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FeedbackMature B-cell non-Hodgkin lymphomas (NHLs) represent a heterogeneous group of hematologic malignancies of lymphoid cells. The diagnostic approach to mature B-cell lymphomas generally involves biopsy and phenotyping by flow cytometry and/or immunohistochemistry (IHC), as well as cytogenetic studies.
Quick Answers for Clinicians
A comprehensive patient history and physical exam, including a full skin examination and attention to node-bearing areas, the liver, and the spleen, are recommended for the initial workup of B-cell lymphomas. Initial laboratory testing should include a CBC with differential, lactate dehydrogenase (LDH) testing, a comprehensive metabolic panel, and uric acid testing. HIV and hepatitis C virus (HCV) testing may be added for at-risk patients. Additionally, a bone marrow biopsy with aspirate, a positron emission tomography (PET)/computed tomography (CT) scan and/or chest/abdominal/pelvic CT scan, and an echocardiogram may be informative and help guide treatment. Additional testing may be indicated based on the suspected classification. Molecular studies (B-cell clonality testing) may be helpful when the differential diagnosis includes a B-cell lymphoma versus a benign/reactive process (chronic inflammatory infiltrate).
Epstein-Barr virus (EBV) is associated with endemic and sporadic variants of Burkitt lymphoma (BL), a subset of diffuse large B-cell lymphomas (DLBCLs) in adults, a subset of posttransplant lymphoproliferative disorders, and some lymphomas in other immunocompromised patients. EBV testing is recommended in certain circumstances to guide diagnosis and proper treatment. Studies have demonstrated a strong association between hepatitis C virus (HCV) seropositivity and B-cell lymphomas, particularly DLBCL and marginal zone lymphoma. Testing is recommended in high-risk patients and in patients with splenic marginal zone lymphoma. HIV infection can lead to development of AIDS-related lymphomas. AIDS-related B-cell lymphomas include DLBCL, BL, primary central nervous system lymphoma, plasmablastic lymphoma, and primary effusion lymphoma. Human herpes virus 8 (HHV-8) is also associated with AIDS-related DLBCL and primary effusion lymphoma. Helicobacter pylori is associated with gastric extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT). Treatment with antibiotics can lead to tumor remission. Other bacteria less commonly associated with marginal zone lymphomas include Chlamydia psittaci, Campylobacter jejuni, and Borrelia burgdorferi.
The National Comprehensive Cancer Network (NCCN) B-Cell Lymphomas and Pediatric Aggressive Mature B-Cell Lymphomas guidelines provide detailed recommendations for specific B-cell lymphomas, including follicular lymphoma, marginal zone lymphomas, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphomas, Burkitt lymphoma (BL), AIDS-related B-cell lymphomas, lymphoblastic lymphoma, posttransplant lymphoproliferative disorders, and Castleman disease, and also provide pediatric-specific guidelines for DLBCL and BL. The World Health Organization (WHO) and the European Organisation for Research and Treatment of Cancer (EORTC) have provided detailed information about the classification of various B-cell lymphomas.
Several published indices are available to assess prognosis for the various B-cell lymphomas. Proper classification is also important for appropriate treatment and prognostication, given that many of these lymphomas have different biologic behavior and outcomes. Prognostic indices for follicular lymphoma include the Follicular Lymphoma International Prognostic Index (FLIPI-1 and FLIPI-2). These prognostic indices include factors such as age, Ann Arbor stage, number of nodal sites involved, hemoglobin level, serum lactate dehydrogenase (LDH) level, size of largest involved lymph node, beta-2 microglobulin level, and bone marrow involvement to predict prognostic risk. A simpler index includes just baseline serum beta-2 microglobulin and LDH levels. Prognostic indices for diffuse large B-cell lymphoma (DLBCL) include the International Prognostic Index (IPI) and, for patients <60 years old, the revised IPI (R-IPI). The scores are based on stage of disease, serum LDH, Eastern Cooperative Oncology Group (ECOG) performance status, and the number of extranodal sites. A National Comprehensive Cancer Network (NCCN) IPI has been developed and stratifies patients into four risk groups using similar factors. Genetic risk factors should also be considered when determining prognostic risk.
Classification
The World Health Organization (WHO) has proposed a classification for B-cell lymphomas that integrates morphologic, immunophenotypic, genetic, and clinical features. The table below details a selected subset of the current WHO classifications for B-cell lymphomas, separated into clinical subtypes covered by the National Comprehensive Cancer Network (NCCN) B-cell lymphomas guidelines.
| Disease Type | Classifications |
|---|---|
| Indolent |
Follicular lymphoma Marginal zone lymphomas
Lymphoplasmacytic lymphoma
|
| Aggressive |
Mantle cell lymphoma DLBCL BL |
|
BL, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; MALT, mucosa-associated lymphoid tissue |
|
Indications for Testing
The presentation of B-cell lymphomas varies widely among patients but may involve lymphadenopathy, fatigue, fever, recurrent infections, hepatosplenomegaly, eosinophilia, skin rash, leukemic manifestations, extranodal masses, or increased white blood cell (WBC) count. Phenotyping is necessary in most situations, and genetic studies may also be recommended. Specific diagnosis is important to determine proper medical management.
Laboratory Testing
Tissue Biopsy
Excisional or incisional biopsy is typically required for initial evaluation. Fine needle aspiration (FNA) is generally not adequate for the initial diagnosis of lymphoma, and histologic grading cannot be performed on an FNA specimen. Core needle biopsies are not optimal but can sometimes be used. If the lymph node of interest is not accessible for excisional or incisional biopsy, a combination of FNA, core biopsy, and ancillary testing may be sufficient for diagnosis.
Lymphoma Phenotyping
Phenotyping by flow cytometry can identify surface antigens on cells that can indicate the cell of origin and any immunophenotypic aberrancies. Flow cytometry is generally performed on peripheral blood, bone marrow, or tissue samples. The NCCN recommends investigation of the following markers in the initial evaluation of B-cell lymphomas: CD45, CD3, CD5, CD19, CD10, CD20, CD30, CD4, CD8, CD7, CD2, CD23, CD43, CD103, TDT, kappa, lambda, CD13, CD33, CD1a, cytoplasmic CD3, CD22, and myeloperoxidase. Investigation of additional markers may be useful to characterize specific classifications.
IHC staining may also be utilized for lymphoma phenotyping. The most commonly used stains offered at ARUP are cyclin D1, Pax-5, Ki-67, MUM1-IRF4, HHV8, EBV, ALK-1, CD3, CD5, CD10 (CALLA), CD19, CD20, CD21 (dendritic cell), CD22, CD23, CD25, CD30 (Ki-1), CD79A, CD138 (Syndecan-1), BCL-2, kappa and lambda light chains, and TdT. Other available stains include BOB-1, caspase-3, CD15, CD43, CD45, CD45RA-MT2, CD45RO, c-Myc, DBA.44, and Oct-2. For more information, refer to ARUP’s Immunohistochemistry Stain Offerings.
B-Cell Clonality Screening
Polyclonal immunoglobulin heavy chain (IgH) and immunoglobulin kappa light chain (IgK) gene rearrangements are characteristic of benign or reactive disease, whereas monoclonal rearrangements may indicate a lymphoma, although they are not entirely specific and can also be seen with infections, autoimmune diseases, and other sources of chronic inflammation. Conversely, a negative result does not exclude a lymphoma diagnosis. As such, B-cell clonality testing can be helpful but should be supported by cytologic and immunophenotypic testing to detect abnormal B-cell populations. This testing should be performed if morphology and immunophenotyping aren’t sufficient to determine the nature of the lymphoproliferative process. This testing also can be useful to monitor disease status after treatment.
Molecular Genetics
Genetic tests to detect somatic mutations or structural abnormalities are often informative and, in some cases, are essential for accurate and precise diagnosis and prognostic assessment of B-cell lymphomas. In general, fluorescence in situ hybridization (FISH) and karyotyping are the best modalities for detecting translocations, whereas polymerase chain reaction (PCR) is useful for detecting and quantifying point mutations.
| Disease | Genetics |
|---|---|
| High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, and BL | MYC, BCL2, BCL6 |
| BL | MYC t(8;14), t(2;8), t(8;22) |
| DLBCL | BCL6, BCL2, IRF4/MUM1 |
| Follicular lymphoma | BCL2 t(14;18)(q32;q21), IRF4/MUM1 |
| Hairy cell leukemia | BRAF V600E |
| Mantle cell lymphoma | BCL1 (CCND1) t(11;14)(q13;q32) |
| Marginal zone lymphoma (nodal, MALT, splenic) | MYD88 |
| MALT (gastric) | t(11;14), t(14;18), t(1;14), t(3;14), t(11:18) |
MYC, BCL2, and BCL6 Gene Rearrangements
B-cell lymphomas with two or three recurrent chromosomal breakpoint aberrations (rare) are referred to as double- or triple-hit lymphomas. These usually involve the MYC oncogene in association with BCL2 and/or BCL6 gene rearrangements and are classified by WHO as a “high-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements.” It is important to identify these high-grade lymphomas because they tend to manifest aggressive behavior and respond poorly to traditional chemotherapy.
IRF4/DUSP22 Gene Rearrangement
Large B-cell lymphoma with IRF4 rearrangement is a recently described, low-grade entity that occurs most commonly in children and young adults. 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, but patients do well when treated. Large B-cell lymphomas with IRF4 rearrangement should also be distinguished from CD10-negative, IRF4/MUM1-positive follicular lymphoma, which is associated with DLBCLs in older adults.
ARUP Laboratory Tests
Aids in evaluation of hematopoietic neoplasms (ie, leukemia, lymphoma)
Specimens include bone marrow, whole blood, tissue, or fluid
Use to monitor therapy in patients with established diagnosis of hematopoietic neoplasms
Flow Cytometry
Aids in the diagnosis of B-cell lymphoproliferative disorders
Capillary Electrophoresis
Aids in the diagnosis of aggressive large B-cell lymphoma with intermediate features between BL and DLBCL and confirmation of suspected double-hit lymphoma, in conjunction with clinical, morphologic, and flow cytometric data
Fluorescence in situ Hybridization (FISH)
Use to diagnose BL or DLBCL with features intermediate between BL and DLBCL, in conjunction with clinical, morphologic, and flow cytometric data
Negative result does not rule out BL or B-cell lymphomas with features intermediate between BL and DLBCL that involve MYC with other translocation partners, such as t(2;8) or t(8;22)
Fluorescence in situ Hybridization (FISH)
Aids in the diagnosis of aggressive large B-cell lymphoma with intermediate features between BL and DLBCL and confirmation of suspected double-hit lymphoma, in conjunction with clinical, morphologic, and flow cytometric data
Detects all MYC rearrangements, including t(8;14), t(2;8), and t(8;22) rearrangements; does not identify translocation partner
Fluorescence in situ Hybridization (FISH)
Aids in the diagnosis of aggressive large B-cell lymphoma with intermediate features between BL and DLBCL and confirmation of suspected double-hit lymphoma, in conjunction with clinical, morphologic, and flow cytometric data
Can also be found in follicular lymphoma
Fluorescence in situ Hybridization (FISH)
Aids in the diagnosis of aggressive large B-cell lymphoma with intermediate features between BL and DLBCL and confirmation of suspected double-hit lymphoma
MYC and/or BCL2 overexpression can be due to other mechanisms not detected by this test
Specimen type: formalin-fixed paraffin-embedded tissue
Fluorescence in situ Hybridization (FISH)
Aids in the diagnosis of aggressive large B-cell lymphoma with intermediate features between BL and DLBCL and confirmation of suspected double-hit lymphoma
Probes in this panel include MYC, BCL2, IGH, BCL6
MYC and/or BCL2 overexpression can be due to other mechanisms not detected by this test
Chromosome alterations outside probe region are not detected
Specimen type: fresh tissue
Fluorescence in situ Hybridization (FISH)
Use to detect IRF4/DUSP22 rearrangements, which can contribute to diagnosis and prognosis in large B-cell lymphomas with IRF4 rearrangement
Fluorescence in situ Hybridization (FISH)
Use to order individual or multiple oncology FISH probes if standard FISH panels are not desired
Fluorescence in situ Hybridization (FISH)
Alternate test to detect prognostically important genomic abnormalities in chronic lymphocytic leukemia (CLL)
Probes include ATM (11q22.3), chromosome 12 centromere (trisomy 12), D13S319 (13q14.3), and p53 (17p13.1)
Panel only detects prognostically important imbalances (gain or loss of DNA) in the chromosomes of interest
Chromosome alterations outside the regions complementary to these FISH probes will not be detected
Ideal testing is when significant disease is present
Fluorescence in situ Hybridization (FISH)
Aids in the diagnosis of mantle cell lymphoma if cyclin testing is noninformative
Fluorescence in situ Hybridization (FISH)
Use to confirm diagnosis of hairy cell leukemia and monitor tumor burden
Polymerase Chain Reaction
Useful in distinguishing lymphoplasmacytic lymphoma (LPL) from other low-grade B-cell lymphoproliferative disorders and for monitoring patients with LPL diagnosis and previously identified MYD88 L265P mutation
Real-time Polymerase Chain Reaction
Use to determine risk group in newly diagnosed CLL
Polymerase Chain Reaction/Sequencing
Preferred test at time of diagnosis for detecting prognostically important genomic abnormalities in leukemias/lymphomas and solid tumors involving loss/gain of DNA, loss of heterozygosity
Use to monitor disease progression and response to therapy
Panel only detects prognostically important imbalances (gain or loss of DNA) in the chromosomes of interest
Chromosome alterations outside the regions complementary to these FISH probes will not be detected
Ideal testing is when significant disease is present
Genomic Microarray (Oligo-SNP Array)
Medical Experts
References
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NCCN - Oncology B-Cell Lymphomas Version 1.2021
National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology, B-cell lymphomas, Version 1.2021. [Updated: Jan 2021; Accessed: Jan 2021]
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WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Rev 4th 2017
Swerdlow S, Campo E, Jaffe E, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Revised 4th ed. International Agency for Research on Cancer, 2017.
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NCCN - Oncology Pediatric Aggressive Mature B-Cell Lymphomas Version 2.2020
National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology, pediatric aggressive mature B-cell lymphomas, Version 2.2020. [Updated: April 2020; Accessed: Jan 2021]
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Willemze R, Cerroni L, Kempf W, et al. The 2018 update of the WHO-EORTC classification for primary cutaneous lymphomas. Blood. 2019;133(16):1703-1714.
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Evans PA, Pott Ch, Groenen PJ, et al. Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia. 2007;21(2):207-214.
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Swerdlow SH, Campo E, Pileri SA, et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood. 2016;127(20):2375-2390.
For additional IHC tests that may be useful in the diagnosis or differential diagnosis of B-cell lymphomas, refer to ARUP’s Immunohistochemistry Stain Offerings.