Genomic Testing for Hematopoietic Neoplasms AHS- M2182

Description of Procedure or Service

Hematologic cancers impact the normal production and function of blood cells. These cancers often begin in bone marrow, where stem cells develop into white blood cells, red blood cells, or platelets. Hematologic cancers such as myeloid and lymphoid neoplasms occur when there is an uncontrolled growth of abnormal cells which overtake the development of normal blood cells. An overabundance of abnormal cells interferes with the regular functions of normal cells (ACCC, 2022). Myeloid neoplasms occur when there is a proliferation of myeloid cells that have originated from a primitive stem cell (NCBI, 2023b). In contrast, lymphoid neoplasms are composed of a lymphocytic cell population which is usually malignant (clonal) by molecular genetic and/or immunophenotypic analysis (NCBI, 2023a).

For guidance on flow cytometry in hematopoietic neoplasms, please refer to AHS-F2019-Flow Cytometry.

For guidance on minimal/measurable residual disease (MRD) in hematopoietic neoplasms, please refer to AHS-M2175-Minimal Residual Disease.

Related Policies:

AHS-F2019 Flow Cytometry

AHS-M2101 Mutation Analysis in Myeloproliferative Neoplasms

AHS-M2175 Minimal Residual Disease

***Note: This Medical Policy is complex and technical. For questions concerning the technical language and/or specific clinical indications for its use, please consult your physician.

Policy

BCBSNC will provide coverage for genomic testing for hematopoietic neoplasms when it is determined to be medically necessary because the medical criteria and guidelines shown below are met.

Benefits Application

This medical policy relates only to the services or supplies described herein. Please refer to the Member's Benefit Booklet for availability of benefits. Member's benefits may vary according to benefit design; therefore member benefit language should be reviewed before applying the terms of this medical policy.

When genomic testing for hematopoietic neoplasms is covered

1. For the initial diagnosis and profiling of hematopoietic neoplasms, the following testing is considered medically necessary:
   1. Bone marrow cytogenetics and/or fluorescent in situ hybridization (FISH) of bone marrow samples.
   2. Multigene panel testing.
2. For the differential diagnosis of chronic myeloid leukemia (CML) or acute lymphoblastic leukemia (ALL) or for optimal risk stratification and treatment planning for individuals diagnosed with B-cell ALL (B-ALL), one time qualitative or quantitative RT-PCR testing on blood or bone marrow for identification of the BCR-ABL1 fusion gene transcript type, including determination of transcript size (e.g., p190 vs. p210), is considered medically necessary.
3. For individuals diagnosed with acute lymphoblastic leukemia (ALL) or pediatric ALL (PEDALL) who are under surveillance, the following tests are considered medically necessary once every 3 to 6 months for at least 5 years:
   1. Bone marrow cytogenetics.
   2. Bone marrow FISH.
   3. Multigene panel testing.
4. For all individuals with Philadelphia chromosome positive ALL (Ph+ ALL), BCR-ABL1 transcript-specific quantification is considered medically necessary at the following frequency:
   1. Once per month for individuals who have detectable levels of BCR-ABL1 transcript.
   2. Once every three months for individuals who have undetectable levels of BCR-ABL1 transcript.
5. For individuals diagnosed with acute myeloid leukemia (AML) who had cytogenetic abnormalities at diagnosis, cytogenetic or FISH testing is considered medically necessary at the following time points:
   1. Fourteen to twenty-one days after the start of therapy to document hypoplasia.
      1. When hypoplasia is documented, repeat analysis seven to fourteen days later to clarify persistence of leukemia.
      2. When hypoplasia is not documented, repeat analysis at the time of hematologic recovery to document remission.
   2. By 42 days post-treatment with either standard-dose or high-dose cytarabine induction for poor-risk AML, regardless of the degree of hematologic recovery.
6. For individuals with AML who have experienced relapsed/refractory disease after the completion of post-remission therapy, multigene panel testing is considered medically necessary.
7. For optimal risk stratification and treatment planning for individuals diagnosed with chronic lymphocytic leukemia (CLL) or small lymphocytic leukemia (SLL), one time testing with any of the following tests (when not already performed during initial diagnosis) is considered
   1. FISH to detect +12; del(11q); del(13q); del(17p).
   2. TP53 mutation testing.
   3. CpG-stimulated metaphase karyotype for complex karyotype.
   4. Immunoglobulin heavy chain variable region gene (IGHV) mutation testing.
8. For individuals diagnosed with CML who are pursuing or who are actively undergoing TKI therapy, bone marrow cytogenetics is considered medically necessary at the following time points:
   1. To establish cytogenetic changes.
   2. When there is a failure to reach response milestones.
   3. When there is any sign of loss of hematologic response.
   4. When there is any sign of loss of complete cytogenetic response (CCyR) or its molecular response correlate, defined as an increase in BCR-ABL1 transcript to >1%.
9. Evaluation of BCR-ABL kinase domain point mutations in patients with CML is considered medically necessary at the following time points:
   1. When the individual has chronic phase CML.
   2. When there is failure to reach response milestones.
   3. When there is any sign of loss of hematologic response.
   4. When there is any sign of loss of CCyR or its molecular response correlate, defined as an increase in BCR-ABL1 transcript to 1%.
   5. When there is a 1-log increase in BCR-ABL1 transcript levels and a loss of MMR.
   6. When the disease progresses to accelerated or blast phase.
10. For individuals with CML who are undergoing treatment discontinuation with TKI therapy and who remain in MMR after discontinuation of therapy, qPCR on blood or bone marrow for the BCR-ABL1 fusion gene transcription is considered medically necessary at the following frequency:
    1. Every month for the first 6 months following discontinuation.
    2. Once every two months in months 7-12 following discontinuation.
    3. Once every three months beginning 12 months following discontinuation of therapy, as long as the individual remains in MMR.

When genomic testing for hematopoietic neoplasms is not covered

1. For individuals with CML, the use of FISH to monitor response to TKI therapy is considered not medically necessary.
2. Simultaneous testing of both bone marrow and blood for monitoring purposes is considered not medically necessary.
3. For the diagnosis or prognosis of individuals with confirmed acute leukemia, global/gene specific methylation, microRNA (miRNA) expression, or gene expression analysis is considered not medically necessary.
4. For diagnosis or prognosis of myeloid or lymphoid neoplasms, all other testing not addressed above is considered not medically necessary.

Notes:

Note: For 2 or more gene tests being run on the same platform, please refer to AHS-R2162 Reimbursement Policy.

Policy Guidelines

Acute Lymphoblastic leukemia (ALL)

Acute lymphocytic leukemia occurs when a bone marrow cell develops a mutation in its genetic material. ALL can be of B-cell precursor (BCP) lineage (BCP-ALL) or, less commonly, T-cell precursor lineage (T-ALL). However, “Both comprise multiple subtypes commonly defined by structural chromosomal alterations that are initiating lesions, with secondary somatic (tumor-acquired) DNA copy number alterations and sequence mutations that contribute to leukemogenesis. Chromosomal alterations include aneuploidy and chromosomal rearrangements that result in oncogene deregulation or expression of chimeric fusion genes. The prevalence of these alterations varies according to age and identification is important for diagnosis, risk classification, and, for some lesions, targeted therapy. Common genomic features of BCR-ABL1–like ALL are alterations of B-lymphoid transcription factor genes (particularly IKZF1 deletions) and genetic alterations deregulating cytokine receptor and tyrosine kinase signaling. These include rearrangements and mutation of CRLF2 (approximately 50%), rearrangements of ABL-class tyrosine kinase genes (12%), rearrangements of JAK2 (7%) and the erythropoietin receptor gene (EPOR; three to ten percent), mutations activating JAK-STAT signaling (11%) and Ras signaling (NRAS, KRAS, PTPN11, and NF1; six percent), and less common kinase alterations (FLT3, NTRK3, BLNK, TYK2, and PTK2B)” (Iacobucci & Mullighan, 2017).

Acute myeloid leukemia (AML)

Acute myeloid leukemia is the most common acute leukemia in adults (80%), with a median age at diagnosis of 65 years. An AML diagnosis in adults is generally associated with a poor prognosis. This disease is much less common in children younger than ten years of age, as less than ten percent of acute leukemias are diagnosed as AML in this age group (Schiffer & Gurbaxani, 2024; Siegel et al., 2017; Yamamoto & Goodman, 2008).

A clinical presentation of AML includes symptoms related to complications of pancytopenia, including weakness and fatigability, infections of variable severity, and hemorrhagic findings (Schiffer & Gurbaxani, 2024). Analysis of gene sequencing of AML cases generally reveal more than ten significant gene mutations; many of which are thought to participate in leukemogenesis (CGARN, 2013). The most common gene mutations are as follows: FLT3 (28%), NPM1 (27%), DNMT3A (26%), IDH1 or IDH2 (20%), NRAS or KRAS (12%), RUNX1 (10%), TET2 (8%), TP53 (8%), CEBPA (6%), and WT1 (6%). Mutations impacting signal activation are frequent (60% of cases); the most common of which are mutations in FLT3 (Stock & Thirman, 2022).

The second most common mutation in AML (27%) is of nucleophosmin (NPM1), a ubiquitously expressed phosphoprotein that normally shuttles between the nucleus and cytoplasm (Falini et al., 2005; Stock & Thirman, 2022). NPM1 mutations are associated with improved outcomes although the mechanism is not known. Concurrent mutations (such as an FTL3 mutation) may influence prognosis, but generally NPM1 patients without concurrent mutations have better prognoses (Schiffer & Uy, 2024).

The CCAAT/enhancer binding protein alpha (CEBPA) gene mutation is also common in AML. CEBPA encodes a transcription factor necessary for myeloid differentiation. This mutation is one of the two mutations associated with familial leukemia and consists of about ten percent of AML cases. Familial AML with a CEBPA mutation has a phenotype similar to those of “sporadic AML with biallelic CEBPA mutations,” but most of the current data revolves around the assessment of CEBPA double mutations. CEBPA single mutations and hypermethylated CEBPA requires further study (Schiffer & Uy, 2024).

Isocitrate dehydrogenase (IDH) 1 and 2 mutations comprise approximately 15% of AML cases. These mutations are mutually exclusive with Tet Methylcytosine Dioxygenase 2 (TET2) and Wilms' tumor 1 (WT1) mutations but are commonly seen with NPM1 and DNA methyltransferase 3A (DNMT3A) mutations. Data on the prognoses of these mutations is varied (Schiffer & Uy, 2024).

The KIT mutations also comprise about six percent of AML cases. KIT encodes the receptor for a stem cell factor, and prognoses are varied (Castells & Akin, 2021; Schiffer & Uy, 2024). Some researchers suggest that of all KIT mutations, the D816 mutation is the most unfavorable prognostic factor in AML patients (Yui et al., 2017).

Approximately eight percent of AML cases consist of WT1 gene mutations. WT1 encodes a transcriptional regulator for genes involved in maturation and growth. Again, the prognosis of this mutation is mixed (Schiffer & Uy, 2024), although some researchers strongly support the theory that WT1 mutations are associated with poor AML prognoses (Hou et al., 2010). The WT1 mutation status of AML patients may also change during disease progression. New research has suggested that after allogenic stem cell transplantation, AML relapse could be due to a gain in WT1 gene alterations and a “high mutation load” (Vosberg et al., 2018).

The ASXL Transcriptional Regulator 1 (ASXL1) and ASXL Transcriptional Regulator 2 (ASXL2) may also be mutated in AML cases. ASXL1 has an unclear function, but it is speculated to be related to histone post-translational modifications. The frequency of ASXL1 is varied, as estimates range from six percent to 30%. Furthermore, ASXL1 mutations are mutually exclusive with NPM1 mutations, and ASXL2 mutations are associated with RUNX1 mutations (also known as AML1 or CBFA2) (Schiffer & Uy, 2024).

The DNMT3A gene amounts to 20%-22% of AML cases. This gene plays a role in epigenetic modifications for development and differentiation. Mutations in this gene affect hematopoietic stem cell differentiation. Prognoses of this gene mutation have been mixed (Schiffer & Uy, 2024).

Tumor protein 53 (TP53) and RAS and may also be present in AML cases and may be accompanied by other genetic abnormalities. RAS regulates cell signal transduction, and its mutation leads to a constitutionally active growth stimulus whereas TP53 encodes a transcriptional activator of growth inhibitory genes (Frucht & Lucas, 2023; Rai & Stilgenbauer, 2023).

Gene expression profiling and microRNA expression profiling may also contribute to assessment and management of AML. Gene expression profiling has been used to differentiate between risk groups based on cytogenetic evaluation whereas microRNA profiling evaluates the regulation of gene expression. However, neither technique is used regularly in clinical practice as these techniques have yet to be widely validated (Schiffer & Uy, 2024).

Chronic Lymphoblastic Leukemia (CLL)

In Western countries, CLL is the most common form of leukemia; in the United States, it accounts for 25 to 30 percent of all leukemias (Rai et al., 2024). CLL and SLL (small lymphocytic leukemia) are mature B cell neoplasms characterized by a progressive accumulation of monoclonal B lymphocytes. It should be noted that “CLL is considered to be identical (ie, one disease with different manifestations) to the nonHodgkin lymphoma SLL. The malignant cells seen in CLL and SLL have identical pathologic and immunophenotypic features. The term CLL is used when the disease manifests primarily in the blood, whereas the term SLL is used when involvement is primarily nodal” (Rai et al., 2024). It is also worth mentioning that “In less than 10 percent of patients, CLL/SLL presents primarily in the lymph nodes as a non-Hodgkin lymphoma (ie, SLL)” and that “this presentation accounts for less than 5 percent of all nonHodgkin lymphomas” (Rai et al., 2024).

The CLL exomes carry approximately “5–20 somatically acquired mutated genes per individual case, much fewer than many other solid cancers. Importantly, the somatically acquired mutation status of sequence variants in genes, like other studies of genomic changes in cancer, necessitates confirmation of the absence of these candidate mutations in paired non-tumorous DNA (T-cell–, buccal cell– or skinderived DNA). Mutations in TP53 are of major clinical relevance, are often associated with del17p and gain in frequency over time. TP53 mutated and associated del17p states substantially lower response rates, remission duration, and survival in CLL. Mutations in NOTCH1 and SF3B1 are recurrent, often associated with progressive CLL” (Amin & Malek, 2016).

Chronic myeloid leukemia

The first human malignancy in which a specific chromosomal defect, known as the minute or Philadelphia (Ph) chromosome, could be linked to pathogenetic events of leukemogenesis (Nowell & Hungerford, 1960). The Ph chromosome translocation (t(9;22) (q34;q11.2)) fuses the breakpoint cluster region protein (BCR) gene from chromosome 22 with the Abelson murine leukemia viral oncogene homolog 1 (ABL1) proto-oncogene from chromosome nine in a head-to-tail manner to form the transcriptionally active BCR-ABL fusion gene (Schrijver & Zehnder, 2022). The fusion of BCR at the 5’ side in ABL alters the tightly regulated function of the Src homology 3 (SH3) domain, disabling control over the tyrosine kinase enzyme. The resulting chimeric BCR-ABL protein has constitutively elevated tyrosine phosphokinase activity (Kurzrock et al., 2003) that activates a number of downstream signaling molecules, including PI3K, AKT, JNK, RAS and STAT5 (Ren, 2005). This then disrupts cellular signal transduction pathways, leading to issues in the regulation of both apoptosis and cell proliferation (Warmuth et al., 1999), ultimately leading to factor-independent and leukemogenic cell growth (Van Etten, 2023).

Detection of the Ph chromosome is the hallmark of CML and is found in up to 95 percent of patients (Leoni & Biondi, 2015). In approximately five percent of CML cases, the Ph chromosome cannot be detected, and BCR-ABL1 formation is attributed to microscopically undetectable translocations or variant complex translocations involving a third chromosome (Schrijver & Zehnder, 2022). Independent of which other chromosomes are involved in variant translocations, the generation of the BCR-ABL fusion gene is the “fundamental cause of Ph-positive leukemias” (Van Etten, 2023), as the 210-KDa fusion protein BCR-ABL is essential for initiation, maintenance, and progression of CML (Ren, 2005). Testing for BCR-ABL1 detects both the Ph chromosome and fusion gene or its transcripts. The BCR-ABL1 transcript is the RNA copy made by the cell.

The FMS-like tyrosine kinase 3 (FLT3) is a transmembrane tyrosine kinase receptor that stimulates cell proliferation upon activation. Both internal tandem duplications (ITDs) of different lengths and point mutations in the activating loop of the kinase domain result in ligand-independent activation of the FLT3 receptor and a proliferative signal. A FLT3-ITD mutation has been shown to have a poor prognosis in contrast to FTL3 point mutations in the activation loop of the kinase domain. Higher ratios of mutated alleles compared to wild-type alleles confer worse prognoses (Schiffer, 2023).

The BCR-ABL1, refers to the fusion gene resulting from a reciprocal translocation that joins the ABL1 gene from chromosome 9 to the BCR gene on chromosome 22 and is necessary for the development of chronic myeloid leukemia (Tachibana et al., 2022) (Van Etten, 2023). This reciprocal translocation also generates a shortened derivative chromosome 22, known as the Philadelphia (Ph) chromosome (Schrijver & Zehnder, 2022). The Ph chromosome is a diagnostic hallmark, present in 95% of people with CML and approximately three to five percent children and 25%–40% adults with acute lymphoblastic leukemia (ALL) (Leoni & Biondi, 2015). An aggressive form of cancer resulting from the neoplastic transformation of lymphoid precursors characterized by the presence of too many lymphoblasts or lymphocytes in the bone marrow and peripheral blood (PDQ, 2023). Predominately a childhood disease, approximately 60% of cases were diagnosed in patients younger than 20 years of age (Pui, 2011).

Analytic Validity

There is very limited published literature on the analytic validity and clinical validity of genetic testing for FLT3 and NPM1 mutations in AML. However, the analytic validity of PCR in general is extremely high (Leonard, 2016). Other tools, such as flow cytometry and next generation sequencing (NGS) have also been used for AML prognostic and diagnostic purposes.

Ampasavate et al. (2019) have developed a quantitative protocol and flow cytometry-based method for monitoring an anti-FLT3 interaction. The FLT3 biomarker has been previously identified as a poor prognostic marker for AML patients. This method can rapidly identify intact FLT3 on the leukemic cell surface. “The results demonstrated good linearity (r2 > 0.99)”; further, “when compared with Western blotting results, FLT3 protein expression levels in leukemia patient's bone marrow samples were demonstrated in the same trend” (Ampasavate et al., 2019). The researchers state that this technique is reliable, rapid, effective and “provided a practical analysis of FLT-3 biomarker levels which is valuable for physician decision in acute leukemia treatment” (Ampasavate et al., 2019).

Alonso et al. (2019) have researched the utility of a 19-gene NGS panel for AML diagnostic purposes. This targeted NGS panel was studied in a cohort of 162 patients with AML. The authors note that “The assay yielded a median read depth >2000×, with 88% of on-target reads and a mean uniformity >93% without significant global strand bias. The method was sensitive and specific, with a valid performance at the clinical variant allele frequency cutoff of 3% for point mutations and five percent for insertions or deletions.” The researchers conclude that this is a “reliable and reproducible method” for AML diagnoses (Alonso et al., 2019).

Molecular testing for the diagnosis of CML confirms typical findings in the blood and bone marrow by the demonstration of the Ph chromosome, the BCR-ABL1 fusion gene or the BCR-ABL1 fusion mRNA. Molecular testing techniques include conventional cytogenetics, fluorescence in situ hybridization (FISH) analysis and reverse transcription polymerase chain reaction (RT-PCR) (Van Etten, 2022). Conventional cytogenetic karyotyping is no longer the diagnostic modality of choice due to its requirements for a highly skilled staff, culturing of cells, long turnaround time, and lower sensitivity (5-10%). Despite this, conventional cytogenetics are still the gold standard, and “should be performed” especially at diagnosis to detect additional clonal abnormalities (Yeung et al., 2016). FISH is more sensitive (0.1-5%) than karyotyping and can be performed on peripheral blood in addition to bone marrow and tissue. FISH can detect certain very rare translocations not usually detectable by the vast majority of commercial and laboratory-developed RT-PCR assays, but FISH is highly specific to the targeted region and may miss other chromosomal changes. Quantitative RT-PCR is the most sensitive technique currently available (0.001-0.01% sensitivity).

On July 22, 2016 the FDA approved the QuantideX qPCR BCR-ABL IS Kit as an in vitro nucleic acid amplification test for the quantitation of BCR-ABL1 and ABL1 transcripts in total RNA from whole blood of diagnosed t(9;22) positive Chronic Myeloid Leukemia (Tachibana et al.) patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2 as a class II device with special controls (FDA, 2016). Brown et al. (2019) performed a study to describe the analytical validation of this kit. They were able to find that “the test has a limit of detection of MR molecular response] 4.7 (0.002% IS) and a linear range from MR0.3 (50%IS) to MR4.7 (0.002%IS) for both Major transcripts. Single-site and multisite precision studies demonstrated a maximum SD of 0.13 MR (30% CV within the assay range between MR0.7 and MR3.7).”

Clinical Utility and Validity

The clinical utility of testing fallows for further risk stratification, prognostication, and guide management decisions in patients with AML. Several studies have concluded that FLT3 and NPM1 mutation testing in cytogenetically normal AML is useful for prognosis and treatment decision making (DeZern et al., 2011; Pastore et al., 2014; Willemze et al., 2014).

Devillier et al. (2015) sought to identify biological and prognostic subgroups based on genetic mutations in AML patients. A total of 125 AML patients with myelodysplasia-related changes (“MRC”) were evaluated. The authors focused on the 26 patients with ASXL1 mutations and 28 with TP53 mutations. The ASXL1 mutation cohort was found to have a higher proportion of marrow dysgranulopoiesis and an overall survival (OS) rate that was below average for wild-types (14% for ASXL1 mutants, 37% for wild-types). The TP53 cohort was found to have a “complex karyotype” and predicted a poor outcome with unfavorable cytogenetic risk AML. Both mutations were found to be an independent factor associated with shorter OS (Devillier et al., 2015).

Bolouri et al. (2018) examined 993 children’s genetic data from the Children's Oncology Group (Khoury et al.) AML trials to characterize the molecular landscape of AML. The authors found that certain somatic variants, such as MBNL1, were “disproportionately prevalent” in children compared to adults. However, certain variants common in adults such as TP53, were not found in children. Other mutations such as NRAS and KRAS were “frequent” in pediatric AML. The authors concluded that their results “highlight the need for and facilitate the development of age-tailored targeted therapies for the treatment of pediatric AML” (Bolouri et al., 2018).

Jongen-Lavrencic et al. (2018) conducted a study of 482 patients 18 to 65 years with newly diagnosed AML. Targeted next-generation sequencing (NGS) was carried out at diagnosis and after induction therapy (during complete remission). At least one mutation was detected in 430 (89.2%) patients, and mutations persisted in 51.4% of those patients during complete remission. The detection of minimal residual disease was associated with a significantly increased relapse rate than no detection. Persistent DTA mutations (mutations in DNMT3A, TET2, and ASXL1) were not correlated with an increased relapse rate. Overall, the authors concluded, “A comparison of sequencing with flow cytometry for the detection of residual disease showed that sequencing had significant additive prognostic value. Among patients with AML, the detection of molecular minimal residual disease during complete remission had significant independent prognostic value with respect to relapse and survival rates, but the detection of persistent mutations that are associated with clonal hematopoiesis did not have such prognostic value within a 4-year time frame” (Jongen-Lavrencic et al., 2018).

Kuwatsuka et al. (2018) evaluated the genetic background of 103 young adults and their subsequent clinical outcomes. The 103 cases included mutations in FLT3-ITD, KIT, CEBPA, NRAS, KRAS, WT1, MLL-PTD, and NPM1. Overall, FLT3-ITD and NPM1 mutations were associated with a greater mortality risk. NPM1 mutations conferred a 100% survival rate in the absence of FLT3-ITD mutations, but FLT3-ITD conferred only a 35% survival without NPM1 mutations (Kuwatsuka et al., 2018).

Zhu et al. (2017) assessed the effect of gene mutations on the subsequent cytogenetic aberrations. A total of 560 patients were enrolled, and the authors examined the following alterations: “CEBPA, NPM1, FLT3, C-KIT, NRAS, WT1, DNMT3A, MLL-PTD and IDH1/2, as well as expression levels of MECOM, ERG, GATA2, WT1, BAALC, MEIS1 and SPI1.” The investigators found that the expression levels of MECOM, MEIS1, and BAALC influenced cytogenetic aberration. Further, FLT3, C-KIT, and NRAS mutations all contained a “conversed” expression profile of MEIS1, WT1, GATA2, and BAALC expression. The investigators also noted “FLT3, DNMT3A, NPM1 and biallelic CEBPA represented the mutations associated with the prognosis of AML in our group” (Zhu et al., 2017).

Papaemmanuil et al. (2016) examined the relationship between genotype and pathophysiology in AML. A total of 1540 patients with 5234 driver mutations across 76 genes were studied. The authors found three genomic subcategories in addition to the currently defined AML subgroups: mutations in genes encoding chromatin, RNA splicing regulators or both (such as ASXL1 or RUNX1), TP53 mutations, chromosomal aneuploidies or both (unusual karyotypes and TP53), and IDH2 mutations. The authors noted that “patients with chromatin–spliceosome and TP53–aneuploidy AML had poor outcomes.” The NPM1 cohort was the largest of the sample (27%, 436 patients) and 319 of those patients also carried a DNA methylation or hydroxylmethylation gene, such as IDH1/2 or TET2. The authors also noted that NPM1-DNMT3A–NRASG12/13 had an “unexpectedly benign” prognosis, and the NPM1 subgroup’s prognoses were largely determined by the context in which the NPM1 mutation occurred (such as in NRAS, IDH, and so on) (Papaemmanuil et al., 2016).

Sperr et al. (2016) evaluated the effect of a genetic mutation and karyotype on the efficacy of treatment for elderly patients. A total of 192 patients over 60 years old were enrolled, and 115 of these patients achieved “complete hematologic remission (CR).” The authors stated that NPM1 mutations (NPM1mut) and karyotype were the only independent predictors of survival, also noting that NPM1mut showed a prognostic impact on both normal (CN) and non-chromosomal (Mkneg) karyotypes. The authors concluded that “elderly patients with CN/Mkneg-NPM1mut or core binding factor AML can achieve long term median continuous CR when treated with intensive induction and consolidation therapy whereas most elderly patients with CN/Mkneg-NPM1wild-type or CN/Mkpos AML may not benefit from intensive chemotherapy” (Sperr et al., 2016).

Heiblig et al. (2019) assessed the impact of NPM1 subtypes on treatment outcomes. One hundred seventy-five patients were examined. The authors found that out of the NPM1 AML cases, 73% (128) were “Type A” mutations (TCTG at exon 12) and 27% (47) were “Non Type-A mutations” (Type B: CATG and Type D: CGTG). The Type-A mutations were found to achieve minimal residual disease (MRD) earlier than non-type A mutations. However, non-type A mutations achieved better rates of medial survival (Heiblig et al., 2019).

D’Adda et al. (2019) evaluated the effect of the BCR-ABL transcript on efficacy of TKIs. The BCR-ABL1 fusion gene may cause CML pathogenesis due to several breakpoints; the most common occur around exon 13 and 14 of the BCR gene and cause the formation of e13a2 and e14a2 transcripts (Greenfield et al., 2019). Out of 173 sampled patients, 67 had the e13a2 transcript, and 106 had the e14a2 version. The patients with the e14a2 version were more likely to achieve a deep molecular response to TKIs (sustained or otherwise). After 68 months, the sustained deep molecular response (sDMR) rate was 39.6% for e14a2 patients compared to 19.6% for e13a2 patients. Overall, the maximum rate of sDMR for e13a2 patients was 37%, after 60 months. Furthermore, only two patients (three percent) with the e13a2 transcript achieved treatment-free remission (TFR) whereas 25 of e14a2 patients achieved TFR (23%) (D’Adda et al., 2019).

Xu et al. (2020) have analyzed data from 220 normal karyotype AML pediatric patients. Participants were selected from the Cancer Genome Atlas database. It was found that 12.7% of these patients had WT1 mutations, and that “the WT1-mutated group suffered lower rates of complete remission (CR) (P < 0.001 and P < 0.001, respectively) but higher rates of minimal residual disease (MRD) (P = 0.003 and P = 0.021, respectively) after both one and two courses of induction chemotherapy” (Xu et al., 2020). Patients with WT1 mutations also had significantly worse event-free and overall survival rates (P=0.007 and P<0.001, respectively) (Xu et al., 2020).

Sasaki et al. (2020) studied the impact of ASXL1, DNMT3A, JAK2, TET2, and TP53 mutations on survival in newly diagnosed acute myeloid leukemia (AML) patients. The 421 bone marrow aspirates were studied using NGS for these mutations with a minimum variant allele frequency (VAF) of 5%. "A total of 71 patients (17%) had ASXL1 mutations, 104 patients (25%) had DNMT3A mutations, 16 patients (4%) had JAK2 mutations, 82 patients (20%) had TET2 mutations, and 86 patients (20%) had TP53 mutations." The median VAF of ASXL1 was 34.31% (range, 1.17%‐58.62%), DNMT3A was 41.76% (range, 1.02%‐91.66%), JAK2 was 46.70% (range, 10.4%‐71.7%), TET2 was 42.78% (range, 2.26%‐95.32%), and TP53 was 45.47% (range, 1.15%‐93.74%). In patients with these mutations, the median overall survival was 11 months. In patients without these mutations, the overall survival was 27 months. The authors conclude that "The VAF of ASXL1, DNMT3A, JAK2, TET2, TP53, and NPM1 mutations is associated with worse prognosis in patients with newly diagnosed AML" (Sasaki et al., 2020).

Duncavage et al. (2021) investigated the clinical utility and accuracy of whole-genome sequencing (WGS) for the purpose of risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). The results from 263 patients were compared with findings from cytogenetic analysis and targeted sequencing. When conducting the WGS, they found that all 40 recurrent translocations and 91 copy-number alterations found on cytogenetic analysis were identified on WGS. There were also new clinically reportable genomic events among 17.0% of the patient sample. The standard AML risk groups, “as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes.” WGS was also able to classify patients with inconclusive cytogenetic analysis results into risk groups. This demonstrates that WGS could increase the diagnostic yield of AML and MDS and supplement the accuracy of cytogenetic analysis (Duncavage et al., 2021).

In a retrospective study, Yang reported the clinical significance of BCR-ABL1 mutations in patients with Philadelphia chromosome-positive CML who underwent allogeneic hematopoietic cell transplantation. Of 315 patients in this study, point mutations were detected in 152 patients. One hundred and one of these 152 patients (66%) had T315I mutations and 51 had mutations other than T315I. The patients were followed 38 months later and overall survival at three years was worse in patients with the mutations than the non-mutation group, especially in the chronic phase of CML. Overall survival in the non-T315I group was significantly worse than that in the no-mutation group. The authors conclude that "mortality risk was significantly higher in patients with the BCR-ABL1 mutation than in patients without the mutation” (Tachibana et al., 2022).

Guidelines and Recommendations

World Health Organization (WHO)

In a revision of the fourth edition on Classification of Tumors of Hematopoietic and Lymphoid Tissues, World Health Organization (WHO) incorporated new molecular genetic findings and clinical data into its classification of acute leukemias. WHO expanded on the prognostic significance of various gene mutations for each AML subtype. For example, for the AML with recurrent genetic abnormalities, inv(3) (q21.3; q26.2) does not represent a fusion gene, but rather a repositioning of a distal GATA2 enhancer leading to activation of MECOM (EVI1) expression and GATA2 haploinsufficiency. The AML with CEBPA mutation is defined based on biallelic mutation instead of single mutations because of prognostic significance. The provisional two categories are also added such as AML with RUNX1 for de novo AML without preexisting cytogenetic abnormalities associated with MDS and AML with BCR-ABL1 fusion gene. AML with NPM1 or biallelic CEBPA mutations and multilineage dysplasia are now considered separately instead of being a part of AML with myelodysplasia-related changes because of a lack of prognostic significance. The complete list for acute myeloid neoplasms 2016 WHO classification is shown in Figure 1 (Arber et al., 2016).

In the absence of JAK2, CALR, and MPL mutations, the presence of another clonal marker is included as one of the major diagnostic criteria for PMF. Additional mutation in ASXL1, EZH2, TET2, IDH1, IDH2, SRSF2 and SF3B1 genes are noted to be of use in determining the clonal nature of the disease.

In the fifth edition of the World Health Organization Classification of Hematolymphoid Tumors: Myeloid and Histiocytic/Dendritic Neoplasms, WHO places greater emphasis in “the separation of AML with defining genetic abnormalities from AML defined by differentiation”. The latter eschews the previously ambiguous categorical term AML NOS, and other notable changes include the “elimination of the 20% blast requirement for AML types with defining genetic abnormalities (with the exception of AML with BCR::ABL1 fusion and AML with CEBPA mutation)” and “the introduction of a section on AML with other defined genetic alterations, a landing spot for new and/or uncommon AML subtypes that may (or may not) become defined types in future editions of the classification” (Khoury et al., 2022).

Their list of acute myeloid leukemias is presented below.

Acute myeloid leukaemia, defined by differentiation

Acute myeloid leukaemia with minimal differentiation

Acute myeloid leukaemia without maturation

Acute myeloid leukaemia with maturation

Acute basophilic leukaemia

Acute myelomonocytic leukaemia

Acute monocytic leukaemia

Acute erythroid leukaemia

Acute megakaryoblastic leukaemia

Acute myeloid leukaemia with defining genetic abnormalities

Acute promyelocytic leukaemia with PML:RARA fusion

Acute myeloid leukaemia with RUNX1:RUNX1T1 fusion

Acute myeloid leukaemia with CBFB :: MYH11 fusion

Acute myeloid leukaemia with DEK-NUP214 fusion

Acute myeloid leukaemia with RBM15 :: MRTFA fusion

Acute myeloid leukaemia with BCR-ABLT fusion

Acute myeloid leukaemia with KMT2A rearrangement

Acute myeloid leukaemia with MECOM rearrangement

Acute myeloid leukaemia with NUP98 rearrangement

Acute myeloid leukaemia with NPM7 mutation

Acute myeloid leukaemia with CEBPA mutation

Acute myeloid leukaemia, myelodysplasia-related

Acute myeloid leukaemia with other defined genetic alterations

Moreover, in defining acute myeloid leukemia, myelodysplasia-related (AML-MR), WHO offers the following abnormalities and mutations of interest:

Cytogenetic and molecular abnormalities defining acute myeloid leukaemia, myelodysplasia-related.

Defining cytogenetic abnormalities

Complex karyotype [23 abnormalities)

5q deletion or loss of 5q due to unbalanced translocation

Monosomy 7, 7q deletion, or loss of 7q due to unbalanced translocation

11q deletion

12p deletion or loss of 12p due to unbalanced translocation

Monosomy 13 or 13q deletion

17p deletion or loss of 17p due to unbalanced translocation

Isochromosome 17q

idic(X)(q13)

Defining somatic mutations

ASXL1

BCOR

EZH2

SF3B1

SRSF2

STAG2

U2AF1

ZRSR2

The WHO published guidelines on the classification of myeloid neoplasms and acute leukemia. In the recent revision of the fourth edition on Classification of Tumors of Hematopoietic and Lymphoid issues, World Health Organization (WHO) incorporated new molecular genetic findings and clinical data into its classification of acute leukemias (Arber et al., 2016).

They state that: “With regard to chronic myeloid leukemia, BCR-ABL1+, most cases of CML in chronic phase can be diagnosed from peripheral blood (Papaemmanuil et al.) findings combined with detection of t (9;22) (q34.1; q11.2) or, more specifically, BCR-ABL1 by molecular genetic techniques. However, a bone marrow (BM) aspirate is essential to ensure sufficient material for a complete karyotype and for morphologic evaluation to confirm the phase of disease. In the era of tyrosine-kinase inhibitor (TKI) therapy, newly diagnosed patients may have a nearly normal lifespan, but regular monitoring for BCR-ABL1 burden and for evidence of genetic evolution and development of resistance to TKI therapy is essential to detect disease progression” (Arber et al., 2016).

They also introduced a provisional classification of ALL: B-ALL with translocations involving tyrosine kinases or cytokine receptors (“BCR-ABL1–like ALL”).

“This newly recognized entity is assuming increasing importance because of its association with an adverse prognosis and responses of some cases to TKI therapies; however, it has been difficult to define in the clinical setting. It was originally described separately by different groups who demonstrated a series of cases of poor-prognosis childhood ALL with gene expression profiles similar to those seen in cases of ALL with BCR-ABL1, though different algorithms applied to the same sets of cases did not classify all cases the same way.

The cases with translocations involving tyrosine kinase genes involve many different genes including ABL1 (with partners other than BCR), as well as other kinases including ABL2, PDGFRB, NTRK3, TYK2, CSF1R, and JAK2. Over 30 different partner genes have been described. Some patients, especially those with EBF1-PDGFRB translocations, have shown remarkable responses to TKI therapy, even after failing conventional therapy” (Arber et al., 2016).

College of American Pathologists (CAP) and American Society of Hematology (ASH)

Following recent progress in molecular genetic findings and 2016 WHO classification of acute leukemias, the College of American Pathologists (CAP) and the American Society of Hematology (ASH) have formed an expert panel to review and establish guidelines for appropriate laboratory testing. The published guideline provides twenty-seven guideline statements ranging from recommendations on what testing is appropriate for the diagnostic and prognostic evaluation of leukemias to where the testing should be performed and how results should be reported. The appropriate molecular genetic testing for AML is discussed starting from 16th guideline statement.

The expert panel strongly recommends testing for FLT3-ITD in adult and pediatric patients with suspected or confirmed AML of any type. They also recommend testing for other mutational analysis that could include, but not limited to, IDH1, IDH2, TET2, WT1, DNMT3A, and/or TP53 for prognostic and/or therapeutic purposes (Statement 16).

In the 17th guideline statement, expert panel strongly recommends testing for KIT mutation in adult patients with confirmed core-binding factor (CBF) AML (AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 or inv(16)(p13.1q22) /t(16;16)(p13.1;q22); CBFB-MYH11). It is only an expert consensus opinion for testing KIT mutation in pediatric patients with confirmed core binding factor AML (AML with t(8;21)(q22;q22.1); RUNX1- RUNX1T1 or inv(16)(p13.1q22) /t(16;16)(p13.1;q22); CBFB-MYH11) which is not a strong recommendation (Statement 17).

The strong recommendation is also given for patients other than those with confirmed core binding factor AML, APL, or AML with myelodysplasia-related cytogenetic abnormalities that testing is needed for mutational analysis for NPM1, CEBPA, and RUNX1 (Statement 19).

In the 20th guideline statement, expert panel is providing no recommendation on either for or against the use of global/gene specific methylation, microRNA (miRNA) expression, or gene expression analysis for diagnosis or prognosis in patients with confirmed acute leukemia.

Finally, in their last statement, expert panel strongly recommends the use of current WHO terminology for the final diagnosis and classification of acute leukemias (Arber et al., 2017).

American Society of Clinical Oncology (ASCO)

The ASCO has announced an endorsement of the 2017 CAP and ASH guideline regarding the initial diagnostic work-up of acute leukemia (de Haas et al., 2019). The ASCO supports all twenty-seven guideline statements. The statements relevant to this policy are noted in the 2017 CAP and ASH guidelines above.

National Comprehensive Cancer Network (NCCN)

Acute myeloid leukemia

For the initial evaluation of AML in individuals 18 years of age or older, the NCCN guidelines recommends bone marrow core biopsy and aspirate analyses (including immunophenotyping by immunohistochemistry stains plus flow cytometry) and analysis for chromosomal structural variations using cytogenetics, FISH, or whole genome sequencing. During the initial evaluation for AML, the NCCN also recommends “molecular analyses (ASXL1, c-KIT, FLT3 [ITD (internal tandem duplication) and TKD (tyrosine kinase domain)], NPM1, CEBPA [biallelic], IDH1, IDH2, RUNX1, TP53, and other mutations. A variety of gene mutations are associated with specific prognoses (category 2A) and may guide medical decision-making (category 2B). Other genetic lesions may have therapeutic significance.

The field of genomics in myeloid malignancies and related implications in AML are evolving rapidly. Mutations should be tested in all patients. Multiplex gene panels and targeted next-generation sequencing (NGS) analysis are recommended for the ongoing management of AML and various phases of treatment” (NCCN, 2023c).

Per the NCCN, the “importance of obtaining adequate samples of marrow or peripheral blood at diagnosis for full karyotyping and FISH cytogenetic analysis for the most common abnormalities cannot be overemphasized. Although FISH studies for common cytogenetic abnormalities may allow for rapid screening to identify either favorable- or unfavorable-risk groups, additional tests are needed to provide a full picture of the genetic factors that contribute to risk” (NCCN, 2023c).

As a part of monitoring during therapy for individuals with AML, the NCCN recommends a bone marrow aspirate/biopsy 14-21 days after the “start of therapy to document hypoplasia. If hypoplasia is not documented or indeterminate, repeat biopsy in 7–14 days to clarify persistence of leukemia. If hypoplasia, then repeat biopsy at time of hematologic recovery to document remission. If cytogenetics were initially abnormal, include cytogenetics as part of the remission documentation.” Furthermore, as a follow up to both standard-dose cytarabine induction therapy or high-dose cytarabine induction for poor-risk AML, the NCCN recommends that all patients should have bone marrow aspirate and biopsy by day 42 post-treatment, “regardless of the degree of hematologic recovery. When performed, BM aspirate and biopsy should include cytogenetic and molecular studies, as appropriate” (NCCN, 2023c).

As a part of surveillance and therapy for individuals with relapsed/refractory disease after the completion of consolidation, the NCCN recommends comprehensive genomic profiling to determine mutation status of actionable genes.

For the evaluation or workup for blastic plasmacytoid dendritic cell neoplasm in individuals 18 years of age or older, the NCCN recommends cytogenetic analysis (karyotype and/or FISH) and molecular analysis “(most common aberrations include: ASXL1, IDH1-2, IKZF1-3, NPM1, NRAS, TET1-2, TP53, U2AF1, ZEB2)”.

Chronic Myeloid Leukemia

The NCCN’s recommendations for CML include the following table:

Table 6. 2022 ELN risk classification by genetics at initial diagnosis*