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We analyzed protein expression of cyclin D1, cyclin D2, and cyclin D3 using high-resolution enzymatic amplification staining and flow cytometry in the neoplastic cells from 80 patients with CD5+ B-cell lymphoproliferative disorders. The D cyclins were expressed differentially in chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), and mantle cell lymphoma (MCL) with strong staining of cyclin D1 and D2 in MCL, strong staining of cyclin D1 but weak staining of cyclin D2 in 4 of 5 PLLs, and low-level staining for both cyclins in most CLLs. No correlation between cyclin D1 and D2 and growth rates or CD38 expression was observed. However, cyclin D1 levels were significantly higher in ZAP-70+ CLL cases, although no association between ZAP-70 and cyclin D2 was detected. The results indicate that flow cytometric analysis of D cyclins may help in classification of CD5+ B-cell lymphoproliferative disorders.

Immunophenotyping of small B-cell neoplasms (SBCNs) may have a critical role in diagnosis. However, there are few data addressing whether the immunophenotypes of SBCNs in bone marrow (BM) and peripheral blood (PB) are representative of those in other tissue sites. We compared the immunophenotypic features of concurrently analyzed lymph node (LN) and BM/PB specimens using multiparameter flow cytometry. Fifty-five SBCNs were identified: 27 follicular lymphomas (FLs), 16 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLLs), and 12 mantle cell lymphomas (MCLs). Major (presence vs absence) or minor (alteration of intensity) variations in expression of individual antigens between LN and BM/PB were observed in up to 25% of cases within a particular SBCN category. All FLs and CLL/SLLs maintained characteristic immunophenotypes in BM/PB. Potentially misleading variations included 1 case of MCL that failed to express CD5 in BM and likely would have been immunophenotypically misclassified as a marginal zone lymphoma and another MCL that expressed moderate CD23 in PB and would have required additional studies for precise classification. The remaining major and minor variations would not have affected interpretation.

We evaluated anti-CD79b for its usefulness in the diagnosis of B-cell chronic lymphoproliferative disorders (BCLPDs), particularly chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). We analyzed 100 BCLPDs for CD5, CD19, CD20, CD23, CD79b, and surface immunoglobulin light chain (sIg) expression by 4-color flow cytometry. CD20, CD79b, and sIg expression were quantified. Correlational analysis and univariable and multivariable logistic regression models were used to determine the best combination of antigens for the immunophenotypic classification of CLL vs other BCLPDs. Positive and statistically significant Spearman pairwise correlations between CD20, CD79b, and sIg fluorescence intensity were demonstrated. In the simplest models in which a single variable was considered, cutoff points were chosen that gave misclassification rates for CLL of 16% for CD79b, 19% for sIg, and 18% for CD20. Low-intensity CD79b, CD20, and sIg are associated highly with CLL. A panel containing CD5, CD19, CD23, and sIg allowed correct classification of most cases. Addition of CD20 or CD79b improved diagnostic accuracy; CD79b was slightly better than CD20. CD79b seems to be a useful addition to a standard flow cytometry panel for the evaluation of BCLPDs.

Chronic lymphocytic leukemia (CLL) is recognized as a distinct entity. However, morphologic and immunophenotypic heterogeneity exist. Twenty-six patients with CLL were studied to investigate whether an association exists among peripheral blood karyotype, morphology and immunophenotype. Clonal cytogenetic abnormalities were detected in 14 patients (53%), using conventional karyotyping techniques in addition to fluorescence in situ hybridization (FISH) for chromosome 12. By FAB guidelines, 7 of the 8 patients (88%) with trisomy 12 had mixed cell morphology compared to only 3 of 18 (17%) without trisomy 12 (P = .004). One patient (12%) with trisomy 12 had lymphocyte morphology typical for CLL. Six of the eight (75%) with trisomy 12 had atypical immunophenotype including one or more of the following: strong CD20 expression, strong surface light chain expression, or absence of CD23 expression. Only 2 of the 18 patients (11%) without trisomy 12 had atypical immunophenotype (P = .005). None of the three patients with clonal structural abnormalities of chromosome 13q14 had mixed cell morphology or atypical immunophenotype. One of the 12 patients (8%) without clonal cytogenetic abnormalities had mixed cell morphology and one had atypical immunophenotype. This study suggests that a correlation exists among karyotype, morphology, and immunophenotype in CLL, and that CLL subgroups can be identified based on laboratory parameters. Although normal karyotypes or clonal structural abnormalities of 13q14 are associated with morphology and immunophenotype considered typical for CLL, trisomy 12 is associated with mixed cell morphology and atypical immunophenotype. These findings may have implications for evaluating variation in both disease course and response to emerging therapies.

Monoclonal antibody FMC7 detects subgroups of B-cell leukemias that have arisen from cells in late stages of B-cell maturation. FMC7 was studied by flow cytometry on cell samples from 192 patients with a diagnosis of chronic lymphocytic leukemia (CLL) or lymphoma. The leukemic cells from 16 patients were reactive with this antibody. These 16 cases were evaluated for other surface markers, morphology of cells, and clinical characteristics. Of the 16 patients, 14 had cells that strongly expressed surface immunoglobulin (SIg). This is atypical of CLL cells, which characteristically show weak expression of SIg. Eleven cases had kappa and five had lambda light chain. All patients' cells had consistently brighter CD20 expression than that of CD19. Fourteen patients had expression of CD5 on their leukemic cells. One patient had more than 55% prolymphocytes, meeting the criteria of prolymphocytic leukemia (PLL), two patients had CLL in prolymphocytic transformation (CLL/PL), and two other patients were classified as having a paraimmunoblastic variant of small lymphocytic lymphoma based on a high number of paraimmunoblasts and on the histologic features. Another nine patients had immature lymphoid cells distinct from prolymphocytes or paraimmunoblasts on morphologic study. The immature cells were variable in size, and the nuclear chromatin was less clumped than that of prolymphocytes. The histologic diagnoses in four of these cases were consistent with mantle cell lymphoma. Splenomegaly was observed in 11 patients (69%), and 11 patients had advanced Rai 3 or 4 disease. Among 10 patients treated with fludarabine, five responded to therapy. Monoclonal antibody FMC7 is useful for identifying a group of atypical variants of CLL, PLL, and other B-cell lymphomas in leukemic phase that can be easily confused with CLL. Careful attention to the cell morphology and histologic features is important for the differential diagnosis of FMC7-positive, B-cell lymphoproliferative diseases.

Classification of non-Hodgkin's lymphomas in bone marrow biopsies may be hampered by several factors, including histotechnical artifacts. The authors compared the initial morphologic characteristics of B-cell lymphomas and leukemias in uniformly plastic-embedded biopsies of lymph nodes and bone marrow, using the Kiel classification and International Working Formulation (WF). Fifteen of 48 cases with multiple positive biopsies showed major differences, with 9 cases resulting in a discrepant malignancy grade. A discordant low grade was found in the bone marrow of seven cases. Two patients with rapidly progressive bone marrow insufficiency had a reversed pattern. One patient with a composite lymphoma also had disseminated high-grade lymphoma. In 4 of 22 patients with a follicle center cell lymphoma, bone marrow histologic characteristics suggested immunocytoma. The available data suggest that most differences between bone marrow and lymph node biopsies are caused by tumor heterogeneity. Morphologic evaluation of this heterogeneity may be of help in the treatment of individual patients.