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Recommendations regarding management of essential thrombocythaemia rely on studi e s done before the discovery of the CALR mutation. On May 20, 2020, the Europ e a n LeukemiaNet annual meeting was held with the goal to identify unmet clin i c a l needs in myeloproliferative neoplasms. Because patients with a CALR mutation have specific clinica l characteristics, treatment of CALR-mutated essential thrombocythaemia was considered an unmet clinical need by the Europ e a n LeukemiaNet. The elaboration of a consensus document with recommendations according to current evidence was proposed as a solution for resolving uncertainties in the treatment of CALR-mutated essential thrombocythaemia. A steering committee comprising f o u r Europ e a n LeukemiaNet members was t h e n formed and a panel of ten experts in the field was recruited. T h e experts proposed 51 potential unmet clinical needs in the management of CALR-mutated essential thrombocythaemia and were as k e d to score the relevance of each topic. Those topics that obtained the highest scores as relevant unmet clinical needs were identified, including antiplat e l e t therapy in patients at low risk, definition of extreme thrombocytosis and its management in patients at low risk, indications of cytoreduction and tar g e t s of therapy, first-line treatment of choice in young patients (1500 x 109 platelets per L) with pegylated interfe r o n alfa being the preferred option for younger patients; both hydroxycarbamide and anagrelide might be given to patients ineligible for pegylated interferon alfa; and treatment algorithms for patients with high-risk pregnancies should not be changed according to genotype. The European LeukemiaNet proposes to use these recommendations in the routine management of patients wit h CALR-mutated essential thrombocythaemia, and designing new clinica l studies in this field might be useful.

The median age of chronic myeloid leukemia (CML) patients is ∼ 60 years, and age is still considered an important prognostic factor, included in Sokal and EURO risk scores. However, few data are available about the long-term outcome of older patients treated with imatinib (IM) frontline. We analyzed the relationship between age and outcome in 559 early chronic-phase CML patients enrolled in 3 prospective clinical trials of Gruppo Italiano Malattie Ematologiche dell'Adulto CML Working Party, treated frontline with IM, with a median follow-up of 60 months. There were 115 older patients (≥ 65 years; 21%). The complete cytogenetic and major molecular response rates were similar in the 2 age groups. In older patients, event-free survival (55% vs 67%), failure-free survival (78% vs 92%), progression-free survival (62% vs 78%), and overall survival (75% vs 89%) were significantly inferior (all P < .01) because of a higher proportion of deaths that occurred in complete hematologic response, therefore unrelated to CML progression (15% vs 3%, P < .0001). The outcome was similar once those deaths were censored. These data show that response to IM was not affected by age and that the mortality rate linked to CML is similar in both age groups. This trial was registered at www.clinicaltrials.gov as #NCT00514488 and #NCT00510926.

BACKGROUND: An increased risk of thrombosis has been reported in primary immune thrombocytopenia (ITP) and with the use of thrombopoietin (TPO) receptor agonists, on the basis of population studies using administrative databases. OBJECTIVES: To evaluate if the incidence of venous and arterial thrombosis in patients with primary ITP is higher than a clinically significant cut-off set at of 3% and 6.4%. PATIENTS/METHODS: We undertook a retrospective multicenter investigation in a large cohort of patients requiring at least one treatment for ITP, enrolled from the major tertiary Italian centers treating ITP. A total of 986 patients were analyzed. RESULTS: During a 3888 patient-year follow-up, 43 first thrombotic events occurred: 28 arterial and 15 venous, resulting in a cumulative incidence of 3.2% for arterial (95% CI, 2.0-5.0) and 1.4% (95% CI, 0.8-2.5) for venous thrombosis at 5 years. The annualized rates for 100 patient-years were 1.14 (95% CI, 0.84-1.54), 0.39 (95% CI, 0.23-0.65) and 0.71 (95% CI, 0.49-1.04) for total, venous and arterial thrombosis. Splenectomy, performed in 136 patients (13.7%), increased thrombotic risk (HR = 3.5, 95% CI) compared with non-splenectomized patients. Age > 60 years, more than two risk factors for thrombosis at diagnosis and steroid use were independently associated with an increased risk of thrombosis. CONCLUSIONS: In this study, we demonstrate that the 5-year cumulative incidence of venous and arterial thrombosis in ITP is well below the predefined thresholds. Venous and arterial thromboembolism are not frequent complications in ITP, except in particular settings, such as in splenectomized and elderly patients. © 2014 International Society on Thrombosis and Haemostasis.

For almost 10 years imatinib has been the therapeutic standard of chronic myeloid leukemia. The introduction of other tyrosine kinase inhibitors (TKIs) raised a debate on treatment optimization. The debate is still heated: some studies have protocol restrictions or limited follow-up; in other studies, some relevant data are missing. The aim of this report is to provide a comprehensive, long-term, intention-to-treat, analysis of 559 newly diagnosed, chronic-phase, patients treated frontline with imatinib. With a minimum follow-up of 66 months, 65% of patients were still on imatinib, 19% were on alternative treatment, 12% died and 4% were lost to follow-up. The prognostic value of BCR-ABL1 ratio at 3 months (⩽10% in 81% of patients) was confirmed. The prognostic value of complete cytogenetic response and major molecular response at 1 year was confirmed. The 6-year overall survival was 89%, but as 50% of deaths occurred in remission, the 6-year cumulative incidence of leukemia-related death was 5%. The long-term outcome of first-line imatinib was excellent, also because of second-line treatment with other TKIs, but all responses and outcomes were inferior in high-risk patients, suggesting that to optimize treatment results, a specific risk-adapted treatment is needed for such patients.

This publication results from a year-lasting (2014-2015) collaboration between the Polytechnic University of Turin and Eindhoven Technical University’s Chair of Rational Architecture, guided by Professor Christian Rapp together with Haike Apelt as Dutch Supervisors and Professor Silvia Malcovati as Italian Supervisor.

Spleen enlargement, present in 10-20% of Essential Thrombocythemia (ET) patients at diagnosis, is a feature clinically easy to assess, confirmable by echography with a very low chance of misinterpretation. Nonetheless, the clinical and prognostic role of splenomegaly has been seldom evaluated. From 1979 to 2013, 1297 ET patients retrospectively collected in the database of the Lazio Cooperative Group and Bologna University Hospital were evaluable for spleen enlargement at diagnosis and included in the analysis. On the whole, spleen was enlarged in 172/1297 (13.0%) patients; in most cases (94.8%) splenomegaly was mild (≤5 cm). Patients with splenomegaly were younger, predominantly male, presented higher platelet count and JAK2V617F allele burden and had a lower incidence of concomitant cardiovascular risk factors. At least one thrombotic event during follow-up occurred in 97/1,125 (8.6%) patients without spleen enlargement compared to 27/172 (15.7%) patients with spleen enlargement (P = 0.003). Despite comparable use of cytoreductive/antiplatelet therapies in the two groups, the cumulative risk of thrombosis at 5 years was significantly higher in patients with baseline splenomegaly (9.8% versus 4.4% in patients without splenomegaly, P = 0.012). In multivariate analysis exploring risk factors for thrombosis, splenomegaly retained its negative prognostic role, together with previous thrombosis, leucocyte count and male gender. Baseline splenomegaly seems to be an independent additional risk factor for thrombosis in nonstrictly WHO-defined ET patients. This data could be useful in the real-life clinical management of these patients.

Definite spectra of point mutations in the Bcr-Abl kinase domain (KD) are known to confer resistance to the tyrosine kinase inhibitors (TKIs) imatinib (IM), dasatinib (DAS) and nilotinib (NIL) in Philadelphia-positive (Ph+) leukemias. Until the recent advent of next-generation sequencing (NGS) technologies, no method was available that could conjugate high sensitivity, possibility to screen for any known or unknown sequence variation and accurate quantitation of mutated subclones. Because of these key technical limitations, the clinical relevance of early detection of small mutated subclones could never be fully elucidated. We have set up and optimized a Bcr-Abl KD mutation screening assay taking advantage of the Roche 454 NGS technology on a GS Junior instrument, that allows parallel pyrosequencing of a hundred thousand clonally amplified DNA molecules of 400 bp average length. We have thus designed 4 partially overlapping amplicons covering the whole KD of the Bcr-Abl transcript (a.a. 240–520) to be generated by nested RT-PCR using sequence-specific primers conjugated with multiplex identifiers – allowing to pool and sequence different samples from one or multiple patients (pts) in a single run. The assay proved capable to identify, characterize and quantitate sequence variations in samples from pts already known to harbour mutations as assessed by conventional Sanger sequencing with 100% concordance. Given that the number of sequence reads generated in each run is relatively constant, the lower detection limit is inversely correlated with the number of samples that can simultaneously be analyzed. Sensitivity could thus be easily modulated – analyzing a single sample per run allowed to routinely achieve lower detection limits ranging between 0.02 and 0.05% (that is, to detect as little as 2 to 5 mutated Bcr-Abl transcripts in a total of 10,000 Bcr-Abl transcripts). With this target sensitivity, we first retrospectively analyzed samples collected at the time of diagnosis from selected chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) cases known to have developed a T315I mutation 3 to 9 months after TKI start. We found that the T315I could not always be traced back to the time of diagnosis. We also found that, both in CML and in Ph+ ALL pts, several low-level mutations (in the range of 0.05 to 1%) with apparently no clinical relevance (e.g., never reported in association with TKI resistance) were routinely detectable – and appeared not to be predictors of a higher level of genetic instability. More interestingly, when used for mutation monitoring of de novo Ph+ ALL pts enrolled in clinical trials with DAS or NIL, NGS could highlight emerging resistant clones far earlier than D-HPLC or conventional sequencing. Also, some IM-resistant CML and Ph+ ALL pts who had been scored as unmutated by conventional sequencing could be shown by NGS to harbour DAS- or NIL-resistant mutations at 1–10% level – a phenomenon that is known to happen because of the clonal deselection resulting from the temporary lack of any TKI selective pressure in the interval between IM discontinuation and DAS or NIL start. Analyses will be presented in detail. We can conclude that: 1) the 454 NGS technology on the GS Junior instrument is a reliable and cost-effective method to perform Bcr-Abl KD mutation screening of Ph+ leukemia pts – with a target sensitivity of 0,1%, 4 samples can simultaneously be analyzed with costs comparable to those of conventional Sanger sequencing and the advantage to quantitatively follow the dynamics of mutated subclones over time; 2) in line with a recent report, small mutated subclones with apparently no clinical relevance can be detected both in newly diagnosed CML and Ph+ ALL pts before TKI start – which further underlines that mutation screening of pts at diagnosis is uninformative, if not misleading; 3) during TKI therapy, the high sensitivity of NGS allows to detect emerging Bcr-Abl mutant subclones earlier than D-HPLC or conventional sequencing; this is particularly relevant: i) for mutation monitoring of Ph+ ALL pts, that are highly prone to develop resistance and mutations while on TKI therapy, and ii) in IM-resistant CML and Ph+ ALL pts, where NGS may uncover the presence of DAS- or NIL-insensitive mutations, and may thus help choosing the second-line therapeutic strategy most likely to be successful.

Background. The expression of BCR-ABL gene in Chronic Myeloid Leukemia (CML) is necessary for malignant transformation but the biologic basis of the progression from Chronic Phase (CP) to Blast Crisis (BC) is poorly understood. Aim. Identifying the genomic imbalances involved in the transition into BC, before clinical features, may provide diagnostic markers of progression; so we used SNP arrays to perform a high-resolution mapping of BC CML patients genomes. Methods. We analyzed 11 patients affected by BC CML disease. Genomic DNA were extracted from bone marrow or peripheral blood mononuclear cells archived both at the time of diagnosis and progression. SNP array-based karyotyping was carried out using Affymetrix GeneChip Human Mapping arrays. Copy number (CN) analysis was performed using Hapmap normal individuals as reference set and two different softwares. Results. After exclusion of genomic copy number variations (CNVs), the following results were achieved: five patients showed huge amplifications and deletions, ranging from 30Mb to 160Mb, on chromosomes 9, 7, 3 and 6. We also found several heterozygous micro-deletions and micro-amplifications spreading all over the genome. This analysis has identified abnormalities in genes involved in apoptosis (e.g., GADD45A, FOXO3A, GAS6), DNA damage response (e.g., MYST as known as Hmof, XRCC2), tumor suppression (e.g., C/EBPdelta, LATS1), chromatin regulation (e.g., HDAC9), and genes belonged to ABC transporters (e.g., ABCB1), ras family and transcriptional/translational factors (e.g., ETV1). Moreover were found copy number changes (gain/loss) in genes associated with malignancy, in particular TNFRSF17, MET, IGFBPL1, EVI1, PTENP1. Other alterations affected key pathways including cell cycle regulation and WNT signaling. Conclusions. The use of the genomic tool Genome-Wide Human SNP array allowed us to identify, at submicroscopic level, genetic lesions in patients affected by CML in BC. Our results will be further validated by real-time PCR for the altered genes involved key pathways, while sequencing and mutation analysis will be performed on the remaining allele of putative tumor suppressor genes to identify their residual activity. All these validations and the increased number of analyzed patients will provide new insights into the genetic profiling that lead disease progression from CP to BC and consequently new opportunities to develop specific target therapies.