Key Points
- Molecular testing to select targeted and conventional therapies for patients with colorectal cancer (CRC) has been the focus of a number of recent studies and is becoming standard practice for management of patients with CRC.
- Current evidence-based recommendations for the molecular testing of CRC tissues to guide EGFR-targeted therapies and conventional chemotherapy regimens were developed through collaboration of four societies: American Society for Clinical Pathology (ASCP), College of American Pathologists (CAP), Association for Molecular Pathology (AMP), and American Society of Clinical Oncology (ASCO).
Diagnosis
- Colorectal carcinoma patients being considered for anti-EGFR therapy must receive RAS mutational testing. Mutational analysis should include KRAS and NRAS codons 12, 13 of exon 2; 59, 61 of exon 3; and 117 and 146 of exon 4 (“expanded” or “extended” RAS). (R-C/A-B-H/I)
- BRAF p.V600 (BRAF c. 1799 (p.V600) mutational analysis should be performed in colorectal cancer tissue in patients with colorectal carcinoma for prognostic stratification. (R-A/Ina-B/H-I/L)
- BRAF p.V600 mutational analysis should be performed in deficient MMR tumors with loss of MLH1 to evaluate for Lynch Syndrome risk. Presence of a BRAF mutation strongly favors a sporadic pathogenesis. The absence of BRAF mutation does not exclude risk of Lynch syndrome. (R-A/Ina-B/H-I/L)
- Clinicians should order mismatch repair status testing in patients with colorectal cancers for the identification of patients at high risk for Lynch syndrome and/or prognostic stratification. (R-A/I-B/H-I/L)
- There is insufficient evidence to recommend BRAF c.1799 p.V600 mutational status as a predictive molecular biomarker for response to anti-EGFR inhibitors. (NR-Ins-U-Ins)
- There is insufficient evidence to recommend PIK3CA mutational analysis of colorectal carcinoma tissue for therapy selection outside of a clinical trial. (NR-Ins-U-Ins)
Note: Retrospective studies have suggested improved survival with post-operative aspirin use in patients whose colorectal carcinoma harbors a PIK3CA mutation.
- There is insufficient evidence to recommend PTEN analysis [expression by immunohistochemistry (IHC) or deletion by fluorescence in situ hybridization (FISH)] in colorectal carcinoma tissue for patients who are being considered for therapy selection outside of a clinical trial. (NR-Ins-U-Ins)
- Metastatic or recurrent colorectal carcinoma tissues are the preferred specimens for treatment predictive biomarker testing and should be used if such specimens are available and adequate. In their absence, primary tumor tissue is an acceptable alternative, and should be used. (EC-Ina/Ins-B/H-L)
- Formalin fixed paraffin embedded tissue is an acceptable specimen for molecular biomarker mutational testing in colorectal carcinoma. Use of other specimens (e.g. cytology specimens) will require additional adequate validation, as would any changes in tissue processing protocols. (EC-Ina/Ins-B/H-L)
- Laboratories must use validated colorectal carcinoma molecular biomarker testing methods with sufficient performance characteristics for the intended clinical use. Colorectal carcinoma molecular biomarker testing validation should follow accepted standards for clinical molecular diagnostics tests. (S-C/A-B-H/I)
- Performance of molecular biomarker testing for colorectal carcinoma must be validated in accordance with best laboratory practices. (S-C/A-B-H/I)
- Laboratories must validate the performance of IHC testing for colorectal carcinoma molecular biomarkers (currently IHC testing for MLH1, MSH2, MSH6, and PMS2) in accordance with best laboratory practices. (S-C/A-B-H/I)
- Laboratories must provide clinically appropriate turnaround times and optimal utilization of tissue specimens by using appropriate techniques (e.g. multiplexed assays) for clinically relevant molecular and immunohistochemical biomarkers of colorectal cancer. (EC-Ina/Ins-B/H-L)
- Molecular and IHC biomarker testing in colorectal carcinoma should be initiated in a timely fashion based upon the clinical scenario and in accordance with institutionally accepted practices. (EC-Ina/Ins-B/H-L)
Note: Test ordering can occur on a case-by-case basis or by policies established by the medical staff.
- Laboratories should establish policies to ensure efficient allocation and utilization of tissue for molecular testing, particularly in small specimens. (EC-Ina/Ins-B/H-L)
- Members of the patient’s medical team, including pathologists, may initiate colorectal carcinoma molecular biomarker test orders in accordance with institutionally accepted practices. (EC-Ina/Ins-B/H-L)
- Laboratories that require send out of tests for treatment predictive biomarkers should process and send colorectal carcinoma specimens to reference molecular laboratories in a timely manner. (EC-Ina/Ins-B/H-L)
Note: It is suggested that a benchmark of 90% of specimens should be sent out within 3 working days.
- Pathologists must evaluate candidate specimens for biomarker testing to ensure specimen adequacy taking into account tissue quality, quantity, and malignant tumor cell fraction. Specimen adequacy findings should be documented in the patient report. (EC-Ina/Ins-B/H-L)
- Laboratories should use colorectal carcinoma molecular biomarker testing methods that are able to detect mutations in specimens with at least 5% mutant allele frequency, taking into account the analytical sensitivity of the assay (limit of detection or LOD) and tumor enrichment (e.g. microdissection). (EC-Ina/Ins-B/H-L)
Note: It is recommended that the operational minimal neoplastic carcinoma cell content tested should be set at least 2 times the assay’s LOD.
- Colorectal carcinoma molecular biomarker results should be made available as promptly as feasible in order to inform therapeutic decision-making, both prognostic and predictive. (EC-Ina/Ins-B/H-L)
Note: It is suggested that a benchmark of 90% of reports be available within 10 working days from date of receipt in the molecular diagnostics laboratory.
- Colorectal carcinoma molecular biomarker testing reports should include a results and interpretation section readily understandable by oncologists and pathologists. Appropriate Human Genome Variation Society (HGVS) and Human Genome Organisation (HUGO) nomenclature must be used in conjunction with any historical genetic designations. (EC-Ina/Ins-B/H-L)
- Laboratories must incorporate colorectal carcinoma molecular biomarker testing methods into their overall laboratory quality improvement program, establishing appropriate quality improvement monitors as needed to assure consistent performance in all steps of the testing and reporting process. In particular, laboratories performing colorectal carcinoma molecular biomarker testing must participate in formal proficiency testing programs, if available, or an alternative proficiency assurance activity. (S-C/A-B-H/I)
Table 1. Outcomes of RAS Mutations and Anti-EGFR Therapy
Table 2. Concordance Rates Between Primary and Metastatic Lesionsa
| Genes Tested (n) | Concordance Rate (%) |
|---|---|
| KRAS (117) | 91 |
| KRAS, NRAS, BRAF (84) | 98.8 |
| PIK3CA (117) | 94 |
| PIK3CA (84) | 92.8 |
| PTEN immunohistochemistry | 66 |
a Summary of two randomized clinical trials where comparison of mutation in KRAS, NRAS, BRAF, and PIK3CA was performed for paired primary tumor and metastatic lesions. Immunohistochemistry for PTEN was done in Cejas et al. (Curr Cancer Drug Targets 12:124-131, 2012). In the study by Cejas et al. metastases were synchronous or metachronous. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and mutational analysis was performed with a polymerase chain reaction–direct sequencing assay. KRAS mutations were detected in 42% of metastatic lesions and 39% of primary tumors. In the study by Vakiani et al. (J Clin Oncol . 30:2956-2962, 2012) DNA was extracted from frozen tissue, and the iPLEX assay (Agena Bioscience, San Diego, CA) was used to examine the following mutations: KRAS 12, 13, 22, 61, 117, and 146; NRAS 12, 13, and 61; BRAF 600; and PIK3CA 345, 420, 542, 545, 546, 1043, and 1047.
