In chronic lymphocytic leukaemia, SLAMF1 deregulation is associated with genomic complexity and independently predicts a worse outcome.

In a series of 349 patients with chronic lymphocytic leukaemia (CLL), we found lower levels of signalling lymphocytic activation molecule family member 1 (SLAMF1) expression in cases with highly complex karyotypes, as defined by the presence of five or more chromosomal abnormalities (CK5; P < 0·001) and with major chromosomal structural abnormalities (P < 0·001). SLAMF1 downregulation was significantly associated with advanced Binet Stage (P = 0·001), CD38 positivity (P < 0·001), high ß2 -microglobulin levels (P < 0·001), immunoglobulin heavy chain variable region gene (IGHV) unmutated status (P < 0·001), 11q deletion (P < 0·001), tumour protein p53 (TP53) disruption (P = 0·011) and higher risk CLL International Prognostic Index categories (P < 0·001). Multivariate analysis showed that downregulated SLAMF1 levels had independent negative prognostic impact on time-to-first treatment (P < 0·001) and overall survival (P < 0·001).

Biological significance and prognostic/predictive impact of complex karyotype in chronic lymphocytic leukemia.

The complex karyotype (CK) is an established negative prognostic marker in a number of haematological malignancies. After the introduction of effective mitogens, a growing body of evidence has suggested that the presence of 3 or more aberrations by conventional banding analysis (CBA) is associated with an unfavorable outcome in chronic lymphocytic leukemia (CLL). Thus, the importance of CBA was recognized by the 2018 guidelines of the International Workshop on CLL, which proposed the introduction of CBA in clinical trials to validate the value of karyotype aberrations. Indeed, a number of observational studies showed that cytogenetic aberrations and, particularly, the CK may have a negative independent impact on objective outcome measures (i.e. time to first treatment, progression free survival, time to chemorefractoriness and overall survival) both in patients treated with chemoimmunotherapy and, possibly, in patients receiving novel mechanism-based treatment. Here, we set out to present the scientific evidence supporting the significance of CK as a prognostic marker in CLL and to discuss the biological basis showing that the CK is a consequence of genomic instability.

In chronic lymphocytic leukaemia with complex karyotype, major structural abnormalities identify a subset of patients with inferior outcome and distinct biological characteristics.

Complex karyotype (CK) is a negative prognostic factor in chronic lymphocytic leukaemia (CLL). However, CK is a heterogeneous cytogenetic category. Unbalanced rearrangements were present in 73·3% of 90 CLL patients with CK (i.e. ≥3 chromosome aberrations in the same clone), and were associated with a shorter overall survival (P = 0·025) and a shorter time to first treatment (P = 0·043) by multivariate analysis. Patients with unbalanced rearrangements presented a distinct mRNA expression profile. In conclusion, CLL patients with unbalanced rearrangements might represent a subset of very high-risk CLL patients with distinct clinical and biological characteristics.

An extensive molecular cytogenetic characterization in high-risk chronic lymphocytic leukemia identifies karyotype aberrations and TP53 disruption as predictors of outcome and chemorefractoriness.

We investigated whether karyotype analysis and mutational screening by next generation sequencing could predict outcome in 101 newly diagnosed chronic lymphocytic leukemia patients with high-risk features, as defined by the presence of unmutated IGHV gene and/or 11q22/17p13 deletion by FISH and/or TP53 mutations. Cytogenetic analysis showed favorable findings (normal karyotype and isolated 13q14 deletion) in 30 patients, unfavorable (complex karyotype and/or 17p13/11q22 deletion) in 34 cases and intermediate (all other abnormalities) in 36 cases. A complex karyotype was present in 21 patients. Mutations were detected in 56 cases and were associated with unmutated IGHV status (p = 0.040) and complex karyotype (p = 0.047). TP53 disruption (i.e. TP53 mutations and/or 17p13 deletion by FISH) correlated with the presence of ≥ 2 mutations (p = 0.001) and a complex karyotype (p = 0.012). By multivariate analysis, an advanced Binet stage (p < 0.001) and an unfavorable karyotype (p = 0.001) predicted a shorter time to first treatment. TP53 disruption (p = 0.019) and the unfavorable karyotype (p = 0.028) predicted a worse overall survival. A shorter time to chemorefractoriness was associated with TP53 disruption (p = 0.001) and unfavorable karyotype (p = 0.025). Patients with both unfavorable karyotype and TP53 disruption presented a dismal outcome (median overall survival and time to chemorefractoriness of 28.7 and 15.0 months, respectively). In conclusion, karyotype analysis refines risk stratification in high-risk CLL patients and could identify a subset of patients with highly unfavorable outcome requiring alternative treatments.

Identifying High-Risk Chronic Lymphocytic Leukemia: A Pathogenesis-Oriented Appraisal of Prognostic and Predictive Factors in Patients Treated with Chemotherapy with or without Immunotherapy.

Chronic lymphocytic leukemia (CLL) displays an extremely variable clinical behaviour. Accurate prognostication and prediction of response to treatment are important in an era of effective first-line regimens and novel molecules for high risk patients. Because a plethora of prognostic biomarkers were identified, but few of them were validated by multivariable analysis in comprehensive prospective studies, we applied in this survey stringent criteria to select papers from the literature in order to identify the most reproducible prognostic/predictive markers. Each biomarker was analysed in terms of reproducibility across the different studies with respect to its impact on time to first treatment (TTFT), progression free survival (PFS), overall survival (OS) and response to treatment. We were able to identify the following biomarkers as the most reliable in guiding risk stratification in the daily clinical practice: 17p-/TP53 mutations, IGHV unmutated configuration, short telomeres and 11q-. However, the method for measuring telomere length was not validated yet and 11q- was predictive of inferior OS only in those patients who did not receive FCR-like combinations. Stage and lymphocytosis were predictive of shorter TTFT and age, high serum thymidine kinase levels and poor performance status were predictive of shorter OS. Using our criteria no parameter was found to independently predict for inferior response to treatment.

Extensive next-generation sequencing analysis in chronic lymphocytic leukemia at diagnosis: clinical and biological correlations.

BACKGROUND: In chronic lymphocytic leukemia (CLL), next-generation sequencing (NGS) analysis represents a sensitive, reproducible, and resource-efficient technique for routine screening of gene mutations. METHODS: We performed an extensive biologic characterization of newly diagnosed CLL, including NGS analysis of 20 genes frequently mutated in CLL and karyotype analysis to assess whether NGS and karyotype results could be of clinical relevance in the refinement of prognosis and assessment of risk of progression. The genomic DNA from peripheral blood samples of 200 consecutive CLL patients was analyzed using Ion Torrent Personal Genome Machine, a NGS platform that uses semiconductor sequencing technology. Karyotype analysis was performed using efficient mitogens. RESULTS: Mutations were detected in 42.0 % of cases with 42.8 % of mutated patients presenting 2 or more mutations. The presence of mutations by NGS was associated with unmutated IGHV gene (p = 0.009), CD38 positivity (p = 0.010), risk stratification by fluorescence in situ hybridization (FISH) (p < 0.001), and the complex karyotype (p = 0.003). A high risk as assessed by FISH analysis was associated with mutations affecting TP53 (p = 0.012), BIRC3 (p = 0.003), and FBXW7 (p = 0.003) while the complex karyotype was significantly associated with TP53, ATM, and MYD88 mutations (p = 0.003, 0.018, and 0.001, respectively). By multivariate analysis, the multi-hit profile (≥2 mutations by NGS) was independently associated with a shorter time to first treatment (p = 0.004) along with TP53 disruption (p = 0.040), IGHV unmutated status (p < 0.001), and advanced stage (p < 0.001). Advanced stage (p = 0.010), TP53 disruption (p < 0.001), IGHV unmutated status (p = 0.020), and the complex karyotype (p = 0.007) were independently associated with a shorter overall survival. CONCLUSIONS: At diagnosis, an extensive biologic characterization including NGS and karyotype analyses using novel mitogens may offer new perspectives for a better refinement of risk stratification that could be of help in the clinical management of CLL patients.

Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia: Clinical and biologic correlations.

To clarify whether karyotype aberrations (KA) involving regions not covered by the standard fluorescence in situ hybridization (FISH) panel have independent prognostic relevance, we evaluated KA by conventional cytogenetics in a learning cohort (LC; n = 166) and a validation cohort (VC; n = 250) of untreated chronic lymphocytic leukemia (CLL) patients. In the VC, novel mitogens were used to improve metaphase generation and TP53, NOTCH1, and SF3B1 mutations were assessed. KA undetected by FISH were found in 35 and 35% of the cases in the LC and VC, respectively. In addition to FISH, KA allowed reclassification of 23 and 26% of cases in the LC and VC, respectively, into a higher cytogenetic risk group. By multivariate analysis, both in the LC and VC, KA other than isolated 13q deletion correlated with a shorter time to first treatment (TFT; P < 0.001 and 0.003, respectively), while a complex karyotype predicted a worse overall survival (OS, P = 0.015 and 0.010, respectively). In the VC, where a comprehensive biologic assessment was performed, a shorter TFT was also predicted by stage (P < 0.001), IGHV mutational status (P = 0.05), and del(17p)/TP53 mutations (P = 0.033) while stage (P = 0.023) and del(17p)/TP53 mutations (P = 0.024) independently predicted a shorter OS. FISH results did not independently impact on TFT and OS, in the LC and VC cohorts; this was also the case for NOTCH1 and SF3B1 mutations in the VC. We suggest that in CLL, conventional karyotyping with novel mitogens could be more effective than FISH for the detection of KA allowing for a more precise refinement of prognosis.

Cytogenetic and molecular cytogenetic profile of bone marrow-derived mesenchymal stromal cells in chronic and acute lymphoproliferative disorders.

The possibility that human mesenchymal stromal cells (hMSC) may derive from the malignant clone in hematological malignancies (HM) is a controversial issue. In order to clarify hMSC origin and disclose possible cytogenetic heterogeneity in hMSC belonging to different patients, bone marrow (BM)-derived hMSC samples from chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) were expanded in culture, characterized by flow cytometry, and screened by conventional cytogenetic analysis and fluorescent in situ hybridization for the presence of possible cytogenetic aberrations, related or not to the hematopoietic neoplastic clone. Our data showed that the presence of cytogenetic aberrations in successfully expanded HM-MSC stromal layers derives from the persistence of contaminating hemopoietic cells (HC), which is greatly supported by in vitro culture conditions that could mimic in vivo microenvironmental niche. Interestingly, the presence of binucleated HM-MSC maintaining a diploid numerical setting has been also detected, while aneuploidies were observed more frequently in mononucleated HM-MSC from patients with an altered karyotype than in patients with a normal karyotype and controls. In conclusion, here, we showed that in ALL and in CLL, the BM-MSC has a normal karyotype, thus supporting a distinct origin from hematopoietic cells (HC). The presence of in vitro hMSC aneuploidy is associated with lymphoid neoplasias carrying chromosome abnormalities, suggesting that hMSC should be characterized before clinical application. The adequacy of hMSC cytogenetic characterization here proposed could represent a "prerequisite" to standardize the hMSC analysis before their use in the autologous setting and cellular therapy.

Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia with "normal" FISH: correlations with clinicobiologic parameters.

It is unclear whether karyotype aberrations that occur in regions uncovered by the standard fluorescence in situ hybridization (FISH) panel have prognostic relevance in chronic lymphocytic leukemia (CLL). We evaluated the significance of karyotypic aberrations in a learning cohort (LC; n = 64) and a validation cohort (VC; n = 84) of patients with chronic lymphocytic leukemia with "normal" FISH. An abnormal karyotype was found in 21.5% and 35.7% of cases in the LC and VC, respectively, and was associated with a lower immunophenotypic score (P = .030 in the LC, P = .035 in the VC), advanced stage (P = .040 in the VC), and need for treatment (P = .002 in the LC, P = < .0001 in the VC). The abnormal karyotype correlated with shorter time to first treatment and shorter survival in both the LC and the VC, representing the strongest prognostic parameter. In patients with chronic lymphocytic leukemia with normal FISH, karyotypic aberrations by conventional cytogenetics with novel mitogens identify a subset of cases with adverse prognostic features.

Clonal evolution including 14q32/IGH translocations in chronic lymphocytic leukemia: analysis of clinicobiologic correlations in 105 patients.

To better define the significance of clonal evolution (CE) including 14q32 translocations involving the immunoglobulin heavy chain gene (IGH) in chronic lymphocytic leukemia (CLL), 105 patients were analyzed sequentially by fluorescence in situ hybridization (FISH) with the following panel of probes: 13q14/D13S25, 11q22/ATM, 17p13/TP53, #12-centromere and 14q32/IGH break-apart probe. CE was observed in 15/105 patients after 24-170 months (median 64). Recurring aberrations at CE were 14q32/IGH translocation in seven patients; other aberrations were 17p -, 11q -, biallelic 13q - and 14q32 deletion. CE was detected in 15/58 pre-treated patients; in contrast, none of 47 untreated patients developed CE (p < 0.0001). In two cases the appearance of 14q32/IGH translocation was first detected in the bone marrow (BM) or in the lymph node (LN) and 13-58 months later in the peripheral blood (PB). ZAP70 + and high-risk cytogenetics predicted for the occurrence of CE with borderline statistical significance (p = 0.055 and 0.07, respectively). Shorter time to first treatment (TTT) and time to chemorefractoriness (TTCR) were noted in 15 patients with CE when compared to patients without CE (TTT: 35 vs. 71 months, p = 0.0033 and TTCR: 34 vs. 86 months, p = 0.0046, respectively). Survival after the development of CE was 32 months (standard error 8.5). We arrived at the following conclusions: (i) 14q32/IGH translocation may represent one of the most frequent aberrations acquired during the natural history of CLL and (ii) it may be detected earlier in BM or LN samples; (iii) CE including 14q32/IGH translocation occurs in pre-treated patients with short TTT and TTCR; (iii) survival after CE is relatively short.

Employment of oligodeoxynucleotide plus interleukin-2 improves cytogenetic analysis in splenic marginal zone lymphoma.

To compare the efficiency of novel mitogenic agents and traditional mitosis inductors, 18 patients with splenic marginal zone lymphoma (SMZL) were studied. Three cultures using oligodeoxynucleotide (ODN) plus interleukin-2 (IL-2), or TPA, or LPS were setup in each patient. Seventeen/18 cases with ODN + IL2 had moderate/good proliferation (94, 4%) as compared with 10/18 cases with TPA and LPS (55%) (P = .015); 14/18 (77, 7%) cases with ODN + IL2 had sufficient good quality of banding as compared with 8/18 cases (44, 4%) with TPA and LPS. The karyotype could be defined from ODN + IL2-stimulated cultures in all 18 patients, 14 of whom (77, 7%) had a cytogenetic aberration, whereas clonal aberrations could be documented in 9 and in 3 cases by stimulation with LPS and TPA, respectively. Recurrent chromosome aberrations in our series were represented by aberrations of chromosome 14q in 5 patients, by trisomy 12 and 7q deletion in 4 cases each, and by abnormalities involving 11q and 13q in two cases each. These findings show that stimulation with ODN + IL2 offers more mitotic figures of better quality and results in an increased rate of clonal aberrations in SMZL, making this method ideal for prospective studies aiming at the definition of the prognostic impact of cytogenetic aberrations in this disorder.

Chromosome 14q32 translocations involving the immunoglobulin heavy chain locus in chronic lymphocytic leukaemia identify a disease subset with poor prognosis.

Immunophenotypic studies, fluorescence in situ hybridization (FISH) and conventional karyotyping were used to define the clinicobiological significance of 14q32 translocations involving the immunoglobulin gene locus (14q32/IGH) in 252 chronic lymphocytic leukaemia (CLL) patients. The following regions were studied: 13q14, centromere 12, 6q21; 11q22/ATM; 17p13/TP53, 14q32/IGH. Patients were classified as group 1 (favourable, i.e. 13q-single or normal), group 2 (intermediate risk, i.e. +12, 6q-, 1-2 anomalies), group 3 (unfavourable, i.e. 17p-, 11q-, complex karyotype), or group 4 (14q32/IGH translocation). Endpoints were treatment-free survival (TFS) and overall survival (OS). One hundred and ten patients were included in group 1, 99 in group 2, 25 in group 3 and 18 in group 4. 14q32/IGH translocation partners were identified in eight cases (BCL2 in five cases, BCL11A, CCND3 and CDK6 in one case each). group 4 showed shorter TFS versus groups 2 and 1 (25% patients treated at 2 months vs. 12 (P = 0.02) and 20 months (P = 0.002), respectively) and shorter OS (25% patients dead at 18 months versus 50 (P = 0.0003) and >60 months (P < 0.0001) respectively. The 14q32/IGH translocation maintained prognostic significance at multivariate analysis on TFS (P = 0.025) and OS (P < 0.001), along with advanced stage and CD38+. These findings show that the 14q32/IGH translocation predicts for an unfavourable outcome in CLL and that this cytogenetic subset might be included as a separate entity in a hierarchical cytogenetic classification of CLL.

Validation of an interphase fluorescence in situ hybridization approach for the detection of MLL gene rearrangements and of the MLL/AF9 fusion in acute myeloid leukemia.

To validate a 2-step FISH assay for the identification of the t(9;11)(p22;q23), 96 acute myeloid leukemias were studied by cytogenetic analysis, FISH and molecular biology. After a first FISH step using an MLL probe, 24/27 cases with 11q23 break showed MLL rearrangement. Southern blotting confirmed FISH data. In the second step, 24 cases with MLL rearrangement were studied using MLL and AF9 probes: 17/18 cases with t(9;11) showed MLL/AF9 fusion. In 6 patients with 11q23/MLL rearrangements other than t(9;11), FISH confirmed MLL involvement and excluded AF9 involvement. This is a reliable method for the identification of MLL/AF9 fusion in interphase cells, allowing for a reclassification of cases with suboptimal chromosome morphology. The frequency of deletion surrounding MLL and AF9 breakpoint is low.

Abnormalities of chromosomes 1p34-36, 4p16, 4q35, 9q11-32 and +7 represent novel recurrent cytogenetic rearrangements in chronic lymphocytic leukemia.

Karyotypes were studied in over 250 cases of CLL seen at our Institution and 12 cases with a previously undescribed chromosome abnormality were identified. Cytogenetic and clinicobiological features in these patients are described. Fluorescence in situ hybridization using probes for the detection of +12 and deletions of 13q14, 17p13, and 11q22-23 was performed. Hematologic and clinical data were reviewed and a review of the literature was performed. Twelve patients were found carrying the following aberrations in the stemline: abnormalities at 1p34 (n = 2), 4p16 (n = 2), 4q35 (n = 2), 9q11-32 (n = 4) and +7 (n = 2). Trisomy 12 was found in 3 cases, whereas no case carried 13q-, 11q-, 17p-. Our data showed that (i) aberrations involving 1p34 and 4p16 as isolated chromosome anomalies were preferentially associated with early stage disease; (ii) 4q35 anomalies were associated with a relatively aggressive disease, atypical morphology and with monoclonal gammopathy; (iii) rearrangements of 9q were characterized by atypical morphology and aggressive disease with splenic involvement; (iv) +7 be may associated with +12. 1p34-36; 4p16; 4q35; 9q and chromosome 7 represent novel recurrent rearranged sites in CLL, with a 0.5-3% incidence. Transformation in these patients seemingly occured through a cytogenetic route not involving the classical CLL-associated chromosome regions. These chromosome rearrangements may be associated with peculiar hematologic features.

"In vitro" evaluation of bone marrow angiogenesis in myelodysplastic syndromes: a morphological and functional approach.

In myelodysplastic syndromes (MDS), ineffective hematopoiesis and cytopenia may arise either from increased susceptibility to apoptosis of hematopoietic cells, or possibly from alterations of bone marrow stroma that contributes to defective development of marrow lineages. In order to test the significance of the endothelial growth in MDS patients, two in vitro assays are presented in this study: a long-term culture method for the detection of human bone marrow endothelial colonies (CFU-En) (77 patients) and a human primary model for the evaluation of the influence of a "bone marrow conditioned medium" on the formation of new vessels in a culture matrix ("angio-kit assay") (in 24 out 77 patients). The in vitro growth pattern of bone marrow CFU-En as well as the generation of microvessels in the angio-kit system was increased in RA, RARS and RAEB in comparison with controls. No CFU-En were detected in RAEB-T. The occurrence of large islands, formed by clusters of endothelial cells, unable to generate microcapillaries was also reported. Chromosomal abnormalities did not correlate with the angiogenetic pattern. These in vitro assays may constitute an useful approach to the analysis of angiogenesis in MDS.

Late appearance of the 11q22.3-23.1 deletion involving the ATM locus in B-cell chronic lymphocytic leukemia and related disorders. Clinico-biological significance.

BACKGROUND AND OBJECTIVES: Chromosome 11q22.3-23.1 deletions involving the ataxia-teleangiectasia mutated (ATM) locus (11q-/ATM+/-) are detected at diagnosis in 10-20% of cases of B-cell chronic lymphocytic leukemia (CLL) and are associated with a relatively aggressive disease. The aim of this study was to ascertain whether 11q-/ATM+/- may appear late during the course of the disease and to analyze its possible correlation with disease evolution. DESIGN AND METHODS: Eighty-two patients with CLL and related disorders, i.e. CLL/PL and prolymphocytic leukemia (PLL), without 11q- at diagnosis were sequentially ascertained at 1-2 year intervals by conventional cytogenetic analysis (CCA) and fluorescence in situ hybridization (FISH), using an ATM-specific probe. RESULTS: Eight patients acquired a submicroscopic 11q deletion 13-43 months after diagnosis: the diagnosis at presentation was CLL in 3 cases, CLL/PL in 3 cases and PLL in 2 cases. A 13q14 deletion preceded the development of 11q- in four patients; additional aberrations included +12 (three cases), 17p13 deletion and 6q21 deletion (one case each). The acquisition of the 11q deletion was more frequently found in those patients presenting with CLL/PL and PLL than typical CLL (p=0.0016) and with splenomegaly (p=0.003). Follow-up data showed that karyotype evolution (p=0.009) and cytological transformation (p<0.001) were associated with the acquisition of this cytogenetic lesion. The variables predicting for a shorter survival in this series included the 11q deletion (p=0.03), along with other classical clinicobiological parameters (performance status, advanced stage, splenomegaly, elevated serum beta2 microglobulin and lactate dehydrogenase levels. INTERPRETATION AND CONCLUSIONS: a) Submicroscopic 11q deletion involving the ATM locus may, in some instances, represent a secondary change in CLL, CLL/P and PLL, suggesting that sequential FISH analyses are necessary to detect this chromosome anomaly in some patients; b) the acquisition of 11q-/ATM deletion may play a role in determining cytological transformation and disease progression of CLL and related disorders.