The Challenges of Precision Medicine and New Advances in Molecular Diagnostic Testing in Hematolymphoid Malignancies: Impact on the VHA

For follow-up assessment of CML patients’ response to TKI treatment, qPCR for BCR-ABL1 should be tested with a peripheral blood sample or a bone marrow sample every 3 months.⁴ A peripheral blood sample is more commonly used because it is conveniently obtained. Early molecular response as indicated by a BCR-ABL1 transcript ratio of < 10% on the International Scale at 3 months, has a strong prognostic value.⁵ Major molecular response as indicated by a BCR-ABL1 transcript ratio of < 0.1% on the International Scale at 12 to 18 months is also highly prognostic.⁵

After the peripheral blood sample becomes negative for BCR-ABL1 by qPCR, testing bone marrow samples may be considered. If important treatment response benchmarks are not achieved, or response is lost with rising BCR-ABL1 levels (TKI resistance), ABL1 kinase domain mutation analysis as well as repeat FISH (to assess for copy number multiplication) should be performed to guide further management. Patients with the ABL1 T315I mutation are resistant to all first-line TKIs but may respond to later third-generation TKIs.⁶

BCR-ABL1–negative MPNs include PV, ET, and PMF. Bone marrow morphology remains the cornerstone of ET and PMF diagnosis. The discovery of JAK2, CALR, and MPL mutations has contributed to how these disorders are diagnosed.^7-12 Besides providing the clonality proof that is crucial for diagnosis, the molecular markers influence the prognosis. The JAK2 (p.V617F) or less common JAK2 exon 12 mutations, which are detected in more than 95% of PV cases, are used as molecular markers to confirm diagnosis.⁷ Further, the JAK2 (p.V617F), CALR (exon 9), and MPL (exon 10) mutations are detected in ET (~60%, 25%, and 3%-5%, respectively) and PMF (~55%, 30%, and 5%, respectively).¹² If ET or PMF is suspected clinically, first JAK2 (p.V617F) mutation analysis should be performed, then CALR mutation analysis, and finally MPL mutation analysis. Although novel gain-of-function JAK2 and MPL mutations were recently discovered in triple-negative ET (negative for canonical mutations in JAK2, CALR, and MPL) and PMF by whole exome sequencing,¹³ clinical testing is not readily available. Besides its utility in the initial diagnosis of ET and PMF, the JAK2 or CALR mutation assay also may be considered for bone marrow transplantation follow-up (Table).¹⁴

Despite the continuing debate on the classification of eosinophilic myeloid disorders, the discovery of the FIP1L1-PDGFRA fusion represents a major milestone in the understanding of these disorders.^15,16 Unlike PDGFRB (5q33) and FGFR1 (8p11) rearrangements, which can be detected with routine chromosomal analysis (cytogenetics), the cryptic FIP1L1-PDGFRA fusion must be detected with FISH (for CHIC2 deletion) or RT-PCR analysis. It should be pointed out that, as most eosinophilia is reactive or secondary, molecular testing for FIP1L1-PDGFRA fusion is indicated only when primary hypereosinophilia or hypereosinophilic syndrome (HES) is suspected. This is particularly the case in the following hypereosinophilia accompanying conditions: CML-like morphology, but BCR-ABL1–negative; chronic myelomonocytic leukemia (CMML)–like morphology with a normal karyotype; and new onset of cardiac damage or dysfunction.¹⁷

Primary eosinophilic myeloid disorders with PDGFRA or PDGFRB rearrangements can be treated with TKIs (eg, imatinib). Next-generation sequencing may be considered in cases of presumed HES when there is no identifiable karyotypic or FISH abnormality. Recent studies have found that cases of HES with somatic mutations indicating clonality had adverse clinical outcomes similar to those of cases of chronic eosinophilic leukemia.¹⁸

The discovery of CSF3R mutations offers a new molecular marker for the diagnosis of chronic neutrophilic leukemia (CNL), an MPN.¹⁹ The CSF3R (p.T618I) mutation or another activating CSF3R mutation is now used as a diagnostic criterion for CNL. Identification of specific CSF3R mutations may have therapeutic implications as well. The test should be ordered only for patients with clinical and morphologic findings suggestive of CNL; reactive neutrophilic leukocytosis (eg, infection, inflammation) should be ruled out before the test is ordered.

Myelodysplastic Syndrome

Myelodysplastic syndrome is a group of clonal bone marrow disorders characterized by ineffective hematopoiesis, manifested by morphologic dysplasia in ≥ 1 hematopoietic lineages and peripheral cytopenias (hemoglobin level, < 10 g/dL; platelet count, < 100×10³/µL; absolute neutrophil count, < 1.8×10³/µL). Diagnosis and classification of MDS depend mainly on the degree of morphologic dysplasia and blast percentages, as determined by examining well-prepared cellular bone marrow aspirate smears and/or biopsy touch preparations and peripheral blood smears.

Conventional karyotyping is an essential part of the diagnostic workup for all presumptive cases of MDS and is of both diagnostic and prognostic importance.²⁰ About 60% of MDS cases have recurrent cytogenetic abnormalities, which can be detected with conventional karyotyping. If a high-quality cytogenetic analysis cannot be performed (eg, the bone marrow sample is inadequate), or if quick turnaround is required, an alternative FISH panel may be used to detect some of the common MDS-associated chromosomal abnormalities (eg, 5q deletion, 7q deletion/monosomy 7, +8, 20q deletion).²¹ Sequencing with FISH also can be useful for assessing MRD by detecting a previously identified chromosomal abnormality.

Targeted sequencing of a limited number of genes can detect mutations in the vast majority of patients with MDS. The most commonly mutated genes in MDS are SF3B1, TET2, SRSF2, ASXL1, DNMT3A, RUNX1, U2AF1, TP53, and EZH2. Mutations in SRSF2 cause RNA splicing abnormalities. In addition, mutations in TP53, EZH2, RUNX1, and ASXL1 are associated with poor prognosis,^22,23 whereas mutations in SF3B1 confer better event-free survival.²⁴ Despite these developments, the HMG subcommittee agreed that NGS-based mutation panels are not cost-effective for the VA population at this time and should not be included in a MDS workup. Only in rare situations and when clinically indicated (to change disease classification or patient management) should evaluation for specific gene mutations be considered—for instance, the SF3B1 mutation for patients with probable MDS with ring sideroblasts, if ring sideroblasts are < 15%.²⁵

Myelodysplastic/Myeloproliferative Neoplasms

Myelodysplastic/myeloproliferative neoplasms are a group of myeloid neoplasms with clinical, laboratory, and morphologic features that overlap both MDS and MPN. In MDS/MPN, the karyotype is often normal or shows abnormalities in common with MDS.

In cases of unexplained monocytosis for which there is clinical concern for CMML, morphologic evaluation and conventional chromosomal karyotyping should be performed after other secondary causes and known myeloproliferative and myelodysplastic entities have been excluded. If concomitant hypereosinophilia is present and the karyotype is normal, FISH or PCR-based assay should be performed to rule out FIP1L1-PDGFRA rearrangements. BCR-ABL1, PDGFRB, FGFR1, and t(8;9)/PCM1-JAK2 rearrangements typically are detected with high-quality cytogenetic analysis and thus do not require targeted molecular assays. Although certain gene mutations (eg, SRSF2, TET2, ASXL1, CBL) are commonly detected in CMML, the HMG subcommittee does not recommend sequencing-based mutation panels, as there is insufficient information for testing for prognostic or treatment stratification.

If MDS/MPN with ring sideroblasts and thrombocytosis is suspected on the basis of the clinical and morphologic criteria, molecular tests for the JAK2 (p.V617F) and SF3B1 mutations may be considered in an effort to help confirm the diagnosis.

Atypical CML is a rare MDS/MPN subtype that is now better characterized molecularly with SETBP1 and/or ETNK1 mutations, which are detectable in up to a third of cases. If clinical suspicion is high, sequencing may be diagnostically helpful.

Lymphoid Neoplasms

Chronic Lymphocytic Leukemia

In CLL, recurrent chromosomal abnormalities (eg, deletions of 13q, trisomy 12, deletions of 11q, deletions of 17p) have clear prognostic value and can be detected with FISH. Other prognostic information, such as somatic mutation of immunoglobulin heavy chain variable (IgHV) genes, TP53 mutations, SF3B1, and NOTCH1 mutation, are mostly derived from PCR-based assays. The discovery of recurrently mutated genes in CLL has increased with the use of highly sensitive sequencing methods constructing a more detailed landscape of CLL at genetic, epigenetic, and cellular levels. A recent literature review summarizes the vast heterogeneity of CLL with recurrent pathogenetic findings in MYD88, SF3B1, TP53, ATM, and NOTCH1 signaling pathways.²⁶ The treatment of CLL is rapidly evolving, and many clinical trials are proposing a change from the “watch and wait” paradigm to treatment upon initial presentation based on molecular findings. Additional testing based on new treatment options from current clinical trials will be recommended.