Lymphoma Phenotyping

During evaluation of peripheral lymphocytosis (absolute lymphocytosis >4,000/µL), the possibility of a malignant disorder requires evaluation. Laboratory studies include cell surface phenotyping of lymphocytes.

Diagnosis

Indications for Testing

  • Evaluation of peripheral lymphocytosis (absolute lymphocytosis >4,000/µL)
  • Unexplained lymphadenopathy

Laboratory Testing

  • Rule out other disorders associated with lymphocytosis
  • If lymphoproliferative disorder remains a significant possibility after clinical evaluation, cell surface phenotyping of lymphocytes should be performed
    • Usually performed on peripheral blood using flow cytometry
      • Technique provides percentage of lymphocytes positive for a particular antigen and density of antigens
      • Normal peripheral blood lymphocytes consist of approximately 10% B-cells, 80% T-cells and 10% NK-cells
    • Most commonly used markers (CD = cluster designation)
      • B-cell – CD10, CD19, CD20, CD22, CD23, CD24, CD79b, CD103, Pax-5, kappa, lambda, CD200, cytoplasmic kappa, cytoplasmic lambda
      • T-cell – CD1, CD2, CD3, CD4, CD5, CD7, CD8, TCR α-β, TCR γ-δ, cytoplasmic CD3
      • 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

Histology

Prognosis

Differential Diagnosis

Background

Classification

WHO Classification of Mature Lymphoid, Histiocytic, and Dendritic Neoplasms, 2016

Clinical Presentation

  • Nonspecific symptoms frequently in initial presentation
    • Malaise, fatigue, weight loss, fever
  • Adenopathy – may be first presenting symptom; may be bulky
  • Related syndromes – autoimmune hemolytic anemia
  • Cutaneous – skin rashes in cutaneous lymphomas
  • Gastrointestinal
    • Hepato/splenomegaly
    • Common site of extranodal disease

ARUP Lab 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

Some hematopoietic neoplasms do not show phenotypic abnormalities and therefore may not be detected by flow cytometry

Poor cell viability may adversely affect antigens and impede the ability to properly identify neoplastic cells

Flow results cannot be used alone to diagnose malignancy; should be interpreted in conjunction with morphology, clinical information, and other necessary ancillary tests for a definitive diagnosis

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

Aid in the diagnosis of lymphoproliferative disorders

Aid in differentiating malignant from reactive lymphoid proliferations

Equally sensitive for both kappa and lambda restricted populations

Clinical sensitivity: >95% for mature B-cell non-Hodgkin lymphomas

Analytical sensitivity: clone must represent at least 1-5% of population examined

Analytic specificity: >98%

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)

Aid in the diagnosis of T-cell lymphoproliferative disorders

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

Detect chromosome abnormalities associated with lymphoma

Specific FISH probes related to lymphoma must be specified and include MYC (c-Myc) rearrangements, t(11;14) (IGH-CCND1), t(14;18) (IGH-BCL2), IGH rearrangement with unknown partner, ALK rearrangements, and BCL6 rearrangements

ARUP Oncology FISH Probes menu

Fresh tissue specimen required

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 nor 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

Limitations 

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 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

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

Does not identify translocation partner

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

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

Diagnose, prognose, and monitor hematopoietic neoplasms

Repeat testing as clinically indicated to monitor disease progression

Diagnose mantle cell lymphoma (MCL) in conjunction with morphology and immunohistochemical studies

FFPE tissue specimens only

Alternate test to detect prognostically important genomic abnormalities in CLL

Specific genomic abnormalities tested for are ATM deletion, 13q deletion, p53 deletion, and trisomy 12

Limit of detection is probe dependent and ~1-5% in interphase nuclei

Repeat testing as clinically indicated to monitor disease progression

Aid in diagnosis of MCL if cyclin testing is noninformative

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

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

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

Reflex pattern: if MYC (8q24) gene rearrangements 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

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

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

Probes in this panel include MYCBCL2IGH, BCL6

Confirm diagnosis of hairy cell leukemia (HCL)

Monitor tumor burden in patients with HCL

Analytical sensitivity: 0.2% mutant allele

Limit of detection is 0.2% mutant allele

Determine risk group in newly diagnosed CLL

Assay is designed for use with a confirmed diagnosis of CLL and includes sequencing

Use of this assay for all other diagnoses will terminate after amplification and will not include sequencing

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

Not intended to detect minimal residual disease

Primarily for evaluating lymphocyte function in patients with suspected cellular immune dysfunction, such as primary and secondary immunodeficiencies

Other uses include monitoring lymphocyte recovery and competence after hematopoietic stem cell transplantation and monitoring lymphocyte function during immunosuppressive therapy

Primarily for evaluating T-cell function in patients with suspected cellular immune dysfunction, such as primary and secondary immunodeficiencies

Other uses include monitoring lymphocyte recovery and competence after hematopoietic stem cell transplantation and monitoring lymphocyte function during immunosuppressive therapy

Not a first-level test; order after lymphocyte proliferation, mitogen induced, by flow cytometry has been performed

Immunohistochemistry Stain Offering
Related Tests

Primarily for evaluating recall antigen responses in patients with suspected cellular immune dysfunction, such as primary and secondary immunodeficiencies

Other uses include monitoring lymphocyte recovery and competence after hematopoietic stem cell transplantation and monitoring lymphocyte function during immunosuppressive therapy

Do not order for patients younger than 3 months unless clinical history of candidiasis is present

Primarily for evaluating lymphocyte function in patients with suspected cellular immune dysfunction, such as primary and secondary immunodeficiencies

Other uses include monitoring lymphocyte recovery and competence after hematopoietic stem cell transplantation and monitoring lymphocyte function during immunosuppressive therapy

Do not use for diagnosis of infectious mononucleosis

Order to detect Epstein-Barr virus (EBV) in individuals suspected of having EBV-related disease

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®