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.

Complete remission of Sweet's syndrome after azacytidine treatment for concomitant myelodysplastic syndrome.

Sweet's syndrome is a rare condition with potentially disabling implications, characterized by painful skin lesions due to neutrophilic dermal infiltration and systemic inflammatory symptoms. A significant proportion of cases is malignancy associated. Hematologic neoplasms, particularly acute myeloid leukemia and myelodysplastic syndromes, are the most commonly associated malignant conditions. Here, we describe the first case of clinical remission of refractory Sweet's syndrome following hypomethylating therapy with azacytidine in a patient with myelodysplastic syndrome who concurrently obtained a complete hematologic response.

Genetic subclonal complexity and miR125a-5p down-regulation identify a subset of patients with inferior outcome in low-risk CLL patients.

The majority of patients with chronic lymphocytic leukemia (CLL) and favorable prognostic features live for long periods without treatment. However, unexpected disease progression is observed in some cases. In a cohort of untreated CD38- CLL patients with normal FISH or isolated 13q- we found that, by fluorescence in situ hybridization (FISH), 16/28 cases presented, within immunomagnetic sorted CD38+ cells, genetic lesions undetectable in the CD38- fraction. These patients showed a shorter time to first treatment (TTFT, p=0.0162) in comparison to cases without FISH lesions in CD38+ cells. Patients with FISH abnormalities in CD38+ cells showed a distinctive microRNA profile, characterized by the down-regulation of miR-125a-5p both in the CD38- and CD38+ populations. In an independent cohort of 71 consecutive untreated CD38- CLL with normal FISH or isolated 13q-, a lower miR125a-5p expression was associated with a shorter TTFT both in univariate and multivariate analysis (p=0.003 and 0.016, respectively) and with a higher prevalence of mutations (7/12 vs 0/8, p=0.015) as assessed by next-generation sequencing. In conclusion, our data showed previously unrecognized subclonal heterogeneity within the CD38+ fraction of CD38- CLL patients with low-risk FISH findings and suggested an association between down-regulated miR-125a-5p expression, genetic complexity and worse outcome.

Delirium in acute promyelocytic leukemia patients: two case reports.

BACKGROUND: Delirium is a frequently misdiagnosed and inadequately treated neuropsychiatric complication most commonly observed in terminally ill cancer patients. To our knowledge this is the first report describing delirium in two patients aged less than 60 years and enrolled in an intensive chemotherapeutic protocol for acute promyelocytic leukemia. CASE PRESENTATION: Two female Caucasian acute promyelocytic leukemia patients aged 46 and 56 years developed delirium during their induction treatment with all-trans retinoic acid and idarubicin. In both cases symptoms were initially attributed to all-trans retinoic acid that was therefore immediately suspended. In these two patients several situations may have contribute to the delirium: in patient 1 a previous psychiatric disorder, concomitant treatments with steroids and benzodiazepines, a severe infection and central nervous system bleeding while in patient 2 steroid treatment and isolation. In patient 1 delirium was treated with short-term low-doses of haloperidol while in patient 2 non-pharmacologic interventions had a beneficial role. When the diagnosis of delirium was clear, induction treatment was resumed and both patients completed their therapeutic program without any relapse of the psychiatric symptoms. Both patients are alive and in complete remission as far as their leukemia is concerned. CONCLUSIONS: We suggest that patients with acute promyelocytic leukemia eligible to intensive chemotherapy should be carefully evaluated by a multisciplinary team including psychiatrists in order to early recognize symptoms of delirium and avoid inadequate treatments. In case of delirium, both pharmacologic and non-pharmacologic interventions may be considered.

Clinical heterogeneity of de novo 11q deletion chronic lymphocytic leukaemia: prognostic relevance of extent of 11q deleted nuclei inside leukemic clone.

Deletion on the long arm of chromosome 11 occurs in 5-20% of chronic lymphocytic leukaemia (CLL) patients. We analysed clinical-biological characteristics of 131 CLL patients carrying 11q deletion documented before therapy (de novo 11q deleted CLL). De novo 11q deleted CLL were characterized by high frequencies of unmutated immunoglobulin variable heavy genes, multiple fluorescence in situ hybridization aberrations and lymph node involvement. Factors significantly associated with shorter time to first treatment (TTFT) were advanced Binet stages, high white blood cell count, increased ß2 -microglobulin levels, 17p in addition, splenomegaly and more extensive lymphadenopathy. We found that patients with <25% 11q deleted nuclei (n = 22) experienced longer TTFT compared with patients with ≥25% 11q deleted nuclei (n = 87; median TTFT, 40 vs. 14 months, p = 0.011) and also showed better response to treatments (complete response, 50% vs. 21%, p = 0.016). The variables identified by multivariate analysis as independently associated with reduced TTFT were advanced Binet stages [hazard ratio (HR) 4.69; p < 0.001] and ≥25% 11q deleted nuclei (HR 4.73; p = 0.004). De novo 11q deleted CLLs exhibit variable clinical outcome. The percentage of deleted nuclei inside leukemic clone should be included in the prognostic definition of therapy-naïve 11q deleted CLL patients.

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.

An Italian retrospective study on the routine clinical use of low-dose alemtuzumab in relapsed/refractory chronic lymphocytic leukaemia patients.

Low-dose alemtuzumab has shown a favourable toxicity profile coupled with good results in terms of efficacy in relapsed/refractory chronic lymphocytic leukaemia (CLL). We conducted a multicentre retrospective study on the routine clinical use of low-dose alemtuzumab in this patient setting. One hundred and eight relapsed/refractory CLL patients from 11 Italian centres were included in the analysis. All patients had an Eastern Cooperative Oncology Group performance status ≤2 and the majority (84%) had adenopathies <5 cm. Low-dose alemtuzumab was defined as a total weekly dose ≤45 mg and a cumulative dose ≤600 mg given for up to 18 weeks. The overall response rate was 56% (22% complete remissions). After a median follow-up of 42.2 months, the median overall survival and progression-free survival were 39.0 and 19.4 months, respectively. In univariate analysis, response was inversely associated with lymph node (P = 0.01) and spleen (P = 0.02) size, fludarabine-refractoriness (P = 0.01) and del(11q) (P = 0.009). Advanced age and del(17p) were not associated with a worse outcome. Cumulative dose of alemtuzumab was not associated to response. Toxicities were usually mild and manageable; severe infections occurred in seven patients (7%) during therapy. This retrospective analysis confirms that low-dose alemtuzumab is a valid and currently used therapeutic option for the treatment of relapsed/refractory CLL.

Magnetic resonance imaging to identify classic and nonclassic forms of ferroportin disease.

The ferroportin-related disorder is an increasingly recognized cause of hereditary iron overload. Based on the in vitro behavior of different ferroportin mutant subsets, it was suggested that different forms of the disorder might exist in humans. We used MRI to address this question in vivo in 22 patients from four different pedigrees carrying different ferroportin mutations: A77D, N144H, G80S and Val 162del. We found that, based on the iron status of spleen and bone macrophages, two different forms of the disease can be identified: a classic, common form, characterized by hepatocyte, splenic macrophage and bone marrow macrophage iron retention in patients carrying the A77D, G80S and Val 162del ferroportin variants; a rarer non-classic form, associated with liver iron overload but normal spleen and bone marrow iron content in patients with the N144H mutation. The two forms are likely caused by lack- or gain-of-protein function, respectively. Interestingly, in treated patients with the classic form, the spleen and the spine show appreciable iron accumulation even when serum ferritin is normal and liver iron content low. In conclusion, MRI is a useful non-invasive diagnostic tool to categorize and diagnose the disorder, monitor the status of iron depletion and gain insights on its natural history and management.