MolDX: Next-Generation Sequencing for Hematologic Malignancies and Suspected Hematologic Malignancies

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.”

Title XVIII of the Social Security Act, §1833(e), prohibits Medicare payment for any claim lacking the necessary documentation to process the claim.

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 Clinical Laboratory Services, §80.1.1 Certification Changes

This policy describes and clarifies coverage for Lab-Developed Tests (LDTs) and FDA-approved or cleared clinical laboratory tests utilizing Next-Generation Sequencing (NGS) in cancer. This policy’s scope is specific for hematologic malignancies and suspected hematologic malignancies. It is exclusive of solid tumor testing, circulating tumor DNA (ctDNA) testing, and other cancer-related indications for NGS such as germline testing, minimal residual disease, or response to therapy.

Criteria for Coverage

A Next-Generation Sequencing (NGS) service is covered for patients with hematologic malignancies or suspected hematologic malignancies when ALL the following requirements are met:

1. The patient has one of the following:
1. a confirmed hematologic malignancy in accordance with current national or international consensus diagnostic guidelines (i.e., International Consensus Classification of Myeloid and Lymphoid Neoplasms (ICC); the World Health Organization Classification of Haematolymphoid Tumours (WHO)), and is undergoing evaluation at the time of initial diagnosis, disease progression/transformation (representing an a priori change in genetic content), or clinical relapse OR
2. an undiagnosed but highly suspected hematologic malignancy in accordance with current national or international consensus guidelines (as above), AND a standard evaluation to rule out benign or reactive causes (e.g., infectious, inflammatory etiology) has been performed, as documented in the medical record.
2. At least one of the following is true:
1. Molecular profiling is required for definitive disease classification according to the current WHO or ICC frameworks, and classification has not yet been fully established by other non-NGS genetic testing; OR
2. Molecular profiling is necessary to direct immediate clinical management, inclusive of targeted therapy selection, decisions regarding treatment intensity, or determination of hematopoietic stem cell transplantation eligibility, as established by national clinical practice guidelines (such as those from the National Comprehensive Cancer Network (NCCN)), and has not yet been established by other non-NGS testing.
3. The patient has not been previously tested for the same or similar genetic content for clinically actionable genetic information for the same intended use.
4. The test is being used in a patient who is part of the population in which the test was analytically validated AND according to the intended use of the test.
5. The assay performed includes at least the minimum genes and genomic positions required for the identification of clinically relevant FDA-approved therapies with a companion diagnostic biomarker as well as other biomarkers known to be necessary for clinical decision-making for its intended use that can be reasonably detected by the test. Because these genes and variants will change as the literature and drug indications evolve, they are listed separately in associated documents such as the MolDX^® Technical Assessment (TA) forms.
6. The test has completed a Technical Assessment (TA) by MolDX^® to verify that analytical validity (AV), clinical validity (CV), and clinical utility (CU) standards are met for the stated indications of the test.

NGS testing in solid tumors has become a routine component of the work-up process¹; the results can uncover the genomic mechanisms of cancer that have diagnostic, predictive, and prognostic utility and improve clinical management.² Understanding the mechanisms of disease and targeting treatment based on those aberrant processes (i.e., targeted therapies) has improved patient outcomes in many tumor types and is the basis of “Precision Medicine”.³ NGS has the ability to capture abundant genomic data efficiently and relatively cheaply compared to prior methodologies. Its use has become standard of care through inclusion in many professional consensus management guidelines, such as those from the NCCN.⁴

In addition to solid tumors, NGS profiling has demonstrated significant impact in the diagnosis and management of hematologic malignancies. A critical distinction is that hematologic malignancies do not conform to traditional solid tumor staging systems (e.g., Tumor Node Metastasis, TNM) or exhibit anatomic localization. Instead, hematologic malignancies are characterized by widespread systemic involvement, clonal evolution, and a high propensity for rapid disease acceleration or transformation.

Advances in sequencing technology and improvements in large scale integrated data analysis have led to identification of new tumor subtypes and altered signaling pathways with potential for targeted therapeutics. Molecular genetics is the underpinning of the two most recent and distinct classification schemes for hematologic malignancies, namely the International Consensus Classification of Myeloid and Lymphoid Neoplasms (ICC) and the World Health Organization Classification of Haematolymphoid Tumours, 5^th edition (WHO).^5,6 Although the two classifications diverge in many respects, both integrate genomic information for diagnosis, risk stratification, and therapy selection.

NGS Test Description

NGS is not a specific test, but a methodology utilized to capture genomic information. Unlike Sanger sequencing (the prior standard technology) that typically provides information for a single DNA target, NGS allows for massively parallel interrogation of millions of DNA molecules concurrently.^7,8 This allows for simultaneous capture of many relevant genomic targets, usually by utilizing technologies such as PCR amplification or hybrid capture. As such, NGS tests for use in cancer are often comprised of gene panels, either relevant to a specific tumor type or condition, or a larger panel of genes that can be used for multiple tumor types or conditions.

NGS tests can vary significantly for many reasons. While NGS defines a broad methodology for massively parallel sequencing, different technologies are available that have different strengths, weaknesses, and technical limitations or liabilities.⁹ Platforms utilize different chemistries, signal amplification, and detection methods. Gene panels may include only the portions of genes that contain the most critical clinically relevant information (targeted), or be comprehensive, containing entire exonic (coding) gene regions and intronic (non-coding) regions, or even sequence RNA for detecting gene fusions or expression levels.

Downstream from the analytic processes mentioned above, the bioinformatics used to process and assess the resultant sequencing reads and identify variants/mutations can yield different results. These software tools must align the resultant sequencing file (e.g., FASTQ format) with a reference genome, resulting in a Binary Alignment Map / Sequence Alignment Map (BAM/SAM) output, and then identify variants from the reference (typically creating a variant call file (VCF)).¹⁰ Once such variants are identified, they must be assessed for validity and subsequently evaluated for clinical relevance. The types of genomic information reported can vary, as tests can uncover a myriad of genomic alterations such as single nucleotide variants (SNVs), insertions/deletions (INDELs), copy number alterations (CNAs; ranging from amplifications/deletions at a single locus to full chromosome gains/losses), and gene fusions/translocations. The resultant information can also be used to calculate additional relevant information, such as tumor mutation burden (TMB) or the presence of microsatellite instability (MSI). All of these variant classes have demonstrated clinical utility, although TMB and MSI are more relevant to solid tumors.

In summary, NGS testing in cancer comprises a large heterogeneous group of assays that are substantially different from each other. Additionally, NGS testing is highly complex and requires expertise from handling the specimen, to running complex equipment, to understanding the bioinformatics, to interpreting the findings and creating an actionable medical report.

Two types of tests are considered for coverage: “hotspot” tests and “comprehensive genomic profiling” (CGP) tests. These tests can detect any combination of the previously described variant types; in general, hotspot tests are limited to SNVs and small INDELs, whereas CGPs can also detect CNAs, larger INDELs and/or gene fusions/translocations, as well as MSI status and TMB when relevant.

Professional Society Clinical Practice Guidelines

Guidelines for validating clinical NGS tests for use in cancer have been published in a joint effort by the Association for Molecular Pathology (AMP) and the College of American Pathologists (CAP).¹¹ Guidelines for employing bioinformatics pipelines for NGS testing have also been published by these groups,¹⁰ along with guidelines for interpreting somatic variants in these panels in collaboration with the American Society of Clinical Oncology (ASCO).¹²

National clinical practice guidelines, including those from the NCCN, ASCO, and the American Society of Hematology (ASH) recommend or incorporate molecular testing for diagnosis, risk stratification, and treatment selection across myeloid and lymphoid malignancies, as well as histiocytic neoplasms.^13-23

Clinical Utility in Hematologic Malignancies

NGS-based testing of hematologic malignancies has become the standard of care.²³ NGS has demonstrated the ability to improve diagnostic accuracy and classification of hematologic malignancies, provide risk stratification and guide therapy selection, facilitating informed and risk-adapted clinical management. This can be seen across the spectrum of hematologic malignancies, including myeloid, lymphoid, and histiocytic neoplasms.^13-24 The following discussion will touch upon the clinical utility of NGS in each of these disease categories.

Myeloid stem cell disorders are a heterogenous group of malignancies with clinical and genomic overlap. According to the WHO classification, myeloid disorders can be classified as myelodysplastic neoplasms (MDS), myeloproliferative neoplasms (MPN), overlap myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and AML (acute myeloid leukemia).⁵ These disorders can comprise a continuum of tumor evolution that ultimately results in AML in many instances.²⁵ Clonal hematopoiesis may also be detected in elderly patients without overt evidence of myeloid neoplasia. Due to the increased risk of developing myeloid neoplasia, such patients require close monitoring. NGS is an important tool in the evaluation of patients who meet objective, documented clinical diagnostic thresholds (such as persistent cytopenias) to establish a baseline clinical pathway at the time of suspected diagnostic transformation.

Molecular evaluation of AML and other myeloid neoplasms routinely integrates NGS panels along with DNA-based cytogenomic assays (e.g., microarrays, fluorescence in situ hybridization (FISH)) to ensure comprehensive genomic profiling.²⁶ Whole genome sequencing (WGS) and genome-wide next-generation cytogenetic approaches have also demonstrated clear proficiency as an alternative to conventional cytogenetic analysis.^27,28 As a core laboratory technology, NGS, with its ability to identify numerous relevant biomarkers concurrently, demonstrates unique clinical utility in establishing definitive diagnosis, prognostic risk stratification, and identification of targeted therapeutic options in myeloid malignancies.^29-31

Lymphoid malignancies comprise a heterogeneous group of neoplasms with substantial clinical, pathologic, and genomic overlap. According to ICC/WHO classifications, lymphoid malignancies include precursor lymphoid neoplasms (B- and T-lymphoblastic leukemia/lymphoma), mature B-cell neoplasms (such as diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and chronic lymphocytic leukemia/small lymphocytic lymphoma CLL/SLL, among others), plasma cell neoplasms (such as multiple myeloma and the monoclonal gammopathies), mature T- and NK-cell neoplasms, and Hodgkin lymphoma (HL).^5,32,33

Molecular genetics now underpins the modern diagnostic and classification framework for lymphoid malignancies.^5,33,34 Molecular profiling by NGS panels is incorporated alongside traditional modalities such as morphology, flow cytometry, cytogenetics, and immunohistochemistry. Incorporation of NGS at the time of initial diagnosis has been shown to improve diagnostic precision, identify prognostically relevant molecular features, and inform evidence-based treatment selection, while reducing diagnostic ambiguity and the need for repeat or sequential tissue sampling that can be invasive, costly, and delay patient care.^24,35

A recent study by Strasser et al (2025) chronicled the clinical validation of an NGS panel in 226 patients with suspected mature B-cell lymphoma with potential bone marrow involvement across multiple clinically relevant scenarios. The assay demonstrated reliable detection of WHO-classified markers including BRAF mutations in hairy cell leukemia, MYD88/CXCR4 mutations in lymphoplasmacytic lymphoma, and the deliberate absence of BRAF mutations in splenic B-cell lymphoma with prominent nucleoli. NGS successfully identified mutations in previously undiagnosed cases and led to diagnostic reclassification. In cases where conventional cytogenetics were uninterpretable, NGS detected pathogenic variants in 61% of cases, successfully compensating for inconclusive karyotypic findings. NGS also outperformed cytogenetics in cases with limited morphological assessment, identifying relevant mutations in 70% of cases compared to just 30% via cytogenetics (p=0.0256).³⁶ These findings highlight how NGS enhances diagnostic accuracy by complementing traditional methods, refining WHO-classified subtypes, and resolving ambiguous cytogenetic or morphologic presentations.³⁶ As observed by Bommier et al. (2021), the integration of multi-gene NGS panels yields actionable diagnostic modifications across multiple hematologic malignancies, reclassifying 15% of small B-cell lymphomas, 13% of large B-cell lymphomas, and 11% of cases with unclassified subtypes.³⁷

Histiocytic neoplasms are rare hematologic disorders, representing less than 1% of all soft tissue and lymph node cancers. They are characterized by accumulation of myeloid-dendritic cell-derived neoplastic cells accompanied by a prominent background inflammatory infiltrate. These disorders are highly heterogeneous in presentation, varying from mild and localized to severe disseminated disease.^17,38,39 Multiple organs can be involved, including bone, nervous, endocrine, respiratory, dermatologic and cardiac systems, among others, posing a diagnostic challenge that can lead to a significant delay in diagnosis and treatment. While there are over 100 subtypes of histiocytoses, the most common histiocytic neoplasms in adults (namely Erdheim-Chester Disease, Rosai-Dorfman disease and Langerhans cell histiocytosis) share overlapping clinical presentations and occasional co-occurrence. Accordingly, NGS panels are recommended by national consensus guidelines to facilitate definitive diagnosis, inform prognosis and risk stratification, and identify targeted therapies.¹⁷

Genomic testing in hematologic malignancies is important for diagnosis, risk stratification, and therapy determinations:

Diagnosis:

Myeloid Disorders

In the setting of MDS, MPN, and MDS/MPN, establishing a definitive diagnosis of a clonal myeloid disorder must occur prior to initiation of therapy. Across all of these disorders, diagnostic classification relies on a multi-parametric testing approach integrated with clinical and morphologic findings.⁴⁰ In other contexts, identifying specific somatic mutations directly supports a diagnosis of a clonal myeloid disease (versus a benign/reactive condition) when there is a high clinical index of suspicion and/or morphologic findings are low-grade or subtle.⁴¹ NGS detection of clonal hematopoiesis with mutation identification in patients with unexplained cytopenias of ≥ 4 months duration is a strong predictor of who has or will have a myeloid disorder.⁴² When a patient harbors a mutation with a variant allele frequency (VAF) ≥10% or exhibits two or more mutations identified on a multi-gene NGS panel, the positive predictive values for a myeloid neoplasm are 0.86 and 0.88, respectively.⁴³ Additionally, molecular profiling can help differentiate myeloid neoplasms from conditions with overlapping histologic findings, such as aplastic anemia, while simultaneously assessing the likelihood of transformation into MDS.⁴⁴

In AML, specific recurrent aberrations such as NPM1 mutations or core binding factor (CBF) fusions define distinct, molecularly-driven disease under ICC/WHO Classification. Importantly, under these contemporary classification frameworks, both schemes have moved away from the historical ≥20% diagnostic blast criterion. Under WHO classification, the identification of disease-defining molecular abnormalities such as NPM1 mutations, RUNX1::RUNX1T1 fusions, or CBFB::MYH11 fusions establishes a definitive diagnosis of AML, regardless of blast count. In contrast, under the ICC framework, the blast threshold is lowered to ≥10% in the presence of defining genetic abnormalities, with cases exhibiting <10% blasts classified as MDS.^5,33

Furthermore, in the setting of a suspected myeloid neoplasm, well defined mutations in genes such as JAK2, SRSF2, CSF3R, U2AF1, SF3B1, ZRSR2, ASXL1, EZH2, STAG2, and BCOR strongly indicate an underlying stem cell disorder.⁴⁵ Therefore, genomic profiling provides the molecular-based diagnostic classification mandated by current WHO and ICC frameworks for myeloid workups.^5,33

To achieve correct disease classification of BCR::ABL1 negative myeloproliferative neoplasms (MPNs), contemporary WHO and ICC classifications integrate molecular driver status with peripheral blood thresholds and morphologic findings.^32,33 For Essential Thrombocythemia (ET), both classification schemes support a platelet threshold of ≥450 ×10⁹/L alongside the determination of JAK2, CALR, and MPL mutational status. For Polycythemia Vera (PV) diagnostic criteria rely on specific combinations of elevated hemoglobin/hematocrit, bone marrow panmyelosis, a JAK2 mutation, and subnormal serum EPO levels.⁴⁶ While bone marrow examination remains a core morphological requirement for most MPN cohorts, especially considering the existence of driver-negative or “triple-negative" cases lacking standard driver mutations, molecular profiling serves as a critical diagnostic anchor, particularly when BM histology is ambiguous. Consequently, for these diagnostically challenging or driver-negative cohorts, multi-gene NGS panel testing provides the genomic data necessary to identify non-canonical mutations and differentiate early clonal MPNs not only from reactive, non-neoplastic blood dyscrasias, but also from alternative hematologic malignancies on the differential diagnosis.

Furthermore, while sequential single-gene reflex testing may be appropriate and guideline-compliant, current NCCN Guidelines explicitly endorse molecular testing using a multi-gene NGS panel that includes JAK2, CALR, and MPL during the initial diagnostic workup for many BCR-ABL-negative MPNs. Per the most recent NCCN Guidelines, molecular testing on blood or bone marrow with multigene panel testing is recommended as part of the initial workup.⁴⁷ By evaluating driver mutations concurrently alongside mutations needed for prognostication, multi-gene NGS provides timely mutational prognostication that directly impacts clinical management regarding risk stratification, monitoring for leukemic transformation, and therapeutic selection.⁴⁷

Lymphoid Disorders

Accurate diagnosis and classification of B- and T-cell lymphoid malignancies is essential prior to initiation of therapy. Lymphoma misdiagnosis remains a significant problem, and while there is generally high concordance between community and lymphoma specialists for common B-cell lymphomas, it is substantially lower (<50%) for rarer subtypes such as Burkitt’s lymphoma (BL) or peripheral T-cell lymphoma.⁴⁸ A misdiagnosis rate of 40% was found in a recent analysis of 2,291 lymphoid neoplasm cases classified according to the 2017 WHO classification criteria, with the most common error being misclassification among lymphoma subtypes (61% of misdiagnoses). All 2291 cases were reviewed by two hematopathologist experts and classified according to the 2017 revised WHO classification criteria, supplemented with immunohistochemistry (IHC), molecular biology and genetic information as needed. The diagnostic discordance between primary and expert review was evaluated.⁴⁹

Diagnostic classification is comprised of a multipronged approach integrating clinical features, morphologic assessment, immunophenotyping, cytogenetics, and molecular findings. In the setting of subtle or equivocal morphologic or immunophenotypic findings, molecular profiling can distinguish a malignancy diagnosis from a benign or reactive process. Undertreatment due to misclassification can have severe consequences.⁴⁸

According to the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) Guidelines for Diagnosis, Indications for Treatment, Response Assessment, and Supportive Management of CLL, the diagnosis of CLL requires the presence of ≥5 × 10^9/L B-lymphocytes in the peripheral blood, sustained for at least 3 months.⁵⁰ Furthermore, the presentation of progressive constitutional B-symptoms (unexplained fever, drenching night sweats, or unintentional weight loss within 6 months), progressive lymphadenopathy, or organomegaly acts as an urgent clinical trigger of disease acceleration, clonal evolution, or an underlying occult lymphoma subtype are established by NCCN guidelines.⁵¹

Standard morphology and flow cytometry can yield inconclusive or indeterminate results.⁵² In the setting of a deteriorating clinical presentation, multi-gene NGS sequence panels can provide the resolution required to capture distinct somatic driver alterations (including but not limited to TP53, NOTCH1, SF3B1, MYD88, or BIRC3 mutations) necessary to establish clonal architecture, secure a definitive disease classification, and avoid diagnostic delays or unnecessary sequential invasive biopsies.⁵³

Aggressive B-Cell Lymphomas

A timely and precise diagnosis is key for patients with lymphoma and histopathological discrimination of Burkitt Lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and high grade B-cell lymphoma (HGBL) is challenging. Patients with an inaccurate diagnosis and diagnostic revision from DLBCL to BL after starting DLBCL-directed therapy experience significantly inferior progression-free survival compared to those diagnosed accurately at onset, underscoring the need for timely diagnostic precision.⁵⁴ NGS reliably differentiates DLBCL from BL based on distinct mutational landscapes. Specifically, BL is characterized by the hallmark IG::MYC rearrangement (found in 80% of cases) alongside recurrent mutations in the ID3–TCF3–CCND3 pathway; while these pathway mutations occur at a lower overall frequency in adults compared to pediatric cohorts (approximately 63% vs 87%, respectively), their presence forms a distinct and specific molecular profile readily identifiable on standard NGS panels to differentiate BL from standard DLBCL.^55,56 Furthermore, within this aggressive B-cell lymphoma spectrum, NGS-based assays equipped to evaluate copy number alterations can detect the characteristic chromosome 11q proximal gain / distal loss pattern that identifies large/high-grade B-cell lymphoma with 11q aberration- classified as HGBCL with 11q aberration (HGBCL-11q) by the WHO and Large B-cell lymphoma with 11q alteration (LBCL-11q) by the ICC. While this entity mimics BL morphologically, it lacks MYC rearrangements, is genetically distinct from BL, and requires a clinical management strategy distinct from standard DLBCL.⁵⁷

In cases of confirmed DLBCL where the subtype remains ambiguous after standard workup, NGS can resolve these features by identifying distinct mutational profiles.⁵⁸ This includes identifying mutations in MYD88 (particularly L265P) and CD79B mutations in activated B-cell-like tumors or EZH2 and BCL2 alterations in germinal center-derived tumors, leading to resolution of ambiguous morphologic or immunophenotypic features.⁵⁸

Differentiation of Small B-Cell Malignancies

Mutational profiling is helpful in the differential diagnosis and classification of small B-cell lymphomas presenting with atypical or overlapping immunophenotypes, such as CD5-positive marginal zone lymphoma versus mantle cell or chronic lymphocytic leukemia/small lymphocytic lymphoma. The diagnostic utility of this modality is evidenced by the distinct mutational signatures uncovered during genomic profiling; specifically, the presence of structural rearrangements or copy number changes involving CCND1, CCND2, or CCND3 helps to definitively confirm a diagnosis of MCL. This includes challenging cases that are cyclin D1–negative by IHC or cryptic by conventional FISH. In contrast, the detection of somatic mutations in NOTCH1, SF3B1, and XPO1 strongly supports a diagnosis of CLL/SLL, whereas mutations in BRAF, KLF2, NOTCH2, and PTPRD favor marginal zone lymphoma.³⁴

T-Cell Lymphomas

Molecular profiling provides diagnostic resolution for mature T-lymphoid neoplasms, which frequently present with prominent reactive inflammatory backgrounds that can obscure malignant populations. For example, in nodal T-follicular helper (TFH) cell lymphoma the WHO category encompassing angioimmunoblastic T-cell lymphoma (AITL), follicular type, and not-otherwise-specified (NOS) subtypes (termed follicular helper T-cell lymphoma by the ICC) morphological assessment is frequently inconclusive. The detection of characteristic mutations in genes such as RHOA, DNMT3A, TET2, IDH2, or PLCG1 supports a diagnosis of a clonal T-cell malignancy over reactive T-cell expansions.^54,59 Similarly, in T-cell large granular lymphocytic leukemia (LGLL), somatic gain-of-function mutations in STAT3 (most commonly Y640F, D661V, D661Y, and N647I; present in approximately 40% of patients) and in STAT5B (notably Y665F and N642H; found in a smaller fraction of cases) represent highly specific molecular findings. These mutational profiles support a definitive diagnosis of LGLL and successfully distinguish clonal disease from reactive T-cell expansions, clonal cytotoxic T-cell proliferations of uncertain significance, and overlapping chronic lymphoproliferative disorders of natural killer cells.^60,61 Both the WHO and ICC recognize these STAT3/STAT5B alterations as diagnostically supportive criteria, and their identification is increasingly required to establish clinical trial eligibility and guide targeted therapy.

Histiocytic Neoplasms

The characteristics of Erdheim-Chester Disease (ECD), Langerhans Cell Histiocytosis (LCH), and Rosai-Dorfman Disease (RDD) span multiple organ systems, inducing those of the bone, nervous endocrine, respiratory, dermatologic, and cardiac systems.⁶² The clinical presentation can mimic other conditions, resulting in a significant diagnostic challenge.

Recurrent genetic alterations have been identified in various histiocytic neoplasms. These alterations predominantly consist of somatic missense mutations, indels, and fusions in genes related to the mitogen-activated protein kinase (MAPK) signaling pathway.^39,63-66 As a result, many histiocytic disorders are now recognized as clonal neoplastic diseases characterized by the constitutive activation of the MAPK pathway.⁶⁶ Therefore, NGS profiling for somatic variants within the RAS-RAF-MAPK-ERK and PI3K-AKT pathways establishes the requisite molecular criteria for definitive disease classification and directly informs systemic targeted therapy selection. In addition, fusion testing for BRAF, ALK, RET, and NTRK1 rearrangements is recommended in cases of clinical suspicion for histiocytosis when NGS panel does not reveal BRAF or other MAPK mutations.¹⁷ Molecular profiling is similarly useful to resolve diagnostic ambiguity in patients who present with mixed histiocytic neoplasms (MXH), wherein two or more disorders co-occur.⁶⁷

Risk Stratification

Myeloid Disorders

NGS testing is used to identify a category of AML patients who typically do not respond to standard induction and consolidation chemotherapy. Rather, these patients are at a high risk for relapse, disease progression, and poor outcomes and would benefit from strategies other than traditional induction and consolidation regimens, in particular hematopoietic stem cell transplant (HSCT).⁶⁸ There are now more than 50 genes that can render diagnostic and risk stratification information across the spectrum of myeloid stem cell disorders.^69-71 The appropriate therapy can be administered in patients only after an accurate diagnosis is rendered.

According to evidence-based guidelines from the American Society for Transplantation and Cellular Therapy (ASTCT), HSCT is considered a valuable therapeutic option in high-risk myeloid disorders and acute leukemia inpatients who lack mutations that are associated with poor outcome in the transplant setting.^72-75 In these situations, NGS testing can help refine who is unlikely to benefit from HSCT.

The initial genomic architecture captured by NGS at diagnosis provides the risk-stratification needed for prediction of early treatment response and post-transplantation relapse risk. Additionally, NGS provides advantages to other methods in its sensitivity and in its ability to overcome clonal evolution and detect new mutations.^76,77

Lymphoid Disorders

NGS testing provides important prognostic and risk stratification information across the spectrum of B- and T-cell lymphoid malignancies. Traditional clinical indices and morphologic features alone fail to fully capture the biologic heterogeneity of lymphoid neoplasms, resulting in substantial variability in treatment response, relapse risk, and overall outcomes among patients with otherwise similar clinicopathologic features. Genomic profiling can identify molecular subgroups with distinct clinical behavior and therapeutic vulnerabilities, enabling more precise risk assessment.

Aggressive and Indolent B-Cell Lymphomas

In accordance with the NCCN diagnostic framework, documentation of critical genomic markers is essential for evaluating disease acceleration, risk stratification, and high-risk clinical presentations in DLBCL. Of note, somatic mutations in PLCG2 harbor prognostic utility for identifying Richter transformation and characterizing aggressive DLBCL that arises from BTK-inhibitor-treated CLL/SLL.^51,78-80 Furthermore, WHO/ICC criteria establish that DLBCL associated with chronic inflammation is a distinct, aggressive entity, with genomic studies demonstrating that mutations in TP53 are frequently observed in this variant.^81,82 When a TP53 mutation occurs concurrently with a MYC rearrangement, it denotes an adverse clinical course and signifies a worse prognosis. Additionally, KMT2D and TP53 mutations demonstrate increased frequency within dark zone gene expression signatures, as well as large B-cell lymphomas characterized by concurrent MYC and BCL2 or BCL6 rearrangements (double-hit lymphomas).⁸³ Furthermore, mutations in B2M, CREBBP, EZH2, MYD88 L265P, SOCS1, and TNFRSF14 stratify aggressive B-cell entities from more indolent pathologies. Comprehensive documentation of these prognostic markers through a multigene NGS panel can facilitate accurate risk stratification and inform therapeutic planning.⁵¹

NCCN B-cell lymphoma guidelines indicate identification of TP53 mutations as an essential prognostic test in classical MCL required for selection of appropriate treatment. TP53 mutation is strongly associated with an unfavorable prognosis and chemoresistance when patients are treated with conventional front-line regimens including autologous stem cell transplantation.^84,85 Given the poor outcomes associated with conventional therapy in this cohort, participation in a clinical trial evaluating novel targeted agents or cellular therapies is strongly recommended.⁵¹

Chronic Lymphocytic Leukemia/Small lymphocytic lymphoma (CLL/SLL)

In CLL/SLL, NGS enables sensitive detection of high-risk mutations with direct prognostic and therapeutic implications. TP53 mutations are associated with resistance to chemoimmunotherapy, early relapse, and inferior survival. Generally, the frequency of TP53 mutations is low at diagnosis (5-10% of patients, depending on the method used), it is slightly higher in cohorts of patients entering frontline treatment (10–20%), and further increases in later disease stages, predominantly in chemoimmunotherapy (CIT)-treated patients and Richter transformation (up to 50%).^86-88 IGHV mutation status is also a predictive biomarker for identifying patients that may benefit the most from chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab. Assessment of these biomarkers at the time of requirement for treatment is recommended by most current guidelines for CLL management.⁸⁹ The latest recommendations for TP53 analysis in CLL, published by the European Research Initiative on CLL (ERIC) state that based on the current knowledge of the relevance of low-burden TP53-mutated clones, a specific minimum VAF cut-off for reporting TP53 mutations is no longer recommended.⁹⁰ Crucially, evaluating these low-VAF variants via an NGS panel prevents the diagnostic gaps introduced by lower-sensitivity standard Sanger sequencing or standalone PCR methods. Furthermore, NCCN Guidelines establish that mutations in ATM, NOTCH1, and SF3B1 serve as independent adverse prognostic indicators that predict a significantly shorter time-to-first-treatment (TTFT) and inferior overall survival (OS). The presence of ATM, BIRC3, and NOTCH1 mutations correlates with a markedly shorter progression-free survival (PFS) and a reduced time-to-next-treatment (TTNT) when patients are subjected to standard chemoimmunotherapy. Furthermore, NOTCH1 mutational status identifies a subpopulation of patients with higher risk for Richter transformation into aggressive high-grade lymphomas.¹⁵

Mature T-Cell Malignancies

The risk stratification value of biomarker evaluation extends to T-cell lymphomas, where characterizing ALK gene rearrangements represents a primary prognostic requirement to stratify divergent risks in Anaplastic Large Cell Lymphomas (ALCLs). ALK-positive ALCL, frequently driven by the t(2;5)(p23;q35) translocation resulting in an NPM1::ALK gene fusion, is independently associated with a highly favorable long-term prognosis, whereas standard ALK-negative variants exhibit an inherently aggressive clinical course and high risk of early treatment failure. Within ALK-negative pathologies, rearrangements involving DUSP22 define a molecular variant demonstrating a favorable, indolent course, whereas structural rearrangements involving the TP63 gene (such as the TBL1XR1::TP63 fusion) define an high-risk subset characterized by an aggressive course.²¹

Histiocytic Neoplasms

Molecular profiling provides objective risk stratification for histiocytic neoplasms by defining distinct genomic subgroups that correlate with disease dissemination and high-risk clinical presentations. The presence of BRAF p.V600E mutation correlates with multicentric disease with organ involvement in patients with adult clonal histiocytosis.⁹¹ In addition, Lang et al⁹² followed patients in a study that included 254 patients, age ≥18 years, with biopsy-proven LCH. MAPK/PI3K pathway alterations were observed in 77.6% (n = 197) of the patients. BRAF p.V600E mutation was the most common (30.7%, n = 78), followed by BRAF_indel (18.1%, n = 46) and MAP2K1 mutations (12.6%, n = 32). The proportion of BRAF_indel was much higher in patients with multisystem involvement than single-system disease (24.5% vs. 6.6%, P < 0.001). Overall, BRAF_indel was associated with inferior overall survival and progression-free survival. BRAF_indel was highly correlated with multisystem LCH and was associated with worse outcomes. NGS is preferred to differentiate these BRAF deletions as standard clinical PCR assays are generally unable to distinguish them.

Furthermore, histiocytic disorders such as ECD are associated with a high frequency of concomitant myeloid malignancies, including MPNs, myelodysplastic syndromes, and mixed MDS/MPNs. Multi-gene NGS profiling is instrumental in uncovering these co-occurring, occult bone marrow malignancies, providing risk assessment that single-gene testing cannot replicate, thereby preventing fragmented care and avoiding multiple overlapping diagnostic workflows.⁹³

Targeted Therapies

Myeloid Disorders

FDA-approved targeted therapies are increasingly directed against specific somatic molecular aberrations in AML, including the FLT3 inhibitors midostaurin, gilteritinib, and quizartinib, as well as the IDH1/IDH2 inhibitors ivosidenib and enasidenib.^94-97 Identifying these driver mutations is clinically mandatory to clear patients for immediate companion targeted interventions at initial diagnosis or clinical relapse. Furthermore, numerous active biomarker-driven clinical trials are currently evaluating novel targeted therapeutic strategies for individuals presenting with high-risk myeloid configurations, underscoring the clinical utility of comprehensive genomic profiling over fragmented single-gene assays to ensure timely access to precision oncology.

Lymphoid Disorders

NGS-based genomic profiling plays a critical role in identifying patients with lymphoid malignancies who are most likely to benefit from specific therapeutic approaches (often, but not always targeted therapies) and in avoiding treatments unlikely to be effective or potentially causing harm. As the therapeutic landscape for lymphoid malignancies has expanded beyond cytotoxic chemotherapy, treatment selection increasingly depends on identification of specific molecular alterations and biologic subgroups that are not detectable by morphology, immunophenotyping, or cytogenetics alone.

For example, PLCG2 mutations signal therapeutic resistance and disease progression/evolution and can be found during Richter transformation or the development of DLBCL arising from CLL/SLL previously treated with Bruton's tyrosine kinase inhibitors (BTKi).^78,79 In CLL/SLL, detection of TP53 mutations or 17p deletion is essential for therapeutic selection, as these alterations predict resistance to chemoimmunotherapy and direct the use of targeted agents such as BTK inhibitors or BCL2 inhibitors.

Mutations in key cellular pathways drive secondary resistance to targeted therapies in B-cell malignancies. For patients progressing on covalent Bruton's tyrosine kinase inhibitors (cBTKi), resistance is frequently heralded by acquired BTK p.C481 and BTK p.A428D mutations. In addition, BTK p.L528 or p.T474 alterations are detected in patients experiencing disease progression despite treatment with both cBTKis and next-generation, noncovalent (reversible) BTK inhibitors (ncBTKi). Beyond primary kinase mutations, downstream alterations in CARD11 and PLCG2 serve as molecular signals identified in patients whose disease has progressed on BTK inhibitors broadly. Finally, in the context of anti-apoptotic targeting, resistance to the BCL2 inhibitor venetoclax is characteristically driven by the emergence of BCL2 p.G101V and p.D103Y mutations, which disrupt drug binding and allow for ongoing cell survival.¹⁵ Detection of these mutations supports timely therapeutic transition.

In Hairy Cell Leukemia, NGS-based identification of the defining BRAF p.V600E mutation (present in >95% of classical cases) predicts sensitivity to BRAF-directed therapy, and BRAF plus MEK inhibitor combinations have demonstrated activity in relapsed or refractory BRAF p.V600E-mutant HCL.¹⁶ Identification of MAP2K1 mutations in BRAF p.V600E-negative HCL-variant further refines therapeutic decision-making, given the distinct clinical behavior and therapeutic sensitivities of this subset.³⁰

Genomic profiling, including FISH and targeted sequencing can facilitate identification of high-grade B-cell lymphoma (HGBL) with double-hit or triple-hit biology, specifically HGBL with MYC and BCL2 rearrangements (with or without BCL6), wherein double-hit biology supports selection of intensified induction regimens. Conversely, the NCCN guidelines clarify that HGBL with concurrent MYC and BCL6 rearrangements may demonstrate clinical outcomes equivalent to DLBCL Not Otherwise Specified (NOS) and can be managed as such.

Targeted therapy selection in T-cell lymphomas similarly benefits from genomic profiling. The NCCN guidelines specifically note that mutations in TET2, IDH2, RHOA, and DNMT3A are highly prevalent in nodal TFH cell lymphoma and that this pathway has been preliminarily associated with higher rates of response to histone deacetylase (HDAC) inhibitors and other epigenetic modifiers.²¹ In addition, JAK2 inhibition is noted to have utility in informing therapeutic strategy in JAK2 rearranged T-cell lymphomas.

Histiocytic Neoplasms

Advances in molecular genetics have led to the identification of key driver mutations that dictate the use of targeted therapies. For patients presenting with ECD, BRAF inhibitors are preferred for BRAF p.V600E mutated disease, whereas MEK inhibitors are preferred for patients with a MAP kinase pathway mutation, or no other detectable/actionable mutation. Additionally, specialized tyrosine kinase and pathway inhibitors demonstrate clinical utility under the following genotype-directed frameworks: ALK inhibitors can be useful in patients with ALK fusions, NTRK inhibitors for patients with NTRK fusions, mTOR inhibitors for patients with PIK3CA mutations, selpercatinib for patients with RET fusions and pexidartinib for patients with CSF1R mutations.^17,98-107

An extensive body of peer-reviewed literature establishes the clinical utility of genetic and genomic profiling in the diagnosis, risk stratification, and management of patients with hematologic malignancies. NGS methodologies demonstrate clear clinical utility by improving diagnostic accuracy, refining disease classification, and reducing diagnostic ambiguity in cases presenting with overlapping morphologic or immunophenotypic features. Molecular profiling identifies distinct genomic subgroups with unique prognoses and therapeutic vulnerabilities, directly enabling risk-adapted treatment strategies, informed HSCT selection, and precise clinical trial matching. This clinical utility is documented across the full spectrum of hematolymphoid neoplasms, including myeloid, lymphoid, and histiocytic categories. Consequently, multi-gene NGS panel testing is considered reasonable and necessary for use in Medicare beneficiaries with hematologic malignancies and suspected hematologic malignancies when performed in accordance with the objective criteria outlined in this LCD.

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