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Abstract

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.

Keywords

colorectal cancer metastasis molecular testing predictive biomarker targeted therapy

Introduction

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in Canada and worldwide, accounting for approximately 10% of all cancer diagnoses.^1,2 Mortality rates for CRC have continued to decline over the past 40 years, which has likely been driven by implementation of cancer screening programs and access to improved therapies. However, 5-year survival rates remain at 67%, with rates as low as 11% for those with stage IV disease at diagnosis.³ Approximately 20% of patients with newly diagnosed CRC present with metastases and an additional 50% of patients initially diagnosed with stage I–III disease will progress to metastatic disease, where surgical control is difficult.⁴

Chemotherapy remains the backbone for management of metastatic CRC (mCRC), consisting of a combination of fluoropyrimidine agents with either irinotecan [5-fluorouracil, leucovorin, irinotecan (FOLFIRI)] or oxaliplatin [5-fluorouracil, leucovorin, oxaliplatin (FOLFOX)]. Over the last 20 years, several therapies targeting pathways that contribute to mCRC pathogenesis entered the treatment paradigm, including monoclonal antibodies and tyrosine kinase inhibitors against the epidermal growth factor receptor (EGFR; cetuximab and panitumumab), vascular endothelial growth factor (VEGF; bevacizumab, regorafenib, and ramucirumab), and BRAF kinase (encorafenib). This coincided with an improved understanding of the biologic heterogeneity of CRC and the relationship between genomic alterations within the tumor and response to targeted therapies.

The first predictive genomic biomarker to be established as part of standard of care testing for patients with mCRC was the KRAS gene, which if mutated at specific codons, negated the benefit from anti-EGFR agents.⁵ A Canadian guidance document was published in 2011, which outlined recommendations for KRAS testing in mCRC⁶; however, other genomic biomarkers have since become important to inform the exclusion or 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)

Table 1.

Grading strength of recommendations based on GRADE system.⁷

Designation	Description	Rationale
Strong recommendation	Recommend for or against a particular molecular testing practice for colorectal cancer (can include must or should)	Supported by convincing or adequate strength of evidence, high or intermediate quality of evidence, and clear benefit that outweighs any harms
Recommendation	Recommend for or against a particular molecular testing practice for colorectal cancer (can include should or may)	Some limitations in strength of evidence (adequate or inadequate) and quality of evidence (intermediate or low), balance of benefits and harms, values, or costs, but panel concludes that there is sufficient evidence and/or benefit to inform a recommendation
Expert consensus opinion	Recommend for or against a particular molecular testing practice for colorectal cancer (can include should or may)	Serious limitations in strength of evidence (inadequate or insufficient), quality of evidence (intermediate or low), balance of benefits and harms, values, or costs, but panel consensus is that a statement is necessary
No recommendation	No recommendation for or against a particular molecular testing practice for colorectal cancer	Insufficient evidence or agreement of the balance of benefits and harms, values, or costs to provide a recommendation

The guideline development and literature search were focused on answering the following questions:

What tumor biomarkers are important to inform treatment selection in mCRC?

What tumor biomarkers have emerging actionability in mCRC?

What are the optimal methods for performing tumor biomarker testing in mCRC?

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).

Figure 1.

Summary of recommendations for testing of predictive tumor biomarkers in metastatic colorectal cancer.

Table 2.

Summary of recommendations and grading for tumor biomarker testing in metastatic colorectal cancer.

Statements	Grading
Minimum standard of care for tumor biomarker testing
All patients with mCRC must have their tumor samples analyzed for: 1. Mutations in KRAS and NRAS genes (extended RAS), including at minimum codons 12 and 13 of exon 2, 59 and 61 of exon 3, and 117 and 146 of exon 4, to inform treatment decisions regarding anti-EGFR monoclonal antibodies: • In combination with chemotherapy in the first-line setting for left-sided primaries • In later lines of therapy 2. Mutations in BRAF V600: • For prognostic assessment • To inform treatment planning with anti-EGFR therapy • To inform treatment planning with combination BRAF inhibitor and anti-EGFR therapy in the second-line and beyond setting 3. MMR/MSI (if not previously assessed) • To inform treatment decisions regarding immune checkpoint inhibitors • To determine need for genetic testing for Lynch syndrome	Strong recommendation
Extended biomarker testing options
4. Testing for NTRK gene fusions should be considered in patients with previously treated mCRC: • To inform eligibility for treatment with TRK inhibitors when no satisfactory treatment options are available	Recommendation
5. Testing for HER2 gene copy number variations may be considered in patients with previously treated mCRC: • To inform eligibility for treatment with HER2-targeted therapy	Expert clinical opinion
6. Broad molecular testing, including but not limited to NTRK, HER2, and TMB, may be considered to inform eligibility for clinical trials in patients with refractory mCRC	Expert clinical opinion
7. There is insufficient evidence to support routine testing of TMB to inform treatment decisions with immune checkpoint inhibitors	No recommendation
Biomarker testing methodologies and reporting
8. Biomarker testing for mCRC must be validated in accordance with best laboratory practices and be performed by an accredited laboratory that conforms to quality guidelines and routinely participates in proficiency testing, such as that offered by the College of American Pathologists	Strong recommendation
9. Biomarker testing results should be reported to the medical oncologist by the time of first consultation to inform first-line treatment decisions	Strong recommendation
10. Biomarker testing reports should conform to existing guidelines (American College of Medical Genetics, College of American Pathologists, Canadian College of Medical Geneticists), be understandable to medical oncologists, and should include description of testing method, sample adequacy, specific alteration detected with classification, and interpretation of results	Strong recommendation
11. Metastatic, recurrent, or primary CRC tissue are all acceptable specimens for IHC or molecular testing in mCRC; however, a new biopsy may be considered if the only available sample for testing is an FFPE tissue block older than 5 years from the primary diagnosis	Recommendation
12. Testing methods must be validated for FFPE. Testing on additional materials such as alcohol-fixed specimens may be performed but should be validated according to local practices	Recommendation
13. Multi-gene NGS panel testing should be considered to optimize turnaround time, utilization of tissue specimen, detection of actionable biomarkers, and to keep pace with evolving biomarker standards	Recommendation
14. When multi-gene panels are used, identification of alterations in genes outside of the minimum standard recommendations for mCRC should be reported to the medical oncologist	Expert clinical opinion

CRC, colorectal cancer; EGFR, epidermal growth factor receptor; FFPE, formalin-fixed, paraffin-embedded; HER2, human epidermal growth factor receptor 2; mCRC, metastatic colorectal cancer; MMR, mismatch repair; MSI, microsatellite instability; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; TMB, tumour mutational burden; TRK, tropomyosin receptor kinases.

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.

Table 3.

Summary of international guidelines on biomarker testing and treatment for metastatic colorectal cancer.

Guideline	Date published/literature search date range	Biomarker summary
Guideline	Date published/literature search date range	KRAS/NRAS	BRAF	MMR/MSI	Other
Canadian Expert Group consensus recommendations: KRAS testing in colorectal cancer. Aubin et al.⁶	August 2011 Reference range: 2007–2010	• Testing of codon 12/13 of KRAS as negative predictive biomarker for anti-EGFR therapy • Requested at start of second-line therapy for mCRC • No mention of NRAS	• BRAF is a negative prognostic factor, but not clear as predictive biomarker • Not recommended	• Not discussed	• Testing of PTEN, amphiregulin. epiregulin, PIK3CA not recommended
Molecular biomarkers for the evaluation of colorectal cancer: guideline from the ASCP, CAP, AMP, and ASCO Sepulveda et al.⁸	February 2017 Reference range: January 2008–February 2015	• KRAS and NRAS exons 2, 3, 4 recommended in patients considering anti-EGFR therapy as it is a negative predictive biomarker	• BRAF V600 mutation analysis recommended to be performed for prognostic stratification • BRAF V600 recommended to be performed in dMMR tumors with loss of MLH1 to evaluate Lynch syndrome risk • Insufficient evidence to recommend BRAF V600 mutational status as predictive biomarker for response to anti-EGFR therapy	• Recommend MMR status testing in CRC for identification of patients with high risk for Lynch syndrome and/or prognostic stratification	• Insufficient evidence to recommend PIK3CA, PTEN testing
ESMO consensus guidelines for the management of patients with metastatic colorectal cancer Van Cutsem et al. 2016⁹	July 2016 Reference range: 1991–2015	• KRAS and NRAS exons 2, 3, 4 recommended in all patients at time of diagnosis of mCRC as it is a negative predictive biomarker for anti-EGFR therapy	• BRAF mutation status is recommended to be assessed alongside RAS status (at diagnosis of mCRC) for prognostic assessment (and/or potential selection for clinical trials)	• Role of MSI as an independent prognostic biomarker in mCRC is unclear • MSI testing in mCRC can assist in genetic counseling • MSI strong predictive biomarker for benefit with immune checkpoint inhibitors	• Emerging biomarkers not recommended for routine patient management outside of a clinical trial setting: PIK3CA (exon 20), PTEN loss by IHC, amphiregulin, epiregulin, TGF-alpha, EGFR protein expression, amplification, copy number variations and mutations in ectodomain, HER2 amplifications/mutations, HER3 and MET receptor overexpression
Pan-Asian adapted ESMO consensus guidelines for the management of patients with metastatic colorectal cancer: a JSMO–ESMO initiative endorsed by CSCO, KACO, MOS, SSO, and TOS Yoshino et al.¹⁰	January 2018 Reference range: 2005–2017	• Same as ESMO	• Same as ESMO except V600E mutation specified	• Same as ESMO except: • Specified both IHC testing for MMR and PCR testing for MSI could be used to inform genetic counseling • Specified tumor MMR testing has strong predictive value for use of checkpoint inhibitors	• Same as ESMO except detection of mutations in PIK3CA, exon 20 deemed optional
UK colorectal cancer NICE guideline (NG151)¹¹	January 2020 Reference range: Up to 2018	• KRAS and NRAS testing is recommended as a negative predictive marker for anti-EGFR therapy	• BRAF V600E testing is recommended as a negative predictive marker for anti-EGFR therapy	• Recommended for all newly diagnosed patients with CRC	• N/A
Cancer Council Australia¹²	October 2017	• KRAS and NRAS mutation testing is recommended for patients with advanced CRC who are being considered for anti-EGFR therapy	• BRAF mutation testing is recommended in patients with advanced CRC as emerging evidence suggests poor response to anti-EGFR treatment	• dMMR testing is recommended in all CRC patients to identify Lynch syndrome	• N/A
NCCN guidelines version 3.2021 colon cancer¹³	September 2021 References up to 2020	• KRAS and NRAS exons 2, 3, 4 recommended in all patients with mCRC as it is a negative predictive biomarker for anti-EGFR therapy	• BRAF mutation genotyping (or IHC) is recommended in all patients with mCRC as BRAF V600E mutation makes response to anti-EGFR therapy highly unlikely unless given with a BRAF inhibitor	• MMR or MSI testing is recommended in all newly diagnosed patients with CRC	• HER2 testing via IHC, FISH, or NGS is noted as an option • NTRK fusion testing via multiple methods is noted as an option (although IHC positive requires confirmation) in patients with wild-type KRAS, NRAS, and BRAF and potentially who are dMMR/MSI-H

AMP, Association for Molecular Pathology; ASCO, American Society of Clinical Oncology; ASCP, American Society for Clinical Pathology; CAP, College of American Pathologists; CRC, colorectal cancer; CSCO, Chinese Society of Clinical Oncology; dMMR/MSI-H, mismatch repair deficiency/microsatellite instability high; EGFR, epidermal growth factor receptor; ESMO, European Society for Medical Oncology; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; JSMO, Japanese Society of Medical Oncology; KACO; Korean Association for Clinical Oncology; MMR, mismatch repair; MOS, Malaysian Oncological Society; MSI, microsatellite instability; NCCN, National Comprehensive Cancer Network; NGS, next-generation sequencing; NICE, The National Institute for Health and Care Excellence; PCR, polymerase chain reaction; SSO; Singapore Society of Oncology; TOS, Taiwan Oncology Society.

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^5,16–26 (Table 4). These findings have strong biologic plausibility given that RAS is an important molecule in the mitogen-activated protein kinase (MAPK) signaling pathway which functions downstream of EGFR. Indeed, in cellular models of CRC, mutations leading to activated KRAS proteins have demonstrated evasion of the MAPK signal-suppressing effects of EGFR inhibitors.¹⁸

Table 4.

Clinical trials of anti-EGFR therapies with reported outcomes by RAS and BRAF mutation status.

Trial, design	Treatment arms	Population	Biomarkers evaluated	Number of patients	Results by biomarker status (HR or OR; [95% CI])
CO.17 Karapetis et al.⁵ Loree et al.²⁷ Ph III	CET + BSC versus BSC	Advanced CRC no remaining SOC available N = 572	KRAS/NRAS codons 12, 13, 59, 61, 117, 146 BRAF V600E	KRASm_{(ex 2)} n = 164 KRASwt_{(ex 2)} n = 230 RASm_{(ex 2,3,4):} n = 213 BRAFm_(V600E) n = 15 RAS/BRAFwt n = 97	KRASwt (ex 2) PFS: HR_{(CET + BSC versus BSC)} = 0.40 [0.30–0.54]* OS: HR_{(CET + BSC versus BSC)} = 0.55; [0.41–0.74]* KRASm (ex 2) PFS: HR_{(CET + BSC versus BSC)} = 0.99; [0.73–1.35] OS: HR_{(CET + BSC versus BSC)} = 0.98; [0.70–1.37] RASm (ex 2, 3, 4) PFS: HR_{(CET + BSC versus BSC)} =1.04; [0.79–1.37] OS: HR_{(CET + BSC versus BSC)} = 0.91; [0.68–1.23] BRAFm (V600E) PFS: HR_{(CET + BSC versus BSC)} = 0.75; [0.26–2.19] OS: HR_{(CET + BSC versus BSC)} = 0.71; [0.22–2.27] RAS/BRAFwt (ex 2, 3, 4 and V600E) PFS: HR_{(CET + BSC versus BSC)} = 0.25; [0.15–0.41]* OS: HR_{(CET + BSC versus BSC)} = 0.51; [0.32–0.81]*
OPUS Bokemeyer et al.²⁴ Ph II	CET + FOLFOX4 versus FOLFOX4	First-line EGFR-expressing mCRC N = 337	KRAS/NRAS codons 12, 13, 59, 61, 117, 146	KRASwt_{(ex 2)} n = 179 KRASm_{(ex 2)} n = 136 RASwt_{(ex 2,3,4)}: N = 87 RASm_{(ex 2,3,4)}: n = 167 RASm_{(KRAS ex 2 wt)}: n = 31	KRASwt (ex 2) ORR: OR_{(CET + FOLFOX4 versus FOLFOX4)} = 2.55 [1.38–4.72] PFS: HR_{(CET + FOLFOX4 versus FOLFOX4)} = 0.57 [0.38–0.86]* KRASm (ex 2) ORR: OR_{(CET + FOLFOX4 versus FOLFOX4)} = 0.46 [0.23–0.92] PFS: HR_{(CET + FOLFOX4 versus FOLFOX4)} = 1.72 [1.10–2.68] RASwt (ex 2, 3, 4) ORR: OR_{(CET + FOLFOX4 versus FOLFOX4)} = 3.33 [1.36–8.17] PFS: HR_{(CET + FOLFOX4 versus FOLFOX4)} = 0.53 [0.27–1.04] RASm (ex 2, 3, 4) ORR: OR_{(CET + FOLFOX4 versus FOLFOX4)} = 0.58 [0.31–1.08] PFS: HR_{(CET + FOLFOX4 versus FOLFOX4)} = 1.54 [1.04–2.29] RASm (KRAS ex 2 wt) ORR: OR_{(CET + FOLFOX4 versus FOLFOX4)} = 1.50 [0.34–6.53] PFS: HR_{(CET + FOLFOX4 versus FOLFOX4)} = 0.77 [0.28–2.08] Overall survival: No significant difference across any subgroups
CRYSTAL Van Cutsem et al.²² Ph III	CET + FOLFIRI versus FOLFIRI	First-line EGFR-expressing mCRC N = 1198	KRAS/NRAS codons 12, 13, 59, 61, 117, 146	KRASwt_{(ex 2)} n = 666 RASwt_{(ex 2,3,4):} N = 367 RASm_{(ex 2,3,4):} n = 460 RASm_{(KRAS ex 2 wt):} n = 63	KRASwt _{(ex 2)} PFS: HR_{(CET + FOLFIRI versus FOLFIRI)} = 0.70 [0.56–0.87]* OS: HR_{(CET + FOLFIRI versus FOLFIRI)} = 0.80 [0.67–0.95]* RASwt (ex 2, 3, 4) PFS: HR_{(CET + FOLFIRI versus FOLFIRI)} = 0.56 [0.41–0.76]* OS: HR_{(CET + FOLFIRI versus FOLFIRI)} = 0.69 [0.54–0.88]* RASm (ex 2, 3, 4) PFS: HR_{(CET + FOLFIRI versus FOLFIRI)} = 1.10 [0.85–1.42] OS: HR_{(CET + FOLFIRI versus FOLFIRI)} = 1.05 [0.86–1.28] RASm (KRAS ex 2 wt) PFS: HR_{(CET + FOLFIRI versus FOLFIRI)} = 0.81 [0.39–1.67] OS: HR_{(CET + FOLFIRI versus FOLFIRI)} = 1.22 [0.69–2.16]
OPUS + CRYSTAL pooled analysis Bokemeyer et al.²⁸	See above	See above	BRAF V600E	KRASwt_{(ex 2)}/BRAFwt n = 730 KRASwt_{(ex 2)}/BRAFm n = 70	KRASwt (ex 2)/BRAFwt PFS: HR_{(CET + Chemo versus Chemo)} = 0.64 [0.52–0.79]* OS: HR_{(CET + Chemo versus Chemo)} = 0.84 [0.71–1.00]* KRASwt (ex 2)/BRAFm PFS: HR_{(CET +Chemo versus Chemo)} = 0.67 [0.34–1.29] OS: HR_{(CET + Chemo versus Chemo)} = 0.62 [0.36–1.06]
COIN Maughan et al.²⁹ Ph III	CET + FOLFOX versus FOLFOX (versus intermittent chemotherapy) N = 1630	First-line advanced CRC	KRAS codons 12, 13, 61 NRAS codons 12, 61 BRAF codons 594, 600	KRASwt n = 729 KRASm n = 565 KRASwt/BRAFm n = 102 RASwt/BRAFwt n = 581	KRASwt (ex 2/codon 61) PFS: HR_{(CET + FOLFOX versus FOLFOX)} = 0.96 [0.82–1.12] OS: HR_{(CET + FOLFOX versus FOLFOX)} = 1.04 [0.87–1.23] KRASm (ex 2/codon 61) OS: HR_{(CET + FOLFOX versus FOLFOX)} = 0.98 [0.81–1.17] KRASwt (ex 2/codon 61)/BRAFm OS: HR_{(CET + FOLFOX versus FOLFOX)} = 1.18 [0.76–1.81] KRASwt (ex 2/codon 61)/BRAFwt OS: HR_{(CET + FOLFOX versus FOLFOX)} = 1.02 [0.83–1.24]
NORDIC-VII Tveit et al.³⁰ Guren et al.³¹ Ph III	FLOX versus CET + FLOX versus CET + intermittent FLOX N = 566	First-line mCRC	KRAS codons 12, 13, 61, 117, 146 NRAS codons 12, 13, 61, 146 BRAF codon 600	KRASwt_{(ex 2)} n = 303 KRASm_{(ex 2)} n = 195 RASwt/BRAFwt n = 192	KRASwt (ex 2) PFS: HR_{(CET + FLOX versus FLOX)} = 1.07 [0.79–1.45] OS: HR_{(CET + FLOX versus FLOX)} = 1.14 [0.80–1.61] KRASm (ex 2) PFS: HR_{(CET + FLOX versus FLOX)} = 0.71 [0.50–1.03] OS: HR_{(CET + FLOX versus FLOX)} = 1.03 [0.68–1.57] RASwt (ex 2, 3, 4)/BRAFwt PFS: HR_{(CET + FLOX versus FLOX)} = 1.06 [0.73–1.55] OS: HR_{(CET + FLOX versus FLOX)} = 1.07 [0.74–1.55]
CALGB/SWOG 80,405 Innocenti et al.³² Ph III	CET + Chemo versus BEV + Chemo versus CET + BEV + Chemo N = 2326 N = 843 with mutational analysis	First-line advanced, protocol amendment to restrict enrollment to KRAS ex 2 wt	KRAS codons 12, 13, 61, 117, 146 NRAS codons 12, 13, 61 BRAF codons 600, 601	RASwt n = 572 RASm n = 266 BRAFwt n = 743 BRAFm n = 100	RASwt PFS: HR_{(BEV + Chemo versus CET + Chemo)} = 0.90 [0.73–1.12] OS: HR_{(BEV + Chemo versus CET + Chemo)} = 0.91 [0.72–1.16] RASm PFS: HR_{(BEV + Chemo versus CET + Chemo)} = 0.77 [0.56–1.06] OS: HR_{(BEV + Chemo versus CET + Chemo)} = 1.07 [0.77–1.48] BRAFwt PFS: HR_{(BEV + Chemo versus CET + Chemo)} = 0.90 [0.74–1.09] OS: HR_{(BEV +Chemo versus CET + Chemo)} = 1.02 [0.83–1.25] BRAFm PFS: HR_{(BEV + Chemo versus CET + Chemo)} = 0.58 [0.33–1.02] OS: HR_{(BEV + Chemo versus CET + Chemo)} = 0.67 [0.37–1.20]
FIRE-3 Stintzing et al.³³ Heinemann et al.³⁴ Ph III	CET + FOLFIRI versus BEV + FOLFIRI N = 593	First-line mCRC, protocol amendment to restrict enrollment to KRAS ex 2 wt	KRAS/NRAS codons 12, 13, 59, 61, 117, 146 BRAF codons 466, 600	RASwt_{(ex 2,3,4)} n = 400 RASm_{(ex 2,3,4)} N = 188 BRAFm n = 48	RASwt (ex 2, 3, 4) PFS: HR_{(CET + FOLFIRI versus BEV + FOLFIRI)} = 0.96 [0.79–1.18] OS: HR_{(CET + FOLFIRI versus BEV + FOLFIRI)} = 0.76 [0.62–0.94]* RASm (ex 2, 3, 4) PFS: HR_{(CET + FOLFIRI versus BEV + FOLFIRI)} = 1.27 [0.94–1.70] OS: HR_{(CET + FOLFIRI versus BEV + FOLFIRI)} = 1.05 [0.78–1.42] BRAFm PFS: HR_{(CET + FOLFIRI versus BEV + FOLFIRI)} = 0.84 [0.47–1.51] OS: HR_{(CET + FOLFIRI versus BEV + FOLFIRI)} = 0.79 [0.43–1.46]
20020408 Amado et al.¹⁶ Patterson et al.³⁵ Ph III	PAN + BSC versus BSC N = 463	Relapsed, EGFR-expressing mCRC	KRAS/NRAS codons 12, 13, 59, 61, 117, 146	KRASwt_{(ex 2)} n = 184 RASwt_{(ex 2,3,4)} n = NR RASm_{(ex 2,3,4)} n = NR	KRASwt (ex 2) PFS: HR_{(PAN + BSC versus BSC)} = 0.45 [0.34–0.59]* RASwt (ex 2, 3, 4) PFS: HR_{(PAN + BSC versus BSC)} = 0.38 [0.27–0.56]* RASm (ex 2, 3, 4) PFS: HR_{(PAN + BSC versus BSC)} = 0.98 [0.73–1.31]
20100007 Kim et al.³⁶ Ph III	PAN + BSC versus BSC N = 377	Chemo-refractory KRAS exon 2 wt mCRC	KRAS/NRAS codons 12, 13, 59, 61, 117, 146 BRAF ex 15	KRASwt_{(ex 2)} n = 377 RASm_{(KRAS ex 2 wt)} n = 54 RASwt_{(ex 2,3,4)} n = 270 RASwt_{(ex 2,3,4)}/BRAFwt n = 242 RASwt_{(ex 2,3,4)}/BRAFm n = 20	KRASwt (ex 2) PFS: HR_{(PAN + BSC versus BSC)} = 0.54 [0.43–0.67]* OS: HR_{(PAN + BSC versus BSC)} = 0.74 [0.59–0.93]* RASm (KRAS ex 2 wt) PFS: HR_{(PAN +BSC versus BSC)} = 1.03 [0.56–1.90] OS: HR_{(PAN + BSC versus BSC)} = 0.99 [0.49–2.00] RASwt (ex 2, 3, 4) PFS: HR_{(PAN + BSC versus BSC)} = 0.45 [0.35–0.59]* OS: HR_{(PAN + BSC versus BSC)} = 0.72 [0.55–0.94]* BRAFwt/RASwt (ex 2, 3, 4) PFS: HR_{(PAN + BSC versus BSC)} = 0.45 [0.34–0.60]* OS: HR_{(PAN + BSC versus BSC)} = 0.75 [0.57–0.99]* BRAFm/RASwt (ex 2, 3, 4) PFS: HR_{(PAN + BSC versus BSC)} = 0.28 [0.07–1.08] OS: HR_{(PAN + BSC versus BSC)} = 0.39 [0.10–1.51]
20050181 Peeters et al.²⁵ Ph III	PAN + FOLFIRI versus FOLFIRI N = 1186	Previously treated mCRC	KRAS/NRAS codons 12, 13, 59, 61, 117, 146 BRAF codon 600	KRASwt_{(ex 2)} n = 597 KRASm_{(ex 2)} n = 486 RASwt_{(ex 2,3,4)} n = 421 RASm_{(ex 2,3,4)} n = 593 RASm_{(KRAS ex 2 wt)} n = 107 RASwt_{(ex 2,3,4)}/BRAFwt n = 376 RASwt_{(ex 2,3,4)}/BRAFm n = 45	KRASwt (ex 2) PFS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.73 [0.59–0.90]* OS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.85 [0.70–1.04] KRASm (ex 2) PFS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.85 [0.68–1.06] OS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.94 [0.76–1.15] RASwt (ex 2, 3, 4) PFS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.70 [0.54–0.91]* OS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.81 [0.63–1.03] RASm (ex 2, 3, 4) PFS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.86 [0.70–1.05] OS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.91 [0.76–1.10] RASm (KRAS ex 2 wt) PFS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.89 [0.56–1.42] OS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.83 [0.53–1.29] BRAFwt/RAS wt (ex 2, 3, 4) PFS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.68 [0.51–0.90]* OS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.83 [0.64–1.07] BRAFm/RAS wt (ex 2, 3, 4) PFS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.69 [0.32–1.49] OS: HR_{(PAN + FOLFIRI versus FOLFIRI)} = 0.64 [0.32–1.28]
PICCOLO Seymour et al.³⁷ Ph III	PAN + IRI versus IRI versus (IRI + ciclosporin) N = 1198	Advanced, chemoresistant, relapsed CRC, protocol amendment to restrict enrollment in PAN + IRI arm for KRAS wt only	KRAS/NRAS codons 12, 13, 59, 61, 117, 146 BRAF codon 600 PIK3CA ex 9, 20	wt_{(RAS, BRAF, PIK3CA)} n = 323 m_{(RAS, BRAF, PIK3CA)} n = 137 BRAFm n = 68	Wild-type (RAS, BRAF, PIK3CA) PFS: HR_{(PAN + IRI versus IRI)} = 0.68 [0.53–0.86]* OS: HR_{(PAN + IRI versus IRI)} = 0.92 [0.73–1.16] Mutated (RAS, BRAF, PIK3CA) PFS: HR_{(PAN ++ IRI versus IRI)} = 1.20 [0.83–1.74] OS: HR_{(PAN + IRI versus IRI)} = 1.64 [1.14–2.34] BRAFm PFS: HR_{(PAN + IRI versus IRI)} = 1.40 [0.82–2.39] OS: HR_{(PAN + IRI versus IRI)} = 1.84 [1.10–3.08]
PRIME Douillard et al.²⁶ Ph III	PAN + FOLFOX4 versus FOLFOX4 N = 1183	First-line mCRC	KRAS/NRAS codons 12, 13, 59a, 61, 117, 146	KRASwt_{(ex 2)} n = 656 KRASm_{(ex 2)} n = 440 RASwt_{(ex 2,3,4)} N = 512 RASm_{(ex 2,3,4)} n = 548 RASm_{(KRAS ex 2 wt)} n = 108	KRASwt (ex 2) PFS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 0.80 [0.66–0.97]* OS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 0.83 [0.70–0.98]* KRASm (ex 2) PFS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 1.29 [1.04–1.62] OS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 1.16 [0.94–1.41] RASwt (ex 2, 3, 4) PFS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 0.72 [0.58–0.90]* OS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 0.77 [0.64–0.94]* RASm (ex 2, 3, 4) PFS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 1.31 [1.07–1.60] OS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 1.21 [1.01–1.45] RASm (KRAS ex 2 wt) PFS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 1.28 [0.79–2.07] OS: HR_{(PAN + FOLFOX4 versus FOLFOX4)} = 1.39 [0.91–2.13]
PEAK Rivera et al.³⁸ Ph II	PAN + mFOLFOX6 versus BEV + mFOLFOX6 N = 285	First-line, KRAS ex 2 wt mCRC	KRAS/NRAS codons 12, 13, 59, 61, 117, 146 BRAF codon 600	RASwt_{(ex 2,3,4)} n = 170 BRAFwt n = 156	RASwt (ex 2, 3, 4) PFS: HR_{(PAN + FOLFOX6 versus BEV + FOLFOX6)} = 0.68 [0.48–0.96]* OS: HR_{(PAN + FOLFOX6 versus BEV + FOLFOX6)} = 0.76 [0.53–1.11] RASwt (ex 2, 3, 4)/BRAF wt PFS: HR_{(PAN +FOLFOX6 versus BEV + FOLFOX6)} = 0.61 [0.42–0.88]* OS: HR_{(PAN + FOLFOX6 versus BEV + FOLFOX6)} = 0.70 [0.48–1.04]
PARADIGM Yoshino et al.³⁹ Ph III	PAN + mFOLFOX6 versus BEV + mFOLFOX6 N = 802	First-line, KRAS/NRAS ex 2/3/4 wt mCRC	KRAS/NRAS codons 12, 13, 59, 61, 117, 146	RASwt_{(ex 2,3,4)} n = 802	RASwt (ex 2, 3, 4) PFS: HR_{(PAN + FOLFOX6 versus BEV + FOLFOX6)} = 1.01 [0.87–1.18] OS: HR_{(PAN + FOLFOX6 versus BEV + FOLFOX6)} = 0.84 [0.72–0.98]*

Denotes statistically significant reduction in risk for anti-EGFR therapy arm

BEV, bevacizumab; BSC, best supportive care; CET, cetuximab; Chemo, chemotherapy; CI, confidence interval; CRC, colorectal cancer; EGFR, epidermal growth factor receptor; ex, exon; FLOX, bolus fluorouracil/folinic acid and oxaliplatin; FOLFIRI, 5-fluorouracil, leucovorin, irinotecan; FOLFOX, 5-fluorouracil, leucovorin, oxaliplatin; HR, hazard ratio; IRI, irinotecan; m, mutated; mCRC, metastatic colorectal cancer; NR, not reported; OR, odds ratio; OS, overall survival; PAN, panitumumab; PFS, progression-free survival; SOC, standard of care; wt, wild-type.

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 post hoc analyses of clinical trials and meta-analyses have confirmed these trends showing mutations in KRAS and NRAS in codons 12 and 13 of exon 2, 59 and 61 of exon 3, and 117 and 146 of exon 4 are negative predictors for response to EGFR targeted therapies^41,42 (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).^43–45 Incorporation of cetuximab or panitumumab with FOLFIRI or FOLFOX are now standard of care first-line treatment options in Canada for patients with mCRC who have left-sided primary tumors and are RAS wild-type.^46,47 Some clinicians may choose to avoid upfront anti-EGFR therapy in combination with chemotherapy in patients with resectable liver metastases, based on the New EPOC data, but this remains a controversial area.⁴⁸

In addition to serving as a biomarker to exclude patients from receiving anti-EGFR therapy, therapies targeting the KRAS G12C mutation, which occur in 3–4% of CRCs,⁴⁹ are under investigation. This includes the small molecule inhibitors sotorasib and adagrasib, which bind specifically to the inactive GDP-bound state of KRAS G12C mutant proteins. Early phase trials have reported overall response rates (ORRs) of 7 and 22%, for these agents as monotherapy in relapsed mCRC, respectively.^50,51 The phase III KRYSTAL-10 study evaluating adagrasib plus cetuximab versus chemotherapy in patients with relapsed advanced CRC and KRAS G12C mutations is ongoing.⁵²

BRAF V600 testing

The BRAF protein is a serine/threonine protein kinase functioning downstream of RAS in the MAPK signaling pathway. Activating V600 mutations in the BRAF gene are considered mutually exclusive with RAS mutations and occur in approximately 10% of mCRC cases. BRAF V600E mutations tend to be enriched in right-sided tumors and tumors with high microsatellite instability (MSI-H).^53–55 Compared with BRAF wild-type CRC, tumors harboring BRAF V600E mutations have been independently correlated with worse survival and rapid disease progression following first-line chemotherapy.^54–56

The perceived value of BRAF mutation analysis has evolved over the last 15 years. Guidelines from European Society for Medical Oncology (ESMO) and ASCO published in July 2016 and February 2017, respectively, acknowledge the prognostic value of BRAF V600E mutations; however, they stated that there was insufficient evidence to conclude that patients with BRAF-mutated CRC do not benefit from anti-EGFR therapies, and therefore should not be used as a predictive biomarker^8,9 (Table 3). This statement is based on the difficulty in discerning the predictive value of BRAF V600E mutations due to low mutational prevalence and association with other poor prognostic features. In addition, a meta-analysis by Rowland et al. pooling data from eight RCTs, showed a lack of PFS benefit with anti-EGFR therapies in BRAF-mutated patients [hazard ratio (HR) 0.86 (95% CI: 0.61–1.21)] and a significant PFS improvement in BRAF wild-type patients [HR 0.62 (95% CI: 0.50–0.77)]; however, the interaction test to detect a difference was just outside the threshold of significance (p = 0.07).⁵⁷ Other groups have argued that although not statistically significant, the p-value of the interaction test is clinically relevant,⁵⁸ and the body of evidence to support the lack of benefit to anti-EGFR therapies in BRAF-mutated mCRC, including a series of individual studies and meta-analyses, is equivalent, if not superior, to that of RAS mutations outside of KRAS exon 2⁵⁹ (Table 4). Assessment of BRAF mutation status is recommended in guidelines published by The National Institute for Health and Care Excellence (NICE), National Comprehensive Cancer Network (NCCN), and Cancer Council Australia to select patients most likely to respond to anti-EGFR therapies (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.^61,62 For this reason, combination therapies targeting multiple points along the MAPK pathway have been investigated in BRAF V600-mutated CRC. The phase II SWOGS1406 study in relapsed mCRC demonstrated that the addition of the BRAF inhibitor vemurafenib to irinotecan and cetuximab resulted in improved PFS, ORR, and disease control rate for patients with BRAF V600E mutations compared with cetuximab and irinotecan alone.⁶³ A phase I study of the BRAF and MEK inhibitors, dabrafenib and trametinib also demonstrated activity in patients with BRAF V600E-mutated mCRC.⁶⁴ Results from the pivotal phase III BEACON study led to the Health Canada approval of encorafenib (BRAF inhibitor) plus cetuximab for patients with previously-treated BRAF V600E-mutated mCRC. This study examined encorafenib in combination with cetuximab, with or without the MEK inhibitor binimetinib versus investigator’s choice of irinotecan or FOLFIRI plus cetuximab.⁶⁵ At a median follow-up of 12.8 months, both the doublet and triplet encorafenib regimens demonstrated superior OS compared to the control arm (median OS 9.3 months for both arms versus 5.9 months for control; HR 0.60, 95% CI: 0.47–0.75 for triplet versus control and HR 0.61, 95% CI: 0.48–0.77 for doublet versus control).

Encorafenib combination therapies are also being investigated in the first-line setting for patients with BRAF V600E-mutated mCRC. This includes the phase II ANCHOR study, which met its primary endpoint with an ORR of 47.8% for encorafenib, binimetinib, and cetuximab and a median PFS and OS of 5.8 and 17.2 months, respectively.⁶⁶ The phase III BREAKWATER trial evaluating encorafenib plus cetuximab with or without chemotherapy for first-line treatment of BRAF V600E-mutated mCRC is ongoing.⁶⁷

Mismatch repair deficiency/microsatellite instability testing

Alterations in genes responsible for DNA mismatch repair (MMR) lead to changes in the length of short, tandemly repeated DNA motifs – a genomic phenotype termed microsatellite instability (MSI). Less than one-third of CRC cases with MMR deficiency (dMMR)/MSI-H have germline mutations in MMR genes (MLH1, MSH2, PMS2, and MSH6) which are linked to an inherited condition of cancer susceptibility called Lynch syndrome.⁸ International guidelines recommend testing for MMR status in all patients with CRC to inform the need for cascade testing of family members and subsequent risk-reduction strategies in those identified with Lynch syndrome.(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.^71–75 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%.^77–79 Results from the pivotal phase III KEYNOTE-177 trial led to the Health Canada approval of pembrolizumab monotherapy as first-line treatment for patients with dMMR/MSI-H mCRC. In this trial, pembrolizumab treatment resulted in significantly prolonged PFS compared with the control arm of FOLFOX or FOLFIRI with or without bevacizumab or cetuximab (median, 16.5 versus 8.2 months; HR 0.60, 95% CI: 0.45–0.80; p = 0.0002).⁸⁰ At a median follow-up of 44 months, there was also a trend for prolonged OS with pembrolizumab (median not reached versus 36.7 months; HR 0.74, 95% CI: 0.53–1.03; p = 0.0359); however, statistical significance was likely not met due to the high rate of patients receiving subsequent immune checkpoint inhibitors (60%).⁸¹

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 multi-gene 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%),^92,93 methods to improve cost-effectiveness of testing should be considered. A Canadian consensus statement on biomarker testing and treatment of patients with cancers harboring NTRK fusions proposes that costs may be reduced by first screening patients for TRK protein expression via IHC, followed by confirmation of NTRK gene fusion using NGS.⁹⁴ Costs may further be reduced by identifying subgroups of patients where NTRK gene fusions are enriched. Since NTRK gene fusions are typically mutually exclusive to other oncogenic drivers such as RAS and BRAF mutations,^92,95 and across multiple clinical trials, 76–89% of patients with TRK-fusion positive CRC were also dMMR/MSI-H,^92,95–98 RAS and BRAF wild-type, dMMR/MSI-H CRCs may be an ideal target population for routine NTRK testing. The NCCN guidelines recommend limiting NTRK testing to this subpopulation, which account for less than 5% of patients with mCRC.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,^100–102 and their occurrence is enriched in RAS and BRAF wild-type CRCs.¹⁰³ HER2 amplifications do not appear to be correlated with worse survival in CRC¹⁰⁴; however, evidence from small, retrospective studies show that HER2 amplifications are correlated with poorer response to anti-EGFR therapies.^105–108 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 HER2-positive 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 HER2-amplified 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

Table 5.

Clinical trials evaluating HER2-targeted therapies in patients with HER2-amplified mCRC.

Trial name, Phase	Treatment arms	Study population	HER2 requirements	Outcomes
HERACLES^102,109 Ph II	Trastuzumab + lapatinib	KRAS exon 2 wt HER2-positive Metastatic CRC Refractory to standard therapy N = 35	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	ORR: 28% Median PFS: 4.7 months (95% CI: 3.7–6.1) Median OS: 10.0 months (95% CI: 7.9–15.8)
MOUNTAINEER^114,115 Ph II	Trastuzumab + tucatinib Tucatinib monotherapy	RAS wt HER2-amplified Metastatic CRC Previously treated with 5FU, oxaliplatin, irinotecan, and an anti-VEGF antibody N = 22	HER2 amplification detected by NGS, FISH, or IHC (3+ or 2+ and amplified by FISH)	ORR: 55% Median PFS: 6.2 months (95% CI: 3.5–NE) Median OS: 17.3 months (95% CI: 12.3–NE)
MyPathway¹¹⁶ Ph II basket study	Trastuzumab + pertuzumab	HER2-amplified Metastatic CRC Treatment refractory N = 57	HER2 amplification detected by NGS, FISH, or IHC (3+ or 2+ and amplified by FISH)	ORR: 32% CR: 2% (one patient) Median PFS: 2.9 months (95% CI: 1.4–5.3)
TAPUR¹¹⁷ Ph II basket study	Trastuzumab + pertuzumab	HER2-positive Advanced CRC No standard treatment options N = 28	HER2 amplification detected by NGS, FISH, or IHC and specific HER2 mutations	DCR: 50% ORR: 25%
DESTINY-CRC01¹¹³ Ph II	Trastuzumab deruxtecan	RAS/BRAF wt HER2-positive Metastatic CRC ⩾2 prior therapies N = 78	Cohort A: IHC 3+ or IHC 2+ and FISH positive Cohort B: IHC 2+ and ISH negative Cohort C: IHC 1+	ORR: 45% in Cohort A No response in Cohorts B or C Median PFS: 6.9 months (95% CI: 4.1–NE)
DESTINY-CRC02 (NCT04744831) Ph II	Trastuzumab deruxtecan	HER2-positive Locally advanced, unresectable, or metastatic CRC Previously treated Projected N = 120	HER2 3+ by IHC or HER2 2+ and positive ISH	Primary endpoint: ORR by BICR

BICR, blinded independent central review; CI, confidence interval; CR, complete response; DCR, disease control rate; FISH, fluorescence in situ hybridization; 5FU, 5-fluorouracil; IHC, immunohistochemistry; NE, not evaluable; NGS, next-generation sequencing; ORR, overall response rate; OS, overall survival; PFS, progression-free survival; VEGF, vascular endothelial growth factor; wt, wild-type.

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%).^119,120 The TAPUR and MyPathway basket studies allow 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 multi-gene 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 trials¹³⁴; however, this may be due to the varying sensitivities of different HER2 mutations to anti-HER2 monotherapy.¹³⁵ In addition, only clinical trials of anti-HER2 combination therapies, not monotherapy, have demonstrated efficacy in HER2-expressing mCRC.¹³⁶ Dual HER2-targeted therapy has demonstrated anti-tumor activity in preclinical studies using xenograft models of HER2-mutated mCRC¹³⁷ and anti-HER2 combination regimens continue to be evaluated in clinical trials for HER2-positive patients with mCRC (NCT05350917, NCT03457896, NCT04639219, and NCT04579380).

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.^139–144 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.^145–148 Thus, combination regimens including PI3K pathway inhibitors are underway (NCT04753203, NCT04495621, NCT02861300, 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, NCT03461952, 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, transition to NGS panel testing should be considered.

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 formalin-fixation 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.^159–162 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. Meta-analyses 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 first-line 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.^{6,8,170–172} For samples requiring send-out 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).^174,175 Stating the testing method 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.^176–182 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.^184–190 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.

Footnotes

Acknowledgements

The authors acknowledge medical writing support, provided by Sarah Doucette of IMPACT Medicom Inc., which was funded through sponsorship from Pfizer Canada and ThermoFisher. Medical writing services included project management, writing of the preliminary draft, consolidation of author revisions, and coordination of manuscript submission.

Declarations

References

Canadian Cancer Statistics Advisory Committee. Canadian cancer statistics 2021, cancer.ca/Canadian-Cancer-Statistics-2021-EN (2021, accessed 26 January 2022).

Sung

Ferlay

Siegel

RL.

Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021; 71: 209–249.

Canadian Cancer Statistics Advisory Committee. Canadian cancer statistics 2018, cancer.ca/Canadian-Cancer-Statistics-2018-EN (2018, accessed 26 January 2022).

Jin

Hubbard

JM.

Optimizing biologic sequencing in metastatic colorectal cancer: first line and beyond. Curr Oncol 2019; 26: S33–S42.

Karapetis

Khambata-Ford

Jonker

, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. New Engl J Med 2008; 359: 1757–1765.

Aubin

Gill

Burkes

, et al. Canadian Expert Group consensus recommendations: KRAS testing in colorectal cancer. Curr Oncol 2011; 18: e180–e184.

Guyatt

Oxman

Vist

, et al. GRADE: an emerging consensus on rating quality of evidence and strength of recommendations. BMJ 2008; 336: 924–926.

Sepulveda

Hamilton

Allegra

CJ.

Molecular biomarkers for the evaluation of colorectal cancer. J Mol Diagn 2017; 19: 187–225.

Van Cutsem

Cervantes

Adam

, et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann Oncol 2016; 27: 1386–1422.

10.

Yoshino

Arnold

Taniguchi

, et al. Pan-Asian adapted ESMO consensus guidelines for the management of patients with metastatic colorectal cancer: a JSMO-ESMO initiative endorsed by CSCO, KACO, MOS, SSO and TOS. Ann Oncol 2018; 29: 44–70.

11.

National Institute for Health and Care Excellence. NICE guideline [NG151] Colorectal cancer, https://www.nice.org.uk/guidance/ng151/chapter/Recommendations#molecular-biomarkers-to-guide-systemic-anti-cancer-therapy (2021, accessed 29 March 2022).

12.

Cancer Council Australia. Clinical guidelines: optimal molecular profiling, https://wiki.cancer.org.au/australia/Clinical_question:Molecular_profiling_of_CRC#Evidence_summary_and_recommendations (accessed 12 December 2021; published 7 November 2017).

13.

National Comprehensive Cancer Network. Clinical practice guidelines in oncology: colon cancer, version 1.2022, https://www.nccn.org/professionals/physician_gls/pdf/colon.pdf (accessed 29 March 2022; published 25 February 2022).

14.

Jonker

O’Callaghan

Karapetis

, et al. Cetuximab for the treatment of colorectal cancer. New Engl J Med 2007; 357: 2040–2048.

15.

Van Cutsem

Peeters

Siena

, et al. Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy-refractory metastatic colorectal cancer. J Clin Oncol 2007; 25: 1658–1664.

16.

Amado

Wolf

Peeters

, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008; 26: 1626–1634.

17.

Lièvre

Bachet

J-B

Boige

, et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol 2008; 26: 374–379.

18.

Benvenuti

Sartore-Bianchi

Di Nicolantonio

, et al. Oncogenic activation of the RAS/RAF signaling pathway impairs the response of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies. Cancer Res 2007; 67: 2643–2648.

19.

Di Fiore

Blanchard

Charbonnier

, et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by cetuximab plus chemotherapy. Br J Cancer 2007; 96: 1166–1169.

20.

De Roock

Piessevaux

De Schutter

, et al. KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab. Ann Oncol 2008; 19: 508–515.

21.

Van Cutsem

Köhne

C-H

Hitre

, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. New Engl J Med 2009; 360: 1408–1417.

22.

Van Cutsem

Lenz

H-J

Köhne

C-H

, et al. Fluorouracil, leucovorin, and irinotecan plus cetuximab treatment and RAS mutations in colorectal cancer. J Clin Oncol 2015; 33: 692–700.

23.

Bokemeyer

Bondarenko

Hartmann

, et al. Efficacy according to biomarker status of cetuximab plus FOLFOX-4 as first-line treatment for metastatic colorectal cancer: the OPUS study. Ann Oncol 2011; 22: 1535–1546.

24.

Bokemeyer

Köhne

Ciardiello

, et al. FOLFOX4 plus cetuximab treatment and RAS mutations in colorectal cancer. Eur J Cancer 2015; 51: 1243–1252.

25.

Peeters

Oliner

Price

, et al. Analysis of KRAS/NRAS mutations in a phase III study of panitumumab with FOLFIRI compared with FOLFIRI alone as second-line treatment for metastatic colorectal cancer. Clin Cancer Res 2015; 21: 5469–5479.

26.

Douillard

J-Y

Oliner

Siena

, et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. New Engl J Med 2013; 369: 1023–1034.

27.

Loree

Dowers

, et al. Expanded low allele frequency RAS and BRAF V600E testing in metastatic colorectal cancer as predictive biomarkers for cetuximab in the randomized CO.17 trial. Clin Cancer Res 2021; 27: 52–59.

28.

Bokemeyer

Van Cutsem

Rougier

, et al. Addition of cetuximab to chemotherapy as first-line treatment for KRAS wild-type metastatic colorectal cancer: pooled analysis of the CRYSTAL and OPUS randomised clinical trials. Eur J Cancer 2012; 48: 1466–1475.

29.

Maughan

Adams

Smith

CG.

Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. Lancet 2011; 377: 2103–2114.

30.

Tveit

Guren

Glimelius

, et al. Phase III trial of cetuximab with continuous or intermittent fluorouracil, leucovorin, and oxaliplatin (Nordic FLOX) versus FLOX alone in first-line treatment of metastatic colorectal cancer: the NORDIC-VII study. J Clin Oncol 2012; 30: 1755–1762.

31.

Guren

Thomsen

Kure

, et al. Cetuximab in treatment of metastatic colorectal cancer: final survival analyses and extended RAS data from the NORDIC-VII study. Br J Cancer 2017; 116: 1271–1278.

32.

Innocenti

, et al. Mutational analysis of patients with colorectal cancer in CALGB/SWOG 80405 identifies new roles of microsatellite instability and tumor mutational burden for patient outcome. J Clin Oncol 2019; 37: 1217–1227.

33.

Stintzing

Miller-Phillips

Modest

, et al. Impact of BRAF and RAS mutations on first-line efficacy of FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab: analysis of the FIRE-3 (AIO KRK-0306) study. Eur J Cancer 2017; 79: 50–60.

34.

Heinemann

von Weikersthal

Decker

, et al. FOLFIRI plus cetuximab or bevacizumab for advanced colorectal cancer: final survival and per-protocol analysis of FIRE-3, a randomised clinical trial. Br J Cancer 2021; 124: 587–594.

35.

Patterson

Peeters

Siena

, et al. Comprehensive analysis of KRAS and NRAS mutations as predictive biomarkers for single agent panitumumab (pmab) response in a randomized, phase III metastatic colorectal cancer (mCRC) study (20020408). J Clin Oncol 2013; 31: 3617–3617.

36.

Kim

Elme

Park

, et al. Final analysis of outcomes and RAS/BRAF status in a randomized phase 3 study of panitumumab and best supportive care in chemorefractory wild type KRAS metastatic colorectal cancer. Clin Colorectal Cancer 2018; 17: 206–214.

37.

Seymour

Brown

Middleton

, et al. Panitumumab and irinotecan versus irinotecan alone for patients with KRAS wild-type, fluorouracil-resistant advanced colorectal cancer (PICCOLO): a prospectively stratified randomised trial. Lancet Oncol 2013; 14: 749–759.

38.

Rivera

Karthaus

Hecht

, et al. Final analysis of the randomised PEAK trial: overall survival and tumour responses during first-line treatment with mFOLFOX6 plus either panitumumab or bevacizumab in patients with metastatic colorectal carcinoma. Int J Colorectal Dis 2017; 32: 1179–1190.

39.

Yoshino

Watanabe

Shitara

, et al. Panitumumab (PAN) plus mFOLFOX6 versus bevacizumab (BEV) plus mFOLFOX6 as first-line treatment in patients with RAS wild-type (WT) metastatic colorectal cancer (mCRC): Results from the phase 3 PARADIGM trial. J Clin Oncol 2021; 40: LBA1–LBA1.

40.

Serebriiskii

Connelly

Frampton

, et al. Comprehensive characterization of RAS mutations in colon and rectal cancers in old and young patients. Nat Commun 2019; 10: 3722–3733.

41.

Sorich

Wiese

Rowland

, et al. Extended RAS mutations and anti-EGFR monoclonal antibody survival benefit in metastatic colorectal cancer: a meta-analysis of randomized, controlled trials. Ann Oncol 2015; 26: 13–21.

42.

Adelstein

B-A

Dobbins

Harris

, et al. A systematic review and meta-analysis of KRAS status as the determinant of response to anti-EGFR antibodies and the impact of partner chemotherapy in metastatic colorectal cancer. Eur J Cancer 2011; 47: 1343–1354.

43.

Cao

D-D

H-L

X-M

, et al. The impact of primary tumor location on efficacy of cetuximab in metastatic colorectal cancer patients with different Kras status: a systematic review and meta-analysis. Oncotarget 2017; 8: 53631–53641.

44.

Tejpar

Stintzing

Ciardiello

, et al. Prognostic and predictive relevance of primary tumor location in patients with RAS wild-type metastatic colorectal cancer: retrospective analyses of the CRYSTAL and FIRE-3 trials. JAMA Oncol 2017; 3: 194–201.

45.

Venook

Niedzwiecki

Innocenti

, et al. Impact of primary (1º) tumor location on overall survival (OS) and progression-free survival (PFS) in patients (pts) with metastatic colorectal cancer (mCRC): analysis of CALGB/SWOG 80405 (alliance). J Clin Oncol 2016; 34: 3504–3504.

46.

Eli Lilly Canada Inc. Product monograph: PrERBITUX^® (cetuximab), https://pi.lilly.com/ca/erbitux-ca-pm.pdf (2020, accessed 21 March 2022).

47.

Amgen Canada Inc. Product monograph including patient medication information: ^PrVectibix^® panitumumab for injection, https://www.amgen.ca/-/media/Themes/CorporateAffairs/amgen-ca/amgen-ca/documents/products/en/vectibix_pm.pdf (2021, accessed 21 March 2022).

48.

Bridgewater

Pugh

Maishman

, et al. Systemic chemotherapy with or without cetuximab in patients with resectable colorectal liver metastasis (New EPOC): long-term results of a multicentre, randomised, controlled, phase 3 trial. Lancet Oncol 2020; 21: 398–411.

49.

Araujo

Souza

Leite

, et al. Molecular profile of KRAS G12C-mutant colorectal and non-small-cell lung cancer. BMC Cancer 2021; 21: 193.

50.

Hong

Fakih

Strickler

, et al. KRASG12C inhibition with sotorasib in advanced solid tumors. New Engl J Med 2020; 383: 1207–1217.

51.

Weiss

Yaeger

Johnson

, et al. LBA6 KRYSTAL-1: adagrasib (MRTX849) as monotherapy or combined with cetuximab (Cetux) in patients (Pts) with colorectal cancer (CRC) harboring a KRASG12C mutation. Ann Oncol 2021; 32: S1294–S1346.

52.

Tabernero

Bendell

Corcoran

, et al. P-71 KRYSTAL-10: a randomized phase 3 study of adagrasib (MRTX849) in combination with cetuximab vs chemotherapy in patients with previously treated advanced colorectal cancer with KRASG12C mutation. Ann Oncol 2021; 32: S121.

53.

Clarke

Kopetz

ES.

BRAF mutant colorectal cancer as a distinct subset of colorectal cancer: clinical characteristics, clinical behavior, and response to targeted therapies. J Gastrointest Oncol 2015; 6: 660–667.

54.

Chu

Johnson

Kugathasan

, et al. Population-based screening for BRAF (V600E) in metastatic colorectal cancer reveals increased prevalence and poor prognosis. Clin Cancer Res 2020; 26: 4599–4605.

55.

Seligmann

Fisher

Smith

, et al. Investigating the poor outcomes of BRAF-mutant advanced colorectal cancer: analysis from 2530 patients in randomised clinical trials. Ann Oncol 2017; 28: 562–568.

56.

Modest

Ricard

Heinemann

, et al. Outcome according to KRAS-, NRAS- and BRAF-mutation as well as KRAS mutation variants: pooled analysis of five randomized trials in metastatic colorectal cancer by the AIO colorectal cancer study group. Ann Oncol 2016; 27: 1746–1753.

57.

Rowland

Dias

Wiese

MD.

Meta-analysis of BRAF mutation as a predictive biomarker of benefit from anti-EGFR monoclonal antibody therapy for RAS wild-type metastatic colorectal cancer. Br J Cancer 2015; 112: 1888–2189.

58.

Cremolini

Di Maio

Petrelli

, et al. BRAF-mutated metastatic colorectal cancer between past and future. Br J Cancer 2015; 113: 1634–1635.

59.

van Brummelen

EMJ

de Boer

Beijnen

, et al. BRAF mutations as predictive biomarker for response to anti-EGFR monoclonal antibodies. Oncologist 2017; 22: 864–872.

60.

Kopetz

Desai

Chan

, et al. Phase II pilot study of vemurafenib in patients with metastatic BRAF-mutated colorectal cancer. J Clin Oncol 2015; 33: 4032–4038.

61.

Prahallad

Sun

Huang

, et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature 2012; 483: 100–103.

62.

Corcoran

Ebi

Turke

, et al. EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer Discov 2012; 2: 227–235.

63.

Kopetz

Guthrie

Morris

, et al. Randomized trial of irinotecan and cetuximab with or without vemurafenib in BRAF-mutant metastatic colorectal cancer (SWOG S1406). J Clin Oncol 2021; 39: 285–294.

64.

Corcoran

Atreya

Falchook

, et al. Combined BRAF and MEK inhibition with dabrafenib and trametinib in BRAF V600-mutant colorectal cancer. J Clin Oncol 2015; 33: 4023–4031.

65.

Tabernero

Grothey

Van Cutsem

, et al. Encorafenib plus cetuximab as a new standard of care for previously treated BRAF V600E-mutant metastatic colorectal cancer: updated survival results and subgroup analyses from the BEACON study. J Clin Oncol 2021; 39: 273–284.

66.

Van Cutsem

Tabernero

Taieb

, et al. ANCHOR CRC: results from a single-arm, phase 2 study of encorafenib, binimetinib plus cetuximab in previously untreated BRAF V600E–mutant metastatic colorectal cancer. In: ESMO world congress on gastrointestinal cancer, Lugano, Switzerland, 30 June–3 July 2021, p. S222.

67.

Kopetz

Grothey

Yaeger

, et al. BREAKWATER: randomized phase 3 study of encorafenib (enco) + cetuximab (cetux) ± chemotherapy for first-line (1L) treatment (tx) of BRAF V600E-mutant (BRAFV600E) metastatic colorectal cancer (mCRC). J Clin Oncol 2021; 39: ts3619.

68.

Battaglin

Naseem

Lenz

H-J.

Microsatellite instability in colorectal cancer: overview of its clinical significance and novel perspectives. Clin Adv Hematol Oncol 2018; 16: 735–745.

69.

Vilar

Gruber

SB.

Microsatellite instability in colorectal cancer: the stable evidence. Nat Rev Clin Oncol 2010; 7: 153–162.

70.

Popat

Hubner

Houlston

RS.

Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol 2005; 23: 609–618.

71.

Sherman

Schuitevoerder

Chan

CHF

, et al. Metastatic colorectal cancers with mismatch repair deficiency result in worse survival regardless of peritoneal metastases. Ann Surg Oncol 2020; 27: 5074–5083.

72.

van der Heide

Turaga

Chan

CHF

, et al. Mismatch repair status correlates with survival in young adults with metastatic colorectal cancer. J Surg Res 2021; 266: 104–112.

73.

Wensink

Bond

Kucukkose

, et al. A review of the sensitivity of metastatic colorectal cancer patients with deficient mismatch repair to standard-of-care chemotherapy and monoclonal antibodies, with recommendations for future research. Cancer Treat Rev 2021; 95: 102174.

74.

Wensink

Elferink

MAG

May

. Survival of patients with deficient mismatch repair metastatic colorectal cancer in the pre-immunotherapy era. Br J Cancer 2021; 124: 399–406.

75.

Tougeron

Sueur

Zaanan

, et al. Prognosis and chemosensitivity of deficient MMR phenotype in patients with metastatic colorectal cancer: an AGEO retrospective multicenter study. Int J Cancer 2020; 147: 285–296.

76.

Gelsomino

Barbolini

Spallanzani

, et al. The evolving role of microsatellite instability in colorectal cancer: a review. Cancer Treat Rev 2016; 51: 19–26.

77.

Durham

Smith

KN.

Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017; 357: 409–413.

78.

Uram

Wang

PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med 2015; 372: 2509–2520.

79.

Kim

Van Cutsem

, et al. Phase II open-label study of pembrolizumab in treatment-refractory, microsatellite instability-high/mismatch repair-deficient metastatic colorectal cancer: KEYNOTE-164. J Clin Oncol 2020; 38: 11–19.

80.

André

Shiu

K-K

Kim

, et al. Pembrolizumab in microsatellite-instability-high advanced colorectal cancer. New Engl J Med 2020; 383: 2207–2218.

81.

Andre

Shiu

K-K

Kim

, et al. Final overall survival for the phase III KN177 study: pembrolizumab versus chemotherapy in microsatellite instability-high/mismatch repair deficient (MSI-H/dMMR) metastatic colorectal cancer (mCRC). J Clin Oncol 2021; 39: 3500–3500.

82.

Overman

Lonardi

Wong

KYM

, et al. Durable clinical benefit with nivolumab plus ipilimumab in DNA mismatch repair-deficient/microsatellite instability-high metastatic colorectal cancer. J Clin Oncol 2018; 36: 773–779.

83.

Lenz

H-J

Van Cutsem

Luisa Limon

, et al. First-line nivolumab plus low-dose ipilimumab for microsatellite instability-high/mismatch repair-deficient metastatic colorectal cancer: the phase II CheckMate 142 study. J Clin Oncol 2022; 40: 161–170.

84.

Hechtman

JF.

NTRK insights: best practices for pathologists. Mod Pathol 2022; 35: 298–305.

85.

Pulciani

Santos

Lauver

, et al. Oncogenes in solid human tumours. Nature 1982; 300: 539–542.

86.

Hong

DuBois

Kummar

, et al. Larotrectinib in patients with TRK fusion-positive solid tumours: a pooled analysis of three phase 1/2 clinical trials. Lancet Oncol 2020; 21: 531–540.

87.

Doebele

Drilon

Paz-Ares

, et al. Entrectinib in patients with advanced or metastatic NTRK fusion-positive solid tumours: integrated analysis of three phase 1–2 trials. Lancet Oncol 2020; 21: 271–282.

88.

Kopetz

The emergence of targetable pathways in colorectal cancer. Clin Adv Hematol Oncol 2021; 19: 774–783.

89.

Bormann Chung

Lee

Barritault

, et al. Evaluating targeted next-generation sequencing assays and reference materials for NTRK fusion detection. J Mol Diagn 2022; 24: 18–32.

90.

Pfarr

Kirchner

Lehmann

, et al. Testing NTRK testing: Wet-lab and in silico comparison of RNA-based targeted sequencing assays. Genes Chromosomes Cancer 2020; 59: 178–188.

91.

Tsao

Torlakovic

Stockley

, et al. CANTRK: a Canadian multi-centre NTRK gene fusion testing validation in solid tumors project. In: Proceedings of the Association for Molecular Pathology annual meeting, Virtual Program, 16–20 November 2020, abstract ST07.

92.

Chou

Fraser

Ahadi

, et al. NTRK gene rearrangements are highly enriched in MLH1/PMS2 deficient, BRAF wild-type colorectal carcinomas: a study of 4569 cases. Mod Pathol 2020; 33: 924–932.

93.

Lasota

Chłopek

Lamoureux

, et al. Colonic adenocarcinomas harboring NTRK fusion genes: a clinicopathologic and molecular genetic study of 16 cases and review of the literature. Am J Surg Pathol 2020; 44: 162–173.

94.

Bebb

Banerji

Blais

, et al. Canadian consensus for biomarker testing and treatment of TRK fusion cancer in adults. Curr Oncol 2021; 28: 523–548.

95.

Pietrantonio

Di Nicolantonio

Schrock

, et al. ALK, ROS1, and NTRK rearrangements in metastatic colorectal cancer. J Natl Cancer Inst 2017; 109: 1–10.

96.

Rosen

Goldman

Hechtman

, et al. TRK fusions are enriched in cancers with uncommon histologies and the absence of canonical driver mutations. Clin Cancer Res 2020; 26: 1624–1632.

97.

Berlin

Hong

Deeken

, et al. Efficacy and safety of larotrectinib in patients with TRK fusion gastrointestinal cancer. J Clin Oncol 2020; 38: 824–824.

98.

Cocco

Benhamida

Middha

, et al. Colorectal carcinomas containing hypermethylated MLH1 promoter and wild-type BRAF/KRAS are enriched for targetable kinase fusions. Cancer Res 2019; 79: 1047–1053.

99.

Yarden

Sliwkowski

MX.

Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001; 2: 127–137.

100.

Ingold Heppner

Behrens

Balschun

, et al. HER2/neu testing in primary colorectal carcinoma. Br J Cancer 2014; 111: 1977–1984.

101.

The Cancer Genome Atlas. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012; 487: 330–337.

102.

Sartore-Bianchi

Trusolino

Martino

, et al. Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (HERACLES): a proof-of-concept, multicentre, open-label, phase 2 trial. Lancet Oncol 2016; 17: 738–746.

103.

Richman

Southward

Chambers

, et al. HER2 overexpression and amplification as a potential therapeutic target in colorectal cancer: analysis of 3256 patients enrolled in the QUASAR, FOCUS and PICCOLO colorectal cancer trials. J Pathol 2016; 238: 562–570.

104.

WH.

Does overexpression of HER-2 correlate with clinicopathological characteristics and prognosis in colorectal cancer? Evidence from a meta-analysis. Diagn Pathol 2015; 10: 144.

105.

Sartore-Bianchi

Amatu

Porcu

, et al. HER2 positivity predicts unresponsiveness to EGFR-targeted treatment in metastatic colorectal cancer. Oncologist 2019; 24: 1395–1402.

106.

Martin

Landi

Molinari

, et al. HER2 gene copy number status may influence clinical efficacy to anti-EGFR monoclonal antibodies in metastatic colorectal cancer patients. Br J Cancer 2013; 108: 668–675.

107.

Raghav

Loree

Morris

, et al. Validation of HER2 amplification as a predictive biomarker for anti-epidermal growth factor receptor antibody therapy in metastatic colorectal cancer. JCO Precis Oncol 2019; 3: 1–13.

108.

Sawada

Nakamura

Yamanaka

, et al. Prognostic and predictive value of HER2 amplification in patients with metastatic colorectal cancer. Clin Colorectal Cancer 2018; 17: 198–205.

109.

Tosi

Sartore-Bianchi

Lonardi

, et al. Long-term clinical outcome of trastuzumab and lapatinib for HER2-positive metastatic colorectal cancer. Clin Colorectal Cancer 2020; 19: 256–262.e2.

110.

Sartore-Bianchi

Lonardi

Aglietta

, et al. Central nervous system as possible site of relapse in ERBB2-positive metastatic colorectal cancer: long-term results of treatment with trastuzumab and lapatinib. JAMA Oncol 2020; 6: 927–929.

111.

Aprile

De Maglio

Menis

, et al. HER-2 expression in brain metastases from colorectal cancer and corresponding primary tumors: a case cohort series. Int J Mol Sci 2013; 14: 2370–2387.

112.

Mitra

Clark

Shih

, et al. Enrichment of HER2 amplification in brain metastases from primary gastrointestinal malignancies. Oncologist 2019; 24: 193–201.

113.

Siena

Di Bartolomeo

Raghav

, et al. Trastuzumab deruxtecan (DS-8201) in patients with HER2-expressing metastatic colorectal cancer (DESTINY-CRC01): a multicentre, open-label, phase 2 trial. Lancet Oncol 2021; 22: 779–789.

114.

Strickler

Zemla

, et al. Trastuzumab and tucatinib for the treatment of HER2 amplified metastatic colorectal cancer (mCRC): initial results from the MOUNTAINEER trial. Ann Oncol 2019; 30: v200.

115.

Strickler

Cercek

, et al. MOUNTAINEER: open-label, phase II study of tucatinib combined with trastuzumab for HER2-positive metastatic colorectal cancer (SGNTUC-017, trial in progress). J Clin Oncol 2021; 39: TPS153.

116.

Meric-Bernstam

Hurwitz

Raghav

KPS

, et al. Pertuzumab plus trastuzumab for HER2-amplified metastatic colorectal cancer (MyPathway): an updated report from a multicentre, open-label, phase 2a, multiple basket study. Lancet Oncol 2019; 20: 518–530.

117.

Gupta

Garrett-Mayer

Halabi

, et al. Pertuzumab plus trastuzumab (P + T) in patients (Pts) with colorectal cancer (CRC) with ERBB2 amplification or overexpression: results from the TAPUR study. J Clin Oncol 2020; 38: 132–132.

118.

Yoshino

Di Bartolomeo

Raghav

KPS

, et al. Trastuzumab deruxtecan (T-DXd; DS-8201) in patients (pts) with HER2-expressing metastatic colorectal cancer (mCRC): final results from a phase 2, multicenter, open-label study (DESTINY-CRC01). J Clin Oncol 2021; 39: 3503–3505.

119.

Wolff

Hammond

MEH

Hicks

, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 2013; 31: 3997–4013.

120.

Bartley

Washington

Colasacco

, et al. HER2 testing and clinical decision making in gastroesophageal adenocarcinoma: guideline from the College of American Pathologists, American Society for Clinical Pathology, and the American Society of Clinical Oncology. J Clin Oncol 2017; 35: 446–464.

121.

Marabelle

Fakih

Lopez

, et al. Association of tumour mutational burden with outcomes in patients with advanced solid tumours treated with pembrolizumab: prospective biomarker analysis of the multicohort, open-label, phase 2 KEYNOTE-158 study. Lancet Oncol 2020; 21: 1353–1365.

122.

Fabrizio

George

Jr Dunne

, et al. Beyond microsatellite testing: assessment of tumor mutational burden identifies subsets of colorectal cancer who may respond to immune checkpoint inhibition. J Gastrointest Oncol 2018; 9: 610–617.

123.

Meiri

Garrett-Mayer

Halabi

, et al. Pembrolizumab (P) in patients (Pts) with colorectal cancer (CRC) with high tumor mutational burden (HTMB): results from the Targeted Agent and Profiling Utilization Registry (TAPUR) study. J Clin Oncol 2020; 38: 133–133.

124.

Fukuoka

Hara

Takahashi

, et al. Regorafenib plus nivolumab in patients with advanced gastric or colorectal cancer: an open-label, dose-escalation, and dose-expansion phase Ib trial (REGONIVO, EPOC1603). J Clin Oncol 2020; 38: 2053–2061.

125.

Chen

Jonker

Loree

, et al. Effect of combined immune checkpoint inhibition vs best supportive care alone in patients with advanced colorectal cancer: the Canadian Cancer Trials Group CO.26 study. JAMA Oncol 2020; 6: 831–838.

126.

Loree

Topham

Kennecke

, et al. Tissue and plasma tumor mutation burden (TMB) as predictive biomarkers in the CO.26 trial of durvalumab + tremelimumab (D + T) versus best supportive care (BSC) in metastatic colorectal cancer (mCRC). J Clin Oncol 2021; 39: 61–61.

127.

Valtorta

Misale

Sartore-Bianchi

, et al. KRAS gene amplification in colorectal cancer and impact on response to EGFR-targeted therapy. Int J Cancer 2013; 133: 1259–1265.

128.

Favazza

Parseghian

Kaya

, et al. KRAS amplification in metastatic colon cancer is associated with a history of inflammatory bowel disease and may confer resistance to anti-EGFR therapy. Mod Pathol 2020; 33: 1832–1843.

129.

Jones

Renfro

Al-Shamsi

, et al. Non-V600 BRAF mutations define a clinically distinct molecular subtype of metastatic colorectal cancer. J Clin Oncol 2017; 35: 2624–2630.

130.

Yaeger

Kotani

Mondaca

, et al. Response to anti-EGFR therapy in patients with BRAF non-V600-mutant metastatic colorectal cancer. Clin Cancer Res 2019; 25: 7089–7097.

131.

Johnson

Loree

Jacome

, et al. Atypical, non-V600 BRAF mutations as a potential mechanism of resistance to EGFR inhibition in metastatic colorectal cancer. JCO Precis Oncol 2019; 3: 1–10.

132.

Ross

Fakih

Ali

, et al. Targeting HER2 in colorectal cancer: the landscape of amplification and short variant mutations in ERBB2 and ERBB3. Cancer 2018; 124: 1358–1373.

133.

Randon

Yaeger

Hechtman

, et al. EGFR amplification in metastatic colorectal cancer. J Natl Cancer Inst 2021; 113: 1561–1569.

134.

Hyman

Piha-Paul

Won

, et al. HER kinase inhibition in patients with HER2- and HER3-mutant cancers. Nature 2018; 554: 189–194.

135.

Ahcene Djaballah

Daniel

Milani

, et al. HER2 in colorectal cancer: the Long and winding road from negative predictive factor to positive actionable target. Am Soc Clin Oncol Educ Book 2022; 42: 1–14.

136.

Siena

Sartore-Bianchi

Marsoni

, et al. Targeting the human epidermal growth factor receptor 2 (HER2) oncogene in colorectal cancer. Ann Oncol 2018; 29: 1108–1119.

137.

Kavuri

Jain

Galimi

, et al. HER2 activating mutations are targets for colorectal cancer treatment. Cancer Discov 2015; 5: 832–841.

138.

Cathomas

PIK3CA in colorectal cancer. Front Oncol 2014; 4: 35.

139.

De Roock

Claes

Bernasconi

, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 2010; 11: 753–762.

140.

Sartore-Bianchi

Martini

Molinari

, et al. PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-targeted monoclonal antibodies. Cancer Res 2009; 69: 1851–1857.

141.

Perrone

Lampis

Orsenigo

, et al. PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients. Ann Oncol 2009; 20: 84–90.

142.

Sood

McClain

Maitra

, et al. PTEN gene expression and mutations in the PIK3CA gene as predictors of clinical benefit to anti-epidermal growth factor receptor antibody therapy in patients with KRAS wild-type metastatic colorectal cancer. Clin Colorectal Cancer 2012; 11: 143–150.

143.

Prenen

De Schutter

Jacobs

, et al. PIK3CA mutations are not a major determinant of resistance to the epidermal growth factor receptor inhibitor cetuximab in metastatic colorectal cancer. Clin Cancer Res 2009; 15: 3184–3188.

144.

Mao

Yang

, et al. PIK3CA exon 20 mutations as a potential biomarker for resistance to anti-EGFR monoclonal antibodies in KRAS wild-type metastatic colorectal cancer: a systematic review and meta-analysis. Ann Oncol 2012; 23: 1518–1525.

145.

Motta

Cabezas-Camarero

Torres-Mattos

, et al. Personalizing first-line treatment in advanced colorectal cancer: present status and future perspectives. J Clin Transl Res 2021; 7: 771–785.

146.

Rosen

Goldman

Hubbard

, et al. 382 phase Ib study of oral dual-PI3K/mTOR inhibitor GDC-0980 in combination with capecitabine and mFOLFOX6 + bevacizumab in patients with advanced solid tumors and colorectal cancer. Eur J Cancer 2014; 50: 122–123.

147.

Doi

Fuse

Yoshino

, et al. A phase I study of intravenous PI3K inhibitor copanlisib in Japanese patients with advanced or refractory solid tumors. Cancer Chemother Pharmacol 2017; 79: 89–98.

148.

Damodaran

Zhao

Deming

, et al. Phase II study of copanlisib in patients with tumors with PIK3CA mutations: results from the NCI-MATCH ECOG-ACRIN trial (EAY131) subprotocol Z1F. J Clin Oncol 2022; 40: 1552–1561.

149.

Morschhauser

Machiels

J-P

Salles

, et al. On-target pharmacodynamic activity of the PI3K inhibitor copanlisib in paired biopsies from patients with malignant lymphoma and advanced solid tumors. Mol Cancer Ther 2020; 19: 468–478.

150.

Liu

Predictive molecular markers for the treatment with immune checkpoint inhibitors in colorectal cancer. J Clin Lab Anal 2022; 36: e24141.

151.

Wang

Zhao

Wang

Y-N

, et al. Evaluation of POLE and POLD1 mutations as biomarkers for immunotherapy outcomes across multiple cancer types. JAMA Oncol 2019; 5: 1504–1506.

152.

College of American Pathologists. Laboratory accreditation program, https://www.cap.org/laboratory-improvement/accreditation/laboratory-accreditation-program (accessed 26 March 2022).

153.

Colorectal Cancer Canada. An environemental scan of molecular porfiling across Canada, https://www.colorectalcancercanada.com/what-we-do/our-programs/ (accessed 28 March 2022; published 7 April 2021).

154.

Yip

Christofides

Banerji

, et al. A Canadian guideline on the use of next-generation sequencing in oncology. Curr Oncol 2019; 26: 241–254.

155.

Drilon

Wang

Arcila

, et al. Broad, hybrid capture-based next-generation sequencing identifies actionable genomic alterations in lung adenocarcinomas otherwise negative for such alterations by other genomic testing approaches. Clin Cancer Res 2015; 21: 3631–3639.

156.

Pennell

Mutebi

Zhou

Z-Y

, et al. Economic impact of next generation sequencing vs sequential single-gene testing modalities to detect genomic alterations in metastatic non-small cell lung cancer using a decision analytic model. J Clin Oncol 2018; 36: 9031–9031.

157.

Perdrizet

Stockley

Law

, et al. Non-small cell lung cancer (NSCLC) next generation sequencing (NGS) using the Oncomine Comprehensive Assay (OCA) v3: integrating expanded genomic sequencing into the Canadian publicly funded health care model. J Clin Oncol 2019; 37: 2620–2620.

158.

Bhullar

Barriuso

Mullamitha

, et al. Biomarker concordance between primary colorectal cancer and its metastases. EBioMedicine 2019; 40: 363–374.

159.

Watanabe

Hashida

Yamamoto

, et al. Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods. Exp Ther Med 2017; 14: 2683–2688.

160.

Dobrovic

Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem 2015; 61: 64–71.

161.

Ademà

Torres

Solé

, et al. Paraffin treasures: do they last forever? Biopreserv Biobank 2014; 12: 281–283.

162.

Guyard

Boyez

Pujals

, et al. DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks. Virchows Arch 2017; 471: 491–500.

163.

Bertheau

Cazals-Hatem

Meignin

, et al. Variability of immunohistochemical reactivity on stored paraffin slides. J Clin Pathol 1998; 51: 370–374.

164.

Grillo

Pigozzi

Ceriolo

, et al. Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections. Histochem Cell Biol 2015; 144: 93–99.

165.

Hanna

King

Thibodeau

, et al. Mortality due to cancer treatment delay: systematic review and meta-analysis. BMJ 2020; 371: m4087.

166.

Biagi

Raphael

Mackillop

, et al. Association between time to initiation of adjuvant chemotherapy and survival in colorectal cancer: a systematic review and meta-analysis. JAMA 2011; 305: 2335–2342.

167.

Voskoboynik

Bae

Ananda

, et al. An initial watch and wait approach is a valid strategy for selected patients with newly diagnosed metastatic colorectal cancer. Ann Oncol 2012; 23: 2633–2637.

168.

Lee

Y-H

Kung

P-T

Wang

Y-H

, et al. Effect of length of time from diagnosis to treatment on colorectal cancer survival: a population-based study. PLoS One 2019; 14: e0210465.

169.

Shiu

K-K

Andre

Kim

, et al. KEYNOTE-177: phase III randomized study of pembrolizumab versus chemotherapy for microsatellite instability-high advanced colorectal cancer. J Clin Oncol 2021; 39: 6–6.

170.

Lindeman

Cagle

Aisner

, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Mol Diagn 2018; 20: 129–159.

171.

Wong

Gonzalez

Salto-Tellez

, et al. RAS testing of colorectal carcinoma—a guidance document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Group. J Clin Pathol 2014; 67: 751–757.

172.

Cheema

Banerji

Blais

, et al. Canadian consensus recommendations on the management of MET-altered NSCLC. Curr Oncol 2021; 28: 4552–4576.

173.

Sheffield

Beharry

Diep

, et al. Point of care molecular testing: community-based rapid next-generation sequencing to support cancer care. Curr Oncol 2022; 29: 1326–1334.

174.

Richards

Aziz

Bale

, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17: 405–424.

175.

College of American Pathologists. Template for reporting results of biomarker testing of specimens from patients with carcinoma of the colon and rectum, https://documents.cap.org/protocols/ColoRectal.Bmk_1.3.0.0.REL_CAPCP.pdf (2021, accessed 28 March 2022).

176.

Gray

Quirke

Handley

, et al. Validation study of a quantitative multigene reverse transcriptase-polymerase chain reaction assay for assessment of recurrence risk in patients with stage II colon cancer. J Clin Oncol 2011; 29: 4611–4619.

177.

Yothers

O’Connell

Lee

, et al. Validation of the 12-gene colon cancer recurrence score in NSABP C-07 as a predictor of recurrence in patients with stage II and III colon cancer treated with fluorouracil and leucovorin (FU/LV) and FU/LV plus oxaliplatin. J Clin Oncol 2013; 31: 4512–4519.

178.

Venook

Niedzwiecki

Lopatin

, et al. Biologic determinants of tumor recurrence in stage II colon cancer: validation study of the 12-gene recurrence score in cancer and leukemia group B (CALGB) 9581. J Clin Oncol 2013; 31: 1775–1781.

179.

Salazar

Roepman

Capella

, et al. Gene expression signature to improve prognosis prediction of stage II and III colorectal cancer. J Clin Oncol 2011; 29: 17–24.

180.

Kopetz

Tabernero

Rosenberg

, et al. Genomic classifier ColoPrint predicts recurrence in stage II colorectal cancer patients more accurately than clinical factors. Oncologist 2015; 20: 127–133.

181.

Kennedy

Bylesjo

Kerr

, et al. Development and independent validation of a prognostic assay for stage II colon cancer using formalin-fixed paraffin-embedded tissue. J Clin Oncol 2011; 29: 4620–4626.

182.

Niedzwiecki

Frankel

Venook

, et al. Association between results of a gene expression signature assay and recurrence-free interval in patients with stage II colon cancer in cancer and leukemia group B 9581 (alliance). J Clin Oncol 2016; 34: 3047–3053.

183.

Mlecnik

Bifulco

Bindea

Multicenter international society for immunotherapy of cancer study of the consensus immunoscore for the prediction of survival and response to chemotherapy in stage III colon cancer. J Clin Oncol 2020; 38: 3638–3651.

184.

Cao

Chang

, et al. Genotyping of circulating tumor DNA reveals the clinically actionable mutation landscape of advanced colorectal cancer. Mol Cancer Ther 2019; 18: 1158–1167.

185.

Choi

Kato

Fanta

, et al. Genomic profiling of blood-derived circulating tumor DNA from patients with colorectal cancer: implications for response and resistance to targeted therapeutics. Mol Cancer Ther 2019; 18: 1852–1862.

186.

Holm

Andersson

Osterlund

, et al. Detection of KRAS mutations in liquid biopsies from metastatic colorectal cancer patients using droplet digital PCR, Idylla, and next generation sequencing. PLoS One 2020; 15: e0239819.

187.

Liu

Zhao

Guo

, et al. Dynamic monitoring of HER2 amplification in circulating DNA of patients with metastatic colorectal cancer treated with cetuximab. Clin Transl Oncol 2020; 22: 928–934.

188.

Nakamura

Okamoto

Kato

, et al. Circulating tumor DNA-guided treatment with pertuzumab plus trastuzumab for HER2-amplified metastatic colorectal cancer: a phase 2 trial. Nat Med 2021; 27: 1899–1903.

189.

Nakajima

Kotani

Bando

, et al. REMARRY and PURSUIT trials: liquid biopsy-guided rechallenge with anti-epidermal growth factor receptor (EGFR) therapy with panitumumab plus irinotecan for patients with plasma RAS wild-type metastatic colorectal cancer. BMC Cancer 2021; 21: 674.

190.

Lyskjær

Kronborg

Rasmussen

, et al. Correlation between early dynamics in circulating tumour DNA and outcome from FOLFIRI treatment in metastatic colorectal cancer. Sci Rep 2019; 9: 11542.

191.

Chakrabarti

Xie

Urrutia

, et al. The promise of circulating tumor DNA (ctDNA) in the management of early-stage colon cancer: a critical review. Cancers 2020; 12: 2808.

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

Abstract

Keywords

Introduction

Guideline development

Minimum biomarker testing standards in mCRC

Extended RAS testing (including KRAS and NRAS)

BRAF V600 testing

Mismatch repair deficiency/microsatellite instability testing

Extended biomarker testing options

NTRK testing

HER2 testing

Tumor mutational burden testing

Other emerging predictive genomic alterations

Biomarker testing methodologies and reporting

Testing methods and specimens

Turnaround time and reporting

Summary and future directions

Footnotes

Acknowledgements

Declarations

References