B cell malignancies by FC Monique Lee

Outline

• Markers used for B cell immunophenotyping• Mature B cell neoplasm• Plasma cell neoplasm• Immature B cell neoplasm

Flow immunophenotyping

1. Identify cells from different lineages2. Determine maturity of cells3. Detect abnormal cells through different

antigen expression 4. Document phenotype of abnormal population5. Diagnosis of distinct disease entity6. Prognostic information through

immunophenotypic information, identifying targets for directed therapy

Uses of Flow in B cell LPD• Identification of clonal restriction ▫ Kappa/ lambda 0.5-3.0 in blood/ bone marrow 1.2-2.7 in lymph nodes Clonal kappa or lambda

• Identification of B cell lineage▫ CD19/ 20

• Identification of mature/ immature neoplasm▫ Tdt/ CD45

• Identification of WHO type of neoplasm

κ/λ interpretation• Light chain restriction in nonclonal populations▫ Lambda Ig restricted nonclonal B cells in childhood

tonsillar specimens & multicentric Castleman disease ▫ Clonal populations in florid follicular hyperplasia e.g.

HIV positive patients • Evaluation of κ/λ ratio will not identify small clonal

populations admixed with reactive polyconal B cells▫ More sensitive to separately evaluate populations of

cells with distinct phenotype and/or size▫ BUT reactive B cells may include small subsets of

phenotypically identical cells

Problems with κ/λ staining• Weak antigen density• Cytophilic antibodies bound to Fc receptor• Nonspecific binding of detection antibodies ▫ Adherence of antibody to “stick” cells e.g.

Damaged or dying cells• Large amounts of competing soluble antibody in

plasma absorbs out antibody• Washing required to remove serum κ/λ• Mouse serum pre-incubation to block FcR▫ 37 degrees for 30 minutes

Caution: use of flow in B cell lineage

• Sampling error• Admixture of normal cells• Loss of cells in preparation or through cell death• Definition of abnormal к/λ ratio• Loss of cell surface marker of equivalent stage in

normal differentiation▫ CD20 in large cell

• Variation in immunophenotype▫ MCL/ CLL

• Paucity of markers for certain stages of differentiation

B cell maturation

Junior, DM., et al. (2010). Immune system- part II basis of the immunological response mediated by T and B lymphocytes. Bras J Rheumatol. 50(5):552-80.

ICPMR, 2012

Craig, FE., et al. (2008). Flow cytometric immunophenotyping for haematologic neoplasms. Blood. 111:3941-67.

Craig, FE., et al. (2008). Flow cytometric immunophenotyping for haematologic neoplasms. Blood. 111:3941-67.

Panel used in B cell LPD

• CD19• CD20• Kappa/ lambda• CD5• CD23• CD10• Tdt/ CD34/ intracyto CD79a (immature cells)• CD25/ CD103/ FMC7/ CD11c (HCL)• CD38/ CD138/ CD56 (plasma cells)

CD19• Expression restricted to B cells• Staining intensity often moderately bright▫ Altered intensity seen in some B cell neoplasms e.g. FL

often weaker intensity CD19• Appears on lymphoblasts committed to B cell

lineage• Expressed on all stages of maturation including

normal plasma cells • Expressed by both B cell malignancies▫ BUT many plasma cell neoplasms are CD19 negative

CD20

• Expressed by most mature B cells▫ Expression more restricted than CD19▫ CD20 absent from most immature B cells,

increases in intensity during B cell maturation• Normal B cell haematogones▫ Gradual acquisition CD20 and CD22- in

synchrony with loss of CD34 and CD10 VS B-ALL: abnormal CD20 expression out of

synchrony with CD34 and CD10

CD20• Mature B cells: bright CD20 staining• Normal plasma cells: CD20 negative• Most plasma cell neoplasms: CD20 negative• Weakly expressed on subset of mature T cells• Staining intensity in B cells & B cell neoplasm:▫ Follicular centre B cells: CD20 bright ▫ Follicular lymphoma: CD20 brighter than normal

B cells▫ CLL: CD20 weak ▫ HCL: CD20 bright

Immunoglobulin heavy & light chains

• B cell maturation: acquisition of cytoplasmic µ heavy chain followed by complete surface Igcomposed of heavy & light chains

• Normal B cells lacking surface Ig▫ Lymphoblasts▫ Plasma cells

Lack surface Ig, contains abundant cytoplasmic Ig▫ Thymic B cells

• B cell neoplasm▫ Single clone of kappa or lambda expressing cells▫ CLL: low intensity sIg (low density of membrane BCR

complex)

Mature lymphoid neoplasms• Immunoglobulin light chain class restriction▫ Non-clonal restriction: Childhood tonsils, multicentric

Castleman▫ Clonal, non-neoplastic restriction: florid follicular

hyperplasia in HIV • Aberrant B cell antigen expression▫ Presence of antigens not normally expressed by B cells (e.g.

CD13, CD33) Aberrant myeloid antigens less frequent in mature B cell

neoplasms than ALL• Abnormal expression of antigens not typically present • Altered staining intensity for B-lineage-associated

antigens

WHO classification mature B• Chronic lymphocytic leukaemia/ small lymphocytic

leukaemia• Lymphoplasmacytic lyphoma• Mantle cell lymphoma• B cell prolymphocytic leukaemia• Follicular lymphoma • Diffuse large B cell lymphoma• Burkitt lymphoma• Marginal zone lymphoma• Hair cell lymphoma• Plasma cell myeloma/ plasmacytoma

Craig, F., et al. (2008). Flow cytometric immunophenotyping for haematologic neoplasms. Blood. 111:3941-67.

CD5+10-

• CD5 expressed on population of normal B cells• Does not on its own establish presence of

neoplasm

• CLL• Mantle cell lymphoma• Prolymphocytic leukaemia• Less frequently▫ MZL, DLBCL, LPL

Case 1

CLL/SLL• Typical phenotype▫ Surface Ig weak▫ CD45 slightly low▫ CD5 positive (weaker than T cell/ stronger typical)▫ CD19 weak▫ CD20 weak▫ CD23+ (moderate to strong)▫ CD200 positive▫ CD22/ CD79a weak▫ FMC-7 negative

• Should test for CD38; prognostic marker

Prognostic markers

• CD38 expression is independent marker of poor prognosis in CLL/SLL▫ 30% positive cells: cut-off for positive CD38

• ZAP 70 indicative of unmutated IgVH and poor prognosis▫ Technically difficult, reliable determination

challenging, not performed

CLL transformation

• CD20 brighter• CD22/ CD79a brighter• CD23 less bright• CD5 weaker• Immunoglobulins stronger• Richters syndrome▫ Transformed cells may replicate CLL phenotype/

plasmacytoid CD138

CLL Transformation

• Entire CD20 positive population is λ restricted

• Subpopulation 10%▫ Slightly CD20 brighter, CD5

weaker, CD23 equivaocal▫ Also lambda restricted▫ ? Transformed CLL cells

ICPMR, 2012

Case 2

Mantle cell lymphoma• Typical phenotype▫ CD5 positive weak▫ CD20 moderate to bright▫ Surface immunoglobulin moderate to bright▫ CD23 negative or weak▫ cD200 absent▫ FMC-7 positive

• Variable immunophenotype▫ E.g. CD23+, blastic CD5-, CD1o+

• Additional studies▫ Cyclin-D1 overexpression

Paraffin section immunohistochemistry▫ Translocation t(11;14)(q13;q32)

Cytogenetics▫ CCND1 gene rearrangement t(11;14)

FISH

B cell prolymphocytic leukaemia

• Large, round cells with abundant basophilic cytoplasm and single central nucleolus

• More aggressive course than CLL• Typical phenotype▫ High levels of cyIgM expression ▫ CD20, CD22, CD79b high▫ FMC7 positive▫ CD5 weakly positive ▫ CD23 negative

CD5-CD10+• CD10 is expressed by ▫ Immature B cells▫ Normal follicular centre B cells▫ Precursor B lymphoblastic leukaemia/ lymphoma▫ Subsets of mature T cells▫ Precursor T cell lymphoblasts, neutrophils, some non-

haematolymphoid cells• Malignancies▫ Precursor B- cell ALL▫ Burkitt lymphoma▫ Diffuse large B cell lymhoma▫ Follicular lymphoma ▫ Hairy cell leukaemia

DLBCL• Most common lymphoma in the west• Heterogeneous category▫ CD10+ 20-40% of DLBCL Ddx from Burkitt lymphoma and high grade

follicular lymphoma can be difficult à morphology ▫ Pleomorphic large cells▫ Variable genotype, may include one or more of: MYC, BCL-2, BCL-6

• Genotype▫ T(14;18)(q32;q21): 20% of DLBCL, 70-95% of FL

DLBCL

• Increased forward/ side scatter• CD19 positive (sometimes negative)• CD20 positive• CD10 positive in germinal centre type• CD5 positive in 10%• CD21 positive ~30%• Immunoglobulin positive 50%• CD45/ HLADR negative occasionally• Irregular antigen expression

Case

Follicular lymphoma

• CD20 strong• CD19 weaker • Immunoglobulin strong• CD10 positive (40-80%)• CD23 positive • CD5 negative• Variation: follicular vs interfollicular (less

CD10/38)

Case

Burkitt lymphoma

• CD20 strong• CD19 positive• CD10 positive• CD45 normal or weak• Immunoglobulin strong• TdT negative• CD34 negative

Burkitt lymphoma• Can be hard to differentiate from DLBCL• BL▫ More uniform population of intermediate size cells with

basophilic cytoplasm▫ Often cytoplasmic vacuoles▫ Variable numbers of nucleoli▫ Ki-67 index approaching 100%▫ Isolated c-MYC rearrangement

• DLBCL▫ More heterogeneous, pleomorphic large cells▫ Lower Ki-67 proliferative index▫ Variable genotype, may include rearrangements of c-MYC,

BCL-2 or BCL-6 (may be more than one)

CD5-10-

• Diverse group, includes ▫ DLBCL▫ MZL▫ HCL▫ LPL▫ CD10- FL▫ CD5- MCL

Case

Hairy cell lymphoma• Typical phenotype▫ Increased side scatter▫ CD20 strong▫ CD22 bright▫ CD11c strong▫ CD25 positive▫ CD103 positive▫ Surface immunoglobulin intermediate to bright▫ FMC-7 positive▫ CD23, CD5, CD10 negative

Hairy cell lymphoma

• Variations▫ CD10+ small subset (10%) Consider if CD20, 22, sIg bright, CD38-, FMC-7-▫ CD103-, CD25-, CD23+

• HCL variant (HCLv)▫ Higher WBC▫ Lack accompanying monocytopenia▫ Prominent nucleoli▫ CD25 negative

Case

Marginal zone lymphoma • Distinguishing phenotype▫ Usually CD5 and CD10 negative▫ CD19 and CD20 positive▫ CD23 negative▫ Surface immunoglobulin positive ▫ Plasmacytic differentiation in significant subset

CD138, bright CD38▫ CD5+ in ~5%

• Morphology▫ Predominantly small cells▫ Lack proliferation centres characteristic of CLL/SLL▫ Infiltration around & into residual benign follicular germinal

centres• Genetics▫ T(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21)

Marginal zone lymphoma

• Exclude other small lymphoid B cell neoplasms▫ CD10- FL▫ CD5- MCL▫ CD5- HCL Differentiation may be difficult MZL often CD11c+, may be 103+▫ Usually weaker variable staining for CD11c▫ Lack combination of CD103, CD11c, CD25▫ Lack bright staining for CD20 and CD22

Splenic marginal zone lymphoma

• CD20 strong• Immunoglobulin positive strong• Varying expression of CD11c, CD25, CD103• Hairy cell like but do not have all the markers

Lymphoplasmacytic lymphoma

• Small cells, subsets with plasmacyticdifferentiation

• Less well-defined• Difficult to distinguish from MZL and B cell

lymphoid neoplasms with plasmacyticdifferentiation

• 60-80% CD5-, CD10-, CD23-▫ Many express CD11c & CD25, but usually CD103-

• 5% are CD5+

Lymphoplasmacytic lymphoma

• CD19 positive• CD20 positive• Occasionally CD10 positive• Occasionally CD 23 positive • Plasma cell component- CD38/ CD138

CD5+CD10+

• Includes subtypes of:▫ DLBCL▫ FL▫ MCL▫ CLL/SLL▫ Burkitt lymphoma

Plasma cell disorders• Flow cytometry useful for:▫ Identification of abnormal plasma cells▫ Distinguishing between lymphoid and plasma cell

neoplasms▫ Prognostic information

• Plasma cell neoplasia▫ Plasmacytoma▫ Plasma cell myeloma and variants▫ Plasma cell leukaemia▫ Amyloidosis▫ Immunoglobulin light & heavy chain diseases

Plasma cell disorders

Craig, FE., et al. (2008). Flow cytometric immunophenotyping for haematologic neoplasms. Blood. 111:3941-67.

Plasma cell disorders• CD38 bright and CD138 commonly used to identify

plasma cells▫ Evaluating both provides most sensitive and specific

means of detecting plasma cells• CD38 (at lower intensity) also expressed on▫ Haematagones▫ Some mature B cells▫ Activated T cells▫ Myeloid cells

• CD138 restricted to plasma cells and some carcinoma cells▫ Less sensitive for detecting plasma cells than CD38

Plasma cell disorders

• CD56 aberrant expression identified in most patients with myeloma▫ Lack of CD56 associated with worse prognosis

• Most PCN CD19-, CD20-▫ Normal plasma cells CD19+, CD20-

• CD28 associated with reduced event-free survival (not overall prognosis)

• Lack of CD27 and CD45 associated with adverse prognosis

Plasma cells MGUS Multiple myeloma

CD19 negative CD19 negative

Ig negative Ig negative

CD45 low CD45 low

CD56 negative CD56 positive

CD138 CD138 positive

CD38 high CD38 strong

CD10 positive

CD117 positive

Immature B cell neoplasms

• Identification of immature B lymphoid cells▫ CD34▫ TdT▫ Lack surface immunoglobulin ▫ Lack CD20

• B cell lineage ▫ CD19: highest sensitivity & specificity▫ cCD22: sensitive and specific B lineage marker▫ cCD79a: stains some precursor T cell ALL & AML

B lymphoblastic leukaemia• Immature B cell, usually ▫ CD19 positive▫ CD45 low ▫ CD10 positive▫ TdT positive ▫ CD20 positive – 50%▫ Intra CD79a▫ Intra CD22

• Other common markers:▫ Myeloid CD13/ CD33, CD58/ CD38

B ALL classification

• Pro-B (BI)▫ CD19+, cyCD79a+, sIg-, CD10-, CD20-

• Common B (BII)▫ CD19+, cyCD79a+, CD10+, Ig-

• Pre-B (BIII)▫ CD19+, cyCD79a+, cyIgM+, sIg-, CD20+

• Mature B (BIV)▫ CD19+, cyCD79a+, sIg+

Orfaeo, et al. (2006). Diagnosis of haematological malignancies: new applications for flow cytometry. Haematology- the European hematology association program. 2(1):6-13.

References • Junior, DM., et al. (2010). Immune system- part II basis of the

immunological response mediated by T and B lymphocytes. Bras J Rheumatol. 50(5):552-80.

• Craig, FE., et al. (2008). Flow cytometric immunophenotyping for haematologic neoplasms. Blood. 111:3941-67.

• Orfaeo, et al. (2006). Diagnosis of haematological malignancies: new applications for flow cytometry. Haematology- the European hematology association program. 2(1):6-13.

• Craig, FE. (2007). Flow cytometric evaluation of B cell lymphoid neoplasms. Clin Lab Med 487-512.

• Past ICPMR lectures

• Acknowledgement: Daniel Orellana, RPAH