Coverage Policy Manual - Arkansas Blue Cross and Blue Shield

Somatic Biomarker Testing (including Liquid Biopsy) for Targeted Treatment in Metastatic Colorectal Cancer (KRAS, NRAS, BRAF, and HER2)

Description:

Cetuximab (Erbitux®, ImClone Systems) and panitumumab (Vectibix®, Amgen) are monoclonal antibodies that bind to the EGFR, preventing intrinsic ligand binding and activation of downstream signaling pathways vital for cancer cell proliferation, invasion, metastasis, and stimulation of neovascularization.

The RAS-RAF-MAP kinase pathway is activated in the EGFR cascade. RAS proteins are G proteins that cycle between active (RAS-GTP) and inactive (RAS-GDP) forms, in response to stimulation from a cell surface receptor such as EGFR, and act as a binary switch between the cell surface EGFR and downstream signaling pathways. The KRAS gene can harbor oncogenic variants that result in a constitutively activated protein, independent of EGFR ligand binding, rendering antibodies to the upstream EGFR ineffective. Approximately 40% of colorectal cancers (CRCs) have KRAS variants in codons 12 and 13 in exon 2. Another protooncogene that acts downstream from KRAS–NRAS harbors oncogenic mutations in codons 12, 13, or 61 that result in constitutive activation of the EGFR-mediated pathway. These variants are less common compared with KRAS, detected in 2% to 7% of CRC specimens. It is unclear whether NRAS variants predict poor response due to anti-EGFR monoclonal antibody therapy or are prognostic of poor CRC outcomes in general. A third protooncogene, BRAF, encodes a protein kinase and is involved in intracellular signaling and cell growth and is a principal downstream effector of KRAS. BRAF mutations occur in less than 10% to 15% of CRCs and appear to be a marker of poor prognosis. KRAS and BRAF variants are considered to be mutually exclusive.

Cetuximab and panitumumab have FDA marketing approval for treatment of metastatic CRC in the refractory disease setting. FDA approval for panitumumab indicates that panitumumab is not indicated for the treatment of patients with KRAS or NRAS variant-positive disease in combination with oxaliplatin-based chemotherapy (Amgen, 2015).

A large body of literature has shown that metastatic CRC tumors with a variant in exon 2 (codon 12 or 13) of the KRAS gene do not respond to cetuximab or panitumumab therapy. More recent evidence has shown that variants in KRAS outside exon 2 (ie, in exons 3 [codons 59 and 61] and exon 4 [codons 117 and 146]) and variants in NRAS exon 2 (codons 12 and 13), exon 3 (codons 59 and 61), and exon 4 (codons 117 and 146) also predict a lack of response to these monoclonal antibodies. Variant testing of these exons outside the KRAS exon 2 is referred to as extended RAS testing.

Human epidermal growth factor receptor 2 (HER2) is a member of the HER (EGFR) family of tyrosine kinase receptors and has no specific ligand. When activated, it forms dimers with other EGFR family members. Amplification of HER2 is detected in approximately 3% of patients with CRC, with higher prevalence in RAS/BRAF-wild type tumors (5% to 14%). In addition to its role as a predictive marker for HER2-targeted therapy, HER2 amplification/overexpression is being investigated as a predictor of resistance to EGFR-targeting monoclonal antibodies.

Mismatch repair deficiency (dMMR) and high levels of microsatellite instability (MSI-H) describe cells that have alterations in certain genes involved in correcting errors made when DNA is replicated. Tumors with dMMR are characterized by a high tumor mutational load and potential responsiveness to anti-PD-L1-immunotherapy. Deficiency in MMR is most common in CRC, other types of gastrointestinal cancer, and endometrial cancer, but it may also be found in other cancers including breast cancer. Testing of MSI is generally performed using polymerase chain reaction (PCR) for 5 biomarkers, although other biomarker panels and next generation sequencing are sometimes performed. High MSI is defined as 2 or more of the 5 biomarkers showing instability or more than 30% of the tested biomarkers showing instability depending on what panel is used. Microsatellite instability testing is generally paired with immunohistochemistry assessing lack of protein expression from 4 DNA mismatch repair genes thereby reflecting dMMR.

Tumor mutational burden (TMB), a measure of gene mutations within cancer cells, is an emerging biomarker of outcomes with immunotherapy in multiple tumor types. Initially, assessments of TMB involved whole exome sequencing. More recently, targeted next generation sequencing panels are being adapted to estimate TMB. Currently FoundationOne CDx is the only U.S. Food and Drug Administration (FDA) approved panel for estimating TMB, but others are in development.

Normal and tumor cells release small fragments of DNA into the blood, which is referred to as cell-free DNA. Cell-free DNA from nonmalignant cells is released by apoptosis. Most cell-free tumor DNA is derived from apoptotic and/or necrotic tumor cells, either from the primary tumor, metastases, or circulating tumor cells. Unlike apoptosis, necrosis is considered a pathologic process and generates larger DNA fragments due to incomplete and random digestion of genomic DNA. The length or integrity of the circulating DNA can potentially distinguish between apoptotic and necrotic origin. Circulating tumor DNA can be used for genomic characterization of the tumor.

Typically, the evaluation of RAS mutation status requires tissue biopsy. Circulating tumor DNA (ctDNA) testing is proposed as a non-invasive alternative.

Detection of ctDNA is challenging because ctDNA is diluted by nonmalignant circulating DNA and usually represents a small fraction (<1%) of total ctDNA. Therefore, more sensitive methods than the standard sequencing approaches (eg, Sanger sequencing) are needed.

Highly sensitive and specific methods have been developed to detect ctDNA, for both single nucleotide variants (eg BEAMing [which combines emulsion polymerase chain reaction with magnetic beads and flow cytometry] and digital polymerase chain reaction) and copy-number variants. Digital genomic technologies allow for enumeration of rare variants in complex mixtures of DNA.

Approaches to detecting ctDNA can be considered targeted, which includes the analysis of known genetic mutations from the primary tumor in a small set of frequently occurring driver mutations, or untargeted without knowledge of specific variants present in the primary tumor, which includes array comparative genomic hybridization, next-generation sequencing, and whole exome and genome sequencing. Targeted testing may impact therapy selection.

Circulating tumor cell assays usually start with an enrichment step that increases the concentration of circulating tumor cells, either by biologic properties (expression of protein markers) or physical properties (size, density, electric charge). Circulating tumor cells can then be detected using immunologic, molecular, or functional assays.

A number of liquid biopsy tests related to targeted treatment of metastatic colorectal cancer have been developed.

Examples of Liquid Biopsy Tests Related to Targeted Treatment of Metastatic Colorectal Cancer:

Target Selector™ ctDNA EGFR Kit, manufactured by Biocept, is a circulating tumor DNA test
FoundationOne Liquid (Previously FoundationAct), manufactured by Foundation Medicine, is a circulating tumor DNA test
Guardant360®, manufactured by Guardant Health, is a circulating tumor DNA test
Velox™ , manufactured by IV Diagnostics, is a circulating tumor cell test
PlasmaSELECT™, manufactured by Personal Genome Diagnostics, is a circulating tumor DNA test
OncoBEAM, manufactured by Sysmex Inostics, is a circulating tumor DNA test
Theranostics, manufactured by Circulogene, is a circulating tumor DNA test

Regulatory Status

KRAS, NRAS, and BRAF mutation analyses using polymerase chain reaction (PCR) methodology are commercially available as laboratory-developed tests. Such tests are regulated under the Clinical Laboratory Improvement Amendments (CLIA). Premarket approval from FDA is not required when the assay is performed in a laboratory that is licensed by CLIA for high-complexity testing.

Coding

Effective in 2012, there are specific CPT codes for KRAS or BRAF mutation analysis.

81210: BRAF (v-raf murine sarcoma viral oncogene homolog B1) (eg, colon cancer), gene analysis, V600E variant

81311 NRAS (neuroblastoma RAS viral [v ras] oncogene homolog) (eg, colorectal carcinoma), gene analysis, variants in exon 2 (eg, codons 12 and 13) and exon 3 (eg, codon 61)

81275: KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene) (eg, carcinoma) gene analysis, variants in codons 12 and 13

Additionally, code 81403 would be reported for KRAS testing for variant(s) in exon 3 (eg, codon 61).

Prior to 2012, multiple codes describing genetic analysis would likely be used to report this testing (eg, codes from 83890-83912).

Related Policies:

Policy # 2017007 Cetuximab (Erbitux™)

Policy #2017024 Panitumumab (Vectibix™)

Policy # 2023023 Somatic Biomarker Testing for Immune Checkpoint Inhibitor Therapy (BRAF, MSI/MMR, PD-L1, TMB)

Policy/
Coverage:

Effective October 2023

Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria

KRAS, NRAS, BRAF, or HER2 variant analysis of tumor tissue meets member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for individuals with metastatic colorectal cancer (mCRC) to select individuals for treatment with FDA-approved therapies.

Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria

KRAS, NRAS, BRAF, or HER2 variant analysis for any use not addressed in this or other policies does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, any other use of KRAS, NRAS, BRAF, or HER2 variant analysis not addressed in this or other policies is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

Circulating tumor DNA testing (liquid biopsy) to guide treatment in individuals with metastatic colorectal cancer does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, circulating tumor DNA testing (liquid biopsy) to guide treatment in individuals with metastatic colorectal cancer is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

Effective October 2022 through September 2023

Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria

KRAS variant analysis of tumor tissue meets member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for individuals with metastatic colorectal cancer (mCRC) to select individuals for treatment with FDA-approved therapies.

NRAS variant analysis of tumor tissue meets member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for individuals with metastatic colorectal cancer to select individuals for treatment with FDA-approved therapies.

BRAF variant analysis of tumor tissue meets member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for individuals with metastatic colorectal cancer who are found to be wild-type on KRAS and NRAS variant analysis to guide management decisions, and to select individuals for treatment with FDA-approved therapies.

Mismatch repair/microsatellite instability testing meets member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes to select individuals for treatment with FDA-approved therapies.

Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria

KRAS variant analysis for any use not addressed in this or other policies does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, any other use of KRAS variant analysis not addressed in this or other policies is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

NRAS variant analysis for any use not addressed in this or other policies does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, any use of NRAS variant analysis not addressed in this or other policies is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

BRAF variant analysis for any use not addressed in this or other policies does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, any use of BRAF variant analysis not addressed in this or other policies is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

Mismatch repair/microsatellite instability variant testing for any use not addressed in this or other policies does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, any use of mismatch repair/microsatellite instability variant analysis not addressed in this or other policies is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

Human epidermal growth factor receptor 2 testing to predict treatment response to immunotherapy in individuals with metastatic colorectal cancer does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, human epidermal growth factor receptor 2 testing to predict treatment response to immunotherapy in individuals with metastatic colorectal cancer is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

Tumor mutational burden testing to predict response to immunotherapy in individuals with metastatic colorectal cancer does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, tumor mutational burden testing to predict response to immunotherapy in individuals with metastatic colorectal cancer is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

Effective Prior to October 2022

Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria

KRAS Variant Analysis meets member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for patients with metastatic colorectal cancer (mCRC) to predict nonresponse prior to planned therapy with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies cetuximab or panitumumab.

NRAS variant meets member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for patients with metastatic colorectal cancer to predict nonresponse prior to planned therapy with anti-EGFR monoclonal antibodies cetuximab or panitumumab.

BRAF Variant Analysis meets member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for patients with metastatic colorectal cancer who are found to be wild-type on KRAS and NRAS variant analysis to guide management decisions.

Mismatch repair/microsatellite instability testing meets member benefit certificate primary coverage criteria to predict treatment response to pembrolizumab:

for first-line treatment of patients with unresectable or metastatic colorectal cancer; OR
in patients with colorectal cancer that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan; OR
in patients with colorectal cancer tumors that have progressed following prior treatment and who have no satisfactory alternative treatment options.

Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria

KRAS variant analysis for any use not addressed in this or other policies does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For contracts without primary coverage criteria, any other use of KRAS variant analysis not addressed in this or other policies is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

NRAS variant analysis for any use not addressed in this or other policies does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For contracts without primary coverage criteria, any other use of NRAS variant analysis not addressed in this or other policies is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

BRAF variant analysis for any use not addressed in this or other policies does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, any other use of BRAF variant analysis not addressed in this or other policies is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

Mismatch repair/microsatellite instability testing for any use not addressed in this or other policies does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, any use of mismatch repair/microsatellite instability analysis not addressed in this or other policies is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

Human epidermal growth factor receptor 2 testing to predict treatment response to immunotherapy in patients with metastatic colorectal cancer does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, human epidermal growth factor receptor 2 testing to predict treatment response to immunotherapy in patients with metastatic colorectal cancer is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

Tumor mutational burden testing to predict response to immunotherapy in patients with metastatic colorectal cancer does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, tumor mutational burden testing to predict response to immunotherapy in patients with metastatic colorectal cancer is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

Circulating tumor DNA testing (liquid biopsy) to guide treatment in patients with metastatic colorectal cancer does not meet Primary Coverage Criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, circulating tumor DNA testing (liquid biopsy) to guide treatment in patients with metastatic colorectal cancer is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

Effective Prior to September 2021

Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria

KRAS Mutation Analysis

KRAS mutation testing meets member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes in patients with metastatic colorectal cancer (mCRC) to determine whether the tumor is likely to respond to therapy with cetuximab or panitumumab. Results of this testing with documentation of normal (wild-type) KRAS gene and negative for KRAS mutation will be required for coverage of either drug.

NRAS Mutation Analysis

NRAS variant meets member benefit certificate primary coverage criteria for patients with metastatic colorectal cancer to predict nonresponse prior to planned therapy with anti-EGFR monoclonal antibodies cetuximab or panitumumab.

Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria

Treatment of KRAS mutation positive mCRC

Treatment of metastatic colorectal cancer that is KRAS mutation positive with cetuximab or panitumumab does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes. For contracts without primary coverage criteria, treatment of metastatic colorectal cancer that is KRAS mutation positive with cetuximab or panitumumab is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

KRAS Mutation Analysis

Any other use of KRAS mutation testing not addressed in this or other policies does not meet Primary Coverage Criteria that there be scientific evidence of effectiveness in improving health outcomes.

For contracts without Primary Coverage Criteria, any other use of KRAS mutation testing not addressed in this or other policies is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

NRAS Mutation Analysis

Any other use of NRAS mutation testing not addressed in this or other policies does not meet Primary Coverage Criteria that there be scientific evidence of effectiveness in improving health outcomes.

For contracts without Primary Coverage Criteria, any other use of NRAS mutation testing not addressed in this or other policies is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

BRAF Mutation Analysis

BRAF mutation analysis to predict nonresponse to anti-EGFR monoclonal antibodies cetuximab and panitumumab in the treatment of metastatic colorectal cancer does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, BRAF mutation analysis is considered investigational to predict nonresponse to anti-EGFR monoclonal antibodies cetuximab and panitumumab in the treatment of metastatic colorectal cancer. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

KRAS, NRAS, and BRAF variant analysis using circulating tumor DNA or circulating tumor cell testing (liquid biopsy) to guide treatment for patients with metastatic colorectal cancer does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes. For members with contracts without primary coverage criteria, KRAS, NRAS, and BRAF variant analysis using circulating tumor DNA or circulating tumor cell testing (liquid biopsy) to guide treatment for patients with metastatic colorectal cancer is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

Effective Prior to January 2019

Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria

KRAS Mutation Analysis

NRAS Mutation Analysis

Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria

Treatment of KRAS mutation positive mCRC

KRAS Mutation Analysis

NRAS Mutation Analysis

BRAF Mutation Analysis

Effective January 2015- December 2017

Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria

KRAS Mutation Analysis

KRAS mutation testing meets primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes in patients with metastatic colorectal cancer (mCRC) to determine whether the tumor is likely to respond to therapy with cetuximab or panitumumab. Results of this testing with documentation of normal (wild-type) KRAS gene and negative for KRAS mutation will be required for coverage of either drug.

Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria

Treatment of KRAS mutation positive mCRC

KRAS Mutation Analysis

Any other use of KRAS mutation testing not addressed in this or other policies does not meet Primary Coverage Criteria that there be scientific evidence of effectiveness in improving health outcomes.

For contracts without Primary Coverage Criteria, any other use of KRAS mutation testing not addressed in this or other policies is considered investigational and is not covered. Investigational services are an exclusion in the member benefit certificate.

NRAS Mutation Analysis

NRAS mutation analysis to predict nonresponse to anti-EGFR monoclonal antibodies cetuximab and panitumumab in the treatment of metastatic colorectal cancer does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.

For members with contracts without primary coverage criteria, NRAS mutation analysis is considered investigational to predict nonresponse to anti-EGFR monoclonal antibodies cetuximab and panitumumab in the treatment of metastatic colorectal cancer. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

BRAF Mutation Analysis

Rationale:

KRAS Mutation Analysis

RCTs

RCTs have performed nonconcurrent subgroup analyses of the efficacy of epidermal growth factor receptor (EGFR) inhibitors in patients with wild-type (WT) versus mutated KRAS in metastatic colorectal cancer (CRC). Data from these trials have consistently shown a lack of clinical response to cetuximab and panitumumab in patients with mutated KRAS, with tumor response and prolongation of progression-free survival (PFS) observed only in WT KRAS patients.

Amado et al performed a subgroup analysis of KRAS tumor mutations in a patient population that had previously been randomly assigned to panitumumab versus best supportive care as third-line therapy for chemotherapy-refractory metastatic CRC (Amado, 2008). The original study was designed as a multicenter, RCT but was not blinded because of expected skin toxicity related to panitumumab administration.3 Patients were randomly assigned 1:1 to receive panitumumab or best supportive care. Random assignment was stratified by Eastern Cooperative Oncology Group (ECOG) performance status (0 or 1 vs 2) and geographic region. Crossover from best supportive care to the panitumumab arm was allowed in patients who experienced disease progression. Of the 232 patients originally assigned to best supportive care alone, 176 crossed over to the panitumumab arm, at a median time to crossover of 7 weeks (range, 6.6-7.3).

Of the 463 patients in the original study, 427 (92%) were included in the KRAS subgroup mutation analysis. A central laboratory performed the KRAS mutational analysis in a blinded fashion, using formalin-fixed, paraffin-embedded (FFPE) tumor sections and a validated KRAS mutation kit (DxS Ltd., Manchester, England) that identifies 7 somatic mutations located in codons 12 and 13 using real-time polymerase chain reaction (PCR). KRAS mutation status could not be determined in 36 patients because tumor samples were not available or DNA was insufficient or poor quality for analysis. Forty-three percent of the KRAS-evaluable patients had KRAS-mutated tumors, with similar distribution of KRAS mutation types between treatment arms.

Patient demographics and baseline characteristics were balanced between the WT and mutated groups (MT) for panitumumab versus best supportive care including patient age, sex, and ECOG performance status. The interaction between mutational status and PFS was examined, controlling for randomization factors. PFS and tumor response rate was assessed radiographically every 4 to 8 weeks until disease progression using Response Evaluation Criteria in Solid Tumors (RECIST) criteria by blinded, central review. In the KRAS-assessable population, 20% of patients had a treatment-related grade 3 or 4 adverse event. The relative effect of panitumumab on PFS was significantly greater among patients with WT KRAS, compared with patients with MT KRAS in whom no benefit from panitumumab was observed. No responders to panitumumab were identified in the MT group, indicating a 100% positive predictive value for nonresponse in the mutant group.

In a third trial, the randomized, phase 2 OPUS trial, the intention-to-treat (ITT) population consisted of 337 patients randomly assigned to C and folinic acid [leucovorin], 5-FU, oxaliplatin (FOLFOX) versus FOLFOX alone in the first-line treatment of metastatic CRC (Bokemeyer, 2009). A 10% higher response rate (assessed by independent reviewers) was observed in the population treated with C, but no difference in PFS was seen between the two groups. The researchers then reevaluated the efficacy in the 2 treatment arms with consideration of KRAS mutational status of the patients’ tumors. Of the original ITT population, 233 subjects had evaluable material for KRAS testing, and 99 (42%) were KRAS mutant. The demographics or baseline characteristics were similar between the WT and MT groups, including patient age, sex, ECOG performance status, involved disease sites, and liver-limited disease. The study showed that the addition of C to FOLFOX resulted in a significant improvement in response rate and PFS only in the WT KRAS group.

In the CAIRO2 study, Tol et al analyzed tumor samples from 528 of 755 previously untreated patients with metastatic CRC who were randomly assigned to receive capecitabine, oxaliplatin and bevacizumab (CB regimen, n=378), or the same regimen plus C (CBC regimen, n=377) (Tol, 2009). A KRAS mutation was found in 40% of tumors (108 from patients in the CB group and 98 from the CBC group). Patients with KRAS mutations treated with C had significantly shorter PFS than the KRAS WT patients who received C (8.1 vs 10.5 months, respectively, p=0.04). In addition, patients who had MT KRAS tumors who received C had significantly shorter PFS than patients with MT KRAS tumors who did not receive C (8.1 vs 12.5 months, respectively, p=0.003) and overall survival (OS) (17.2 vs 24.9 months, respectively, p=0.03). For patients with WT tumors, no significant PFS differences were reported between the 2 groups. Overall, patients treated with C who had tumors with a mutated KRAS gene had significantly decreased PFS compared with C-treated patients with WT KRAS tumors or patients with mutated KRAS tumors in the CB group.

Karapetis et al analyzed tumor samples from 394 of 572 patients (69%) with CRC who were randomly assigned to receive C plus BSC (n=287) versus BSC alone (n=285) for KRAS mutations and assessed whether mutation status was associated with survival (Karapetis, 2008). The patients had advanced CRC, had failed chemotherapy and had no other standard anticancer therapy available. Of the tumors that were evaluated (198 from the C group, 196 from the BSC group), 41% and 42% had a KRAS mutation, respectively. In OS (median, 9.5 months vs 4.8 months, respectively; HR for death, 0.55; 95% confidence interval [CI], 0.41 to 0.74; p<0.001) and PFS (median, 3.7 months vs 1.9 months, respectively; HR for progression to death, 0.40; 95% CI, 0.30 to 0.54; p<0.001). For patients with MT KRAS tumors, no significant differences were reported between those treated with C versus BSC alone with respect to OS (HR=0.98, p=0.89) or PFS (HR=0.99, p=0.96).

Douillard et al reported the results of a multicenter, phase 3 trial in which patients with no prior chemotherapy for metastatic CRC, ECOG performance status of 0 to 2, and available tissue for biomarker testing were randomly assigned 1:1 to receive panitumumab-FOLFOX4 versus FOLFOX4 (Douillard, 2010). The primary end point was PFS; OS was a secondary end point. Results were prospectively analyzed on an ITT basis by tumor KRAS status. KRAS results were available for 93% of the 1183 patients randomly assigned. In the WT KRAS group, panitumumab-FOLFOX4 significantly improved PFS compared with FOLFOX4 alone (median PFS, 9.6 vs 8.0 months, respectively; HR=0.80; 95% CI, 0.66 to 0.97; p=0.02). A nonsignificant increase in OS was also observed for panitumumab-FOLFOX4 versus FOLFOX4 (median OS=23.9 vs 19.7 months, respectively; HR=0.83; 95% CI, 0.67 to 1.02; p=0.072). In the mutant KRAS group, PFS was significantly reduced in the panitumumab-FOLFOX4 arm versus the FOLFOX4 arm (HR=1.29; 95% CI, 1.04 to 1.62; p=0.02), and median OS was 15.5 months versus 19.3 months, respectively (HR=1.24; 95% CI, 0.98 to 1.57; p=0.068). Adverse event rates were generally comparable across arms with the exception of toxicities known to be associated with anti-EGFR therapy. The study demonstrated that panitumumab-FOLFOX4 was well-tolerated and significantly improved PFS in patients with WT KRAS tumors.

The CRYSTAL trial demonstrated that the addition of cetuximab to a combined first-line chemotherapy regimen of irinotecan, infusional fluorouracil and leucovorin (FOLFIRI) statistically significantly reduced the risk of disease progression and increased the chance of response in patients with metastatic CRC that was KRAS WT, compared with chemotherapy alone (Van Cutsem, 2009). An updated analysis of the CRYSTAL trial reported increased follow-up time and an increased number of patients evaluable for tumor KRAS status and considered the clinical significance of the tumor mutation status of BRAF in the expanded population of patients with KRAS WT tumors (Van Cutsem, 2011). Subsequent to the initial published analysis, which had a cutoff for OS of December 2007, and an associated overall median duration of follow-up of 29.7 months, additional tumor analysis allowed for the typing of an additional 523 tumors for KRAS mutation status, representing an increase in the ascertainment rate from 45% of ITT population patients in the original analysis to 89% (540 to 1063) in the current analysis, with mutations detected in 37% of tumors. The updated analysis of OS was carried out with a new cutoff date of May 2009, giving an overall median duration of follow-up of 46 months. The addition of cetuximab to FOLFIRI in patients with KRAS WT disease resulted in significant improvements in OS (median, 23.5 vs. 20.0 months; HR=0.796; p=0.009), PFS (median, 9.9 vs 8.4 months; HR=0.696; p=0.001), and response (rate 57.3% vs 39.7%; odds ratio [OR], 2.069; p<0.001) compared with FOLFIRI alone. Significant interactions between KRAS status and treatment effect were noted for all key efficacy end points. KRAS mutation status was confirmed as a powerful predictive biomarker for the efficacy of cetuximab plus FOLFIRI. BRAF V600E mutations were detected in 60 (6%) of 999 tumor samples evaluable for both BRAF and KRAS. In all but 1 case, BRAF mutations were identified in tumors that were WT for KRAS. The impact of BRAF tumor mutation status in relation to the efficacy of cetuximab plus FOLFIRI was examined in the population of patients with KRAS WT disease (n=625). No evidence was reported for an independent treatment interaction by tumor BRAF mutation status. The authors concluded that BRAF mutation status was not predictive of treatment effects of cetuximab plus FOLFIRI but that BRAF tumor mutation was a strong indicator of poor prognosis for all efficacy end points compared with those whose tumors were WT.

Peeters et al reported the results of a phase 3 study in which 1186 patients with metastatic CRC were randomized to receive panitumumab with FOLFIRI versus FORFIRI alone as second-line treatment (Peeters, 2010). The study end points were PFS and OS, which were independently tested and prospectively analyzed by KRAS status. KRAS status was available for 91% of patients: 597 (55%) with WT KRAS tumors and 486 (45%) with MT KRAS tumors. In the WT KRAS subpopulation, when panitumumab was added to chemotherapy, a significant improvement in PFS was observed (HR=0.73; 95% CI, 0.59 to 0.90; p=0.004); median PFS was 5.9 months for panitumumab-FOLFIRI versus 3.9 months for FOLFIRI. A nonsignificant trend toward increased OS was observed; median OS was 14.5 months versus 12.5 months, respectively (HR=0.85, 95% CI, 0.70 to 1.04; p=0.12); response rate was improved to 35% versus 10% with the addition of panitumumab. In patients with MT KRAS, no difference was reported in efficacy. Adverse events were comparable across arms. The authors concluded that panitumumab plus FOLFIRI significantly improved PFS and is well-tolerated as second-line treatment in patients with WT KRAS metastatic colorectal cancer (mCRC).

Maughan et al reported the results of a phase 3, multicenter trial (MRC COIN trial) which randomized patients with advanced CRC who had not received previous chemotherapy to oxaliplatin and fluoropyrimidine chemotherapy (arm A) or the same combination plus cetuximab (Maughan, 2011). The comparison between arms A and B (for which the primary outcome was OS) was in patients with KRAS WT tumors.

Baseline characteristics were well-balanced between the trial groups. Analysis was by ITT and treatment allocation was not masked. Further analysis with respect to other mutations, including BRAF, was done; 1630 patients were randomly assigned to treatment groups (815 to standard therapy, 815 to the addition of cetuximab). Tumor samples from 1316 (81%) of patients were used for somatic mutation analyses; 43% had KRAS mutations. In patients with KRAS WT tumors, OS did not differ between treatment groups (median survival, 17.9 months in the control group vs 17.0 months in the cetuximab group (HR=1.04; 95% CI, 0.87 to 1.23; p=0.67). BRAF mutations were detected in 8% of patients; BRAF did not show any evidence of a benefit from the addition of cetuximab. Contrary to other trials that have assessed KRAS mutation status and the benefit of the addition of cetuximab to the regimen of WT KRAS patients, this trial did not show a benefit of the addition of cetuximab to oxaliplatin-based chemotherapy.

Systematic Reviews

Qiu et al conducted a meta-analysis of 22 studies on the predictive and prognostic value of KRAS mutations in metastatic CRC patients treated with cetuximab (Qui, 2010). The overall KRAS mutation rate was 38% (829/2188 patients). The results of the meta-analysis were consistent with previous reports on the use of cetuximab and KRAS mutation status, that patients with tumors that harbor mutant-type KRAS are more likely to have a worse response, PFS and OS when treated with cetuximab when compared with those with WT KRAS.

Dahabreh et al conducted a systematic review of RCTs that assessed the use of KRAS mutation testing as a predictive biomarker for treatment of advanced CRC with cetuximab and panitumumab (Dahabreh, 2011). The authors concluded that, compared with patients with WT KRAS, KRAS mutations are consistently associated with reduced OS and PFS and increased treatment failure rates among patients with advanced CRC who are treated with anti- EGFR antibodies.

A pooled analysis of the CRYSTAL and OPUS RCT data was performed to further investigate the findings of these trials in patients with KRAS WT tumors, using extended survival data and following an enhancement in the ascertainment rate of KRAS and BRAF tumor mutation status.14 Pooled individual patient data from each study were analyzed for OS, PFS and best objective response rate (ORR) in patients evaluable for KRAS and BRAF mutation status. Treatment arms were compared according to mutation status using log-rank and Cochran-Mantel-Haenszel tests. In 845 patients with KRAS WT tumors, adding cetuximab to chemotherapy led to a significant improvement in OS (HR= 0.81; p=0.006), PFS (HR=0.66; p<0.001), and ORR (OR=2.16; p<0.001). BRAF mutations were detected in 70 of 800 (8.8%) evaluable tumors. No significant differences were found in outcome between the treatment groups in these patients. However, prognosis was worse in each treatment arm for patients with BRAF tumor and OPUS studies confirms the consistency of the benefit obtained across all efficacy end points from adding cetuximab to first-line chemotherapy in patients with KRAS WT mCRC. It further suggests that BRAF mutation does not appear to be a predictive biomarker in this setting, but is a marker of poor prognosis.

Single-Arm Studies (Cetuximab or Panitumumab)

In addition to the 3 randomized trials outlined here, a number of single-arm studies retrospectively evaluated KRAS mutational status and treatment response in patients with metastatic CRC (Benvenuti, 2007; De Rook, 2008; Di Fiore, 2007; Khambata-Ford, 2007); Lievre, 2008). Overall they showed similar nonresponse to anti-EGFR monoclonal antibodies in patients with MT KRAS tumors. Two of these single-arm studies also reported a difference in PFS and OS (De Roock, 2008; Lievre, 2008).

NRAS Mutation Analysis

No RCT evidence is available to evaluate an effect of NRAS mutation status alone on anti-EGFR therapies in CRC. Evidence is available on RAS mutations (KRAS and NRAS) in tumor samples from patients enrolled in the PRIME RCT (Panitumumab Randomized Trial in Combination with Chemotherapy for Metastatic Colorectal Cancer to Determine Efficacy) (Douillard, 2013). In a prospective-retrospective analysis, a total of 108 of 641(17%) tumor specimens that did not harbor KRAS mutations in exon 2 had mutations in other RAS exons, including NRAS (exons 2 or 4) and KRAS (exons 3 and 4). Among the WT KRAS exon in 2 patients (n=656), OS was significantly better with panitumumab added to FOLFOX4 (n=325, median 23.8 months) versus FOLFOX4 alone (n=331, median 19.4 months, p=0.03). Among patients with no KRAS exon 2 mutation but with one type of RAS mutation, median OS with panitumumab-FOLFOX4 was shorter (n=51, 17.1 months) than with FOLFOX4 alone (n=57, 17.8 months) (p=0.01). These data suggest mutation in a RAS gene exon other than KRAS exon 2 negatively affects anti-EGFR therapy. However, the investigators do not discriminate specific types of RAS mutations, so it is not possible to relate NRAS to these results. Furthermore, the numbers of patients involved are very small, further limiting conclusions.

Tumor specimens (n=288 of 320) from a second RCT3 were analyzed using massively parallel multigene sequencing (next-generation sequencing) to investigate whether EGFR pathway mutations predicted response to monotherapy with panitumumab compared with BSC (Peeters, 2013). This analysis showed that NRAS was mutated in 14 of 282 (5%) samples with available data. Among patients with WT KRAS (codons 12, 13, 61) and WT NRAS (n=138), treatment with panitumumab was associated with improved PFS (HR=0.39; 95% CI, 0.27, 0.56; p<0.001) compared with BSC. Among those with WT KRAS but mutated NRAS (n=11), treatment with panitumumab was no longer associated with longer PFS (HR=1.94; 95% CI, 0.44, 8.44; p=0.379). A treatment interaction analysis was suggestive but not significantly indicative of an interaction between the presence of mutated NRAS and poorer outcome (p==0.076). The authors suggest their data are consistent with a hypothesis that NRAS mutations may limit the efficacy of anti-EGFR therapy. However, because the prevalence of NRAS mutations is low, their true predictive or prognostic value is unclear.

A retrospective consortium analysis reported results of centrally performed high-throughput mass spectrometric mutation profiling of CRC specimens gathered from 11 centers in 7 European countries (De Roock, 2010). Patients had been treated with panitumumab alone, cetuximab alone, or cetuximab plus chemotherapy. Among 747 of 773 samples with data, KRAS was mutated in 299 (40%), including codons 12, 13, 61, and 146. By contrast, NRAS mutations were identified in 17 of 644 (2.6%) samples with data, primarily in codon 61. KRAS and NRAS mutations were mutually exclusive. Among WT KRAS samples from patients treated with cetuximab plus chemotherapy, NRAS mutation was associated with an ORR of 7.7% (1 of 13) compared with WT NRAS (ORR=38%, p=0.013). However, there were no significant differences between NRAS mutants and WT in median PFS (14 versus 26 weeks, p=0.055) or OS (38 versus 50 weeks, p=0.051). Similar to the results previously reported, the results for this analysis show a very low prevalence of NRAS mutations and are inconclusive as to whether NRAS mutation is predictive of non-response to anti-EGFR therapy or is a prognostic indicator of poor outcomes of CRC.

The rarity of NRAS mutations reported in the studies previously outlined in this Policy is also shown in a study that used PCR and pyrosequencing (Qiagen, Valencia, CA) to assess tumor samples from individuals who developed CRC and were identified within the databases of 2 prospective cohort studies: the Nurses ‘Health Study and the Health Professionals Follow-Up Study (Irahara, 2010). Among 225 CRC specimens, NRAS mutations were identified in 5 (2.2%). Because of the low frequency of NRAS mutations, they were not associated with any clinical or pathologic features or with patient survival.

A recent meta-analysis (MA) evaluated the predictive value of NRAS mutations on clinical outcomes of anti-EGFR therapy in CRC (Therkildsen, 2014). The MA included data from 3 studies included in this policy (Douillard, 2013; Peeters, 2013; De Roock, 2010). The investigators suggest that the pooled analyses showed a trend toward poor odds ratio (OR) based on 17 events, but significant effects on PFS (HR=2.30; 95% CI, 1.30 to 4.07) and OS (HR=1.85; 95% CI, 1.23 to 2.78) among patients with WT KRAS. These results are limited by the small pool of mutations, permitting no conclusions as to whether NRAS mutations have an effect on anti-EGFR therapy.

BRAF Mutation Analysis

A meta-analysis of BRAF mutation and resistance to anti-EGFR monoclonal antibodies in patients with metastatic CRC was performed (Mao, 2011). The primary end point of eligible studies was ORR, defined as the sum of complete and partial tumor response (CR, PR). A total of 11 studies (Cappuzzo, 2008; Di Nicolantonio, 2008; Freeman, 2008; Laurent-Puig, 2009; Loupakis, 2009; Molinari, 2009; Moroni, 2005; Perrone, 2009; Sartore-Bianchi, 2009; Tol, 2009) reported sample sizes ranging from 31 to 259 patients. All studies were conducted retrospectively (1 study was a nonconcurrent analysis of response in a population previously randomized (Tol, 2009). Anti-EGFR therapy was given as first-line treatment in one study and as second-line or greater in the other (Peeters, 2010). In 2 studies, the anti-EGFR monoclonal antibody was given as monotherapy, and in 9 studies, patients received various chemotherapies. Seven studies were performed in unselected patients (ie, KRAS mutational status was unknown) totaling 546 patients, for whom 520 were assessable for tumor response. In the unselected population, a BRAF mutation was detected in 8.8% of patients, and the ORR for patients with mutant BRAF was 29.2% (14/48) and for WT BRAF was 33.5% (158/472; p=0.048). Four studies were performed in patients with WT KRAS metastatic CRC. BRAF mutational status was performed on 376 KRAS WT tumors. BRAF mutation was detected in 10.6% (n=40) of primary tumors. Among the 376 analyzed, all patients were assessable for tumor response. ORR of patients with mutant BRAF was 0% (0/40), whereas the ORR of patients with WT BRAF was 36.3% (122/336). Only 3 studies presented data on PFS and OS; and therefore, a pooled analysis was not performed. The authors conclude that although the meta-analysis provided evidence that BRAF mutation is associated with lack of response to anti-EGFR monoclonal antibodies in WT KRAS metastatic CRC, the number of studies and number of patients included in the meta analysis were relatively small and that large studies are needed to confirm the results of the meta-analysis using homogenous metastatic CRC patients with assessors blinded to the clinical data.

Mao et al conducted a meta-analysis of BRAF mutation V600E and resistance to anti-EGFR monoclonal antibodies in patients with metastatic CRC (Mao, 2011). Eleven studies were included, with sample sizes ranging from 31 to 259; all of the studies were retrospective analyses. Seven of the studies included unselected patients, and 4 included only patients with WT KRAS. The primary end point was ORR. In the 7 studies with unselected patients, BRAF mutational status was performed successfully on 546 mCRC. BRAF mutation was detected in 8.8% of primary tumors. The ORR of mCRC patients with mCRC with mutant BRAF was 29.2% versus 33.5% in patients with WT BRAF. In the 4 studies that included patients with WT KRAS, BRAF mutational status was performed successfully on 376 KRAS WT mCRC. BRAF mutations were detected in 10.6% of primary tumors. The ORR of patients with mutant BRAF was 0.0%, whereas the ORR of patients with WT BRAF was 36.3%. The authors concluded that the results of their meta-analysis provided evidence that BRAF mutation is associated with lack of response in WT KRAS mCRC treated with anti-EGFR monoclonal antibodies.

Phillips et al analyzed the data from 4 studies that reported tumor response and survival in patients with mCRC treated with anti-EGFR monoclonal antibodies as related to BRAF mutational status (Phillips, 2010). Di Nicolantonio et al looked retrospectively at 113 patients with mCRC who had received cetuximab or panitumumab (Di Nicolantonio, 2008). None of the BRAF-mutated tumors responded to treatment (0/11), whereas 32.4% (22/68) of the BRAF WT did. Loupakis et al retrospectively assessed 87 patients receiving irinotecan and cetuximab (Loupakis, 2009). Of the 87 patients in the study, BRAF was mutated in 13 cases, and none of them responded to chemotherapy, compared with 32% (24/74) with WT BRAF who did. In the CAIRO2 study, a retrospective analysis of BRAF mutations was performed in 516 available tumors from patients previously randomized to CB regimen or the same regimen plus cetuximab (CBC regimen) (Tol, 2009). A BRAF mutation was found in 8.7% (n=45) of the tumors. Patients with a BRAF mutation had a shorter median PFS and OS compared with WT BRAF tumors in both treatment arms. The authors concluded that a BRAF mutation is a negative prognostic marker in patients with mCRC and that this effect, in contrast with KRAS mutations, is not restricted to the outcome of cetuximab treatment. In the CRYSTAL trial, Van Cutsem et al randomized 1198 patients with untreated mCRC to FOLFIRI with or without cetuximab (Van Cutsem, 2009). A recent analysis of BRAF mutations in this patient population and the influence on outcome was presented at the 2010 American Society of Clinical Oncology (ASCO) Gastrointestinal Cancers Symposium (Peeters, 2014). The authors showed that of the KRAS WT/BRAF-mutated patients, the OS for FOLFIRI plus cetuximab and FOLFIRI alone was 14.1 and 10.3 months, respectively (p=0.744). Although this was not statistically significant, it showed a trend toward improved OS, PFS, and response, suggesting that KRAS WT/BRAF-mutant patients may benefit from anti-EGFR therapy. This unpublished analysis is the first to show a possible benefit of anti-EGFR therapy in patients with BRAF-mutant tumors.

De Roock et al reported the effects of 4 mutations, including BRAF, on the efficacy of cetuximab and chemotherapy in chemotherapy-refractory metastatic CRC in 773 primary tumor samples (De Roock, 2010). Tumor samples were from fresh frozen or FFPE tissue, and the mutation status was compared with retrospectively collected clinical outcomes including objective response, PFS, and OS. BRAF mutations were found in 36 of 761 tumors (4.7%). In patients with WT KRAS, carriers of BRAF mutations had a significantly lower response rate (8.3% or 2 of 24 patients) than BRAF WT (38.0% or 124 of 326 patients; OR=0.15; 95% CI, 0.02 to 0.51; p=0.001). PFS for BRAF-mutated versus WT was a median of 8 weeks versus 26 weeks, respectively (HR=3.74; 95% CI, 2.44 to 5.75; p<0.001) and OS median 26 weeks versus 54 weeks, respectively (HR=3.03; 95% CI, 1.98 to 4.63; p<0.001).

An updated analysis of the CRYSTAL trial reported increased follow-up time and an increased number of patients evaluable for tumor KRAS status and considered the clinical significance of the tumor mutation status of BRAF in the expanded population of patients with KRAS WT tumors (Van Cutsem, 2011). The impact of BRAF tumor mutation status in relation to the efficacy of cetuximab plus FOLFIRI was examined in the population of patients with KRAS WT disease (n=625). No evidence was reported for an independent treatment interaction by tumor BRAF mutation status. The authors concluded that BRAF mutation status was not predictive of treatment effects of cetuximab plus FOLFIRI but that BRAF tumor mutation was a strong indicator of poor prognosis for all efficacy end points compared with those whose tumors were WT.

At the latest review of this policy (December 2013), no new clinical trials were identified on the use of BRAF mutation analysis to guide use of anti-EGFR therapy in patients with metastatic CRC.

Summary of Evidence

Clinical trial data show that patients with KRAS-mutated metastatic colorectal cancer (CRC) do not benefit from cetuximab or panitumumab, either as monotherapy or in combination with other treatment regimens. These data support the use of KRAS mutation analysis of tumor DNA before considering use of cetuximab or panitumumab in a treatment regimen. Identifying patients whose tumors express mutated KRAS will avoid exposing patients to ineffective drugs and unnecessary drug toxicities and expedites the use of alternative therapies. Thus, KRAS mutation analysis may be considered medically necessary to predict nonresponse to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies in the treatment of metastatic CRC.

Another member of the RAS family of protooncogenes, NRAS, can harbor mutations in codons 12, 13 and 61 that constitutively activate the EGFR-mediated signaling pathway as do specific mutations in KRAS. Thus, the NRAS oncogene also may have an impact on outcomes of anti-EGFR treatments for CRC. Compared with KRAS, NRAS mutations are extremely rare. Although NRAS mutations account for approximately 15% of all RAS mutations, they are found in perhaps 2% to 7% of all CRC. As a consequence of the low prevalence of NRAS mutations in CRC, it is difficult to assess their effect on cancer behavior or therapy. Current clinical evidence is sparse and inconclusive as to whether NRAS mutations are prognostic markers for poor outcomes similar to BRAF (discussed next) or may be like KRAS mutations, acting as predictive markers for poor response to anti-EGFR therapy. Given these uncertainties, NRAS mutation analysis is considered investigational to predict nonresponse to anti-EGFR monoclonal antibodies in the treatment of metastatic CRC.

The data for patients with metastatic CRC and a BRAF mutation have shown consistently that a BRAF mutation is a poor prognostic marker, as it is associated with shorter progression-free survival and overall survival, regardless of treatment. Most of the data for a BRAF mutation predicting response to anti-EGFR therapy are limited by small numbers of patients and conflicting results among studies. No evidence is available from a randomized trial designed specifically to assess the clinical utility of BRAF mutation analysis in guiding management of patients with metastatic CRC. The large, randomized CRYSTAL trial, with nonconcurrent subgroup analyses of BRAF mutations in patients previously randomized, reported the impact of BRAF tumor mutation status in relation to the efficacy of cetuximab plus FOLFIRI in the population of patients with KRAS wild-type (WT) disease. No evidence was reported for an independent treatment interaction by tumor BRAF mutation status, and the trial showed that BRAF mutation status was not predictive of treatment effects of cetuximab plus folinic acid [leucovorin], 5-fluorouracil (5-FU), and irinotecan (FOLFIRI). BRAF tumor mutation was a strong indicator of poor prognosis for all efficacy end points compared with those whose tumors were BRAF WT. Thus, BRAF mutation analysis is considered investigational to predict nonresponse to anti-EGFR monoclonal antibodies in the treatment of metastatic CRC.

National Cancer Institute PDQ® Clinical Trials Registry

A search of the National Cancer Institute PDQ® clinical trials registry on November 3, 2014, did not identify any phase 3 clinical trials in which BRAF, NRAS, or KRAS mutation testing was used to guide management of CRC patients.

National Comprehensive Cancer Network

The National Comprehensive Cancer Network (NCCN) guidelines (http://www.nccn.org/professionals/physician_gls/pdf/colon.pdf, v2.2015) on the treatment of colon cancer recommend that tumor KRAS and NRAS gene status testing be performed for all patients with metastatic colon cancer. Testing should be performed on archived specimens of primary tumor or a metastasis at the time of diagnosis of metastatic disease. The guidelines indicate that cetuximab and panitumumab are appropriate only for patients with a tumor that expresses the WT KRAS and NRAS gene. The guidelines further state that if the tumor harbors the WT KRAS and NRAS gene, the clinician should consider testing for BRAF mutation status.

NCCN guidelines also state that patients with a BRAF V600E mutation appear to have a poorer prognosis. However, evidence is insufficient to guide the use of anti-EGFR therapy in the first-line setting with active chemotherapy based on BRAF V600E mutation status. Limited evidence suggests lack of antitumor activity from anti-EGFR monoclonal antibodies in the presence of a BRAF V600E mutation when used after a patient has progressed on first-line therapy.

An evidence review published in 2013 by the American College of Medical Genetics and Genomics, Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group, states that evidence is insufficient to support the clinical validity or utility of testing CRC specimens for NRAS mutations to guide patient management (EGAPP Working Group, 2013). In the same review, EGAPP found no guidelines on NRAS testing from any other U.S. group. The EGAPP recommendations align with the Policy statements in this Policy document.

2016 Update

A literature search conducted through November 2016 did not reveal any new information that would prompt a change in the coverage statement. The key identified literature is summarized below.

NRAS Mutations

Randomized Controlled Trials

RCTs have performed non-concurrent subgroup analyses of the efficacy of EGFR inhibitors in patients with wild-type versus mutated RAS in metastatic CRC.

In 2015, Peeters and colleagues reported the influence of RAS mutation status in a prospective/retrospective analysis of a randomized, multicenter phase 3 trial of panitumumab plus FOLFIRI versus FOLFIRI alone as second-line therapy in patients with metastatic CRC (Peeters, 2015). If a tumor was wild-type KRAS exon 2, extended RAS mutations beyond KRAS exon 2 was performed (KRAS exons 3 and 4; NRAS exons 2, 3, and 4; BRAF exon 15). Primary end points were PFS and OS. RAS mutations were obtained in 85% of the specimens from the original trial; 18% of wild-type KRAS exon 2 tumors harbored other RAS mutations. For PFS and OS, the HR for panitumumab plus FOLFIRI versus FOLFIRI alone more strongly favored panitumumab in the wild-type RAS population than in the wild-type KRAS exon 2 population (PFS HR= 0.70; 95% CI, 0.54 to 0.91; p=0.007 vs PFS HR=0.73; 95% CI, 0.59 to 0.90; p=0.004; OS HR=0.81; 95% CI, 0.63 to 1.03; p=0.08 vs OS HR=0.85; 95% CI, 0.70 to 1.04; p=0.12). Patients with RAS mutations were unlikely to benefit from panitumumab. Among RAS wild-type patients, the ORR was 41% in the panitumumab plus FOLFIRI group and 10% in the FOLFIRI group.

In 2015, van Cutsem and colleagues reported results of a prospective/retrospective extended RAS mutation analysis in tumor samples from the randomized phase 3 CRYSTAL trial, which compared FOLFIRI to FOLFIRI plus cetuximab in wild-type KRAS exon 2 patients (van Cutsem, 2015). Mutation status was available in 430 (64.6%) of 666 patients from the trial. A pooled analysis of RAS mutations, other than KRAS exon 2, found a lack of benefit from the addition of cetuximab to FOLFIRI for median PFS (7.4 months vs 7.5 months; p=NS) and median OS (16.4 months vs 17.7 months; p=NS). Patients with tumors that had no RAS mutations experienced significant benefit in median PFS (9.9 months vs 8.4 months; p<0.05) and median OS (23.5 months vs 20 months; p<.05) with the addition of cetuximab to chemotherapy.

National Comprehensive Cancer Network

The National Comprehensive Cancer Network (NCCN) guidelines on the treatment of colon cancer strongly recommend that KRAS and NRAS tumor gene status testing be performed for all patients with metastatic colon cancer (v.2.2016) (NCCN, 2016). Testing should be performed on archived specimens of primary tumor or a metastasis at the time of diagnosis of metastatic disease. The guidelines indicate that cetuximab and panitumumab are appropriate only for patients with a tumor that expresses wild-type KRAS and NRAS genes. The guidelines further state if the tumor harbors wild-type KRAS and NRAS mutations, the clinician should consider testing for BRAF mutation status.

NCCN guidelines also state that patients with a BRAF V600E mutation appear to have a poorer prognosis. However, evidence is insufficient to guide the use of anti-EGFR therapy in the first-line setting with active chemotherapy based on BRAF V600E mutation status, and testing for BRAF is currently optional but not necessary part of decision-making on use of anti-EGFR agents. Limited evidence suggests lack of antitumor activity with anti-EGFR monoclonal antibodies in the presence of a BRAF V600E mutation when used after a patient has progressed on first-line therapy.

2019 Update

Annual policy review completed with a literature search using the MEDLINE database through December 2018. No new literature was identified that would prompt a change in the coverage statement.

2020 Update

Annual policy review completed with a literature search using the MEDLINE database through December 2019. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.

The FoundationACT ctDNA assay, the predecessor of FoundationOne Liquid, was compared to tissue biopsy using the FoundationOne assay in one manufacturer sponsored study (Li, 2019). The researchers reported results on the subset of 51 patients with KRAS, NRAS, and BRAF variants. Positive percent agreement was 80% for all time points for short variants and increased to 90% for cases in which tissue and liquid biopsy were measured less than 270 days apart. Clinical validity studies were limited by unclear reporting of blinding, use of convenience rather than consecutive samples, and variation in the timing of sample collection. There are no published studies reporting clinical outcomes or clinical utility.

2021 Update

Annual policy review completed with a literature search using the MEDLINE database through December 2020. No new literature was identified that would prompt a change in the coverage statement.

2022 Update

Annual policy review completed with a literature search using the MEDLINE database through December 2021. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.

Evidence for the effectiveness of pembrolizumab in patients with MSI-H/dMMR metastatic CRC comes from the KEYNOTE-177 trial, reported by Andre et al (Andre, 2020).

The trial demonstrated a statistically significant improvement in PFS for patients randomized to pembrolizumab compared with chemotherapy (HR=0.60; 95% CI 0.45 to 0.80; p=.0002). At the time of the PFS analysis, the overall survival data were not mature (66% of the required number of events for the OS final analysis). The median follow-up time was 32.4 months (range: 24.0 to 48.3 months). The independent data monitoring committee recommended the continued masking of overall survival data until 190 deaths for the final analysis of overall survival have been observed or 12 months have elapsed since the last data review.

Hainsworth et al reported results of MyPathway, an open-label, phase 2, nonrandomized basket trial of targeted treatment in 251 patients with various advanced refractory solid tumors harboring genetic alterations (Hainsworth, 2018). The cohort included 37 patients with HER2-ampified/overexpressed metastatic CRC. Treatment with trastuzumab plus pertuzumab produced partial response in 14 patients (38%; 95% CI, 23% to 55%) and the median duration of response was 11 months (range1 to 16+ months; 95% CI, 2.8 months to not estimable).

In an open-label, phase 2 trial of trastuzumab deruxtecan, objective response, the primary outcome, was observed in 24 of 53 patients with HER2-positive metastatic CRC (45.3%; 95% CI 31.6 to 59·6) after a median follow-up of 27.1 weeks (interquartile range [IQR] 19.3 to 40.1) (Siena, 2021). One (2%) patient had a complete response, and 23 (43%) had a partial response. Median PFS was 6.9 months (4.1 to not evaluable). Median OS had not been reached at data cutoff (95% CI.74 months to not evaluable).

Preliminary evidence has suggested that HER2 amplification/overexpression may be predictive of nonresponse to EGFR-targeted therapy (Sartore, 2019).

Marabelle et al reported the association of high TMB to response to pembrolizumab in patients with solid tumors enrolled in a prespecified exploratory analysis of the KEYNOTE-158 study (Marabelle, 2020). High TMB was defined as >10 mutations per megabase according to the FoundationOne CDx panel. The proportion of patients with an objective response in the TMB-high group was 29%. At a median follow-up of approximately 3 years, the median duration of response was not reached in the TMB-high group and was 33.1 months in the non-TMB-high group. Notably, TMB-high status was associated with improved response irrespective of programmed death-ligand 1 (PD-L1). Median PFS and OS did not differ between the high and non-high TMB groups. Objective responses were observed in 24 (35%; 95% CI 24 to 48) of 68 participants who had both TMB-high status and PD-L1-positive tumors (ie, PD-L1 combined positive score of ≥1) and in 6 (21%; 8 to 40) of 29 participants who had TMB-high status and PD-L1-negative tumors. Study eligible cancers were limited to anal, biliary, cervical, endometrial, mesothelioma, neuroendocrine, salivary, small-cell lung, thyroid, and vulvar. Because no patients with colorectal cancer were included in these analyses, it is not possible to draw conclusions about the clinical validity and utility of TMB in this group of patients.

2023 Update

Annual policy review completed with a literature search using the MEDLINE database through December 2022. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.

Evidence for the effectiveness of pembrolizumab in patients with MSI-H/dMMR metastatic CRC comes from the KEYNOTE-177 trial, reported by Andre et al (Andre, 2020). The trial demonstrated a statistically significant improvement in PFS for patients randomized to pembrolizumab compared with chemotherapy (HR 0.60; 95% confidence interval [CI], 0.45 to 0.80; p=.0002). Final results were reported by Diaz et al (Diaz, 2022). Median PFS was 16.5 months (95% CI, 5.4 to 38.1) with pembrolizumab versus 8.2 months (6.1 to 10.2) with chemotherapy (HR 0.59, 95% CI 0.45 to 0.79). Treatment-related adverse events of grade 3 or worse occurred in 33 (22%) of 153 patients in the pembrolizumab group versus 95 (66%) of 143 patients in the chemotherapy group.

Preliminary evidence has suggested that HER2 amplification/overexpression may be predictive of nonresponse to EGFR-targeted therapy (Sartore-Bianchi, 2019; Huang, 2022).

2024 Update

Annual policy review completed with a literature search using the MEDLINE database through December 2023. No new literature was identified that would prompt a change in the coverage statement.

CPT/HCPCS:

0037U	Targeted genomic sequence analysis, solid organ neoplasm, DNA analysis of 324 genes, interrogation for sequence variants, gene copy number amplifications, gene rearrangements, microsatellite instability and tumor mutational burden
0111U	Oncology (colon cancer), targeted KRAS (codons 12, 13, and 61) and NRAS (codons 12, 13, and 61) gene analysis utilizing formalin fixed paraffin embedded tissue
0239U	Targeted genomic sequence analysis panel, solid organ neoplasm, cell free DNA, analysis of 311 or more genes, interrogation for sequence variants, including substitutions, insertions, deletions, select rearrangements, and copy number variations
0242U	Targeted genomic sequence analysis panel, solid organ neoplasm, cell-free circulating DNA analysis of 55-74 genes, interrogation for sequence variants, gene copy number amplifications, and gene rearrangements
0338U	Oncology (solid tumor), circulating tumor cell selection, identification, morphological characterization, detection and enumeration based on differential EpCAM, cytokeratins 8, 18, and 19, and CD45 protein biomarkers, and quantification of HER2 protein biomarker–expressing cells, peripheral blood
0448U	Oncology (lung and colon cancer), DNA, qualitative, nextgeneration sequencing detection of single-nucleotide variants and deletions in EGFR and KRAS genes, formalin-fixed paraffinembedded (FFPE) solid tumor samples, reported as presence or absence of targeted mutation(s), with recommended therapeutic options
81210	BRAF (B Raf proto oncogene, serine/threonine kinase) (eg, colon cancer, melanoma), gene analysis, V600 variant(s)
81275	KRAS (Kirsten rat sarcoma viral oncogene homolog) (eg, carcinoma) gene analysis; variants in exon 2 (eg, codons 12 and 13)
81276	KRAS (Kirsten rat sarcoma viral oncogene homolog) (eg, carcinoma) gene analysis; additional variant(s) (eg, codon 61, codon 146)
81311	NRAS (neuroblastoma RAS viral [v ras] oncogene homolog) (eg, colorectal carcinoma), gene analysis, variants in exon 2 (eg, codons 12 and 13) and exon 3 (eg, codon 61)
81403	Molecular pathology procedure, Level 4 (eg, analysis of single exon by DNA sequence analysis, analysis of >10 amplicons using multiplex PCR in 2 or more independent reactions, mutation scanning or duplication/deletion variants of 2-5 exons) ANG (angiogenin, ribonuclease, RNase A family, 5) (eg, amyotrophic lateral sclerosis), full gene sequence ARX (aristaless-related homeobox) (eg, X-linked lissencephaly with ambiguous genitalia, X-linked mental retardation), duplication/deletion analysis CEL (carboxyl ester lipase [bile salt-stimulated lipase]) (eg, maturity-onset diabetes of the young [MODY]), targeted sequence analysis of exon 11 (eg, c.1785delC, c.1686delT) CTNNB1 (catenin [cadherin-associated protein], beta 1, 88kDa) (eg, desmoid tumors), targeted sequence analysis (eg, exon 3) DAZ/SRY (deleted in azoospermia and sex determining region Y) (eg, male infertility), common deletions (eg, AZFa, AZFb, AZFc, AZFd) DNMT3A (DNA [cytosine-5-]-methyltransferase 3 alpha) (eg, acute myeloid leukemia), targeted sequence analysis (eg, exon 23) EPCAM (epithelial cell adhesion molecule) (eg, Lynch syndrome), duplication/deletion analysis F8 (coagulation factor VIII) (eg, hemophilia A), inversion analysis, intron 1 and intron 22A F12 (coagulation factor XII [Hageman factor]) (eg, angioedema, hereditary, type III; factor XII deficiency), targeted sequence analysis of exon 9 FGFR3 (fibroblast growth factor receptor 3) (eg, isolated craniosynostosis), targeted sequence analysis (eg, exon 7) (For targeted sequence analysis of multiple FGFR3 exons, use 81404) GJB1 (gap junction protein, beta 1) (eg, Charcot-Marie-Tooth X-linked), full gene sequence GNAQ (guanine nucleotide-binding protein G[q] subunit alpha) (eg, uveal melanoma), common variants (eg, R183, Q209) Human erythrocyte antigen gene analyses (eg, SLC14A1 [Kidd blood group], BCAM [Lutheran blood group], ICAM4 [Landsteiner-Wiener blood group], SLC4A1 [Diego blood group], AQP1 [Colton blood group], ERMAP [Scianna blood group], RHCE [Rh blood group, CcEe antigens], KEL [Kell blood group], DARC [Duffy blood group], GYPA, GYPB, GYPE [MNS blood group], ART4 [Dombrock blood group]) (eg, sickle-cell disease, thalassemia, hemolytic transfusion reactions, hemolytic disease of the fetus or newborn), common variants HRAS (v-Ha-ras Harvey rat sarcoma viral oncogene homolog) (eg, Costello syndrome), exon 2 sequence KCNC3 (potassium voltage-gated channel, Shaw-related subfamily, member 3) (eg, spinocerebellar ataxia), targeted sequence analysis (eg, exon 2) KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2) (eg, Andersen-Tawil syndrome), full gene sequence KCNJ11 (potassium inwardly-rectifying channel, subfamily J, member 11) (eg, familial hyperinsulinism), full gene sequence Killer cell immunoglobulin-like receptor (KIR) gene family (eg, hematopoietic stem cell transplantation), genotyping of KIR family genes Known familial variant not otherwise specified, for gene listed in Tier 1 or Tier 2, or identified during a genomic sequencing procedure, DNA sequence analysis, each variant exon (For a known familial variant that is considered a common variant, use specific common variant Tier 1 or Tier 2 code) MC4R (melanocortin 4 receptor) (eg, obesity), full gene sequence MICA (MHC class I polypeptide-related sequence A) (eg, solid organ transplantation), common variants (eg, 001, 002) MT-RNR1 (mitochondrially encoded 12S RNA) (eg, nonsyndromic hearing loss), full gene sequence MT-TS1 (mitochondrially encoded tRNA serine 1) (eg, nonsyndromic hearing loss), full gene sequence NDP (Norrie disease [pseudoglioma]) (eg, Norrie disease), duplication/deletion analysis NHLRC1 (NHL repeat containing 1) (eg, progressive myoclonus epilepsy), full gene sequence PHOX2B (paired-like homeobox 2b) (eg, congenital central hypoventilation syndrome), duplication/deletion analysis PLN (phospholamban) (eg, dilated cardiomyopathy, hypertrophic cardiomyopathy), full gene sequence RHD (Rh blood group, D antigen) (eg, hemolytic disease of the fetus and newborn, Rh maternal/fetal compatibility), deletion analysis (eg, exons 4, 5, and 7, pseudogene) RHD (Rh blood group, D antigen) (eg, hemolytic disease of the fetus and newborn, Rh maternal/fetal compatibility), deletion analysis (eg, exons 4, 5, and 7, pseudogene), performed on cell-free fetal DNA in maternal blood (For human erythrocyte gene analysis of RHD, use a separate unit of 81403) SH2D1A (SH2 domain containing 1A) (eg, X-linked lymphoproliferative syndrome), duplication/deletion analysis TWIST1 (twist homolog 1 [Drosophila]) (eg, Saethre-Chotzen syndrome), duplication/deletion analysis UBA1 (ubiquitin-like modifier activating enzyme 1) (eg, spinal muscular atrophy, X-linked), targeted sequence analysis (eg, exon 15) VHL (von Hippel-Lindau tumor suppressor) (eg, von Hippel-Lindau familial cancer syndrome), deletion/duplication analysis VWF (von Willebrand factor) (eg, von Willebrand disease types 2A, 2B, 2M), targeted sequence analysis (eg, exon 28)
81404	Molecular pathology procedure, Level 5 (eg, analysis of 2-5 exons by DNA sequence analysis, mutation scanning or duplication/deletion variants of 6-10 exons, or characterization of a dynamic mutation disorder/triplet repeat by Southern blot analysis) ACADS (acyl-CoA dehydrogenase, C-2 to C-3 short chain) (eg, short chain acyl-CoA dehydrogenase deficiency), targeted sequence analysis (eg, exons 5 and 6) AQP2 (aquaporin 2 [collecting duct]) (eg, nephrogenic diabetes insipidus), full gene sequence ARX (aristaless related homeobox) (eg, X-linked lissencephaly with ambiguous genitalia, X-linked mental retardation), full gene sequence AVPR2 (arginine vasopressin receptor 2) (eg, nephrogenic diabetes insipidus), full gene sequence BBS10 (Bardet-Biedl syndrome 10) (eg, Bardet-Biedl syndrome), full gene sequence BTD (biotinidase) (eg, biotinidase deficiency), full gene sequence C10orf2 (chromosome 10 open reading frame 2) (eg, mitochondrial DNA depletion syndrome), full gene sequence CAV3 (caveolin 3) (eg, CAV3-related distal myopathy, limb-girdle muscular dystrophy type 1C), full gene sequence CD40LG (CD40 ligand) (eg, X-linked hyper IgM syndrome), full gene sequence CDKN2A (cyclin-dependent kinase inhibitor 2A) (eg, CDKN2A-related cutaneous malignant melanoma, familial atypical mole-malignant melanoma syndrome), full gene sequence CLRN1 (clarin 1) (eg, Usher syndrome, type 3), full gene sequence COX6B1 (cytochrome c oxidase subunit VIb polypeptide 1) (eg, mitochondrial respiratory chain complex IV deficiency), full gene sequence CPT2 (carnitine palmitoyltransferase 2) (eg, carnitine palmitoyltransferase II deficiency), full gene sequence CRX (cone-rod homeobox) (eg, cone-rod dystrophy 2, Leber congenital amaurosis), full gene sequence CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1) (eg, primary congenital glaucoma), full gene sequence EGR2 (early growth response 2) (eg, Charcot-Marie-Tooth), full gene sequence EMD (emerin) (eg, Emery-Dreifuss muscular dystrophy), duplication/deletion analysis EPM2A (epilepsy, progressive myoclonus type 2A, Lafora disease [laforin]) (eg, progressive myoclonus epilepsy), full gene sequence FGF23 (fibroblast growth factor 23) (eg, hypophosphatemic rickets), full gene sequence FGFR2 (fibroblast growth factor receptor 2) (eg, craniosynostosis, Apert syndrome, Crouzon syndrome), targeted sequence analysis (eg, exons 8, 10) FGFR3 (fibroblast growth factor receptor 3) (eg, achondroplasia, hypochondroplasia), targeted sequence analysis (eg, exons 8, 11, 12, 13) FHL1 (four and a half LIM domains 1) (eg, Emery-Dreifuss muscular dystrophy), full gene sequence FKRP (fukutin related protein) (eg, congenital muscular dystrophy type 1C [MDC1C], limb-girdle muscular dystrophy [LGMD] type 2I), full gene sequence FOXG1 (forkhead box G1) (eg, Rett syndrome), full gene sequence FSHMD1A (facioscapulohumeral muscular dystrophy 1A) (eg, facioscapulohumeral muscular dystrophy), evaluation to detect abnormal (eg, deleted) alleles FSHMD1A (facioscapulohumeral muscular dystrophy 1A) (eg, facioscapulohumeral muscular dystrophy), characterization of haplotype(s) (ie, chromosome 4A and 4B haplotypes) GH1 (growth hormone 1) (eg, growth hormone deficiency), full gene sequence GP1BB (glycoprotein Ib [platelet], beta polypeptide) (eg, Bernard-Soulier syndrome type B), full gene sequence (For common deletion variants of alpha globin 1 and alpha globin 2 genes, use 81257) HNF1B (HNF1 homeobox B) (eg, maturity-onset diabetes of the young [MODY]), duplication/deletion analysis HRAS (v-Ha-ras Harvey rat sarcoma viral oncogene homolog) (eg, Costello syndrome), full gene sequence HSD3B2 (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2) (eg, 3-beta-hydroxysteroid dehydrogenase type II deficiency), full gene sequence HSD11B2 (hydroxysteroid [11-beta] dehydrogenase 2) (eg, mineralocorticoid excess syndrome), full gene sequence HSPB1 (heat shock 27kDa protein 1) (eg, Charcot-Marie-Tooth disease), full gene sequence INS (insulin) (eg, diabetes mellitus), full gene sequence KCNJ1 (potassium inwardly-rectifying channel, subfamily J, member 1) (eg, Bartter syndrome), full gene sequence KCNJ10 (potassium inwardly-rectifying channel, subfamily J, member 10) (eg, SeSAME syndrome, EAST syndrome, sensorineural hearing loss), full gene sequence LITAF (lipopolysaccharide-induced TNF factor) (eg, Charcot-Marie-Tooth), full gene sequence MEFV (Mediterranean fever) (eg, familial Mediterranean fever), full gene sequence MEN1 (multiple endocrine neoplasia I) (eg, multiple endocrine neoplasia type 1, Wermer syndrome), duplication/deletion analysis MMACHC (methylmalonic aciduria [cobalamin deficiency] cblC type, with homocystinuria) (eg, methylmalonic acidemia and homocystinuria), full gene sequence MPV17 (MpV17 mitochondrial inner membrane protein) (eg, mitochondrial DNA depletion syndrome), duplication/deletion analysis NDP (Norrie disease [pseudoglioma]) (eg, Norrie disease), full gene sequence NDUFA1 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 1, 7.5kDa) (eg, Leigh syndrome, mitochondrial complex I deficiency), full gene sequence NDUFAF2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, assembly factor 2) (eg, Leigh syndrome, mitochondrial complex I deficiency), full gene sequence NDUFS4 (NADH dehydrogenase [ubiquinone] Fe-S protein 4, 18kDa [NADH-coenzyme Q reductase]) (eg, Leigh syndrome, mitochondrial complex I deficiency), full gene sequence NIPA1 (non-imprinted in Prader-Willi/Angelman syndrome 1) (eg, spastic paraplegia), full gene sequence NLGN4X (neuroligin 4, X-linked) (eg, autism spectrum disorders), duplication/deletion analysis NPC2 (Niemann-Pick disease, type C2 [epididymal secretory protein E1]) (eg, Niemann-Pick disease type C2), full gene sequence NR0B1 (nuclear receptor subfamily 0, group B, member 1) (eg, congenital adrenal hypoplasia), full gene sequence PDX1 (pancreatic and duodenal homeobox 1) (eg, maturity-onset diabetes of the young [MODY]), full gene sequence PHOX2B (paired-like homeobox 2b) (eg, congenital central hypoventilation syndrome), full gene sequence PLP1 (proteolipid protein 1) (eg, Pelizaeus-Merzbacher disease, spastic paraplegia), duplication/deletion analysis PQBP1 (polyglutamine binding protein 1) (eg, Renpenning syndrome), duplication/deletion analysis PRNP (prion protein) (eg, genetic prion disease), full gene sequence PROP1 (PROP paired-like homeobox 1) (eg, combined pituitary hormone deficiency), full gene sequence PRPH2 (peripherin 2 [retinal degeneration, slow]) (eg, retinitis pigmentosa), full gene sequence PRSS1 (protease, serine, 1 [trypsin 1]) (eg, hereditary pancreatitis), full gene sequence RAF1 (v-raf-1 murine leukemia viral oncogene homolog 1) (eg, LEOPARD syndrome), targeted sequence analysis (eg, exons 7, 12, 14, 17) RET (ret proto-oncogene) (eg, multiple endocrine neoplasia, type 2B and familial medullary thyroid carcinoma), common variants (eg, M918T, 2647_2648delinsTT, A883F) RHO (rhodopsin) (eg, retinitis pigmentosa), full gene sequence RP1 (retinitis pigmentosa 1) (eg, retinitis pigmentosa), full gene sequence SCN1B (sodium channel, voltage-gated, type I, beta) (eg, Brugada syndrome), full gene sequence SCO2 (SCO cytochrome oxidase deficient homolog 2 [SCO1L]) (eg, mitochondrial respiratory chain complex IV deficiency), full gene sequence SDHC (succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa) (eg, hereditary paraganglioma-pheochromocytoma syndrome), duplication/deletion analysis SDHD (succinate dehydrogenase complex, subunit D, integral membrane protein) (eg, hereditary paraganglioma), full gene sequence SGCG (sarcoglycan, gamma [35kDa dystrophin-associated glycoprotein]) (eg, limb-girdle muscular dystrophy), duplication/deletion analysis SH2D1A (SH2 domain containing 1A) (eg, X-linked lymphoproliferative syndrome), full gene sequence SLC16A2 (solute carrier family 16, member 2 [thyroid hormone transporter]) (eg, specific thyroid hormone
88363	Examination and selection of retrieved archival (ie, previously diagnosed) tissue(s) for molecular analysis (eg, KRAS mutational analysis)

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