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 lymphoproliferative disorders • Discuss recent studies to overcome limitations and shortcomings – New markers – New methods Incidence of B-cell , 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 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 (WBC = 12,720/μl, 60% ) CD45+, CD19+, CD20 dim+, CD5+, CD11c dim+, CD23+, sIg lambda+

CD10-, sIg kappa-

 Chronic lymphocytic /small lymphocytic

[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

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-

(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 ) 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 , 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… component:

CD38+, CD138+ cIg kappa+

CD19-, CD56-, cIg lambda-

 Lymphoplasma- cytic lymphoma (MYD88 L265P)

[Older adults; BM; WMG = BM + IgM ; 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-

(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.Mason et Pathol2017; 148:33Clin J Am

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% al.Mason et Pathol2017; 148:33Clin J Am

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 (, pentostatin) HCL-V MAP2K1 Purine analogs + (anti-CD20), anti-CD22 , or anti-CD52 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

• 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-

/leukemia ( 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+, Bcl-2- DHL +/↓ +/↓ - +/- - - CD38, CD45↓, TdT-, Myc+, Bcl-6+, Bcl-2+ Conventional flow cytometric analysis: problems and limitations

• Flow cytometric analysis is expensive and labor intensive • Reactive, polyclonal background cells • CD5 -negative, CD10-negative B–LPDs (lack of good markers for differential diagnosis) • Neoplasms may exhibit phenotypic variation • Definitive (clonality) markers may be unclear • Interpretation of the immunophenotype may be subjective (poor reproducibility)

 additional markers (for example, CD23, CD79b, CD43, CD103, CD200, CD1d) and approaches may be helpful New methods for flow cytometric analysis

• Cytoplasmic and nuclear antigens – cIg in MM, TdT in ALL, bcl-2 in FL, MPO in AML, cCD3 in T-ALL • Markers of clonality by expression usage – TCR β chain usage in T-NHL • Scoring systems diagnosis and subclassification – CLL, AML-M0 vs. ALL, MDS • Customized panels for minimal residual disease – CLL, ALL, AML, MM • Enzymatic amplification of staining – catalyzed tyramide reporter deposition cyclin D1 in MCL • Analysis of neoplasms not traditionally studied by flow cytometric immunophenotyping (epithelial neoplasms) – Ber-EP4 • Multiplex analysis • Value-based (cost effective) analysis • New (automated) approaches for data analysis Multiplexing: London Health Science Center custom design flow cytometry tube (12 markers, 10 fluorochromes, 14 parameters)

Antigen Fluorochrome

CD8 FITC

Kappa FITC

CD4 PE

Lambda PE

CD19 ECD

CD56 PE-Cy5.5

CD3 PE-Cy7

CD20 APC

CD10 APC-Alexa700 Excitation: 633nm Near-IR Viability Dye Emission 780nm

CD5 Pacific Blue

CD45 Krome Orange

Hedley et al. Cytometry B 2015; 88B:361 Multiplexed 12-marker, 10-color flow cytometric analysis

10-color, 15-Ab, 17-parameter panel for LPDs:

CD4/κ, CD8/λ, CD3/CD14, CD38, CD20/CD56, CD10, CD19, CD5, CD57/CD23, CD45

Rajab et al. Int J Lab Hematol 2017; 39(Suppl 1):76 Value-base flow cytometry testing of chronic lymphoproliferative disorders

• Retrospective analysis of peripheral blood samples submitted for flow cytometric analysis for LPD to develop a cost-effective testing strategy (multicenter study) – 8/70 cases (11%) with normal range absolute counts were positive – 2/20 cases (10%) with patients younger than 45 years were positive –  diagnostic algorithm for patients with low pre-test probability of a LPD (triage panel: CD3, CD4, CD5, CD19, sIg κ, sIg λ) and high pre-test probability of LPD (triage panel + additional B cell marker tube) –  reduction of 40% in use

• Statistical analysis (classification tree approach; University of Calgary) – Patients < 50 yrs with an absolute lymphocyte count <5,000/μl were nearly always negative (98.2%) for monoclonal B cells – Patient’s >50 years with an absolute lymphocyte count <5,000/μl and high ferritin level (450 µg/liter; acute phase reactant) were nearly always negative (97.1%) for monoclonal B cells –  26% of cases were correctly predicted as negative with greater than 97% accuracy (recommend that testing should not be performed) Oberly et al. Am J Clin Pathol 2014; 142:411 Healey et al Leuk Lymphoma 2015; 56:2619 Peripheral blood samples submitted for flow cytometric analysis for LPD: absolute lymphocyte count and patient age

8/110 = 7.2% 2/110 = 1.8%

Oberly et al. Am J Clin Pathol 2014; 142:411 New (data analysis) approaches

• Standardization of antibody panels, instrument settings, and data files, based on multicenter studies – Euroflow consortium • Standardized, lyophilized reagent tubes for uniformity of multi-institutional analysis – Texas Medical Center (Texflo) • Automated analysis: advanced, algorithmic approaches to population gating and data analysis of existing diagnostic marker panel – CLL vs. Mantle cell lymphoma (Zare et al.) 1

2

1. SamSPECTRAL clustering algorithm 2. FeaLect method (~Bolasso algorithm) to identify flow cytometry features most useful to discriminate CLL and MCL

A. Weng, University of British Columbia, Am J Clin Pathol 2012; 137:75 Automated analysis of flow cytometric findings to distinguish CLL/SLL from MCL

70 cases CLL/SLL 44 cases MCL

Am J Clin Pathol 2012; 137:75 Identification of discriminative ratios (CD20/CD23, FMC7/CD23, and CD20/CD11c) =0 =1

 composite diagnostic predictor with values of 0-1 (67/70 (96%) of CLL cases) vs. 2-3 (44/44 (100%) of MCL cases)

Am J Clin Pathol 2012; 137:75 Summary: Flow cytometric analysis of B-cell lymphoproliferative disorders

• B- cell antigen expression (CD19, CD20, CD22) • Monoclonal immunoglobulin κ or λ light chain expression (or absence of surface immunoglobulin) • CD5+/CD10-, CD5-/CD10+, CD5-/CD10- categories and exceptions • Presence of cells with abnormal light scatter characteristics (high forward scatter or side scatter) • New markers and approaches to overcome limitations and shortcomings of conventional flow cytometric analysis

Correlation with clinical, morphologic, and laboratory findings