Lymphoma Phenotyping

  • Diagnosis
  • Algorithms
  • Background
  • Lab Tests
  • References
  • Related Content

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

Lymphoma Leukemia Phenotyping Testing Algorithm

During evaluation of peripheral lymphocytosis (absolute lymphocytosis >4,000/µL), the possibility of a malignant disorder requires evaluation.

WHO Classification of Lymphoid Neoplasms, 2008

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

Indications for Laboratory Testing

Tests generally appear in the order most useful for common clinical situations.
Click on number for test-specific information in the ARUP Laboratory Test Directory

Leukemia/Lymphoma Phenotyping by Flow Cytometry 2008003
Method: Flow Cytometry

Limitations

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

B-Cell Clonality Screening (IgH and IgK) by PCR 2006193
Method: Polymerase Chain Reaction/Capillary Electrophoresis

Limitations

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)

T-Cell Clonality by Next Generation Sequencing 2008409
Method: Massively Parallel Sequencing

Limitations

Clonal TCRG gene rearrangements below the limit of detection will not be reported

T-Cell Clonality by Flow Cytometry Analysis of TCR V-Beta 0093199
Method: Flow Cytometry

Limitations

Tests only for TCR α-β receptors; if identification of TCR γ-δ receptors is desired, PCR testing is recommended

Chromosome FISH, Interphase 2002298
Method: Fluorescence in situ Hybridization

Limitations

Fresh tissue specimen required

IGH-BCL2 Fusion, t(14;18) by FISH 2001536
Method: Fluorescence in situ Hybridization

Limitations

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

IGH-MYC Fusion t(8;14) by FISH 2001538
Method: Fluorescence in situ Hybridization

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

MYC (8q24) Gene Rearrangement by FISH 2002345
Method: Fluorescence in situ Hybridization

Limitations

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

BCL6 (3q27) Gene Rearrangement by FISH 2010107
Method: Fluorescence in situ Hybridization

Limitations

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

Chromosome Analysis, Bone Marrow 2002292
Method: Giemsa Band

Follow Up

Repeat testing as clinically indicated to monitor disease progression

Cyclin D1, SP4 by Immunohistochemistry 2003842
Method: Immunohistochemistry

Chromosome FISH, CLL Panel 2002295
Method: Fluorescence in situ Hybridization

Limitations

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

Follow Up

Repeat testing as clinically indicated to monitor disease progression

IGH-CCND1 Fusion, t(11;14) by FISH 2007226
Method: Fluorescence in situ Hybridization

Limitations

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

Aggressive B-Cell Lymphoma FISH Reflex, Tissue 2012710
Method: Fluorescence in situ Hybridization

Limitations

Interpretation of results requires correlation with morphology and immunophenotype

MYC and/or BCL2overexpression can be due to other mechanisms not detected by this test

Chromosome alterations outside probe region are not detected

Lymphoma (Aggressive) Panel by FISH 2002650
Method: Fluorescence in situ Hybridization

Limitations

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

BRAF V600E Mutation Detection in Hairy Cell Leukemia by Real-Time PCR, Quantitative 2007132
Method: Polymerase Chain Reaction

Limitations

Limit of detection is 0.2% mutant allele

IGHV Mutation Analysis by Sequencing 0040227
Method: Polymerase Chain Reaction/Sequencing

Limitations

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

ZAP-70 Analysis by Flow Cytometry 0092392
Method: Flow Cytometry

Limitations

Cell viability must be >98%

Results should not be used for diagnostic purposes and should always be correlated with morphologic and clinical information

Additional Tests Available

Epstein-Barr Virus by PCR 0050246
Method: Qualitative Polymerase Chain Reaction

Comments

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

Sensitive and specific qualitative test for detecting EBV in plasma, serum, and cerebrospinal fluid

IGH-CCND1 (BCL-1/JH) Translocation, t(11;14) by PCR 0055557
Method: Polymerase Chain Reaction

Comments

Aid in diagnosis of MCL if cyclin testing is uninformative

Blood, bone marrow, fresh frozen tissue, and FFPE tissue specimens are acceptable

Analytical sensitivity – 1 in 105

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

Guidelines

NCCN Clinical Practice Guidelines in Oncology, Hodgkin Lymphoma. National Comprehensive Cancer Network. Fort Washington, PA [Accessed: Nov 2015]

NCCN Clinical Practice Guidelines in Oncology, Non-Hodgkin's Lymphomas. National Comprehensive Cancer Network. Fort Washington, PA [Accessed: Jun 2015]

General References

Bahler D. Flow Cytometric Analysis. In Kjeldsberg C. Practical Diagnosis of Hematologic Disorders, 5th ed. Chicago: ASCP Press, 2006.

Craig F, Foon K. Flow cytometric immunophenotyping for hematologic neoplasms. Blood. 2008; 111(8): 3941-67. PubMed

Ochs R, Bagg A. Molecular genetic characterization of lymphoma: application to cytology diagnosis. Diagn Cytopathol. 2012; 40(6): 542-55. PubMed

Skoog L, Tani E. B cell neoplasms. Monogr Clin Cytol. 2009; 18: 19-37. PubMed

Skoog L, Tani E. T cell neoplasms. Monogr Clin Cytol. 2009; 18: 38-48. PubMed

Sun T. Chapters 3-6. In Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms, 2nd ed. Philidelphia, PA: Lippincott Williams and Wilkins, 2011.

Swerdlow S, Campo E, Harris N, Jaffe E, Pileri S, Harald S, Thiele J, Vardiman J. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed. Lyon, France: International Agency for Research on Cancer, 2008.

References from the ARUP Institute for Clinical and Experimental Pathology®

Bahler D, Hartung L, Hill S, Bowen G, Vonderheid E. CD158k/KIR3DL2 is a useful marker for identifying neoplastic T-cells in Sézary syndrome by flow cytometry. Cytometry B Clin Cytom. 2008; 74(3): 156-62. PubMed

Bahler D, Kim B, Gao A, Swerdlow S. Analysis of immunoglobulin V genes suggests cutaneous marginal zone B-cell lymphomas recognise similar antigens. Br J Haematol. 2006; 132(5): 571-5. PubMed

Bentz J, Rowe L, Anderson S, Gupta P, McGrath C. Rapid detection of the t(11;14) translocation in mantle cell lymphoma by interphase fluorescence in situ hybridization on archival cytopathologic material. Cancer. 2004; 102(2): 124-31. PubMed

Bohling S, Jenson S, Crockett D, Schumacher J, Elenitoba-Johnson K, Lim M. Analysis of gene expression profile of TPM3-ALK positive anaplastic large cell lymphoma reveals overlapping and unique patterns with that of NPM-ALK positive anaplastic large cell lymphoma. Leuk Res. 2008; 32(3): 383-93. PubMed

Cairo M, Gerrard M, Sposto R, Auperin A, Pinkerton R, Michon J, Weston C, Perkins S, Raphael M, McCarthy K, Patte C, FAB LMB96 International Study Committee. Results of a randomized international study of high-risk central nervous system B non-Hodgkin lymphoma and B acute lymphoblastic leukemia in children and adolescents. Blood. 2007; 109(7): 2736-43. PubMed

Cairo M, Raetz E, Lim M, Davenport V, Perkins S. Childhood and adolescent non-Hodgkin lymphoma: new insights in biology and critical challenges for the future. Pediatr Blood Cancer. 2005; 45(6): 753-69. PubMed

Capriotti E, Vonderheid E, Thoburn C, Wasik M, Bahler D, Hess A. Expression of T-plastin, FoxP3 and other tumor-associated markers by leukemic T-cells of cutaneous T-cell lymphoma. Leuk Lymphoma. 2008; 49(6): 1190-201. PubMed

Cessna M, Hartung L, Tripp S, Perkins S, Bahler D. Hairy cell leukemia variant: fact or fiction. Am J Clin Pathol. 2005; 123(1): 132-8. PubMed

Chen L, Delgado J, Jensen P, Chen X. Direct expansion of human allospecific FoxP3+CD4+ regulatory T cells with allogeneic B cells for therapeutic application. J Immunol. 2009; 183(6): 4094-102. PubMed

Cooney-Qualter E, Krailo M, Angiolillo A, Fawwaz R, Wiseman G, Harrison L, Kohl V, Adamson P, Ayello J, Ven C, Perkins S, Cairo M, Children's Oncology Group. A phase I study of 90yttrium-ibritumomab-tiuxetan in children and adolescents with relapsed/refractory CD20-positive non-Hodgkin's lymphoma: a Children's Oncology Group study. Clin Cancer Res. 2007; 13(18 Pt 2): 5652s-5660s. PubMed

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Everton K, Abbott D, Crockett D, Elenitoba-Johnson K, Lim M. Quantitative proteomic analysis of follicular lymphoma cells in response to rituximab. J Chromatogr B Analyt Technol Biomed Life Sci. 2009; 877(13): 1335-43. PubMed

Gerrard M, Cairo M, Weston C, Auperin A, Pinkerton R, Lambilliote A, Sposto R, McCarthy K, Lacombe M, Perkins S, Patte C, FAB LMB96 International Study Committee. Excellent survival following two courses of COPAD chemotherapy in children and adolescents with resected localized B-cell non-Hodgkin's lymphoma: results of the FAB/LMB 96 international study. Br J Haematol. 2008; 141(6): 840-7. PubMed

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Hartung L, Bahler D. Flow cytometric analysis of BCL-2 can distinguish small numbers of acute lymphoblastic leukaemia cells from B-cell precursors. Br J Haematol. 2004; 127(1): 50-8. PubMed

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Jasionowski T, Hartung L, Greenwood J, Perkins S, Bahler D. Analysis of CD10+ hairy cell leukemia. Am J Clin Pathol. 2003; 120(2): 228-35. PubMed

Kiel M, Velusamy T, Betz B, Zhao L, Weigelin H, Chiang M, Huebner-Chan D, Bailey N, Yang D, Bhagat G, Miranda R, Bahler D, Medeiros J, Lim M, Elenitoba-Johnson K. Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in splenic marginal zone lymphoma. J Exp Med. 2012; 209(9): 1553-65. PubMed

Leventaki V, Rodic V, Tripp S, Bayerl M, Perkins S, Barnette P, Schiffman J, Miles R. TP53 pathway analysis in paediatric Burkitt lymphoma reveals increased MDM4 expression as the only TP53 pathway abnormality detected in a subset of cases. Br J Haematol. 2012; 158(6): 763-71. PubMed

Lones M, Heerema N, Le Beau M, Sposto R, Perkins S, Kadin M, Kjeldsberg C, Meadows A, Siegel S, Buckley J, Abromowitch M, Kersey J, Bergeron S, Cairo M, Sanger W. Chromosome abnormalities in advanced stage lymphoblastic lymphoma of children and adolescents: a report from CCG-E08. Cancer Genet Cytogenet. 2007; 172(1): 1-11. PubMed

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Szankasi P, Reading S, Vaughn C, Prchal J, Bahler D, Kelley T. A quantitative allele-specific PCR test for the BRAF V600E mutation using a single heterozygous control plasmid for quantitation: a model for qPCR testing without standard curves. J Mol Diagn. 2013; 15(2): 248-54. PubMed

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Medical Reviewers

Last Update: January 2016