flow cytometry of t -cell lymphoproliferative disorders...lymphadenopathy* cd3+, cd5+,...
TRANSCRIPT
Flow cytometry of T-cell lymphoproliferative disorders
David M. Dorfman, M.D., Ph.D.Department of Pathology
Brigham and Women’s Hospital and Harvard Medical School
Boston, MA
Objectives
• Outline basic principles of T cell neoplasia classification and diagnosis
• Review T cell maturation and normal T cell subsets
• Discuss important immunophenotypic patterns in neoplasms of immature and mature T cells
2
Uncommon: <10% of lymphoid neoplasms in the U.S., 15%-25% of ALL
Difficult to recognize/diagnose
Aggressive, in general
T-cell neoplasms are:
M.D. Anderson Cancer Center, Houston; Blood 1997; 89:4514-4520
1,595 diffuse BCL288
Hôpital Saint-Louis, Paris; Blood 1998; 92:76-82
Incidence of T-cell neoplasms, United States
Subtype Incidence rate 2011-2012 per 100,000 New cases, 2016
Lymphoid neoplasms 34.4 136,960
Lymphoid neoplasms, B 29.0 117,470
B-LL/L 1.4 4,930
CLL/SLL 5.1 20,980FL 3.4 13,960DLBCL 6.3 27,650
MM 5.9 24,280
Lymphoid neoplasms, T/NK 2.1 6.7% 8,380
T-LL/L 0.3 18% 1,070
T-PLL <0.1 160T-LGL 0.2 670ATL/L <0.1 180SS <0.1 70
PTCL, NOS 0.4 1,660AITL 0.1 530ALCL, ALK+ 0.2 830
Teras et al. CA Cancer J Clin 2016; 66:443-459 (North American Association of Central Cancer Registries)
WHO classification Revised 4th Edition 2017
Precursor lymphoid neoplasms T- and NK-lymphoblastic leukemia/lymphoma
Mature T and NK neoplasms T-cell prolymphocytic leukemiaT-cell large granular lymphocytic leukemiaChronic lymphoproliferative disorder of NK cellsAggressive NK-cell leukemiaAdult T-cell leukemia/lymphoma
EBV-positive T-cell and NK-cell lymphoproliferative disorders of childhood (4 subtypes)
Extranodal NK-/T-cell lymphoma, nasal typeIntestinal T-cell lymphomas (4 subtypes)Hepatosplenic T-cell lymphomaSubcutaneous panniculitis-like T-cell lymphomaBreast implant–associated anaplastic large-cell lymphoma
Mycosis fungoidesSezary syndromePrimary cutaneous CD30+ T-cell lymphoproliferative disorders (2 subtypes)Primary cutaneous T-cell lymphomas, rare subtypes (4 subtypes)
Peripheral T-cell lymphoma, NOSAngioimmunoblastic T-cell lymphoma and other nodal lymphomas of TFH originAngioimmunoblastic T-cell lymphoma Follicular T-cell lymphomaNodal peripheral T-cell lymphoma with TFH phenotype
Anaplastic large-cell lymphoma, ALK+Anaplastic large-cell lymphoma, ALK-
Provisional entities are listed in italics.
Leukemic/disseminated
Extranodal
Cutaneous
Nodal
γδ T cells
T-PLL
NK/T cells
Th1, Th2,
TFH, Treg
Knowles, Neoplastic Hematopathology, 2001
CD4-, CD8-
Normal T-cell development
T-ALLPTCL
CD4
CD8
T-cell neoplasia: Diagnostic approach
Morphologic findings
Immunophenotypic findings (flow cytometry/immuno-histochemistry)
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Cytogenetic findings
Molecular diagnostic findings (TCR gene rearrangement)
New molecular diagnostic findings: gene expression array/ DNA sequence
+ correlation with other clinical and laboratory findings
Immunophenotypic criteria for the diagnosis and subtyping of T-cell NHL
• Markers of T cell immaturity : TdT, CD1a, CD34, CD4/CD8 -/- or +/+ (blasts (T-LL) vs. prolymphocytes (T-PLL)
• Aberrant expression of pan-T cell markers (CD2, CD3, CD5, CD7) (Most T-cell neoplasms of mature T-cell origin)
• T helper (CD4+) vs. T supressor/cytotoxic (CD8+) derivation(Most T-cell neoplasms vs. T-LGL leukemia)
• Alpha-beta (αβ) vs. gamma-delta (γδ) T cell derivation(Most T cell neoplasms vs. γδ T-cell neoplasms)
• Tumor-associated cell markers; e.g. FDCs in AITL• Activation/differentiation state markers
- CD30 in ALCL, - FOXP3 in ATLL- PD-1 in AITL
Case 1: 47 year old man with lymphadenopathy*
CD3+, CD5+, CD7-population that is CD4+, CD8-
*the patient could have presented with similar findings in peripheral blood and/or bone marrow
+ atypical lymphoid infiltrate in lymph node(bone marrow, peripheralblood)
T-NHL
Flow cytometric evidence of aberrant expression of pan-T-cell markers in peripheral T-cell neoplasms
Jamal et al. Am J Clin Pathol 2001; 116:512
[aberrant expression in 46 of 50 cases (92%)]
CD3 CD5 CD7
• T-NHL with ↑forward scatter, sCD3-, CD4-, CD8- (AITCL case)
• T-NHL with dim sCD3, partial CD2+, CD7- (not shown; PTCL, NOS) Jamal et al. Am J Clin Pathol 2001; 116:512
• T-NHL with ↑forward scatter, ↑CD3+, ↓CD4+, ↑CD45+ (PTCL, NOS)
• T-NHL with dim CD3+, CD7-, CD4+, CD8- (PTCL, NOS)
Jamal et al. Am J Clin Pathol 2001; 116:512
Caveat: There are non-neoplastic patterns of absence/loss of T-cell markers
• HIV infection with CD4 T-cell depletion
• Aberrant T-cell populations of unclear significance (Hamburg; Flammiger et al. Leuk Lymph 2015; 56:639)
• Down regulation of CD7 in RA, inflammatory dermatoses, and with aging
• Down regulation of CD7 in viremias
Down-regulation of pan-T-cell antigens, particularly CD7, in acute infectious mononucleosis
Weisberger et al. Am J Clin Pathol 2003; 120:49-55
• No pan-T cell antigen deletion• Dominant population of activated CD8+ cytotoxic-suppressor
cells which have a role in suppression of viral replication and cytotoxic activity against virally infected B cells
• May be seen in other viremias, incl. HIV
• CD4+/CD8+
• CD3+, CD7-
• CD3+, CD5dim+, CD8+ (T-LGL)
• CD3-, CD2+, CD7+(NK)
Non-neoplastic T cell subsetsin peripheral blood and bone marrow
NK cells:
sCD3-, CD4-, CD5-
CD2+, CD7+, CD8var+
CD16+, CD56+, CD57 var+, CD94+
90% CD16 bright, CD56 dim10% CD16 dim, CD56 bright
1. Chronic LPD of NK-cells: rare; >2000/μl, 6 mos.; adults, 60 years; ddx: autoimmunity, viral infections, dasatinib; CD3-, CD16+, CD56-/↓, CD57+, CD94+; STAT3 mutations in 30%; indolent clinical course
2. Aggressive NK-cell leukemia: rare; young adults, Asians; EBV+; ddx: NK/T cell lymphoma; fever, cytopenias, HSM; CD2+, CD3-, CD16+, CD56+, CD57-; fulminant clinical course
NK-cell neoplasms (leukemic/disseminated; innate immune system)
CD
56
CD16FSC
SSC
Jamal et al. Am J Clin Pathol 2001; 116:512
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˩
˥
˩
˥
˩
Peripheral blood γδ T-cellsAnother non-neoplastic T cell subset
↑ CD3+, CD4-, CD8-, ↓ CD5 +, ↓ CD7+
1-10%
Roden et al. APLM 2008; 132:1774-1780
Peripheral blood γδ T-cells
CD3+, CD4-, CD8-/dim (vs. NK)
↑ CD3 in 6/62*↓/- CD5 in 16/62↓ CD7 in 3/62+ CD16
* >5% of lymphocytes and >200 cells/ul(usually <10% of lymphocytes and <500 cells/ul)
*
γδ T cell lymphoma:
CD2+ CD3+ CD7+, CD56 dim+, TCR γδ+
CD4-, CD5-, CD8-, CD57-, TCRαβ-
[rare, adolescents and adults, extranodal, skin, aggressive]
Flow cytometric assay for T-cell clonality using antibodies to the variable region of the TCR β chain
Directly conjugated antibodies to the variable region of the TCR βchain (which can identify 19 of 25 V-β families = 70% of the normal repertoire) are utilized three at a time in 8 flow cytometric assay mixes to assess V-β family usage in cases of suspected T-LPD
Pathologic V-β restriction was observed in 24 of 27 T-LPD cases and 2 of 16 non-neoplastic cases (sensitivity = 89%, specificity = 88%) in comparison with PCR (Beck et al., AJCP 2003; 120:785); in another study 23 of 29 T-LPD cases (79%) were identified by V-β restriction (Morice et al., AJCP 2004; 121:373)
Morice et al. AJCP 2004; 121:373-383.
TCR-Vβ flow cytometric analysis to assess T-cell clonality [IO Test (Beckman Coulter)]
clonality: >40% positive for a specific Vβor >70% not positive for any Vβ
polyclonal T cells T-cell large granular lymphocytic leukemia
angioimmunoblastic T cell lymphoma
D. Wu, University of Washington, Am J Clin Pathol 2016; 145:467
Multiplexed single tube TCR-Vβ flow cytometric analysis to assess T-cell clonality
cutaneous T cell lymphoma
82%
90
%
null96%
Case 2. 67 year old woman with leukocytosis (WBC = 56,000/μl)
Specific neoplastic T cell immunophenotypic patterns
Small to medium-sized cells; cytoplasmic blebs; small cell variant (20%), cerebriform variant (5%)
• No loss of pan-T cell markers, including CD7 (vs. ATLL, MF/SS)• TdT-, CD34-, CD1a- (vs. T-LL)• CD4+/CD8- (60%) or CD4+/CD8+ (25%) or CD4-/CD8+ (15%)
T cell prolymphocytic leukemia
T-cell prolymphocytic leukemia
• 2% of non-acute leukemia in adults; 3% of T-NHL
• Elderly; HSM, lymphadenopathy, anemia, thrombocytopenia, WBC > 100,000/ul
• Small to medium-sized cells; cytoplasmic blebs; small cell variant (20%), cerebriform variant (5%)
• CD2,CD3, CD5,CD7+; TdT-, CD34-, CD1a-, CD10-; 60% CD4+CD8-, 25% CD4+CD8+, 15% CD4-CD8+ (versus ATLL, MF/SS and T-LL/LL)
• inv14q(q11;q32) in 80-90% (TCL1 oncogenes)↑TCL1 protein expression (↑cell survival [via AKT signaling]); chromosome 8 abnormalities in 70-80%
• Aggressive clinical course; median survival < 1 year
Chen and Cherian. Immunophenotypic characterization of T-cell prolymphocytic leukemia. AJCP 2013; 140:727-735
20 patients with T-PLL
Case 3. 45 year old man with persistent neutropenia, relative leukocytosis
• Circulating large lymphocytes with azurophilic cytotoxic granules
• Intrasinusoidal bone marrow/splenic infiltrates
• Clonal T cellsCD8
CD3+CD8+CD16+CD57+ T cells with ↓/-CD5 and/or CD7
T-LGL leukemia Cytotoxic markers: TIA-1, granzyme, or perforin
T-cell large granular lymphocytic (LGL) leukemia• 2-3% of non-acute leukemia in adults
• Persistent >6 mos ↑LGLs (2,000-20,000/ul) in p.b. (+BM, liver spleen) or lower if cells are clonal and with clinical and hematologic features, such as RA or cytopenias
• Median age 57 years, M:F 45%/55%; 70% 10 year survival (infections)
• Clinically indolent, severe neutropenia (84%) ± anemia (29%), recurrent infections (39%), RA and other autoimmune diseases (28%) >> B-LPDs (5%)
• Circulating large lymphocytes with cytotoxic granules
• CD3+CD4-CD8+TCRαβ (80-90%), usually CD57+, CD16+, with TCR gene rearrangement, and ↓CD5
• Somatic mutations in STAT3 in 28-75% and STAT5B (uncommon, aggressive clinical course); (STAT3 in 30-48% of NK-LGL leukemia)
• Pathogenesis model: Initial cytotoxic T cell response to chronic antigen stimulation STAT3 activation/mutation monoclonal population produces inflammatory cytokines (cytopenias, autoimmunity), cytotoxic granules (cytopenias, autoimmunity), clonal survival and expansion (Lamy et al., Blood 2017; 1082-1094)
Case 4. 42 year old man presented with abdominal pain, HSM, inguinal, axillary, and cervical LAD, and rapidly rising white blood cell count (from 20,000/ul to 32,000/ul in three days).
Subsequent serologic testing was reactive for HTLV1/2 by immunoblot and positive for HTLV1 antigens by Western blot.
Diffuse, pleomorphic infiltrate, including polylobated ‘flower’ cells
• CD2+, CD3+ (slightly ↓), CD4+, CD5+ • CD7-, CD8-• CD25+
Adult T cell leukemia/lymphoma
Adult T-cell leukemia/lymphoma
• HTLV-1 endemic regions; adults, M:F = 1.5:1
• Multiple clinical presentations that can involve PB, BM, lymph nodes, HSM, skin; lytic bone lesions, hypercalcemia: acute (60%), lymphomatous (20%), chronic (15%), smoldering (5%), cutaneous
• Diffuse, pleomorphic infiltrate, flower cells
• Flow cytometry- CD2+, CD3+, CD4+- CD7- in 20/30 cases- CD25+ in 13/30 cases- CD3 can be dim positive (14/30 cases; Yokote et al. AJCP 2005; 124:199
204.)
• FOXP3, a transcription repressor and marker of CD4+CD25+ regulatory T cells, is expressed in 17/25 (68%) of ATLL cases, but not other T cell lymphomas (Roncador et al. Leukemia 2005; 19:2247-2253) suppression of immune response (opportunistic infections, viral infections)
• Multistep oncogenesis involving multiple genetic events (Watanabe, Blood 2017; 1071-1081).
Case 5: A 57-year-old man with recently diagnosed mycosis fungoides presented with WBC = 7,300/ul with 39% lymphocytes, including 16% atypical cells, and lymphadenopathy
• CD2+, CD3+, CD4+, CD5+, CD7-, CD8-, CD26-• CD4:CD8 = 10.7:1
Sézary Syndrome
Sézary Syndrome
• Triad: Erythroderma, lymphadenopathy, Sézary cells in peripheral blood
• Rare; adults; poor prognosis (2-4 year median survival)
• Aberrant T cell antigen staining: CD7 (8/11 cases) > CD2 (5/11) > CD3 (4/11) >
CD5 (3/11) [Morice et al. AJCP 2006; 125:364-374.]
• Diagnostic criteria [B2 high blood tumor burden]
- Demonstration of a T cell clone in peripheral blood and
- Absolute Sézary cell count of ≥ 1,000/ul (or 20% of lymphocytes) or
- ↑CD3+ or CD4+ cells with CD4/CD8 > 10 or
- ↑CD4+ cells with an abnormal phenotype (≥ 40% showing loss of CD7 or ≥
30% showing loss of CD26 suggested)
• Ddx: HTLV-1(-) versus ATLL; loss of pan-T antigens versus T-PLL
Revised ISCL-EORTC staging and classification of MF/SS. Blood 2007; 110:1713-1722.
Herling et al. Blood 2004; 104:328-335
↑TCL1,
CD7+
CD7-,CD25+
CD7-, CD26-
CD3+, CD8+, CD57+CD5↓, CD7↓, CD16+
γδ T cells
(also NK cell neoplasms: chronic or aggressive)
[not blasts]
Blasts: T-ALL, ETP-ALLCD3+, TdT+, MPO-
• CD2+ CD3- CD4+• CD5+ CD7+/- CD8-• CD10+ non-B cells
Case 6: 52 year old man with lower extremity rash, cervical and inguinal lymphadenopathy
*
CXCL13PD-1
Angioimmunoblastic T cell lymphoma
Angioimmunoblastic T-cell lymphoma
• Middle aged to elderly adults; M=F; advanced disease; immune dysregulation, including hyperactivity and immunodeficiency; EBV infection
• architectural effacement; paracortical polymorphous infiltrates; increased vasculature and FDC networks
• Proliferation of neoplastic CXCL-13+, PD-1+ TFH cells (CD10+, Bcl-6+); EBV+ B cells present in >75% of cases
• Flow cytometric evaluation (Yuan et al. Hum Pathol 2005; 36:784-791)- CD2+, CD3+, CD4+- CD7- in 5/8 cases; decreased CD3 in 1/8 cases; decreased CD5 in 1/8 cases - CD10+ T cells in 6/8 cases
• Increased CD3-/dim CD4+ T cells in TFH NHLs, including AITL (Alikhan et al. Modern Pathology 2016)
Increased CD3-/dim CD4+ T-cells in TFH NHLs
Present in 11/15 AITL (mean 19.5% of lymphocytes; range: 3%-71.8%)
Present in 9/10 other TFH NHLs (29.3% of lymphocytes; range: 7.9%-62%)
<3% of lymphocytes in 25/26 reactive lymph nodes (and 15 NLPHL and 8 cHL) Alikhan et al. Modern Pathology 2016
Case 7: A 41 year old man presented with lymphadenopathy*
↑FS, ↑SS, CD45+, CD30+, CD4+, CD8- Anaplastic large cell lymphoma
CD
45 P
erC
P
CD30 FITC
Juco et al. AJCP 2003; 119:205-212.
Flow cytometric analysis of ALCL(Juco et al. AJCP 2003;119:205-212.)
• Neoplastic cells constituted 4%-84% of the sample
• Increased forward and side scatter vs. normal lymphocytes
• CD45+, CD30+ neoplastic cells in 19/19 cases; CD30 flow cytometric analysis
was employed after morphologic and initial flow cytometric analysis
• Loss of pan-T cell antigens in 18/19 cases: CD2+ (12/17); CD4+
(12/17), CD3+ (6/19), CD7+ (6/19), CD5+ (5/19), CD8+ (4/19)
• Myeloid antigen expression was observed in 12/19 cases: CD13, CD15, or CD33
• ALK positivity was observed in 3/9 cases
• Similar findings in Shen et al. Oncology Letters 2013; 5:515-520 and
Kesler et al. Am J Clin Pathol 2007; 128:314-322.
Flow cytometry of mature T-cells: important patterns• Loss/decrease/absence of pan-T cell markers in T-NHL (and reactive states
[CD7])
• Normal T cell subsets include NK, γδ, T-LGL, CD7-, and CD4+/CD8+ cells
• Specific neoplastic patterns
- T-PLL (CD4+ or CD4+CD8+ or CD8+)
- T-LGL (CD8+, CD16+, CD57+, ↓CD5 )
- AITCL/TFH lymphomas (CD10+ non-B cells, CD3-/dim,CD4+ T cells)
- ATLL (CD4+, CD7-, CD25+)
- Sézary syndrome (CD4+, CD7-, CD26-)*
- ALCL (large cells, CD30+, myeloid Ag+)*
* gating on large cells by forward and side scatter
**correlation with clinical, laboratory, and morphologic findings