Lymphoma Phenotyping

  • Diagnosis
  • Algorithms
  • Background
  • Lab Tests
  • References
  • Related Topics
  • Videos

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

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
  • 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

    Related Tests

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

    Ochs RC, 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 DW, Hartung L, Hill S, Bowen GM, Vonderheid EC. 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 DW, Kim BK, Gao A, Swerdlow SH. 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 JS, Rowe LR, Anderson SR, Gupta PK, McGrath CM. 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 SD, Jenson SD, Crockett DK, Schumacher JA, Elenitoba-Johnson KS J, Lim MS. 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 MS, Gerrard M, Sposto R, Auperin A, Pinkerton R, Michon J, Weston C, Perkins SL, 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 MS, Raetz E, Lim MS, Davenport V, Perkins SL. 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 EC, Thoburn CJ, Wasik MA, Bahler DW, Hess AD. 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 MH, Hartung L, Tripp S, Perkins SL, Bahler DW. Hairy cell leukemia variant: fact or fiction. Am J Clin Pathol. 2005; 123(1): 132-8. PubMed

    Chen LC, Delgado JC, Jensen PE, 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 RA, Wiseman G, Harrison L, Kohl V, Adamson PC, Ayello J, Ven Cvande, Perkins SL, Cairo MS, 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

    Deffenbacher KE, Iqbal J, Sanger W, Shen Y, Lachel C, Liu Z, Liu Y, Lim MS, Perkins SL, Fu K, Smith L, Lynch J, Staudt LM, Rimsza LM, Jaffe E, Rosenwald A, Ott GK, Delabie J, Campo E, Gascoyne RD, Cairo MS, Weisenburger DD, Greiner TC, Gross TG, Chan WC. Molecular distinctions between pediatric and adult mature B-cell non-Hodgkin lymphomas identified through genomic profiling. Blood. 2012; 119(16): 3757-66. PubMed

    Everton KL, Abbott DR, Crockett DK, Elenitoba-Johnson KS J, Lim MS. 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 MS, Weston C, Auperin A, Pinkerton R, Lambilliote A, Sposto R, McCarthy K, Lacombe MT, Perkins SL, 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

    Gunn SR, Mohammed MS, Gorre ME, Cotter PD, Kim J, Bahler DW, Preobrazhensky SN, Higgins RA, Bolla AR, Ismail SH, de Jong D, Eldering E, van Oers MH J, Mellink CH M, Keating MJ, Schlette EJ, Abruzzo LV, Robetorye RS. Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia. J Mol Diagn. 2008; 10(5): 442-51. PubMed

    Hartung L, Bahler DW. 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

    Heerema NA, Bernheim A, Lim MS, Look T, Pasqualucci L, Raetz E, Sanger WG, Cairo MS. State of the Art and Future Needs in Cytogenetic/Molecular Genetics/Arrays in childhood lymphoma: summary report of workshop at the First International Symposium on childhood and adolescent non-Hodgkin lymphoma, April 9, 2003, New York City, NY. Pediatr Blood Cancer. 2005; 45(5): 616-22. PubMed

    Ho AK, Hill S, Preobrazhensky SN, Miller ME, Chen Z, Bahler DW. Small B-cell neoplasms with typical mantle cell lymphoma immunophenotypes often include chronic lymphocytic leukemias. Am J Clin Pathol. 2009; 131(1): 27-32. PubMed

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

    Kiel MJ, Velusamy T, Betz BL, Zhao L, Weigelin HG, Chiang MY, Huebner-Chan DR, Bailey NG, Yang DT, Bhagat G, Miranda RN, Bahler DW, Medeiros J, Lim MS, Elenitoba-Johnson KS J. 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 SR, Bayerl MG, Perkins SL, Barnette P, Schiffman JD, Miles RR. 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 MA, Heerema NA, Le Beau MM, Sposto R, Perkins SL, Kadin ME, Kjeldsberg CR, Meadows A, Siegel S, Buckley J, Abromowitch M, Kersey J, Bergeron S, Cairo MS, Sanger WG. 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

    Lundell R, Hartung L, Hill S, Perkins SL, Bahler DW. T-cell large granular lymphocyte leukemias have multiple phenotypic abnormalities involving pan-T-cell antigens and receptors for MHC molecules. Am J Clin Pathol. 2005; 124(6): 937-46. PubMed

    Miles RR, Cairo MS, Satwani P, Zwick DL, Lones MA, Sposto R, Abromovitch M, Tripp S, Angiolillo AL, Roman E, Davenport V, Perkins SL. Immunophenotypic identification of possible therapeutic targets in paediatric non-Hodgkin lymphomas: a children's oncology group report. Br J Haematol. 2007; 138(4): 506-12. PubMed

    Miles RR, Raphael M, McCarthy K, Wotherspoon A, Lones MA, Terrier-Lacombe MJ, Patte C, Gerrard M, Auperin A, Sposto R, Davenport V, Cairo MS, Perkins SL, SFOP/LMB96/CCG5961/UKCCSG/NHL 9600 Study Group. Pediatric diffuse large B-cell lymphoma demonstrates a high proliferation index, frequent c-Myc protein expression, and a high incidence of germinal center subtype: Report of the French-American-British (FAB) international study group. Pediatr Blood Cancer. 2008; 51(3): 369-74. PubMed

    Newell JO, Cessna MH, Greenwood J, Hartung L, Bahler DW. Importance of CD117 in the evaluation of acute leukemias by flow cytometry. Cytometry B Clin Cytom. 2003; 52(1): 40-3. PubMed

    Palomero T, Sulis MLuisa, Cortina M, Real PJ, Barnes K, Ciofani M, Caparros E, Buteau J, Brown K, Perkins SL, Bhagat G, Agarwal AM, Basso G, Castillo M, Nagase S, Cordon-Cardo C, Parsons R, Zúñiga-Pflücker JCarlos, Dominguez M, Ferrando AA. Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia. Nat Med. 2007; 13(10): 1203-10. PubMed

    Patte C, Auperin A, Gerrard M, Michon J, Pinkerton R, Sposto R, Weston C, Raphael M, Perkins SL, McCarthy K, Cairo MS, FAB/LMB96 International Study Committee. Results of the randomized international FAB/LMB96 trial for intermediate risk B-cell non-Hodgkin lymphoma in children and adolescents: it is possible to reduce treatment for the early responding patients. Blood. 2007; 109(7): 2773-80. PubMed

    Perkins SL, Pickering D, Lowe EJ, Zwick D, Abromowitch M, Davenport G, Cairo MS, Sanger WG. Childhood anaplastic large cell lymphoma has a high incidence of ALK gene rearrangement as determined by immunohistochemical staining and fluorescent in situ hybridisation: a genetic and pathological correlation. Br J Haematol. 2005; 131(5): 624-7. PubMed

    Preobrazhensky SN, Bahler DW. Optimization of flow cytometric measurement of ZAP-70 in chronic lymphocytic leukemia. Cytometry B Clin Cytom. 2008; 74(2): 118-27. PubMed

    Schumacher JA, Crockett DK, Elenitoba-Johnson KS J, Lim MS. Proteome-wide changes induced by the Hsp90 inhibitor, geldanamycin in anaplastic large cell lymphoma cells. Proteomics. 2007; 7(15): 2603-16. PubMed

    Sjostrom C, Seiler C, Crockett DK, Tripp SR, Johnson KS J Elenit, Lim MS. Global proteome profiling of NPM/ALK-positive anaplastic large cell lymphoma. Exp Hematol. 2007; 35(8): 1240-8. PubMed

    Smock KJ, Nelson M, Tripp SR, Sanger WG, Abromowitch M, Cairo MS, Perkins SL, Children's Oncology Group. Characterization of childhood precursor T-lymphoblastic lymphoma by immunophenotyping and fluorescent in situ hybridization: a report from the Children's Oncology Group. Pediatr Blood Cancer. 2008; 51(4): 489-94. PubMed

    Smock KJ, Perkins SL, Bahler DW. Quantitation of plasma cells in bone marrow aspirates by flow cytometric analysis compared with morphologic assessment. Arch Pathol Lab Med. 2007; 131(6): 951-5. PubMed

    Smock KJ, Yaish HM, Cairo MS, Lones MA, Willmore-Payne C, Perkins SL. Mantle cell lymphoma presenting with unusual morphology in an adolescent female: a case report and review of the literature. Pediatr Dev Pathol. 2007; 10(5): 403-8. PubMed

    Szankasi P, Reading S, Vaughn CP, Prchal JT, Bahler DW, Kelley TW. 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

    Termuhlen AM, Smith LM, Perkins SL, Lones M, Finlay JL, Weinstein H, Gross TG, Abromowitch M. Disseminated lymphoblastic lymphoma in children and adolescents: results of the COG A5971 trial: a report from the Children's Oncology Group. Br J Haematol. 2013; 162(6): 792-801. PubMed

    Tward JD, Cachoeira CV, Salama ME, Lee RJ, Bowen G, Perkins SL, Glenn M, Confer M, Gaffney DK. Survival and recurrence in nonmycosis fungoides primary cutaneous lymphoma. Cancer J. 2009; 15(1): 87-92. PubMed

    Yang DT, Dunphy CH, Tripp SR, Lagoo AS, Perkins SL. Nodular lymphocyte predominant Hodgkin lymphoma at atypical locations may be associated with increased numbers of large cells and a diffuse histologic component. Am J Hematol. 2008; 83(3): 218-21. PubMed

    Yang DT, Greenwood JH, Hartung L, Hill S, Perkins SL, Bahler DW. Flow cytometric analysis of different CD14 epitopes can help identify immature monocytic populations. Am J Clin Pathol. 2005; 124(6): 930-6. PubMed

    Medical Reviewers

    Last Update: February 2016