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

˥

˩

˥

˩

˥

˩

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