Adding 2nd MoAB Improves Correlation Between ZAP-70, Mutational Status

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[3277] Flow Cytometric Detection of ZAP-70 in Chronic Lymphocytic

Leukemia: Addition of a Novel Second Monoclonal Antibody Improves

Correlation with IgVH Mutation Status. Session Type: Poster Session

528-III

L. nsen, Dan , Rajyalakshmi Luthra, le G.

Department of Hematopathology, UT M.D. Cancer

Center, Houston, TX, USA

Background: Expression of ZAP-70 is associated with unmutated

immunoglobulin heavy chain variable region genes and poor prognosis

in B-cell CLL. Reliable clinical flow cytometric assays have been

difficult to establish, as many anti-ZAP-70 antibodies show a

suboptimal signal-to-noise ratio, and an imperfect correlation with

IgVH mutation status.

Methods: We studied fresh peripheral blood or bone marrow specimens

from forty-three patients with CLL/SLL. Samples were stained for

intracytoplasmic ZAP-70 using a standard monoclonal antibody,

1E7.2/PE (Caltag, Burlingame, CA), and the novel monoclonal antibody

J13-1164/PE (kindly provided by BD Biosciences, San Diego, CA).

Threshholds were set using isotype-matched negative control

antibodies. Mutational status of the IgVH genes was assessed by RT-

PCR, followed by standard Sanger DNA sequencing. Divergence from

germline IgVH segments (IMGT database) was calculated using VBASE,

with 2% or less changes over codons 1-94 of IgVH regarded as

unmutated.

Results: Staining with J13-1164 revealed a clear apparent cutpoint

in levels of positivity, with 26/43 (60%) of the CLL cases showing

>49% of cells above the isotype threshhold. Most of the remaining

cases (15/43, 35%) showed <26% of cells positive. The 1E7.2/PE

reagent yielded a similar number of positive cases, with 28/43 (65%)

showing >20% positive cells (the cutpoint suggested by Rassenti et

al., N Engl J Med 2004, 351:893). However, staining with 1E7.2 was

more difficult to interpret, as many cases had levels of positivity

that were close to the cutpoint, with 15/43 (35%) showing 10-30% of

cells positive. Both antibodies showed similar degrees of

correlation with IgVH mutation status, with 72-75% of unmutated

cases showing the expected positivity for ZAP-70, and 57-64% of

mutated cases negative for ZAP-70 (see Table 1).

The two antibodies yielded concordant results in about half of cases

(21/43), and the concordant cases showed a strong correlation with

IgVH mutation status: 15/16 cases positive with both antibodies had

unmutated genes, and 4/5 cases negative with both antibodies had

mutated genes (see Table 2). The cases with discordant staining

between the two antibodies were a mixture of cases with mutated or

unmutated IgVH genes.

Conclusions: The J13-1164 anti-ZAP-70 antibody provides more easily

interpretable results in routine clinical use than the 1E7.2 clone,

with brighter staining in many cases and a more distinct difference

between positive and negative cases in our series. Compared to the

1E7.2 clone, the J13-1164 clone shows a similar degree of

correlation with the IgVH mutation status. A combination of both

antibodies provides the strongest correlation with IgVH mutation

status, with 19/21 concordant cases showing the expected staining

pattern.