Mature

B-cell neoplasms

Fiona E. Craig, MD
(Case examples courtesy Adam Seegmiller, MD)
 Flow cytometry for
mature B-cell neoplasms
•  DisBnguish normal/reacBve and neoplasBc cells:
–  Immunoglobulin light chain restricBon
–  Abnormal (not normal) immunophenotypes
•  Phenotypes associated with B-cell neoplasms e.g.
chronic lymphocyBc leukemia
•  Need for addiBonal studies, such as immuno-
histochemical stains, cytogeneBcs, molecular
•  PotenBal therapeuBc markers
 Normal B-cell immunophenotype

•  Immature bone marrow B-cell precursors

(hematogones)
–  CharacterisBc maturaBon paLern CD20 and CD10
–  Most immature subset CD34+and TdT+
–  Also CD45 dim+, CD38 bright+
Plasma cells
•  Mature B-lymphoid cells
•  Plasma cells Hematogones

Mature B-
lymphoid cells
 Normal B-cell immunophenotype

•  Mature B-cells:
•  CD45(bright), low side-light scaLer (lymphoid cells)
•  Pan B-cell anBgen expression: CD19, CD20, CD22,
CD79a
•  Surface immunoglobulin: kappa or lambda, with a
polytypic mixture of kappa+ and lambda+ cells
•  Subsets in different sites:
– TransiBonal, Naïve, Memory
– Germinal center B-cells in lymphoid Bssues
 Normal B-cell immunophenotype

•  Plasma cells:
•  CD45(dim to negaBve), variable side-light scaLer
•  Plasma cell-associated anBgens:
– CD38 (bright), CD138
•  Pan B-cell anBgen expression: CD19, CD79a
•  Surface immunoglobulin negaBve
•  Cytoplasmic immunoglobulin: kappa or lambda,
with a polytypic mixture of kappa+ and lambda+
cells
 B-cell differenBaBon
Hematogone
Transi3onal
B-cell

Naïve B-cell Germinal

center B-cell Plasma cell
(reac3ve)

An3gen
s3mula3on Memory
B-cell
(protec3ve)
 Flow cytometry for
mature B-cell neoplasms
•  DisBnguish normal/reacBve and neoplasBc cells:
–  Immunoglobulin light chain expression
–  Abnormal immunophenotype
•  IdenBfy phenotype associated with a disease enBty
or group e.g. chronic lymphocyBc leukemia
•  Determine need for addiBonal studies, such as
immunohistochemical stains, cytogeneBcs,
molecular
•  IdenBfy potenBal therapeuBc markers
 DisBnguishing normal / neoplasBc B-cells

Lambda
SSC
Polytypic
B cells

FSC Kappa

Lambda
SSC
Monotypic
B cells

Ig light chain analysis:

1.  Gate B cells as precisely as possible
2.  Use a bivariate kappa/lambda plot FSC Kappa
3.  Use generous kappa and lambda
gates (preferably not quadrants)
4.  Kappa:lambda should be 1-2:1
 Light Chain Pi`alls

•  Background polytypic B cells

Lambda

Lambda
CD10
Kappa CD20 Kappa
Kappa:Lambda = 4.3:1
Lambda

Lambda
CD19

Kappa FSC Kappa

Kappa:Lambda = 0.6:1
 Light Chain Pi`alls

•  Non-specific staining:
–  Cytophillic staining (non-B-cells)
•  Monocytes, NK-cells and dendriBc cells
•  Dead and dying cells
•  Gate on viable B-cells
–  Bound serum immunoglobulins & other proteins:
•  Staining with both anB-kappa and anB-lambda
•  Blocking of reagent anBbody binding
•  Washing, warming, and incubate immune serum
•  Cytoplasmic staining
 Light Chain Pi`alls

•  Dim / absent light chain expression:

–  CharacterisBc of some normal populaBons:
•  Immature B cells (hematogones), plasma cells, some
germinal center B-cells
–  Occurs approximately 25% B-cell lymphoma
–  Try washing, warming, and incubaBng immune
serum, and cytoplasmic staining
–  Try alternate reagent polyclonal versus
monoclonal detecBon anBbodies
–  Look for other indicators of neoplasia, such as
abnormal immunophenotype
 Light Chain Pi`alls

•  Dim or negaBve light-chain expression

Lambda

Lambda
Kappa Kappa
Lambda

Lambda
CD10

Kappa CD20 Kappa

 Light Chain Pi`alls

•  Light chain restricted non-neoplasBc B-cells:

–  Florid follicular hyperplasia (also clonal)
–  Marginal zone hyperplasia (lambda restricted, but
not clonal)
–  CSF B-cells in mulBple sclerosis
•  Light chain restricted B-cells of uncertain significance:
–  Monoclonal B lymphocytosis
–  In situ follicular neoplasia
–  In situ mantle cell neoplasia
 DisBnguishing normal /neoplasBc B-cells

•  Immunoglobulin light chain expression

•  Abnormal immunophenotype:
–  Altered expression of expected B-cells anBgens
e.g. CD19(dim)
–  Gain of anBgens not normally expressed by B-cells
e.g. CD13, CD2, CD7, CD8
–  Expansion of a subset of cells or abnormal
distribuBon of cells
 Abnormal immunophenotype

•  Determined by comparison with phenotype of

appropriate non-neoplasBc B-cells e.g. CD20
expression usually brighter on germinal center B-
cells
•  Lack or decreased expression e.g. CD19(dim)
common in follicular lymphoma
•  Increased expression e.g. CD10(bright) follicular
lymphoma
•  Gain unexpected anBgens e.g. bcl-2+ germinal center
B-cells in follicular lymphoma
 Flow cytometry for
mature B-cell neoplasms
•  DisBnguish normal/reacBve and neoplasBc cells:
–  Immunoglobulin light chain restricBon
–  Abnormal (not normal) immunophenotypes
•  Phenotypes associated with B-cell neoplasms e.g.
chronic lymphocyBc leukemia
•  Need for addiBonal studies, such as immuno-
histochemical stains, cytogeneBcs, molecular
•  PotenBal therapeuBc markers
 Flow cytometry for mature
B-cell neoplasms
•  A standard series of quesBons for any lymphoid
populaBon…
–  What lineage?
–  Mature or immature?
–  Benign or neoplasBc?
–  Cell size? Small, medium or large
–  Phenotype?
•  Algorithm for disease enBty or possible diseases
 What lineage?
B lineage: CD19 vs. SSC T lineage: CD3 vs. SSC

Other B-cell markers: Other T-cell markers:

CD20 CD2 (also NK)
CD22 CD5
iCD79a CD7 (also NK)
 What lineage?

•  Possible pi`alls:
–  Abnormal lack of staining for pan B-cell anBgens:
•  CLL with CD20(dim)
•  Aberrant decrease or loss of anBgen expression
–  Loss following therapy e.g. anBbody or CAR-T
–  Expression of “pan-B-cell associated anBgens by
other cells e.g. AML with t(8;21), NK-cells
–  Aberrant expression of anBgens associated with
other cell types e.g. CD5, CD8
 Mature or Immature?

Lambda
SSC
Plasma cells

Hematogones
CD38

FSC Kappa
Mature

Lambda
SSC
B cells

SSC
FSC Kappa
Mature B cells

Lambda
SSC
CD45

Hematogones
(or blasts)

SSC FSC Kappa

 Size?
CLL/SLL
Mantle cell lymphoma

SSC
Smaller Follicular lymphoma
Marginal Zone Lymphoma
Lymphoplasmacy3c lymphoma
FSC

Hairy cell leukemia

SSC

Medium BurkiP lymphoma

FSC
SSC

Larger Diffuse large B-cell lymphoma

FSC
 Mature B-cell neoplasm classificaBon
Small / Medium mature B-lineage neoplasms

CD5+/CD10- CD5-/CD10+ CD5-/CD10-

CLL/SLL Follicular Marginal Zone
Mantle Cell Burkitt (med. size) Lymphoplasmacytic
Hairy Cell (med. Size)

CD23+/CD200+ CD23-/CD200-
CLL/SLL Mantle Cell
 When flow cytometry is not enough

•  Mantle cell lymphoma:

–  Cyclin-D1 immunohistochemical stain
–  t(11;14), CCND1 / IgH
•  BurkiL lymphoma and other high-grade B-cell
lymphoma:
–  t(8;14), MYC +/- BCL2 or BCL-6 gene
rearrangement
•  LymphoplasmacyBc lymphoma:
–  MYD88
 Flow cytometry for
mature B-cell neoplasms
•  DisBnguish normal/reacBve and neoplasBc cells:
–  Abnormal (not normal) immunophenotypes
–  Immunoglobulin light chain restricBon
•  Phenotypes associated with B-cell neoplasms e.g.
chronic lymphocyBc leukemia
•  Need for addiBonal studies, such as immuno-
histochemical stains, cytogeneBcs, molecular
•  PotenBal therapeuBc markers:
–  CD20, CD19, CD22, CD38…