Immunophenotype of B-cell chronic lymphocytic leukemia: practical issues

1. The diagnosis of B-cell chronic lymphocytic leukemia (B-CLL) relies on

the immunophenotype
The diagnosis of B-CLL is not done until an adequate/compatible immunophenotype is
established. Peripheral blood study is sufficient. The panel of monoclonal antibodies
included in the study should be able to make the differential diagnosis with other
lymphoproliferative B-cell disorders.

2. Not only the immunophenotype but also B-cell count.

For most of usual markers, the immunophenotype of B-CLL is identical to that of CLL-
type monoclonal B-cell lymphocytosis (MBL). Therefore, in the absence of other clinical or
laboratory findings, the differential diagnosis between them is based on B-cell count. At
this moment, the definition of MBL needs a B-cell count <5,000/µl.

3. Markers to study: diagnosis, prognosis and minimal residual disease.

a. Markers for diagnosis

No single marker is enough to establish the diagnosis of B-CLL. Therefore, a minimum
number of monoclonal antibodies (moAb) must be evaluated. The selection should include
pan B-cell markers (CD19, CD20, CD22, CD79b, and surface immunoglobulin light chains),
and markers for differential diagnosis with other B-cell chronic lymphoproliferative
diseases (CD5, CD23, CD10, and CD11c). However, there is no global consensus about
which minimum number of which moAb are indispensable for diagnosing B-CLL yet.

In 1994, Proffessor E. Matutes proposed a scoring system based on the evaluation of 5

parameters: CD5, CD23, FMC7, intensity of kappa/lambda chains, and CD22/CD79b. B-CLL
score would range between 5 (typical B-CLL cases) and 3 (less typical B-CLL cases). Scores
0-2 would exclude the diagnosis of B-CLL. More recently, the Euroflow group designed a
larger panel of moAbs in an attempt to reach the diagnosis of the different WHO
categories, and also improve the identification of overlapping entities. Using an 8-color
flow cytometer, they make a simultaneous evaluation of 8 to 12 parameters distributed in
2 different tubes as a start. Markers studied include all the moAbs studied by E. Matutes
with the exception of FMC7 and CD22, and incorporate others as CD43, CD81, CD38, CD20,
and CD200.

Diagnosing B-CLL it is usually an easy issue whenever the B-cell population exhibits the
characteristic immunophenotype (see Box below): small lymphocytes, CD19+, CD5+
(homogeneous weak or high, or heterogeneous), CD23+, CD22/CD79blow, surface µ
chainlow, monoclonal light chain expressionlow, CD20low, CD200+/high, CD43+low, CD11c-/+low,
FMC7-, CD10-, CD103-. Problems arise when dealing with non-characteristic
immunophenotypes, including some of the Dr. Matutes score 3 (or even 4) cases. Special
“uncomfortable” cases would be those lacking CD5 or CD23 expression, or showing strong
positivity for CD20, CD22/CD79b, or light chains.

From a practical point of view, the immunophenotype should be able to exclude other
B-cell lymphoproliferative disorders, specifically those with different prognosis and/or
therapy: mantle cell lymphoma (MCL) and hairy cell leukemia (HCL). Rare cases of HCL may
express CD5, but usually, there is no problem in distinguishing these 2 entities (see Box
below). On the other hand, some MCL cases might be particularly difficult to distinguish
from atypical B-CLL. According to Euroflow, the most informative markers for
distinguishing between B-CLL and MCL are CD23, CD38, CD20, sIgM, CD79b and FMC7.
However, clinical guides still require information from other techniques as necessary to
establish the diagnosis of MCL (molecular Bcl-1 rearrangement, immunohistochemistry
cyclin D1 positivity or specific karyotype/FISH).

The immunophenotype is usually informative enough to distinguish B-CLL and

follicular B-NHL, even in CD10 negative cases. On the other hand, it is less informative to
recognize prolymphocytic leukemia secondary to B-CLL or development of a Richter´s
syndrome. For other lymphoproliferative B-cell disorders (marginal zone lymphoma or
lymphoplasmocytoid lymphoma), the Euroflow group proposes the study of a larger
number of markers.

b. Markers for prognosis

Classical markers for adverse prognosis of B-CLL are CD38, CD49d and ZAP-70. Most
studies consider bad prognosis a B-CLL with at least 30% of positive cells for CD49d or
CD38, although this last marker it is not clear and not easy to determine. Things are less
clear for ZAP-70 since there is still much controversy about the best flow cytometry
protocol to apply, including cut-off points, clones, and definition of negative internal
controls. According to recent reports, differential expression of “new” markers (LAIR-1
(CD305), CCR6 and CXCR5) might be associated with some poor-risk chromosomal
abnormalities.

c. Minimal residual disease (MRD)

Several studies confirm the idea that the goal therapy in B-CLL should be to reach a low
level of MRD. A recent report “emphasizes the prognostic significance of the quality of
 remission after therapy because patients with less than 1x10-4 MRD were associated with
longer progression-free survival and overall survival”.

General rules for a correct use of flow cytometry for the search of MRD must also be
followed in B-CLL: a good quality of the sample, acquisition of a high number of cells, and
identification of a cluster of cells (10-100) with the immunophenotypic features selected.
In B-CLL, it is also particularly important to obtain information about therapy protocols
including monoclonal antibodies (i.e rituximab) in order to include (or exclude) that
antigen in the panel of reagents studied. Information about the B-CLL immunophenotype
at diagnosis might be also interesting, but literature encourages the use of consensus
protocols. There are several 4-color panels proposed (all of them including CD5) for
evaluation of either peripheral blood or bone marrow. The most cited combinations for
the identification of MRD in CLL are: CD22low/CD81low/CD19+/CD5+ and
CD43high/CD79blow/CD19+/CD5+.

4. Just B-CLL?
Finally, do not forget that coexistence of more than one clonal B-cell population is a
relatively frequent finding in patients with chronic lymphoproliferative disorders (4.8%),
ranging between 3.4% and 13.8% of patients diagnosed with B-CLL. Different surface Ig
light chain or different levels of the same sIg, associated or not with phenotypic
differences, will help to identify these B-cell populations.

BOX 1

Characteristic immunophenotype data in each disease.

B-CLL CD19+, CD5+, CD20low, CD22low/neg, CD79blow/neg, CD23+, CD43low, CD200+/++,sIgκ/λlow, sIgMlow, FMC7-, CD10-, CD11c+low/neg,CD103- .

MCL CD19+, CD5+, CD20high, CD22high, CD79b+, CD23-, CD43high, CD200low/neg,sIgκ/λhigh, sIgMhigh, FMC7+, CD10-, CD11c-,CD103-.

HCL CD19+, CD5-, CD20high, CD22high, CD79b+, CD23-, CD43-, CD200+/high,sIgκ/λhigh, multiple sIg isotypes, FMC7+, CD10-, CD11chigh,CD103+.

high/low expression define categories of intensity of expression for each antigen compared to antigen surface density of normal B cells.
KEY ISSUES

1-For diagnosing B-CLL: identify >5.000 monoclonal B-cells in peripheral blood with a
compatible immunophenotype.

2- The characteristic immunophenotype: CD19+, CD5+, CD20low, CD22low/neg, CD79blow/neg,

CD23+, CD43low, CD200+/++,sIgk/llow, sIgMlow, FMC7-, CD10-, CD11c+low/neg,CD103- .

3- If the immunophenotype is not characteristic: discard other B-cell lymphoproliferative

disorders, especially MCL and HCL.

4- Include markers related to prognosis.

5- Check association with other lymphoproliferative B-cell disorders.

6- MRD in B-CLL is associated with progression-free survival and overall survival. Use consensus
panels for its search.

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