Flow cytometric analysis of B-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 • Review basic principles of flow cytometric immunophenotypic analysis of B cell lymphoproliferative disorders • Discuss recent studies to overcome limitations and shortcomings – New markers – New methods Incidence of B-cell neoplasms, United States Subtype Incidence rate 2011-2012 New cases, 2016 per 100,000 Lymphoid neoplasms 34.4 136,960 Lymphoid neoplasms, B 29.0 93.3% 117,470 B-LL/L 1.4 82.2% 4,930 CLL/SLL 5.1 20,980 FL 3.4 13,960 DLBCL 6.3 27,650 MM 5.9 24,280 Lymphoid neoplasms, T/NK 2.1 8,380 T-LL/L 0.3 1,070 T-PLL <0.1 160 T-LGL 0.2 670 ATL/L <0.1 180 Teras et al. CA Cancer J Clin 2016; 66:443-459 (North American AssociationTeras of et Central al. CA Cancer Cancer Registries) J Clin 2016; 66:443-459 (North American Association of Central Cancer Registries) SS <0.1 Teras et al. CA Cancer J Clin70 2016; 66:443-459 (North American Association of Central Cancer Registries) 94% WHO revised 4th ed., 2017 Flow cytometric analysis of B-cell lymphoproliferative disorders • B-cell antigen expression (CD19, CD20, CD22) • Monoclonal surface immunoglobulin κ or λ light chain expression (or absence of surface immunoglobulin) • Expression of additional B-cell antigens or other antigens, including abnormal expression levels • Presence of cells with abnormal light scatter characteristics ( high forward scatter or side scatter) B-ALL MCL FL, HL MZL, CLL, MM LPL DLBCL DLBCL WHO revised 4th ed. Case 1. 61 year old woman with lymphocytosis (WBC = 12,720/μl, 60% lymphocytes) CD45+, CD19+, CD20 dim+, CD5+, CD11c dim+, CD23+, sIg lambda+ CD10-, sIg kappa- Chronic lymphocytic leukemia/small lymphocytic lymphoma [Older adults; ≥5000/ul; PB, BM, lymphoid tissues] CLL/SLL CD20 bright+ reactive B cells neoplastic B cells CD20 dim+ Subpopulation gating based on differential staining for pan-B-cell markers (CD19, CD20, CD22) can be helpful to distinguish neoplastic B-cell populations from reactive, polyclonal background cells Normal Lymph Node B-CLL/SLL Follicular lymphoma reactive neoplastic reactive neoplastic Mantle cell lymphoma Follicular lymphoma Marginal zone lymphoma Huang et al, Ohio State University, Am J Clin Pathol 2005; 123:826-832 Case 2. 67 year old man with lymphadenopathy underwent a staging bone marrow biopsy CD19+, CD20+, CD5+, sIg lambda+ CD10-, CD11c-, CD23-, sIg kappa- Mantle cell lymphoma (t(11;14) CCND1) [older adults; LNs>spleen>BM>extranodal; 3-5 yr survival] CD20 x CD5 CLL MCL sIg κ x sIg λ CD23 x CD79b 13 Dr. M. Linden, University of Minnesota Percentage of CLL/SLL cases deviating from classical antigenic patterns Classic antigenic pattern % of cases deviating CD20 dim positive 11-38% CD22 dim positive 0-8% CD23 negative 3-5% FMC7 negative (CD20 epitope) 7-14% CD79b negative 5-18% Surface immunoglobulin dim positive 5-42% CD5 positive ? S. Kroft and A. Harrington, Clin Lab Med 2017; 37: 697 CD200 (Ig superfamily membrane glycoprotein) distinguishes CLL/SLL from Mantle cell lymphoma CLL/SLL 79/79+ MCL 0/14+ + >20%+ Palumbo et al., Catania, Leuk Res 2009; 33:1212 Case 3. A 54-year-old woman with a history of non-Hodgkin’s lymphoma presented with inguinal lymphadenopathy. An FNA was performed. CD19+, CD20+, CD10+, CD38+, sIg kappa+ CD5-, sIg lambda- F ollicular lymphoma (t(14;18) BCL2) [Older adults; LN>spleen>BM>PB] Wang and Zu Arch Pathol Lab Med 2017; 141:1236 CD5+, CD10+ B-cell neoplasms CD5+, CD10+ Mantle cell lymphoma Coexpression of CD5 and CD10 occurs in <1% of B cell lymphomas, including DLBCL, follicular lymphoma, mantle cell lymphoma, CLL/SLL, other small cell B cell lymphomas, and precursor B lymphoblastic leukemia/lymphoma, and is of uncertain clinical significance. [Dong et al. B-cell lymphomas with coexpression of CD5 and CD10. Am J Clin Pathol 2003; 119:218-230.] Surface immunoglobulin-negative B-LPDs B cells before (left) and after (right) 37C incubation and increased light chain reagent (Harrington and Kroft. Clin Lab Med 2017; 37:697) Biclonal B-cell lymphoproliferative disorders account for <5% of cases (23/477 in study cited), and include CLL/SLL, aCLL, HCL, LPL, SMZL, FL, LCL Sanchez et al. Blood 2003; 102:2994 Case 4. A 58 year old woman presented with splenomegaly, and anemia CD45+, CD19+, CD20 dim+, CD23 var+, sIg kappa+ CD5-, CD10-, CD11c-, sIg kappa- Marginal zone lymphoma [Older adults; spleen>PB / MALT / LN; indolent] , HCL-v Wang and Zu Arch Pathol Lab Med 2017; 141:1236 Case 5. 55 year old man with weakness, fatigue, progressive neuropathy, anemia, IgM kappa paraprotein (2.77 g/dl) CD19+, CD20+, sIg kappa+ CD5-, CD10-, CD11c-, CD23-, sIg lambda- and… Plasma cell component: CD38+, CD138+ cIg kappa+ CD19-, CD56-, cIg lambda- Lymphoplasma- cytic lymphoma (MYD88 L265P) [Older adults; BM; WMG = BM + IgM monoclonal gammopathy; indolent] Case 6. A 43-year-old woman presented with slight leukopenia and thrombo- cytopenia. WBC= 4,200/ul with 59% lymphocytes, 4% atypical lymphocytes with cytoplasmic projections. Bone marrow examination revealed 65% lymphocytes, some with cytoplasmic projections. negative control CD19+, CD20+, sIg kappa+, CD11c+, CD25+, CD103+ CD5-, CD10- Hairy cell leukemia (BRAF V600E) [Older adults; BM, splenic red pulp, PB] CD200 expression in B-cell lymphoproliferative disorders by flow cytometric analysis *HCL CD1d is a MHC class I-liCD1d is a* MHC class I-like CD1d is a MHC class I-like cell surface cell surface glycoprotein expressed in a wide glycoprotein expressed in a wide range range of cells, with increased expression in of cells, with increased expression in resting, naïve, and marginal zone B cells vs. resting, naïve, and marginal zone B activated and memory B cells cellsExpression vs. activatedin CLL/SLL is andless than memory in MCL; B expression cells in MZL but not LPL/WMG ke cell surfaceExpression glycoprotein in CLL/SLL expressed is less than in in a MCL; wide expression in MZL but not LPL/WMG range of cells, with increased expression+ in resting, naïve, and marginal zone B cells vs. activated and memory B cells Expression in CLL/SLL is less than in MCL; expression in MZL but not LPL/WMG +HCL-V Pillai et al Am J Clin Pathol 2013; 140:536-543 CD200 and CD1d expression in CD5-, CD10- B-cell lymphoproliferative disorders Hairy cell leukemia Hairy cell leukemia- variant Mason et al. Am Am J Clin 148:33 2017; Pathol et Mason al. Lympho- plasmacytic lymphoma Marginal zone lymphoma CD11c CD103 CD200 CD1d Pattern of CD200 and CD1d expression in CD5-, CD10- B-cell lymphoproliferative disorders + + -- + - - + 100.00% 80.00% Neg Dim 60.00% Pos Bright 40.00% Am J Clin 148:33 2017; Pathol et Mason al. 20.00% 0.00% HCL HCLv LPL MZL +/+: 94% sensitive and 98% specific for HCL +/-: 60% sensitive and 97% specific for LPL -/+: 41% sensitive and 100% specific for MZL CD5-negative, CD10-negative B-cell lymphoproliferative disorders Cytogenetics/Molecular Treatment HCL BRAF V600E; MAP2K1 Purine analogs (cladribine, pentostatin) HCL-V MAP2K1 Purine analogs +Rituximab (anti-CD20), anti-CD22 , or anti-CD52 immunotherapy LPL MYD88 Rituximab ± (multi-agent) chemotherapy MZL -7q (SMZL), NOTCH2, MLL2, KLF2, Rituximab ± (multi-agent) chemotherapy PTPRD (NMZL) ? + anti-CD200 immunotherapy in HCL, LPL/WMG Case 7. 59 year old woman with a history of DLBCL, now with WBC = 69,110/ul with 57% atypical cells CLL/SLL CD19+, CD20+, sIg kappa+ CD5-, CD10-, sIg lambda- Large B cell lymphoma (5-10% are CD5+ de novo or arising from CLL/SLL) [Elderly and younger; nodal and extranodal>BM; 60-65% 5 year survival] Expression of T-cell markers in B-LPDs (Tsuyama et al. Oncotarget 2017; 8:33487-33500) • CD2, CD3, CD4, CD5, CD7, CD8 expression was evaluated in 501 B- LPDs, including 225 DLBCLs, by flow cytometry • T-cell markers other than CD5 were expressed in 27/501 patients (5%), all large B cell lymphomas: 25 DLBCL and 2 IVLBCL • CD8 > CD7 > CD2 > CD4; 8 cases had >1 T-cell marker; no CD3+ cases • CD5 was present in 31/225 DLBCLs (15%); CD5 was coexpressed with other T-cell markers in 5/31 CD5+ DLBCLs (16%) • Poorer survival in CD5+ DLBCL vs. CD5- DLBCL, but no differences in survival with expression of other T-cell markers • CD8 previously reported in CLL (0.5-3% of cases) Case 8. 46 year old man with a right parapharyngeal mass and leukocytosis (WBC = 17,830/ul; 14% lymphocytes, 30% atypical lymphocytes CD45+, CD19+, CD20+, CD10+, CD38+, sIg kappa+ CD5-, sIg lambda- Burkitt lymphoma/leukemia (MYC translocation) [Children and young adults; extranodal>LN; highly aggressive but curable] Bright CD38 staining is an indicator of MYC rearrangement MYC rearrangement Numerical MYC aberrations Normal MYC 106 cases of CD10+ high grade lymphomas Maleki et al. Leuk Lymph 2009;50:1054-57. Burkitt lymphoma vs. Double-hit lymphoma with MYC and BCL translocations Burkitt lymphoma: CD19+, CD20+, CD10+, CD38 bright+ Double-hit lymphoma: Roth et al. Oncol Res 2016; 23:137 CD45↓, CD19+, CD20↓, CD10+, CD38+ Immunophenotypic findings for mature B-cell neoplasms 1, 2 3,4 5 6 7 8 9, 10+ Pan-B sIg CD5 CD10 CD23 CD11c other (CD19/ Κ v. λ CD20) CLL/SLL +/↓ +/↓ + - + v CD79b-, CD200+ MCL + + + - - - CD79b+, CD200-, Cyclin D1+, Sox 11+ Follicular + + - + v - CD38, Bcl-2+, Bcl-6+ MZL + + - - v v CD200-, CD1d+ LPL + + - - - - CD200+, CD1d-, cIg+ plasma cells HCL + + - - - + CD25+, CD103+, CD200+, CD1d+ HCL-V + + - - - + CD25-, CD103+, CD200-, CD1d- DLBCL + +/- -/+ +/- v Bcl-2+, Bcl-6+/- Burkitt + + - + - - CD38+, TdT-, Myc+, Bcl-6+,