Molecular Cytogenetics Analysis of 13q14 Biallelic Deletion in Chronic Lymphocytic Leukemia: A Study on 250 Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1454-1454
Author(s):  
Alessandro Gozzetti ◽  
Giulia Papini ◽  
Rosaria Crupi ◽  
Adele Frasconi ◽  
Francesco Forconi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Chromosomal Abnormality ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Strong Association ◽  
Lymphocytic Leukemia ◽  
The Other ◽  
Median Percentage ◽  
Mutational Status ◽  
13Q Deletion

Abstract Abstract 1454 Deletion 13q14 (13q-) detected by fluorescence in situ hybridization (FISH) is the most frequent chromosomal abnormality in chronic lymphocytic leukemia (CLL). When 13q- is detected as sole abnormality has a good prognosis, while aggressive outcome is registered when 13q- is combined with other chromosomal abnormalities such as del 11q or del 17p. A recent study evidenced also that patients with higher percentage of 13q-deleted cells (>70%) are at higher risk for aggressive disease. Some studies evidenced that 13q- deletion size (involving D13S319 +/−Rb1) seems to matter in terms of time to treatment (TTT) and prognosis (OS). Few studies evaluated so far the incidence and prognosis of a biallelic 13q- deletion, i.e. the deletion of both alleles of the minimal deleted region (MDR) of chromosome 13q.In particular prognosis has been reported controversial. We analyzed at single institution 250 CLL patients by FISH in order to evaluate the incidence and prognosis of biallelic 13q- by using probes for D13S319 and RB1 that map to DLEU2/MIR15A/MIR16-1 and RB1 loci. Results were correlated in terms of TTT and OS with IGHV mutational status (mutated vs unmutated), RAI/BINET stage, CD38 positivity and/ or ZAP-70 positivity, beta-2M, LDH, other chromosomal abnormality (+12, del17p, del11q). Deletion 13q was considered present if >10% of nuclei were deleted out of 300 nuclei scored by two different and independent observers. All biallelic cases were confirmed by FISH using a probe for LSI-D13S319 and 13q34 to exclude false positive results. 135/250 (52%) patients presented a monoallelic del 13q. 45/135 (32%) presented a monoallelic del of RB1 while 20/135 (15%) cases presented a biallelic 13q-.12/20 (80%) presented a biallelic 13q- as sole abnormality, while 8/20 presented a 13q- associated with other cytogenetic abnormalities (one 17p-, five 11q-, two +12). The median percentage of 13q-deleted cells was 50% (range 14–86). Median age was 65 (range 50–85), M/F 12/8; 80% of the patients were RAI stage 0–1, while only 10% were RAI stage 4. No differences were seen in patients with biallelic deletion of 13q when LDH, b2M, ZAP-70,IGHV were considered. CD38 was negative in 16/20 patients. Regarding the MDR of chromosome 13q, 11/20 patients presented a biallelic del of D13S319, while 5/20 had a biallelic deletion of RB1; 5/20 patients presented a mono+biallelic del of D13S319 while 3/20 a mono+biallelic deletion of RB1. When we analyzed clinical and biological characteristics comparing patients with biallelic13q-,monoallelic 13q- and with no 13q-, we did not find differences in terms of: stage (RAI-Binet), P=0.2,P=0.9; B2 M, P=0.4; LDH,P=0.1; CD38 positivity, P=0.2; ZAP-70, P=0,1; IGHV M vs UM,P=0.65; P53 mutated vs wild type, P=0.1; del 11q was significantly associated more with biallelic 13q-, P=0.02. TTT and OS were not significantly different between biallelic 13q- patients and the other two groups (164 vs 212 vs 211, P=0.9). 8/20 patients with biallelic 13q- have been treated, all 5 patients carrying also a 11q- received treatment, the other 3 patients had: 1patient RB1 deletion in 92% of cells and 2 deletion 13q- in>75% of cells. In conclusion, biallelic 13q- are present in about 8% of all cases of CLL and in 15% of 13q- patients. A strong association with del 11q was found and this correlated with disease progression and treatment.CD38 was negative in the majority of patients with biallelic 13q-. RB1 was deleted in 32% of 13q- patients. No differences were found in terms of clinical characteristics, TTT and OS with monoallelic 13q-. Disclosures: No relevant conflicts of interest to declare.

Extensive Intraclonal Diversification in a Subgroup of Chronic Lymphocytic Leukemia Patients with Stereotyped IGHV4-34/IGKV2-30 B cell Receptors: Implications for Ongoing Interactions with Antigen.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2337-2337
Author(s):  
Lesley-Ann Sutton ◽  
Efterpi Kostareli ◽  
Anastasia Hadzidimitriou ◽  
Nikos Darzentas ◽  
Athanasios Tsaftaris ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
B Cell Receptors ◽  
Valuable Insight ◽  
Cell Receptors ◽  
Mutational Status ◽  
The Impact

Abstract Abstract 2337 Poster Board II-314 Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen (Ag) recognition through the clonotypic B cell receptors (BCRs). However, it is still unclear whether Ag involvement is restricted to the malignant transformation phase or whether the putative Ag(s) may continuously trigger the CLL clone. Valuable insight into these issues may be gleaned from the study of intraclonal diversification (ID) within the immunoglobulin (IG) genes through ongoing somatic hypermutation (SHM). Definitive data regarding ID within IG genes in CLL remains limited and conflicting. In the present study we systematically explored the presence of ID within IG genes of CLL, not only at cohort level but also in subgroups defined by BCR stereotypy and IG gene mutational status. We thus conducted a large-scale subcloning study of both IG heavy and light variable genes, in a total of 1496 and 1008 subcloned sequences from 71 and 56 CLL cases, respectively. The analysis was intentionally biased to cases expressing IGHV4-34/IGKV2-30 IGs (subset #4) and IGHV3-21/IGLV3-21 IGs (subset #2) that exhibit distinctive, subset-biased SHM patterns. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and at least 14 colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample (ii) confirmed mutation (CM) - a mutation observed more than once among subcloned sequences from the same sample. Analysis of heavy chain sequences revealed that 40% (28/71) of cases carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, whilst 32% (23/71) of cases carried only UCMs. The remaining 28% (20/71) of cases carried sets of identical IGHV-D-J subcloned sequences. Although most cases showed no or low levels of ID, an intense and, likely, functionally driven ID was evident in selected cases, especially those belonging to subset #4. The distinct ID in subset #4 was statistically significant when compared to all other groups defined by IGHV gene usage and mutation status, BCR stereotypy or heavy chain isotype. Subsequent analysis of the clonotypic light chains revealed that the impact of ID was generally low, with the outstanding exception again relating to subset #4. In fact, of 22 IGKV-J rearrangements exhibiting CMs, 11 (50%) utilized the IGKV2-30 gene and notably 10/11 (91%) of these were expressed by subset #4 cases. In such cases, the expressed IGKV2-30 gene was affected by an active and precisely targeted ID, analogous to their partner IGHV4-34 gene. These findings suggest that the SHM mechanism may continuously operate in certain subsets of CLL patients, particularly those cases expressing stereotyped IGHV4-34/IGKV2-30 BCRs typical of subset #4. In such cases, the observed ID patterns attest to the very precise targeting of the SHM process and may be considered as evidence for a “stereotyped response” to an active, ongoing interaction with Ag(s). Disclosures: No relevant conflicts of interest to declare.

Analysis of Stereotyped IGHV Distribution In a Series of 1133 Chronic Lymphocytic Leukemia Patients: The Experience of a Multicenter Italian Study Group

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2423-2423
Author(s):  
Francesco Maura ◽  
Giovanna Cutrona ◽  
Massimo Gentile ◽  
Serena Matis ◽  
Monica Colombo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Pairwise Alignment ◽  
Immunoglobulin Heavy Chain ◽  
Lymphocytic Leukemia ◽  
Study Group ◽  
Data Set ◽  
Italian Study ◽  
Mutational Status

Abstract Abstract 2423 Chronic lymphocytic leukemia (CLL) is characterized by an extremely variable clinical course. Mutational status of the immunoglobulin heavy-chain variable (IGHV) region defines two disease subsets with different prognosis. A fraction of CLL cases carries highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3). We performed sequence analysis to characterize IGHV regions in a panel of 1133 CLL patients investigated by a multicenter Italian study group. A total of 1148 rearrangements were identified; the analysis of stereotyped subsets was performed based on previously reported criteria (Messmer et al, J Exp Med 2004; Stamatopuolos et al, Blood 2007). Specifically, we compared all our sequences with those found in three different publicly available data sets (Stamatopoulos et al, Blood 2007; Murray et al, Blood 2008 and Rossi et al, 2009 Clin Cancer Res). In addition, a pairwise alignment within all sequences was performed in order to discover novel potential subsets (HCDR3 identity > 60%). Based on the 2% cut-off used to discriminate between Mutated (M) and Unmutated (UM) cases, 777 sequences (67.59%) were classified as M, while 371 sequences (32.3%) as UM. The most represented IGHV genes within mutated cases were IGHV4-34 (104/118) and IGHV3-23 (85/96), whereas IGHV1-69 (97/112) was the most frequently used in the UM group. Interestingly, the IGHV3-21 gene, reported to be frequently expressed in CLL patients from Northern Europe, was present in only a small fraction of cases (24; 2.07%), confirming a previous finding reported by Ghia et al (Blood 2005) in a smaller panel. In our series, stereotyped HCDR3 sequences were found in 407/1148 (35.45%) patients, 177 of whom were M and 230 were UM cases. Overall, we observed that stereotyped sequences were significantly associated with UM IGHV status (Fisher's exact test, P<0.0001). Among the 407 stereotyped HCDR3 sequences, 345 belong to the clusters reported by Murray et al and 14 to those described by Rossi et al., 2009 Clin Cancer Res. The most frequent stereotyped subsets identified in our panel were #1 (35 cases), #7 (28 cases), #4 (24 cases), #3 and #9 (16 cases), #28 (13 cases), and #2 (12 cases), together with subsets #5, #8, #10, #12, #13, #16 and #22 (all ranging from 6 to 9 cases). Finally, we were able to identify by auto-matching analysis 48 sequences potentially specific for 23 novel putative stereotype subsets. In our series we identified 407/1148 (35.45%) stereotyped HCDR3 sequences. The percentage was higher than that reported by Stamatopoulos et al and Murray et al. This discrepancy may partially be due to the different approach used in our analysis, namely the matching to a general data set including all published stereotyped subsets instead of the auto-matching performed by those Authors. We demonstrated a significant association between IGHV status and stereotyped sequences and confirmed the finding that #1 is the most frequent subset identified so far. Finally, we were able to identify a series of 23 novel putative subsets that will require further confirmation. Disclosures: No relevant conflicts of interest to declare.

Vegf and bFGF Single-Nucleotide Polymorphisms In Chronic Lymphocytic Leukemia: Impact On Susceptibility

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5287-5287
Author(s):  
Sandra Ballester ◽  
Begoña Pineda ◽  
Eduardo Tormo ◽  
Blanca Navarro ◽  
Ariadna Perez ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Lymphocytic Leukemia ◽  
P Value ◽  
Nucleotide Polymorphisms ◽  
Exact Test ◽  
Cd38 Expression ◽  
Mutational Status ◽  
The Individual

Abstract Background B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical outcome. Recent studies have identified a number of different molecular prognostic markers (including mutational status of the IgVH gene, ZAP70 and CD38 expression) that allow to discriminate patients in prognostic subgroups. However, different expression patterns of angiogenic factors as VEGF, VEGFR1 and bFGF have been related with B-CLL susceptibility and treatment requirements. We have analyzed the polymorphisms: -710 C/T in VEGFR1, rs1109324, rs1547651, rs3025039 (936C/T) and rs833052 in VEGF and rs1449683 (223 C/T) in bFGF in order to determine the possible association with susceptibility in B-CLL. Methods Peripheral blood samples from 230 B-CLL patients and 476 healthy controls were genotyped using probes TaqMan SNP Genotyping Assays. Samples were providing from the Hospital Clinic of Valencia. Four SNPs in the VEGF gene, one SNP in the bFGF gene and one SNP in the VEGFR1 gene were evaluated. Statistical analysis was performed using SNPStats program (Catalan Institute of Oncology) and Fisher's exact test was applied to evaluate the significance. Results We have observed an increased frequency of the T allele in the rs1449683 SNP [OR 1.62 (95% CI: 0.98-2.66) p-value =0.063] and in the rs1547651 SNP [OR 0.72 (95% CI: 0.51-1.03), p-value=0.072] in our B-LLC patients when compared to control subjects. Moreover we observed that T allele carriers of rs3025039 (VEGF) have a significant protective effect concerning this disease [OR 0.59 (95% CI: 0.39-0.89) p-value=0.009]. Conclusion Our data indicate an increased frequency of the T allele in polymorphisms rs1449683 (bFGF) and rs1547651 (VEGF) in the group of patients, which possibly account for the individual susceptibility to develop B-CLL. On the other hand the data provided suggest that the T allele of VEGF rs3025039 is likely important genetic marker of susceptibility to B-CLL. Further studies regarding the role of pro-angiogenic markers in B-CLL would be beneficial to help elucidate pathogenic pathways in this disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 98% IGHV Gene Identity Is Still the Optimal Cutoff to Predict the Prognosis of Chronic Lymphocytic Leukemia Patients in China

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5545-5545
Author(s):  
Yi Xia ◽  
Ke Shi ◽  
Qian Sun ◽  
Chun Qiao ◽  
Huayuan Zhu ◽  
...  
Keyword(s):  
Prognostic Factor ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Study Cohort ◽  
Optimal Cutoff ◽  
Entire Cohort ◽  
Mutational Status ◽  
Significant Difference

Abstract Background: Immunoglobulin heavy chain variable region (IGHV) has been an important prognostic factor for chronic lymphocytic leukemia (CLL) for decades. 98% being a cut-off value for IGHV is a mathematical choice and researches on the best cut-off value have never been stopped. Chinese CLL patients are known to differ from Caucasian CLL patients on both clinical and genetical features. However, the optimal cutoff for IGHV mutational status has not yet been studied in this particular ethnic group. Method: We carried out a study on 595 Chinese CLL patients in order to find out whether 98% is the best cut-off value for IGHV in Chinese CLL patients. Genomic DNA from peripheral blood or bone marrow was subjected to PCR amplification following the IGH Somatic Hypermutation Assay v2.0 protocol (InVivoScribe). Sequences were aligned to ImMunoGeneTics/V-QUEry and Standardization (IMGT/-VQUEST) database. Result: 600 sequences were received after IGHV rearrangement sequencing. IGHV3-23, IGHV4-34, IGHV3-7, IGHV4-39 and IGHV1-69 were the most frequently used IGHV genes. 352 (58.7%) cases were IGHV-mutated while 248 (41.3%) cases were IGHV-unmutated if the classical 98% classification by ERIC was used. In order to determine the optimal cut-off value, we used 1% as the interval to divide the entire cohort into 7 groups according to the mutational rate, which were <95%, 95%-95.99%, 96%-96.99%, 97%-97.99%, 98%-98.99%, 99%-99.99% and 100% respectively. Binet A patients had a relatively indolent course of disease and cases with different IGHV mutational rates had no significant differences in time to first treatment (TTFT) apart from truly unmutated (100%) cases. For the whole study cohort, significant difference appeared at 98% interval (P<0.001 and P=0.005 for TTFT and OS respectively) while intervals less than 98% had no significant difference compared with the <95% group. Similarly, there was no clear dissimilarities among 98%, 99% and 100% intervals (Table 1a and b). All the other prognostic factors including del(17p), del(11q), TP53 mutation, MYD88 mutation, NOTCH1 mutation, SF3B1 mutation, CD38, ZAP-70, Binet staging, gender, β2-microglobulin and EBV-DNA were differently distributed between group <98% and group ³98%, but not among subgroups in ³98%. In multivariate analysis, the 98% IGHV was also an independent prognostic factor for TTFT and OS. Conclusion: 98% is the optimal cutoff value for IGHV mutational status to predict the prognosis of CLL patients in China. Table 1. Table 1. Disclosures No relevant conflicts of interest to declare.

SNP-Arrays: a Routine Cytogenetic Tool In Chronic Lymphocytic Leukemia ?.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4611-4611
Author(s):  
Alban Godon ◽  
Franck Genevieve ◽  
Malgorzata Truchan-Graczyk ◽  
Laurence Baranger ◽  
Virginie Eclache ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
Lymphocytic Leukemia ◽  
Primary Data ◽  
Assay Sensitivity ◽  
Snp Arrays ◽  
Median Size ◽  
Trisomy 12

Abstract Abstract 4611 Background: Chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia in Western countries, and is characterized by a highly variable clinical course. Interphase fluorescent in situ hybridization (I-FISH) has been able to identify chromosomal abnormalities in ~80% of B-CLL, including deletions at 13q, 11q, 17p and trisomy 12, which has proven to be prognostic indicators for disease progression and survival. Although recent immunostimulatory methods have substantially improved analysis via conventional metaphase cytogenetics (CC), detection of chromosome changes is limited by the low mitotic activity of CLL cells in vitro. High-density single nucleotide polymorphism (SNP) arrays are commercially available technologies, which allow genome-wide detection of allelic copy number gains or losses, and loss-of-heterozygosity (LOH) regions. Aims: To assess whether SNP-arrays are more sensitive than I-FISH and CC to detect specific chromosomal abnormalities associated with prognosis in B-CLL, i.e. del13q, del11q, del17p and tri12. Methods: Blood samples from 24 patients with B-CLL at diagnosis were tested in parallel by I-FISH, CC and SNP-array. FISH: blood smear samples were hybridized with 4 probes, in order to detect deletions at 17p13.1, 11q22.3, 13q14.3, and trisomy 12 [respectively LSI p53, ATM, D13S319 and centromeric CEP12 probes, Abbott]. In normal lymphocytes, an average of 6.7% nuclei showed one signal (truncated nuclei), and we defined the cut-off level for detection of a deletion at 11% (mean+3SD). The cut-off for detection of tri12 was defined at 5% (mean+3SD). CC: blood lymphocytes are cultivated for 72 hours with immunostimulants (DSP30 and IL-2) and metaphases analyzed according to standard procedures. SNP-arrays: DNA samples (200 ng) were hybridized on the Illumina HumanCNV370-quad v3 BeadChips, which assess 373,397 markers with a median marker spacing of 4.9 kb (mean 7.8 kb). The I-Scan system was used to scan the BeadChips (primary data). GenomeStudio 1010 v1 and CNVPartition 2.4.4 package were used to process primary data and identify chromosomal deletions/amplifications and LOH regions. Results: I-FISH identified deletions at 13q (15-95% of nuclei; mean=51%), 11q (35-54%; mean=43%) and trisomy 12 (32-49%; mean=37%) in 17 (71%) [including 2 cases of biallelic deletions], 3 (12.5%) and 4 (17%) cases, respectively. No del17p was detected. Five B-CLL cases presented associated FISH abnormalities. SNP-arrays identified all changes (100%) detected by I-FISH (del13q, median size: 13 Mb – range, 0.49–50 Mb; del11q, median size: 39 Mb – range, 34.8–42 Mb]. However, SNP-arrays showed 5 additional deletions at 13q14.3. Three patients had cryptic deletions (~52 to 82 kb), not detected by the FISH LSI D13S319 probe (~130 kb) or CC, and two others had large deletions (1.1 and 33.3 Mb) but with one signal below the cut-off of 11% by I-FISH (therefore considered as negative- both had normal CC). In these two cases, tumor cell enrichment before I-FISH allowed the detection of the deletion. In one case with del13q and tri12 (30% of nuclei by FISH), an additional del11q was also detected by SNP-array and CC (in 5/24 mitoses). The size of this deletion was 37.8 Mb and involved the cytogenetic bands 11q14.1 to 11q23.2 (including ATM). In addition, SNP-arrays enabled to define more precisely the size and location of the abnormalities. For instance, in one case with tri12 (as identified by the centromeric FISH probe), a partial trisomy of chromosome 12 short arm was indeed detected by SNP-array. No 17p abnormality was detected by SNP-array (either deletion or LOH). Only one of the 24 samples had no abnormality by SNP, I-FISH or CC for the loci studied. Conclusions: Despites using a relatively low density SNP-array (~370,000 markers), a higher sensitivity of Illumina BeadChips was observed. “SNP+/FISH negative” cases can be explained by either cryptic deletions or low leukemic cell content (< cut-off level). “SNP+/CC negative” cases can be explained by either a higher resolution, or the presence of normal mitosis in excess following stimulation. Higher density SNP-arrays (> 5 million markers) may be used to improve the assay sensitivity, especially regarding del13q14 which seems to be present in a great majority of our cases. Our study confirms usefulness of SNP-arrays for prognostic evaluation of B-CLL patients at diagnosis, and suggests the use of this assay as a routine procedure. Disclosures: No relevant conflicts of interest to declare.

BIRC3 disruption and Copy Number Aberrations in Chronic Lymphocytic Leukemia (CLL) Patients with 11q Deletion

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3295-3295
Author(s):  
Ilaria Del Giudice ◽  
Silvia Bonina ◽  
Marilisa Marinelli ◽  
Luciana Cafforio ◽  
Sara Raponi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Copy Number ◽  
Poor Prognosis ◽  
Conflicts Of Interest ◽  
Progression Free Survival ◽  
Chromosome Analysis ◽  
Lymphocytic Leukemia ◽  
The Other ◽  
11Q Deletion

Abstract Introduction. Chronic lymphocytic leukemia (CLL) with 11q deletion has been associated to a poor prognosis, but the clinical course of patients carrying this lesion is variable. This aberration, most often monoallelic, is present in 10-17% of newly diagnosed CLL and in 20-30% of patients with progressive or chemorefractory disease. The minimal deleted region (MDR) (2-3 Mbp) is located on the 11q22.3-q23.1 region and includes ATM. Moreover, 30-40% of 11q- CLL have also an inactivating ATM mutation on the other allele. The deleted region on 11q can also include BIRC3, a gene that is often deleted or mutated in advanced/chemorefractory stages of the disease. Although BIRC3 disruption has been associated to a poor prognosis, its prognostic implications in addition to ATM deletion are not well defined. The aim of this study was to perform a copy number aberration (CNA) and gene sequencing analyses on a cohort of CLL patients with 11q- in order to identify subgroups with potential prognostic relevance based on: i) the inclusion of BIRC3 in the deleted region; ii) the presence of BIRC3 mutation; iii) the presence of other CNAs. Methods. The study has included 55 untreated CLL patients followed at our Institution or enrolled in GIMEMA clinical trials (2003-2013). Genomic DNA was extracted from peripheral blood samples. CNA analysis was performed by genomic hybridization on the CytoScan HD array (Affymetrix), which contains more than 2.6 x 106 markers for copy number analysis and 750.000 SNPs. Data were analyzed using both Partek Genomics Suite and ChAS (Chromosome Analysis Suite, Affymetrix) software. The resulting CNAs were verified by visual examination of the plotted copy number profiles. BIRC3 mutations (exons 6-9) were evaluated by Sanger sequencing. Time to first treatment (TFT) was calculated from the date of diagnosis to the date of first therapy or last follow-up; progression-free survival (PFS) from the date of first therapy to the date of progression, death or last follow-up. Results. Baseline characteristics of the 55 cases were as follows: median age at diagnosis 59 years (range 39-84), male gender in 81.8% of patients, progressive disease in 62%. All patients showed 11q- by FISH (median 80%, range 25-99% of nuclei); germline IGHV were present in 96.4% of cases; TP53 deletion in 1 case and TP53 mutation in none; NOTCH1 mutation in 4/40 cases; SF3B1 mutation in 5/40 (all mutually exclusive with only 1 case having both SF3B1 and BIRC3 mutations). By CytoScan HD array, the size of 11q- was very variable, ranging from 0.36 Mbp to 65.14 Mbp; the MDR was located on 11q22.3 region, encompassing 4 genes (ACAT1, ATM, CUL5, NPAT). BIRC3 was included in the deleted region in 45/55 cases (81.8%) and was mutated in 4/54 (7.5%), being always deleted on the other allele. Beside 11q-, 51 cases (92.7%) showed several additional CNAs (average 4.9, range 1-14 per patient), with 5 recurrent lesions: 2p gain in 11 cases, del4(p15.2) in 6, del19(p13.3) in 6, 8q gain in 5 and del4(q22.1) in 4. BIRC3 deletion was not associated to the number of additional CNAs nor to specific CNA. After a follow-up of 59.6 months (range 7.4-229.7), 40 of 47 evaluable patients have received treatment (median TFT 15.8 months, range 0-167). BIRC3 deleted cases (n=37) showed a TFT not significantly different from WT cases (n=10). Conversely, BIRC3 mutation was associated to a shorter TFT (p <0.0001), 0.5 vs 19.3 months of WT cases. Notably, 3/4 cases with the biallelic BIRC3 disruption had a marked hyperleukocytosis at diagnosis (212.4-302.8 x 109L). The presence of additional CNAs was associated to a shorter, though not significant, TFT considering either >3 CNAs larger than 5 Mb (n=14) or >10 CNAs (n=5), or the presence of 2p gain, del4(p15.2), del19(p13.3) or 8q gain. So far, 22 patients have been evaluated for PFS after first-line therapy (median 28.7 months). BIRC3 deleted cases (n=17) were not associated to a shorter PFS compared to WT cases (n=5), in line with the results from Rose-Zerilli et al (Haematologica 2014). Conclusions. Among CLL with 11q-: 1) BIRC3 deletion involves more than 80% of cases, whilst the mutation is rare (7.5%); 2) BIRC3 deletion is not associated to a higher genomic complexity; 3) BIRC3 deletion does not seem to influence TFT or PFS of 11q- CLL; 4) BIRC3 mutation is strongly associated to a short TFT; 5) BIRC3 biallelic lesions can be associated to a marked hyperleucocytosis at diagnosis and immediate need of treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Incidence and Implications of Skin Cancers in Cancercare Manitoba Chronic Lymphocytic Leukemia (CLL) Clinic Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4359-4359
Author(s):  
Sara Beiggi ◽  
Mohammad Pannu ◽  
Versha Banerji ◽  
Dhali H.S. Dhaliwal ◽  
Spencer B. Gibson ◽  
...  
Keyword(s):  
Skin Cancer ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
P Value ◽  
Close Monitoring ◽  
Mutational Status ◽  
Skin Cancers ◽  
Increased Risk

Abstract Background:We previously conducted a population-based study on chronic lymphocytic leukemia (CLL) in Manitoba, which showed that second cancers are twice as common and skin cancers eight times as common in this disease, as compared to an age- and sex-matched control population and patients with follicular lymphoma. It is postulated that this is related to immunosuppression, secondary to the disease and chemo-immunotherapy. Here we set out to investigate rates and types of skin cancers in CLL patients and how these influence the outcome of CLL patients. Methods: Newly diagnosed CLL patients attending the CancerCare Manitoba CLL Clinic from the January 1st, 2002 until December 31st, 2012 were selected for this study. Patients were followed until December 31, 2014. Cox Proportional Hazard models were constructed to predict hazard's ratios (HR) and 95% confidence intervals (95% CI) for survival as well as risk of non-cutaneous malignancies. Association between skin cancer and CLL prognostic markers were investigated by Fisher's Exact test, Student's t-test and logistic regression analysis. P-value <0.05 was considered statistically significant. Statistical analysis was performed using SAS Studio 3.5. Results: There were 582 CLL patients in this study. The median age was 67 years (range 36-99 years) with a M:F ratio of 1.6:1. This compares with a median age of 71.5 years and a M:F ratio of 1.3:1 in the Manitoba CLL population. The median follow-up for the study was 5.8 years (range 0.1-13.0 years). There were 131 (23%) CLL patients with at least one skin cancer; 73 (56%) had their first skin cancer before the diagnosis of CLL and 58 (44%) after. Rates of first skin cancer diagnoses were constant before CLL diagnosis (5.2 per 1000 CLL cases), but began to increase three years prior to the CLL diagnosis (10.2 per 1000 CLL cases) and continued to increase after the CLL diagnosis (22.7 per 1000 CLL cases). There were a total of 368 skin cancers; 208 (57%) were basal cell carcinomas (BCC), 92 (25%) were squamous cell carcinomas (SCC), 47 (13%) were Bowen's disease, 18 (5%) were melanomas, and three (1%) were Merkel cell carcinomas. Interestingly, multiple skin cancers with varying histologies occurred in almost half the patients. When the total number of skin cancers/year was assessed, the number started to increase seven years before the CLL diagnosis and continued to increase yearly after the CLL diagnosis. Within the follow-up period, 154 (27%) patients died, with the major causes of death being CLL and second malignancies. However, the presence of skin cancers did not appear to influence survival. There were a total of three deaths due to skin cancers; two patients died of melanoma and one from BCC. However, the presence of a skin cancer, in CLL cases without a history of a solid tumor, increased the risk of a non-cutaneous malignancy by seven-fold (HR 7.55, 05% CI 3.92 - 14.53, p<0.0001). The presence of a skin cancer prior to the diagnosis of CLL did not predict CLL aggressiveness at diagnosis, as evaluated by Rai stage, Zap-70 or CD38 status, immunoglobulin levels or IGHV mutational status. However, for those patients developing their first skin cancer after the CLL diagnosis, the risk of developing a skin cancer correlated with the unmutated IGHV status (HR 1.54, 95% CI 1.01 - 2.34, p=0.0462) and baseline CD38 positivity (HR 1.58, 95% CI 1.02 - 2.44, p=0.0405). Interestingly, the risk of developing skin cancer was not increased by chemotherapy. Discussion: In summary, with a median follow-up of 5.8 years, 23% of patients had a skin cancer, half before the diagnosis of CLL and half after the CLL diagnosis. The incidence of skin cancers increased prior to the diagnosis of CLL, indicating that immunosuppression possibly preceded the diagnosis of CLL by years. The increased risk of developing skin cancers in patients with unmutated IGHV and CD38 positivity indicates that CLL patients with a more aggressive disease are more likely to develop skin cancer, probably due to a more pronounced immune deficiency. The diagnosis of skin cancer in CLL patients was associated with a seven-fold increased risk of developing a solid tumour. These results underscore the need for close monitoring and active surveillance of CLL patients for skin and other cancers throughout their disease course, by clinicians experienced in skin and other malignancies. Disclosures No relevant conflicts of interest to declare.

Distinct IGHV Repertoire Features In Chinese Chronic Lymphocytic Leukemia Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5279-5279
Author(s):  
Yi Xia ◽  
Wei Xu ◽  
Lei Fan ◽  
Chun Qiao ◽  
Li Wang ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Western World ◽  
Antigenic Difference ◽  
Mutational Status ◽  
Ighv Gene ◽  
Variable Heavy ◽  
Gene Usage

Abstract Mounting evidence indicates that immunoglobulin variable heavy-chain (IGHV) repertoire and mutational status in chronic lymphocytic leukemia (CLL) patients are prognostically relevant. However, rare data is available in Chinese CLL population. Our group investigated 270 Chinese CLL patients for their IGHV sequences and discovered significant differences between Chinese and European CLL patients. First of all, 169 (62.6%) patients in our group got mutated IGHV and 101 (37.4%) were unmutated, rendering a considerable higher percentage of mutated subgroup compared with European patients (55%) (Figure 1). While IGVH3 is still the most frequently used IGVH gene in Chinese CLL patients (135/270, 50%), discrepancy occurs in the usage of IGVH1 gene, which only presents in 13.7% (37/274) patients in our cohort whereas 23.79% for European (Figure 2). Regarding IGHV subgroups, IGHV3-23 and IGHV4-34 are more often used in Chinese CLL patients (10.7% and 10.4%, respectively). Remarkably, IGHV1-69, the most prevalent IGHV subgroup in European CLL patients (12.81%), only accounts for 5.2% (14/270) Chinese cases.Figure 1Higher percentage of mutated IGHV in Chinese CLL patientsFigure 1. Higher percentage of mutated IGHV in Chinese CLL patientsFigure 2Different IGHV gene usage between Chinese and European CLL patients, with IGVH1 gene accounts for 23.79% of European CLL patients and for only 13.70% of Chinese CLL patients.Figure 2. Different IGHV gene usage between Chinese and European CLL patients, with IGVH1 gene accounts for 23.79% of European CLL patients and for only 13.70% of Chinese CLL patients.Figure 3IGVH1-69 is the most prevalent IGHV gene among European CLL patients(12.81%), however, only 5.20% Chinese CLL patients use VH1-69. IGVH4-39 and IGVH4-59 are more often used in Chinese CLL patients (7.80% vs 3.73% and 5.60% vs 2.75%, respectively).Figure 3. IGVH1-69 is the most prevalent IGHV gene among European CLL patients(12.81%), however, only 5.20% Chinese CLL patients use VH1-69. IGVH4-39 and IGVH4-59 are more often used in Chinese CLL patients (7.80% vs 3.73% and 5.60% vs 2.75%, respectively). We further studied the distribution of stereotyped BCR in our cohort. Thirty-eight patients (14.07%) with stereotyped BCR that belonged to 21 subsets were identified, with 1 to 7 sequences contained each. Among them, subset 1 and subset 8 are the most common types with 6 and 7 cases respectively. Three new subsets were discovered (Table 1). Notably, only 1 case belonged to subset 2, the subset with largest group size in western world. Hence, we conclude that Chinese CLL patients show unique IGHV repertoire features compared to patients from western countries. While the mechanism within remains unknown, the discrepancy might due to antigenic difference in geographically remote areas.Table 1Three new subsets of BCR stereotypy in Chinese CLL patientsNO.IGHVIGHDIGHJM/UMIdentityHCDR3 AA sequenceLengthNovel 1NJ-15IGHV4-59*083-22*016*03UM100,00%ARGNYYDSSGYYYVGYYYYYMDV23NJ-31IGHV4-59*013-22*016*03UM99,65%ARGDYYDSSGYYYVGYYYYYMDV23Novel 2NJ-186IGHV3-23*013-22*014*02M96.60%AKGYRDNYDGDQSSVFDS18NJ-23IGHV3-23*012-21*014*02M96,53%AKGYRDNYDGDQSSVFDS18Novel 3NJ-36IGHV4-34*016-6*015*02M93,33%AKLMAGRPNWFDP13NJ-123IGHV4-34*016-6*015*02M91,67%AKLMAGRPNWFDP13 Disclosures: No relevant conflicts of interest to declare.

ZAP-70 expression is associated with enhanced ability to respond to migratory and survival signals in B-cell chronic lymphocytic leukemia (B-CLL)

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3584-3592 ◽  
Author(s):  
Sarah J. Richardson ◽  
Christine Matthews ◽  
Mark A. Catherwood ◽  
H. Denis Alexander ◽  
B. Sean Carey ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Actin Polymerization ◽  
Chromosomal Abnormalities ◽  
Lymphocytic Leukemia ◽  
Cellular Migration ◽  
Mutational Status ◽  
Cell Chronic Lymphocytic Leukemia

Molecular markers like IgVH mutational status, chromosomal abnormalities, and CD38 and ZAP-70 expression have prognostic value in B-cell chronic lymphocytic leukemia (B-CLL). These may be pathogenetic because of the coincidental expression of ZAP-70 and increased B-cell receptor (BCR) signaling and the signaling function of CD38 in CLL. This study shows that ZAP-70+ CLL B cells respond in vitro more readily than ZAP-70– CLL and normal B cells to chemokine migratory signals through enhanced surface CCR7 expression (P = .009; P < .001) and increased responsiveness to its ligands CCL19 and CCL21, demonstrated by F-actin polymerization (P < .05) and cellular migration (P < .01). In addition, ZAP-70+ CLL cells exhibit sustained ERK phosphorylation/activation following stimulation with CXCL12 (SDF1-α, a survival factor produced by stromal cells) compared with ZAP-70– cells (P = .004). Following coculture with nurse-like cells, the survival of ZAP-70+ but not ZAP-70– CLL cells is significantly enhanced by the addition of CXCL12 (P < .05), an effect that is partially blocked by the MEK inhibitor PD98059. These advantageous migratory and survival responses may promote easier access to and greater proliferation in pseudo-germinal centers and explain in part the more progressive nature of ZAP-70+ disease.

DETECTION of CHROMOSOMAL 14q32 ABNORMALITIES IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4385-4385
Author(s):  
Mariangela Mura ◽  
Alessandra Trojani ◽  
Silvia Soriani ◽  
Alessandra Tedeschi ◽  
Milena Lodola ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Small Sample Size ◽  
Variable Region ◽  
Small Sample ◽  
Lymphocytic Leukemia ◽  
Chromosome 14 ◽  
Chromosomal Breakage ◽  
Cpg Oligodeoxynucleotide

Abstract Abstract 4385 Introduction Conventional cytogenetic and FISH studies with the standard panel (13q14, 11q22.3, 17p13, 12, 6q23) are used to detect chromosomal abnormalities of clinical significance in patients with chronic lymphocytic leukemia (B-CLL). Recently, translocations and 5'IGH deletions involving chromosome14q32 are associated with B-CLL. Aim Our aim was to investigate the presence and the characteristics of chromosome 14q32 aberrations in a group of patients with B-CLL. Patients and Methods A total of 58 patients with B-CLL were investigated by conventional cytogenetic, in addition the cultures were stimulated with CpG oligodeoxynucleotide plus IL2. FISH studies with 14q32/IGH break-apart probe designed to detect chromosomal breakage of the IGH (14q32) locus, were done for 59 patients. Results Chromosomal abnormalities were detected in 60.3% of cases by cytogenetic. 14 patients showed complex cariotype while trisomy 12 was the most frequent anomaly found in 8 patients. Among the twelve other patients several chromosomal aberrations were noted: del(13)(q14), del(13)(q12q21), del(13)(q12q14), del(13)(q13q32), +21, t(11;13)(q23;q14), +12 t(1;4)(q31;p14), t(3;14)(p21;q22), del (11)(q12) +21, del(6)(q21), inv3(?p13q21), del(11)(q12). Abnormalities of chromosome 14 were found in 23.8% of patients. Deletion of 5'IGH, corresponding to the variable IGH segment, was the most frequent anomaly found in 8 patients. Interesting, a 3'IGH deletion was detected in two patients, while only one patient showed a complete deletion of chromosome 14 (47,XXY,add14q32). Three patients showed 14q32 translocations involving the IGH locus. Conclusions Based on our findings, deletions of the variable region of the IGH gene (IGHv) and 14q32/IGH translocations are involved in B-CLL. As these preliminary data are based on small sample size, our goal will be to study the cytogenetic profile of a large number of CLL patients. Future studies will permit the identification of 14q32 translocations and the type and the frequency of IGH rearrangements. Finally, chromosome 14 abnormalities will be correlated to other cytogenetic and FISH abnormalities, and associations with known prognostic markers, such as IGVH mutation status and ZAP-70 expression, will be investigated. Disclosures: No relevant conflicts of interest to declare.

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