Sara Martinelli

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

Abstract 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.

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.

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 β2‐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.

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 <5cm. 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.

Response to ibrutinib of refractory life-threatening autoimmune hemolytic anemia occurring in a relapsed chronic lymphocytic leukemia patient with 17p deletion

Autoimmune hemolytic anemia (AIHA) is one of the most common disease-specific complications of chronic lymphocytic leukemia (CLL), occurring in 3–10% of patients, with a higher incidence in advanced stages or in cases with an unmutated configuration of the variable portion of the immunoglobulin heavy chain gene (IGHV).[1] AIHA is mediated most frequently by IgG auto-antibodies produced by nonmalignant B cells, or by IgM antibodies in <10% of cases.[2] Activated CLL cells may bind and present the erythrocyte protein band 3 and activate CD4þ cells in a HLA-DR-dependent way, playing a role as possible initiators of the hemolytic process.[3] Furthermore, CLL cells have been shown capable of causing regulatory T-cell dysfunction which may contribute to the development of this complication.[4] Even though AIHA had no independent effect on survival in two studies,[5,6] this complication has been shown to be more frequent in CLL subsets with unfavorable prognostic features, i.e. 11q-, 17p-, CD38þ [6] and patients with severe anemia refractory to steroids and alkylating agents have been described.[5] In steroid-refractory patients, CLL-directed treatment including rituximab, with or without cytotoxic agents, or alemtuzumab has been shown to induce response in some cases.[1] Chemorefractory disease and/or inability to tolerate chemoimmunotherapy represent a major problem in patients not responding to steroids who may succumb to infections, AIHA-related complications [5] or disease progression. Hence, effective and tolerable treatment represents an unmet need in refractory CLL-associated AIHA. In March 2011, a 62-year-old male was referred to our institution with a diagnosis of Rai stage II asymptomatic CLL and abnormal bilirubin excretion (Gilbert syndrome) with indirect bilirubin levels ranging between 2 and 3 mg/dl. Hematologic work-up was performed as previously reported,[7] documenting a typical morphology and immunophenotype and negativity for CD38 and ZAP-70. The karyotype was normal and no aberration was detected by fluorescence in situ hybridization (FISH). The Ig heavy chain variable (IGHV) region was mutated; no mutations of exons 4–9 of the TP53 gene were detected. In October 2012, the patient presented disease progression with fatigue, peripheral edema, hyperleukocytosis (lymphocytes 195,000/ml), adenopathies and symptomatic splenomegaly (8 cm below the left costal margin). Clonal evolution with 17p deletion in 22% of the cells was documented. Fludarabine, cyclophosphamide and rituximab (FCR regimen) treatment was started and soon after the second cycle, the patient presented with dyspnea and jaundice. Hemoglobin (Hb) levels were low (Hb 6.5 g/ dl) with high reticulocyte count. Elevated serum lactate dehydrogenase (LDH) levels (694 U/l normal values 240–480 U/l) and indirect bilirubin levels (5.08 mg/dl; normal values 0.10–1.00 mg/dl), low haptoglobulin levels (5 mg/dl; normal values 30–200 mg/dl) and strong positivity for IgG (5þ) on direct antiglobulin test (DAT) were consistent with a diagnosis of AIHA. High-dose methylprednisolone and rituximab were administered along with intravenous (iv) Ig (1 g/kg for two consecutive days). Due to persistent hemolysis after 4 weeks, the patient was treated with bendamustine and rituximab for a total of six cycles. Normalization of Hb levels and hemolysis-associated laboratory parameters was observed over a 3-month period and a partial response (PR) according to NCI criteria [8] was documented at the end of treatment, with a normalization of blood

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.

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

Abstract 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.

Diagnostic work-up for clinical and prognostic assessment of acute leukaemia

SummaryAcute leukaemia is a neoplastic disorder of haematopoietic precursors characterized by a rapid clinical course and a relatively high early death rate in spite of recent therapeutic progress. Prompt and reliable diagnostic and prognostic assessment has a favourable impact on patient outcome. Diagnostic suspicion relies on signs and symptoms of bone marrow failure or quali-/quantitative abnormalities of blood cells, which are accurately detected by modern automated counters. Cytomorphological examination of a stained blood and bone marrow film plays a key role in the diagnostic process, with relevant additional information coming from immunophenotyping of blast cells. Cytogenetic and molecular genetic data are the basis of prognostic stratification. In this context, a coordinated intervention of specialized laboratories combining solid expertise in each field involved in the diagnostic work-up is essential; at the same time, the availability of all the required technologies at the same hospital structure would probably lack efficiency due to the low number of tests performed. The organization of laboratory networks, either at regional or national level, especially in the context of clinical trials, may offer a great opportunity for centralization of more sophisticated technologies in reference laboratories, each highly specialized in a particular field of investigation and all interconnected. The challenge for laboratory haematology is the reorganization of clinical and scientific activity according to this model, without loosing educational potential in favour of new generations of medical doctors, haematologists, biologists and laboratory technicians.RiassuntoLa leucemia acuta è una malattia neoplastica dei precursori emopoietici caratterizzata da una rapida evolutività clinica, con un tasso di mortalità precoce che si mantiene elevato nonostante i continui progressi terapeutici. La tempestività di un corretto inquadramento diagnostico secondo criteri classificativi internazionali e di una precisa stratificazione prognostica ha un impatto clinico dimostrato in termini di outcome. Il sospetto diagnostico può essere formulato sulla base di sintomi o segni, in genere associati alla soppressione della normale emopoiesi, oppure sulla base di alterazioni emocromocitometriche, quantitative o qualitative, sensibilmente rilevate dai moderni contatori automatici. La valutazione morfologica dello striscio di sangue periferico e della citologia midollare rappresenta un momento fondamentale nell’iter diagnostico e può ricevere contributi di rilievo dall’indagine immunofenotipica. La stratificazione prognostica si fonda su dati citogenetici e molecolari sempre più specifici e dettagliati. In questo contesto, l’integrazione tra le diverse competenze cliniche e laboratoristiche coinvolte nel processo diagnostico acquista grande importanza; allo stesso tempo però la disponibilità presso ogni centro delle risorse strumentali necessarie e di personale altamente qualificato nei diversi settori si scontra con esigenze di efficienza economica. Lo sviluppo, a livello locale e nazionale, di reti di laboratori a elevata specializzazione in ognuno dei differenti ambiti diagnostici e di ricerca, consente una proficua ottimizzazione delle risorse. La sfida che si propone quindi all’Ematologia di laboratorio è quella di una riorganizzazione dell’attività clinica e scientifica secondo un modello che soddisfi elevati standard di accuratezza diagnostica ed efficienza economica, senza sacrificare le esigenze formative delle nuove generazioni di medici, specialisti ematologi, biologi e tecnici di laboratorio.