Cytogenetic Analysis of Normal Human B Cells Following CpG Stimulation: Implications for Interpretation of CpG Induced CLL Metaphase Analysis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3124-3124
Author(s):  
Xiaosheng Wu ◽  
Grzegorz S. Nowakowski ◽  
Stephanie A. Smoley ◽  
Bonnie A. Arendt ◽  
Mark A. Peterson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Clinical Utility ◽  
Chromosomal Abnormalities ◽  
Karyotype Analysis ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Cytogenetic Abnormalities ◽  
Trisomy 12 ◽  
Clonal Abnormalities

Abstract BACKGROUND: Karyotype analysis of metaphase cells for discovering genome-wide cytogenetic abnormalities has played an important role in prognosis and counseling of patients with chronic lymphocytic leukemia (CLL). Due to the low proliferative rate of CLL B cells, karyotype analysis has been of limited clinical utility in this disease. Thus, fluorescence in situ hybridization (FISH) on interphase cells has replaced karyotype analysis for the assessment of recurrent genetic changes in CLL. Recently, immunostimulatory CpG-oligodeoxynucleotides (CpG) have been shown to induce primary CLL B cell proliferation via engagement of toll-like receptor 9 (TLR9), thereby rendering karyotype analysis feasible and providing for a useful and reliable method to discover new cytogenetic abnormalities in CLL B cells. However, we and others have shown that CpG stimulation of CLL and normal B cells also induces robust expression of activation induced cytidine deaminase (AID), a mutagenic enzyme that is linked to the development of cytogenetic abnormalities. It is therefore plausible that cytogenetic abnormalities observed following CpG stimulation of CLL B cells may not reflect de novo abnormalities but rather reflect CpG induced-AID mediated cytogenetic alterations. We began to address this possibility by assessing the effects of CpG stimulation on the karyotypes of normal blood B cells. HYPOTHESIS: Since CpG stimulation induces AID expression in CLL and normal B cells, we hypothesize that: robust B cell activation by CpG could result in cytogenetic abnormalities thereby contributing to lymphomagenesis; and if CpG induces cytogenetic abnormalities that are linked to disease initiation and/or progression, such information would be of great value in the interpretation of cytogenetic data obtained from CpG-stimulated CLL B cells. METHODS: We performed interphase FISH analysis on freshly isolated blood B cells, and metaphase karyotype analysis on CpG stimulated B cells from 17 healthy blood donors. Pure (>98%) B cells obtained by negative selection, were hybridized at isolation with a probe set to identify deletion 6q, 11q, 13q, and 17p, trisomy 12, and IGH gene rearrangements, and cells were also stimulated for 5 days with 10 μg/ml CpG (CpG 2006, synthesized in-house) plus IL-2 and IL-15 for metaphase analysis. At least 20 post-CpG metaphase spreads were analyzed for each sample and abnormalities were considered clonal using the accepted convention (gains or translocations are present in two or more cells and loss present in at least 3 cells). The expression of AID in the B cells pre- and post-CpG stimulation was assessed by RT-PCR. RESULTS: FISH analysis of freshly isolated B cells showed that all 17 subjects were normal. After CpG stimulation, all samples examined exhibited strong AID expression as revealed by RT-PCR. By karyotype analysis, none of the 17 CpG stimulated samples showed evidence of clonal abnormalities. However, one or more nonclonal chromosomal abnormalities were observed in 7 out of 17 (41.2%) samples. Among those 7 samples, 4 samples had 1 abnormal metaphase out of a minimum of 20 examined while the other 3 samples exhibited two or more cells, each with distinct abnormalities. Some of those nonclonal abnormalities have also been observed in CLL (i.e., trisomy 3, trisomy 12 and 14q32 rearrangements). This is a much higher incidence of nonclonal abnormalities than has been seen with peripheral blood cells cultured by other methods. CONCLUSION: Our data have significance for the biology of CLL and for clinical practice. First, our data suggest that signaling through TLR9, and perhaps other receptors that likewise induce robust B cell AID expression, may represent a natural mechanism by which B lineage cells acquire chromosomal abnormalities during normal immune responses that may predispose toward neoplastic transformation. Second, given that none of the CpG-induced metaphases in normal B cells resulted in clonal abnormalities in the 5 day timeframe, our data also validate the clinical utility of CpG as a valuable tool for the cytogenetic analysis of CLL B cells since only clonal abnormalities are currently scored. However, it remains possible that non-clonal abnormalities seen in CpG-stimulated CLL B cells may be relevant to the disease process if future work shows that CpG stimulation uncovers recurring non-clonal defects of chromosomes/genes associated with tumorigenic events.

Cytogenetic Abnormalities In a Cohort of 171 Patients with Waldenström Macroglobulinemia Before Treatment: Clinical and Biological Correlations

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 801-801 ◽  
Author(s):  
Florence Nguyen-Khac ◽  
Julie Lejeune ◽  
Elise Chapiro ◽  
Aurore Grelier ◽  
Sarah Mould ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Protein Concentration ◽  
Chromosomal Abnormalities ◽  
Response Duration ◽  
Lower Percentage ◽  
Time To Progression ◽  
Advisory Committees ◽  
Cytogenetic Abnormalities ◽  
Trisomy 12

Abstract Abstract 801 The genetic bases of Waldenström Macroglobulinemia (WM) are poorly understood. Because of the difficulty in obtaining tumor metaphases for karyotype studies, few recurrent chromosomal abnormalities have been reported in WM. We have studied a cohort of 171 untreated WM patients, enrolled in a prospective randomized trial from the French Cooperative Group on Chronic Lymphocytic Leukemia and Waldenstrom Macroglobulinemia (FCG-CLL/WM) comparing the efficacy of fludarabine to that of chlorambucil, by conventional cytogenetic (CC) and Fluorescence in situ hybridization (FISH). CC was systematically performed on bone marrow or peripheral blood, and FISH analysis carried out using 8 probes CEP4, CEP12, 13q14, 11q22 (ATM), 17p13 (TP53), IGH, BCL2 Abbott, 6q21 Q-Biogene, on metaphases and interphase nuclei. The sex ratio was 2.1M/1F, the average age at inclusion was 66.9 years [40-89]. The mean percentage of lymphoplasmacytic cells was 53% [8-97]. Out of 140/171 successful CC, 65 (46.4%) showed clonal abnormalities. Out of 65 abnormal CC, 19 (29.2%) were complex, with at least 3 chromosomal changes, and 22 (33.8%) showed translocations (balanced and unbalanced). Using CC and FISH, we observed 29/132 (22%) 6q deletion, 18/141 (12.8%) 13q14 deletion, 9/80 (11.2%) trisomy 18, 11/135 (8.1%) TP53 deletion, 10/133 (7.5%) trisomy 4, 10/132 (7.6%) ATM deletion, 4/118(3.4%) IGH rearrangement, and 4/136 (2,9%)trisomy 12. Chromosomal abnormalities were compared to adverse characteristics described by Morel et al (ISSWM, Blood 2009,113(8),4163-70): advanced age (> 65 years), hemoglobin < 11.5g/dl, platelet count < 100×109/l, b2-microglobulin > 3 mg/l, and serum monoclonal protein concentration > 7g/dl. Patients with 6q deletion had significantly more frequently albumin < 3.5g/dl (15/29 (51.7%) vs 24/103 (23.3%), p=0.005), b2microglobuline > 3 mg/l (20/29 (68.9%) vs 48/103 (46.6%), p=0.04). Similarly, patients with trisomy 4 had significantly more frequently b2microglobuline > 3 mg/l (9/10 (90%) vs 59/123 (47.9%), p=0.02). Of note, all patients with trisomy 4 had M-protein concentration > 2 g/dl (10/10 (100%) vs 73/123 (59.3%), p=0.02). Finally, there were significantly more patients with age > 65 years, when ATM deletion was observed (9/10 (90%) vs 65/122 (53.2%), p=0.04). Cytogenetic abnormalities did not influence the response rate but in multivariate analysis, TP53 deletion was associated with a shorter time to progression (15months (m) versus 35m, p=0.0007) and 6q deletion with a longer time to progression (55m versus 24m, p=0.04) in responding patients. Cytogenetic abnormalities in WM differ from those commonly reported in other B-cell neoplasms and confirm the originality of this disease. The 6q deletion is frequent compared to chronic lymphocytic leukaemia (CLL) or marginal zone lymphoma (MZL) and 13q14 deletion is rare compared to CLL. In our series trisomy 12 is rare compared to atypical-CLL and MZL. Involvement of the IGH locus, which is frequent in multiple myeloma or lymphoplasmocytic lymphoma, is rare in WM. Finally trisomy 4 is present in WM but not reported in other B-cell malignancies. The 6q deletion is the most frequent reported cytogenetic abnormality in WM. We found 22% cases with deletions of 6q21, a lower percentage compared with the literature [38-54%].Conversely to previous publications, 6q deletion was associated with a longer response duration to treatment and did not influence the overall survival. These discrepancies could be explained by the heterogeneity of the previous published series, mixing untreated patients and treated patients in various ways. In our trial, all patients were analyzed before randomization. As in CLL patients, TP53 deletion seems to be associated with a shorter response duration. However, regarding the small numbers of patients with cytogenetic abnormalities, these results must be confirmed in larger series. Disclosures: Leblond: ROCHE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MUNDIPHARMA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Deletions of Chromosomes 6q and 13q, and Trisomy 4 Are the Most Common Cytogenetic Abnormalities in Waldenstrom Macroglobulinemia. Preliminary Results of a Multicentric Study.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1771-1771
Author(s):  
Florence Nguyen-Khac ◽  
Elise Chapiro ◽  
Sarah Mould ◽  
Carole Barin ◽  
Agnes Daudignon ◽  
...  
Keyword(s):  
B Cell ◽  
Recent Observation ◽  
Partial Trisomy ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Multicentric Study ◽  
Cytogenetic Abnormalities ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Trisomy 12

Abstract The genetic bases of Waldenstrom Macroglobulinemia (WM) are poorly understood. We have studied a cohort of 139 untreated WM patients, enrolled in a prospective randomized trial from the French Cooperative Group on Chronic Lymphocytic Leukemia and Waldenstrom Macroglobulinemia (FCG-CLL/WM), by conventional cytogenetic (CC) and Fluorescence in situ hybridization (FISH). The sex ratio was 2.2M/1F, the average age at inclusion was 66 years [40–85]. The mean percentage of lymphoplasmacytic cells was 53% [8–97]. CC was systematically performed on bone marrow or peripheral blood, and FISH analysis carried out using 7 probes CEP4, CEP12, 13q14, 11q22 ( ATM), 17p13 (TP53), IGH Abbott, 6q21 Q-Biogene, on metaphases and interphase nuclei. Out of 110/137 successful karyotypes, 37% showed clonal abnormalities (9 additional cases had numerical changes of sexual chromosomes). Among abnormal karyotypes, there were 14 (34%) 6q deletions, 5 (12%) trisomy 4/partial trisomy 4, 4 (10%) trisomy 18, 3 (7%) trisomy 12. Using FISH, deletions of 6q21 were observed in 23/86 cases (27%), 13q14 in 12/89 cases (13%), TP53 9/85 cases (11%), ATM 6/81 cases (7%). Trisomy 4 was present in 9/82 cases (11%), and trisomy 12 in 4/86 cases (5%). No rearrangement of IGH was observed in the first 27 analyzed cases. The 6q deletion is the most frequent reported cytogenetic abnormality in WM. We found 27% with deletions of 6q21 (FISH), a low percentage compared to the literature [39–54%]. This could be explained either by the difference in the probe used or by the absence of selection of lymphoplasmacytic cells before cytogenetic analyses. Interestingly, we confirmed our recent observation that trisomy 4 was frequent in WM (12% if partial trisomy 4 was included). Furthermore we observed in this large series a frequent 13q14 deletion (13%). In conclusion, cytogenetic abnormalities in WM differ from those commonly reported in other B-cell neoplasms and confirm the originality of this disease. 6q deletion is frequent compared to CLL or marginal zone lymphoma (MZL) and 13q14 deletion is rare compared to CLL. In our series trisomy 12 is rare compared to atypical-CLL and MZL. We didn’t observe cytogenetic involvement of the IGH locus, which is frequent in multiple myeloma or lymphoplasmocytic lymphoma. Finally trisomy 4 is present in WM but not reported in other B-cell malignancies. Searches for correlations with clinical and other biological parameters are ongoing.

Karyotype Results From CpG Oligodeoxynucleotide Stimulated Chronic Lymphocytic Leukemia (CLL) Cultures Are Consistent Among Laboratories: a CLL Research Consortium (CRC) Study.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1614-1614
Author(s):  
Nyla A. Heerema ◽  
John C. Byrd ◽  
Paola Dal Cin ◽  
Marie L Dell' Aquila ◽  
Prasad Koduru ◽  
...  
Keyword(s):  
B Cell ◽  
Prospective Trial ◽  
Pokeweed Mitogen ◽  
Fish Analysis ◽  
Cytogenetic Abnormalities ◽  
Interphase Fish ◽  
Cpg Oligodeoxynucleotide ◽  
Research Consortium ◽  
Clonal Abnormalities

Abstract Abstract 1614 Poster Board I-640 Background Cytogenetic abnormalities in B-cell CLL are important prognostic indicators for predicting disease progression and treatment response. Until recently, only interphase cytogenetics has been readily applicable to clinical use in CLL due to the inability of traditional mitogens to promote effective metaphase cytogenetic identification. Recently, cytokine or CpG oligonucleotide stimulation has been utilized to promote efficient metaphase analysis. One concern with this approach is the actual clonality and reproducibility of such clones when examined by different cytogenetic laboratories. The CLL Research Consortium (CRC) conducted a prospective trial to test (1) whether CpG would enhance detection of abnormal clones and (2) whether the CRC cytogenetic laboratories would achieve similar results when testing the same patient samples. Methods Initially, one laboratory compared stimulation of CLL cells using CpG versus pokeweed mitogen (PWM) + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the same samples. To test the reproducibility of CpG stimulation, fresh blood samples from 1 normal control and 12 CLL patients were collected at one site and distributed (blinded to phenotype and prior cytogenetic results) to 5 CRC cytogenetic laboratories where they were cultured for 3 days using CpG stimulation, harvested, banded and analyzed utilizing standard laboratory procedures. When possible, each laboratory analyzed 20 metaphases per case. Interphase FISH analysis for CLL (including probes for chromosomes 6q, 11q, 12, 13q, 17p, and IGH/CCND1) was also done on each sample. Results In the PWM+TPA versus CpG comparison, more cases were found to have clonal abnormalities with CpG stimulation (147 of 229, 64%) than with PWM+TPA (110 of 229, 48%; P=0.0005). All clonal abnormalities detected in the PWM+TPA cultures were also observed in the CpG cultures. In the 5-laboratory CpG comparison, the results were compiled and compared to the concurrent FISH results. The CpG karyotypes were concordant with the FISH results. The control case had a normal CpG-stimulated karyotype in all 5 laboratories. Three cases exhibited a complex karyotype; one of these also exhibited 11q-, one had 17p-, and one had neither. The cytogenetic descriptions varied modestly among labs, but all identified the complex clones. Every case including the control exhibited some nonclonal abnormal cells, but no pattern was detected among the 5 labs. When an abnormal clone was found by only 1 or 2 labs, it was present in only a very small number of cells. A small 13q- clone was observed in 2 cases in 1 and 2 labs (3 of 20 and 5 of 50 cells, respectively), and was confirmed by interphase FISH. A single lab found 2 of 30 cells with a t(12;15) in one case. Conclusions Abnormal clones in CLL are more readily detected with CpG stimulation than with traditional B-cell mitogens. The clonal abnormalities revealed by CpG stimulation are consistent with the “gold standard” interphase FISH results, and are reproducible among different cytogenetic laboratories. We have no evidence that CpG oligonucleotides create new clonal B-cell populations in vitro during 3 days of cell culture. CpG stimulation in vitro reveals clonal cytogenetic abnormalities and complexity in CLL that should be evaluated as potential independent prognostic parameters. Disclosures Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Association between the proliferative rate of neoplastic B cells, their maturation stage, and underlying cytogenetic abnormalities in B-cell chronic lymphoproliferative disorders: analysis of a series of 432 patients

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 5130-5141 ◽  
Author(s):  
Sandra Quijano ◽  
Antonio López ◽  
Ana Rasillo ◽  
Susana Barrena ◽  
Maria Luz Sánchez ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoproliferative Disorders ◽  
Lymphocytic Leukemia ◽  
Maturation Stage ◽  
Cytogenetic Abnormalities ◽  
M Phase ◽  
The Impact

Abstract Limited knowledge exists about the impact of specific genetic abnormalities on the proliferation of neoplastic B cells from chronic lymphoproliferative disorders (B-CLPDs). Here we analyze the impact of cytogenetic abnormalities on the proliferation of neoplastic B cells in 432 B-CLPD patients, grouped according to diagnosis and site of sampling, versus their normal counterparts. Overall, proliferation of neoplastic B cells highly varied among the different B-CLPD subtypes, the greatest numbers of proliferating cells being identified in diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Compared with normal B cells, neoplastic B-CLPD cells showed significantly increased S + G2/M-phase values in mantle cell lymphoma (MCL), B-chronic lymphocytic leukemia (B-CLL), BL, and some DLBCL cases. Conversely, decreased proliferation was observed in follicular lymphoma, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM), and some DLBCL patients; hairy cell leukemia, splenic marginal zone, and MALT-lymphoma patients showed S + G2/M phase values similar to normal mature B lymphocytes from LN. Interestingly, in B-CLL and MCL significantly higher percentages of S + G2/M cells were detected in BM versus PB and in LN versus BM and PB samples, respectively. In turn, presence of 14q32.3 gene rearrangements and DNA aneuploidy, was associated with a higher percentage of S + G2/M-phase cells among LPL/WM and B-CLL cases, respectively.

Gene Expression Profiling of Peripheral T-Cell Lymphoma (PTCL, NOS) and Comparison to Diffuse Large B-Cell Lymphoma (DLBCL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2277-2277
Author(s):  
Daruka Mahadevan ◽  
Catherine Spier ◽  
Kimiko Della Croce ◽  
Susan Miller ◽  
Benjamin George ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lymph Node ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Real Time ◽  
Expression Profiling ◽  
Cell Lymphoma ◽  
Rt Pcr

Abstract Background: WHO classifies NHL into B (~85%) and T (~15%) cell subtypes. Of the T-cell NHL, peripheral T-cell NHL (PTCL, NOS) comprises ~6–10% with an inferior response and survival to chemotherapy compared to DLBCL. Gene Expression Profiling (GEP) of DLBCL has provided molecular signatures that define 3 subclasses with distinct survival rates. The current study analyzed transcript profiling in PTCL (NOS) and compared and contrasted it to GEP of DLBCL. Methods : Snap frozen samples of 5 patients with PTCL (NOS) and 4 patients with DLBCL were analyzed utilizing the HG-U133A 2.0 Affymetrix array (~18,400 transcripts, 22,000 probe sets) after isolating and purifying total RNA (Qiagen, RNAeasy). The control RNA samples were isolated from normal peripheral blood (PB) B-cell (AllCell, CA), normal PB T-cell (AllCell, CA) and normal lymph node (LN). Immunohisto-chemistry (IHC) confirmed tumor lineage and quantitative real time RT-PCR was performed on selected genes to validate the microarray study. The GEP data were processed and analyzed utilizing Affymetrix MAS 5.0 and GeneSpring 5.0 software. Our data were analyzed in the light of the published GEP of DLBCL (lymphochip and affymtrix) and the validated 10 prognostic genes (by IHC and real time RT-PCR). Results : Data are represented as “robust” increases or decreases of relative gene expression common to all 5 PTCL or 4 DLBCL patients respectively. The table shows the 5 most over-expressed genes in PTCL or DLBCL compared to normal T-cell (NT), B-cell (NB) and lymph node (LN). PTCL vs NT PTCL vs LN DLVCL vs NB DLBCL vs LN COL1A1 CHI3L1 CCL18 CCL18 CCL18 CCL18 VNN1 IGJ CXCL13 CCL5 UBD VNN1 IGFBP7 SH2D1A LYZ CD52 RARRES1 NKG7 CCL5 MAP4K1 Of the top 20 increases, 3 genes were common to PTCL and DLBCL when compared to normal T and B cells, while 11 were common when compared to normal LN. Comparison of genes common to normal B-cell and LN Vs DLBCL or PTCL and normal T-cell and LN Vs PTCL or DLBCL identified sets of genes that are commonly and differentially expressed in PTCL and/or DLBCL. The 4 DLBCL patients analyzed express 3 of 10 prognostic genes compared to normal B-cells and 7 of 10 prognostic genes compared to normal LN and fall into the non-germinal center subtype. Quantitative real time RT-PCR on 10 functionally distinct common over-expressed genes in the 5 PTCL (NOS) patients (Lumican, CCL18, CD14, CD54, CD106, CD163, α-PDGFR, HCK, ABCA1 and Tumor endothelial marker 6) validated the microarray data. Conclusions: GEP of PTCL (NOS) and DLBCL in combination with quantitative real time RT-PCR and IHC have identified a ‘molecular signature’ for PTCL and DLBCL based on a comparison to normal (B-cell, T-cell and LN) tissue. The categorization of the GEP based on the six hallmarks of cancer identifies a ‘tumor profile signature’ for PTCL and DLBCL and a number of novel targets for therapeutic intervention.

B- Prolymphocytic Leukemia Shows Heterogeneous IgVH Mutational Status and Expression of ZAP-70 and CD38.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2004-2004
Author(s):  
Ilaria Del Giudice ◽  
Zadie Davis ◽  
Nnenna Osuji ◽  
Nilima Parry-Jones ◽  
Estella Matutes ◽  
...  
Keyword(s):  
Overall Survival ◽  
B Cell ◽  
Peripheral Blood ◽  
Fish Analysis ◽  
Laboratory Findings ◽  
Prolymphocytic Leukemia ◽  
Mutational Status ◽  
Number Of Patients ◽  
Trisomy 12 ◽  
Igvh Mutational Status

Abstract B-cell prolymphocytic leukemia (B-PLL) is a rare lymphoid leukemia with an aggressive course. Diagnosis is based on morphology (>55% peripheral blood prolymphocytes), immunophenotype and histology when available, as there is no cytogenetic hallmark for this disease. There are scanty data regarding the mutational status of the variable region of immunoglobulin heavy chain (IgVH) genes and ZAP-70 expression in B-PLL. Davi et al (Blood1996;88:970–6) showed that 9 of 11 B-PLL cases had mutated IgVH genes (<98% homology) with a preferential use of the V3-23 and V4-34 genes, accounting for more than half of the B-PLL repertoire.We analyzed the mutational status of IgVH genes and ZAP-70 expression in 16 B-PLL cases and correlated the findings with clinical and biological features such as CD38 expression, chromosome abnormalities detected by FISH and overall survival. There were 7 females and 9 males, with a median age of 71 years (range 42–91). All except one were untreated at the time of the study. The diagnosis was established by peripheral blood morphology and immunophenotype (CLL score ≤ 3) in all cases and bone marrow and spleen histology in 6. Mantle cell lymphoma was excluded by the absence of t(11;14) by FISH in 12 out of 12 cases tested, including 4 which were CD5 positive. IgVH mutational status was performed by PCR (cut off >98% homology) and ZAP-70 expression was evaluated by 4-colour flow cytometry. Seven of 13 cases (54%) in which IgVH mutational status was evaluable, showed unmutated IgVH genes (99.7–100% homology) and 6 (46%) mutated IgVH genes (90.1–97.4% homology). V3-23 and V4-34 genes were used in one third of the cases. ZAP-70 was expressed (≥ 20% CD19+ cells) in 7 of 10 evaluable cases and CD38 (≥ 30% of CD19+ cells) in 7 of 11 cases. FISH analysis showed delp53 in 6/13 (46%) and del(13)(q14) in 3/11 (27%) cases; trisomy 12 was absent in 7 cases tested. Correlation between IgVH status and other features is shown in the table. There was a prevalence of delp53 (83%) among the unmutated group, while ZAP-70 expression did not correlate with IgVH mutational status. Median overall survival was 41 months. Six patients (4 mutated, 2 unmutated) are alive at 50 months (range 8-112) from diagnosis, 9 (5 unmutated and 2 mutated) died at 17 months (range 0–55) and 1 was lost to follow-up at 4 months. The number of patients is too small to conclude whether IgVH mutational status and/or ZAP-70 expression have a significant impact on survival. In summary, B-PLL is heterogeneous with respect to IgVH mutational status as in most chronic B-cell disorders, with equal representation of unmutated and mutated cases. P53 deletion is more common in the unmutated subgroup and most of the cases express ZAP-70 and CD38. Laboratory findings according to mutational status ZAP (>/=20%) CD38 (>/=30%) Del p53 Del (13)(q14) Trisomy 12 Unmutated (7) 4/5 1/4 5/6 2/5 0/3 Mutated (6) 2/3 4/5 1/5 0/4 0/4

Analysis of B-CLL with Discordant ZAP-70 Expression and IgVH Mutational Status.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1194-1194 ◽  
Author(s):  
Remi Letestu ◽  
Magali Le Garff-Tavernier ◽  
Dominique Vaur ◽  
Michel Ticchioni ◽  
Fanny Baran-Marszak ◽  
...  
Keyword(s):  
B Cells ◽  
Somatic Mutations ◽  
Staining Procedure ◽  
Fish Analysis ◽  
Prognostic Information ◽  
Proliferation Markers ◽  
Cd38 Expression ◽  
Mutational Status ◽  
Trisomy 12 ◽  
Igvh Mutational Status

Abstract The clinical course of CLL is heterogeneous and presence or absence of IgVH somatic mutations has been correlated with stable and evolutive disease respectively. ZAP-70 protein expression was shown to be associated with unmutated IgVH genes in CLL and was proposed as a surrogate for mutational status. Recent studies of large number of cases have shown various percentage of discrepancy with respect to ZAP-70 expression and IgVH mutational status. As aggressive disease is not always associated with unmutated IgVH genes we aimed at better characterize the ZAP-70 discordant cases by determining the other prognostic factors such as cytogenetics, expression of CD38 and proliferation markers (thymidine kinase and sCD23). We investigated 292 patients with previously untreated B-CLL. Although several antiZAP-70 antibodies adapted to flow cytometry (FCM) are commercially available, staining procedure and interpretation of the results still remain controversial. Therefore, ZAP-70 expression was determined in the four participating centers by the same FCM method using the 2F3.2 mAb with indirect staining, and expression of the results was standardized. Moreover, ZAP-70 expression was investigated by RQ-PCR in 62 cases and by Western blot in 61 further cases on isolated B cells. The results obtained with these techniques were correlated with FCM data. ZAP-70 was found expressed in 141 cases, among which 38 cases (27%) exhibited ≥2% somatic mutations. Conversely, among the 151 ZAP-70 negative cases, only 6 cases (4%) were found unmutated (≥ 98% VH homology). We focused on the characteristics of the mutated ZAP-70 positive cases. Only 26/38 were in stage A at diagnosis. Incidence of CD38 expression >10% B cells was low (7/28 cases). Analysis of VH sequences pointed to the frequency of VH3-21 usage in 8/38 cases (21%) as compared to an expected 3% frequency in the French population. FISH analysis identified one case with del11q22.3, one case with del17p and two cases with trisomy 12. Del13q14 was present in half of cases. Proliferation markers were significantly higher in these cases than in ZAP-70 negative mutated cases, even among stage A patients. Follow-up of these patients is still too short for significant event free survival as compared with other groups of patients but the number of evolutive Binet stage A patients and advanced B and C stages was already higher than among the ZAP negative mutated cases. In conclusion, in our hands, ZAP-70 was almost always expressed in unmutated cases (103/109). Among mutated cases, ZAP-70 expression was present in 38/183 cases (21%), and brought prognostic information, independently of CD38 or chromosomal alterations. The correlation between ZAP-70 and proliferation markers suggests that ZAP-70 expression may result in a survival advantage of the malignant cells independently of the mutational status.

Classical and Molecular Cytogenetic Abnormalities in 124 Pediatric Patients with Acute Lymphocyte Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4419-4419
Author(s):  
Yihuan Chai ◽  
Hui Lv ◽  
Jun Lu ◽  
Peifang Xiao ◽  
Jianqing Li
Keyword(s):  
Bone Marrow ◽  
Multiplex Pcr ◽  
Cytogenetic Analysis ◽  
Chromosomal Abnormalities ◽  
Molecular Diagnostic ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Childhood All ◽  
Cytogenetic Abnormalities ◽  
Conventional Cytogenetic Analysis

Abstract In childhood acute lymphocyte leukemia (ALL), cytogenetics plays an important role in diagnosis, allocation of treatment and prognosis. On base of the conventional cytogenetic analysis, molecular methods have inproved our ability to accurately and rapidly risk-stratify patient with childhood ALL in the last few years. Our aim was to assess the demography of cytogenetic abnormalities in childhood ALL. The study sample consisted of 124 newly diagnosed ALL patients younger than 16 years, who were diagnosed at the Department of Pediatric Hematology/Oncology, Soochow University Children’s Hospital. The diagnosis and FAB subtypes of ALL was determined by Wright-Giemsa-stained bone marrow smears and cytochemicalstaining. Immunophenotyping of the bone marrow samples was performed by flow cytometry. Multiplex reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to detect the 29 most common leukemia translocations for routine molecular diagnostic hematopathology practice, and complement the information gained from conventional cytogenetic analysis. Cytogenetic analysis was successful in 112 of 124 children with ALL. Sixty-eight (60%)of them had clonal chromosomal abnormalities. Numerical imbalances consisted of hyperdipoild(>47 chromosomes, 36 cases), hypodipoild(<46 chromosomes, 14 cases), pseudodiploidy(18 cases). Chromosomal translocations were observed in 13 patients by conventional cytogenetic analysis. Three cases were found positive for t (4;11), 3 cases for t (9;22), 1 case for t (1;19) and 6 cases for other rare translocations. RT-PCR analysis detected 116 of the 124 ALL patients. Thirteen cases of TEL-AML1, 10 cases of rearrangement in the MLL gene, 4 cases of E2A-PBX1, 4 cases of E2A-HLF, 3 cases of BCR-ABL, 2 cases of TLS-ERG, 32 cases of HOX11, were detected by RT-PCR in B-lineage leukemias. SIL-TAL1 had been found in 4 of 7 of T-lineage leukemias. Sixty-eight cases of ALL show chromosomal aberrations. Multiplex PCR positivity was detected in 59(50%)of the 116 ALL patients studied. Multiplex PCR combined with chromosome analysis uncovered Chromosomal abnormalities in 95 of 124(77%) of ALL patients and supplemented each other in detecting Chromosomal abnormalities. It provides reliable evidence for the diagnosis, classification and prognosis.

Influence of Chromosomal Aberrations on Response to First Line Therapy in B-Cell Chronic Lymphocytic Leukemia: Interim Analysis of PALG-CLL3 Randomized Study Comparing Cladribine Plus Cyclophosphamide vs Fludarabine Plus Cyclophosphamide.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2046-2046
Author(s):  
Tadeusz Robak ◽  
Jerzy Z. Blonski ◽  
Ewa Wawrzyniak ◽  
Aleksandra Palacz ◽  
Joanna Gora-Tybor ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Chromosomal Aberrations ◽  
Interim Analysis ◽  
Chromosomal Abnormalities ◽  
Complete Response ◽  
Lymphocytic Leukemia ◽  
Lower Probability ◽  
Trisomy 12 ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Impact of cytogenetic abnormalities on treatment with different purine nucleoside analogs in patients (pts) with B-cell chronic lymphocytic leukemia (B-CLL) is largely unknown. One of objectives of PALG-CLL3 trial, comparing cladribine plus cyclophosphamide (CC) with fludarabine plus cyclophosphamide (FC) in previously untreated progressive B-CLL, was to verify the response to treatment in subsets of pts characterized by common cytogenetic abberrations. Chromosomal abnormalities were assessed using fluorescence in situ hybridization (FISH) on interphase nuclei of lymphocytes on whole blood smears prior to the start of the study treatment. Pts were screened for trisomy 12, deletions (del) 11q, del 13q and del 17p using DNA probes: CEP12, LSI: ATM, D13S319 and p53 (Vysis), respectively. For the purpose of the present interim analysis complete cytogenetic results were available in 133 pts out of 423 pts included to the study. In this group the chromosomal aberrations were detected in 102 pts (77%) including single abnormalities observed in 69 pts (52%) and two or more aberrations in 33 pts (25%). Thirty-one pts (23%) exhibited a normal interphase FISH pattern. The most frequent single abnormality was del 13q found in 38 pts (29%), while del 17p, trisomy 12 and del 11q were identified in 14 pts (11%), 11 pts (8%), and 6 pts, (5%), respectively. The most frequently observed associations of chromosomal aberrations were: del 13q with del 11q (11 pts, 8%) and del 13q with del 17p (10 pts, 8%). Four pts (3%) revealed three chromosomal abnormalities including association of trisomy 12/del 11q/del 13q in two pts, trisomy 12/del 11q/del 17p in one pt and del 11q/del 13q/del 17p in one pt. Overall, treatment was completed and response assessed in 113 out of 133 pts with known FISH pattern. In this group of pts del 17p was the only chromosomal abnormality that correlated significantly with treatment outcome. Pts with del 17p (21, 19%) had lower probability to achieve a complete response (CR) (0.044). Interestingly, in independent analyses of both treatment arms, the negative impact of 17p was seen in pts treated with FC (p=0.002), but not in pts treated with CC (p=0.6). Moreover, comparing response rates between treatment arms we found that CC was superior to FC in terms of complete response in pts with del 17p (57% CR in CC v 14% CR in FC arm, p=0.04). In conclusion, chromosomal abnormalities can be detected in majority B-CLL pts requiring treatment. Our preliminary results suggest that CC combination may have some advantage in terms of CR achievement in B-CLL pts harboring del 17p.

Two Distinct Subsets of dic(9;12)(p12;p11.2) among Children with B-Cell Precursor Acute Lymphoblastic Leukemia (ALL): PAX5-ETV6 and ETV6-RUNX1 Rearrangements: A Report from the Children’s Oncology Group.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1439-1439 ◽  
Author(s):  
Julie M. Gastier-Foster ◽  
Andrew J. Carroll ◽  
Denise Ell ◽  
Richard Harvey ◽  
I-Ming Chen ◽  
...  
Keyword(s):  
B Cell ◽  
Gene Rearrangement ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Cytogenetic Abnormality ◽  
Fish Analysis ◽  
Oncology Group ◽  
Rt Pcr ◽  
Cell Precursor ◽  
Pediatric All

Abstract The dic(9;12)(p12;p11.2) has been described as a rare cytogenetic abnormality in pediatric precursor B-cell ALL. Initial studies suggested that the rearrangement is associated with a favorable outcome, and recent studies demonstrated the presence of a PAX5-ETV6 fusion gene was associated with this cytogenetic abnormality. Twenty cases with a cytogenetic dic(9;12) were identified in the Children’s Oncology Group (COG) cytogenetics databases. FISH analysis with the ETV6-RUNX1 (TEL-AML1) probes was done on 12 of these samples. Five cases were positive for fusion, indicating a cryptic t(12;21)(p13;q22), and also had loss of the ETV6 probe from the chromosome 12 not involved in the t(12;21). Seven cases were negative for fusion and had loss of an ETV6 signal, although one of the latter had a diminished ETV6 signal identified. To determine whether both PAX5-ETV6 and ETV6-RUNX1 rearrangements occurred in some patients, a diagnostic sample from each patient was analyzed by RT-PCR for the PAX5-ETV6 and ETV6-RUNX1 fusion genes. Primers from exon 3 of PAX5 and exon 3 of ETV6 were used for the PAX5-ETV6 analysis and from exon 5 of ETV6 and exon 4 of RUNX1 for the ETV6-RUNX1 analysis. Of the 20 cases, only 8 were RT-PCR positive for the PAX5-ETV6 fusion with the above primers; however, an additional 2 were RT-PCR positive with alternate primers, and all 10 of these were negative for the ETV6-RUNX1 fusion by RT-PCR. Of the remaining 10 patients, 9 were RT-PCR positive for the ETV6-RUNX1 fusion, including all of the ETV6-RUNX1 cases positive by FISH. The gene rearrangement associated with the dic(9;12) in these cases is not known. One patient was negative for both fusions by RT-PCR, negative by FISH for ETV6-RUNX1 rearrangement, yet had loss of an ETV6 signal. No cytogenetic differences could be seen between the 2 groups, either in the appearance of the dic(9;12) or in the other abnormalities identified. These results demonstrate the presence of two mutually exclusive dic(9;12) rearrangements in pediatric ALL; one associated with ETV6-RUNX1 rearrangement and one resulting in PAX5-ETV6 fusion. Both PAX5-ETV6 and ETV6-RUNX1 rearrangements are associated with a favorable prognosis. However, molecular analysis of the dic(9;12) patients must be performed to determine whether the dicentric chromosome results in PAX5-ETV6 fusion or whether the case has ETV6-RUNX1 fusion.

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