Additional lesions identified by genomic microarrays are associated with an inferior outcome in low-risk chronic lymphocytic leukaemia patients.

We explored the relevance of genomic microarrays (GM) in the refinement of prognosis in newly diagnosed low-risk chronic lymphocytic leukaemia (CLL) patients as defined by isolated del(13q) or no lesions by a standard 4 probe fluorescence in situ hybridization (FISH) analysis. Compared to FISH, additional lesions were detected by GM in 27 of the 119 patients (22.7%). The concordance rate between FISH and GM was 87.4%. Discordant results between cytogenetic banding analysis (CBA) and GM were observed in 45/119 cases (37.8%) and were mainly due to the intrinsic characteristics of each technique. The presence of additional lesions by GM was associated with age > 65 years (p = 0.047), advanced Binet stage (p = 0.001), CLL-IPI score (p < 0.001), a complex karyotype (p = 0.004) and a worse time-to-first treatment in multivariate analysis (p = 0.009). Additional lesions by GM were also significantly associated with a worse time-to-first treatment in the subset of patients with wild-type TP53 and mutated IGHV (p = 0.025). In CLL patients with low-risk features, the presence of additional lesions identified by GM helps to identify a subset of patients with a worse outcome that could be proposed for a risk-adapted follow-up and for early treatment including targeted agents within clinical trials.

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.

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.

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.

Combined interphase fluorescence in situ hybridization elucidates the genetic heterogeneity of T-cell acute lymphoblastic leukemia in adults.

BACKGROUND: Molecular lesions in T-cell acute lymphoblastic leukemias affect regulators of cell cycle, proliferation, differentiation, survival and apoptosis in multi-step pathogenic pathways. Full genetic characterization is needed to identify events concurring in the development of these leukemias. DESIGN AND METHODS: We designed a combined interphase fluorescence in situ hybridization strategy to study 25 oncogenes/tumor suppressor genes in T-cell acute lymphoblastic leukemias and applied it in 23 adult patients for whom immunophenotyping, karyotyping, molecular studies, and gene expression profiling data were available. The results were confirmed and integrated with those of multiplex-polymerase chain reaction analysis and gene expression profiling in another 129 adults with T-cell acute lymphoblastic leukemias. RESULTS: The combined hybridization was abnormal in 21/23 patients (91%), and revealed multiple genomic changes in 13 (56%). It found abnormalities known to be associated with T-cell acute lymphoblastic leukemias, i.e. CDKN2A-B/9p21 and GRIK2/6q16 deletions, TCR and TLX3 rearrangements, SIL-TAL1, CALM-AF10, MLL-translocations, del(17)(q12)/NF1 and other cryptic genomic imbalances, i.e. 9q34, 11p, 12p, and 17q11 duplication, del(5)(q35), del(7)(q34), del(9)(q34), del(12)(p13), and del(14)(q11). It revealed new cytogenetic mechanisms for TCRB-driven oncogene activation and C-MYB duplication. In two cases with cryptic del(9)(q34), fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction detected the TAF_INUP214 fusion and gene expression profiling identified a signature characterized by HOXA and NUP214 upregulation and TAF_I, FNBP1, C9orf78, and USP20 down-regulation. Multiplex-polymerase chain reaction analysis and gene expression profiling of 129 further cases found five additional cases of TAF_I-NUP214-positive T-cell acute lymphoblastic leukemia. CONCLUSIONS: Our combined interphase fluorescence in situ hybridization strategy greatly improved the detection of genetic abnormalities in adult T-cell acute lymphoblastic leukemias. It identified new tumor suppressor genes/oncogenes involved in leukemogenesis and highlighted concurrent involvement of genes. The estimated incidence of TAF_I-NUP214, a new recurrent fusion in adult T-cell acute lymphoblastic leukemias, was 4.6% (7/152).

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.

Prognostic impact of genetic characterization in the GIMEMA LAM99P multicenter study for newly diagnosed acute myeloid leukemia.

BACKGROUND: Recent advances in genetic characterization of acute myeloid leukemia indicate that combined cytogenetic and molecular analyses provide better definition of prognostic groups. The aim of this study was to verify this prospectively in a large group of patients. DESIGN AND METHODS: Genetic characterization was prospectively carried out in 397 patients with acute myeloid leukemia (median age, 46 years) receiving uniform treatment according to the LAM99P protocol of the Italian GIMEMA group. The impact of genetic markers on response to therapy and outcome was assessed by univariate and multivariate analyses. RESULTS: For induction response, conventional karyotyping identified three groups with complete remission rates of 92%, 67% and 39% (p<0.0001). Complete remission rates in NPM1 mutated (NPM1+) and wild-type (NPM1-) groups were 76% and 60%, respectively, for the whole population and 81% and 61% in the group with normal karyotype (p<0.001 and p=0.026, respectively). Multivariate analysis indicated that low risk karyotype and NPM1+ were independent factors favorably affecting complete remission. Multivariate analysis of overall and disease-free survival among 269 patients who achieved complete remission showed a significant impact of karyotype on both estimates and of FLT3 status on disease free-survival (FLT3-ITD vs. FLT3 wild-type, p=0.0001). NPM1 status did not significantly influence disease free-survival in either the whole population or in the patients with a normal karyotype in this series, probably due to the low number of cases analyzed. CONCLUSIONS: These results reiterate the prognostic relevance of combining cytogenetic and mutational analysis in the diagnostic work up of patients with acute myeloid leukemia.

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.

Methylenetetrahydrofolate reductase C677T and A1298C gene variants in adult non-Hodgkin's lymphoma patients: association with toxicity and survival.

BACKGROUND AND OBJECTIVES: Common methylenetetrahydrofolate reductase gene variants (MTHFR C677T and A1298C) have been described to have opposite effects on cancer patients. They may reduce cancer susceptibility and increase drug-related toxicity when folate antagonists (e.g. methotrexate) are utilized. We analyzed 110 patients with high-grade non-Hodgkin's lymphoma (NHL), 68 of whom were eligible for a chemotherapy combination containing methotrexate (MACOP-B) and 42 for chemotherapy without methotrexate (CHOP). DESIGN AND METHODS: Patients were genotyped by polymerase chain reaction and stratified by MTHFR variants. These data were related to the toxicity (WHO grade GO-4) that the patients suffered and their survival. Overall 64 cases (58.2%) developed some form of toxicity and 23 (20.9%) had grade 3/4 toxicity. RESULTS: When considering toxicity of any grade (grade 1-4), the 677TT genotype was significantly over-represented among cases with mucositis (OR=4.85; 95% CI, 1.47-15.97; p=0.009) and those with hepatic toxicity (OR=3.43; 95% CI, 0.99-11.86; p=0.052). Sub-analyses in the group treated with MACOP-B showed a slight increase in the risk of developing mucositis (OR=5.22; 95% CI, 1.20-27.27; p=0.03), and a strong increase in the risk of hepatic toxicity (OR=7.08; 95% CI, 1.38-36.2; p=0.019) and thrombocytopenia (OR=7.69, 95% CI 1.0-58.94; p=0.05). Interestingly, compared to the risk of developing toxicity of any grade, the risk of developing severe (grade 3/4) mucositis was almost doubled in the whole group of cases with 677TT (OR=8.13; 95% CI 1.61-41.04; p=0.011) and dramatically increased in the MACOP-B-treated cases with this gene variant (OR=24.6; 95% CI 2.49-87.41; p=0.001). There were significant results for 1298CC cases exclusively for mucositis (any grade, OR=5.33; 95% CI, 1.25-22.70; p=0.023 and OR=9.15; 95% CI, 1.14-73.41; p=0.037; for the whole group and the MACOP-B-treated group, respectively). Similarly, the risk of 1298CC patients developing severe mucositis increased (OR=9.24; 95% CI, 1.47-58.0; p=0.017 and OR=11.53; 0.93-143.18; p=0.057; in the whole group and in the MACOP-B-treated group, respectively). Event-free survival analysis revealed a lower probability of event-free survival at 5 years for 677T-carriers (log-ranks, p=0.05 and p=0.07 in the whole group and in the MACOP-B-treated group, respectively). More significant results were obtained when 1298CC cases were excluded from the reference group (log-ranks, p=0.03 and p=0.04, respectively). No significant associations were found in the CHOP-treated group. INTERPRETATION AND CONCLUSIONS: Our data suggest that MTHFR gene variants play a critical role in NHL outcome, possibly by interfering with the action of methotrexate with significant effects on toxicity and survival. Genotyping of folate pathway gene variants might be useful to enable reduction of chemotherapy toxicity and/or to improve survival by indicating when dose adjustments or alternative treatments are necessary.

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.

Dual-color split signal fluorescence in situ hybridization assays for the detection of CALM/AF10 in t(10;11)(p13;q14-q21)-positive acute leukemia.

We developed dual-color split fluorescence in situ hybridization (FISH) assays to detect AF10 and/or CALM rearrangements. Among nine cases of acute leukemia with translocation breakpoints at 10p13 and 11q14-21, a CALM/AF10 rearrangement was found in seven and was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) in all. In 2/7 cases, FISH detected CALM/AF10 in extramedullary leukemic infiltrations in the mediastinum and breast. As expected, FISH was less sensitive than RT-PCR for disease monitoring of CALM-AF10 positive cases. This new FISH assay reliably discriminates between MLL/AF10 and CALM/AF10 genomic rearrangements, identifies variant and complex CALM/AF10 translocations and detects the CALM/AF10 rearrangement in extramedullary leukemic infiltrations.

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.

"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.

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.

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.

rHuEpo administration in patients with low-risk myelodysplastic syndromes: evaluation of erythroid precursors' response by fluorescence in situ hybridization on May-Grunwald-Giemsa-stained bone marrow samples.

The issue of whether, in patients affected by myelodysplastic syndromes (MDS), haematological response to cytokines, particularly to recombinant human erythropoietin (rHuEpo), is a phenomenon related to the stimulation of normal haemopoietic cells or to the differentiation of cells belonging to the abnormal clone remains an open question. To assess the pattern of response to rHuEpo treatment of bone marrow (BM) cells, we evaluated in 13 low-risk MDS patients with known cytogenetic abnormalities the number of cytogenetically normal and abnormal cells by conventional cytogenetic analysis (CCA) and by a fluorescence in situ hybridization (FISH) technique, enabling the simultaneous visualization of FISH chromosomal abnormalities in morphologically and immunophenotypically identifiable BM elements. Patients responding to rHuEpo presented a lower number of abnormal metaphases at diagnosis in comparison with patients who did not respond (22.74% vs 76.23%, P = < 0.001). This was confirmed by the combined morphological FISH analysis, showing that, before treatment, BM samples from patients responding to rHuEpo had a lower proportion of both FISH abnormal erythroid (36.48% vs 66.93%, P = 0.002) and myeloid (40.76% vs 67.70%, P = 0.014) elements than unresponsive patients. After rHuEpo treatment, responding patients presented a significantly lower proportion of FISH abnormal erythroid precursors than observed before treatment (16.93%vs 36.48%, P = 0.017). Likewise, in responding patients, a significantly lower proportion of FISH abnormal erythroid elements (16.93% vs 66.30%, P < 0.001) was detected in comparison with unresponsive patients. These findings provide evidence that, in low-risk MDS patients with known cytogenetic abnormalities, response to rHuEpo may be due to the proliferation of karyotypically normal erythroid precursors, possibly representing residual normal erythroid elements.