MolDX: Non-Next Generation Sequencing Targeted Molecular Panel Tests for Predictive Testing in Cancer

Title XVIII of the Social Security Act (SSA), §1862(a)(1)(A), states that no Medicare payment shall be made for items or services that are not reasonable and necessary for the diagnosis or treatment of illness or injury or to improve the functioning of a malformed body member.

42 CFR §410.32(a) Diagnostic x-ray tests, diagnostic laboratory tests, and other diagnostic tests: Conditions

CMS Internet-Only Manual, Pub. 100-02, Medicare Benefit Policy Manual, Chapter 15, §80 Requirements for Diagnostic X-Ray, Diagnostic Laboratory, and Other Diagnostic Tests, §80.1.1 Certification Changes

This policy describes and clarifies coverage for molecular or proteomic panel tests for Non-Next Generation Sequencing Targeted Molecular Panel Tests for Predictive Testing in Cancer. The determination outlines the medical necessity, indications, limitations, and required documentation for biomarker testing to ensure appropriate patient selection and effective utilization.

Criteria for Coverage

Non-Next Generation Sequencing Targeted Molecular Panel Tests for Predictive Testing in Cancer are covered when ALL of the following are met:

1. The patient has a diagnosis of cancer for which molecular testing of the tumor will inform treatment.
2. At least one of the following is true:
1. Testing by next-generation sequencing (NGS) is not feasible or will likely fail based on specimen type or tumor content.
2. NGS is not required by national consensus guidelines to inform medical management decisions AND testing by a non-NGS method will provide a rapid result that allows for the prompt and timely management of the patient.
3. The test accurately identifies known, common, and necessary predictive and actionable biomarkers that can safely preclude unnecessary NGS testing, reducing NGS use in patients for whom the treatment plan would not likely be altered.
3. The patient has not been previously tested by a molecular panel test for the same cancer indication for the same genetic content. NOTE: A negative result (no clinically actionable mutations found) by the non-NGS test may be followed by an NGS test that includes additional necessary non-duplicative genes and genomic positions.
4. The test accurately detects the most common genes and genomic positions required for the identification of clinically relevant FDA-approved therapies with a companion diagnostic biomarker for the given cancer type.
5. The test has been validated in the intended-use population and with the intended-use sample types.
6. The test demonstrates accuracy for measured analytes comparable to NGS testing, with superior turnaround times less than the ASCO-recommended 10 business days from sample acquisition.
7. The test has satisfactorily completed a Technical Assessment (TA) by the Molecular Diagnostic Services Program (MolDX^®) to ensure analytical validity (AV), clinical validity (CV) and clinical utility (CU) standards are met.
8. Clinical validity (CV) of any analytes or profiles must be established through a study published in the peer-reviewed literature for the intended use of the test in the intended population with demonstrated reproducibility across clinical study cohorts. If the test relies on an algorithm, the algorithm must be validated in a cohort that is not a development cohort for the algorithm.
9. Tests utilizing a similar methodology or evaluating a similar analyte(s) to a test for which existing coverage has been established must demonstrate equivalent or superior test performance (i.e., sensitivity and/or specificity) when used for the same indication in the same intended use population.

Notes:

• Reference to specific tests in this LCD does not automatically imply coverage.
• NGS-based panel tests must fulfill criteria outlined in LCDs L38119 and L38121, MolDX: Next-Generation Sequencing for Solid Tumors and L38123 and L38125, MolDX: Next-Generation Sequencing Lab-Developed Tests for Myeloid Malignancies and Suspected Myeloid Malignancies.
• Non-NGS-based tests for the diagnosis of BCR-ABL-negative myeloproliferative neoplasms must fulfill criteria outlined in LCD L39923 and L39927, MolDX: Non-Next Generation Sequencing Tests for the Diagnosis of BCR-ABL Negative Myeloproliferative Neoplasms.

The Importance of Molecularly - Informed Targeted Therapies in Cancer

Molecular testing of tumors has become a standard of care for many cancer types, and an increasing number of molecularly targeted therapies (therapies that exploit discrete targets in the genome) have become available in recent years. For example, approximately 70% of tumors from patients with metastatic lung adenocarcinoma have an actionable driver mutation^1-4 and at least one FDA-approved therapy currently exists that targets each of seven non-small cell lung cancer (NSCLC) oncogenic drivers: Epidermal Growth Factor Receptor (EGFR), anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 (ROS1), B-Raf proto-oncogene (BRAF), rearranged during transfection (RET), mesenchymal-epithelial transition exon 14 (METex14), and neurotrophic tyrosine receptor kinase (NTRK).^5,6

Molecularly targeted therapies have resulted in improved patient management and outcomes for various cancer types.^7-10 For patients with advanced non-squamous NSCLC, a number of studies have found that the availability of genomic results prior to the initiation of treatment is associated with a statistically significant improvement in overall survival (OS).^11-16 Studies have also shown improved time to therapy discontinuation, the avoidance of ineffective therapies (such as immune checkpoint inhibitors (ICPIs) among ALK/EGFR/RET/ROS1-positive patients), and improved progression free survival (PFS) when first-line therapy is initiated on the basis of molecular findings.^14-18 Importantly, when actionable oncogenic driver (AOD) status is unknown and driver-positive patients first receive chemo-ICPIs, patients face the risk of severe immune-related events on subsequent targeted therapy.^18,19 A multisite retrospective observational analysis found that advanced NSCLC patients harboring AODs who were empirically treated with non-tyrosine kinase inhibitor (non-TKI) therapy while awaiting genomic test results had significantly inferior outcomes than those initially treated on the basis of genomic results.¹⁶ Notably, patients who were not treated until molecular results were available fared better despite having begun treatment approximately 25 days later (range: 6-55 days for NGS-tissue test result availability).¹⁶ A large retrospective analysis of a nationwide electronic health record–derived database of patients with advanced NSCLC and an AOD found that those who switched to a targeted therapy within 42-84 days after a biomarker test result had similar outcomes to those who received targeted treatment as a first-line therapy.⁶ Notably, patients with AODs who never received targeted treatment had worse OS and PFS than all cohorts.⁶

In patients with advanced colorectal cancer (CRC), first-line treatment includes the use of targeted therapies informed by AODs, some of the most common being in the BRAF, KRAS, and EGFR genes.^20,21 Importantly, the integration of such targeted agents into combination regimens has improved treatment efficacy and survival outcomes.^20,22 In a study of 3,216 patients with an array of advanced cancers including NSCLC, CRC, and breast cancer, patients who received molecularly-informed targeted therapies had better survival outcomes compared with chemotherapy only (hazard ratio [HR], 0.66, [95% CI, 0.52 to 0.84], P<.001), even after controlling for age and tumor type.¹⁰

Despite the benefits of biomarker-driven targeted therapy and immunotherapy in NSCLC and other cancers, biomarker testing is often underutilized, not comprehensive, or not acted upon.^8,12,23,24 Reasons for this include knowledge gaps surrounding updates in required biomarker testing and available treatments, impatience with the long turnaround time (TAT) of NGS tests prompting “fast-track” approaches to testing, and the procurement of adequate tissue for testing.^23-25 In a retrospective observational chart review study of community-based oncology practices that investigated patients with metastatic NSCLC between April 2018 and March 2020, less than 50% of patients received all five guideline-recommended biomarker tests relevant at the time (EGFR, ALK, ROS1, BRAF, and PD-L1); additionally, only 35% of all patients had testing for all 5 biomarkers prior to first-line treatment initiation.²⁶ A literature review of publications evaluating the clinical impact of NGS in multiple tumor types found that, on average, only 29% of patients who received NGS testing were matched to targeted treatment; differences in OS and PFS were statistically significantly longer only for those patients who had NGS testing and who also received matched therapies.⁸ In a study of patients with NSCLC within the Veterans Affairs National Precision Oncology Program, more than 30% of patients with actionable gene variants found by NGS testing received chemotherapy instead of targeted treatments.²⁷ Another report using a multisource database of commercial and Medicare claims from over 500,000 patients with NSCLC found that only approximately 36% of patients with NSCLC are benefiting from targeted therapies.²⁴ For every 1,000 patients in this study cohort, nearly 50% were lost to precision therapy because of factors associated with getting biomarker test results (including a lack of physician orders for the testing); among those who did receive biomarker test results, 29.2% did not receive appropriate targeted treatments.²⁴

An American Society of Clinical Oncology (ASCO) task force survey of physicians reported that TAT exceeding 2 weeks often resulted in empiric non-targeted therapies for NSCLC.⁵ Nearly half of providers responded that they would be less likely to defer empiric treatment if biomarker results would not be available within 2 weeks, though this was primarily driven by generalists who waited more than 3 weeks for genomic test results. Interestingly, thoracic oncologists were more likely to defer treatment until molecular results were available, even if this meant waiting for more than 3 weeks.⁵ Ninety-three percent of respondents to this survey also reported utilizing multi-gene panels more frequently than single gene assays and most tested for biomarkers associated with earlier (pre-2018) FDA-approved targeted therapies (targeting EGFR, ALK, ROS1 and BRAF) for non-squamous NSCLC; testing for more recently FDA-approved targeted therapies (targeting MET, RET, and NTRK) was less common among generalists and those practicing in the community settings compared with thoracic oncologists and those at academic institutions.⁵

The Utility of NGS vs. Non-NGS Multigene Panel Testing in Tumor Profiling

Clinical guidelines recommend broad-based tumor profiling for a variety of cancers because numerous AODs are often associated with the same tumor type. For patients with metastatic or advanced cancer, ASCO strongly recommends that “multigene panel–based assays should be used if more than one biomarker-linked therapy is approved for the patient's disease.”²⁸ Genomic testing should also be considered to determine candidacy for tumor-agnostic therapies and when considering a treatment for which there are specific “genomic biomarker–based contraindications or exclusions.”²⁸ The National Comprehensive Cancer Network (NCCN) and ASCO guidelines recommend broad-based molecular profiling and biomarker assessment prior to therapy initiation for all patients with non-squamous NSCLC, when clinically feasible.^13,29 For NSCLC, NCCN currently defines broad molecular profiling as “molecular testing that identifies all of the classic actionable biomarkers identified in the algorithm [i.e., ALK, BRAF, EGFR, ERBB2 (HER2), KRAS, METex14skipping, NTRK1/2/3, RET, ROS1] in either a single assay or a combination of a limited number of assays, and optimally also identifies emerging biomarkers (e.g., high-level MET amplifications and FGFR alterations).”¹³

The most comprehensive approach to broad-based testing is NGS. With its expansive capability to interrogate multiple different types of genomic alterations (including single nucleotide variants (SNVs), insertions and deletions (indels), copy number alterations (CNAs) and structural rearrangements), it can provide information regarding essentially all approved targeted therapies. However, there are challenges with NGS-based approaches, including complex and time-consuming laboratory workflows and bioinformatics analytics not immediately accessible to most community laboratories; thus, obtaining results from NGS specimens sent to external reference laboratories often takes weeks after procurement of the specimen.³⁰ This is despite the recommendation by the College of American Pathologists, International Association for the Study of Lung Cancer, and the Association for Molecular Pathology (CAP/IASLC/AMP) guideline and the American Cancer Society National Lung Cancer Roundtable’s (ACS NLCRT) strategic plan which stipulate that genomic results be available within 10 business days after sample receipt in the laboratory.^23,30,31

Additionally, the ACS NLCRT strategic plan explicitly recommends procurement of adequate tissue for comprehensive biomarker testing.^23,30 Testing by NGS requires a higher tissue input, which is often problematic, particularly when tissue is obtained from small samples such as fine needle aspirates (FNAs) or small core needle biopsies (CNBs).^24,32 Furthermore, the CAP/IASLC/AMP guideline states that “Laboratories should use, or have available at an external reference laboratory, clinical lung cancer biomarker molecular testing assays that are able to detect molecular alterations in specimens with as little as 20% cancer cells.”³¹ However, 20-30% of tissue specimens from patients with NSCLC have been reported as inadequate and tumor specimens with less than 20% tumor purity are often considered to be quantity-not-sufficient (QNS) for testing by NGS.²⁵ Moreover, in approximately 10% of CRC samples, the DNA quality extracted from formalin-fixed paraffin embedded (FFPE) samples does not reach quality criteria for amplicon based sequencing.³³

Other testing approaches are available and can provide results more rapidly or when there is not sufficient tissue available to enable testing by NGS. Such approaches may involve multi-gene panels or a single-gene reflexive approach (a tiered approach, based on the prevalence and relative exclusivity of the AODs). For example, in NSCLC, mutations in KRAS account for up to 30% of gene alterations and are associated with a history of smoking whereas those in EGFR occur in approximately 19% of cases and are associated with never-smokers; prevalence of the other known AODs is significantly lower, though they collectively comprise 10-15% of cases.^34,35 Moreover, the most common driver alterations in NSCLC tend to occur exclusively from one another in up to 97% of cases.^13,36-39 For these reasons, NCCN guidelines for NSCLC consider tiered testing approaches, “based on the low prevalence of co-occurring biomarkers” to be acceptable.¹³

Various reflexive testing protocols have been successfully implemented by a number of institutions, resulting in decreased TATs for results, higher detection rates of targeted gene alterations, and improved time to optimal systemic therapy compared to pre-intervention routine ordering practices.^40-42 However, as noted by ASCO, and as reported by the ACS NLCRT strategic plan, “with the increasing number of biomarkers needed to guide therapeutic decisions, sequential testing strategies for single gene abnormalities now run the risk of depleting available tissue and increasing the TAT from initial test requisition to availability of actionable results.”³⁰ A study evaluating tissue stewardship in NSCLC found that the large majority of lung tissue samples submitted for clinical testing were small (70.5% CNBs; 10.0% FNAs) and more than one fifth of CNB samples had <25% tumor content.⁴³ The success of reporting molecular test results decreased as the number of biomarkers increased when multiple single-gene tests were ordered on a given sample.⁴³

Further, the benefits derived from targeted therapies can be realized regardless of test methodology, as long as the tests performed provide accurate results for the most common relevant AODs in a similar timeframe.⁴⁴ In a retrospective multicenter study of 5,688 patients with advanced NSCLC who received care in the community oncology setting, broad-based genomic sequencing (BBGS) directly informed treatment in only a minority of patients (4.5%) and was not independently associated with better survival, compared to “routine” genomic testing (which consisted of testing only for the most common EGFR mutations +/- ALK rearrangements).⁴⁵ Additionally, receipt of targeted treatment and chemotherapy in the first-line setting was similar between routine-tested and BBGS groups. Only in the patients without documented EGFR mutations or ALK rearrangements was the percentage that received targeted treatment as first-line therapy higher in the BBGS group (8.3%) than in the routine-tested (4.7%) group (P < .001).⁴⁵ Notably, during the time period of this study (2011-2016), the only targeted therapies approved for NSCLC were those directed against EGFR and ALK, which may explain the limited utility of multigene testing that was observed at the time; many more targeted therapies have subsequently been approved. Further, a high concordance (98–99.1%) was observed between the EGFR and ALK tests performed in patients who received both BBGS and single-gene testing.⁴⁵ Similarly, a retrospective cohort study on patients diagnosed with advanced NSCLC between 2017 and 2018 using a Korean population-based database also found that NGS for all-comers did not increase survival outcomes.⁴⁶ NGS associated with better survival outcomes only in the subgroup for whom EGFR or ALK inhibitors were not indicated (14.1 versus 9.0 months, log-rank P = 0.006; 95% CI 0.69-0.97).⁴⁶ Notably, EGFR mutations are expected to be observed in a higher proportion (∼40%-60%) of lung adenocarcinoma patients of Asian descent.⁴⁶

More recently, a retrospective comparative cohort study from the University of Pennsylvania found that improved OS was observed irrespective of test comprehensiveness and methods used to identify AODs when molecular test results were available before first-line therapy.¹⁴ This study evaluated patients diagnosed between 2019 and 2020 when “comprehensive” testing was defined as testing for EGFR, ALK, BRAF, ROS1, MET, RET, and NTRK; “incomplete” testing was defined as testing for ≤6 of these genes; additionally, some of the participating labs performed testing for AODs using non-NGS methodologies, including fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR).¹⁴ Other studies that reported improved outcomes among patients who received targeted treatment as first-line therapy also did not evaluate comprehensive testing approaches; in fact, some only focused on testing for EGFR or ALK gene alterations.^11,17 Taken together, these findings reinforce the importance of testing for the most common AODs prior to the administration of first-line therapy and affirm that multi-gene testing approaches other than NGS can be implemented successfully to optimize patient outcomes.

Importantly, therapy with tyrosine kinase inhibitors can induce rapid and profound clinical improvement and delays in appropriate treatment have been associated with inferior outcomes.⁴⁷ Multiple reports have described the rapid improvement (within days) of local and systemic symptoms after beginning targeted therapy with a TKI in patients with NSCLC.^48-52 A delay of >3-weeks in the initiation of ALK-inhibitor therapy has been associated with a >2-fold higher risk of death (adjusted hazard ratio [HR] 2.05, 95 % CI [1.13, 3.71]) in NSCLC patients harboring a rearrangement in the ALK gene.⁵³ Delayed treatment initiation for a variety of solid tumors has also been associated with absolute increased risk of mortality ranging from 1.2–3.2% per week in curative settings such as early-stage breast, lung, renal and pancreas cancers.⁵⁴ Therefore, rapid testing and treatment of these alterations, particularly in symptomatic patients and in those with an extensive disease burden, may expedite clinical improvement and improve overall survival.^47,52-54

Non-NGS Targeted Molecular Panel Tests

Though generally simpler to perform, traditional molecular tests have been limited in the types of genomic alterations they can detect. For example, traditional PCR and Sanger sequencing approaches are unable to evaluate tumor mutation burden (TMB) as well as many structural genomic alterations. However, different tumor types have different requirements based on the actionability of relevant AODs. While TMB is not considered an actionable biomarker in NSCLC, it is important in other tumor types. NSCLC tumors harbor fusions in the ALK, RET or ROS genes that are also unlikely to be detected by traditional PCR assays, requiring testing by other modalities (i.e. FISH) if NGS is not performed.^13,55 There is therefore an unmet need for tests that can detect druggable genomic alterations, such as fusions, by methods that are rapid and include multigene coverage.

Newer methods, including more advanced PCR-based technologies, have shown the ability to detect the most common AODs beyond the capabilities of traditional PCR. One of these is a high-definition multiplex dPCR panel (HDPCR) (ChromaCode, Carlsbad, CA, USA) that uses amplitude modulation and multi-spectral encoding to increase accuracy and specificity above that of traditional PCR methods.⁵⁶ This HDPCR detects NCCN-informed and clinically actionable results for NSCLC DNA variants and RNA fusions across nine genes in a matter of hours and can be performed using a low mass input, including samples that would be considered as quantity not sufficient (QNS) for NGS.⁵⁶ In an analytical validation and concordance study authored by the manufacturer, comparison of HDPCR results against NGS using 77 remnant FFPE samples from NSCLC patients demonstrated that the HDPCR achieved variant-specific limits of detection (LOD) of 0.8-4.9% (40ng DNA input) and 2.4-10.9% (15ng DNA input) mutant allele fractions (MAF); with a 5 ng RNA input, the test achieved a LOD of 23-101 positive partition counts for RNA targets.⁵⁶ Overall accuracy was greater than 99% after resolution of discordant results, with a target-specific positive percent agreement (PPA) and negative percent agreement (NPA) of 71.4% (ALK) - 100.0% and 98.9%-100.0%, respectively; of the six discordant results that aligned with NGS, 50.0% were novel fusions outside the inclusivity of the HDPCR NSCLC Panel and the panel missed 3 ALK fusions, one of which was a novel isoform.⁵⁶ Based on known prevalence of the genetic alterations and the performance of the test, the panel was predicted to miss 2 calls per 1000 NSCLC samples (primarily due to the presence of rare variants). In a separate multi-center proof-of-concept study, the assay reported an overall 100% PPA and a 98.5% NPA compared with a sequencing-based assay in a cohort of 62 patient FFPE samples; however, there were no ALK positive human specimens and, of the contrived samples, there were 3 false positive ALK fusions.⁵⁷ The HDPCR also rescued actionable information in 10 samples that failed to sequence.⁵⁷ Additional studies have also reported on the performance of this test and its ability of this method to rescue samples of low quality and quantity that would not be evaluable by NGS.^58,59 Notably, in a study using stored aliquots of nucleic acid post-extraction, in addition to false negative results, the assay produced a number of false positive results in EGFR L858R, as well as ALK, ROS1, and RET fusions that were mitigated (converted to true negative results) after assay optimization with updated thresholds.⁵⁸ The optimized assay has been further evaluated by the University of Louisville using clinical specimens, where the assay’s concordance with NGS was found to be 100%; however, this was based on only 5 positive (including for EGFR L858R and ROS1) and 53 negative specimens.⁶⁰ Further, the assay’s failure rate in that study was 19.4%, thought to be due to specimen integrity issues related to the archived samples tested.⁶⁰

ASPYRE (Allele-Specific Pyrophosphorolysis REaction) Lung (Biofidelity, Cambridge, UK), is a multi-step enzymatic amplification method that enables the concurrent detection of 114 genetic alterations (77 detected by DNA and 37 by RNA, in a single workflow) across 11 genes from paired DNA (20 ng) and RNA (6 ng) derived from FFPE lung tissue.^61-63 The LOD is ≤3% for SNVs and indels, 100 copies for fusions, and 200 copies for MET exon 14 skipping. The test can be performed on samples with >10% tumor purity and has a TAT from sample extraction to analysis of approximately two days. A concordance study with NGS authored by the manufacturer reported an analytical accuracy of grouped (contrived and clinical) samples at 100% and specificity at 100%.⁶¹ To date, the publications reporting on the performance of this test have been authored solely by the manufacturer.

On the other hand, there is significant literature inclusive of external validations, reported for the various Idylla assays (Biocartis, Mechelen, Belgium). One of these assays is the Idylla EGFR Mutation Assay, which qualitatively detects 51 different EGFR oncogene mutations in FFPE NSCLC tissue samples, including exon 18 (G719A/C/S), exon 19 (deletions), exon 20 mutations (T790M, S768I, and insertions), and exon 21 (L858R and L861Q).⁶⁴ A multicenter, multinational evaluation of 449 archived FFPE samples from NSCLC biopsies and resection specimens compared the performance of Idylla EGFR against routine reference methods (including NGS, Sanger sequencing, pyrosequencing, mass spectrometry, and PCR-based assays), and observed an overall concordance of 97.59% (95% CI, 95.63%–98.69%) for on-target variants; NPA was 96.26% (95% CI, 92.80%–98.09%) and a PPA was 99.01% (95% CI, 96.46%–99.73%).⁶⁴ Notably, a result was obtained in 98.9% of the cases, including those with <10 mm² of tissue area.⁶⁴ In a retrospective analysis of 119 patients with NSCLC following a diagnostic algorithm that included immunohistochemistry (IHC) for PD-L1/ALK/ ROS1, Idylla real-time PCR for EGFR/KRAS/BRAF mutations, and NGS, there were no discordant results observed between the Idylla platform and NGS.⁶⁵ Of the ten patients who received targeted treatment matching the genomic alterations detected, 5 had EGFR mutations detected by Idylla, while 2 had ALK-rearranged tumors and 3 had MET and NTRK alterations identified by NGS.⁶⁵ Notably, the study did not include the Idylla Gene Fusion Assay which also identifies MET and NTRK alterations. Additional studies evaluating Idylla EGFR against a reference NGS test have reported overall sensitivities, specificities, and limits of detection of 97.5%-98.5%, 96.3%-100%, and 2%-5% variant allele frequency (VAF), respectively, with false negatives attributed to low tumor content in the sample and mutations in regions outside the detection range of the test.^66-70

However, sensitivities and specificities have varied across individual variants. In a single-center feasibility study for facilitating rapid testing using the Idylla EGFR Assay as a screening method prior to NGS, Memorial Sloan Kettering Cancer Center (MSKCC) found a 98.6% concordance between the methods in 1249 samples.⁶⁹ Comprehensive NGS detected several targetable mutations not included in the Idylla design, including mutations in exon 20 insertions and targetable (albeit rare) EGFR exon 19 insertions (known to be highly variable in size/sequence/location, and therefore difficult to capture with a PCR)and the resistance mutations, C797S and G724S.^69,71 Testing success rates for small biopsies and cytology samples were 94.29% and 93.71%, respectively, higher than those prior to transitioning to the reflexive testing approach.⁶⁹ Sensitivity of the Idylla EGFR for the resistance-associated EGFR T790M mutation is also reportedly low and unreliable, ranging between 65.5%-74%.^68,69,72 Idylla EGFR has also shown poor specificity for the rare but clinically important exon 20 S768I variant, with an 80% false positive rate.⁷³ Studies have highlighted that the reliability and validity of results depend heavily on sample quality and preparation, with multiple preanalytical factors affecting performance, including suboptimal sample collection, handling procedures, variations in tissue fixation methods, and the use of stained tissues.^69,73 Overall, the test has performed well for on-target EGFR variants other than T790M and the rare exon 20 S768I, and has demonstrated its utility as part of a reflexive testing strategy.⁶⁹

Importantly, testing for the range of genes required in NSCLC and other cancers requires the use of more than one Idylla assay (e.g. Idylla EGFR, BRAF, KRAS and Gene Fusion Assays) and, even with the use of all these, may still not detect all of the actionable genes required for testing.^13,21 However, testing that includes both assays is expected to detect the most common relevant gene alterations in in a matter of hours. As previously noted, Idylla EGFR covers the most common EGFR mutations in the hotspots of exons 18-21; meanwhile, Idylla Gene Fusion detects fusions in ALK, ROS1, RET and NTRK and skipping of MET exon 14 through fusion-specific detection and expression imbalance analysis.^74,75 In two separate multicenter European studies using archival NSCLC tissue specimens, the Idylla Genefusion Panel was compared with earlier results of routine reference technologies including FISH, IHC, RT-PCR and NGS, and demonstrated the following sensitivities/specificities: 87% and 96.1%/98% and 99.6% for ALK, 82% and 96.7%/99.0% and 99% for ROS1, 94% and 100%/100% and 99.3% for RET fusion, and 84% and 92.5%/100% and 99.6% for MET exon 14 skipping.^76,77 In a study by MSKCC that evaluated fusions across multiple solid tumors, the test demonstrated a sensitivity of 97% (28/29), 100% (31/31), 92% (22/24), 81% (22/27), and 100% (20/20) for ALK, RET, ROS1, and NTRK1/2/3 rearrangements and MET exon 14 skipping alterations, respectively, with 100% specificity for all. Concordant results were achieved in specimens archived up to 5 years, with >10% tumor, and inputs of at least 9 mm2 (surgical specimens) or 9000 cells (cytologic cell blocks).⁵² In another concordance study against NGS published by the Mayo Clinic, the test demonstrated 100% sensitivity in detecting fusions of ALK, ROS1, RET, NTRK1, and MET exon 14 skipping and 83% sensitivity for NTRK2/3 fusions; specificity was 100% for detecting fusions of ROS1, RET, NTRK2/3, and MET exon 14 skipping and 98% for ALK.⁷⁵ Testing was successful with biopsy and surgical FFPE tissues, cell blocks from FNAs and pleural fluids (down to 5% tumor content, 18 mm² tissue), cytology smears (≥300 cells), and previously extracted RNA (≥20 ng).⁷⁵

The Idylla platform includes assays applicable to additional cancer indications. For example, the Idylla KRAS Mutation Test covers 21 KRAS mutations in exons 2-4, and the Idylla NRAS-BRAF Mutation Test covers 18 mutations in NRAS exons 2-4 and 5 mutations in BRAF codon 600.⁷⁴ Therefore, performance of these tests has been evaluated for cancers other than NSCLC. For example, for patients with CRC, NCCN guidelines recommend molecular testing for RAS (KRAS and NRAS) and BRAF mutations, individually or as part of a broad-based panel.²¹ Importantly, the role of activating KRAS mutations as intrinsic resistance markers for therapy with anti–EGFR-targeted antibodies (cetuximab or panitumumab) in patients with CRC is well established.^78,79 Testing for these genes also aligns with the ASCO recommendation that genomic testing should be performed in patients with advanced solid tumors when there are defined resistance markers for a treatment under consideration.²⁸

In a retrospective study of 44 archived FFPE CRC tissue samples, Idylla detected all mutations previously identified by NGS in KRAS (G12C, G12D, G12V, G13D, Q61K, Q61R, A146T), NRAS (G12V, G13R, Q61H), and BRAF (V600E), with a TAT of 2 hours.⁸⁰ Similar to testing in NSCLC, the tests performed for CRC required the use of multiple assays (one for KRAS and another for NRAS-BRAF-EGFR S492R), as one assay would not provide the minimum evidence-based testing required. The sensitivity of the Idylla assays allows for a minimal sample requirement of one 10-μm FFPE tissue section per cartridge and a minimum tumor content of 10%. Additionally, unlike most molecular methods currently available for somatic mutation testing, Idylla does not require separate sample preprocessing steps such as deparaffinization, FFPE tissue digestion, or DNA extraction. Additionally, the closed cartridge system minimizes the potential for contamination.

Finally, a validation study by the University of Texas MD Anderson Cancer Center evaluated four Idylla assays designed to detect alterations in KRAS, EGFR, and BRAF, and gene fusions and expression imbalances in ALK, ROS1, RET, NTRK1/2/3, and MET exon 14. Their testing focused on archived cytology and small biopsy specimens from NSCLC, thyroid carcinomas, and melanomas, because these are cancer types with guideline-recommended targeted therapies that are commonly encountered in the cytopathology laboratory where an FNA may be the only specimen available. Accuracy (compared to NGS) across the four cartridge types for KRAS, EGFR, BRAF and gene fusions was 100%, 94%, 100%, and 94%, respectively.⁸¹ In the two discordant EGFR assay cases, the known EGFR mutations confirmed by orthogonal testing were detected by Idylla, but an additional false-positive EGFR p.S768I (at a high cycle threshold) was detected; this is a known issue with the rare p.S768I variant. The manufacturer has since issued software updates to change the cutoff values for a false “mutation detected” call. In the two discordant gene fusion cases, an orthogonally confirmed ALK fusion failed to be detected and an NTRK3 fusion was detected but as “equivocal.” The cellularity and tumor area of both discordant samples were relatively low and near the LOD of the assay.⁸¹ Analytical sensitivity for all assays ranged from 1% to 5% VAF.⁸¹ The authors highlight that “in NSCLC, we expect 60%–80% of cases to yield actionable results, depending on the patient population. In thyroid carcinoma, BRAF activating mutations are reported to occur in 50%–80% of cases. Additionally, depending on the subtype of thyroid carcinoma, gene fusions can be detected in a small percentage of cases or in up to 80% of cases. In melanoma, approximately 50% of patients will carry BRAF p.V600E mutations, whereas rare cases may be driven by gene fusions, such as NTRK1/2/3”.⁸¹ Given the high yield of expected positive results, the robust performance of the test and the fact that the samples can be directly processed and analyzed by the instrument without the need for separate nucleic acid extraction, the authors conclude that these tests demonstrate the feasibility of incorporating same-day molecular testing as part of rapid on-site evaluation (ROSE) procedures in the cytopathology laboratory.⁸¹

These “next generation” non-NGS molecular panel assays, like all laboratory tests, have limitations. The platforms are predicated on the prior identification of target sequences, constraining their ability to detect low-prevalence genomic alterations and novel fusions that have not been previously characterized. In such cases, reflex testing negative cases using NGS would provide a broader capability to capture rarer mutations and fusion events. Further, these are qualitative assays that do not quantify tumor VAF. However, VAF is generally not a requirement for therapeutic decision-making. Further, these platforms do not measure TMB and microsatellite instability (MSI) but these are not always required for informing the initial treatment of many cancer types. Finally, these platforms are generally designed to target genes with established clinical relevance according to current guidelines. Consequently, they may not include variants associated with therapeutics approved for other cancer indications which may be prescribed off-label. Nevertheless, the rapid TAT of these PCR-based panels for guideline-recommended biomarkers do not preclude (and do not significantly delay) reflex testing using comprehensive NGS panels if no actionable variants are detected.

Tumor profiling by NGS is currently the most comprehensive approach to tumor profiling, given that most genomic alterations can be identified using this method. However, testing by NGS involves a complex workflow, implementation and analysis, as well as a longer time to results, particularly when specimens must be sent to reference laboratories. Newer technologies have been developed that can provide a rapid result for some of the most common targetable genomic alterations in many cancer types. Compared to NGS, these approaches also have simpler workflows and can be implemented more broadly in clinical practices.

Studies have repeatedly demonstrated the importance of broad molecular testing to inform the use of targeted therapies in cancer; however, this benefit need not be derived solely from testing by NGS. In fact, NGS-based genomic testing has been underutilized and has not always resulted in an improvement in patient outcomes. Prompt testing and clinical management informed by test results are paramount. This is true regardless of test methodology, so long as the test performed can identify the most common AODs with the appropriate accuracy, sensitivity and specificity in the intended-use patient population. Given that advancements in the technology of non-NGS tests (e.g. “next generation” PCR) can also be highly multiplexed and provide accurate results, it is reasonable that such tests be utilized as a first-line option for patients who may benefit from the rapid initiation of therapies based on molecular testing.

Further, many specimen types, including cytology specimens and small core needle biopsies, often do not provide a sufficient volume or percentage of tumor required for testing by NGS. However, these same specimen types have been successfully tested using alternate methods including PCR, providing clinical benefit for patients who might not otherwise have the option to be tested by NGS. For all these reasons, this contractor finds non-NGS multi-gene panel testing to be reasonable and necessary (R&N), according to the criteria outlined herein, for patients who may benefit from rapid results that can inform therapy. This approach should minimize the depletion of precious tissue when specimens are limited. Moreover, it is not expected to result in unnecessarily lengthy TATs, given that results from rapid methodologies such as PCR are achieved rapidly and reflexing to NGS can occur expediently, for those cases in which the initial methods have yielded negative results.

The Summary of Evidence illustrates the complex issues applicable to testing for different tumor types. Prevalence of the various AODs in the specific tumor type and patient population, along with their exclusivity from other AODs, are important factors in determining whether panel testing by methodologies other than NGS should be utilized. For some tumor types and populations, the most common mutations can be accurately detected by these advanced non-NGS methods, rendering a lesser incremental yield from testing by NGS. Nevertheless, given that rarer clinically relevant biomarkers, including tumor-agnostic alterations, may not be detected by non-NGS methods, NGS can still serve an important role in cases that are negative by non-NGS panels.

This contractor also recognizes that some of the issues pertaining to the lengthier TAT by NGS are due to suboptimal workflow issues in test ordering and specimen accessioning/processing that should be remedied. All efforts should be made to minimize unnecessary delays in sample testing and reporting. This contractor also recognizes that physicians should try to maximize the tissue obtained for diagnosis, as well as prediction/prognosis, whenever possible. The ability of some methods to use a lesser specimen input should not be regarded as an acceptable reason for not obtaining the most appropriate amount of specimen possible.

In conclusion, while an NGS-based genomic testing approach is often preferred due to its comprehensiveness, some clinical situations warrant rapid testing and molecularly informed management. In many cases, non-NGS panel testing will provide a positive result that will obviate the need for further testing. In this regard, accurate test performance with a low false positive rate is key such that positive calls should not require confirmation by secondary testing (e.g. with NGS), enabling clinicians to initiate treatment in a timely manner. Reduced sensitivity is only acceptable for rarer targets or those that may not be included on the panel; their lack is expected to be mitigated by reflex testing to a comprehensive NGS in negative cases.

Finally, this contractor considers the testing service in its entirety, and notes that multiple assays may be required to constitute a singular service.

N/A

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• Other (LCD Request from Laboratory)

• Non-Next Generation Sequencing
• Targeted Molecular Panel Tests
• Predictive Testing