Balanced and unbalanced translocations in a multicentric series of 2843 patients with chronic lymphocytic leukemia.

Chromosomal translocations in chronic lymphocytic leukemia (CLL) are very rare, and therefore systematic analysis of large series of cases is needed to allow the identification of recurrent rearrangements, breakpoints involved, and target genes. The aims of the present study were to identify new translocations and their clinical impact and to establish their frequency in a large cohort of 2843 CLL patients. By conventional cytogenetics 250 translocations were identified in 215 (7.5%) patients, 186 (74%) were apparently balanced and 64 (26%) were unbalanced. All chromosomes were involved in translocations, except Y chromosome. The chromosomes more frequently translocated were in decreasing frequency chromosomes 14, 18, 13, 17, 1, 6, 2, 3, 8, and 11. Translocations were found in the karyotypes either as the unique chromosomal abnormality (27%), associated with another alteration (24%), or as a part of a complex karyotype (CK, 48%). A large proportion of rearranged breakpoints involved genes related to CLL such as IGH (14q32), RB1, MIR15A, MIR16-1 (13q14), BCL2 (18q21), IGL (22q11.2), TP53 (17p13), IRF4 (6p25-p23), ATM (11q22), and CDK6 (7q21). Overall, 76 novel CLL translocations were identified, including a recurrent t(8;11)(p21;q21-23). Whole-genome sequencing and/or copy-number microarray data of 24 cases with translocations confirmed all rearrangements, enabled refinement of 3 karyotypes and all breakpoints at gene level. The projected survival and time to first treatment significantly decreased linearly with the number of translocations. In summary, this study allowed to establish the frequency of translocations (7.5%) and to identify new translocations in a cohort of 2843 CLL patients.

Prognostic impact of chromosomal translocations in myelodysplastic syndromes and chronic myelomonocytic leukemia patients. A study by the spanish group of myelodysplastic syndromes.

Chromosomal translocations are rare in the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). With the exception of t(3q), translocations are not explicitly considered in the cytogenetic classification of the IPSS-R and their impact on disease progression and patient survival is unknown. The present study was aimed at determining the prognostic impact of translocations in the context of the cytogenetic classification of the IPSS-R. We evaluated 1,653 patients from the Spanish Registry of MDS diagnosed with MDS or CMML and an abnormal karyotype by conventional cytogenetic analysis. Translocations were identified in 168 patients (T group). Compared with the 1,485 patients with abnormal karyotype without translocations (non-T group), the T group had a larger proportion of patients with refractory anemia with excess of blasts and higher scores in both the cytogenetic and global IPSS-R. Translocations were associated with a significantly shorter survival and higher incidence of transformation into AML at univariate analysis but both features disappeared after multivariate adjustment for the IPSS-R cytogenetic category. Patients with single or double translocations other than t(3q) had an outcome similar to those in the non-T group in the intermediate cytogenetic risk category of the IPSS-R. In conclusion, the presence of translocations identifies a subgroup of MDS/CMML patients with a more aggressive clinical presentation that can be explained by a higher incidence of complex karyotypes. Single or double translocations other than t(3q) should be explicitly considered into the intermediate risk category of cytogenetic IPSS-R classification.

Refining the Breakpoints of Three New Translocations Identified in Myelodysplastic Syndromes.

Recurrent translocations are uncommon in myelodysplastic syndromes (MDS). Three new recurrent translocations, namely der(12)t(3;12)(q13;p13), t(11;13;22)(q13;q14;q12) and der(17)t(13;17)(q21;p13), identified by conventional cytogenetics (CC) in 4 MDS patients, were further characterized using a panel of commercial and homemade fluorescence in situ hybridization (FISH) probes. The goal of this study was to determine the precise breakpoints and to identify genes that could be related with the neoplastic process. Half of the breakpoints (4/8) were precisely identified and in the remaining half they were narrowed to a region ranging from 14 to 926 kb. All the studied breakpoints had interstitial or terminal deletions ranging from 536 kb to 89 Mb, and only those 7 Mb were detected by CC. The genes located in or around the breakpoints described in our study have not been previously related to MDS. The deleted regions include the ETV6 and RB1 genes, among others, and exclude the TP53 gene. FISH studies were useful to refine the breakpoints of the translocations, but further studies are needed to determine the role of the involved genes in the neoplastic process.

Clonal evolution in chronic lymphocytic leukemia: analysis of correlations with IGHV mutational status, NOTCH1 mutations and clinical significance.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized with highly variable clinical course. The most common chromosomal abnormalities in CLL, using conventional and molecular cytogenetics, are trisomy 12, del(13)(q14), del(11)(q22-23), del(17)(p13), and del(6)(q21). Whereas the prognostic marker such as IGHV mutational status remains stable during course of the diseases, chromosomal aberrations may be acquired over time. The aim of this study was to determine the incidence, and biological significance of clonal evolution (CE) using conventional and molecular cytogenetics and its relationship with prognostic markers such as CD38, ZAP70, and the mutational status of IGHV and NOTCH1. One hundred and forty-three untreated CLL patients were included in the study. The median time interval between analyses was 32 months (range 6-156 months). Forty-seven patients (33%) had CE as evidenced by detection of new cytogenetic abnormalities during follow-up. CE was not correlated with high expression of ZAP70, unmutated IGHV genes or NOTCH1 mutations. Multivariate analysis revealed that CE and IGHV mutation status had a significant impact on TFS. The combination of conventional and molecular cytogenetics increased the detection of CE, this phenomenon probably being a reflection of genomic instability and conferring a more aggressive clinical course.

Reciprocal translocations in myelodysplastic syndromes and chronic myelomonocytic leukemias: review of 5,654 patients with an evaluable karyotype.

The infrequency of translocations in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemias (CMML) makes their identification and reporting interesting for the recognition of the recurrent ones and the genes involved in these neoplasias. The aims of this study were to identify new translocations associated with MDS and CMML and to establish their frequency in a cohort of 8,016 patients from the Spanish Group of MDS database. The karyotype was evaluable in 5,654 (70%) patients. Among those, 2,014 (36%) had chromosomal abnormalities, including 213 (10%) translocations identified in 195 patients. The translocations were balanced in 183 (86%) cases and unbalanced in 30 (14%) cases. All chromosomes were found to be involved in translocations, with the single exception of the Y chromosome. The chromosomes most frequently involved were in decreasing frequency: 3, 1, 7, 2, 11, 5, 12, 6, and 17. Translocations were found in karyotypes as the unique chromosomal abnormality (33%), associated with another chromosomal abnormality (11%), as a part of a complex karyotype (17%), and as a part of a monosomal karyotype (38%). There were 155 translocations not previously described in MDS or CMML and nine of them appeared to be recurrent.

Refining the diagnosis and prognostic categorization of acute myeloid leukemia patients with an integrated use of cytogenetic and molecular studies.

Significant progress in the understanding of the genetic basis of acute myeloid leukemia (AML) has been made during the last 30 years. The aim of the present study was to assess whether the detection of recurrent gene rearrangements by fluorescent in situ hybridization (FISH) studies and NPM1 and FLT3 gene mutations by molecular studies added clinically relevant information to the karyotype in 113 AML patients. Thus, FISH and molecular studies were found to add new information in 22 and 55% of the patients, respectively, particularly in cases with normal karyotype (NK) or when a cytogenetic analysis failed. Patients with NK changed their genetic risk group to favorable in 27 and 29% of cases using FISH and molecular biology studies, respectively. Our results demonstrate that molecular biology and FISH studies provide relevant information in AML and should be routinely performed.

Different distribution of NOTCH1 mutations in chronic lymphocytic leukemia with isolated trisomy 12 or associated with other chromosomal alterations.

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in Western countries. Chromosomal abnormalities commonly found using conventional cytogenetics and FISH are del(11)(q22-23), trisomy 12, del(13)(q14), and del(17)(p13). Trisomy 12 is the most frequent numerical abnormality in CLL. It can appear isolated or associated with other chromosomal aberrations, including t(14;18)(q32;q21) and trisomy 18. The aim of this study was to determine whether CLL patients with isolated trisomy 12 or associated with other chromosomal alterations have different clinico-pathological features, including a different distribution NOTCH1 mutation. Patients were classified into four groups: Group 1, isolated trisomy 12 (n=14); Group 2, trisomy 12 plus trisomy 18 (n=4); Group 3, trisomy 12 plus t(14;18) (n=8); and Group 4: patients with trisomy 12 plus other abnormalities not involving BCL2 (n=28). The Binet stage and expression of ZAP70 were significantly different among cytogenetic groups. NOTCH1 mutations were detected in 6/12 (50%) patients from Group 1, 4/25 (16%) patients from Group 4, and in no patient from groups 2 and 3 (P=0.020). Patients in Group 2 had a more rapid disease progression (median Treatment-free Survival 2 months) as against patients from Groups 1 (50 months), 3 (69 months), or 4 (68 months; P=0.001). These findings indicate that the distribution of NOTCH1 mutations in CLL with trisomy 12 is heterogeneous and that the presence of additional chromosomal abnormalities such as trisomy 18 could change the prognosis of these patients.

A new genetic abnormality leading to TP53 gene deletion in chronic lymphocytic leukaemia.

The analysis of chromosomal abnormalities provides significant prognostic information in patients with chronic lymphocytic leukaemia (CLL), a disease with a highly heterogeneous clinical course. Chromosomal abnormalities commonly found are trisomy 12, del(13)(q14), del(11)(q22-23), del(17)(p13) and del(6)(q21). Translocations are present in some patients and affect regions recurrently involved in CLL. This report describes the clinical and pathological characteristics of four CLL patients showing a new recurrent chromosomal abnormality dic(8;17)(p11;p11), that implied loss of the TP53 gene in all cases. In addition, TP53 gene was mutated in three out of four patients. Mechanically, Low Copy Repeats (LCR) in 17p12 and 8p11 may explain the origin of the translocation by non-allelic homologous recombination (NAHR). Isolated dic(8;17)(p11;p11) in patients with mutated IGHV genes status may not have the same prognostic impact as other mutations or deletions affecting the TP53 gene. Larger series are needed to better evaluate the clinical impact of this chromosomal aberration during the course of the disease.

Conventional and molecular cytogenetic studies to characterize 32 complex variant Philadelphia translocations in patients with chronic myeloid leukemia.

BCR/ABL1 gene fusion is the hallmark of chronic myeloid leukemia (CML), and is generated in 5-10% of patients by a variant translocation involving 9q34, 22q11.2 and one or more additional genomic regions. The objective of the present study was to characterize, by conventional and molecular cytogenetics, 32 complex variant Philadelphia (Ph) translocations present at diagnosis in patients with CML. The chromosomes most frequently involved were 1 and 5, and the breakpoint most frequently involved was 12p13. The q-chromosome arm was more frequently involved (60%) than the p-arm. The breakpoints were located in the G-light bands in the majority of cases (85%). Additional chromosomal abnormalities were observed in 6 out of 32 (19%) patients. In conclusion, the combination of conventional and molecular cytogenetics studies has allowed us to: i) Detect and quantify the BCR/ABL1 fusion gene; ii) characterize the complex variant translocations and detect cryptic translocations; iii) confirm that the breakpoints are commonly localized in the G-light bands; (iv) confirm that the genesis of variant translocations could be via either the one-step or two-step mechanisms; and v) to report new cases of complex variant translocations.

Clinical and biological significance of isolated Y chromosome loss in myelodysplastic syndromes and chronic myelomonocytic leukemia. A report from the Spanish MDS Group.

Isolate loss of chromosome Y (-Y) in myelodysplastic syndromes (MDS) is associated to a better outcome but it is also well described as an age-related phenomenon. In this study we aimed to analyze the prognostic impact of -Y in the context of the IPSS-R cytogenetic classification, evaluate the clinical significance of the percentage of metaphases with isolated -Y, and test whether finding -Y may predispose to over-diagnose MDS in patients with borderline morphological features. We evaluated 3581 male patients from the Spanish MDS Registry with a diagnosis of MDS or chronic myelomonocytic leukemia (CMML). -Y was identified in 177 patients (4.9%). Compared with the 2246 male patients with normal karyotype, -Y group showed a reduced risk of leukemic transformation that did not translate into a survival advantage. The overall survival and the risk of leukemic transformation were not influenced by the percentage of metaphases with -Y. The -Y group was not enriched in patients with minor morphologic traits of dysplasia, suggesting that the better outcome in the -Y group cannot be explained by enrichment in cases misdiagnosed as MDS. In conclusion, our results support the current recommendation of classifying patients with -Y within the very good risk category of the IPSS-R for MDS and rule out a selection bias as a possible explanation of this better outcome. An analysis of the molecular basis of MDS with isolated -Y would be of interest as it may provide a biological basis of protection against progression to acute leukemia.

Studies of complex Ph translocations in cases with chronic myelogenous leukemia and one with acute lymphoblastic leukemia.

The BCR/ABL gene fusion, the hallmark of chronic myelogenous leukemia (CML) is generated in 2-10% of patients by a variant Ph translocation involving 9q34, 22q11.2, and one or more additional genomic regions. The objective of this study was the characterization by conventional and molecular cytogenetics of complex variant Ph translocations present at diagnosis. FISH studies were performed in 7 cases using the LSI BCR/ABL ES probe allowing the detection of the fusion BCR/ABL gene on the Ph chromosome in all of them and 9q34 deletions in 2 cases. Three cryptic complex rearrangements were detected by FISH studies. The third and the fourth chromosome regions involved in the 8 complex variant translocations were: 1q21, 1p36, 5q31, 11q13, 12q13, 12p13, and 20q12. In conclusion, FISH studies have been useful in the detection of the BCR/ABL rearrangements and 9q34 deletions, and to identify complex rearrangements that differ from the ones previously established by conventional cytogenetics.

Do we need to do fluorescence in situ hybridization analysis in myelodysplastic syndromes as often as we do?

Although conventional cytogenetics is considered the gold standard to detect chromosomal abnormalities in myelodysplastic syndromes (MDS), fluorescence in situ hybridization (FISH) is being increasingly used additionally. However, the real contribution of FISH analysis in the cytogenetic diagnosis of MDS has not been well defined. The aim of this study was to evaluate whether FISH studies are able to reveal chromosomal abnormalities in MDS patients undetected by conventional cytogenetics. One hundred seventy-four FISH studies were performed on bone marrow samples of 60 patients with MDS. The number of FISH studies in each patient was variable (1-5). FISH studies confirmed the G-banding cytogenetic findings in 99.4% (153/154) of samples and detected cytogenetic abnormalities in 25% (5/20) of cases in which the conventional cytognetic study failed. These results indicate that FISH studies provide relevant information in MSD in which the conventional cytogenetic analysis was unsuccessful but add little value to a normal katyotype in conventional cytogenetic analysis.