Genetic Cancer Susceptibility Panels Using Next Generation Sequencing - CAM 265HB

Next generation sequencing (NGS), also known as massively parallel sequencing, is a type of DNA sequencing technology that sequences many small fragments of DNA in parallel. The wide application of NGS has helped to identify infrequent gene alterations contributing to oncogenesis, cancer progression, metastasis, and tumor complexity (Hulick, 2022).   

Regulatory Status
A search of the FDA Device database on 02/05/2020 for “gene panel” yielded 4 results, last updated 11/30/2017. Additionally, many labs have developed specific tests that they must validate and perform in house. These laboratory-developed tests (LDTs) are regulated by the Centers for Medicare & Medicaid Services (CMS) as high-complexity tests under the Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88). As an LDT, the U.S. Food and Drug Administration has not approved or cleared this test; however, FDA clearance or approval is not currently required for clinical use. 

On Nov. 30, 2017, the FDA approved FoundationOne CDx, by Foundation Medicine, Inc. This device is a next generation sequencing based in vitro diagnostic device for detection of substitutions, insertion and deletion alterations (indels), and copy number alterations (CNAs) in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens (FDA, 2017a).

On June 29, 2017, the FDA approved Praxis Extended RAS Panel, by Illumina, Inc. The Praxis™ Extended RAS Panel is a qualitative in vitro diagnostic test using targeted high-throughput parallel sequencing for the detection of 56 specific mutations in RAS genes [KRAS (exons 2, 3, and 4) and NRAS (exons 2, 3, and 4)] in DNA extracted from formalin‐fixed, paraffin‐embedded (FFPE) colorectal cancer (CRC) tissue samples (FDA, 2017b).

On June 22, 2017, the FDA approved Oncomine Dx Target Test, by Life Technologies Corporation. The Oncomine Dx Target Test is a qualitative in vitro diagnostic test that uses targeted high throughput, parallel-sequencing technology to detect single nucleotide variants (SNVs) and deletions in 23 genes from DNA and fusions in ROS1 from RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples from patients with non-small cell lung cancer (NSCLC) using the Ion PGM Dx System (FDA, 2017c).

On December 19, 2016, the FDA approved FoundationFocus CDxBRCA, by Foundation Medicine, Inc. The FoundationFocus CDxBRCA is a next generation sequencing based in vitro diagnostic device for qualitative detection of BRCA1 and BRCA2 alterations in formalin-fixed paraffin-embedded (FFPE) ovarian tumor tissue. The FoundationFocus CDxBRCA assay detects sequence alterations in BRCA1 and BRCA2 (BRCA1/2) gene (FDA, 2016).

Application of coverage criteria is dependent upon an individual’s benefit coverage at the time of the request.

  1. Pre-test genetic counseling IS REQUIRED.
  2. Genetic cancer susceptibility panels (see Notes 1 – 3) using next generation sequencing is considered MEDICALLY NECESSARY when all the following criteria are met:
    1. The individual displays clinical features and/or has a family history consistent with a hereditary cancer syndrome.
    2. All genes in the panel are relevant based on the personal and family history for the individual being tested.
    3. The results of the genetic test will impact the medical management of the individual.

The following does not meet coverage criteria due to a lack of available published scientific literature confirming that the test(s) is/are required and beneficial for the diagnosis and treatment of a patient’s illness.

  1. For other situations not addressed above, multi-gene panel testing (see Note 4) is considered NOT MEDICALLY NECESSARY.


Note 1: For 5 or more gene tests being run on the same platform, please refer to Reimbursement Policy, CAM 235.

Note 2: Concurrent ordering of multi-gene panel tests for a specific condition IS STRICTLY PROHIBITED; only one multi-gene panel test may be ordered at a time for a specific condition. 

Note 3: Multi-gene panels must contain the genes specified in the AMA CPT coding description. 

Note 4: Current scientific evidence is not yet sufficient to establish how test results from panels which include a broad number of genes may be used to direct treatment decisions and improve health outcomes associated with all genetic sequences included in the panel.

Table of Terminology  




American College of Medical Genetics


Acute lymphoblastic leukaemia


Acute myeloid leukemia


American Society of Clinical Oncology


ASXL transcriptional regulator 1


ATM serine/threonine kinase


BRCA1 associated RING domain 1


BCL6 corepressor


BCR1/ABL1 fusion gene


B-Raf proto-oncogene, serine/threonine kinase


Breast cancer gene 1 or 2


BRCA1 interacting helicase 1




Cbl proto-oncogene


Cadherin 1


Checkpoint kinase 2


Colorectal cancer


Clinical Laboratory Improvement Amendments


Centers for Medicare & Medicaid


Center For Medical Technology Policy


Copy number alterations


Central nervous system


Copy number variation


DEAD-box helicase 41


DNA methyltransferase 3 alpha


Eastern Cooperative Oncology Group


Epidermal growth factor receptor


Epithelial cell adhesion molecule


European Society for Medical Oncology


ETS variant transcription factor 6


Enhancer of zeste 2 polycomb repressive complex 2 subunit


FA complementation group C


Food and Drug Administration


Formalin fixed paraffin embedded


Fibroblast growth factor receptor 1


Fluorescence in situ hybridization


Fms related receptor tyrosine kinase 3


GATA binding protein 2


Genomic instability score


Hereditary nonpolyposis colorectal cancer


Homologous recombination deficiency


Isocitrate dehydrogenase (NADP(+)) 1


Isocitrate dehydrogenase (NADP(+)) 2


Insertion and deletion alterations


Janus kinase 2


KRAS proto-oncogene, GTPase


Laboratory developed tests


Loss of heterozygosity


Large scale state transitions


Multiplex ligation dependent probe amplification


MPL proto-oncogene, thrombopoietin receptor


Mismatch repair


Mononuclear cells


Minimum residual disease


MRE11 homolog, double strand break repair nuclease


Messenger ribonucleic acid


Microsatellite instability


Memorial Sloan Kettering- integrated mutation profiling of actionable cancer targets


MRE11 homolog, double strand break repair nuclease




National Comprehensive Cancer Network


Neurofibromin 1


Next generation sequencing


NRAS proto-oncogene, GTPase


Nucleophosmin 1


Non-small cell lung cancer


Partner and localizer of BRCA2


Polymerase chain reaction


Platelet derived growth factor receptor alpha


Platelet derived growth factor receptor beta


PHD finger protein 6


Protein phosphatase, Mg2+/Mn2+ dependent 1D


Phosphatase and tensin homolog


RAD51 paralog C


RAD51 paralog D


Reticular activating system


RecQ like helicase 4


RAD50 interactor 1


Ribonucleic acid


Receptor tyrosine kinase


RUNX family transcription factor 1


Small cell lung


SET binding protein 1


Splicing factor 3b subunit 1


SLX4 structure-specific endonuclease subunit


SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4


RINT1Single nucleotide variants


Serine and arginine rich splicing factor 2


Signal transducer and activator of transcription 3


Stromal antigen 2


Serine/threonine kinase 11


Telomeric allelic imbalance


Tet methylcytosine dioxygenase 2


Tumour fraction


Tumour mutational burden


Tumor protein p53




U2 small nuclear RNA auxiliary factor 1


Variants of uncertain significance


WT1 transcription factor


X-ray repair cross complementing 2


Zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2

Next generation sequencing (NGS) allows for the rapid sequencing of multiple strands of DNA. It is not limited to one specific type of test; rather, it encompasses numerous technologies that produce swift and high-volume sequencing. NGS can be used to sequence multiple genes, the exome, or even the entire genome. This is opposed to traditional Sanger sequencing, which is more useful for sequencing a specific gene (ACMG, 2012; Hulick, 2022).

Next generation sequencing (NGS) typically includes the following steps: the patient’s DNA is prepared to serve as a template, followed by isolation of DNA fragments on solid surfaces such as small beads, generating sequence data. These results are then compared against a reference genome. Though any DNA sample may be used if the quality and quantity of that sample are sufficient, the methods of library generation and data analysis vary from panel to panel. Evaluating the results of a gene panel typically requires some expertise in bioinformatics. NGS reports data on all variants which are identified. As such, great care must be taken to evaluate these gene variants, especially variants of unknown significance (VUS) and secondary findings (Hulick, 2022; Rehm et al., 2013).

Panels that sequence a specific set of genes are referred to as “targeted panels” and may range from 5 to over 1000 genes. Targeted panels are generally more cost-effective than whole exome or whole genome sequencing and are useful for conditions where a disease has more than one causative gene. For example, nonsyndromic hearing loss may be caused by a variance in one of over 60 genes and sequencing each gene individually would not be cost effective. Many companies have developed a wide variety of gene panels. From the FDA-approved MSK-IMPACT all the way to well-validated proprietary panels, many different options of panel testing are available (Hulick, 2022).

While panel testing can be useful, there are still some instances where exome and genome sequencing may be necessary. While the exome is comprised of all the protein-encoding genes and at least 85% of pathogenic mutations are found within the exome, it only represents ~1.5% – 2% of the genome. This difference makes exome sequencing more cost effective than genome sequencing — the entire exome includes ~30 megabases as compared to the genome’s 3.3 gigabases. However, sequencing an entire genome can still be useful, as pathogenic mutations can sometimes occur in a non-coding region of the genome. One example is mutations outside of the exome resulting in gene regulation dysfunction. Most clinical NGS testing use targeted panels or whole exome sequencing, with whole genome sequencing used only in select cases (Hulick, 2022).

Clinical genomics play a significant part in treatment, diagnosis, and understanding of cancer. Assessment of multiple pathogenic genes has become a widely used technique with the rise of NGS technologies, and the NCCN often recommends genetic panels in certain clinical situations. Some panels may also test for other genetic defects, such as microsatellite instabilities or expression levels of specific proteins. Evaluation of genomic information (somatic changes, inherited germline changes, and so on) is widely prevalent in treatment and diagnosis of numerous types of cancer (Hulick, 2022). Genomic profiling of a tumor can help refine cancer subtype classification and help with identifying patients likely to benefit from systemic therapies, as well as help screen for germline variants influencing heritable cancer risk (Chakravarty & Solit, 2021). 

With the declining costs associated with sequencing and the identification of new genomic biomarkers which are predictive of drug response, multigene NGS-based tumor genomic profiling panels are becoming more commonplace as a component in routine cancer care. Additionally, this genomic profiling can identify that refine or confirm a patient’s cancer subtype diagnosis and provides clinicians with insight into both heritable cancer risk as well as the likelihood of cancer recurrence and death. Not all mutations within the same gene produce the same biological effect, nor do they have the same clinical significance. Thus, it is vital to improve the clinical reporting of these detected variant and to improve the overall knowledge surrounding different variants (Chakravarty & Solit, 2021).  

Analytical Validity
Pathogenic variants and other NGS findings are traditionally confirmed by Sanger sequencing, the gold standard of gene sequencing (> 99.99% accuracy). NGS has been shown to compare favorably to Sanger sequencing. In a study performed by (Strom et al., 2014), 110 single-nucleotide variants (SNVs) were found by NGS. Of these SNVS, 103 met the minimum quality score threshold, set by the lab, of 500, with 7 falling below this threshold. However, 109 of the 110 total SNVs were validated by Sanger sequencing (Strom et al., 2014). Another study focused on the agreement between Sanger sequencing and NGS results, finding that out of 5,800 variants identified by NGS, only 2 did not have cross-method agreement. Overall, the agreement rate was 99.965%. The authors concluded that a single round of Sanger sequencing was “more likely to incorrectly refute a true-positive variant from NGS than to correctly identify a false-positive variant from NGS” (Beck et al., 2016).

D'Haene et al. (2019) designed and validated a custom NGS panel for routine diagnosis of gliomas, including 14 genes (H3F3A, ACVR1, IDH1, PDGFRA, TERT, HIST1H3B, HIST1H3C, EGFR, BRAF, CDKN2A, PTEN, IDH2, TP54, and ATRX) and one codeletion (1p/19). After validation to 52 known glioma samples, the panel was applied to 91 unknown brain lesions. For these brain lesions, a sensitivity of 99.4% and specificity of 100% was achieved. “Orthogonal” methods (such as in situ hybridization and immunohistochemistry) demonstrated high concordance with the panel (D'Haene et al., 2019).

Woodhouse et al. (2020) evaluated the analytical performance of FoundationOne Liquid CDx assay to detect genomic alterations in cancer patients. The assay was evaluated across more than 30 different cancer types in over 300 genes and greater than 30,000 gene variants. "Results demonstrated a 95% limit of detection of 0.40% variant allele fraction for select substitutions and insertions/deletions, 0.37% variant allele fraction for select rearrangements, 21.7% tumor fraction (TF) for copy number amplifications, and 30.4% TF for copy number losses. The false positive variant rate was 0.013% (approximately 1 in 8,000). Reproducibility of variant calling was 99.59%” (Woodhouse et al., 2020). In comparison to in situ hybridization and immunohistochemistry, FoundationOne had an overall 96.3% positive percent agreement and > 99.9% negative percent agreement. "These study results demonstrate that FoundationOne Liquid CDx accurately and reproducibly detects the major types of genomic alterations in addition to complex biomarkers such as microsatellite instability, blood tumor mutational burden, and tumor fraction” (Woodhouse et al., 2020). 

Clinical Utility and Validity 
Next generation sequencing (NGS) has utility in numerous clinical scenarios and is especially useful in situations where multiple genes can cause the same phenotype, where other candidate genes were found to be normal, or where sequencing individual genes would not be timely or cost effective (Hulick, 2022).

Discussions of utility may also revolve around what is done with the findings of a gene panel. For instance, a study by Zehir et al. (2017) focused on the MSK-IMPACT gene panel. This panel of 410 cancer-related genes was used to sequence 10,945 tumors from 10,336 patients. Of these patients, 36.7% (3792/10336) were found to have a “clinically actionable” gene variant, such as TP53 and KRAS. Of these, 527 patients were enrolled in clinical trials (Zehir et al., 2017). NGS has also helped provide diagnostic information to patients. A study focusing on 382 patients with a previously undiagnosed condition used NGS technology to diagnose 98 patients with exome or genome sequencing, allowing for changes in diagnostic testing, treatment, and genetic counseling. A total of 31 new syndromes were defined as well (Splinter et al., 2018).

Surrey et al. (2019) evaluated the clinical utility of a custom NGS panel for pediatric tumors. Sequencing was performed on 367 pediatric cancer samples. The authors found that results from the panel testing were “incorporated successfully into clinical care” for 88.7% of leukemias and lymphomas, 90.6% of central nervous system (CNS) cancers, and 62.6% of non-CNS solid tumors. A diagnosis change occurred in 3.3% of cases, and 19.4% of patients had variants requiring further germline testing (Surrey et al., 2019).

Tayshetye et al. (2020) analyzed the clinical utility of NGS in tumor testing using FoundationOne, a validated NGS genomic profiling test. 157 NGS results were collected of many different tumor types, with 63% being stage IV cancer at the time of testing. With NGS analysis, 185 genes with mutations were found in the RTK/RAS pathway, PI3K pathway, p53 pathway and cell cycle pathway. Overall, 82% of the patients had a mutation that could be treated with an FDA-approved treatment. NGS results were used in treatment decisions for 18% of these patients, and only 7% of the patients initiated therapy based on NGS results. The most common reason for not initiating NGS-based therapy was the lack of an FDA-approved medication used for that specific tumor type, as a major challenge is insurance approval for an off-label indication. The authors state that "while there are numerous potential benefits from the use of NGS, further studies are still needed to determine its full clinical utility” (Tayshetye et al., 2020).

Owattanapanich et al. (2021) analyzed the incidence and clinical impact of molecular genetic aberrations in Thai patients with AML and myelodysplastic syndrome — excess blasts (MDS-EB), as detected by NGS. The authors used a custom NGS panel targeting 42 genes recurrently mutated in myeloid neoplasms and found a median number of 3 mutations, with the most frequent alterations occurring in FLT3 internal tandem duplications (ITD) (28.6%), DNMT3A (24.5%), and TW1 (22.4%). FLT3-ITD was more frequent in the de novo AML group than in the MDS/secondary AML group. In contrast, in the MDS/secondary AML group, ASXL1, ETV6, and SRSF2 mutations were more frequent. Advanced age and TP53 mutations were independent, poor prognostic factors for patients’ survival and the authors note that “genetic landscape of AML patients for each disease type, each age group, and each nation differ.” They note that “personalized treatment based on each molecular mutation in individual patients could improve their treatment responses and long-term survival outcomes” and conclude that a comprehensive genetic investigation should guide the most suitable treatment to improve an individual patient’s outcome (Owattanapanich et al., 2021). 

Ma et al. (2022) enrolled 118 patients with advanced thyroid cancer to investigate the clinical application of NGS in the management of advanced thyroid cancer. The most common molecular alterations that patients had were BRAF V600E (62%) and NRAS (15%) mutation in papillary thyroid cancers; RET alteration (78%) in medullary thyroid cancer; and BRAF V600E (38%) and TP53 (62%) mutations in anaplastic thyroid cancer. Most patients (87%) were found to have actionable alterations, while 57% of patients had at least one Level 1 or 2 alteration for which an FDA-approved drug was available. Overall, a matched therapeutic approach was undergone by 13% of patients. In conclusion, the authors noted a rationalized “need for routine multigene NGS testing or reflex BRAF and RET testing in the management of patients with advanced thyroid cancer” (Ma et al., 2022).

National Comprehensive Cancer Network (NCCN)
Numerous gene panels have been recommended by the NCCN. Cancers, such as breast, ovarian, and leukemia, may be caused by many different gene variants, and the NCCN recommends panels in genetic testing for these conditions. These conditions are as follows:

Acute Lymphoblastic Leukemia (ALL):
The NCCN recommends comprehensive testing by NGS for gene fusions and pathogenic mutations, particularly if known to be BCR-ABL1/Ph-negative or Ph-like. For MRD assessment, the NCCN recommends NGS-based assays to detect clonal rearrangements in immunoglobin heavy chain gene and or T-cell receptor genes. They also note that PCR/NGS methods can detect leukemic cells at a sensitivity threshold of < 1 x 10-6 (< 0.0001%) bone marrow mononuclear cells (MNCs). Assays to detect alternative leukemia-specific fusion genes using NGS are in development but are not yet recommended for MRD quantification outside the context of a clinical trial (NCCN, 2022a).

Acute Myeloid Leukemia (AML):
The NCCN states that NGS analysis may be used “for the ongoing management of AML and various phases of treatment” of gene mutations involved with AML such as c-KIT, FLT3-ITD, FLT3-TKD, NPM1, CEBPA, IDH1/IDH2, RUNX1, ASXL1, TP53, BCR-ABL, and PML-RAR. As a caveat, commercial NGS panels for AML use neoplastic tissue samples and may lack coverage of certain genes and mutations. (NCCN, 2023a).

Breast Cancer:
The NCCN notes that NTRK mutations may be detected with NGS (NCCN, 2022b). 

Central Nervous Cancers:
Evaluation of IDH1 and IDH2 mutations is highly recommended. The most common mutation of IDH1 of R132H is reliably screened by immunohistochemistry, but sequencing (through Sanger or NGS-based assays) of IDH1 and IDH2 may also be highly recommended in the appropriate contexts. NGS is included as a “standard sequencing method” (NCCN, 2023c). 

Chronic Lymphocytic Leukemia/ Small Lymphocytic Leukemia: 
The NCCN recommends assessing Minimal Residual Disease (MRD) using an assay with a sensitivity of 10-4 according to the standardized NGS method (NCCN, 2023d). 

Colon and Rectal Cancer: 
The NCCN recommends that sequencing for RAS and BRAF genes and HER2 amplifications be performed if a patient is suspected or proven to have a metastatic synchronous adenocarcinoma. The NCCN does not recommend any sequencing method over another, listing NGS and Sanger sequencing as possible methods. However, if there is a known RAS/RAF mutation, HER2 testing is not indicated (NCCN, 2023e, 2023o).

Esophageal and Esophagogastric Junction Cancers and Gastric Cancers:
The NCCN recommends the use of NGS for assessing esophageal cancer and gastric cancer when there is limited diagnostic tissue available for testing and the patient is unable to undergo additional procedures. NGS can be considered instead of sequential testing for single biomarkers. NCCN notes that for esophageal and esophagogastric junction cancers and gastric cancer, NGS may be considered as part of the initial workup. For gastric cancer and esophageal squamous cell carcinoma and adenocarcinomas that are unresectable locally advanced, locally recurrent, or metastatic, with a Karnofsky performance score ≥ 60% or an ECOG performance score ≤ 2, NGS may be considered via validated assay. (NCCN, 2022c, 2023f).

Gastrointestinal Stromal Tumors:
The NCCN recommends that all gastrointestinal stromal tumors lacking KIT or PDFRA should be tested for SDH deficiency and alternative driver mutations using NGS. In addition, NGS testing should be performed to identify alternative driver mutations such as BRAF, NF1, NTRK, and FGFR fusions, as these could provide insight for a targeted therapy (NCCN, 2023g). 

Multiple Myeloma:
The NCCN notes NGS as a valid method for informing treatment decisions. They comment that an NGS array on bone marrow may be useful in certain circumstances and that in certain circumstances, it may be useful to consider baseline clone identification and storage of aspirate sample for future MRD testing by NGS. NGS is recommended for follow-up/surveillance as needed in smoldering myeloma (asymptomatic). NGS is also listed as a way to assess minimum residual disease (MRD) and categorize responses to treatment (criterion is based on recommendations from the International Myeloma Working Group) (NCCN, 2023i). 

Myelodysplastic Syndromes:
The NCCN recommends that evaluation of mutations should include NGS panels incorporating the 21 most frequently mutated genes, which are as follows: TET2, DNMT3A, ASXL1, EZH2, SF3B1, SRSF2, U2AF1, ZRSR2, RUNX1, TP53, STAG2, NRAS, CBL, NF1, JAK2, CALR, MPL, ETV6, GATA2, DDX41, IDH1, IDH2, SETBP1, PHF6, BCOR, FLT3, WT1, NPM1, STAT3, PPM1D, and UBA1. 

The NCCN added in version 3.2021 that NGS has low sensitivity for the KIT D816V mutation. In this case, allele-specific PCR is more sensitive and recommended in patients with high clinical suspicion of mast cell disease (NCCN, 2023j). 

Myeloid/Lymphoid Neoplasms with Eosinophilia and Tyrosine Kinase Fusion Genes
The NCCN recommends that “NGS may be used to identify novel fusion gene or cryptic rearrangements when clinical suspicion is high and fluorescence in situ hybridization (FISH) for PDGFRA, PDGFRB, FGFR1, JAK2, ABL1, or FLT3 are negative ... currently, the impact on outcomes of additional mutations detected by NGS is unclear. Further studies are needed to determine the impact of mutations on disease course.” In myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase fusion genes, an NGS myeloid mutation panel is recommended as a general diagnostic workup, though RT-PCR may be preferred over NGS for FTL3. NCCN also notes that “mutations detected by NGS may also provide a means to identify primary (clonal/neoplastic) eosinophilia from secondary (reactive) eosinophilia, including in cases where no rearrangements of PDGFRA, PDGFRB, FGFR1, PCM1-JAK2, ETV6-JAK2, or BCR-JAK2 are detected. Mutations described include TET2, ASXL1, EXH2, or SETBP1 and, recently, activating STAT5 N642H mutations” (NCCN, 2023k).

Myeloproliferative Neoplasms:
The NCCN states that NGS may be useful in establishing clonality in selected circumstances, such as the “triple negative” of non-mutated JAK2, CALR, and MPL. The NCCN also notes that workup may include a multi-gene NGS panel that includes all three of JAK2, CALR, and MPL. After an MPN diagnosis is confirmed, NGS is also recommended for mutational prognosticiation. NCCN also notes that additional molecular testing using a multi-gene NGS panel should be considered to evaluate for higher-risk mutations associated with disease progression in patients with primary myelofibrosis (NCCN, 2023l). 

Ovarian Cancer: 
The NCCN recommends NGS for BRCA1/2 somatic mutations, as clinically indicated (NCCN, 2022f). 

Pancreatic Adenocarcinoma:
The NCCN states that NGS may be used to detect “potentially actionable somatic findings,” such as ALK, NRG1, NTRK, ROS1, FGFR2, RET, BRAF, BRCA1/2, HER2 (amplifications), KRAS, PALB2, MSI and/or MMR deficiency-related genes. Testing on tumor tissue is preferred but cell-free DNA testing can be considered if tumor tissue testing is not feasible (NCCN, 2023m). 

B-Cell Lymphomas:
The NCCN states that NGS may be used if a high suspicion of clonal process remains but other techniques have not clearly identified a clonal process. The NCCN states that an NGS panel including EXH2, TNFRSF14 and STAT6 may be useful “under certain circumstances” for Follicular Lymphoma. For pediatric-type follicular lymphoma in adults, consider NGS for TNFRSF14 and MAP2K1 mutations. NGS may also be useful for “treatment selection” (NCCN, 2023b). 

T-Cell Lymphomas: 
The NCCN also states that “genetic testing, including…NGS… to detect somatic mutations or genetic abnormalities are often informative and, in some cases, essential for an accurate and precise diagnostic and prognostic assessment of T-cell lymphomas." For hepatosplenic T-cell lymphoma, the NCCN finds NGS sequencing panels including STAT3, STA5B, PIK3CD, SETD2, INO80, TET3, and SMARCA2 to be useful for diagnosis under certain circumstances (NCCN, 2022h).

Non-Small Cell Lung Cancer (NSCLC):
The NCCN recommends that testing be performed in a “panel-based approach, most typically performed by next-generation sequencing (NGS)” when feasible. NCCN notes that multiple studies suggest that NGS testing with broad gene coverage may allow for unambiguous determination of clonal relatedness among separate lung modules. RNA-based NGS should be considered in patients without identifiable driver oncogene mutations, especially in “never smokers,” to maximize detection of fusion events. The NCCN mentions NGS as a commonly used method to detect sequence changes such as gene mutations in EGFR, BRAF, and KRAS, skipping events in METex14, insertion events in EGFRex20, and fusion events in ALK and ROS1 (though DNA based NGS may not derdetect ROS1 fusions). For fusion detection, RNA-based NGS is preferable to DNA-based NGS. They note that studies exploring tumor relatedness by testing tissue from separately sampled lesions using broad gene coverage with an NGS approach suggests it may be superior to histopathologic assessment. However, the NCCN notes that NGS may be considered in biomarker analysis but cautions that not all types of alterations will be detected, that any method which investigates sequences other than a subset of highly specific alterations can identify variants of unknown significance (should not be considered as a basis for targeted therapy selection, even if other variants within that same gene are clinically actionable), and to be aware of the nuances of NGS (NCCN, 2022e). 

Prostate Cancer: 
The NCCN recommends NGS cancer predisposition screening for BRCA1, BRCA2, ATM, PALB2, CHEK2, MLH1, MSH2, MSH6, and PMS2. If MSI testing is performed, testing using an NGS assay validated for prostate cancer is preferred (NCCN, 2023n). 

Small Bowel Adenocarcinoma:
Universal microsatellite instability (MSI) testing is recommended in all patients with a history of small bowel adenocarcinoma. NCCN recommends using validated NGS panels to test for MSI (NCCN, 2023p). 

Soft Tissue Sarcoma: 
The NGS is mentioned among the techniques used to identify genetic aberrations in soft tissue sarcoma. Next-generation sequencing may be appropriate for patients who qualify for and who are interested in enrolling in a clinical trial or for patients with refractory disease; additionally, NGS may be a technique for patients with histologies for which NGS could provide clinically actionable information. Patients who require bone biopsies may also benefit from NGS (NCCN, 2023q). 

Systemic Mastocytosis:
The NCCN recommends against NGS panels alone for detection of KIT D816V, citing their low sensitivity (approximately 5%). However, a myeloid mutation panel should be performed on bone marrow (although testing can be done on peripheral blood in the presence of an associated hematologic neoplasm and/or circulating mast cells). Prognostically relevant mutations include TET2, SRSF2, CBL, ASXL1, RUNX1, EZH2, JAK2, and RAS (NCCN, 2022g). 

Genetic/Familial High-Risk Assessment for Colorectal Cancer:
Next generation sequencing (NGS) allows for the sequencing of multiple genes simultaneously (multi-gene testing).The introduction of multi-gene testing for hereditary forms of cancer has rapidly altered the clinical approach to testing at-risk patients and their families. Multi-gene testing can simultaneously analyze sets of genes associated with a specific family cancer phenotype/phenotypes and may include syndrome-specific tests, cancer-specific tests, or comprehensive cancer panels. NCCN states that there are numerous scenarios in which multi-gene testing may be more effective. This includes greater efficiency in testing when more than one gene may explain presentation and family history, a higher chance of providing the patient with a possible explanation for their cause of cancer, competitive cost relative to sequentially testing single genes, and the chance of identifying pathogenic variants in multiple actionable genes that would me missed using cancer syndrome-specific panels, which could ultimately impact screening and management for the individual and their family members. 

The NCCN notes certain cons associated with panel testing, such as slower turn around, the possibility of missing some mutations that would be detected with traditional single-gene analysis, and identification of mutations for more than one gene, which adds complexity that could lead to difficulty in making risk management recommendations. There is also a higher chance of identifying variants of unknown significance, unactionable variants, or variants that do not have a clear course of treatment. The NCCN also identifies two examples of clinical scenarios in which multi-gene testing should not be considered: “1) an individual from a family with a known mutation and there is no other reason for multi-gene testing; 2) the patient’s family history is strongly suggestive of a known hereditary syndrome.”

The NCCN panel recommends NGS as one of three options for patients or families where a colorectal or endometrial tumor is available — specifically, they note that in this situation, a comprehensive tumor NGS panel can be considered for workup and should include, at minimum, the 4 MMR genes and EPCAM, BRAF, MSI, and other known familial cancer genes. 

Overall, the NCCN acknowledges the significant benefits of panel testing, but states that choice of panel and testing is critical.

As a final aside, the NCCN is in agreement with the 2015 ASCO recommendations (NCCN, 2022d). 

Genetic/Familial High-Risk Assessment for Breast, Ovarian, and Pancreatic Cancer:
In this guideline, the NCCN cites similar pros and cons to multi-gene testing as those covered in the previous guidelines for colorectal cancer. They also note that not all genes included on available multi-gene tests are necessarily clinically actionable and multi-gene panel testing increases the likelihood of finding pathogenic/likely pathogenic variants without clear clinical significance. The NCCN notes the following genes as pathogenic/likely pathogenic variants associated with breast/ovarian cancer: BRCA1/2, ATM, BARD1, BRIP1, CDH1, CHEK2, MSH2, MSH6, MLH1, PMS2, EPCAM, NBN, NF1, PALB2, RAD51C, RAD51D, STK11, and . Lower penetrance genes that may be included as part of a multi-gene panel for breast and/or ovarian cancer include FANCC, MRE11A, MUTYH heterozygotes, RECQL4, RAD50, RINT1, SLX4, SMARCA4, and XRCC2 (NCCN, 2023h). 

American Society of Clinical Oncology (ASCO) 
The ASCO released guidelines discussing tumor testing for epithelial ovarian cancer. In it, they recommend germline sequencing of BRCA1/2 “in the context of a multigene panel” that includes “at minimum” the following genes: BRCA1, BRCA2, RAD51C, RAD51D, BRIP1, MLH1, MSH2, MSH6, PMS2, and PALB2 (Konstantinopoulos et al., 2020).

The ASCO published guidelines regarding evaluating susceptibility to pancreatic cancer. In it, they recommend that germline genetic testing be performed using a multigene panel that includes the following genes: APC, ATM, BRCA1/2, CDKN2A, MLH1, MSH2, MSH6, PMS2, EPCAM, PALB2, STK11, TP53. An exception is if a genetic diagnosis has been previously confirmed in a family member; a panel should not be used in this case. Further, ASCO recommends that every patient diagnosed with pancreatic adenocarcinoma should undergo a risk assessment for hereditary syndromes associated with increased risk of pancreatic adenocarcinoma (Stoffel et al., 2018).

In 2020, ASCO published an update to recommendations for pancreatic cancer that reaffirmed the 2018 version and added more information on precision medicine. Genetic testing can lead to therapeutic decisions such as inhibitor therapy and other targeted therapies. The group recommends that early testing for “actionable genomic alterations is recommended for patients who are likely to potential candidates for additional treatment after first-line therapy. Both germline and tumor (somatic) testing are recommended.” In the list, they note MSI, MMR, BRCA mutations (excluding mutations of unknown significance) and NTRK gene fusions as important for testing (Sohal et al., 2020). (Sohal et al., 2020)

American College of Medical Genetics (ACMG)
The ACMG published guidelines on inclusion criteria for genes with “various gene-disease evidence levels.” For confirming a clinical diagnosis, the ACMG stated to include any gene associated (with a “moderate,” “strong,” or “definitive” association) with the disease, if the primary method of diagnosis was a “Disease-focused multigene panel or other non-sequencing-based ancillary assays.” Genes with no emerging evidence or without evidence at all were to be excluded. Genes with emerging evidence should “typically” be excluded, although the ACMG notes some inclusions that may be “meaningful.” The ACMG also states that genes with this level of evidence should be reported with a statement that disease association and inheritance has not been established. 

For panels intended to “Establish genetic diagnosis for clinically complex cases” and that are used for conditions primarily diagnosed through exome/genome sequencing, genes that have evidence levels of “definitive,” “strong,” and “moderate” should be included. Genes of unknown significance should be qualified with a statement that disease association and inheritance have not been completely established (Bean et al., 2019).

The ACMG recommends that the selection of genes and transcripts in any given panel be limited to genes with “sufficient scientific evidence for a causative role in the disease.” Genes without clear evidence of association with the disease should not be included.

The ACMG recommends validating diagnostic testing through another method such as Sanger sequencing.

The ACMG cannot recommend a minimum threshold for “coverage” as many factors of the platform and assay may influence minimum coverage. However, the ACMG recommends that each laboratory independently validate their panel tests (Rehm et al., 2013).

The ACMG released a statement regarding some points to consider for germline findings using NGS in patients undergoing tumor testing. ACMG states that NGS has some limitations that make it harder to identify some types of germline variants, such as genomic rearrangements, large insertions/deletions, or expansion/contraction of repetitive sequences. In addition, the assay and analytical performance varies between laboratories. Therefore, confirmation with an orthogonal method such as PCR, microarray, or multiplex ligation-dependent probe amplification (MLPA) is recommended (Li et al., 2020). 

The ACMG released a 2021 guideline on NGS for constitutional variants in the clinical laboratory, in which they note that diagnostic gene panels are optimal for well-defined clinical presentations that are genetically heterogeneous (have more than one pathogenic variant that can cause the diagnosed phenotype), when these pathogenic variants in the various disease-associated genes account for a significant fraction of cases. They address the fact that incidental findings should not be encountered, but also that broad panels may identify clinically significant findings unrelated to the initial test indication. Panels should be optimized by limiting the test to those genes relevant to a given disease. 

In clinically relevant genomic regions that cannot be assayed reliably by NGS, ancillary assays such as Sanger sequencing of regions with low coverage by NGS, CNV detection, methylation, and repeat expansion. “Disease-targeted gene panels that include these areas should include appropriate additional methodologies to maximize clinical sensitivity” (Rehder et al., 2021).

Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists 
The Joint Commission recommended that somatic variants be categorized by and reported based on their impact on clinical care. The Joint Commission notes that somatic variants include indels, SNVs, fusion genes from genomic rearrangements, and CNVs and should focus on their impact on clinical care. Any variant may be considered a biomarker if it predicts response to therapy, influences prognosis, diagnosis, treatment decisions, or the gene function itself. The Joint Commission proposes four levels for these biomarkers which are as follows: 

  1. "Level A, biomarkers that predict response or resistance to US FDA-approved therapies for a specific type of tumor or have been included in professional guidelines as therapeutic, diagnostic, and/or prognostic biomarkers for specific types of tumors.
  2. Level B, biomarkers that predict response or resistance to a therapy based on well-powered studies with consensus from experts in the field, or have diagnostic and/or prognostic significance of certain diseases based on well-powered studies with expert consensus.
  3. Level C, biomarkers that predict response or resistance to therapies approved by FDA or professional societies for a different tumor type (i.e., off-label use of a drug), serve as inclusion criteria for clinical trials, or have diagnostic and/or prognostic significance based on the results of multiple small studies.
  4. Level D, biomarkers that show plausible therapeutic significance based on preclinical studies, or may assist disease diagnosis and/or prognosis themselves or along with other biomarkers based on small studies or multiple case reports with no consensus.”

The Joint Commission also includes variants in different tiers based on the amount of evidence there is to support its significance. For example, tier 1 variants include significance of levels A and B and tier 2 includes significance of levels C and D. Tier 3 is variants of unknown significance (VUS), such as variants in cancer genes that haven’t been reported in any other cancers. These variants are not typically seen in significant frequencies in the general population. When evaluating these variants, the type of mutation and gene function should be considered. Tier 4 is benign variants or likely benign variants. These alleles are often observed in significant amounts in general populations. Tier 3 variants should be reported while ensuring that the most important information is communicated to the patient (Li et al., 2017).

European Society for Medical Oncology (ESMO) Precision Medicine Working Group
The ESMO released clinical practice guidelines on the use of NGS to diagnose tumors. Overall, ESMO suggests that NGS should be used routinely in patients with metastatic cancers including advanced lung adenocarcinoma, prostate cancer, ovarian cancer, and cholangiocarcinoma. For colon cancer, NGS can be an alternative option to PCR if it does not incur additional costs. Tumor mutational burden (TMB) should be tested in cervical cancer, salivary cancer, thyroid cancers, well-to-moderately differentiated neuroendocrine tumors, and vulvar cancer. Patients with other cancers may decide with their physician to order NGS on a large gene panel, if "pending no extra cost for the public health care system, and if the patient is informed about the low likelihood of benefit” (Mosele et al., 2020). ESMO states that more evidence is still needed to improve understanding on how to use NGS to treat patients based on precision biomarkers. 

Recommendations according to cancer type are summarized below. Recommendations were provided based on the ESCAT scale ranking that calculates the number of patients that would need to be tested with NGS to identify one patient who could be matched to an effective drug. Level I means that the match between drug and genomic alterations has been validated in clinical trials and should drive treatment decision in daily practice. Level II means that alteration has been associated with phase I/phase II trials. Level III means that genome alteration has been validated in another cancer, but not for that specific one. Level IV are hypothetically targetable alterations based on preclinical data (Mosele et al., 2020).

Cancer Type


Lung Adenocarcinoma

“Tumour multigene NGS to assess level I alterations. Larger panels can be used only on the basis of specific agreements with payers taking into account the overall cost of the strategy (drug included) and if they report accurate ranking of alterations. NGS can either be done on RNA or DNA, if it includes level I fusions in the panel.

Squamous cell lung cancer

No current indication for tumour multigene NGS

Breast cancer

No current indication for tumour multigene NGS

Colon cancer

Multigene tumour NGS can be an alternative option to PCR if it does not result in additional cost

Prostate cancer

Multigene tumour NGS to assess level I alterations. Larger panels can be used only on the basis of specific agreements with payers taking into account the overall cost of the strategy and if they report accurate ranking of alterations.

Gastric cancer

No current indication for tumour multigene NGS

Pancreatic cancer

No current indication for tumour multigene NGS

Hepatocellular carcinoma

No current indication for tumour multigene NGS


Multigene tumour NGS could be recommended to assess level I alterations. Larger panels can be used only on the basis of specific agreements with payers taking into account the overall cost of the strategy (drug included) and if they report accurate ranking of alterations. RNA-based NGS can be used.


Tumour multigene NGS can be used in ovarian cancers to determine somatic BRCA1/2 mutations. In this latter case, larger panels can be used only on the basis of specific agreements with payers taking into account the overall cost of the strategy (drug included) and if they report accurate ranking of alterations. Large panel NGS can be used in carcinoma of unknown primary.

It is recommended to determine TMB in cervical cancer, salivary cancer, thyroid cancers, well-to-moderately differentiated neuroendocrine tumours, vulvar cancer, pending drug access (and in TMB-high endometrial and SCL cancers if anti-PD1 antibody is not available otherwise)” (Mosele et al., 2020).


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  25. NCCN. (2022e, December 7, 2021). Non-Small Cell Lung Cancer.
  26. NCCN. (2022f, January 18, 2022). Ovarian Cancer Including Fallopian Tube Cancer and Primary Peritoneal Cancer.
  27. NCCN. (2022g, October 18, 2022). Systemic Mastocytosis.
  28. NCCN. (2022h, December 22, 2021). T-Cell Lymphomas.
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  33. NCCN. (2023e). Colon Cancer.
  34. NCCN. (2023f, December 5, 2022). Esophageal and Esophagogastric Junction Cancers.
  35. NCCN. (2023g). Gastrointestinal Stromal Tumors (GISTS).
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  37. NCCN. (2023i). Multiple Myeloma.
  38. NCCN. (2023j, September 12, 2022). Myelodysplastic Syndromes.
  39. NCCN. (2023k, October 18,2022). Myeloid/Lymphoid Neoplasms with Eosinophilia and Tyrosine Kinase Fusion Genes.
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Coding Section






Hereditary breast cancer-related disorders (e.g., hereditary breast cancer, hereditary ovarian cancer, hereditary endometrial cancer); genomic sequence analysis panel, must include sequencing of at least 10 genes, always including BRCA1, BRCA2, CDH1, MLH1, MSH2, MSH6, PALB2, PTEN, STK11, and TP53



Hereditary breast cancer-related disorders (e.g., hereditary breast cancer, hereditary ovarian cancer, hereditary endometrial cancer); duplication/deletion analysis panel, must include analyses for BRCA1, BRCA2, MLH1, MSH2, and STK11



Hereditary retinal disorders (e.g., retinitis pigmentosa, Leber congenital amaurosis, cone-rod dystrophy), genomic sequence analysis panel, must include sequencing of at least 15 genes, including ABCA4, CNGA1, CRB1, EYS, PDE6A, PDE6B, PRPF31, PRPH2, RDH12, RHO, RP1, RP2, RPE65, RPGR, and USH2A



Hereditary colon cancer disorders (e.g., Lynch syndrome, PTEN hamartoma syndrome, Cowden syndrome, familial adenomatosis polyposis); genomic sequence analysis panel, must include sequencing of at least 10 genes, including APC, BMPR1A, CDH1, MLH1, MSH2, MSH6, MUTYH, PTEN, SMAD4, and STK11



Hereditary colon cancer disorders (e.g., Lynch syndrome, PTEN hamartoma syndrome, Cowden syndrome, familial adenomatosis polyposis); duplication/deletion analysis panel, must include analysis of at least 5 genes, including MLH1, MSH2, EPCAM, SMAD4, and STK11



Hereditary neuroendocrine tumor disorders (e.g., medullary thyroid carcinoma, parathyroid carcinoma, malignant pheochromocytoma or paraganglioma); genomic sequence analysis panel, must include sequencing of at least 6 genes, including MAX, SDHB, SDHC, SDHD, TMEM127, and VHL



Hereditary neuroendocrine tumor disorders (e.g., medullary thyroid carcinoma, parathyroid carcinoma, malignant pheochromocytoma or paraganglioma); duplication/deletion analysis panel, must include analyses for SDHB, SDHC, SDHD, and VHL



Noonan spectrum disorders (e.g., Noonan syndrome, cardio-facio-cutaneous syndrome, Costello syndrome, LEOPARD syndrome, Noonan-like syndrome), genomic sequence analysis panel, must include sequencing of at least 12 genes, including BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, RIT1, SHOC2, and SOS1



Targeted genomic sequence analysis panel, solid organ or hematolymphoid neoplasm, DNA analysis, and RNA analysis when performed, 51 or greater genes (e.g., ALK, BRAF, CDKN2A, CEBPA, DNMT3A, EGFR, ERBB2, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MLL, NPM1, NRAS, MET, NOTCH1, PDGFRA, PDGFRB, PGR, PIK3CA, PTEN, RET), interrogation for sequence variants and copy number variants or rearrangements, if performed



Targeted genomic sequence analysis panel, non-small cell lung neoplasia, DNA and RNA analysis, 23 genes, interrogation for sequence variants and rearrangements, reported as presence/absence of variants and associated therapy(ies) to consider
Proprietary test: Oncomine™ Dx Target Test
Lab/Manufacturer: Thermo Fisher Scientific



Hereditary colon cancer disorders (e.g., Lynch syndrome, PTEN hamartoma syndrome, Cowden syndrome, familial adenomatosis polyposis), genomic sequence analysis panel utilizing a combination of NGS, Sanger, MLPA, and array CGH, with MRNA analytics to resolve variants of unknown significance when indicated (15 genes [sequencing and deletion/duplication], EPCAM and GREM1 [deletion/duplication only])
Proprietary test: ColoNext®
Lab/Manufacturer: Ambry Genetics®



Hereditary breast cancer-related disorders (e.g., hereditary breast cancer, hereditary ovarian cancer, hereditary endometrial cancer), genomic sequence analysis panel utilizing a combination of NGS, Sanger, MLPA, and array CGH, with MRNA analytics to resolve variants of unknown significance when indicated (17 genes [sequencing and deletion/duplication])
Proprietary test: BreastNext®
Lab/Manufacturer: Ambry Genetics®



Hereditary ovarian cancer (e.g., hereditary ovarian cancer, hereditary endometrial cancer), genomic sequence analysis panel utilizing a combination of NGS, Sanger, MLPA, and array CGH, with MRNA analytics to resolve variants of unknown significance when indicated (24 genes [sequencing and deletion/duplication], EPCAM [deletion/duplication only])
Proprietary test: OvaNext®
Lab/Manufacturer: Ambry Genetics®



Hereditary breast cancer-related disorders (e.g., hereditary breast cancer, hereditary ovarian cancer, hereditary endometrial cancer), genomic sequence analysis and deletion/duplication analysis panel (ATM, BRCA1, BRCA2, CDH1, CHEK2, PALB2, PTEN, and TP53)
Proprietary test: BRCAplus
Lab/Manufacturer: Ambry Genetics

   0405U (effective 10/01/2023) Oncology (pancreatic), 59 methylation haplotype block markers, next-generation sequencing, plasma, reported as cancer signal detected or not detected


Medical genetics and genetic counseling services, each 30 minutes face-to-face with patient/family 



Genetic counseling, under physician supervision, each 15 minutes

Procedure and diagnosis codes on Medical Policy documents are included only as a general reference tool for each policy. They may not be all-inclusive. 

This medical policy was developed through consideration of peer-reviewed medical literature generally recognized by the relevant medical community, U.S. FDA approval status, nationally accepted standards of medical practice and accepted standards of medical practice in this community, Blue Cross Blue Shield Association technology assessment program (TEC) and other nonaffiliated technology evaluation centers, reference to federal regulations, other plan medical policies and accredited national guidelines.

"Current Procedural Terminology © American Medical Association. All Rights Reserved" 

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01012024  NEW POLICY

04/30/20024 Moving review date to 7/2024. Review will coincide with Avalon's review date.

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