Typical B-CLL Research Articles

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.

Automated pattern-guided principal component analysis vs expert-based immunophenotypic classification of B-cell chronic lymphoproliferative disorders: a step forward in the standardization of clinical immunophenotyping.

Immunophenotypic characterization of B-cell chronic lymphoproliferative disorders (B-CLPD) is becoming increasingly complex due to usage of progressively larger panels of reagents and a high number of World Health Organization (WHO) entities. Typically, data analysis is performed separately for each stained aliquot of a sample; subsequently, an expert interprets the overall immunophenotypic profile (IP) of neoplastic B-cells and assigns it to specific diagnostic categories. We constructed a principal component analysis (PCA)-based tool to guide immunophenotypic classification of B-CLPD. Three reference groups of immunophenotypic data files—B-cell chronic lymphocytic leukemias (B-CLL; n=10), mantle cell (MCL; n=10) and follicular lymphomas (FL; n=10)—were built. Subsequently, each of the 175 cases studied was evaluated and assigned to either one of the three reference groups or to none of them (other B-CLPD). Most cases (89%) were correctly assigned to their corresponding WHO diagnostic group with overall positive and negative predictive values of 89 and 96%, respectively. The efficiency of the PCA-based approach was particularly high among typical B-CLL, MCL and FL vs other B-CLPD cases. In summary, PCA-guided immunophenotypic classification of B-CLPD is a promising tool for standardized interpretation of tumor IP, their classification into well-defined entities and comprehensive evaluation of antibody panels.

Syndrome de Richter de type pseudohodgkinien. À propos de deux cas et revue de la littérature

We report the clinical and immunohistological features of two cases of chronic lymphocytic leukaemia (CLL) with Hodgkin's transformation. These cases occurred in a 70-year-old man with a three-year history of CLL and in a 76-year-old man with a few months history of CLL. Microscopic examination showed the presence of large tumor cells with the morphological and immunophenotypic features of classical Hodgkin and Reed-Sternberg (R-S) cells, in a background of otherwise typical B-CLL. The transformation of CLL into large B cell lymphoma (Richter's syndrome) is a well-documented phenomenon. Only rarely does CLL transform into Hodgkin's lymphoma, but this diagnosis is often easy and offers few differential diagnoses. The major points of interest lie in the pathogenetic relationship between CLL and Hodgkin's disease, and in the potential clinical implications of this peculiar condition. Literature on the subject indicates that identical IgH gene rearrangements in micromanipulated R-S and CLL cells have been identified in 7/12 cases. In these patients, the R-S and CLL cells belong to the same clonal population, suggesting a progression from the underlying CLL cells. This group appears to have a poor prognosis, identical to classical Richter's syndrome. In other cases, the R-S cells were often Epstein-Barr virus (EBV) positive and did not share the clonal rearrangements identified in CLL cells, suggesting that Hodgkin's disease in these patients could represent a second malignancy, EBV-related and favored by immunosuppression, associated with a better prognosis.

Unsupervised immunophenotypic profiling of chronic lymphocytic leukemia

Proteomics and functional genomics have revolutionized approaches to disease classification. Like proteomics, flow cytometry (FCM) assesses concurrent expression of many proteins, with the advantage of using intact cells that may be differentially selected during analysis. However, FCM has generally been used for incremental marker validation or construction of predictive models based on known patterns, rather than as a tool for unsupervised class discovery. We undertook a retrospective analysis of clinical FCM data to assess the feasibility of a cell-based proteomic approach to FCM by unsupervised cluster analysis. Multicolor FCM data on peripheral blood (PB) and bone marrow (BM) lymphocytes from 140 consecutive patients with B-cell chronic lymphoproliferative disorders (LPDs), including 81 chronic lymphocytic leukemia (CLLs), were studied. Expression was normalized for CD19 totals, and recorded for 10 additional B-cell markers. Data were subjected to hierarchical cluster analysis using complete linkage by Pearson's correlation. Analysis of CLL in PB samples (n = 63) discovered three major clusters. One cluster (14 patients) was skewed toward "atypical" CLL and was characterized by high CD20, CD22, FMC7, and light chain, and low CD23. The remaining two clusters consisted almost entirely (48/49) of cases recorded as typical BCLL. The smaller "typical" BCLL cluster differed from the larger cluster by high CD38 (P = 0.001), low CD20 (P = 0.001), and low CD23 (P = 0.016). These two typical BCLL clusters showed a trend toward a difference in survival (P = 0.1090). Statistically significant cluster stability was demonstrated by expanding the dataset to include BM samples, and by using a method of random sampling with replacement. This study supports the concept that unsupervised immunophenotypic profiling of FCM data can yield reproducible subtypes of lymphoma/chronic leukemia. Expanded studies are warranted in the use of FCM as an unsupervised class discovery tool, akin to other proteomic methods, rather than as a validation tool.

Bi-Allelic Deletion of 13q14 Is Associated with Inferior Progression Free Survival Compared to Mono-Allelic 13q14 Deletion in B-Cell Chronic Lymphocytic Leukaemia.

Isolated mono-allelic deletion of chromosome region 13q14 is seen in 40–60% of patients with typical B-CLL and is associated with good risk disease. Bi-allelic deletion of 13q14 region has an overall incidence of 4–5% in typical B-CLL. It is not known if loss of both allelic regions impacts on disease progression and survival. FISH analysis was performed on 995 patients with CLL. 451 cases were assessed using 13q14 PAC probes 933-e9 and 273–2 (Dr Stilgenbauer, Ulm University, Germany) and 540 cases were assessed with the B-CLL probe set (α12/13q14/13q34 and p53/ATM probe sets, Abbott/Vysis).Normal results for 13q14 were obtained in 501 (50.4%) cases, 450 (45.2%) had a mono-allelic deletion of 13q14 and 44 (4.4%) cases had a bi-allelic deletion of 13q14. Control probes (13q34 region) had normal patterns in all cases tested. Detailed analysis was carried out to test the hypothesis that loss of both allelic regions conferred a poorer prognosis by comparing the 44 cases with bi-allelic deletion of to 162 cases with mono-allelic deletion good risk CLL. Cases with bi-allelic deletion have similar clinical characteristics compared to cases with a mono-allelic deletion. There was no significant difference in Binet stage between patients with a mono-allelic (84% A, 7% B, 9% C) or a bi-allelic deletion of 13q14 (73% A, 13% B, 13% C). However, Binet stage was significantly predicative of outcome independent of the cytogenetic abnormality. Immunophenotypes were compared using the dChip program. Unsupervised hierarchical cluster analysis showed that mono-allelic and bi-allelic deletion 13q14 CLL cases have different protein expression profiles. CD5 and CD38 were more strongly expressed (1.23 fold increase, p = .06 and 2.36 fold increase, p = .02, repsectively), whereas CD22 and CD23 had lower levels of expression (1.61 fold decrease, p = <.001 and 1.75 fold decrease, p = <.001 respectively) in cases with bi-allelic deletion compared to mono-allelic deletion. 12% of cases with bi-allelic deletion also have sub-populations of cells with mono-allelic 13q14 deletions. 29% of cases with bi-allelic deletion (5.8% p53; 11.7% ATM; 13.5% +12) had additional cytogenetic abnormalities compared to just 6.5% of cases with mono-allelic deletion (4.5% p53; 1.1% ATM; 0.6% +12)(p = <.001). The overall survival between patients with a bi-allelic deletion compared to a mono-allelic deletion was not significant but median follow-up is relatively short (25 months). Patients with a bi-allelic deletion were significantly more likely to have disease progression and require treatment (median progression free survival (PFS) 12 months) (p = <.001) compared to patients with a mono-allelic deletion (median PFS 26 months) (progression was defined as time to first theraputic intervention; 58% of cases with mono-allelic deletion have not required treatment and 40% of cases with bi-allelic deletion have not required treatment to date). PFS remained significant when cases with deletions of ATM &p53 and +12 were excluded from analysis suggesting that the poor PFS is due to the bi-allelic deletion of 13q14.In conclusion, we have demonstrated that bi-allelic 13q14 deletion occurs in approximately 5% of B-CLL patients and that it is associated with an inferior progression free survival. The altered immunophenotypic profile associated with bi-allelic 13q14 deletion is intrigueing and further study of this group is warranted.

Analysis of Genetic Lesions in Atypical B-CLL by Pangenomic CGH-Arrays.

Among B-CLPD, characterized primarily by morphology and expression of cell surface markers, is is important to identify patients with CD5+ atypical B-CLL that is regarded as clinically more aggressive than typical B-CLL. However these cases are not well defined. To better characterize the genetic lesion observed in atypical B-CLL we have analysed a cohort of 40 patients by CGH microarrays.Study was made on 5 typical B-CLL and on 35 atypical B-CLL patients with either CD20dim or bright expression that do not express cyclin D1. All the patients with atypical B-CLL will be defined as the presence of an absolute B-CD5-positive lymphocytosis > 4 x 109/l and a RMH score < 4. DNA was extracted using QuiAmp kit according to manufacturer recommendations. 2μg of DNA was used in each hybridization experiment. DNA labeling was performed using Cy3dUTP and Cy5 dUTP respectively for control and tumoral sample respectively. CGH-arrays was performed using the 1 Mb Human genome micro-array from “arraygenomics” that contains 3400 BAC clones fully FISH mapped and end sequenced all printed in triplicate. Each experiment was made using two slides in dye swap method. Cy5 and Cy3 fluorescence intensities spot were quantified using Axon Scanner 4100 and Acuity Software. Data were imported into SpectralWare 2.0 software and Normalise Suite, version 2.0, Profiler from Squire lab.Results: Identification of known and previously uncharacterized copy number alterations (CNAs) in the a-B-CLL cells genome was made in all the cases. The CGH profiles revealed that a-B-CLL genome is highly rearranged harbouring large numbers of distinct copy-number aberrations (75 CNAs among 31 chromosomal regions were found). Some of these CNAs are recurrent across different samples, allowing the definition of minimal common regions (MCR) of amplification or deletion. The size of the CNAs was extremely variable from one Bac probe to complete chromosome gains or losses. Specifically, our dataset included the known gains of chromosome 12 (14 cases), and the known deletion at 11q23, 13q14.3, 17p region but also new region of interest as +3p, 3q22 to 3qter, 4pter to 4q35.2, 5p15, 6p25.3 to 6p22, 8q22 to 8q24, 15q15.3 to 15q26, 17q11 to 17qter, +18 and +19 for gains and 1p35,1, 1p33, 2q22.3, 3p26.3 to 3p21.3, 5q34, 6p25.3, 6q16, 6q25.3 to 6q27, 7q31.3 to 7q32.2, 8p23.3 to 8p12, 10q11.2 to 10q21.1, 10q21.3, 10q23.2 to 10q24, 11q22.3 to 11q24.2, 15q14 to 15q21, 16p11.2 to 16q21, 21q22.1 for the deletions.To further corroborate the above finding, we had performed conventional FISH analysis using known probes for del 13q, del 11q, trisomy 12, del 17p and correlate our results with conventional cytogenetic findings when they were available. In all the cases CGH-arrays findings were confirmed by fish analysis or karyotype. The above findings were confirmed also in few cases using the Agilent's Human Genome CGH Microarray 44K that contains over 40,000 probes. Our first results confirmed BAC arrays results. CGH-arrays appears to be very informative to detect lesion in B-CLPD and show the high frequency of genetic lesions in a-CLL. The biological impact of this lesions by transcriptome analysis on the same sample and the prognosis impact is in progress.

In vitro chemo-sensitivity of B-chronic lymphocytic leukaemia to ara-G

In vitro sensitivity to ara-G of peripheral blood B-CLL lymphocytes from 23 patients was assessed using the flow cytometric TdT assay. All patients had typical B-CLL, according to immunophenotype and morphology, and none had received treatment within 1 year of testing. A wide range of spontaneous apoptosis was recorded. Exposure of the cells to a concentration of ara-G, comparable to plasma levels achieved in a phase I trial, produced significant increases in the rate of apoptosis in 21 out of 23 patients. Prior treatment, stage or lymphocyte doubling time did not influence the effect of ara-G. Ten patients’ samples tested, in parallel, for sensitivity to fludarabine demonstrated a good correlation of response to both drugs. This in vitro evidence of activity against B-CLL suggests that the spectrum of clinically useful action may be broader than previously demonstrated.

Cytogenetic findings in a patient presenting simultaneously with chronic lymphocytic leukemia and acute myeloid leukemia

A case of simultaneous presentation of B-chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) is described. CLL was documented by bone marrow and peripheral blood lymphocytosis with a typical B-CLL immunophenotype. The diagnosis of AML was supported by the presence of bone marrow and circulating blast cells positive for myeloperoxidase and myeloid-associated markers. Although the immunophenotyping and morphocytochemical studies indicated two different cell populations (mature B-CLL lymphocytes and myeloblasts), chromosome aberrations commonly associated with CLL and AML were found simultaneously in the same metaphases obtained from unstimulated 24-hour cultures of peripheral blood cells.

Over-expression of cyclin D1 in chronic B-cell malignancies with abnormality of chromosome 11q13.

Accurate identification of B-cell chronic malignancies is sometimes uncertain, despite careful cytologic and immunophenotypic evaluation. Cytogenetics and molecular biology studies may therefore prove useful, because some of these disorders are associated with non-random abnormalities, such as the t(11;14)(q13;q32) translocation and bcl-1 rearrangement mainly observed in mantle-cell lymphoma (MCL). We studied the expression of cyclin D1 in malignant lymphoid cells from the peripheral blood of 32 patients with various B-cell chronic lymphoproliferative disorders, using Northern blot (NB) and RNA in situ hybridization (ISH). Cytogenetic analysis was informative in 18 cases, and most of the missing karyotype data were from typical B-CLL cases where a t(11;14) is unlikely to be found. Over-expression of cyclin D1 mRNA was observed by both NB and ISH in four samples (MCL; two cases; lymphoplasmacytic lymphoma: one case, unclassified B-cell chronic disorder: one case). In each of these cases there was an abnormality of chromosome 11q13, either a t(11;14)(q13;q32) translocation (three cases) or a del(11)(q13) without evidence of chromosome 14 involvement (one case). Cytogenetic and gene rearrangement studies are not available in all institutions and have some technical pitfalls. Because of its close association with bcl-1 rearrangement and/or t(11;14), the demonstration of cyclin D1 mRNA over-expression either by NB, or, more conveniently, by ISH, may represent additional information which could be of help for the identification of B-cell malignancies.

Phorbol ester-induced hairy cell features on chronic lymphocytic leukemia cells in vitro.

Leukemic cells from eight patients with chronic lymphocytic leukemia were isolated and cultured in the continuous presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) at a concentration of 1.6 x 10(-9) M for 4-10 days. Aliquots of cells were then analyzed at intervals of 24-72 hr for changes in morphology, acid phosphatase staining (AP), and expression of two hairy cell-associated surface antigens, HCL1 (CD22, Leu 14) and HCL3 (CD11c, Leu M5). All cases studied showed typical B-CLL phenotype, and only a small proportion of cells expressed CD22 and CD11c (mean 7% and 4.9%, respectively). TPA treatment induced the coexpression of CD22 (mean 49%) and CD11c (mean 48%) and tartrate-resistant acid phosphatase in seven of eight cases. Morphologically, cells in TPA cultures expressed hairy cell features that were evident in light and electron microscopic studies. Collectively these changes indicate that TPA can induce hairy cell features on CLL cells in vitro, suggesting the later maturational stage of HCL compared with CLL.

Non-radioactive in situ hybridization for the detection and monitoring of trisomy 12 in B-cell chronic lymphocytic leukaemia.

Non-radioactive in situ hybridization (NISH) with a chromosome 12-specific alpha satellite probe was performed on 20 patients with chronic lymphocytic leukaemia (CLL) with normal karyotype (15 cases) or with inadequate mitotic yield (5 cases) from mitogen-stimulated cultures. All patients had over 70% lymphocytes coexpressing the CD5/CD23 antigens. While less than 1% interphase nuclei showed three fluorescent spots in 16/20 patients, evidence of trisomy 12 in 15-25% interphase cells was detected in four patients. According to the FAB classification the diagnosis in these patients was typical B-CLL, stage III (Rai's staging system) in one case, CLL/PLL, stage II and III in two cases, PLL, stage III in one case. In order to confirm these results, NISH was repeated after 1 month in one patient and after 2 years in three patients. All patients had been treated with chemotherapy in the period between the two NISH experiments. In all cases a 1.8-3-fold increase of percentage of trisomic interphase cells was detected. These findings suggest that in B-CLL clones with trisomy 12 may have proliferative advantage over clonal B-lymphocyte without +12 and, possibly, that they may be more resistant to chemotherapy. We conclude that NISH is a sensitive technique allowing for the detection and monitoring of trisomy 12 in a fraction of B-CLL patients with normal karyotype or with no analysable mitoses despite employment of polyclonal B-cell mitogens.