Leonardo Campiotti

JAK2V617F mutation for the early diagnosis of Ph- myeloproliferative neoplasms in patients with venous thromboembolism: a meta-analysis.

Recent studies suggested that JAK2V617F mutation is frequent in patients with splanchnic vein thrombosis (SVT) but not in patients with other venous thromboembolic events (VTE). However, whether screening for the JAK2V617F mutation in VTE patients is justified remains unclear. Therefore, we performed a systematic review to assess the frequency of JAK2 mutation in VTE patients and the role of JAK2V617F mutation in the diagnosis of myeloproliferative neoplasms. MEDLINE and EMBASE databases were searched. Two reviewers independently performed study selection and extracted study characteristics. Pooled odds ratios of case-control studies and weighted mean proportion of the prevalence of JAK2V617F mutation of uncontrolled series were calculated. Twenty-four studies involving 3123 patients were included. Mean prevalence of JAK2 mutation was 32.7% (95% confidence interval, 25.5%-35.9%) in SVT patients. JAK2 mutation was associated with increased risk of SVT (odds ratio, 53.98; 95% confidence interval, 13.10-222.45). Mean prevalence of JAK2 mutation in other VTE patients was low (range, 0.88%-2.57%). Presence of JAK2V617F mutation in SVT patients was associated with a subsequent diagnosis of myeloproliferative neoplasm in many patients. JAK2 mutation is strongly associated with SVT, and routine screening of JAK2 mutation appears to be indicated in these patients.

Recurrent ETNK1 mutations in atypical chronic myeloid leukemia

Despite the recent identification of recurrent SETBP1 mutations in atypical chronic myeloid leukemia (aCML), a complete description of the somatic lesions responsible for the onset of this disorder is still lacking. To find additional somatic abnormalities in aCML, we performed whole-exome sequencing on 15 aCML cases. In 2 cases (13.3%), we identified somatic missense mutations in the ETNK1 gene. Targeted resequencing on 515 hematological clonal disorders revealed the presence of ETNK1 variants in 6 (8.8%) of 68 aCML and 2 (2.6%) of 77 chronic myelomonocytic leukemia samples. These mutations clustered in a small region of the kinase domain, encoding for H243Y and N244S (1/8 H243Y; 7/8 N244S). They were all heterozygous and present in the dominant clone. The intracellular phosphoethanolamine/phosphocholine ratio was, on average, 5.2-fold lower in ETNK1-mutated samples (P < .05). Similar results were obtained using myeloid TF1 cells transduced with ETNK1 wild type, ETNK1-N244S, and ETNK1-H243Y, where the intracellular phosphoethanolamine/phosphocholine ratio was significantly lower in ETNK1-N244S (0.76 ± 0.07) and ETNK1-H243Y (0.37 ± 0.02) than in ETNK1-WT (1.37 ± 0.32; P = .01 and P = .0008, respectively), suggesting that ETNK1 mutations may inhibit the catalytic activity of the enzyme. In summary, our study shows for the first time the evidence of recurrent somatic ETNK1 mutations in the context of myeloproliferative/myelodysplastic disorders.

Microhomologies and interspersed repeat elements at genomic breakpoints in chronic myeloid leukemia.

Reciprocal translocation t(9;22) is central to the pathogenesis of chronic myeloid leukemia. Some authors have suggested that Alu repeats facilitate this process, but supporting analyses have been sparse and often anecdotal. The purpose of this study was to analyze the local structure of t(9;22) translocations and assess the relevance of interspersed repeat elements at breakpoints. Collected data have been further compared with the current models of DNA recombination, in particular the single‐strand annealing (SSA) and the nonhomologous end joining (NHEJ) processes. We developed a protocol for the rapid characterization of patient‐specific genomic junctions and analyzed 27 patients diagnosed with chronic myeloid leukemia. Sequence analysis revealed microhomologies at the junctions of 21 patients of 27, while interspersed repeats were of relevance (P < 0.05) in at least 16 patients. These findings are more frequent than expected and give an indication that the main mechanisms involved in the t(9;22) translocation are the SSA and NHEJ pathways, both playing a role. Furthermore, our report is consistent with microhomologies facilitating the joining of DNA ends in the translocation process, and with both Alu and a variety of other repeat sequences pairing nonhomologous chromosomes during the SSA pathway.

A randomized comparison of caspofungin versus antifungal prophylaxis according to investigator policy in acute leukaemia patients undergoing induction chemotherapy (PROFIL-C study)

BACKGROUND Invasive fungal infections (IFIs) are considered a major problem among patients undergoing acute leukaemia (AL) induction treatment. PROphylaxis of Fungal invasive Infections in Leukaemia-Caspofungin (PROFIL-C) is a multicentre study aiming to assess the comparative yield of using caspofungin versus standard policy (SP) regimens and the overall impact of IFI in routine clinical care conditions. METHODS All AL patients receiving IFI prophylaxis according to local SP were prospectively included in the study by Northern Italy Leukaemia Group (NILG) centres. To allow the comparison of caspofungin versus SP regimens as prophylaxis strategies, caspofungin treatment was assigned via a centralized randomized procedure. The study was registered at http://www.clinicaltrial.gov (NCT00501098). RESULTS Over a 2 year period, 175 patients were included. The overall incidence of IFI was 32/175 (18.3%) [10/175 (5.7%) probable/proven and 22/175 (12.6%) possible], with no statistically significant differences between caspofungin-based versus SP-based regimens [overall: 15/93 (16.1%) versus 17/82 (20.7%), relative risk (RR) 0.78, 95% confidence interval (CI) 0.42-1.46; probable/proven: 7/93 (7.5%) versus 3/82 (3.7%), RR 2.06, 95% CI 0.55-7.7; possible: 8/93 (8.6%) versus 14/82 (17.1%), RR 0.5, 95% CI 0.22-1.14]. Only one IFI-related death was recorded (10%). CONCLUSIONS The incidence and mortality of IFI were lower than expected in this strictly sequential cohort representative of the routine care in the NILG network. The efficacy and safety of caspofungin were similar to other prophylactic regimens.

JAK2-V617F mutation and Philadelphia positive chronic myeloid leukemia.

JAK2 is a tyrosine kinase that plays an important role in the signaling pathways of many hematopoietic growth factor receptors. A single acquired point mutation – V617F – in JAK2 occurs in the great majority of patients with polycythemia vera (PV) and approximately half of the patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET). In contrast, the JAK2-V617F mutation is only rarely found in chronic myeloid leukemia (CML) but, recently, some authors have reported the coexistence of JAK2V617F and BCR/ABL+ in CML patients expressing the p210 BCR–ABL oncoprotein. Here, we report a CML patient with the expression of p210/b2a2 type BCR–ABL transcript and JAK2V617F mutation.

ESC position paper on cardiovascular toxicity of cancer treatments: challenges and expectations—comment

We read with interest the paper published by Raschi et al. about cardiotoxicity of new drugs in cancer treatment [1]. The authors divided new drugs into three groups according to their different therapeutic mechanisms of action. Although this is a sound pharmacological approach, we have to note that, especially for the clinician in everyday practice, an important distinction should be based on the prognosis and the overall survival of patients treated with these drugs, to select a tailored prevention of cardiotoxicity. The large part of new drugs such as tyrosine kinase inhibitors (TKIs), monoclonal antibodies, mTOR inhibitors, and immune checkpoint inhibitors are in fact used in patients affected by advanced cancer, with a reduced life expectancy. An exception is the treatment of chronic myeloid leukemia (CML) with TKIs. These patients have, with current therapies, a life expectancy similar to that of the general population, and apart from selected cases, TKIs should be continued indefinitely [2]. As such, cardiotoxicities due to these drugs can become an important concern for the clinician, and should be a key focus in research and preventive measure. In keeping with these observations, we suggest that, in adjunct to those cited in the review, ponatinib should also be included among TKIs with a proven cardiotoxicity. In 2013, the FDA removed ponatinib from the market for cardiotoxicity until safeguards were instituted to manage toxicity [3]. From 2014, ponatinib is employed in the treatment of resistant CML and Philadelphia-positive acute lymphoblastic leukemia, with good clinical results, but it is still hampered by a relevant cardiotoxicity. The PACE trial reports serious cardiovascular toxicities in as much as 5% of patients treated with ponatinib [4] and the EPIC trail report serious arterial thrombotic events in 7% of patients receiving ponatinib [5]. In general, ponatinib is associated with serious cardiovascular toxicity including arterial thrombosis (8%), myocardial infarction (5%), peripheral arterial occlusive disease (2%), and cerebrovascular events (2%). It should be noted that CML patients treated with TKIs more frequently have arterial thrombosis in comparison with venous events. A possible pathophysiologic explanation is TKIs interaction with endothelial cells. In our opinion, preventive measures of these adverse events should include the employment of low-dose aspirin, as just encoded in other chronic myeloid disorder, such as polycythaemia vera and essential thrombocytosis and we advocate for further research into the role of statins in prevention of cardiovascular adverse events in these patients.

In vitro effects of Apixaban on 5 different cancer cell lines

Background Cancer is associated with hypercoagulability. However, several data suggest that anticoagulant drugs may have an effect on tumor development and progression mediated by both coagulation dependent processes and non-coagulation dependent processes. Therefore, we investigated the in vitro effects of Apixaban on cell proliferation, mortality, cell migration, gene expression and matrix metalloproteinase in 5 different cancer cell lines. Methods The following cancer cell lines, and 2 normal fibroblast cultures (lung and dermal fibroblasts), were studied: OVCAR3 (ovarian cancer), MDA MB 231 (breast cancer), CaCO-2 (colon cancer), LNCaP (prostate cancer) and U937 (histiocytic lymphoma). Proliferation and cell mortality were assessed in control cells and Apixaban treated cultures (dose from 0.1 to 5 μg/ml, 0 to 96-h). Necrosis/Apoptosis (fluorescence microscopy), cell migration (24-h after scratch test), matrix metalloproteinase (MMP) activity and mRNA expression (RT PCR) of p16, p21, p53 and HAS were also assessed. Results High-dose (5 μg/ml) Apixaban incubation was associated with a significantly reduced proliferation in 3 cancer cell lines (OVCAR3, CaCO-2 and LNCaP) and with increased cancer cell mortality in all, except LNCaP, cancer lines. Apoptosis seems to account for the increased mortality. The migration capacity seems to be impaired after high-dose Apixaban incubation in OVCAR3 and CaCO-2 cells. Data on mRNA expression suggest a consistent increase in tumor suppression gene p16 in all cell lines. Conclusions Our data suggest that high-dose Apixaban may be able to interfere with cancer cell in vitro, reducing proliferation and increasing cancer cell mortality through apoptosis in several cancer cell lines.

Acute myeloid leukemia with associated translocation t(15;17) and 11q23/MLL abnormality.

The classification of acute myeloid leukemia (AML) recognizes a subgroup of diseases with recurring genetic abnormalities. Translocation (15;17) is the marker of acute promyelocitic leukemia (APL) and is associated with good prognosis [1]. 11q23/MLL abnormality has been detected in about 4% of AML [2], but never in APL [3]. 11q23/MLL abnormality is related to a previous treatment with topoisomerase inhibitors and also to a monocytic differentiation of blasts [4]. AML with 11q23/MLL has an adverse prognosis, comparable to AML with unfavourable karyotype. In this letter, we describe a case of AML with t(15;17) and t(11;22)(q23;q11.2) abnormalities; as far as we know, this association has not been reported in literature. A 64-year-old woman was admitted to our department because of cytopenia. The patient had no history of myelodysplasia, radiotherapy or chemotherapy, or toxic drug exposure. Clinical examination was negative. Blood cells count showed anaemia (haemoglobin 8.4 g/dL), leucopenia (0.876 10 cells/L) and thrombocytopenia (226 10 cells/L). Myeloid blast cells (8%) were found in peripheral blood smear. Bone marrow examination showed a blastic myeloid infiltration. Blast cells were 35% of nucleated cells and were hypogranular and myeloperossidase positive without similarities to promyelocytic blasts. No Auer’s rod was observed. Blast cells immunophenotype showed strong positivity for CD33, CD13, CD34, CD38 and a moderate expression of CD15 and CD19. HLA-DR, CD10, CD7, CD56 were negative. No morphologic evidence of dyserythropoiesis was detected by bone marrow biopsy. There were no signs of disseminated intravascular coagulation or accelerated fibrinolysis. Cytogenetic analysis revealed the association of t(15;17)(q22;q21) and t(11;22)(q23;q11.2) (WCP 11 and WCP 22 Vysis) abnormalities in all the metaphases analyzed (Figure 1). Reverse transcription polymerase chain reaction (RT-PCR) subsequently confirmed the presence of PML/RARa Bcr1 transcript and fluorescence in situ hybridisation (FISH-painting) confirmed the presence of t(11;22)(q23;q11.2) (Figure 2). Interphase FISH analysis with Vysis LSI MLI Dual Color probe, Break Apart Rearrangement probe showed that MLL gene region (11q23) was involved in the rearrangement (Figure 3). Karyotype and FISHpainting analysis for t(11;22) on lymphocytes were negative, therefore excluding an inherited chromosome translocation. The patient received induction therapy with alltrans-retinoic acid (ATRA) and idarubicin and

SETBP1 induces transcription of a network of development genes by acting as an epigenetic hub

SETBP1 variants occur as somatic mutations in several hematological malignancies such as atypical chronic myeloid leukemia and as de novo germline mutations in the Schinzel–Giedion syndrome. Here we show that SETBP1 binds to gDNA in AT-rich promoter regions, causing activation of gene expression through recruitment of a HCF1/KMT2A/PHF8 epigenetic complex. Deletion of two AT-hooks abrogates the binding of SETBP1 to gDNA and impairs target gene upregulation. Genes controlled by SETBP1 such as MECOM are significantly upregulated in leukemias containing SETBP1 mutations. Gene ontology analysis of deregulated SETBP1 target genes indicates that they are also key controllers of visceral organ development and brain morphogenesis. In line with these findings, in utero brain electroporation of mutated SETBP1 causes impairment of mouse neurogenesis with a profound delay in neuronal migration. In summary, this work unveils a SETBP1 function that directly affects gene transcription and clarifies the mechanism operating in myeloid malignancies and in the Schinzel–Giedion syndrome caused by SETBP1 mutations.SETBP1 variants occur as somatic mutations in several malignancies and as de novo germline mutations in developmental disorders. Here the authors provide evidence that SETBP1 binds to gDNA in AT-rich promoter regions to promote target gene upregulation, indicating SETBP1 functions directly to regulate transcription.

Vitamin D and tyrosine kinase inhibitors in chronic myeloid leukemia

Dear editor, Vitamin D and calcitriol, participate in critical cell functions such as proliferation, apoptosis, and suppression of inflammation, and play a key role in many cancer hallmarks such as angiogenesis, cell invasion, and metastatic spread [1, 2] (see Fig. 1). Vitamin D levels have a prognostic role in diabetes and cardiovascular disease [3]. As for oncology, a prognostic role of vitamin D deficiency in cancer is established, especially in tumors with strong interaction with the immune system such as melanoma [4], follicular lymphoma [5] and in diffuse large B cell lymphoma [6]. Chronic myeloid leukemia (CML) is a myeloproliferative disorder; characterized by a specific genetic signature, the BCR-ABL gene fusion, a permanently active tyrosine kinase [7]. Tyrosine kinase inhibitors (TKIs) are capable of inhibiting the driver mutation, and rapidly became the standard of care. CML is considered the perfect example of a driver mutation disease, but we often forget that, before the advent of TKIs, CML was considered an immunogenic cancer. The first drug that was able to re-establish normal hematopoiesis in a minority of CML patients was interferon α [8]. Given the possible link between vitamin D and the immune system, we dosed vitamin D levels in patients affected by CML treated with TKIs of first or second generation, and compared vitamin D levels in other inpatients. We included patients affected by CML, in treatment at our institution with Imatinib 400 mg daily or dasatinib 100 mg daily who achieved a stable deep molecular response (SDMR). SDMR includes both complete molecular response (CMR), and major molecular response (MMR), defined as undetectable BCR-ABL and a BCR-ABL/ABL1 IS ratio < 0.1% with a 4.5 log sensibility PCR, respectively. Control was provided by other inpatients. Patients affected by diabetes, renal disease, hepatic disease, cancer, patients who underwent parathyroid or thyroid surgery or patients in therapy with steroids were all excluded, from control as well as from the CML group. Blood samples were collected between December 2016 and February 2017. Vitamin D blood levels were measured by a radio-immunoassay for in vitro quantitative measurement of 25-hydroxy vitamin D3 and D2 in serum (DIA source 25 OH Vitamin D total-RIA-CT kit). Vitamin D levels were defined normal if > 30 ng/mL, low from 15 ng/dL to 29 ng/mL, deficient < 15 ng/mL. Data are reported as median and inter-quartile range (nonnormally distributed data), and comparison between groups were made by independent T test or Mann–Whitney test, as appropriate. All CML and control group patients with vitamin D deficiency received oral supplementation in accordance to international guidelines [9, 10]. We studied 26 CML patients and 52 controls. Median age and male to female ratio were balanced between CML and control group. Patients affected by CML were in treatment with imatinib (18 patients) and dasatinib (8 patients). Median duration of TKIs therapy was 6.3 years (range 5.2–10), see Table 1 for further details. The median vitamin D level in CML patients was 12.45 ng/dL (10.25–15.32 IQR) while controls had a median level of 15.9 ng/dL (12.05–21.38 IQR) (p = 0.018, Fig. 1). We analysed vitamin D levels stratifying CML patients based on their response to TKIs therapy. In our CML cohort, 19 patients achieved MMR and 7 patients achieved CMR; vitamin D levels were lower in patients in MMR compared to CMR patients: 11.3 ng/dL (9.3–13 IQR) and 16 ng/dL (15.1–20.1 IQR), respectively (p = 0.001, Fig. 2 and Table 2). * Leonardo Campiotti leonardo.campiotti@uninsubria.it