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

Flow cytometry and morphology are standard for CLL diagnosis. The HMG subcommittee recommends FISH for del(13q14), del(11q), trisomy 12, and del(17p) at time of diagnosis or immediately before therapy initiation. Zeta-chain (ζ-chain) associated protein 70 (ZAP-70) by flow cytometry and IgHV mutation status are optional (use depends on test availability). For high-risk CLL cases, PCR-based or sequencing-based assays should be used to detect the TP53 mutation, especially in CLL patients who are candidates for treatment with recently approved CLL-targeted therapies such as ibrutinib (irreversible inhibitor of Bruton tyrosine kinase) and idelalisib (PI3Kγ inhibitor). Recent studies have shown that NOTCH1 and SF3B1 mutations may have prognostic significance, but routine testing is not recommended at this time.^27-29

Other B-Cell Lymphoproliferative Disorders

Unlike the common molecular changes in CLL, in other mature B-cell lymphomas, chromosomal translocations that juxtapose a variety of different oncogenes next to an Ig gene enhancer usually are—and those that switch regions less commonly are—important initiating events that can be detected with PCR, DNA sequencing, or FISH. In follicular lymphoma (FL), Burkitt lymphoma, marginal zone lymphoma (MZL), and mantle cell lymphoma (MCL), these oncogenes driven by an Ig gene enhancer typically include BCL2, MYC, MALT1, and CCND1 (cyclin D1), respectively. Molecular variants of these lymphomas that lack these classical translocations often activate homologous genes (eg, cyclin D3/CCND3 is activated in variants of MCL).

Morphology, flow cytometry, and IHC are routinely used for diagnosis. In inconclusive cases, Ig gene rearrangement by PCR may be used. The Table summarizes common molecular changes in B-cell lymphomas.

Mantle cell lymphoma. MCL is a non-Hodgkin lymphoma subtype characterized by t(11;14) (q13;q32) translocations that in the majority of cases lead to overexpression of cyclin D1 (BCL1). Recent molecular profiling has identified an MCL variant that is cyclin D1–negative but SOX11-positive and may have a more aggressive clinical course.³⁰ SOX11 regulates PAX5 expression and blocks terminal B-cell differentiation in aggressive MCL.

Lymphoplasmacytic lymphoma. Lymphoplasmacytic lymphoma (LPL), MZL, and CLL/small lymphocytic lymphoma are well-defined clinicopathologic entities. However, distinguishing LPL from MZL and atypical cases of CLL can sometimes be difficult because of overlapping clinical and morphologic features. Recent studies have identified a recurrent L265P mutation in the MYD88 gene in 90% to 95% of LPL cases with IgM paraprotein and in 40% to 50% of the rare non-IgM LPL cases. In contrast, the mutation is much less frequently present in MZL and other low-grade B-cell neoplasms (2%-7%).³¹ Therefore, testing for this abnormality can be a diagnostic aid in these difficult-to-classify cases. In addition, from a therapeutic perspective, presence or absence of MYD88 mutation may prove more significant than presence of a specific paraprotein or histopathologic features. Ibrutinib has shown efficacy in LPL and demonstrates improved response rates in patients with MYD88 mutation compared with that of their mutation-negative counterparts.³² Several MYD88 inhibitors are in clinical trials. This again indicates the need to more accurately identify and subclassify these non-IgM LPL cases to ensure appropriate molecular evaluation.

Hairy cell leukemia. Flow cytometry and morphology are usually sufficient for a hairy cell leukemia (HCL) diagnosis. However, rare cases are difficult to distinguish variant HCL from other mimics. The BRAF V600E mutation recently was described as a disease-defining molecular marker for HCL—present in nearly all HCL cases but virtually absent in HCL mimics. Therefore, detection of the BRAF mutation by IHC stain with specific antibody or PCR analysis is highly sensitive and specific for the diagnosis of HCL.³³

Diffuse large B-cell lymphoma. Recent molecular analysis has created various risk stratification schemata for diffuse large B-cell lymphoma (DLBCL). The HGM subcommittee agrees that well-preserved morphology, IHC, flow cytometry, and FISH-specific markers (BCL2, BCL6, cMYC) are sufficient for diagnostic, prognostic, and therapeutic purposes. Although a wide range of genes have been implicated in the pathogenesis of DLBCL, sequencing and gene expression profiling are not cost-effective at this time and do not add benefit to patient treatment.

The MYD88 L265P mutation has been identified in DLBCL, particularly the activated B-cell-like type and primary central nervous system lymphoma (PCNSL), and may have implications for ibrutinib therapy. PCNSL commonly manifests aggressive clinical behavior and has a poor prognosis. It has been proposed that the MYD88 mutation can be used as a genetic hallmark for PCNSL to distinguish CNS involvement by systemic DLBCL from PCNSL.³⁴

Plasma cell neoplasms. Flow cytometry is acceptable for the diagnosis of plasma cell neoplasms and for residual disease follow-up. Chromosomal karyotype or FISH for IGH/CCND1, IGH/MMSET, and IGH/CMAF dual fusion probes is recommended in conjunction with morphology, IHC, and flow cytometry. In plasma cell myeloma, several genetic mutations can be detected with NGS, including mutations in NRAS, KRAS, TP53, BCL7A, DIS3, and FAM46C.³⁵ Less commonly, BRAF mutations, previously described in melanoma and several other solid tumors, can be detected with DNA sequencing in 4% of multiple myeloma cases, which may prove promising for targeted therapy with BRAF inhibitors. However, current therapeutic decisions are based on genetic and clinical factors, and sequence-based assays are not recommended at this time.

Follicular lymphoma. Cytology, histology, and IHC typically are sufficient for diagnosing FL. In difficult-to-diagnose cases and in cases with scant material, additional tests may help with diagnosis. Eighty to ninety percent of FL cases have t(14;18)(q32;q21), which places the BCL2 gene transcription under the control of the IGH promoter. In addition, about 10% of FL cases have 3q27 aberrancies at the BCL6 gene.^36-38 More recently, cases of FL with bulky inguinal disease negative for IGH-BCL2 and BCL6 translocations were found to have 1p36 deletions. These 1p36-deleted FLs typically have a diffuse pattern and a good prognosis.³⁹ For t(14;18), 3q27, or 1p36, FISH is a sensitive means for detecting these translocations, as is PCR for IGH-BCL2.⁴⁰ There are reports that t(14;18) can be detected in a substantial fraction of otherwise healthy donors at levels and rates that depend on the type of detection test used.^41-43 In addition, between one-fourth and one-third of de novo DLBCLs show t(14;18), and about one-third show BCL6 abnormalities at 3q27. Therefore, these genetic changes are not specific for FL and should not be used to subtype a lymphoma as follicular in origin.

Use of IGH-BCL2 as a marker for MRD is still controversial. Some studies have found that a postinduction and posttransplantation IGH-BCL2-positive finding by PCR predicted relapse.^44,45 However, others studies have not found significance to postinduction IGH-BCL2 positivity.⁴⁶ The NCCN guidelines recommend testing for IGH-BCL2 or BCL6 translocations or 1p36 deletion only if this testing is needed for diagnosis. The guidelines do not recommend using these genetic assays in follow-up biopsies, as the importance of treating early relapse has not been definitively demonstrated.

Therefore, if a lymphoma has morphologic, histologic, and IHC findings consistent with FL, then cytogenetic, FISH, or PCR testing is not needed for diagnosis but may be used as confirmation. Follow-up molecular and cytogenetic testing should be avoided if the original cytogenetic abnormality is unknown. That is, IGH-BCL2 FISH should be performed in follow-up samples only if the original lymphoma is known to contain the translocation. As follow-up genetic testing is of disputed clinical significance even in cases in which the original molecular change is known, the NCCN recommendations for therapy are no different. The HMG subcommittee does not recommend molecular or cytogenetic testing in FL beyond what is required for initial diagnosis.

T-Cell Lymphomas

Mature T-Cell Lymphoma and Leukemia

For mature T-cell lymphoma (TCL) and leukemia, the clinical and morphologic criteria have a very important role in the initial workup. However, IHC immunophenotyping is crucial for definitive diagnosis and subclassification. Flow cytometry is routinely used in diagnosing diseases such as T-cell prolymphocytic leukemia (TPLL), T-cell large granular lymphocytic (LGL) leukemia, and Sézary syndrome. T-cell clonality studies, preferably with BIOMED-II–validated primers against targets such as T-cell receptor γ (TCR-γ) and TCR-β, are commonly used as ancillary tests in the evaluation of TCL and T-cell leukemia. Clonality testing, however, comes with an important caveat: A gene rearrangement study is never a substitute for thorough morphologic and immunophenotypic evaluation. Clonality is not proof of malignancy.

Significant advances in TCL classification have led to revisions and the inclusion of new provisional entities in the 2016 World Health Organization classification of lymphoid neoplasms.⁴⁷ Many of these changes originated in studies of gene expression profiling and the genetic landscape of T-cell neoplasms. Even though subsets of peripheral TCL not otherwise specified (PTCL-NOS) have been recognized on the basis of phenotypic and molecular abnormalities with possible clinical implications, in most cases molecular testing is not part of routine practice. Typically, only a few cytogenetic abnormalities and genetic mutations are used in the evaluation of TCL and T-cell leukemia.