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, 2016per 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 20,980FL 3.4 13,960DLBCL 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 160T-LGL 0.2 670ATL/L <0.1 180SS <0.1 70Teras et al. CA Cancer J Clin 2016; 66:443-459 (North American Association of Central Cancer Registries) Teras et al. CA Cancer J Clin 2016; 66:443-459 (North American Association of Central Cancer Registries) Teras et al. CA Cancer J Clin 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)

WHO revised 4th ed.

B-ALL MCL FL, HL MZL, CLL, MM LPL

DLBCL DLBCL

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]

reactive B cells

neoplastic B cells

CLL/SLL

CD20 bright+

CD20 dim+

Huang et al, Ohio State University, Am J Clin Pathol 2005; 123:826-832

Normal Lymph Node B-CLL/SLL

Mantle cell lymphoma Follicular lymphoma

Follicular lymphoma

Marginal zone lymphoma

reactive

reactive

neoplastic

neoplastic

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

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]

CLL MCL

13Dr. M. Linden, University of Minnesota

CD20 x CD5

sIg κ x sIg λ

CD23 x CD79b

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 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

(CD20 epitope)

Palumbo et al., Catania, Leuk Res 2009; 33:1212

CLL/SLL 79/79+

MCL 0/14+

CD200 (Ig superfamily membrane glycoprotein) distinguishes CLL/SLL from Mantle cell lymphoma

+ >20%+

Case 3. A 54-year-old woman with a history of non-Hodgkin’slymphoma presented with inguinal lymphadenopathy. An FNA was performed.

CD19+, CD20+, CD10+, CD38+, sIg kappa+

CD5-, sIg lambda-

Follicular lymphoma (t(14;18) BCL2)

[Older adults; LN>spleen>BM>PB]

Wang and Zu Arch Pathol Lab Med 2017; 141:1236

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.]

CD5+, CD10+ B-cell neoplasms

CD5+, CD10+ Mantle cell lymphoma

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]

Wang and Zu Arch Pathol Lab Med 2017; 141:1236

, HCL-v

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-, cIglambda-

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.

CD19+, CD20+, sIg kappa+, CD11c+, CD25+, CD103+CD5-, CD10-

Hairy cell leukemia (BRAF V600E) [Older adults; BM, splenic red pulp, PB]

negative control

CD200 expression in B-cell lymphoproliferative disorders by flow cytometric analysis

*HCL

+HCL-V

*

+

Pillai et al Am J Clin Pathol 2013; 140:536-543

CD1d is a MHC class I-liCD1d is a MHC class I-like cell surface glycoprotein expressed in a wide 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

ke cell surface glycoprotein expressed in a wide 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

CD1d is a MHC class I-like cell surface glycoprotein expressed in a wide 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

Marginal zone

lymphoma

Lympho-plasmacytic lymphoma

Hairy cell leukemia

Hairy cell leukemia-

variant

CD11c CD103 CD200 CD1d

CD200 and CD1d expression in CD5-, CD10- B-cell lymphoproliferative disordersM

ason et al. Am J C

lin Pathol 2017; 148:33

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

HCL HCLv LPL MZL

Neg

Dim

Pos

Bright

+/+: 94% sensitive and 98% specific for HCL+/-: 60% sensitive and 97% specific for LPL-/+: 41% sensitive and 100% specific for MZL

+ + - - + - - +

Pattern of CD200 and CD1d expression in CD5-, CD10- B-cell lymphoproliferative disordersM

ason et al. Am J C

lin Pathol 2017; 148:33

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, PTPRD (NMZL)

Rituximab ± (multi-agent) chemotherapy

? + anti-CD200 immunotherapy in HCL, LPL/WMG

CD5-negative, CD10-negative B-cell lymphoproliferative disorders

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]

Maleki et al. Leuk Lymph 2009;50:1054-57.

Bright CD38 staining is an indicator of MYC rearrangement

106 cases of CD10+ high grade lymphomas

MYC rearrangement

Numerical MYC aberrations

Normal MYC

Burkitt lymphoma vs. Double-hit lymphoma with MYC and BCL translocations

CD45↓, CD19+, CD20↓, CD10+, CD38+

Burkitt lymphoma: CD19+, CD20+, CD10+, CD38 bright+Double-hit lymphoma:

Roth et al. Oncol Res 2016; 23:137

Pan-B (CD19/CD20)

sIgΚ v. λ

CD5 CD10 CD23 CD11c other

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+

Immunophenotypic findings for mature B-cell neoplasms1, 2 3,4 5 6 7 8 9, 10+

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 gene 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

Near-IR Viability DyeExcitation: 633nmEmission 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 lymphocyte 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 antibody 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

Oberly et al. Am J Clin Pathol 2014; 142:411

Peripheral blood samples submitted for flow cytometric analysis for LPD: absolute lymphocyte count and patient age

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

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. SamSPECTRAL clustering algorithm2. FeaLect method (~Bolasso algorithm) to identify flow cytometry features most useful to

discriminate CLL and MCL

1

2

A. Weng, University of British Columbia, Am J Clin Pathol 2012; 137:75

70 cases CLL/SLL44 cases MCL

Am J Clin Pathol 2012; 137:75

Automated analysis of flow cytometric findings to distinguish CLL/SLL from MCL

Identification of discriminative ratios (CD20/CD23, FMC7/CD23, and CD20/CD11c)

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

=0 =1

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