Clonality assessment of cutaneous B-cell lymphoid proliferations: a comparison of flow cytometry immunophenotyping, molecular studies, and immunohistochemistry/in situ hybridization and review of the literature.

Cutaneous lymphoid infiltrates are diagnostically challenging. Although ancillary techniques to assess clonality can help distinguish between reactive lymphoid hyperplasia and lymphoma, one of the most widely used techniques in hematopathology, flow cytometry immunophenotyping (FCI), has not been routinely applied to skin specimens. We performed FCI on 73 skin specimens from 67 patients clinically suspected of having a cutaneous B-cell lymphoma (CBCL) and compared the results with those obtained from immunoglobulin heavy chain (IGH) gene molecular studies (58 cases, primarily by polymerase chain reaction) and either immunohistochemistry (IHC) or in situ hybridization to evaluate for light chain restriction (22 and 2 cases, respectively). Sufficient quantity of CD45 (leukocyte common antigen)-positive cells and staining quality were achieved in 88% of cases by FCI, and clonality was detected in 68% of CBCLs versus molecular studies showing sufficient DNA quality in 74% and only 39% clonality detection, and interpretable/contributory IHC results in 84% of cases with 55% clonality detection. Clonality was documented more frequently in secondary rather than primary CBCLs by all 3 techniques. Therefore, FCI is feasible and appears to be more reliable than molecular studies or IHC/in situ hybridization for detecting clonality in CBCLs and can provide additional prognostically and therapeutically relevant information. The exception is cases with plasmacytic differentiation such as marginal zone lymphoma for which IHC might be a superior tool. We have also shown that a large subset of primary cutaneous follicle center lymphomas express CD10 and/or BCL2 by FCI. Recent advances in FCI beg the question of applicability to cutaneous T-cell and NK-cell lymphomas.

High-resolution kinetics of cytokine signaling in human CD34/CD117-positive cells in unfractionated bone marrow.

Cytokine-mediated phosphorylation of Erk (pErk), ribosomal S6 (pS6), and Stat5 (pStat5) in CD34(+)/CD117(+) blast cells in normal bone marrow from 9 healthy adult donors were analyzed over 60 minutes. Treatment with stem cell factor (SCF), Flt3-ligand (FL), IL-3, and GM-CSF and measurement by multiparametric flow cytometry yielded distinctive, highly uniform phosphoprotein kinetic profiles despite a diverse sample population. The correlated responses for SCF- and FL-stimulated pErk and pS6 were similar. Half the population phosphorylated Erk in response to SCF between 0.9 and 1.2 minutes, and S6 phosphorylation followed approximately a minute later (t½(pS6 rise) = 2.2-2.7 minutes). The FL response was equally fast but more variable (t½(pErk rise) = 0.9-1.3 minutes; t½(pS6 rise) = 2.5-3.5 minutes). Stat5 was not activated in 97% of the cells by either cytokine. IL-3 and GM-CSF were similar to each other with half of blast cells phosphorylating Stat5 and 15% to 20% responding through Erk and S6. Limited comparison with leukemic blasts confirmed universal abnormal signaling in AML that is significantly different from normal bone marrow blasts. These differences included sustained signals, a larger fraction of responding cells, and amplification of phosphorylation levels for at least one phosphoprotein. These data support the eventual use of this approach for disease diagnosis and monitoring.

The usefulness of CD26 in flow cytometric analysis of peripheral blood in Sézary syndrome.

The loss of CD26 expression was proposed to be a constant feature of circulating Sézary cells by flow cytometric immunophenotyping (FCIP), but the experience with CD26 is limited. To establish its usefulness, CD26 results were correlated with morphologic, molecular, and immunophenotypic findings. Based on FCIP of 179 samples of peripheral blood, CD26 negativity was found in 59.3% of cases with Sézary syndrome (SS), 33.3% of mycosis fungoides (MF), 14.2% of benign dermatosis (BD), and no control cases. In diagnostic subgroups of SS based on morphologic, molecular, and immunophenotypic criteria, the percentage of CD26- cases varied from 41.1% to 63.6%. The specificity of a CD26- result was inferior to that of T-cell antigen loss in differentiating SS from MF and BD. CD26 offers lower diagnostic performance than previously suggested; however, in addition to the findings of major T-cell antigen loss, it could improve sensitivity of FCIP in patients with SS.

Instability of immunophenotype in plasma cell myeloma.

Little information has been reported describing antigen stability in plasma cell myeloma. In this study, the expression frequency and stability of 2 potential therapeutic targets, CD20 and CD52, along with the frequently aberrantly expressed CD56 antigen, were evaluated by flow cytometric analyses in 56 patients with plasma cell myeloma. Of the 56 patients, 23 (41%) showed immunophenotype change, including CD56 in 6 cases, CD20 in 7 cases, and CD52 in 17 cases. Combined CD56/CD52 change was seen in 3 cases and combined CD20/CD52 in 4 cases. No correlation was found between immunophenotype change and age, sex, stage, plasma cell morphologic features, extent of marrow involvement, time between analyses, type of therapy, or response to therapy. Immunophenotype shift was more common in patients with IgA than in patients with IgG paraprotein. Recognition of lack of stability in immunophenotype may be important, especially in antigen-directed treatment decisions and when specific phenotypes are used to detect residual disease.

CD23+ mantle cell lymphoma: a clinical pathologic entity associated with superior outcome compared with CD23- disease.

Mantle cell lymphoma (MCL) commonly lacks expression of CD23. However, a significant minority of MCLs express CD23, as assessed by flow cytometric immunophenotyping (FCIP). The aims of our study were to investigate the expression of CD23 by FCIP in patients with MCL and to correlate CD23 expression with pathologic and clinical parameters, including outcome. We studied 53 patients with untreated MCL who had CD23 expression determined by FCIP. At diagnosis, 14 MCLs (26%) were CD23+ at all tissue sites, whereas 33 (62%) were CD23-, and 6 (11%) had discordant CD23 expression among different tissue sites. Patients with CD23- MCL had extranodal disease more commonly compared with patients with CD23+ MCL. Moreover, with 57-month median follow-up, the 4-year event-free and overall survival rates for CD23+ MCL were 45% and 75%, respectively, compared with 19% and 51% for CD23- MCL. In multivariate Cox regression analysis, CD23 status and leukemic-phase MCL were the most important factors predicting outcome.

Comparative analysis of flow cytometric techniques in assessment of ZAP-70 expression in relation to IgVH mutational status in chronic lymphocytic leukemia.

We compared 1 subjective and 5 objective flow cytometric methods to evaluate zeta-associated protein (ZAP-70) expression in relation to immunoglobulin heavy-chain variable-region (IgVH) gene mutational status in 154 samples from 125 patients with chronic lymphocytic leukemia (CLL). ZAP-70 expression determined by all methods used correlated with IgVH gene mutational status, but none of them demonstrated high concordance rates. Of the objective methods, ZAP-70 staining determined as a ratio of molecules of equivalent soluble fluorochrome intensity in CLL cells to that in normal B cells (ZAP-70+ staining in IgVH germline cases, 59%; ZAP-70- in IgVH mutated cases, 75%) or T cells (ZAP-70+ in IgVH germline cases, 66%; ZAP-70- in IgVH mutated cases, 57%) provides the best combination for assigning ZAP-70+ status to IgVH germline and ZAP-70- status to IgVH mutated cases. The subjective method based on ZAP-70 expression in natural killer/T cells gave a similar result, but reproducibility between laboratories may be difficult. Further studies on ZAP-70 expression in relation to clinical parameters may address whether ZAP-70 is an independent prognostic marker for CLL.

Flow cytometric analysis of lymphoid enhancer-binding factor 1 in diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma.

OBJECTIVES: Nuclear overexpression of lymphoid enhancer-binding factor 1 (LEF1) assessed by immunohistochemistry has been shown to be highly associated with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) among small B-cell lymphomas. The purpose of this study was to evaluate the utility of flow cytometric analysis of LEF1 in the diagnosis of CLL/SLL. METHODS: Normal peripheral blood was used to validate the test. Flow cytometric analysis of LEF1 was performed in 64 patient samples qualitatively and quantitatively by comparing the staining intensity and the ratios of the median fluorescence intensities (MFIs) of LEF1 in B cells of interest to the internal reference cell populations. The results were correlated with the pathologic diagnosis. RESULTS: Proper sample processing ensured sufficient separation of positive LEF1 staining in T cells from negative staining in normal B and natural killer (NK) cells. Qualitative analysis of patient samples showed that all 25 cases of CLL/SLL but none of the other small B-cell lymphomas were positive for LEF1. Using a B/NK MFI ratio of 1.5 and B/T MFI ratio of 0.45 separated CLL/SLL cases from non-CLL lymphomas. CONCLUSIONS: Flow cytometric analysis of LEF1 is sufficient to differentiate CLL/SLL from other small B-cell lymphomas and may serve as a useful tool in the diagnosis of CLL/SLL.

Pan B-cell markers are not redundant in analysis of chronic lymphocytic leukemia (CLL).

BACKGROUND: The classic immunophenotype for chronic lymphocytic leukemia (CLL) is CD19(+), restricted dim surface expression of kappa or lambda light chain, CD5(+), CD23(+), dim CD20(+), negative FMC7, and negative CD79b. However, the necessity of assaying for all 3 pan B-cell markers (CD20, FMC7, and CD79b) by flow cytometry has not been definitively documented for CLL. METHODS: Qualitative patterns and semi-quantitative assessment of staining intensity for CD20, FMC7 and CD79b were performed in 70 cases with a current or prior diagnosis of CLL or CLL with increased prolymphocytes leukemia (CLL/PL). The concurrent morphology in 66 of 70 specimens was classified as typical CLL in 53 cases, CLL/PL in 10 cases, and large cell lymphoma in 3 cases. RESULTS: Forty percent of the cases varied from the characteristic immunophenotype by having moderate or bright staining of CD20 (36%), FMC7 (7%), and/or CD79b (18%). Discrepant qualitative staining patterns were found between FMC7 and CD20 (21%), CD20 and CD79b (15%), and CD79b and FMC7 (10%). Semiquantitative measurement of staining intensity showed little correlation between CD79b and CD20 or FMC7. Moderate correlation was seen between CD20 and FMC7. No correlation was observed between morphology and intensity of marker expression. CONCLUSIONS: Variable patterns and intensity of staining were seen for FMC7, CD20, and CD79b in this cohort of CLL samples. Dim or negative staining was most consistently seen for FMC7 (93% of specimens). Although FMC7 staining intensity was moderately correlated with CD20, CD79b intensity was poorly correlated with either CD20 or FMC7, and thus, may provide some independent information.

Expansion of a clonal CD8+CD57+ large granular lymphocyte population after autologous stem cell transplant in multiple myeloma.

Clonal expansions of large granular lymphocytes (LGLs) have been identified in patients following stem cell transplants and may represent posttransplant LGL leukemias or reactive immune responses. To differentiate between these 2 possibilities, we assessed peripheral blood and bone marrow of patients with myeloma after autologous stem cell transplant. All patients examined shortly after autologous stem cell transplant had significant increases in the LGLs in the peripheral blood and bone marrow (71% of lymphocytes) as compared with controls (39%). This increase was detectable years after transplant. The LGLs had a reproducible immunophenotype of CD8+CD57+ T cells without phenotypic abnormalities in 19 of 20 patients. Sixty-five percent of the post-autologous stem cell transplant patients had clonal T-cell receptor gene rearrangements in the bone marrow, yet no patients had neutropenia or splenomegaly. Although the LGL expansions were clonal and persistent, the lack of clinical sequelae suggests the clonal LGL expansion is a reactive, potentially beneficial, immune response to autologous stem cell transplant.

Dysregulated angiogenesis in B-chronic lymphocytic leukemia: morphologic, immunohistochemical, and flow cytometric evidence.

BACKGROUND: The extent of enhanced bone marrow angiogenesis in chronic lymphocytic leukemia (CLL) and relationship to proangiogenic factors and prognostic indicators is largely unexplored. METHODS: To further investigate the role of angiogenesis in CLL by evaluating the topography and extent of angiogenesis in a group of CLL bone marrow biopsies, to study the expression of pro and antiangiogenic vascular factors in CLL cells to more precisely document the cell types producing these factors, and to evaluate the role, if any, of localized hypoxia in upregulation of angiogenesis in CLL We used immunohistochemistry (IHC) (n = 21 pts) with antibodies to CD3 and CD20, proangiogenic (VEGF, HIF-1a) and antiangiogenic (TSP-1) factors, and VEGF receptors -1 and -2 to examine pattern/extent of CLL marrow involvement, microvessel density (MVD), and angiogenic characteristics; flow cytometry (FC) was performed on 21 additional cases for VEGF and TSP-1. RESULTS: CLL patients had higher MVD (23.8 vs 14.6, p~0.0002) compared to controls (n = 10). MVD was highest at the periphery of focal infiltrates, was not enhanced in proliferation centers, and was increased irrespective of the presence or absence of cytogenetic/immunophenotypic markers of aggressivity. By IHC, CLL cells were VEGF(+), HIF-1a (+), TSP-1(-), VEGFR-1(+), and VEGFR-2(+). By FC, CLL cells were 1.4-2.0-fold brighter for VEGF than T cells and were TSP-1(-). CONCLUSION: CLL demonstrates enhanced angiogenesis, with increased MVD, upregulated VEGF and downregulated TSP-1. Upregulation of HIF-1a in all CLL cases suggests localized tissue hypoxia as an important stimulant of microvessel proliferation. The presence of VEGF receptors on CLL cells implies an autocrine effect for VEGF. Differences in MVD did not correlate with traditional genetic/immunophenotypic markers of aggressiveness.

Biphenotypic acute leukemia with coexpression of CD79a and markers of myeloid lineage.

Acute leukemias demonstrating immunophenotypic features of more than 1 cell lineage are referred to as acute leukemias of ambiguous lineage in the new World Health Organization classification system. A subtype of leukemia of ambiguous lineage is biphenotypic acute leukemia in which the malignant cell population expresses markers of 2 different lineages, most commonly myeloid and either B- or T-lymphoid lineages. This entity has been defined by a scoring system proposed by the European Group for the Immunological Characterization of Acute Leukemias (EGIL), with various markers assigned a score of 2, 1, or 0.5 depending on their specificity for myeloid or lymphoid lineage. Those cases having a score greater than 2 for the myeloid and either the B- or T-lymphoid lineages are biphenotypic acute leukemia in this system. One marker, CD79a, has been so clearly associated with acute lymphoblastic leukemia (ALL) by some researchers that its expression in the presence of blast markers is considered indicative of B-ALL. We describe an unusual case of acute leukemia meeting the criteria for biphenotypic acute leukemia in which CD79a expression was observed in the blast population.

Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity.

BACKGROUND: Stat5(1) (Signal Transducer and Activator of Transcription 5) is normally phosphorylated and activated by Janus kinases. In cells transformed with BCR/ABL, Stat5 is constitutively activated by promiscuous phosphorylation. Cytometry of intracellular antigens can be used to evaluate cell treatments affecting gene expression, because it precisely provides the fraction of affected cells and the quantitative change in expression. Here, we asked whether we could measure a phosphorylated epitope on Stat5 by cytometry, and whether that measurement would respond to Bcr/Abl inhibition. METHODS: Chronic myelogenous leukemia (CML) cell lines or control Bcr/Abl-negative cells were treated or not with imatinib mesylate, fixed and permeabilized with formaldehyde followed by methanol; reacted with rabbit polyclonal and mouse monoclonal antibodies against an epitope including tyrosine 694 of Stat5a (pSTAT5); reacted with antibodies that mark mitotic cells; counterstained with secondary fluorescent antibodies and 4',6-diamidino-2-phenylindole (DAPI); and then subjected to flow cytometry. Western blotting was performed with pSTAT5 and Stat5 antibodies. RESULTS: Optimal fixation and staining parameters were established for pSTAT5 antibodies with K562 cells. These cells displayed high levels of immunoreactivity with pSTAT5 probes that could be inhibited uniformly with imatinib mesylate in a dose-response and time-dependent manner. The IC50 for downregulation of pSTAT5 immunoreactivity for K562 cells by cytometry was approximately 70 nM. The inhibition half-time was approximately 1 min. At micromolar doses this reactivity remained minimal for up to 7 days. Cultured cells also displayed a population of negative cells that increased under conditions related to cessation of cell growth (media nutrient depletion). This study also showed quantitatively that a rabbit polyclonal antibody that cross-reacted with an additional epitope could be used successfully as a measure of Bcr/Abl activity. CONCLUSION: We have developed a sensitive cytometric assay for Bcr/Abl kinase activity in human hematopoietic cell lines.