Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma ...

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Key Words: Chronic lymphocytic leukemia/small lymphocytic lymphoma; Serum IgM; Plasmacytoid lymphocytes; ZAP-70 .... Nuclear staining of nonneoplastic, reactive T cells served as ..... associated with a variable number of prolymphocytes.


Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Associated With IgM Paraprotein A Clinicopathologic Study of 26 Cases C. Cameron Yin, MD, PhD,1 Pei Lin, MD,1 Dennis A. Carney, MD,2 Beverly C. Handy, MD,3 George Z. Rassidakis, MD, PhD,1 Joan H. Admirand, MD,1 Michael J. Keating, MD,2 and L. Jeffrey Medeiros, MD1 Key Words: Chronic lymphocytic leukemia/small lymphocytic lymphoma; Serum IgM; Plasmacytoid lymphocytes; ZAP-70 DOI: 10.1309/FDGWB5C2MYRYXH2E

Abstract We studied the clinicopathologic, immunophenotypic, and cytogenetic features of 26 patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) associated with serum IgM paraprotein. The study group (16 men; 10 women; median age, 64 years; range, 40-82 years) represents approximately 2.5% of CLL/SLL cases at our institution. The paraprotein level ranged from 1 to 14 g/L (median, 4 g/L). Neoplasms in bone marrow were composed of small round lymphocytes arranged in nodular (n = 6), diffuse (n = 5), interstitial (n = 5), or mixed (n = 10) patterns. All cases were positive for monotypic surface immunoglobulin light chain, IgM/IgD, CD5, CD19, CD20, and CD23. CD11c (14/20 [70%]), CD79b (11/19 [58%]), FMC-7 (11/26 [42%]), CD22 (8/20 [40%]), and ZAP-70 (6/19 [32%]) were expressed in subsets of cases. Of 17 bone marrow specimens assessed by conventional cytogenetics, 6 were abnormal and 11 were diploid. The overall survival of this group (median follow-up, 24 months) was not significantly different from that for an age-, sex-, and stage-matched group of 52 CLL/SLL patients without IgM paraprotein (P = .60). We conclude that CLL/SLL cases with serum IgM paraprotein are similar to other CLL/SLL cases in their clinicopathologic and immunophenotypic features.

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Am J Clin Pathol 2005;123:594-602 DOI: 10.1309/FDGWB5C2MYRYXH2E

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) represents 6.7% of all non-Hodgkin lymphomas, and CLL is one of the most common types of leukemia in the Western world.1 The median age at diagnosis is 65 years, and there is a male/female ratio of 2:1. Although most patients are asymptomatic at diagnosis, some can have systemic (B type) symptoms, hepatosplenomegaly, lymphadenopathy, and cytopenias due to leukemic infiltration of bone marrow and other organs. As defined in the World Health Organization (WHO) classification, CLL/SLL is a neoplasm of small lymphocytes that are positive for monotypic surface immunoglobulin (dim intensity), CD5, CD19, CD20 (dim), and CD23 and usually negative or dimly positive for CD22, CD79b, and FMC-7.1 Serum IgM paraprotein (so-called M component) can be detected in a subset of patients with CLL/SLL by conventional methods such as serum protein electrophoresis and can be detected in a higher number of patients by more sensitive techniques.2 The WHO classification recognizes that a “small M component” can be found in some patients with CLL/SLL, but no mention is made of the frequency of this occurrence or the range of serum paraprotein levels in patients with CLL/SLL. Furthermore, the clinical significance of a serum IgM paraprotein in patients with CLL/SLL is unknown. An earlier study reported that patients with CLL/SLL with IgM paraproteinemia have an inferior survival compared with patients with CLL/SLL without serum paraprotein.3 However, other studies have not confirmed this observation.4 We assessed the clinical, morphologic, immunophenotypic, and conventional cytogenetic findings in a group of patients with CLL/SLL with IgM paraprotein. We also compared the clinical outcome of this group of patients with that © American Society for Clinical Pathology

Hematopathology / ORIGINAL ARTICLE

for a group of patients with CLL/SLL without IgM paraprotein matched for age, sex, and stage of disease.

Materials and Methods Case Selection The files of the Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, from January 1997 to March 2004 were searched for cases of CLL/SLL with IgM paraprotein detected by serum protein electrophoresis and immunofixation. The diagnosis of CLL/SLL was based on morphologic and immunophenotypic findings using the criteria of the WHO classification.1 All cases were composed of a proliferation of predominantly small lymphocytes, with or without morphologic evidence of plasmacytoid differentiation, that were positive for monotypic immunoglobulin light chain, surface IgM/IgD, CD5, CD19, CD20, and CD23. We also selected a group of 52 patients from the same period with CLL/SLL without serum IgM paraprotein that were matched for age, sex, and stage of disease. This group was used for survival analysis and for a comparison of morphologic features in a subset of cases. Serum Protein Electrophoresis and Immunofixation Serum protein electrophoresis and immunofixation were performed using the PARAGON (Beckman Coulter, Brea, CA) or the HYDRAGEL (Sebia, Norcross, GA) system. Total serum IgM and monoclonal IgM (M component) levels were quantified using densitometric scanning. Levels at the time of initial examination and the highest levels during the course of disease were recorded. Morphologic Evaluation H&E-stained slides routinely prepared from formalinfixed, paraffin-embedded bone marrow biopsy and/or aspirate clot specimens and Wright-Giemsa–stained aspirate smears were reviewed. The percentage of lymphocytes, their cytologic features, and the extent and pattern of leukemic infiltration were assessed. In cases that also had lymph node biopsy specimens, the histologic features of these specimens also were reviewed. We also compared the cytologic features of the lymphocytes in CLL/SLL cases associated with serum IgM paraprotein and CLL/SLL cases from the matched control group without serum IgM paraprotein. For the latter group, bone marrow aspirate smears from 19 patients were readily available for analysis. For both groups of patients, 3 observers (P.L., J.H.A., and L.J.M.) quantified the percentage of plasmacytoid lymphocytes in bone marrow aspirate smears by performing manual counts. These smears were reviewed without

knowledge of the presence or level of serum IgM paraprotein. Plasmacytoid lymphocytes were defined as cells with eccentrically situated nuclei and moderately abundant cytoplasm and/or with cartwheel-like (plasmacytoid) chromatin. The percentages of plasmacytoid lymphocytes generated by each pathologist for an individual case were averaged. Flow Cytometric Immunophenotypic Analysis Flow cytometric immunophenotypic analysis was performed using a FACScan or FACSCalibur instrument (Becton Dickinson Biosciences, San Jose, CA). Lymphocytes were gated for analysis using CD45 (peridinin chlorophyll-α protein conjugated) and side scatter and analyzed using a panel of monoclonal antibodies specific for CD3 (fluorescein isothiocyanate [FITC] or phycoerythrin [PE]), CD5 (FITC or PE), CD10 (FITC), CD11c (PE), CD19 (FITC, PE, or allophycocyanin), CD20 (PE), CD22 (FITC), CD23 (PE), CD38 (allophycocyanin), CD79b (PE), FMC-7 (FITC), immunoglobulin light chains (FITC), and surface IgM/IgD (FITC). All antibodies were purchased from Becton Dickinson Biosciences, and negative staining levels for each antibody were set by comparison with an isotype-matched control sample. By using the scoring system proposed by Moreau and colleagues,5 cases were scored based on 5 variables: expression of dim surface immunoglobulin (1 point), CD5 (1 point), and CD23 (1 point); dim or absent CD22/CD79b (1 point); and absent FMC-7 (1 point). Cases with a score of 4 or 5 were considered to have a typical immunophenotype for CLL/SLL. Cases that deviated from this pattern of antigen expression were considered to have an atypical immunophenotype for CLL/SLL. Immunohistochemical Analysis for ZAP-70 Formalin-fixed, paraffin-embedded bone marrow biopsy tissue sections were stained with a monoclonal antibody specific for ZAP-70 (dilution 1:500, Upstate Cell Signaling Systems, Lake Placid, NY) as described previously.6 Detection of the primary antibody was achieved using the LSAB+ kit (DAKO, Carpinteria, CA), which contains secondary biotinylated antibody and streptavidin/horseradish peroxidase complex, according to the manufacturer’s instructions. The chromogen was 3,3'-diaminobenzidine/hydrogen peroxide (DAKO), and slides were counterstained with hematoxylin. Nuclear staining of nonneoplastic, reactive T cells served as an internal control. Conventional Cytogenetics Conventional G-band karyotyping was performed on metaphase cells from samples cultured for 24 or 48 hours using previously described methods.7 Metaphase cells were obtained using a methanol acetic acid fixation method. The karyotype reports were written using the International System for Human Cytogenetic Nomenclature.8 Am J Clin Pathol 2005;123:594-602

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Survival Analysis The outcome for patients was obtained by reviewing medical records and the database of the Department of Leukemia, The University of Texas M.D. Anderson Cancer Center. By using the Kaplan-Meier method, we compared the overall survival of patients with CLL/SLL with serum IgM paraprotein with a matched group of 52 patients with CLL/SLL without serum IgM paraprotein. Analysis was performed using Statistica (StatSoft, Tulsa, OK).

lymphadenopathy. The lymphocytes in this group ranged from 1,400 to 3,500/µL (1.4-3.5 × 109/L; median, 2,200/µL [2.2 × 109/L]). In addition to bone marrow aspiration biopsy specimens, lymph node biopsy specimens were available for this subset of 7 patients. The levels of serum total IgM and IgM paraprotein at initial diagnosis varied widely ❚Table 2❚. During the course of disease, the serum IgM paraprotein level in 2 patients rose, from 1.1 to 21 g/L in one patient and from 3 to 14 g/L in the other patient. The serum IgM paraprotein level remained stable or decreased in the remaining 24 patients.

Results Clinical Findings We identified 26 untreated patients with CLL/SLL with IgM paraprotein from a total of 1,053 untreated patients with CLL/SLL at our institution from January 1997 to March 2004, for a frequency of 2.47%. There were 16 men and 10 women with a median age of 64 years (range, 40-82 years). The clinical features are summarized in ❚Table 1❚. Of the 26 patients, 4 (15%) had systemic symptoms, including fever, fatigue, night sweats, and weight loss. No patients had symptoms related to hyperviscosity. Physical examination revealed generalized lymphadenopathy in 17 patients (65%) and splenomegaly in 9 patients (35%). Laboratory evaluation showed 16 patients (62%) with an elevated WBC count, 15 (58%) with anemia, and 7 (27%) with thrombocytopenia. Serum β2-microglobulin and lactate dehydrogenase levels were elevated in 24 patients (92%) and 5 patients (19%), respectively. Seven patients who did not have peripheral lymphocytosis (lymphocyte count,