Typical CLL Research Articles

Evaluation of Immunophenotypic Markers in Diagnosis and Prognosis of Atypical Chronic Lymphocytic Leukemia

Abstract: BACKGROUND: Atypical chronic lymphocytic leukemia diagnosis (aCLL) constitutes of monoclonal B lymphocytes of more than 5000 with unique morphological and immunophenotypic features different from classical or typical CLL. By immunophenotyping, aCLL is differentiated from typical CLL in the negative expression of either one or two immunophenotypic markers, mostly CD23 and/or CD5, provided that the patient does not have the diagnostic criteria of any other lymphoid neoplasm. aCLL patients are usually presented clinically in an aggressive manner and have worse outcomes compared to classic CLL. MATERIALS AND METHODS: This cross-sectional study includes 36 patients diagnosed as aCLL compared to 36 patients diagnosed as typical CLL. Diagnosis depends on blood film morphology and immunophenotyping by 8-color flow cytometry, the markers that are used for diagnosis depend on Matutes scoring system of CLL with the addition of CD200 and CD43, in addition to prognostic markers CD38 and CD305. The clinical staging system was applied, and the patients were divided into low-, intermediate-, and high-risk groups. Follow-up program for 6–12 months with evaluation of their hematological and clinical response and assessment of their remission state as was instituted in the hematological center. RESULTS: CD5 and CD23 are negative in 16.6% and 33.3% of aCLL patients, respectively, whereas CD200 and CD43 are positively expressed in 100% and 83.33% of aCLL patients, respectively, co-expression percentage of both markers in 83.33%. aCLL cases were commonly presented with high-risk group and usually have markers that are linked to poor prognosis, including negative expression of CD305 in 83.3% and positive CD38 expression in 75% of cases and only 5.56% of patients achieved remission state. CONCLUSIONS: aCLL is characterized by immunophenotypic differences in comparison to typical CLL. A definitive diagnosis needs additional markers, mainly CD200 and CD43; the prognosis of aCLL is worse than classical CLL.

Towards a Better Characterisation of Leukemic Cells in Chronic Lymphocytic Leukaemia: Cell-Size Heterogeneity Reflects Their Activation Status and Migratory Abilities.

Simple SummaryChronic lymphocytic leukaemia (CLL) is a heterogeneous chronic disease characterised by the clonal expansion of mature CD19+CD23+CD5+ B-cells in blood, bone marrow and lymphoid tissue. Despite the CLL tumour cell population showing considerable heterogeneity in cell size, the functional characteristics of leukemic cells that differ in size have not been explored. The results of our study demonstrate that differences in CLL cell size reflect their activation state, polarisation and migratory capacity, with large CLL cells being more activated, polarised and motile than the small CLL cells present in the CLL cell pool. Our data provide evidence of the importance of cell-size heterogeneity within the CLL cell pool and the dynamics of cell-size changes for disease pathogenesis.Chronic lymphocytic leukaemia (CLL) is a genetically, morphologically and phenotypically heterogeneous chronic disease with clinical variability between patients. Whether the significant heterogeneity of cell size within the CLL population contributes to the heterogeneous features of this disease has not been investigated. The present study aimed to characterise the phenotypic and functional properties of two subpopulations of typical CLL cells that differ in cell size: small (s-CLL) and large (l-CLL) CLL cells delineated by forward scatter cytometry. The s-CLL cells were characterised by the CD5lowCXCR4hi phenotype, while the l-CLL cells were characterised by the CD5hiCXCR4dim phenotype and indicated a higher expression of CXCR3, CD20, CD38 and HLA-DR. The l-CLL cells displayed higher migration activity towards CXCL12, a tendency towards a higher proliferation rate and an increased capacity to produce IgM in the presence of CpG compared with s-CLL cells. When stimulated with CpG and CXCL12, l-CLL cells were characterised by a higher polarisation phenotype and motility than s-CLL cells. Our study revealed that the differences in CLL cell size reflected their activation status, polarisation and migratory abilities. Our data provide evidence of the importance of cell-size heterogeneity within a CLL pool and the dynamics of cell-size changes for disease pathogenesis, thus deserving further investigation.

Three Cases of Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) Involving Cerebrospinal Fluid (CSF)

Abstract Background Chronic lymphocytic leukemia (CLL) is a clonal disorder of B lymphocytes, characterized by proliferation of small mature lymphocytes involving the blood, bone marrow, and lymphoid tissues. CLL can rarely involve the central nervous system (CNS), either by involving brain parenchyma or cerebrospinal fluid (CSF). We present a series of three cases with clinically significant involvement of the CNS with CLL. Results During the past 2 years, our medical center managed three patients with CLL who presented with symptomatic CNS involvement, as determined by flow cytometry. The immunophenotypic profile was that of a typical CLL with light chain restricted small B cells positive for CD20 (dim) and coexpressing CD5 and CD23. In addition, two patients had brain involvement by SLL that was confirmed by brain biopsy. Notably, the brain lesions had a mildly elevated Ki-67 proliferative index (10%-30%). Bone marrow was involved in two patients, showing nodular, interstitial, and diffuse patterns. Bone marrow involvement ranged from 60% to 80% and showed very low Ki-67 proliferative index. None of the patients had features suggestive of a CLL transformation. FISH was performed on either bone marrow or CSF and demonstrated that patient 1 had Del11q(ATM) and Dell13q, patient 2 had trisomy 12, and patient 3 had del17(TP53) and IGH rearrangement. All of the patients showed persistent CSF involvement, ranging from 4 to 12 weeks, requiring aggressive treatment with intrathecal chemotherapy. At the end of treatment, all of the patients were clear of CNS involvement as judged by flow cytometry of CSF. Conclusion We report three patients who had persistent involvement of CSF. Only one patient had del17(TP53), a cytogenetic feature that is associated with high-risk CLL. It would be interesting to study clonal evolution of CLL to understand the mechanisms that underlie involvement of the CNS.

CD200 is a useful marker in the diagnosis of chronic lymphocytic leukemia.

The diagnosis of CLL is supported by a typical morphology and immunophenotype and usually does not present difficulties. Nevertheless, some patients with CLL can show an atypical phenotype, this raising the possibility of a lymphoproliferative disorder other than CLL. It has been recently shown that the expression of CD200 could be a rather consistent marker for CLL. The expression of CD200 was investigated in 120 consecutive patients with B-cell chronic lymphoproliferative disorders (B-CLPD) (65 cases diagnosed as typical CLL, 16 atypical CLL, and 39 non-CLL before entering the study) by using multiparametric flow cytometry with four color combinations. CD200 was analyzed as percentage of positive cells (≥30%) and MFIR expression. ROC curves were used to determine the cut-off for the CD200 MFIR. Matutes score (MS) was used as comparator. All 81 (100%) patients classified as CLL and 25 of 39 (64.1%) classified as non-CLL expressed high CD200 expression (≥30%). CD200 expression showed a high sensitivity (100%) and a low specificity (35.9%), and the accuracy was similar to that of Matutes score markers (range: 79.2%-86.7%); except SmIg that was 59.1%. The addition of CD200 to the Matutes score correctly identified 74 of 81 (91.4%) CLL cases including 9 of 16 atypical CLL cases. As per non CLL cases, 37 of 39 (94.9%) were correctly diagnosed by the modified system. Altogether, CD200 improved the diagnostic accuracy of Matutes score from 86.7% to 92.5% (P < .01). These results show that CD200 is a valuable, albeit not specific, CLL diagnostic marker. © 2018 International Clinical Cytometry Society.

IgA Levels Can Predict Survival in CLL Patients: A Retrospective Analysis of 183 Cases in a Brazilian Cohort

Background: Immunosuppression and frequent infections has impact on morbidity and mortality in CLL patients. It is basic related with chronic immune dysregulation or treatment toxicities. Hypogammaglobulin is a frequent event in CLL and can explain some of the infection susceptibility related to the disease. Low IgG levels were associated with a higher frequency of bacterial infections, commonly Streptococcus pneumoniae and Haemophilus influenza. Although hypogammaglobulin can contribute with repeated infections in CLL patients, the prognostic value of this occurrence is controversial. At our center we routinely test Ig levels at time of CLL diagnosis.Methods: A retrospectively analysed was done with 183 patients diagnosed with typical CLL between 2007 to 2013 for clinical and laboratorial features (sex, age, CBC, CD38, Beta-2-microglobulin, albumin, Binet staging, direct coombs test and Ig levels) and survival (overall and time to first treatment). The statistical analysis was done on IBM SPSS v.20. This study received approval on the ethical committee from our institution.Results: Median follow up of this cohort was 52 months. Median age was 65y (34-90) and 98 were male (52.1%). Most of them, 61.1% (n=110) were Binet A, 17.8% (n=32) Binet B and 21.1% (n=38) Binet C. Direct coombs test was positive in 9.9% of cases (n=16). CD38 was positive in 23.3% (n=40). The IgG, IgM and IgA levels were above normal laboratory value in 15.3% (n=29), 6.9% (n=13) and 6.3% (n=12), respectively, and it was below in 26.5% (n=50), 35.6% (n=67) and 36% (n=68), respectively. Twenty-three patients (12.2%) had both low levels of IgG, IgM and IgA and 57.7% had at least one Ig subtype at low level. When we consider only the IgA, 42.3% (n=80) had above or below levels. Albumin level was below 4.5 g/dl, 4.0 g/dl and 3.5 g/dl in 63.2% (n=115), 20.3% (n=37) and 9.3% (n=17), respectively. Beta-2 microglobulin level was higher than 2.5 mg/dl in 55% (n=93). The median overall survival was not reached and 76.8% +- 3.8% were alive at 52 months. On the univariate analysis (Kaplan-meier) of overall survival, high Binet staging (p=0.001), altered (high or low) IgG level (p=0.017), altered IgA level (p<0.001), any low Ig level (p<0.001), positive coombs test (p=0.006), beta-2-microglobulin higher than 2.5 mg/dl (p<0.001) and CD38 membrane expression showed worse prognosis. The multivariate model (cox regression) showed that just IgA at high or low level (p=0.002), coombs (p=0.022) and high Beta-2-microglobulin (p=0.001) had impact on survival. Hundred and eight patients with Binet A were included at time of first treatment survival analysis with median follow up of 58 months. The median TTFT again was not reached, but at 58 months was 58% (+- 5.6%). On the univariate analysis low albumin (p=0.039), IgA high or low levels (p=0.003), any low Ig level (p=0.037) and high Beta-2-microglobulin (p<0.001) were significant, remaining only IgA level (p=0.04) and high beta-2-microglobulin (p=0.006) at multivariate analysis.Conclusion: This analysis suggests that low or high IgA level are associated with a worse prognosis. This could be explained by immune suppression (mucous immune system depression) and higher susceptibility for bacterial infection (mortality cause not evaluated). The fact that this group had a shorter time to first treatment can not be explained by the same reason. Maybe not normal IgA levels itself can be associated with a more aggressive and progressive disease. Despite of all the bias of a retrospective study (loss of follow up, missing values) and lack of FISH and IgVH mutational status at the diagnosis, this data points the importance of Ig level evaluation at the time of diagnosis DisclosuresNo relevant conflicts of interest to declare.

Issue Highlight 2015.

Issue Highlight 2015.

Automated quantification of apoptosis in B-cell chronic lymphoproliferative disorders: a prognostic variable obtained with the Cell-Dyn Sapphire (Abbott) automated hematology analyzer.

B-chronic lymphocytic leukemia CLL, a neoplastic clonal disorder with monomorphous small B lymphocytes with scanty cytoplasm and clumped chromatin, can be morphologically differentiated in typical and atypical forms with different prognosis: Smudge cells (Gumprecht's shadows) are one of the well-known features of the typical CLL and are much less inconsistent in other different types CLPD. Abbott Cell-Dyn Sapphire uses the fluorescence after staining with the DNA fluorochrome propidium iodide for the measurement of nucleated red blood cells (NRBCs) and nonviable cells (FL3+ cell fraction): We have studied the possible correlation between presence and number of morphologically identifiable smudge cells on smears and the percentage of nonviable cells produced by Cell-Dyn Sapphire. 305 blood samples from 224 patients with B-cell lymphoproliferative disorders and 40 healthy blood donors were analyzed by CBC performed by Cell-Dyn Sapphire, peripheral blood smear, and immunophenotype characterization. FL3+ fraction in CLPD directly correlated with the percentage of smudge cells and is significantly increased in patients with typical B-CLL. This phenomenon is much less evident in patients with atypical/mixed B-CLL and B-NHL. In small laboratories without FCM and cytogenetic, smudge cells%, can be utilized as a preliminary diagnostic and prognostic tool in differential diagnosis of CLPD.

The Diagnostic Value of Flow Cytometry Imunophenotyping in an Albanian Patient Population with a Preliminary Clinical Diagnosis of Chronic Lymphocytic Leukemia

Objective: Based on the flow cytometry multiparametric immunophenotyping methodology we studied some useful cell marker criteria needed for the practical differentiation of the chronic lymphocytic leukemia from other chronic limphoproliferative diseases with a leukemic component.Materials and Methods: The applied methodology is a four color flow cytometry multiparametric immunophenotyping technique using EDTA blood samples taken from 84 consecutive patients diagnosed with CLL through a preliminary clinical and white blood cell examination. The following fluorescent stained monoclonal antibodies were used: CD3, CD4, CD5, CD8, CD11c, CD19, CD20, CD23, CD25, FMC7 and kappa/lambda light chains.Results: From the 84 individuals tested, 2 out of them (2.4%) resulted with a abnormal T-cell population while 82 (97.6%) showed a pathological B cell line. 58 (69.1%) patients resulted with typical CLL markers (CD19+CD5+CD23+) while 5 (5.9%) of them presented a non typical chronic lymphocytic leukemia profile (CD19+CD5+CD23-). 19 (22.6%) out of patients displayed an abnormal CD19+CD5- B cell population. A statistically significant correlation was found between the clinical stage of CLL and the positivity for the CD38 marker (p=0.04).Conclusion: Flow cytometry immunophenotyping is a fundamental examination for the final diagnosis of chronic lymphocytic leukemia. The expression of CD38+ in CLL patients stands for a more advanced clinical stage.

The Diagnostic Value of Flow Cytometry Imunophenotyping in an Albanian Patient Population with a Preliminary Clinical Diagnosis of Chronic Lymphocytic Leukemia

Objective: Based on the flow cytometry multiparametric immunophenotyping methodology we studied some useful cell marker criteria needed for the practical differentiation of the chronic lymphocytic leukemia from other chronic limphoproliferative diseases with a leukemic component.Materials and Methods: The applied methodology is a four color flow cytometry multiparametric immunophenotyping technique using EDTA blood samples taken from 84 consecutive patients diagnosed with CLL through a preliminary clinical and white blood cell examination. The following fluorescent stained monoclonal antibodies were used: CD3, CD4, CD5, CD8, CD11c, CD19, CD20, CD23, CD25, FMC7 and kappa/lambda light chains.Results: From the 84 individuals tested, 2 out of them (2.4%) resulted with a abnormal T-cell population while 82 (97.6%) showed a pathological B cell line. 58 (69.1%) patients resulted with typical CLL markers (CD19+CD5+CD23+) while 5 (5.9%) of them presented a non typical chronic lymphocytic leukemia profile (CD19+CD5+CD23-). 19 (22.6%) out of patients displayed an abnormal CD19+CD5- B cell population. A statistically significant correlation was found between the clinical stage of CLL and the positivity for the CD38 marker (p=0.04).Conclusion: Flow cytometry immunophenotyping is a fundamental examination for the final diagnosis of chronic lymphocytic leukemia. The expression of CD38+ in CLL patients stands for a more advanced clinical stage.

Chronic Lymphocytic Leukemia with t(14;18)(q32;q21) As a Sole Cytogenetic Abnormality.

BACKGROUNDChronic lymphocytic leukemia (CLL) is the most common leukemia in adults. The chromosomal abnormality t(14;18)(q32;q21) is most commonly associated with neoplasms of a follicular center cell origin. However, t(14;18) has also been reported in rare cases of CLL.OBJECTIVEWe describe the clinicopathologic, immunophenotypic, conventional, and molecular cytogenetic features of two rare cases proven to be CLL morphologically and immunologically in which t(14;18) was found as the sole cytogenetic abnormality.METHODSMorphologic, flow cytometric analysis and molecular cytogenetic of peripheral blood and/or bone marrow samples were analyzed.RESULTSCytomorphologically, the cells were small mature lymphocytes without any findings that had characteristics of follicular lymphoma (FL) such as indented or clefted nuclei. Immunologic findings were characteristic of typical CLL without expression of CD10. A cytogenetic study revealed the two cases of CLL carrying t(14;18)(q32;q21).CONCLUSIONWe concluded that CLL with t(14;18) is rare and should be differentiated from FL as the therapy is highly diverse between both diseases. Using immunoglobulin heavy chain gene (IGH) probes are important in the workup of patients with suspected CLL and suggest that the IGH probe should be used routinely in all CLL fluorescence in situ hybridization (FISH) panels.

Cyclin D1 (CCND1) mRNA Expression Assessed by Real-Time PCR Is Increased in Patients with Mantle Cell Lymphoma and Contributes to Diagnosis in B-Cell Lymphoma Cases

Abstract 1595 Background:Immunohistochemistry for Cyclin D1 (CCND1) expression is routine in cases suggestive of mantle cell lymphoma (MCL). Most MCL are t(11;14)/IGH-CCND1-positive by FISH. PCR based detection of the fusion transcript is hampered by widespread breakpoints. Only few data is available on quantitative real-time PCR (RQ-PCR) for CCND1 expression measurement. Aims:To assess CCND1 mRNA expression and correlate it with t(11;14) in mature B-cell neoplasms. Methods:We established a RQ-PCR assay for CCND1 mRNA measurement and investigated 451 cases: 142 MCL (in all cases IGH-CCND1 confirmed by FISH), 76 chronic lymphocytic leukemia (43 typical CLL, 33 CLL/PL), 20 hairy cell leukemia (HCL), 13 hairy cell leukemia-variant (HCL-v), 20 splenic marginal zone lymphoma (SMZL), 91 other mature B-cell neoplasms. CCND1 background expression was assessed in 29 pts with other hematological neoplasms and 60 healthy individuals. FISH and/or chromosome banding analysis for the t(11;14) was available in 364 pts.Bone marrow (BM, n=267) or peripheral blood (PB; n=184) samples were analyzed by cytomorphology, multiparameter flow cytometry (MFC), FISH, and RQ-PCR. CCND1 mRNA expression was given by RQ-PCR in comparison to ABL1 mRNA expression (%CCND1/ABL1). Limited dilution of high expressers into cDNA of healthy controls revealed a sensitivity of the assay of up to 0.1 %. Results:IGH-CCND1 translocation carriers had higher %CCND1/ABL1 than those without which hold true in the total cohort (mean±SD, 420.4±740.3 vs 17.8±128.3; p<0.001), separately in all B-cell neoplasms (420.4±740.3 vs. 20.6±140.5; p<0.001), in all CLL (352.9±302.9 vs. 28.8±201.6; p<0.001), and in all SMZL (178.7±236.9 vs. 3.4±4.8; p=n.s.). In 150 IGH-CCND1- positive samples (142 MCL, 3 CLL, 2 SMZL, 3 other mature B-Cell neoplasms) median %CCND1/ABL1 level was 178.2 (range, 1.5–4,152.0). Normalized by the number of FISH-positive cells assuming 100% lymphoma infiltration, IGH-CCND1- positive samples showed a median %CCND1/ABL1 of 445.8 (range, 17.9–4848.5). Therefore, we chose a normalized %CCND1/ABL1 expression of 17.0 as threshold for CCND1 positivity. In total, 131/150 IGH-CCND1- positive samples had %CCND1/ABL1 expression levels above this threshold resulting in a sensitivity of 87.3% (not normalized) for detection of the t(11;14) by CCND1 expression. In the IGH-CCND1-positive samples, %CCND1/ABL1 was higher in PB than BM (p=0.003) corresponding to a higher infiltration degree in PB by MFC (p=0.002). In IGH-CCND1 negative samples (n=214), the median %CCND1/ABL1 was 2.4 (range, 0.0–1,721.5). In total, 204/214 of t(11;14)/IGH-CCND1 negative cases had low CCDN1 expression (<17.0), resulting in a negative predictive value of 95.3%. In the group of other mature B-cell neoplasms median %CCND1/ABL1 was 1.1 (range, 0.1–72.4), in healthy individuals 1.1 (range, 0.0–7.8). Mean %CCND1/ABL1 was higher in MCL than in other mature B-cell neoplasms pts (mean±SD, 392.9±685.3 vs. 51.5±324.1; p<0.001). The mean expression levels were considerably lower in CLL, CLL/PL, SMZL, and other mature B-cell neoplasms, but the maximum values in these subgroups were comparable to MCL, thus reflecting a significant overlap and indicating that IGH-CCND1-positive lymphomas other than MCL can have increased CCND1 expression. HCL and HCL-v showed low CCND1 expression. Of 141 cases with %CCND1/ABL1 values above the threshold of 17.0, in which FISH was available, 131 cases had a t(11;14)/IGH-CCND1 (92.9%), and 10 were translocation-negative. Thus, the specificity of %CCND1/ABL1 to indicate t(11;14) was 92.9%. Performing Spearman rank correlation in MCL, the %CCND1/ABL1 ratio was correlating with % aberrant nuclei by FISH (R=0.688; p<0.001) and lymphoma infiltration by MFC (R=0.700; p<0.001). 34 cases were analyzed during follow up, CCDN1 expression level was stable when untreated, showed 2.5–3.5 log reduction after therapy, and increase at relapse. Conclusions:CCND1 expression is highly correlated with t(11;14)/IGH-CCND1 and significantly increased in MCL as compared to other B-cell lymphoma subtypes. Increased CCND1 expression can also be found in other lymphoma subtypes. CCND1 expression measurement by RQ-PCR may be considered a useful adjunct for diagnosis in pts with suspected MCL or in lymphoma pts with suspected t(11;14)/IGH-CCND1 but ambiguous results by other techniques. This technique may also be useful to monitor therapy response. Disclosures:Bacher:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

Circulating B Cell Clones in Familial Waldenström Macroglobulinemia.

Abstract 2703 Background and Significance:Lymphoplasmacytic lymphoma (LPL) is an indolent but incurable B cell lymphoproliferative characterized by the clonal expansion of plasmacytoid lymphocytes in the lymph nodes and bone marrow. The majority of cases are associated with an IgM isotype paraprotein, and when present, is termed Waldenström macroglobulinemia (WM). WM shows familial aggregation suggesting an inherited risk for disease, and also co-aggregates with CLL in families. In our previous work, we have shown that clonal populations of peripherally circulating B cells can be identified in 18% of unaffected family members from CLL kindreds. Most of these clonal populations have a typical CLL immunophenotype and have been termed CLL-like monoclonal B cell lymphocytosis (MBL). Because CLL and WM have related gene expression profiles and appear to have shared genetic risk, we hypothesized that unaffected family members of WM kindreds would have detectable circulating clonal B cell populations. Further, we undertook systematic flow cytometric screening of familial WM and IgM MGUS cases to determine the prevalence, immunphenotype, and biologic characteristics of circulating malignant B cells. Methods:A diagnosis of LPL / WM or IgM MGUS was determined using standard WHO criteria. All patients and unaffected family members were ascertained at the National Cancer Institute and provided informed consent. Peripheral blood mononuclear cells were isolated using density centrifugation and viably frozen in DMSO containing media. We developed a two tube, nine color flow cytometric assay: the first tube allowed for detection of CLL-like clones based upon co-expression of CD5, CD20, and CD23; the second tube targeted WM populations based upon expression on CD19, CD20, CD25, CD38, and surface IgM. Cell populations were considered clonally restricted if the κ: λ was > 3.0 or < 0.3. Clonal populations were then isolated using fluorescence activated cell sorting (FACS). RNA and genomic DNA were extracted for genetic and genomic studies using phenol: chloroform purification. Results:A total of 155 individuals were analyzed: 54 WM / LPL, 17 IgM MGUS, 1 IgG MGUS, 1 IgA MGUS, 1 NHL, and 81 unaffected family members. Twenty of 54 WM patients had detectable peripherally circulating populations. Thirteen WM patients had no detectable B cells, of these, 11 patients had prior treatment. As such, among the 41 WM patients with a B cell compartment that was analyzable by flow cytometry, 49% (20 of 41) had peripherally circulating B cell clones detected. Four of these 20 cases showed two immunophenotypically distinct clonal B cell populations. The immunophenotype was somewhat heterogenenous: 18 cases expressed surface IgM, CD38 was variable but expressed in most cases, CD25 was not detected in any case, and 4 cases showed a CLL like (CD5+CD20dimCD23+) immunophenotype. Interestingly, we detected peripherally circulating B cell clones in 9 of 17 cases (53%) of IgM MGUS, a proportion nearly identical to that identified in WM / LPL. Three of 9 were “CLL-like” with co-expression of CD5 and CD23, while the majority of clones were CD5negIgM+CD38+. Among unaffected family members, we identified B cell clones in only 4 of 81 (5%). All 4 cases expressed CD5, and 3 showed a CLL-like phenotype, consistent with these individuals having MBL. Among all study subjects, 20 clonal B cell populations of > 104 B cells were FACS purified from 18 different cases: 12 WM, 4 IgM MGUS, 1 IgA MGUS, and 1 unaffected family member. Conclusions:Peripherally circulating B cell clones with an immunophenotype similar to that of LPL can be identified in approximately half of patients with WM / LPL. We observed for the first time that a similar proportion of patients with IgM MGUS have detectable clonal populations with an immunophenotype similar to that observed in WM patients. Genetic and genomic studies to determine the lineage of these populations are currently underway. The frequency of MBL among unaffected family members with WM is lower than that observed in CLL kindreds. CLL-like MBL can be detected at very low numbers because the cell population is immunophenotypically abnormal. The peripherally circulating clones identified in WM / LPL patients have an otherwise normal B cell immunophenotype and can only be detected by light chain restriction. This likely significantly limits the ability of flow cytometry to detect pre-clinical CD5neg IgM expressing clonal populations. Disclosures:No relevant conflicts of interest to declare.

Morphological Atypia Represents Favorable Prognosis of Chronic Lymphocytic Leukemia in Japanese Patients

Abstract 4594 Background:B-chronic lymphocytic leukemia (B-CLL) is rare in Japan. Therefore, only a few data are available on the clinical characteristics of B-CLL for Japanese patients (pts). We reported that the rate of atypical CLL appeared to be higher in Japanese pts than that of Caucasians. Patients and Methods:This nation-wide study was performed from Oct. 2003 to March 2008, and was supported by Japanese Elderly Leukemia and Lymphoma Study Group. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Boards of the core members’ hospitals. The pts with CLL and CLL-like disease were registered. Precursor leukemia and adult T-cell leukemia/lymphoma were excluded. The smears of peripheral blood and bone marrow of all the registered pts were examined by the Diagnosis Committee by using the 4th edition of WHO classification and FAB classification. FAB classification defines typical CLL as prolymphocytes≤10% and atypical CLL including CLL/PLL as 11%≤prolymphocyte<55% and CLL mixed as prolymphocytes<10% and pleomorphic lymphocyte>15%. Mantle cell lymphoma was excluded by means of cytogenetics, fluorescent in situ hybridization of CCND1-IGH, and immunohistochemistry of CCND1. Results:One hundred and thirty-one evaluable pts were registered. There were 96 pts with B-CLL, 2 SLL, 1 B-PLL, 3 hairy cell leukemia, 16 leukemic phase of other B-cell lymphomas. Thirteen pts had NK or T cell leukemia/lymphoma with 7 T-LGL, 4 T-PLL, 1 NK-LGL, and 1 Sezary syndrome. Among B-CLL, there were 62 pts with typical morphology (typical CLL), 18 with atypical morphology (atypical CLL), 16 with intermediate morphology between typical and atypical CLL (intermediate CLL). The differences among 3 subgroups are shown in Table. No differences were found between the 3 morphological subgroups for age, sex, stage (modified Rai and Binet classification), hemoglobin level, platelet counts, the presence of hepatomegaly, splenomegaly and lympoadenopathy, ECOG-PS and the rate of treatment requirement. However, the incidence of CD5 and CD23 expression was higher in typical CLL than in atypical/intermediate morphology groups (P<0.001), i.e. 7% of the pts with atypical CLL showed co-expression of CD5 and CD23. The overall survival (OS) of typical CLL pts was lower than that of intermediate or atypical CLL pts (Figure). Conclusion:Morphological atypia was more frequent in Japanese B-CLL pts, partly because of the low incidence of typical CLL. The atypical/intermediate CLLs were associated with atypical immunophenotype (absence of CD5 and/or CD23) and favorable prognosis. Prospective study is ongoing to evaluate other prognositic factors including the implication of IgVH gene mutation status and ZAP-70 expression in Japan.TableTypicalIntermediateAtypicalPPatients No.621618Age, median6765670.50Sex (male/female)1.301.292.60.47CD20+98.4%100%88.9%0.16CD5+98.4%81.30%77.8%0.003CD5+23+86.5%50.0%7.0%<0.001No treatment45%50%61%0.49Outcome(died)29.8%0%12.5%0.018 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Concurrent chronic lymphocytic leukemia and prolymphocytic leukemia derived from two separate B‐cell clones

An 82-year-old man presented with leukocytosis and splenomegaly. His blood counts showed a white cell count of 134 × 109/l, hemoglobin 96 g/l, and platelet count 86 × 109/l. The blood film showed two distinct populations of lymphocytes, morphologically, 60% typical CLL cells and 40% large prolymphocytes (Image 1). Patient's spleen was palpable 14 cm below the left costal margin. There were mildly enlarged cervical and axillary lymph nodes up to 2 cm but no evidence of localized lymphoma. Blood film shows two populations of CLL and PLL cells. Flow cytometry analysis showed 90% B lymphocytes. Although all the B cells had lambda light chain restriction, they were heterogeneous for CD5 and CD23 expression, including the CD5+/CD23+ B cells typical of CLL phenotype, and the cells lacking CD5 and CD23 consistent with a PLL population. Clonality analysis was performed with use of multiplex PCR (polymerase chain reaction) assays for IgH rearrangement. With this assay, a clonal B-cell population gives rise to PCR product identical in size and independent clones can be distinguished by unique size of the PCR products. The patient's blood sample showed a pattern of two clones of B lymphocytes (Image 2), indicating the two independent clones of CLL and PLL cells. PCR clonality analysis is based on IgH gene rearrangement and two peaks of PCR products indicate two independent clones of B cells. The patient was treated initially with chlorambucil 2 mg daily with a poor response. His white cell count rose quickly to 240 × 109/l in 5 months. He required transfusion for worsening anemia. Further treatment with fludarabine and cyclophosphamide was tried and discontinued due to poor intolerance. This patient presented with a mixed population of CLL and PLL. The genetic relationship between CLL and PLL may be revealed by immunoglobulin isotypes or immunoglobulin gene rearrangements [1]. Similar to diffuse large B-cell lymphoma developing in CLL (Richter syndrome), B-cell PLL may arise from clonal progression of CLL or a second malignancy unrelated to CLL [1-3]. It is likely that this patient had an indolent and undiagnosed CLL, but the duration of CLL before the emerging PLL is unclear. Nonetheless, the finding of clonality analysis indicates PLL derived from an independent clone to that of CLL.

Progressive but Previously Untreated CLL Patients with Greater Array CGH Complexity Exhibit A Less Durable Response to Chemoimmunotherapy

AbstractAbstract 2406The outcome for a given CLL patient is difficult to predict. While there are promising models, they require collation of multiple clinical and laboratory parameters, and it remains to be seen whether they will apply to typical CLL patients in the community. To further dissect out explanations for this dramatic clinical heterogeneity, we sought to understand genomic complexity of clonal B-cells as a possible explanation of clinical variability with specific application to genomic complexity as a predictor of therapeutic response and clinical outcome in CLL. Thus we wished to identified global gains and losses of genetic material in order to define copy-number abnormalities (CNA) in 48 clinically progressive CLL patients who were about to be treated on a chemoimmunotherapy protocol. This protocol was previously reported by us (Blood. 109:2007) and had an induction phase with pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and then responding patients were followed ever three months until relapse. In order to estimate CNA, we employed array-based comparative genomic hybridization (aCGH) using a one-million oligonucleotide probe array format on the leukemic B-cells from the 48 patients entering this trial. In those same patients, the aCGH data were compared to a) FISH detecxtable data using a panel for the common recurring genetic defects seen in CLL and b) to their clinical outcome on this trial. With aCGH we found that 288 CNA were identified (median of 4 per patient; range 0–32) of which 215 were deletions and 73 were gains. The aCGH method identified most of the FISH detected abnormalities with a complete concordance for 17p13.1- deletion (17p-) between aCGH and FISH. We also identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. We found that CLL patients with ≥15 CNA had a significantly worse progression free survival (PFS) than patients with <15 CNA (p=0.004)(figure). Patients with ≥15 CNA also had a shorter duration of response than those with <15 CNA (p=0.0726). Of interest, more complex genomic features were found both in patients with a 17p13.1 deletion and in more favorable genetic subtypes such as 13q14.1. Thus, for 5 patients with >15 CNAs the following FISH patterns were seen: +12/13q14.1-x1/13q14.1 -x2, 13q14.1 ×1 (n=2), and 17p13.1 (n=2). In addition, a 17p- by FISH was positively associated with the number of CNA and total deletion size. The odds of having an overall response decreased by 28% (95% CI: 5–55%; p=0.015) with each additional CNA for the 17p13.1- patients. In addition to defining genomic complexity as the total number of CNA for each patient, we also defined complexity as the sum of the lengths of all interstitial chromosomal gains and losses. When defined as the total size of chromosomal gains or losses, genomic complexity was significantly associated with 17p13.1 and worse overall clinical response. In summary, this analysis utilized the global assessment of copy number abnormalities using a high-resolution aCGH platform for clinically progressive CLL patients prior to initiation of their treatment. One outcome was that we found higher genomic complexity was associated with shorter progression-free survival, reduced duration of response and predicted a poor response to treatment. In addition since we did find genomic complexity in more traditionally favorable FISH categories, such as 13q14.1 type defects, this may explain why some of the latter patients do not fare as well as might be expected even with aggressive chemoimmunotherapy approaches. This study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. [Display omitted] Disclosures:Off Label Use: Pentostatin. Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.

Family-Associated Monoclonal B Lymphocytosis Shows Differences From CLL That Suggest An Indolent Biology.

Abstract 1241Poster Board I-263 Background and SignificanceMonoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts >1.9 × 109 / L, progress to CLL, “low count” MBL (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL. MethodsIndividuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL. ResultsFifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IGVH genes were 3-07 (3 MBL clones), 3-15 (3), and 4-34 (3). No VH1 family gene rearrangements were observed. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12. ConclusionsOur data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL. DisclosuresNo relevant conflicts of interest to declare.

Monoclonal B Cell Lymphocytosis Exhibits Biologic Diversity Similar to Chronic Lymphocytic Leukemia.

Background: Monoclonal B cell lymphocytosis (MBL) is a pre-clinical syndrome characterized by small accumulations of monoclonal B lymphocytes in the peripheral blood. MBL have an immunophenotype similar to CLL: CD5+, CD19+, CD20lo, CD23+, CD79blo, sIglo. The biologic characteristics and clinical implications of MBL remain unclear. We hypothesized that MBL have a biology similar to CLL and represent a pre-neoplastic state of CLL. Characterization and longitudinal evaluation of MBL may provide insight into mechanisms of CLL leukemogenesis.Methods: We defined MBL as a population of CD5+, CD20lo cells that were kappa/lambda restricted and comprised at least 2% of the CD19+ peripheral B cell compartment. Persons with MBL were ascertained by flow cytometric screening of peripheral blood from unaffected members of CLL kindreds. Flow cytometric analysis and fluorescence-activated cell sorting (FACS) were used to determine the surface immunophenotype and molecular characteristics of MBL. MBL cells were bulk-sorted and analyzed using FISH for loci associated with clinical CLL [13q14.3 (D13S319), 17p13.1 (TP53), 11q22.3 (ATM), and 12p11.1-q11 (enumeration probe)]. Single MBL cells were sorted and genomic DNA analyzed by PCR to determine immunoglobulin heavy and light chain sequences.Results: Twelve out of 113 (11%) unaffected family members were found to have MBL. Seven of these individuals provided additional blood for MBL characterization. All 7 MBL subjects had normal complete blood counts and leukocyte differentials. Six of the 7 MBL subjects had MBL counts less than 150 cells/μL. All MBL samples had immunophenotypes similar to CLL. One MBL sample was CD38+/Zap70+, one was CD38+/Zap70−, one was CD38−/Zap70+, and 4 samples were CD38−/Zap70−. The ratio of median fluorescence intensity of CD69:CD71 was low (<1.0) in 6 of the 7 MBL samples, but was 2.8 in the one CD38+/Zap70+ case, consistent with an activated membrane surface phenotype. Next, MBL cells were sorted as single cells and genomic DNA was amplified for direct sequencing of immunoglobulin heavy and light chains. This analysis has been completed in 4 of 7 individuals. Immunoglobulin heavy chain variable (IgVH) region gene usage mirrored that of typical CLL and included examples of IgVH mutated and unmutated sequences. Some MBL were monoclonal, some consisted of related antigen-driven oligoclonal populations, and others consisted of unrelated oligoclonal populations. For example, one person showed three unique clones: a mutated VH4-59 clone (38% of all MBL cells), a mutated VH4-34 clone (5%), and an unrelated unmutated VH4-34 clone (57%). FISH analyses showed mono- or biallelic deletion of 13q14 in four of seven samples. No other loci were deleted or amplified in these 7 MBL subjects. Four individuals provided a second sample of blood for longitudinal MBL analysis. The size of the MBL clone expanded in 3 of these subjects and was unchanged in the 4th subject.Conclusions: This cohort of familial MBL recapitulated much of the biologic diversity of clinical CLL. By several different phenotypic measures, the MBL phenotype was similar to typical CLL. To our knowledge, this is the first report of CD38+, Zap70+, and IgVH unmutated MBL. In contrast to most CLL, we found that many MBL consisted of more than one clonal population. Likewise, we identified evidence of ongoing antigen-driven intraclonal diversification in MBL.

Monoclonal B Cell Lymphocytosis as a Marker of Inherited Predisposition to Chronic Lymphocytic Leukemia.

Background: Segregation analyses suggest that CLL has one of the highest inherited risks among all types of cancer. However, no common germline mutations that confer an inherited predisposition to CLL have been found. Identification of a CLL predisposition gene should lead to improved prevention and treatment strategies. Gene identification studies to date have been limited by few affected patients per family, late age of disease onset, and probable genetic heterogeneity. Identification of a phenotypic marker for carriers of a CLL predisposition gene would dramatically improve the likelihood of finding a causative gene. Monoclonal B-cells with typical CLL immunophenotype (monoclonal B-cell lymphocytosis, MBL) can be identified in normal relatives of patients with overt CLL. We hypothesize that MBL identify unaffected carriers of a CLL predisposition gene.Methods: We used 6-color flow cytometric methods to analyze peripheral blood samples for MBL. We defined MBL as a population of CD5+, CD20lo cells that were kappa/lambda restricted and comprised at least 2% of the CD19+ peripheral B cell compartment. We identified extended families segregating 2 or more members with CLL from patients receiving care at Duke and the Durham VA. We collected peripheral blood from both affected and unaffected family members for complete blood count, flow cytometry for MBL detection, and DNA purification. Genotyping studies were performed using PCR to amplify highly polymorphic short tandem repeat sequences (STR) at candidate loci.Results: We ascertained 16 extended families with CLL and obtained samples from 138 family members. Twelve of the 113 (11%) unaffected participating family members had MBL. In one family, evaluation of 32 members revealed 3 with overt CLL and 5 with MBL. Two distinct inheritance patterns emerge upon review of the families ascertained and analyzed to date. Five families suggested a dominant mode of inheritance of the combined CLL/MBL phenotype: multiple family members with MBL, vertical transmission, and an approximate 50% affection rate for among persons over age 40. In these 5 families, 11 of 64 (17%) unaffected family members had MBL while in the other 11 families no unaffected family members had MBL (0 of 48). In some of the latter 11 families, analysis of obligate carriers of a CLL predisposition gene showed no MBL. We pooled the 5 families with MBL for screening of candidate loci associated with an inherited predisposition to CLL from previously published linkage studies. Using markers at these candidate loci, we excluded linkage to 5q22.1, 6p22, 9q21.33, 10q24.1, 11q13.1, 13q22.2, and 14q32.2.Conclusions: A combined MBL/CLL phenotype appears to be inherited as a dominant trait in some cases of familial CLL. This supports the hypothesis of genetic heterogeneity in the development of CLL. A familial analysis of both CLL and MBL greatly increases the statistical power for genetic analysis, and may allow for families to be pooled for genetic analysis based on the MBL phenotype. Gene identification studies are ongoing.

Defining the 10 Year Risk of Disease Progression in Stage A0 CLL.

80% of patients with CLL present with a lymphocytosis detected on a routine blood count. Many studies have identified risk factors for disease progression but there remains uncertainty about the magnitude of risk and which factors are most useful in determining this risk. To address this we have studied a cohort of 122 stage A0 patients who presented with a lymphocytosis and typical CLL immunophenotype before 1996 and whose CLL has either progressed or remained stable over a 10 year period. All patients were reviewed at least annually. Disease progression was defined as the need for anti-leukemic therapy, an increase in stage or a downward trend in haemoglobin or platelet count, progressive lymphadenopathy or splenomegaly or constitutional symptoms. Karyotypic data, from analysis of G banded metaphases derived fromTPA stimulated lymphocyte cultures, were available on all cases at diagnosis. CD38 and ZAP 70 expression were measured on DMSO frozen cells. VH gene usage and mutational status, and telomere length were assessed on frozen DNA samples, while interphase FISH for del 13q14, del 11q and 17p (ATM and p53 loss) and trisomy 12, was undertaken on fixed cell suspensions. All samples had been collected and stored at, or close to the time of diagnosis.50 patients had progressive disease. Of the 72 patients with stable disease, the lymphocyte count remained below 20x109/l in 53 and rose with a lymphocyte doubling time of > 1 year in the remainding 19 (termed slowly progressive). In univariate analysis, a lymphocyte count of > 20x109/l, unmutated VH genes, high expression of CD38 (cut off 30%) and ZAP70, del 11q, abnormal presenting karyotype and short telomere length all predicted for disease progression. There was no difference in VH gene usage between the progressive and stable groups. Among the 107 patients for whom lymphocyte count, VH gene mutational status, CD38 and ZAP 70 expression are all currently available, the number of poor risk factors in the progressive and stable groups is shown in Table 1. There was no difference in the number of risk factors between the stable and slowly progressive groups. Del 11q was found in 9 progressive cases all of whom had 2 or more risk factors. P53 loss was found in a single patient with stable disease and no risk factors.20/122 patients presented with a lymphocyte count < 5.0x109/l, below which patients have been considered to have monoclonal B cell lymphocytosis. Interestingly, 5 of these developed progressive disease. Using only the lymphocyte count, CD38 and ZAP 70 expression, Table 2 shows the risk of disease progression according to the risk factors present.In summary, once ZAP 70 measurement by flow cytometry is standardized, readily available prognostic tests can provide a quantitative estimate of the risk of disease progression. Performing interphase FISH for 11q and p53 loss in poorer risk cases only may provide additional prognostic information.Table 1No. of adverse factors at presentation. (%)01234GroupProgressive11(25)10(23)11(25)9(20)3(7)Stable/Slowly progressive44(70)16(25)3(5)00Table 2Odds of Progression% of Patients ProgressingNo risk factors0.216.9ZAP70 only0.7442.7Lymphs>200.8947.0CD38 only1.5761.1ZAP70+lymphs3.2476.4ZAP70+CD385.7585.2CD38+lymphs6.8587.3All25.196.2

The t(14;19)(q32;q13) Translocation in B Lymphoproliferative Disorders: A Continuum from Chronic Lymphocytic Leukemia (CLL) to Marginal Zone Lymphoma (MZL)?.

Translocation t(14;19)(q32;q13) juxtaposing BCL3 in 19q13 with IGH in 14q32, is a rare recurrent event found in patients with B-cell malignancies. So far, a few cases of well-documented B-cell neoplasm have been reported. We analyzed 34 patients with t(14;19) and 1 patient with a variant t(2;19)(p11;q13) collected by the Groupe Francophone de Cytogenetique Hematologique on the basis of cytogenetic abnormality. Clinico-biological data, morphological review, immunophenotyping with Matutes' score, conventional karyotype and FISH analysis with BCL3, IGH, CEP12, 13q14, ATM, TP53, 6q21 probes, and IgVH mutational status were recorded. The sex ratio was 22M/13F, the median age at diagnosis was 61 [39–89], the lymphocyte count was > 4×109/l in 96% of patients, and spleen enlargement was found in 47%. 31% (11/35) were morphologically classified CLL, 37% (13/35) atypical CLL, 20% (7/35) MZL; 79% had features of disease progression; 3 of the 4 latter were Diffuse Large B Cell Lymphoma, possibly transformed from MZL. 87% were CD5+, 72% had a Matutes' score < 3. The IgVH genes were unmutated in 9/11 cases. The time to treatment was < 1 year in 68% of patients. The BCL3 locus involvement was confirmed by FISH analysis in all cases. 46% of cases showed complex karyotype. The chromosomal abnormalities associated with t(14;19) were +12 (57%), 6q- (27%), +3 (15%), 11q- (15%), 13q- (13%), 17p- (12%), +18 (12%), 7q- (12%). Comparison with published cytogenetic CLL data shows that deletion 6q was frequent and deletion 13q uncommon. Trisomies 3 and 18, and 7q deletion are less common than in published MZL. The independent analysis of our series of CLL/atypical CLL and MZL gave the same tendency. The chromosomal abnormalities associated with t(14;19) are not specific, but their frequencies are between those of typical CLL and MZL suggesting an intermediary status between the 2 malignancies. The t(14;19) identifies a subgroup of B-disorders CD5+ and Matutes' score < 3, which could be of poor prognosis, based on progressive disease and unmutated status.