• Predict response to EGFR-targeted immunotherapy in patients with metastatic colorectal cancer

Multiple signaling pathways are involved in colorectal cancer pathogenesis. The epidermal growth factor receptor (EGFR) plays a key role in activation of these pathways and is commonly overexpressed in metastatic colorectal cancer (mCRC). Consequently, EGFR is a target of anti-cancer therapies. Two of these drugs, cetuximab and panitumumab, are monoclonal antibodies that block EGFR action. These EGFR immunotherapies, however, are not effective if downstream signaling pathways are activated independent of EGFR; such is the case when gene mutation causes overexpression of enzymes in these pathways. Thus, detecting mutations that lead to activation of signaling pathways downstream from EGFR can predict resistance to EGFR immunotherapy.

For example, KRAS, BRAF, or NRAS mutations can all activate the RAS-RAF-MAPK pathway, which is downstream from EGFR. In chemotherapy-refractory patients, fewer than 10% of patients who harbor 1 of these mutations respond to EGFR immunotherapy.^1,2 The American Society of Clinical Oncology (ASCO) and the National Comprehensive Cancer Network (NCCN) both recommend KRAS mutation testing prior to prescribing EGFR antagonist therapy for patients with mCRC and state that alternative therapy should be prescribed when mutations are detected.^3,4 When KRAS is mutation-negative, the NCCN suggests considering BRAF mutation testing.⁴

PIK3CA mutations activate the PI3K-PTEN-AKT pathway which is downstream from both the EGFR and the RAS-RAF-MAPK pathways. PIK3CA mutations have been associated with resistance to anti-EGFR therapy in some studies^2,5 but not in others.^6,7 The reasons for the discrepancy are not clear.

Mutation Profiling

PIK3CA mutations may overlap with mutations in BRAF, KRAS, and NRAS,² but BRAF, KRAS, and NRAS mutations are almost always mutually exclusive (ie, mutations in only 1 of the 3 genes occur within any individual tumor). These facts suggest that mutation analysis of all 4 of these genes could be clinically useful. Indeed, studies support mutation profiling in CRC tumors: 1) Response rate improved from 24% in cetuximab-treated patients whose mutation status was unknown to 41% in patients who tested negative for mutations in all 4 genes (response rates and outcomes are summarized in the Table)²; and 2)  objective response, progression-free survival (PFS), and overall survival (OS) were progressively worse when 0, 1, and ≥2 mutations were present in patients treated with either cetuximab or panitumumab.⁸ Thus, mutation profiling can effectively select patients who are more likely to respond to anti-EGFR immunotherapy, rule out this treatment for those who are not likely to respond, and predict patient outcomes.^2,4,8

The Quest Diagnostics Colorectal Cancer Mutation Panel includes testing for gene mutations in both EGFR downstream signaling pathways and thus provides increased ability to predict sensitivity or resistance to cetuximab or panitumumab versus single pathway or single gene testing. Although less data are available for panitumumab than for cetuximab, common molecular resistance pathways support mutation profile testing for both drugs.⁹ Individual tests for mutation detection are also available for each gene.

• Patients with colorectal cancer who are candidates for cetuximab or panitumumab therapy

• Polymerase chain reaction (PCR) amplification of

–   Exon 1 (codons 12 and 13) and exon 2 (codon 61) of the KRAS and NRAS genes

–   Exon 1, exon 9 (codon 545), and exon 20 (codon 1047) of the PIK3CA gene (targets >95% of known

mutations)

–   Exons 11, 12, and 15 (codon 599/600) of the BRAF gene (targets >99% of known mutations)

• Forward and reverse, dye-terminator gene sequencing of purified products

• Analytical sensitivity: 10% tumor cells in the background of normal cells. Testing is performed on sample where tumor is enriched.

Mutation in any 1 of the 4 genes is associated with resistance to cetuximab and panitumumab (Figure).

KRAS, BRAF, or NRAS mutations are associated with shorter PFS and OS in patients treated with anti-EGFR immunotherapy relative to those who are mutation-negative.^2,6 BRAF mutations are negative prognostic indicators irrespective of therapy.⁶

Polymorphisms or mutations at locations not tested (see “Method”) that may be associated with drug efficacy or patient outcome will not be detected. Results should be interpreted in conjunction with other laboratory and clinical findings.