B-Cell Lymphomas

B-cell non-Hodgkin lymphomas (NHL) represent a heterogenous group of hematologic malignancies of lymphoid cells. Following biopsy, flow cytometry, immunohistochemical stains, and cytogenetic studies can confirm the diagnosis.

Key Points

Double- and Triple-Hit Lymphomas

B-cell lymphomas with 2 or 3 recurrent chromosomal breakpoint aberrations (rare) are referred to as double- or triple-hit lymphomas. These usually involve the MYC oncogene in association with the BCL2 and/or BCL6 gene rearrangements. These are classified by WHO as a “high-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements” (Swerdlow, WHO, 2016).

It is important to identify these high-grade lymphomas as they tend to manifest aggressive behavior and respond poorly to traditional chemotherapy.

  • Follicular lymphoma
  • DLBCL
  • High-grade lymphomas (rare)
Breakpoints Used to Identify Double- or Triple-Hit Lymphomas
Oncogene Break-apart MYC BCL2 BCL6
Locus 8q24 18q21 3q27
Biology Accelerator of cell proliferation Apoptosis inhibitor Transcription modifier
Cytogenetics (translocations) Any MYC translocation BCL2/IGH t(14;18)(q32;21)
  • BCL6 almost always has a non-Ig translocation partner – BCL6 (3q27)
  • Uncommon partner – BCL6/IGH [t(3;14)(q27;q32)]
Specific lymphomas associated with translocation
  • Burkitt lymphoma
  • Diffuse large B-cell lymphoma (DLBCL)
  • High-grade B-cell lymphomas (HGBL)
  • Follicular lymphoma
  • DLBCL
  • HGBL
  • Follicular lymphoma
  • DLBCL
  • High-grade lymphomas (rare)
Other malignancies associated with translocation B-cell ALL (rare) B-cell ALL (rare) n/a

DIAGNOSTIC TESTING

ARUP Tests

  • Individual probes
    • Chromosome FISH, Interphase 2002298 – order probes separately
  • Panels
    • Aggressive B-Cell Lymphoma Reflex Panel by FISH, Tissue 3001495 – if MYC (8q24) gene rearrangement by FISH is positive, then IGH-BCL2 fusion t(14;18) by FISH is added; if IGH-BCL2 fusion, t(14;18) by FISH is negative, then BCL6 (3q27) gene rearrangement by FISH will be added
    • Lymphoma (Aggressive) Panel by FISH 2002650 – probes include IGH/BCL2, BCL6, and MYC
  • For histologically aggressive B-cell lymphomas with either Burkitt-like or diffuse large cell morphologies in adults demonstrating a CD20+, CD10+ phenotype with high proliferative index OR CD20+ neoplasms with morphologic features indeterminate between DLBCL and Burkitt lymphoma, it is reasonable to test for MYC, BCL2, and BCL6 rearrangements using FISH
    • IGH-BCL2 Fusion, t(14;18) by FISH 3001298
    • IGH-MYC t(8;14) by FISH 3001299
    • MYC (8q24) Gene  Rearrangement by FISH 3001300
    • BCL6 (3q27) Gene Rearrangement by FISH 3001311

References: Aukema SM, 2011; Johnson NA et al, 2009; Salaverria I et al, 2011; Savage et al, 2009; WHO, 2008.

ALL, acute lymphoblastic lymphoma; n/a, not available

Diagnosis

Indications for Testing

  • Adenopathy
  • Fevers/night sweats
  • Recurrent infections
  • Unexplained lymphocytosis or abnormal manual differential

Laboratory Testing

  • Initial testing involves the following
    • CBC with peripheral smear
    • Liver chemistries
    • Lactate dehydrogenase (LD)
    • Serum uric acid, potassium, calcium, phosphorus
    • HIV for at-risk patients
  • Immunophenotyping to identify the lymphoid proliferation malignancy and to categorize lymphoma type
    • See the Lymphoma Phenotyping algorithm for specific test-ordering recommendations
    • May be performed on peripheral blood, bone marrow

Histology

  • Fine-needle aspiration (FNA) not considered adequate – use incisional biopsy specimen (NCCN, 2015)
  • Follow-up testing with bone marrow biopsy
    • ≥2 cm specimen length
    • Classified as involved or not
  • Immunophenotyping (flow cytometry testing) and immunoperoxidase staining of tissue for specific B-cell antigens or clonality with kappa and lambda light-chain staining
  • Immunohistochemistry – useful in conjunction with phenotyping
    • Most commonly used stains – 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 – BOB-1, Bax, caspase-3, CD15, CD43, CD45, CD45RA-MT2, CD45RO, CD74, c-Myc, DBA.44, and Oct-2
  • B-cell clonality studies
    • Locate clonal rearrangements of the immunoglobulin gene in B-cell malignancies
    • Provide information regarding prognosis and treatment recommendations
  • T-cell clonality studies

Genetic Testing

Disease Genetics Comments
High-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements, and Burkitt lymphoma MYC, BCL2, BCL6 Refer to Key Points
Burkitt lymphoma MYC t(8;14), t(2;8), t(8;22)  
Diffuse large B-cell lymphoma (DLBCL) BCL6, BCL2, IRF4/MUM1  
Follicular lymphoma BCL2 t(14;18)(q32;q21), IRF4/MUM1 Recommended methodology – FISH or cytogenetics
Hairy cell leukemia BRAF V600E  
Mantle cell lymphoma BCL1(CCND1)  t(11;14)(q13;q32)  
Marginal zone lymphoma (nodal, MALT, splenic) MYD88 Use to differentiate from lymphoplasmacytic lymphoma
Mucosa-associated lymphoid tissue (MALT) (gastric) t(11;14), t(14;18), t(1;14), t(3;14), t(11:18)

FISH or cytogenetics

PCR useful for t(14;18)

Other Testing

  • Virus testing
    • EBV – posttransplant lymphomas, endemic nasopharyngeal lymphomas
    • HHV8 – AIDS-related lymphomas
  • Bacteria testing
    • Helicobacter – mucosa-associated lymphoid tissue (MALT) lymphoma
  • Kappa and lambda light-chain clonality in situ hybridization
  • Testing prior to treatment with immunosuppressive therapies
    • HBV – surface antigen and core antibodies for all patients considering rituximab
      • Also include HBV e antigen testing for patients with relevant risk factors
    • HCV – for high-risk patients considering rituximab
    • CMV – PCR quantitative

Prognosis

  • International Prognostic Index scoring system
    • Based on pretreatment clinical factors of age (≤60 years); Ann Arbor tumor stage (I or II); number of extranodal sites (≤1); Eastern Cooperative Oncology Group (ECOG) performance status (0 or 1); and serum LD (≤1 times normal) – all scored as 0
    • Patients placed in four risk groups
    • Limited usefulness in follicular lymphoma, mantle cell lymphoma, NK lymphoma, nasal-type lymphoma, hepatosplenic lymphoma, and enteropathy-type lymphoma
  • Follicular Lymphoma International Prognostic Index scoring system
    • Five risk factors (each scored as one point)
      • Blood hemoglobin <12 g/dL
      • Serum LD >normal
      • Ann Arbor tumor stage (III or IV)
      • >4 nodal sites
      • Age >60 years
    • 0-1 risk factor – low risk (5-year survival 90%)
    • 2 risk factors – intermediate risk (5-year survival 78%)
    • 3 risk factors – high risk (5-year survival 53%)
  • All prognostic indices may change in the future as the IPI and FLIPI indices were developed before immunotherapy
  • Genetic mutations (cytogenetics)
    • Follicular lymphoma
      • Adverse prognosis associated with del 17p, trisomy 12 and abnormalities of 6p
    • Diffuse large B-cell lymphoma (DLBCL)
      • Better prognosis associated with t(14;18)
      • Adverse prognosis with MYC oncogene; BCL2 gene, p53(+)
    • Follicular and DLBCL
      • Dismal outcomes for 8q24/MYC in association with 18q21/BCL2 or 3q27/BCL6
    • Double-hit and triple-hit lymphomas
      • Refer to Key Points for double- and triple-hit lymphomas
    • CLL
      • CD38 – mutation status correlates inversely with prognosis

Differential Diagnosis

Monitoring

  • Alemtuzumab (Campath) therapy – risk of CMV reactivation
    • PCR quantitative two to three weeks after start of therapy
  • Rituximab (Rituxan) therapy – high risk of hepatitis reactivation
    • HBV surface antigen and core antibody testing for all patients
      • Add HBV e antigen testing for patients with relevant risk factors
    • HCV testing for at-risk patients

Background

Epidemiology

  • Incidence – represents 80-85% of the cases of non-Hodgkin lymphomas (NHL) diagnosed annually
    • >70,000 new cases of NHL (NCCN, 2014)
  • Age – peaks in 60s
  • Sex – M<F (minimal)

Classification

Mature B-cell Neoplasms (WHO, 2016)

  • Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL)
  • Monoclonal B-cell lymphocytosis
  • B-cell prolymphocytic leukemia
  • Splenic marginal zone lymphoma (MZL)
  • Hairy cell leukemia
  • Splenic lymphoma/leukemia, unclassifiable (provisional entities)
    • Splenic diffuse red pulp small B-cell lymphoma
    • Hairy cell leukemia variant
  • Lymphoplasmacytic lymphoma
    • Waldenström macroglobulinemia
  • Monoclonal gammopathy of undetermined significance (MGUS), IgM
  • Heavy chain diseases
    • Alpha heavy chain disease
    • Gamma heavy chain disease
    • Mu heavy chain disease
  • MGUS, IgG/A
  • Plasma cell neoplasms
  • Solitary plasmacytoma of bone
  • Extraosseous plasmacytoma
  • Monoclonal immunoglobulin deposition diseases
  • Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT)
  • Nodal MZL
    • ​Pediatric nodal marginal zone lymphoma (provisional entity)
  • Follicular lymphoma
    • In situ follicular neoplasia
    • Duodenal-type follicular lymphoma
  • Pediatric-type follicular lymphoma
  • Large B-cell lymphoma with IRF4 rearrangement (provisional entity)
  • Primary cutaneous follicle center lymphoma
  • Mantle cell lymphoma
    • In situ mantle cell neoplasia
  • Diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS)
    • Germinal center B-cell type
    • Activated B-cell type
  • T-cell/histiocyte-rich large B-cell lymphoma
  • Primary DLBCL of the central nervous system
  • Primary cutaneous DLBCL, leg type
  • Epstein-Barr virus (EBV)-positive DLBCL of the elderly
  • EBV-positive mucocutaneous ulcer (provisional entity)
  • DLBCL associated with chronic inflammation
  • Lymphomatoid granulomatosis
  • Primary mediastinal (thymic) large B-cell lymphoma
  • Intravascular large B-cell lymphoma
  • ALK-positive large B-cell lymphoma
  • Plasmablastic lymphoma
  • Primary effusion lymphoma
  • Human herpes virus 8 (HHV8)-positive DLBCL, NOS
  • Burkitt lymphoma
  • Burkittlike lymphoma with 11q aberration (provisional entity)
  • High-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements
  • High-grade B-cell lymphoma, NOS
  • B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma

Risk Factors

Clinical Presentation

Selected Lymphoma Subtypes (Based on WHO, 2016)

ARUP Lab Tests

Primary Tests

Aid in evaluation of hematopoietic neoplasms (ie, leukemia, lymphoma)

Specimens include bone marrow, whole blood, tissue, or fluid

Monitor therapy in patients with established diagnosis of hematopoietic neoplasms

Markers selected based on provided clinical history and/or previous test results

Antigens included

T cell: CD1a, CD2, CD3, CD4, CD5, CD7, CD8, TCR γ-δ, cytoplasmic CD3

B cell: CD10, CD19, CD20, CD22, CD23, CD103, CD200, kappa, lambda, cytoplasmic kappa, cytoplasmic lambda

Myeloid/monocyte: CD11b, CD13, CD14 (Mo2), CD14 (MY4), CD15, CD33, CD64, CD117, myeloperoxidase

Miscellaneous: CD11c, CD16, CD25, CD30, CD34, CD38, CD41, CD42b, CD45, CD56, CD57, CD61, HLA-DR, glycophorin, TdT, bcl-2, CD123, CD138, CD26, CD45, CRLF-2

Use to order individual or multiple oncology FISH probes if standard FISH panels are not desired

Fresh tissue sample required

Indicate names of probes needed for testing

ARUP Oncology FISH Probes menu

Aid in the diagnosis of lymphoproliferative disorders

Aid in differentiating malignant from reactive lymphoid proliferations

Equally sensitive for both kappa and lambda restricted populations

False-negative results may result from specimen inadequacy and mutations affecting primer sites

Detection of clonally rearranged IgH is seen in a subset of T-cell neoplasms (ie, a positive result in the test should not be used to differentiate between T- and B-cell neoplasms)

Diagnose mantle cell lymphoma in conjunction with morphology and immunohistochemical studies

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens only

Stained and returned to client pathologist for interpretation; consultation available if needed

Diagnose follicular lymphoma in conjunction with clinical, morphologic, and flow cytometric data

Most sensitive method to detect IGH-BCL2 fusion in FFPE tissue specimens

Not validated for tissue fixed in alcohol-based or nonformalin fixatives

Negative result does not exclude possibility of translocations involving other partners or rule out follicular lymphoma

Diagnose Burkitt lymphoma (BL) or diffuse large B-cell lymphoma (DLBCL) with features intermediate between BL and DLBCL in conjunction with clinical, morphologic, and flow cytometric data

Requires FFPE tissue

Negative result does not rule out BL or B-cell lymphomas with features intermediate between BL and DLBCL involving MYC with other translocation partners, such as t(2;8) or t(8;22)

IGH-MYC t(8;14) by FISH has not been validated for tissue fixed in alcohol-based or nonformalin fixatives

MYC is not specific for BL or B-cell lymphomas with features intermediate between BL and DLBCL

Diagnose BL or DLBCL with features intermediate between BL and DLBCL 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

Requires FFPE tissue

Negative result does not rule out BL or B-cell lymphomas with features intermediate between BL and DLBCL

MYC (8q24) gene rearrangement by FISH has not been validated for tissue fixed in alcohol-based or nonformalin fixatives

MYC is not specific for BL or B-cell lymphomas with features intermediate between BL and DLBCL

Diagnose BL or DLBCL with features intermediate between BL and DLBCL in conjunction with clinical, morphologic, and flow cytometric data

Sensitive method for detecting BCL6 rearrangements in FFPE, which can contribute to diagnosis and prognostication in B-cell lymphomas

Aid in diagnosis of aggressive large B-cell lymphoma with intermediate features between Burkitt lymphoma and diffuse large B-cell lymphoma (DLBCL) and confirmation of suspected double-hit lymphoma

Specimens – formalin-fixed, paraffin-embedded (FFPE) tissue specimens

Interpretation of results requires correlation with morphology and immunophenotype

MYC and/or BCL2 overexpression can be due to other mechanisms not detected by this test

Chromosome alterations outside probe region are not detected

Aid in diagnosis of aggressive large B-cell lymphoma with intermediate features between Burkitt lymphoma and diffuse large B-cell lymphoma (DLBCL) and confirmation of suspected double hit lymphoma

For reflex test, refer to Aggressive B-Cell Lymphoma FISH Reflex, Tissue

Specimens – bone marrow or whole blood specimens; other specimens may be acceptable

Probes in this panel include MYC, BCL2, IGH, BCL6

Interpretation of results requires correlation with morphology and immunophenotype

MYC and/or BCL2 overexpression can be due to other mechanisms not detected by this test

Chromosome alterations outside probe region are not detected

FFPE and frozen specimens unacceptable

Confirm diagnosis of hairy cell leukemia (HCL)

Monitor tumor burden in patients with HCL

Limit of detection – 0.2% mutant allele

Determine risk group in newly diagnosed CLL

Results should always be correlated with morphologic and clinical information

Samples that do not yield amplification product may contain too few CLL cells (<50% B cells) or express VH genes with high numbers of mutations that may compromise clonal B-cell amplification

Not intended to detect minimal residual disease

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

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

Alternate test to detect prognostically important genomic abnormalities in 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

Aid in diagnosis of mantle cell lymphoma (MCL) if cyclin testing is noninformative

FFPE specimen require

Not validated for tissue fixed in alcohol-based or nonformalin fixatives or decalcified tissue

Negative result does not exclude the possibility of translocations involving other partners

This mutation is not specific for MCL; results should be analyzed in conjunction with morphology, immunohistochemistry, and immunophenotyping results

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid in identifying colorectal adenomas, carcinomas; distinguishes follicular lymphoma from reactive follicles

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid in identifying large cell lymphomas, BL, and Hodgkin lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of lymphoblastic lymphoma, BL, follicular lymphoma, and CML

Aid differential diagnosis of small B-cell lymphomas and subtyping of lymphoblastic leukemias

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell leukemia/lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid in identifying common acute lymphoblastic leukemia, pre-B ALL, CLL, prolymphocytic leukemia, hairy cell leukemia, lymphoma cell leukemia, B-cell lymphomas, including BL, Waldenstrom, macroglobulinemia, and immunoblastic B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid in identifying B-cell CLL, follicular lymphoma, low-grade MALT-type B-cell lymphoma, primary salivary gland and gastric lymphoma, T-cell and histiocyte-rich B-cell lymphoma, angioimmunoblastic T-cell lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, follicular dendritic sarcoma and some Reed-Sternberg cells not expressing other B- or T-cell-associated markers

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid in differentiating small lymphocytic lymphoma (+) and mantle cell lymphoma (-)

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid in identifying acute leukemia of precursor B-cell type, B-cell lymphomas and some myelomas

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Primarily aids the distinction between CLL/SLL and mantle cell lymphoma where CD200 is usually positive in CLL/SLL and negative in mantle cell lymphoma; CD200 is also positive in other B-cell lymphoproliferative disorders

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of multicentric Castleman disease, angioimmunoblastic lymphadenopathies, and Kaposi sarcoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aids in the prognostic determination of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Detect IRF4/DUSP22 rearrangements which can contribute to diagnosis and prognosis in B-cell and T-cell lymphomas

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Useful for cyclin D1-negative MCL that is difficult to distinguish from other small B-cell lymphomas

Highly specific marker for both cyclin D1-positive and negative MCL

Stained and returned to client pathologist for interpretation; consultation available if needed

Aid histologic diagnosis of B-cell lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed

Related Tests

Initial test to evaluate lymphoma

May provide prognostic information

Initial test to evaluate lymphoma

May provide prognostic information

Initial test to evaluate lymphoma

May provide prognostic information

Initial test to evaluate lymphoma

May provide prognostic information

Assay interference (negative) may be observed when high concentrations of N-acetylcysteine (NAC) are present

Negative interference has also been reported with NAPQI (an acetaminophen metabolite) but only when concentrations are at or above those expected during acetaminophen overdose

Order for rituximab monitoring

Diagnose, prognose, and monitor hematopoietic neoplasms

Identify HBV status if considering rituximab therapy

Identify HBV status if considering rituximab therapy

Screen for hepatitis C virus (HCV) infection in at-risk individuals

Screen for IgG antibodies to HCV

Detect and quantify cytomegalovirus (CMV)

Use for at-risk patients

Medical Experts

Contributor

Lamb

Allen N. Lamb, PhD, FACMG
Allen N. Lamb, PhD, FACMG
Retired Former Professor of Clinical Pathology, University of Utah
Retired Former Laboratory Section Chief, Cytogenetics and Genomic Microarray, ARUP Laboratories
Contributor
Contributor

References

Additional Resources
Resources from the ARUP Institute for Clinical and Experimental Pathology®