Lymphoma Phenotyping

Diagnosis

Indications for Testing

• Evaluation of peripheral lymphocytosis (absolute lymphocytosis >4,000/µL)
• Lineage-associated antigens and minimal monoclonal antibody panels for diagnosis

  Lymphoma Surface and Nuclear Antigens
  B-cell	CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD79a, CD79b, CD138, PCA-1, immunoglobulins (IgA, IgG, IgM, IgD, κ, λ)
  T-cell	CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD45RA, CD45RO, TCRαβ, TCRγδ
  NK-cell	CD16, CD56, CD57
  Minimal Monoclonal Antibody Panels for Lymphoma Diagnosis
  B-cell	κ/λ, CD5/CD19, CD23, CD10, FMC7
  T-cell	CD3/CD4, CD3/CD8, CD5, CD7, CD25, CD30
  NK-cell	CD2, CD3/CD4, CD3/CD8, CD16, CD56, CD57

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, FMC7, 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
• B-cell lymphoproliferative disorders

  Probable if immunoglobulin light chain restriction is demonstrated by surface typing of kappa or lambda B-cell CLL or mantle cell lymphomas are suspected if CD5 is positive and CD10 is negative Circulating mantle cell lymphoma can be mistaken morphologically for B-cell CLL Mantle cell lymphoma considered in the following CD20, CD19 – strong intensity Surface immunoglobulin – strongly expressed CD23 – absent Diagnosis Molecular and FISH testing Requires t(11;14) translocation demonstration CLL is more likely when CD20 – weak intensity Surface immunoglobulins –  weakly expressed CD23 – present Circulating germinal center cell-derived lymphoma is probable if CD10 is positive and CD5 is negative Germinal center lymphomas – follicular, BL, diffuse large B-cell lymphoma (DLBCL) Diagnosis Some cases can be confirmed by demonstration of  t(14;18) breakpoint by PCR or FISH testing PCR detects approximately 80% of t(14;18) translocations found in follicular lymphoma FISH is more sensitive for this translocation in fixed tissue FISH can also detect an MYC or BCL6 rearrangement for BL or DLBCL Marginal zone lymphoma should be considered if both CD5 and CD10 are negative Hairy cell leukemia has a characteristic phenotype that is CD5-, CD10-, CD11c+, CD25+ and CD103+ CD103 antigen (also known as B-ly7) is present in virtually all cases CD11c and CD25 are less specific but present in almost all cases of hairy cell leukemia

• T-cell lymphoproliferative disorders

  Most show abnormalities of pan T-cell antigens CD2, 3, 5, or CD7 T-cell disorders Proliferating lymphocytes are usually positive for CD3 Most common form is large granular lymphocytosis Usually show rearrangement of TCR locus Clonality assessed by flow cytometry, PCR or Southern blot Large granular lymphocytosis is suspected if percentage of CD16, CD56 or CD57 positive T-cells is >50% or if absolute count of these cells >2,000/µL Peripheral T-cell lymphoma (NOS) Phenotypic abnormalities Angioimmunoblastic lymphoma has characteristic CD10 and CD4 positive and CD52, CD56 and CD16 negative Anaplastic large cell lymphoma – positive CD30 and ALK(+) Some pan T-cell antigens are frequently deleted Sézary syndrome should be considered if CD7 and CD26 are negative Clinical spectrum and prognosis are variable in both T-cell and NK-cell types Most behave in indolent fashion

  NK-cell lymphoproliferative disorders

  Many show abnormalities of pan NK-cell antigens CD2, CD7, CD16, CD56, and/or CD57

Histology

• Immunohistochemistry – for recommended stains, see individual ARUP Consult topics
  • Acute Lymphoblastic Leukemia
  • Acute Myeloid Leukemia
  • B-Cell Lymphomas
  • Chronic Lymphocytic Leukemia
  • Epstein Barr Virus
  • Ewing Sarcoma
  • Human Immunodeficiency Virus
  • Human T-Lymphotropic Virus Types I, II
  • Myelodysplastic Syndromes
  • Myeloproliferative Neoplasms
  • Plasma Cell Dyscrasias
  • Sézary Syndrome
  • T/NK-Cell Lymphomas
  • Tumor Markers

Prognosis

• Non-Hodgkin B-cell lymphoma

  Indolent CLL/SLL – favorable  Sequencing – >2% mutation of IGHV Flow cytometry – <30% CD38, <20% ZAP 70 Cytogenetics – del(13q) as sole abnormality CLL/SLL – unfavorable Sequencing – ≤2% mutation of IGHV or any IGHV rearrangements involving VH3-21 even if mutated Flow cytometry – ≥30% CD38, ≥20% ZAP 70 Cytogenetics – del(11q), del(17p) Follicular lymphoma Unfavorable – multiple prior therapies; transformation into DLBCL MZL (includes nodal, MALT, and splenic) MALT (extranodal MZL) Gastric MALT associated with Helicobacter pylori infection; good prognosis if infection is eradicated Aggressive DLBCL Unfavorable – known risk factors, including age (>60 years), disease stage, elevated LD, therapy response (ECOG 1496 trial), number of extra-nodal sites Favorable – no risk factors Mantle cell lymphoma Favorable – Ki-67 proliferation fraction <30% Highly aggressive Burkitt lymphoma and lymphoblastic lymphoma (T and B) Unfavorable – elevated LD, concurrent HIV infection AIDS-related B-cell lymphoma (includes Burkitt and PCNSL) Unfavorable – PCNSL, persistent CD4 count <100

  Non-Hodgkin T-cell lymphoma

  All considered aggressive   Peripheral T-cell lymphoma (PTCL) Favorable – ALK-positive ALCL subtype; normal LD level, younger age (<60 years) Unfavorable – AILT, EBER-positive tumors Mycosis fungoides/Sézary syndrome Unfavorable – older age (>56 years), tumor-stage disease or erythrodermic skin involvement, TCR gene rearrangements, extracutaneous disease

  Hodgkin lymphoma

  Unfavorable prognostic factors Mediastinal bulk in early stage disease Presence of B-cell lymphoma symptoms Disease found in ≥3 sites Erythrocyte sedimentation rate of ≥50 Age ≥45 years Male sex Stage IV disease Albumin level <4 g/dL Hemoglobin level <10.5 g/dL Leucocytosis – WBC >15,000/mm Lymphocytopenia – lymphocyte count <8% of WBC count and/or <600/mm

Differential Diagnosis

• EBV
• Chronic infections
• Granulomatous disease
  • M. tuberculosis
  • Sarcoidosis
• Pediatric 
  • Ewing sarcoma
  • Neuroblastoma
  • Wilms tumor
• Lymphadenitis

Clinical Background

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

WHO Classification of Lymphoid Neoplasms, 2008

• Precursor lymphoid neoplasms

  Precursor Lymphoid Neoplasms

  B lymphoblastic leukemia/lymphoma, NOS (not otherwise specified) B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities B lymphoblastic leukemia/lymphoma with t(9/22)(q34;q11.2); BCR-ABL1 B lymphoblastic leukemia/lymphoma with t(v;11q23); MLL rearranged B lymphoblastic leukemia/lymphoma with t(12/21)(p13;q22); TEL-AML1 (ETV6-RUNX1) B lymphoblastic leukemia/lymphoma with hyperdiploidy B lymphoblastic leukemia/lymphoma with hypodiploidy (hypodiploid ALL) B lymphoblastic leukemia/lymphoma with t(5;14)(q31;q32); IL3-IGH B lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3); E2A-PBX1 (TCF3-PBX1) T lymphoblastic leukemia/lymphoma

  Mature B-cell neoplasms

  Mature B-Cell Neoplasms

  Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) B-cell prolymphocytic leukemia Splenic marginal zone lymphoma (MZL) Hairy cell leukemia (HCL) Splenic lymphoma/leukemia, unclassifiable Lymphoplasmacytic lymphoma Waldenström macroglobulinemia Heavy chain diseases Plasma cell myeloma Solitary plasmacytoma of bone Extraosseous plasmacytoma Extranodal MZL of mucosa-associated lymphoid tissue (MALT) Nodal MZL Follicular lymphoma Primary cutaneous follicle center lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma (DLBCL), NOS (not otherwise specified) T-cell/histiocyte rich large B-cell lymphoma Primary DLBCL of the central nervous system (PCNSL) Primary cutaneous DLBCL, leg type Epstein-Barr virus (EBV)-positive DLBCL of the elderly 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 Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease Primary effusion lymphoma Burkitt lymphoma B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma

  Mature T- and NK-cell Neoplasms

  Mature T- and NK-cell Neoplasms

  Precursor T-cell neoplasm T-cell prolymphocytic leukemia T-cell large granular lymphocytic leukemia Chronic lymphoproliferative disorder of NK-cells (provisional entity) Aggressive NK-cell leukemia Epstein-Barr virus (EBV)-positive T-cell lymphoproliferative disorder of childhood Adult T-cell leukemia/lymphoma (ATLL) Extranodal NK/T-cell lymphoma, nasal type Enteropathy-associated T-cell lymphoma Hepatosplenic T-cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Mycosis fungoides Sézary syndrome Primary cutaneous CD30-positive T-cell lymphoproliferative disorders Primary cutaneous gamma-delta T-cell lymphoma Peripheral T-cell lymphoma, NOS (not otherwise specified) Angioimmunoblastic T-cell lymphoma Anaplastic large-cell lymphoma (ALCL), ALK positive ALCL, ALK negative (provisional entity)

  Hodgkin lymphoma

  Hodgkin Lymphoma
  Nodular lymphocyte predominant Hodgkin lymphoma Classical Hodgkin lymphoma Nodular sclerosis classical Hodgkin lymphoma Lymphocyte-rich classical Hodgkin lymphoma Mixed cellularity classical Hodgkin lymphoma Lymphocyte-depleted classical Hodgkin lymphoma

Clinical Presentation

• Frequently nonspecific symptoms – 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
  • Splenomegaly
  • Common site of extranodal disease
• Common hematological antigens in lymphomas

  Common Hematological Antigens in Lymphomas
  Antigen or Cluster Designation (CD)	Application
  CD1a	T-cell lymphoma, particularly T-lymphoblastic lymphoma
  CD2	T-cell lymphoma
  CD3	T-cell lymphoma (immature T-cell neoplasms may have cytoplasmic CD3 without cell surface CD3)
  CD4	Identify T-cell subset in all T-cell lymphomas
  CD5	T/B-cell lymphoma, CLL; may be absent in peripheral T-cell lymphoma (PTCL) and plasmacytoid lymphoma
  CD7	T-cell lymphoma (may be absent in PTCL, adult T-cell leukemia/lymphoma [ATLL], mycosis fungoides)
  CD8	Identify T-cell subset in all T-cell lymphomas
  CD9	Some CLL
  CD10 (CALLA)	B-cell lymphoma (follicular lymphoma, Burkitt lymphoma), angioimmunoblastic T-cell lymphoma (AILT). lymphoblastic lymphomas
  CD11a	May be absent in some B-cell neoplasms
  CD11c	Hairy cell leukemia (HCL), marginal zone lymphoma (MZL), other B-cell neoplasms
  CD14	Chronic myelomonocytic leukemia (CMML)
  CD15	Hodgkin lymphoma
  CD16	NK-cell disorder
  CD18	Some B-cell neoplasms
  CD19	B-cell lymphoma, CLL; usually absent in plasma cell neoplasms
  CD20	B-cell lymphoma, CLL; usually absent in plasma cell neoplasms
  CD21	B-cell lymphoma
  CD22	B-cell lymphoma, HCL
  CD23	B-cell lymphoma, CLL, small lymphocytic lymphoma (SLL); usually absent in mantle cell lymphoma
  CD24	Most mature B-cell neoplasms
  CD25	ATLL, HCL, some T- and B-cell lymphomas, including anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma
  CD26	Usually absent in Sézary syndrome
  CD30	Hodgkin lymphoma, ALCL
  CD38	PTCL, some B-cell lymphomas, myeloma, plasmacytic neoplasms
  CD45	Lymphomas/leukemias (usually reduced/absent in lymphoblastic lymphoma, ALCL, AILT, plasma cell proliferations)
  CD45RA	Follicular lymphoma, some T-cell lymphomas, some mature B-cell lymphomas
  CD45RO	T-cell lymphoma
  CD56	NK-cell disorder, large granular lymphocytic leukemia (LGLL), hematodermic neoplasms (blastic NK-cell lymphoma)
  CD57	NK-cell disorder, LGLL
  CD71	Acute leukemia/lymphoma, intermediate- and high-grade lymphomas, Hodgkin lymphoma
  CD74	B-cell lymphoma
  CDw75	B-cell lymphoma
  CD79a	B-cell lymphoma
  CD79b	B-cell lymphoma
  CD103	HCL, some splenic lymphomas
  CD138	B-cell lymphoma, myeloma
  FMC-7	B-cell lymphoma, mantle cell lymphoma, HCL, splenic lymphoma
  HLA-DR	B-cell neoplasms, Hodgkin lymphoma, PTCL
  PCA-1	Myeloma
  TdT	Lymphoblastic lymphoma
  TCR - α-β	T-cell lymphoma/leukemia (most T-cell neoplasms expressing CD3)
  TCR - γ-δ	T-cell lymphoma/leukemia

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

Click the plus sign to expand the table of additional tests.

Test Name and Number	Comments
Epstein-Barr Virus by PCR 0050246 Method: Qualitative Polymerase Chain Reaction	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

Diagnostic Algorithm

  Lymphoma Leukemia Phenotyping Testing Algorithm