Mature B-Cell Lymphomas

Classification

The World Health Organization (WHO) and the International Consensus Classification (ICC) have classification systems for B-cell lymphomas that integrate morphologic, immunophenotypic, genetic, and clinical features. ^, 

Indications for Testing

The presentation of B-cell lymphomas varies widely among patients but may involve lymphadenopathy, extranodal masses, B symptoms (e.g., fever, night sweats, unintentional weight loss), fatigue, recurrent infections, hepatosplenomegaly, skin rashes, and/or lab abnormalities such as cytopenias. 

Laboratory Testing

Tissue Biopsy

Excisional or incisional biopsy is preferred for definitive diagnosis and histologic grading.  Fine needle aspiration (FNA) is insufficient for the initial diagnosis of lymphoma, and histologic grading cannot be performed on an FNA specimen. Core needle biopsies are an appropriate alternative when excisional or incisional biopsy cannot be performed and may be used in conjunction with FNA and ancillary testing to make a diagnosis.

Lymphoma Phenotyping

Immunophenotyping by flow cytometry and/or IHC is essential for the diagnosis of various subtypes of B-cell lymphomas, as it allows for the identification of surface antigens, which can indicate the cell of origin and any immunophenotypic aberrancies.  Flow cytometry is generally performed on peripheral blood and/or tissue biopsy specimens, but it is occasionally necessary to perform on cerebrospinal fluid (CSF) and bone marrow specimens. The National Comprehensive Cancer Network (NCCN) recommends using morphology and clinical presentation to guide the selection of markers. Commonly ordered markers involved in the initial evaluation of B-cell lymphomas include CD3, CD5, CD10, CD11c, CD19, CD20, CD21, CD22, CD23, CD79a, PAX5, BCL2, BCL6, IRF4/MUM1, cyclin D1, and Ki-67. The NCCN also has specific recommendations regarding additional markers and the workup for B-cell lymphomas. 

B-Cell Clonality Screening

B-cell lymphoproliferative disorders encompass both clonal and nonclonal processes. Given that B-cell lymphomas are clonal expansions of mature or immature B lymphocytes, establishing clonality may be a key part of the diagnosis. ^,  In addition to flow cytometry, clonality may be assessed using B-cell clonality screening, which uses multiplex polymerase chain reaction (PCR) to detect monoclonal rearrangements in immunoglobulin heavy chain (IgH) and immunoglobulin kappa light chain (IgK).  Although monoclonal rearrangements may support the diagnosis of lymphoma, they are not entirely specific and can also occur with infections, autoimmune diseases, and other sources of chronic inflammation. Conversely, a negative result does not exclude a diagnosis of lymphoma. As such, B-cell clonality testing can be helpful but should be supported by morphologic and immunophenotypic testing to detect abnormal B-cell populations. If other methods fail to identify a clonal process and suspicion remains high, NGS may be used. 

Molecular Genetics

Molecular genetic or cytogenetic analysis may be used to detect somatic mutations or chromosomal rearrangements to make a diagnosis, assess prognosis, establish clonality, and/or detect minimal residual disease (MRD) in some B-cell lymphomas. ^,  Many genetic abnormalities (i.e., chromosomal translocations, mutations, copy number variations, etc.) are not specific for a single entity and may be seen in several different types of lymphoma.  In general, FISH and karyotyping are the best modalities for detecting translocations, whereas PCR is useful for detecting and quantifying point mutations.

MRD testing in lymphoma may be used to monitor treatment and detect molecular relapses during periods of clinical remission.  The methodologies used to detect MRD are highly sensitive and specific to the lymphoma subtype and include FISH, real-time quantitative PCR, digital droplet PCR, multicolor flow cytometry, and NGS.

MYC, BCL2, and/or BCL6 Gene Rearrangements

B-cell lymphomas with two or three recurrent chromosomal breakpoint aberrations (rare) are referred to as double- or triple-hit lymphomas. It is important to identify these high-grade lymphomas because they tend to manifest aggressive behavior and respond poorly to traditional chemotherapy.  Double-hit lymphomas involve oncogenic rearrangements targeting MYC and BCL2 genes. The WHO classifies these entities as “diffuse large B-cell lymphoma/high-grade B-cell lymphoma with MYC and BCL2 rearrangements.”  The ICC classifies them as “high-grade B-cell lymphoma, with MYC and BCL2 rearrangements.”  Previously, if a BCL6 rearrangement was present in addition to both MYC and BCL2, the lymphoma was considered to be a triple-hit lymphoma, and if a BCL6 rearrangement was only present with MYC, the lymphoma was a double-hit lymphoma. The WHO notes that the majority of those cases were found to have BLC6::MYC reciprocal translocations and are now considered to be “pseudo-triple-hit” and “pseudo-MYC/BCL6 double-hit,” as the impact of translocation on locus regulation is unknown.  The ICC maintains “high-grade B-cell lymphoma, with MYC and BCL6 rearrangements” as a separate provisional entity. 

IRF4 Rearrangement

Large B-cell lymphoma with IRF4 rearrangement is a low-grade entity that occurs most commonly in children and young adults.  IRF4 translocations define a subset of large B-cell lymphomas. Rearrangement of IRF4 may be confirmed by FISH or karyotyping. ^,  Strong IRF4/DUSP22 expression is usually seen with BCL6 and a high proliferation index. It is important to identify this disease because it is more aggressive than pediatric-type follicular lymphomas (FLs), but patients do well when treated. Large B-cell lymphomas with IRF4 rearrangement should also be distinguished from CD10-negative, IRF4/MUM1-positive FL, which is associated with diffuse large B-cell lymphomas (DLBCLs) in older adults.

Other Genetic Markers

Mutations in MYD88, particularly L265P, are supportive of lymphoplasmacytic lymphoma/Waldenström macroglobulinemia; however, mutations in MYD88 may occur in other lymphomas as well. CCND1 rearrangements are often associated with mantle cell lymphoma (MCL). Cyclin D1 immunohistochemical staining is commonly used as a surrogate marker for CCND1 rearrangements. If morphology or immunophenotype differ from what is expected for MCL, the rearrangement should be confirmed through FISH.