Guiding the global evolution of cytogenetic testing for hematologic malignancies.

Cytogenetics has long represented a critical component in the clinical evaluation of hematologic malignancies. Chromosome banding studies provide a simultaneous snapshot of genome-wide copy number and structural variation, which have been shown to drive tumorigenesis, define diseases, and guide treatment. Technological innovations in sequencing have ushered in our present-day clinical genomics era. With recent publications highlighting novel sequencing technologies as alternatives to conventional cytogenetic approaches, we, an international consortium of laboratory geneticists, pathologists, and oncologists, describe herein the advantages and limitations of both conventional chromosome banding and novel sequencing technologies and share our considerations on crucial next steps to implement these novel technologies in the global clinical setting for a more accurate cytogenetic evaluation, which may provide improved diagnosis and treatment management. Considering the clinical, logistic, technical, and financial implications, we provide points to consider for the global evolution of cytogenetic testing.

Genetic variability profiling of the p53 signaling pathway in chronic lymphocytic leukemia. Individual and combined analysis of TP53, MDM2 and NQO1 gene variants.

TP53 gene disruption, including 17p13 deletion [del(17p)] and/or TP53 mutations, is a negative prognostic biomarker in chronic lymphocytic leukemia (CLL) associated with disease progression, treatment failure and shorter survival. Germline variants in p53 signaling pathway genes could also lead to p53 dysfunction, but their involvement in CLL has not been thoroughly evaluated. The aim of this study was to determine the association of TP53, MDM2 and NQO1 gene variability with clinical and genetic data of CLL patients. Individual genotype and haplotype data of CLL patients were compared with clinical prognostic factors, cytogenetic and molecular cytogenetic findings as well as IGHV and TP53 mutational status. The study included 116 CLL patients and 161 healthy blood donors. TP53 (rs1042522, rs59758982, rs1625895), NQO1 (rs1800566) and MDM2 (rs2279744, rs150550023) variants were genotyped using different PCR approaches. Analysis of genotype frequencies revealed no association with the risk of CLL. TP53 rs1042522, rs1625895 and MDM2 rs2279744 variants were significantly associated with abnormal karyotype and the presence of del(17p). Similarly, these two TP53 variants were associated with TP53 disruption. Moreover, TP53 C-A-nondel and G-A-del haplotypes (rs1042522-rs1625895-rs59758982) were associated with an increased likelihood of carrying del(17p) and TP53 disruptions. MDM2 T-nondel haplotype (rs2279744-rs150550023) was found to be a low risk factor for del(17p) (OR = 0.32; CI: 0.12-0.82; p = 0.02) and TP53 disruptions (OR = 0.41; CI: 0.18-0.95; p = 0.04). Our findings suggest that TP53 and MDM2 variants may modulate the risk to have chromosome alterations and TP53 disruptions, particularly del(17p). To our knowledge this is the first study of several germline variants in p53 pathway genes in Argentine patients with CLL.

Somatic hypermutation profiles in stereotyped IGHV4-34 receptors from South American chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV4-34 is one of the most frequently used genes in CLL patients, which usually display an indolent outcome. In this study, we explored the mutational profile of CLL patients expressing IGHV4-34 within different stereotypes and their association with prognostic factors and clinical outcome. A multi-institutional cohort of unselected 1444 CLL patients was analyzed by RT-PCR and bidirectional sequencing. Cytogenetics and molecular cytogenetics analyses were also performed. We identified 144 (10%) IGHV4-34 expressing cases, 119 mutated (M), 44 of them with stereotyped B-cell receptors. Subset #4 was the most frequent (56.8% of cases) followed by subsets #16 (13.6%), #29 (6.8%), and #201 (2.3%), with different distribution among countries. Analysis of somatic hypermutation profile showed significant differences among stereotyped subsets for G28>D/E, P45>S, E55>Q, and S64>I changes (p < 0.01) and high frequency of disruption of the glycosylation motif in the VH CDR2 region. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 as a sole alteration was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in non-stereotyped vs. stereotyped M-IGHV4-34 patients (p = 0.034). Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of South American CLL patients.

Frontline treatment with the combination obinutuzumab ± chlorambucil for chronic lymphocytic leukemia outside clinical trials: Results of a multinational, multicenter study by ERIC and the Israeli CLL study group.

In recent years, considerable progress has been made in frontline therapy for elderly/physically unfit patients with CLL. The combination of obinutuzumab and chlorambucil (O-Clb) has been shown to prolong progression free survival (PFS, median PFS-31.5 months) and overall survival (OS) compared to chlorambucil alone. More recently, obinutuzumab given in combination with either ibrutinib or venetoclax improved PFS but not OS when compared to O-Clb. In this retrospective multinational, multicenter co-operative study, we evaluated the efficacy and safety of frontline treatment with O ± Clb in unfit patients with CLL, in a "real-world" setting. Patients with documented del (17p13.1)/TP53 mutation were excluded. A total of 437 patients (median age, 75.9 years; median CIRS score, 8; median creatinine clearance, 61.1 mL/min) were included. The clinical overall response rate was 80.3% (clinical complete and partial responses in 38.7% and 41.6% of patients, respectively). Median observation time was 14.1 months and estimated median PFS was 27.6 months (95% CI, 24.2-31.0). In a multivariate analysis, high-risk disease [del (11q22.3) and/or IGHV-unmutated], lymph nodes of diameter > 5 cm, obinutuzumab monotherapy and reduced cumulative dose of obinutuzumab, were all independently associated with shorter PFS. The median OS has not yet been reached and estimated 2-year OS is 88%. In conclusion, in a "real-world" setting, frontline treatment with O-Clb achieves PFS comparable to that reported in clinical trials. Inferior outcomes were noted in patients with del (11q22.3) and/or unmutated IGHV and those treated with obinutuzumab-monotherapy. Thus, O-Clb can be still considered as legitimate frontline therapy for unfit CLL patients with low-risk disease.

Differential expression of long non-coding RNAs are related to proliferation and histological diversity in follicular lymphomas.

Long non-coding RNAs (lncRNAs) comprise a family of non-coding transcripts that are emerging as relevant gene expression regulators of different processes, including tumour development. To determine the possible contribution of lncRNA to the pathogenesis of follicular lymphoma (FL) we performed RNA-sequencing at high depth sequencing in primary FL samples ranging from grade 1-3A to aggressive grade 3B variants using unpurified (n = 16) and purified (n = 12) tumour cell suspensions from nodal samples. FL grade 3B had a significantly higher number of differentially expressed lncRNAs (dif-lncRNAs) with potential target coding genes related to cell cycle regulation. Nine out of the 18 selected dif-lncRNAs were validated by quantitative real time polymerase chain reaction in an independent series (n = 43) of FL. RP4-694A7.2 was identified as the top deregulated lncRNA potentially involved in cell proliferation. RP4-694A7.2 silencing in the WSU-FSCCL FL cell line reduced cell proliferation due to a block in the G1/S phase. The relationship between RP4-694A7.2 and proliferation was confirmed in primary samples as its expression levels positively related to the Ki-67 proliferation index. In summary, lncRNAs are differentially expressed across the clinico-biological spectrum of FL and a subset of them, related to cell cycle, may participate in cell proliferation regulation in these tumours.

Analysis of basal chromosome instability in patients with chronic lymphocytic leukaemia.

Genomic instability is a hallmark of cancer, contributing to tumour development and transformation, being chromosome instability (CIN) the most common form in human cancer. Chronic lymphocytic leukaemia (CLL) is the most frequent adult leukaemia in the Western world. In this study, we have evaluated basal CIN in untreated patients with CLL by measuring chromosome aberrations (CAs) and micronucleus (MN) frequency and their association with different prognostic factors. Seventy-two patients and 21 normal controls were analysed. Cytogenetic and fluorescence in situ hybridisation (FISH) studies were performed. IGHV (immunoglobulin heavy chain variable region) mutational status was evaluated by reverse transcription polymerase chain reaction and sequencing. An increased number of CA in patients compared with controls (P = 0.0001) was observed. Cases with abnormal karyotypes showed increased CA rate than those with normal karyotypes (P = 0.0026), with a particularly highest frequency in cases with complex karyotypes. Among FISH risk groups, a significant low frequency of CA was found in patients with no FISH alterations compared to those with del13q14 and ≥2 FISH alterations (P = 0.0074). When mean CA value (6.7%) was considered, significant differences in the distribution of low and high CA frequency between cases with normal and abnormal karyotypes (P = 0.002) were observed. By MN analysis, higher frequency in patients compared to controls (P = 0.0001) was also found, as well as between cases with ≥2 FISH abnormalities and those with no FISH alterations (P = 0.026). Similarly, significant differences were observed when patients were divided according to mean MN frequency (2.2%; P ≤ 0.04). Interestingly, patients with high MN frequency had shorter time to first treatment than those with low frequency (P = 0.024). Cases with mutated and unmutated IGHV status showed increased CA and MN frequencies compared to controls (P ≤ 0.0007), but no differences between both groups were found. Our results support the strong interaction between CIN and genomic complexity as well as their influence on poor outcome in this pathology.

The kinase inhibitors R406 and GS-9973 impair T cell functions and macrophage-mediated anti-tumor activity of rituximab in chronic lymphocytic leukemia patients.

Small molecules targeting kinases involved in B cell receptor signaling are showing encouraging clinical activity in chronic lymphocytic leukemia (CLL) patients. Fostamatinib (R406) and entospletinib (GS-9973) are ATP-competitive inhibitors designed to target spleen tyrosine kinase (Syk) that have shown clinical activity with acceptable toxicity in trials with CLL patients. Preclinical studies with these inhibitors in CLL have focused on their effect in patient-derived leukemic B cells. In this work we show that clinically relevant doses of R406 and GS-9973 impaired the activation and proliferation of T cells from CLL patients. This effect could not be ascribed to Syk-inhibition given that we show that T cells from CLL patients do not express Syk protein. Interestingly, ζ-chain-associated protein kinase (ZAP)-70 phosphorylation was diminished by both inhibitors upon TCR stimulation on T cells. In addition, we found that both agents reduced macrophage-mediated phagocytosis of rituximab-coated CLL cells. Overall, these results suggest that in CLL patients treated with R406 or GS-9973 T cell functions, as well as macrophage-mediated anti-tumor activity of rituximab, might be impaired. The potential consequences for CLL-treated patients are discussed.

Gene polymorphism profiles of drug-metabolising enzymes GSTM1, GSTT1 and GSTP1 in an Argentinian population.

BACKGROUND: Glutathione S-transferases (GSTs) are drug-metabolising enzymes involved in biotransformation of carcinogens, drugs, xenobiotics and oxygen free radicals. Polymorphisms of GST genes contribute to inter-individual and population variability in the susceptibility to environmental risk factors, cancer predisposition and pharmacotherapy responses. However, data about GST variability in Argentina are lacking. AIM: The purpose was to determine the prevalence of GSTM1, GSTT1 and GSTP1 polymorphisms in the general population from a central region of Argentina and to perform inter-population comparisons. SUBJECTS AND METHODS: GSTM1 and GSTT1 gene deletions and GSTP1 c.313A > G were genotyped by PCR assays in 609 healthy and unrelated Argentinians. RESULTS: The frequencies of variant genotypes in Argentinians were GSTM1-null (45%), GSTT1-null (17%) and GSTP1-GG (11%). GSTM1-present genotype was significantly associated with GSTP1-AG or GSTP1-GG variants (p = 0.037; p = 0.034, respectively). Comparison with worldwide populations demonstrated that the GST distributions in Argentina are similar to those reported for Italy and Spain, whereas significant differences were observed regarding Asian and African populations (p < 0.001). CONCLUSION: This study has determined, for the first time, the normative profile of three pharmacogenetically relevant polymorphisms (GSTM1, GSTT1 and GSTP1) in the largest Argentinian cohort described to date, providing the basis for further epidemiological and pharmacogenetic studies in this country.

SOXC and MiR17-92 gene expression profiling defines two subgroups with different clinical outcome in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a heterogeneous B-cell lymphoid malignancy where most patients follow an aggressive clinical course whereas others are associated with an indolent performance. SOX4, SOX11, and SOX12 belong to SOXC family of transcription factors involved in embryonic neurogenesis and tissue remodeling. Among them, SOX11 has been found aberrantly expressed in most aggressive MCL patients, being considered a reliable biomarker in the pathology. Several studies have revealed that microRNAs (miRs) from the miR-17-92 cluster are among the most deregulated miRNAs in human cancers, still little is known about this cluster in MCL. In this study we screened the transcriptional profiles of 70 MCL patients for SOXC cluster and miR17, miR18a, miR19b and miR92a, from the miR-17-92 cluster. Gene expression analysis showed higher SOX11 and SOX12 levels compared to SOX4 (P ≤ 0.0026). Moreover we found a negative correlation between the expression of SOX11 and SOX4 (P < 0.0001). miR17-92 cluster analysis showed that miR19b and miR92a exhibited higher levels than miR17 and miR18a (P < 0.0001). Unsupervised hierarchical clustering revealed two subgroups with significant differences in relation to aggressive MCL features, such as blastoid morphological variant (P = 0.0412), nodal presentation (P = 0.0492), CD5(+) (P = 0.0004) and shorter overall survival (P < 0.0001). Together, our findings show for the first time an association between the differential expression profiles of SOXC and miR17-92 clusters in MCL and also relate them to different clinical subtypes of the disease adding new biological information that may contribute to a better understanding of this pathology. © 2016 Wiley Periodicals, Inc.

Genomic imbalances and microRNA transcriptional profiles in patients with mycosis fungoides.

Mycosis fungoides is the most common type of primary cutaneous T cell lymphoma. We have evaluated CDKN2A losses and MYC gains/amplifications by FISH analysis, as well as expression of miR-155 and members of the oncogenic cluster miR-17-92 (miR17, miR18a, miR19b, and miR92a) in MF patients with advanced disease. Formalin-fixed paraffin-embedded skin biopsies from 36 patients at diagnosis, 16 with tumoral MF (T-MF), 13 in histological transformation to a large T cell lymphoma (TR-MF), and 7 cases with folliculotropic variant (F-MF), were studied. Twenty cases showed genomic alterations (GAs): 8 (40 %) had CDKN2A deletion, 7 (35 %) showed MYC gain, and 5 (25 %) exhibited both alterations. GAs were more frequently observed in F-MF (p = 0.004) and TR-MF (p = 0.0001) than T-MF. GAs were significantly higher in cases presenting lesions in head, neck, and lower extremities compared to those observed in trunk and upper extremities (p = 0.03), when ≥25 % neoplastic cells were CD30 positive (p = 0.016) as well as in cases with higher Ki-67 proliferation index (p = 0.003). Patients with GAs showed bad response to treatment (p = 0.02) and short survival (p = 0.04). Furthermore, MF patients showed higher miRNA expression compared to controls (p ≤ 0.0223). T-MF showed higher miR17 and miR-18a expression compared to F-MF and TR-MF (p ≤ 0.0387) while miR19b, miR92a, and miR-155 showed increased levels in F-MF and TR-MF with respect to T-MF (p ≤ 0.0360). Increased expression of miR17 and miR19b in GA group compared to cases without alterations (p ≥ 0.0307) was also detected. Our results add new information about genomic imbalances in MF patients, particularly in F-MF, and extend the present view of miRNA deregulation in this disease.

Acquired TERT promoter mutations stimulate TERT transcription in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm with poor prognosis. Acquired telomerase reverse transcriptase gene promoter (TERTp) mutations are among the most frequent somatic non-coding mutations in cancers. In this study, the prevalence of TERTp mutations in 24 MCL and 21 other lymphoid neoplasias (oLN) was investigated. Eight MCL samples (33%) carried TERTp mutations, two homozygous and six heterozygous (seven C228T and one C250T), which directly correlated with higher TERT transcription, mitochondrial DNA copy number, and IGHV mutational status in MCL neoplastic cells. TERTp mutations were not found in oLN. TERTp mutations correlated with more lymphoma proliferation and tumor burden, as suggested by the higher number of lymphoma cells circulating in peripheral blood, and tended to associate with longer MCL telomeres, especially in homozygous mutants, although not statistically significant. Telomere-biology genes were overexpressed in MCL cells in comparison to healthy lymphocytes, but were not influenced by mutation status. The findings described for the first time that acquired TERTp mutations are common in MCL but not in other lymphoid neoplasms. It was also demonstrated that TERTp mutations are associated with higher TERT mRNA expression in MCL cells in vivo and higher tumor burden, suggesting these mutations as a driver event in MCL development and progression.

Telomere shortening associated with increased genomic complexity in chronic lymphocytic leukemia.

Telomeric dysfunction has been proposed as an emerging prognostic factor in chronic lymphocytic leukemia (CLL). We have explored the relationship between telomere length (TL) and chromosome alterations studied by fluorescence in situ hybridization (FISH) and conventional cytogenetics in 107 newly diagnosed CLL patients; 61 normal controls were also evaluated. Results were correlated with clinical parameters and outcome. Absolute TL measurement was carried out on DNA samples by real-time quantitative PCR. A significant telomere shortening in patients compared to controls was observed (p = 0.0001). The analysis taking into account FISH risk groups showed shorter TLs in cases with del11q/17p compared to patients with 13q14 deletion as a single alteration (p = 0.0037), no alterations (NA) (p = 0.028), and cases with abnormal karyotypes (p = 0.014). In addition, a significant TL reduction in cases with two or more anomalies with respect to those with NA (p = 0.033) and with one alteration (p = 0.045), and no differences compared to cases with deletions 11q/17p were observed. Patients with only one anomaly did not show statistical differences with respect to controls; meanwhile, a significant TL reduction in cases with two or more aberrations was observed (p = 0.025). The shortest telomeres were associated to 11q/17p deletion with significant differences compared to the remaining groups (p ≤ 0.045). Significantly shorter treatment free survival in patients with two or more alterations compared to those with NA plus one abnormality was observed (p = 0.0006). Our findings support the association between short TL and chromosome alterations in CLL and indicate the importance of telomere dysfunction in driving genomic instability in this pathology.

SOX11 expression in chronic lymphocytic leukemia correlates with adverse prognostic markers.

The transcription factor SOX11 plays an important role in embryonic neurogenesis and tissue remodeling. Recent studies have shown aberrant expression of SOX11 in various types of aggressive B cell neoplasms. In this study, we have analyzed SOX11 transcription levels in 86 patients with diagnosis of chronic lymphocytic leukemia (CLL). Results were correlated with well-known prognostic factors such as immunoglobulin heavy chain variable (IGHV) gene mutational status, cytogenetics risk groups and clinicopathological characteristics of the disease. Overall, 35 % of cases showed SOX11 expression; meanwhile, the remaining 65 % lacked gene expression. The analysis taking into account the IGHV mutational status showed significant differences in SOX11 transcripts levels between mutated (0.004 ± 0.0001) and unmutated CLL patients (0.405 ± 0.011) (p < 0.0001), as well as a positive correlation between SOX11 mRNA expression and the percentage of IGHV homology (p = 0.0001). Furthermore, significantly lower SOX11 mRNA expression was detected in patients with deletion 13q14 as a single alteration (0.016 ± 0.008) than those observed in cases with deletions 11q/17p (0.35 ± 0.017) (p = 0.02). The correlation of gene expression with clinical evolution showed shorter treatment free survival (p = 0.043) and overall survival (p = 0.047) in SOX11 positive patients compared to SOX11 negative cases. Our findings show for the first time an association between SOX11 expression and some CLL poor prognostic factors. These results suggest SOX11 as a possible biomarker that adds new biological information that could contribute to a better understanding of this pathology.

Cytogenetic Alterations in Multiple Myeloma: Prognostic Significance and the Choice of Frontline Therapy.

Multiple myeloma tumor cells demonstrate multiple and often complex genetic lesions as evaluated by standard cytogenetic/FISH studies. Over the past decade, specific abnormalities have been associated with standard or high-risk clinical behavior and they have become strong prognostic indicators. Further, as evidenced by recent randomized clinical trials, the choice of front-line therapy (transplant vs. no transplant, inclusion of novel drugs such as bortezomib, thalidomide, and lenalidomide) may be able to overcome the adverse effect of high-risk genetic lesions.

Quantitative analysis of CKS1B mRNA expression and copy number gain in patients with plasma cell disorders.

In this study, we have examined CKS1B gene expression and copy number in a total of 114- patients at diagnosis: 83 with multiple myeloma (MM) and 31 with monoclonal gammopathy of undetermined significance (MGUS). Results were correlated with cytogenetics, FISH and clinical characteristic. Significant CKS1B mRNA levels in MM compared to MGUS cases (p<0.048) were detected. In MM, the frequency of 1q21 (CKS1B) copy gain was significantly higher in cases with abnormal karyotype compared to patients with normal karyotype (p=0.021). Global analysis showed a positive correlation between CKS1B expression and 1q21 copy number (p<0.0001). No association between CKS1B mRNA expression and clinical parameters was found. However, a significantly higher level of ß2 microglobulin in cases with 1q21 gains than those without (p=0.0094) was observed. Overall survival was shorter in cases with 1q21 gain compared to those with normal 1q21 region (p=0.0082). Our results suggest a role for CKS1B in the multiple step process of progression of MGUS to MM and show that CKS1B copy gain has a more significant prognostic value than its overexpression. This adverse impact on survival probably reflects the genetic instability associated to chromosome 1q alterations resulting in a more aggressive behavior of the disease.

Expression profile of shelterin components in plasma cell disorders. Clinical significance of POT1 overexpression.

The core complex of telomere-associated proteins, named the shelterin complex, plays a critical role in telomere protection and telomere length (TL) homeostasis. In this study, we have explored changes in the expression of telomere-associated genes POT1, TIN2, RAP1 and TPP1, in patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). A total of 154 patients: 70 with MGUS and 84 with MM were studied. Real-time quantitative PCR was used to quantify gene expression. TL was evaluated by Terminal Restriction Fragments. Our data showed increased expression of POT1, TPP1, TIN2 and RAP1 in MM with respect to MGUS patients, with significant differences for POT1 gene (p=0.002). In MM, the correlation of gene expression profiles with clinical characteristics highlighted POT1 for its significant association with advanced clinical stages, high calcium and ß2-microglobulin levels (p=0.02) and bone lesions (p=0.009). In multivariate analysis, POT1 expression (p=0.04) was a significant independent prognostic factor for overall survival as well as the staging system (ISS) (p<0.02). Our findings suggest for the first time the participation of POT1 in the transformation process from MGUS to MM, and provide evidence of this gene as a useful prognostic factor in MM as well as a possible molecular target to design new therapeutic strategies.

[Expression of SOX11 transcription factor. Its implication in mantle cell lymphoma]. / Expresión del factor de transcripción SOX11. Su implicancia en el linfoma de células del manto.

SOX11, belonging to the family of genes SOXC, is a transcript factor involved in the embryonic neurogenesis and tissue remodeling, also participating in the control of cell proliferation. Its role in lymphomagenesis still remains unknown. Recent studies have shown aberrant SOX11 nuclear protein expression as well as mRNA levels in patients with mantle cell lymphoma (MCL). Although the majority of these lymphomas have an aggressive clinical course, there is a subgroup of patients with an indolent clinical evolution, suggesting a greater heterogeneity of this disease. Currently, there are contradictions regarding the association of SOX11 gene expression and outcome in MCL, while some authors have related the lack of SOX11 expression with good prognosis, others find it associated with an adverse clinical course. This difference in the gene expression could be associated to epigenetic mechanisms such as modifications at the histone level and DNA methylation that would allow the aberrant expression of this gene in some lymphoid neoplasias, including LCM. More knowledge of gene SOX11 in LCM will lead to a greater understanding of those mechanisms involved in the pathogenesis and progression of this lymphoma, also the involvement of SOX11 in these processes.

Structural alterations in chronic lymphocytic leukaemia. Cytogenetic and FISH analysis.

In this study, we described cytogenetics and fluorescence in situ hybridization (FISH) analysis performed in chronic lymphocytic leukaemia (CLL) patients with structural alterations. Results were correlated with clinical characteristics. A total of 38 CLL patients: 16 cases with complex and 22 with simple karyotypes were studied. For comparison of clinical parameters, a control group of 78 CLL patients with normal karyotype and without FISH genomic alterations were also evaluated. We found 38 structural abnormalities not previously described in the literature, 28 (74%) of them were translocations. In cases with complex karyotypes, chromosomes 6, 8 and 13 were the most frequently involved in new alterations (nine each), followed by chromosomes 12, 14 and 15 (six each). Chromosome 8p was particularly involved in losses, being 8p21-pter the commonest region of overlap. Cases with simple karyotypes, showed del(6q) as the most frequent alteration (39%). Del(9)(q11) was recurrent in our series. Analysis of clinical parameters showed significant differences in white blood count (p = 0.005) and platelet count (p = 0.015) between patients with structural alterations and the control group. In addition, patients with structural alterations had a significantly shorter time to first treatment (TFT) (29 months) than the control group (69 months) (p = 0.037). Cases with complex karyotypes had a lower proportion of patients in Rai 0 clinical stage (15.4% vs 75%) (p = 0.005) and higher ß2 microglobulin levels (3.3 vs 2.5 µg/mL) (p = 0.037) than those with simple karyotypes. Furthermore, a shorter TFT (13 months) and overall survival (56 months) in the complex karyotypes group compared with controls (69 and 144 months, respectively) (p = 0.015 and p = 0.005, respectively) were also found. Our results support the importance of cytogenetic analysis for clinical outcome in CLL and suggest that the diversity of genomic alterations is much greater than previously appreciated.