational analysis of NRAS, KRAS, and BRAF genes, as well as determination of MMR/MSI status. In addition, NTRK fusions and HER2 amplifications are actionable in mCRC and testing for these alterations should be considered as part of a multi-gene panel in all patients, or as a single-gene test in appropriately selected patients.
Ongoing clinical trials continue to push a biomarker-driven approach to the selection of therapy for CRC, with new biomarkers expected to be actionable in the coming years. Of particular interest are biomarkers of disease persistence and recurrence. Assays quantifying gene expression are being evaluated as prognostic classifiers for risk of disease recurrence in early-stage CRC. Thus far, assays including Oncotype Dx, ColoPrint, and ColDx have demonstrated some success in independently predicting risk of disease recurrence for patients with stage II/III CRC through gene expression profiling, whereas the ability to predict benefit of adjuvant chemotherapy has been less clear and requires further validation. [176][177][178][179][180][181][182] Immunoscore, a unique scoring system evaluating the proportion of CD3+ and CD8+ immune cells within tumor samples, is also under investigation as a predictor for risk of recurrence in CRC. 183 Liquid biopsies measuring circulating tumor DNA (ctDNA) are of great interest and show promising utility in the metastatic setting as a non-invasive alternative to biopsy-driven biomarker analysis, and they may provide insight on mechanisms of resistance to therapy, response to therapy, and early disease progression. [184][185][186][187][188][189][190] Identification of ctDNA in the plasma of patients with localized CRC is being investigated, with great anticipation, as a surrogate marker of minimal residual disease to predict benefit from adjuvant chemotherapy in stage II CRC in clinical trials including COBRA (NCT0406810) and DYNAMIC-III (ACTRN12617001566325). 191 Together, this highlights the growing importance of molecular testing in CRC and the need for centers to assess current testing workflow, equipment, and personnel, to ensure they are able to keep pace with the quickly evolving technologies necessary for practicing precision medicine in CRC.

Declarations
Ethics approval and consent to participate Not applicable.