Barbara Rocca

World Health Organization classification in combination with cytogenetic markers improves the prognostic stratification of patients with de novo primary myelodysplastic syndromes

This study correlated chromosomal defects with French–American–British (FAB)/World Health Organization (WHO) classification subtypes, proposed a revised International Prognostic Scoring System (IPSS) cytogenetic grouping; and established which classification, when used with the IPSS cytogenetic categories, best predicted clinical outcome in the myelodysplastic syndromes (MDS). A higher prevalence of chromosomal defects and distinct defects were observed in patients with multi‐lineage dysplasia and a blast cell percentage >10%. Abnormalities of the long arm of chromosome 3, del(7)(q31q35), trisomy 8, del(11)(q14q23), del(12p) and 20q‐ could be segregated from their respective IPSS cytogenetic categories and used to develop new cytogenetic subgroups. Clinical parameters, FAB/WHO classification, IPSS score and standard or revised cytogenetic categories were statistically relevant for overall survival (OS) and progression‐free intervals (PFI) and were included within five distinct multivariate models compared by the Akaike Information Criterion. To predict OS, the best models included age, WHO classification and standard or revised IPSS cytogenetic categories; to predict PFI, the best model included the same variables and revised cytogenetic categories. In conclusion, (i) the WHO classification was associated with a more homogeneous cytogenetic pattern than the FAB classification, (ii) WHO classification and standard/revised IPSS cytogenetic categories were much more effective than IPSS for predicting MDS clinical outcome, (iii) revised cytogenetic subgroups predicted PFI more effectively than standard categories.

Reversible pulmonary arterial hypertension likely related to long-term, low-dose dasatinib treatment for chronic myeloid leukaemia

Dasatinib is a potent inhibitor of tyrosine-kinases (TKI), such as CR-ABL, PDGFR, c-kit, and of SRC-family kinases. It is indicated or treatment of Chronic Myeloid Leukaemia (CML) and Ph-positive cute Lymphoblastic Leukaemia after imatinib failure/intolerance, nd has recently obtained authorisation as first-line therapy in CML, ased on results in newly diagnosed patients with chronic phase ML [1]. At the previously recommended dose of 70 mg BID, the ost relevant non-haematological side effect of dasatinib therapy as exudative pleural effusion (PE), which has been observed in up o 14–35% of patients [2], and has been related to an autoimmuneediated mechanism or inhibition of PDGFR, as opposed to fluid etention. The occurrence of grade 3–4 PE, however, has decreased o less than 2% at the presently approved dosage of 100 mg QD 1–4] Other rare adverse events involving the cardiovascular sysem and lungs are cardiac failure, QTc prolongation, pericardial ffusion, pulmonary parenchymal infiltrates and transient increase n right ventricular systolic pressure in conjunction with PE [2]. To ur knowledge, only three cases of reversible pulmonary arterial ypertension (PAH) on dasatinib have been published [5–7]. Here, e describe in detail an additional case of PAH that occurred under asatinib therapy and resolved after drug discontinuation. In April 2006, a 53 year-old female patient (height 152 cm, eight 54 kg) was diagnosed with CML, low Sokal risk. She was non-smoker and had been treating essential hypertension with a alcium-channel blocker and a beta-blocker for 4 years; no other o-morbidities were evident. Imatinib 400 mg daily was started nd complete cytogenetic response was documented 6 months ater. As molecular response was suboptimal, imatinib was escaated to 600 mg daily and in February 2008 dasatinib was started t the dose of 100 mg QD. On baseline evaluation, ECG was normal nd transthoracic Doppler echocardiography showed only minimal itral valve regurgitation. After 1 month, the patient complained of ever without any other symptoms. Infectious diseases were ruled ut. Laboratory tests for connective tissue disorders were negative, hile a low-level transient monoclonal IgGk was detected. On bone arrow evaluation, plasma cells were less than 5%. Chest-X ray as normal and echocardiography was unchanged. No peripheral arge granular lymphocytosis was observed. Fever was attributed o a hypersensitivity reaction and dasatinib was temporally witheld. It was then reintroduced at the reduced dose of 70 mg QD and he fever gradually decreased and disappeared. Complete cytogeetic response was maintained and a major molecular response BCR-ABL/ABL 0.09% according to International Scale) was docuented in February 2009. The patient remained on dasatinib 70 mg D without any problems until September 2010, when she noticed rogressive exertional dyspnoea and fatigue. In October 2010, she omplained of breathlessness, and moderate liver enlargement and

Clinical relevance of cytogenetics in myelodysplastic syndromes

Abstract:  Myelodysplastic syndromes (MDS) are a group of heterogeneous stem cell disorders with different clinical behaviors and outcomes. Conventional cytogenetics (CC) studies have demonstrated that the majority of MDS patients harbor clonal chromosome defects. The probability of discovering a chromosomal abnormality has been increased by fluorescence in situ hybridization (FISH), which has revealed that about 15% of patients with a normal chromosome pattern on CC may instead present cryptic defects. Cytogenetic abnormalities, except for the interstitial long‐arm deletion of chromosome 5 (5q−), are not specific for any French‐American‐British (FAB)/World Health Organization (WHO) MDS subtypes, demonstrate the clonality of the disease, and identify peculiar morphological entities, thus confirming clinical diagnosis. In addition, chromosome abnormalities are independent prognostic factors predicting overall survival and the likelihood of progression in acute myeloid leukemia.

Molecular Genetics of Acute Myeloid Leukemia

Abstract: Recurring chromosomal abnormalities are detected in most patients with acute myeloid leukemia (AML). They may be associated with a distinct AML FAB subtype or may identify distinct clinicobiological entities within the same FAB subtype. Therefore, cytogenetic investigation has a pivotal role in AML diagnosis. In addition, it is one of the most valuable prognostic determinants of the disease, as recently demonstrated. The development of new molecular techniques, such as reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization, has allowed perfect definition of the chromosome regions containing genes with a crucial role in normal hemopoiesis and leukemia. Understanding the action of such genes provides new insights into AML pathogenesis and has led us to envisage new therapeutic options.

Long‐term follow up with conventional cytogenetics and band 13q14 interphase/metaphase in situ hybridization monitoring in monoclonal gammopathies of undetermined significance

Summary. One‐third of patients with monoclonal gammopathy of undetermined significance (MGUS) may progress to multiple myeloma (MM) and may develop a long arm deletion of chromosome 13 (13q–). As the incidence of 13q–, time of development and prognostic impact in MGUS patients is still under debate, we decided to perform serial sequential conventional cytogenetics (CC) and metaphase/interphase fluorescence in situ hybridization (FISH) analyses on bone marrow mononuclear cells obtained from 18 asymptomatic, untreated MGUS patients. Median follow up was 30 months (range 6–72). Interphase FISH identified a 13q14 deletion in five out of 18 patients (on clinical diagnosis in one patient and during the follow up in the remaining four patients). Subsequently, metaphase FISH and CC also identified the deletion in four out of five patients. All five of the patients progressed to MM 6–12 months after 13q– identification, without developing any FISH determined JH rearrangements. MM progression also occurred in two other karyotypically normal patients. We conclude that: (i) the extent of the 13q deletion does not vary during the clinical outcome; (ii)13q– plays a crucial role in MGUS/MM pathogenesis and confers a proliferative advantage to clonal plasma cells being initially demonstrated by interphase FISH and only afterwards by metaphase FISH and CC; and (iii) association of 13q– with t(4;14)(p16.3;q32) remains to be demonstrated. However, a transition from MGUS to MM may also occur in patients with normal karyotypes or other abnormalities, suggesting the possibility of distinct pathogenetic pathways.

Detection of TET2 abnormalities by fluorescence in situ hybridization in 41 patients with myelodysplastic syndrome

TET2 haplo-insufficiency occurs through different molecular mechanisms and is promptly revealed by array comparative genomic hybridization, single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS). Fluorescence in situ hybridization (FISH) can effectively demonstrate TET2 deletions and is often used to validate molecular results. In the present study 41 MDS patients with and without 4q abnormalities were analyzed with a series of bacterial artificial chromosome (BAC) probes spanning the 4q22.3-q25 region. On conventional cytogenetic (CC) studies, a structural defect of the long arm of chromosome 4 (4q) was observed in seven patients. In three, one each with a t(1;4)(p21;q24), an ins(5;4)(q23;q24qter), and a t(4;17)(q31;p13) as the sole chromosomal abnormality, FISH with the RP11-356L5 and RP11-16G16 probes, which cover the TET2 locus, produced one signal only. Unexpectedly, this same result was achieved in 3 of the remaining 34 patients. Thus, a TET2 deletion was observed in a total of six patients (14.6%). TET2 deletion was not correlated with any particular clinical findings or outcome. These findings demonstrate that 1) FISH is an effective and economical method to reveal cryptic abnormalities of band 4q22-q24 resulting in TET2 deletions; 2) in these patients, TET2 deletion is the unifying genetic event; and 3) the different breakpoints within the 4q22-q25 region suggest that deletions are not mediated by repetitive sequences.

Alternative splicing of hTERT: a further mechanism for the control of active hTERT in acute myeloid leukemia

Abstract hTERT component is the key regulator of telomerase. Alternatively spliced variants of hTERT generate different telomerase activity. The goal of the study was to determine the role of different hTERT isoforms in the regulation of telomerase expression in AML patients. Among the 97 studied patients, 45 had a complex karyotype and 52 a normal karyotype. hTERT isoforms expression was determined in bone marrow samples by q-RT-PCR, using SYBR Green I. hTERT expression was lower in AML patients than controls (median 2.5 vs. 10.1, p = .003), though no difference was observed between the complex and normal karyotype (median 3.2 vs. 2.3, p = .37). High trans-dominant negative isoform expression increased the response rate by two. High expression of inactive product (–α – β) was shown to increase the risk of relapse by about three times. In conclusion, our data suggest an intriguing link between the control of hTERT isoforms expression and AML outcome.

‘Real-life’ study of imatinib therapy in chronic phase-chronic myeloid leukemia: A novel retrospective observational longitudinal analysis

Objectives: Imatinib is a cornerstone of treatment of chronic myeloid leukemia. It remains unclear whether transient treatment discontinuation or dose changes affect outcome and this approach has not yet been approved for use outside clinical trials. Methods: We conducted a retrospective single-institution observational study to evaluate factors affecting response in ‘real-life’ clinical practice in 138 chronic myeloid leukemia patients in chronic phase treated with imatinib. We used a novel longitudinal data analytical model, with a generalized estimating equation model, to study BCR–ABL variation according to continuous standard dose, change in dose or discontinuation; BCR–ABL transcript levels were recorded. Treatment history was subdivided into time periods for which treatment was given at constant dosage (total 483 time periods). Molecular and cytogenetic complete response was observed after 154 (32%) and 358 (74%) time periods, respectively. Results: After adjusting for length of time period, no association between dose and cytogenetic complete response rate was observed. There was a significantly lower molecular complete response rate after time periods at a high imatinib dosage. Discussion: This statistical approach can identify individual patient variation in longitudinal data collected over time and suggests that changes in dose or discontinuation of therapy could be considered in patients with appropriate biological characteristics.

Late post-transplant recurrence of Chronic Myeloid Leukaemia after immuno-chemotherapy for secondary Non-Hodgkin's Lymphoma

We read with great interest the paper published by Norkin et al. 1]. They described six cases of very late recurrences of leukaemia nd discussed the possible mechanisms by which late relapses ccur. Two of reported cases concern patients who relapsed 13 and 7 years, respectively, after allogeneic peripheral stem cell translantation for Chronic Myeloid Leukaemia (CML) performed in the re-Imatinib era and in the corresponding “Comments” section the uthors hypothesize that relapses were likely due to loss of immune urveillance. However, specific reasons for immune system failure ere not identified in their cases. We would like to report on a late molecular relapse in a CML atient who received Rituximab-based immuno-chemotherapy for secondary post-transplant Non-Hodgkin’s Lymphoma (NHL). A 7-year-old male was diagnosed with chronic phase CML in 1989 nd proceeded with allogeneic bone marrow transplant from his ully HLA matched brother in May 1990. Myeloablative condiioning regimen consisted of TBI and Cyclophosphamide. T-cell epletion of the graft was not performed. Cyclosporine was used s prophylaxis against Graft Versus Host Disease (GVHD). Eleven ays after transplant the patient developed a grade 2 acute GVHD, reated by standard steroid therapy. Chronic GVHD did not occur nd 100 days after transplant immunosuppressive treatment was apered. The patient obtained a complete molecular response (undeectable BCR-ABL transcript by RQ-PCR; assay sensitivity threshold × 10−5), and yearly testing showed persistent negativity for BCRBL transcript. The patient was well until April 2004, when a diffuse arge B-cell NHL stage III EBV-unrelated was diagnosed. Six courses f Rituximab + CHOP were administered allowing achievement of complete clinical remission. Thereafter, the patient was strictly onitored for both CML and NHL recurrence. In November 2008 18 years after transplantation) RQ-PCR for BCR-ABL mRNA resulted ositive at a very low level, while FISH and cytogenetic analyis were negative for Ph chromosome. BCR-ABL transcript load ncreased slowly over time and in October 2009 it reached the level f 0.19% according to International Scale [2]. Bone marrow was orphologically normal and the patient was still in complete cytoenetic response. At that time, Imatinib treatment was started at 00 mg daily. After 12 months of Imatinib therapy, BCR-Abl mRNA s undetectable. No lymphoma relapse has been observed so far. he donor was serially monitored for BCR-ABL rearrangement in eripheral blood and results were negative. Microsatellites analysis as not performed. Very late recurrences (beyond 10 years) are rare in CML pts given ransplantation in first chronic phase [3], although the true incience could be underestimated as relapses occurring at molecular evel only are not considered. Recently, a patient-specific PCR strat[