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Autophagy protects chronic myeloid leukemia stem cells from tyrosine kinase inhibitors hence supporting the disease persistence under therapy. However, the signals involved in autophagy regulation relative to BCR–ABL1 are still elusive. The autophagic flux proceeding from the inhibition of BCR–ABL1 tyrosine kinase represents a regulatory mechanism of β-catenin stability through events encompassing the activation of calpain, which targets β-catenin for proteasome-independent degradation. Accordingly, its inactivation may contribute to induce autophagy and autophagy induction may, in turn, promote β-catenin autolysosomal degradation to originate a regulatory loop where β-catenin plays a central role in cell decision between life and death. Here we proved that the cytoplasmic accumulation of β-catenin driven by up-regulation of its antagonist Chibby1 is a component of autophagy induction in response to imatinib in BCR–ABL1 + cells opposing the apoptotic death. It is contingent upon ER stress and elevation of free Ca2 + cytosolic concentration and results in the calpain cleavage into a 28 kDa fragment implicated in β-catenin proteasome-independent degradation. More important for BCR–ABL1 + cell survival and proliferation following IM treatment, might be the calpain-mediated cleavage of β-catenin accumulated within the cytoplasmic compartment into a 75 kDa fragment, still owning TCF-dependent transcriptional activity. Such a β-catenin fragment might be crucial for BCR–ABL1 + cell survival following the fusion protein TK inhibition.

Vascular calcification is related to vascular diseases, for example, atherosclerosis, and its comorbidities, such as diabetes and chronic kidney disease. In each condition, a distinctive histological pattern can be recognised that may influence technical choices, possible intra-operative complications, and procedure outcomes, no matter if the intervention is performed by open or endovascular means. This review considers the classification and initiating mechanisms of vascular calcification. Dystrophic and metastatic calcifications, Monckeberg's calcification, and genetic forms are firstly outlined, followed by their alleged initiation mechanisms; these include (a) ineffective macrophage efferocytosis; (b) ectopic osteogenesis driven by modified resident or circulating osteoprogenitors. As in physiological bio-mineralisation, active calcification starts with the deposition of cell derived matrix vesicles into the extracellular matrix. To substantiate this belief, an in depth ultra-structural documentation of hydroxyapatite crystal deposition on such vesicles is provided in an ex-vivo human vascular cell model. Revealing the vesicle composition and phenotype in normal and pathological vascular conditions will be essential for the development of new therapeutic strategies, in order to prevent and treat vascular calcification.

Background/Aims: The increase in the survival rate of patients with chronic renal failure due to substitution treatment prompts an investigation of their quality of life (QoL), a key measure to evaluate the outcomes of chronic disease treatment. To determine whether hemodialysis or peritoneal dialysis provide a better QoL, a systematic meta-analysis was performed. Methods: We searched through the database Cinahl, Medline, PubMed, Scopus and Proquest, including articles published from 2011 until June 2016. We selected articles that compared, through KDQOL-SF 1.3 or 36 questionnaires, QoL among patients undergoing hemodialysis and peritoneal dialysis. The data was collected using Excel Office, and t-test has been performed on independent samples to identify significant differences. Results: Only some of the seven articles found significant differences between the two treatments. One of the studies showed a better QoL for peritoneal dialysis patients, while, on the contrary, two other studies support that the best QoL is in patients receiving hemodialysis. Another article displayed significant difference only for satisfaction in relation to care, better in patients on peritoneal dialysis, and for physical health, better in hemodialysis. Conclusions: The analysis has not led to a unanimous conclusion. Quantitative analysis showed that the only statistically significant difference between the QoL of patients on hemodialysis and peritoneal dialysis regards the effect of kidney disease, which happens to be better in patients undergoing peritoneal dialysis.

Mesenchymal Stem Cells (MSCs) possess important characteristics that could be exploited in therapeutic strategies for Type 1 Diabetes (T1D) and for certain complications of Type 2 Diabetes (T2D). MSCs can inhibit autoimmune, alloimmune and inflammatory processes. Moreover, they can promote the function of endogenous and transplanted pancreatic islets. Furthermore, they can stimulate angiogenesis. MSC functions are largely mediated by their secretome, which includes growth factors, exosomes, and other extracellular vesicles. MSCs have shown a good safety profile in clinical trials. MSC-derived exosomes are emerging as an alternative to the transplantation of live MSCs. MSCs harvested from different anatomical locations (e.g. bone marrow, umbilical cord, placenta, adipose tissue, and pancreas) have shown differences in gene expression profiles and function. Data from clinical trials suggest that umbilical cord-derived MSCs could be superior to bone marrow-derived MSCs for the treatment of T1D. Autologous MSCs from diabetic patients may present abnormal functions. BM-MSCs from T1D patients exhibit gene expression differences that may impact in vivo function. BM-MSCs from T2D patients seem to be significantly impaired due to the T2D diabetic milieu. In this review, we highlight how the harvesting site and donor derivation can affect the efficacy of MSC-based treatments for T1D and T2D.

Objectives and background: According to the World Health Organization (WHO) classification, the diagnosis of Systemic Mastocytosis (SM) relies on bone marrow (BM) examination and is based on a major and four minor criteria. The somatic ‘autoactivating’ point mutation D816V in the KIT receptor gene is one of the minor criteria, founded in the great majority of patients (90%) and it plays a central role in the pathogenesis of the disease. Indolent Systemic Mastocytosis (ISM) is the most common variant of SM, characterized by a very low MC burden and associated with very different clinical pictures. A highly sensitive diagnostic methods for D816V detection are required to assure an appropriate diagnosis and to reduce false-negative results. The recent development of “ultra-deep amplicon sequencing” (UDS) technologies has opened the way to a more accurate characterization of molecular aberrations with higher sensitivity of screening for known and unknown mutations. Our aims were: i) to set-up and optimize a UDS-based mutation screening strategy of the KIT gene on the Roche GS Junior Instrument; ii) to test the sensitivity of our UDS assay to detect the D816V mutation; iii) to investigate the presence of additional KIT mutations in SM. Methods: We decided to take advantage of a next generation sequencing approach to perform an UDS KIT gene mutation analysis on 20 bone marrow (BM) samples from patients whit ISM that were negative for the D816V mutation by Sanger Sequencing which has a sensitivity of 20%. Fusion primers were designed to generate ten partially overlapping amplicon covering the whole KIT transcript (exons 1-21) by RT-PCR. To determine the lower detection limit of our UDS-assay, serial dilutions of the HMC-1 cell line (harboring the D816V mutation) into an unmutated K562 cell line in ratios such as to simulate the following mutation loads were sequenced: 50%, 37.5%, 25%, 12.5%, 5%; 2.5%, 1.25%, 0.5%, 0.25%. Results and significance: UDS of cell line dilutions showed a high accuracy of D816V mutation detection and linearity of mutation calling over the entire range down to 0.25%. The UDS technology allowed to detected the D816V mutation, below the lower detection limit of Sanger Sequencing, with an abundance from 0.5% to 11%, in 12/20 ISM patients. Two additional sequence variations were detected in a large proportion of patients. These two variations included a 3bp in-frame deletion in exon 15 (GenBank X06182.1: c.2164_2166delAGC; p.S715del) found in 11/20 patients and a 12bp in frame-deletion in exon 9 in all patients, whit an abundance ranging from 83% to 97% (GenBank X06182.1: c.1550_1561delGTAACAACAAAG; p.G510_K513del). Previously published studies indicate that the KIT Gly-Asn-Asn-Lys510-513+/- alternatively spliced located immediately downstream to the extracellular KIT domain and KIT Ser715+/-, an interkinase KIT domain, are expressed in normal human hematopoietic cell, leukemic cell lines, acute myeloid leukemia blast and GISTs and represent rather a splice variant of KIT transcript. Interestingly our results showed the presence of the transmembrane domain M541L (GenBank X06182.1: c.1642A>C; p.Met541Leu) KIT-activating mutation in exon 10, with an abundance of 50%, in addition to D816V, in 2/20 ISM. This mutation is known to retain sensitivity to imatinib mesylate. Conclusions: Our preliminary results suggest that our-UDS based KIT gene mutation screening assay might be a reliable and sensitive alternative to conventional sequencing methods for the detection of the D816V. We are now planning to investigate whether the greater sensitivity of UDS allows to detect the D816V mutation in peripheral blood mononuclear cells from patients with a suspected clonal mast cell disorder. These results could represent a starting point to plan other extensive studies to better understand the exact role of KIT receptor alterations in SM. Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Cavo: Celgene: Consultancy, Honoraria, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Systemic mastocytosis (SM) includes a heterogeneous group of disorders ranging from indolent SM to mast cell leukemia (MCL). Somatic mutations in the Kit receptor tyrosine kinase (most frequently, D816V) can be detected in >90% of patients with SM and are thought to play a key pathogenetic role. Nevertheless, morphological and clinical diversity, as well as the fact that some patients are negative for KIT mutations, suggest that the underlying molecular picture is far from being fully elucidated. To shed further light on this issue, we undertook an integrated molecular genetic study of a KIT gene mutation-negative MCL patient who came to our attention in 2012 – a 63 year-old woman diagnosed with MCL, aleukemic variant (50-60% atypical mast-cells in the bone marrow [BM] smear; CD117+/CD2+/CD13+-/CD33+/CD59+ immunophenotype; serum tryptase, 2500 µg/L; no C-findings). The patient had received imatinib for 6 months, with no clinical benefit. The disease, however, had had an overall chronic clinical course for 6 more months until severe anemia occurred. The patient rapidly progressed and died after 21 months from diagnosis. After having obtained written informed consent, we extracted genomic DNA and total RNA from purified MCs isolated from BM at diagnosis and at progression, as well as DNA from saliva, and performed whole exome sequencing (WES) and RNA sequencing on an HiSeq1000 (Illumina, San Diego CA). Cytoscan HD arrays (Affymetrix, Santa Clara CA) were also used to scan for chromosomal gains and losses as well as for loss of heterozigosity (LOH). Among the mutated genes detected by WES, SETD2 stood out among others because two distinct putatively inactivating heterozygous mutations were identified, a frameshift insertion of a C in exon 20 (NM_014159:c.7595_7596insC: p.Gly2515ArgfsTer5) and a nonsense mutation in exon 15 (NM_014159:c.G6753T:p.Glu2234Ter). The two mutations were found to hit distinct alleles, pointing to a loss-of-function event. Western Blotting (WB), however, showed that only the 2234 a.a. Setd2 truncated isoform resulting from the nonsense mutation, losing the highly conserved WW and SRI functional domains, was detectable in the sample. The SETD2 gene encodes a histone methyltransferase nonredundantly responsible for trimethylation of lysine 36 of histone H3, a key hystone mark associated not only with active chromatin but also with transcriptional elongation, alternative splicing, DNA replication and repair. SETD2 gene mutations have been described in a variety of cancers and, more recently, have been found to be cooperating events in acute leukemia initiation and progression. In yeast, deletion of the SRI domain abolishes Set2-RNA polymerase II (PolII) interaction causing transcription elongation defects and abolishes K36 methylation. The truncated SETD2 isoform was actually found to lose the ability to bind RNAPolII, as shown by co-immunoprecipitation. Accordingly, RNA-sequencing showed evidence of spurious transcripts initiated from cryptic promoter-like sequences within genes rather than from canonical promoters. More importantly, WB confirmed that H3K36Me3 was completely abrogated. In line with the recently reported role of SETD2-dependent H3K36Me3 in homologous recombination (HR) repair and genome stability, Cytoscan HD arrays showed that LOH and several gains and losses at many chromosomal loci, undetectable at diagnosis, had been acquired at the time of progression. Haploinsufficiency of PSIP1 (recruiting HR machinery at double strand breaks) at 9p24.3 might have represented a cooperating event. Downmodulation of the Setd2 protein (in the presence of LOH but in the apparent absence of sequence variations other than polymorphisms) and reduced H3K36Me3 levels were detected in two more MCL cases, in which putative cooperative lesions were also identified. Results of WES and high resolution karyotyping of additional SM cases will be presented. Our findings point to epigenetic regulation and/or DNA repair as two candidate pathways deserving further investigation in an attempt to identify novel actors or mechanisms contributing to the pathogenesis and progression of SM, or novel modulators of disease phenotype. They also extend the recent observation that the molecular landscape of SM is much more complex than the initial finding of KIT mutations allowed to imagine. Supported by FP7 NGS-PTL project and Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Speakers Bureau.

Systemic mastocytosis (SM) includes a heterogeneous group of disorders ranging from indolent SM to the rare and aggressive mast cell leukemia (MCL). Somatic mutations in the KIT receptor kinase (most frequently, D816V) can be detected in >90% of patients and are thought to play an important pathogenetic role. Nevertheless, morphological and clinical diversity, as well as the fact that some patients are negative for KIT mutations, suggest that the underlying molecular picture is far from being fully elucidated. To shed further light on this issue, we undertook an integrated molecular genetic study of a KIT gene mutation-negative MCL case who came to our attention in 2012. After having obtained written informed consent, we extracted genomic DNA and total RNA from purified mast cells (MCs) isolated from bone marrow at diagnosis and at progression, as well as DNA from saliva, and performed whole exome sequencing (WES) and RNA-seq on an HiSeq1000 (Illumina, San Diego CA). High resolution karyotyping was also performed with Cytoscan HD arrays (Affymetrix, Santa Clara CA). Among the mutated genes detected in MCs but not in saliva by WES, SETD2 stood out among others because two loss-of-function mutations (a nonsense and a frameshift mutation) inactivating both alleles of the gene were identified. Western Blotting (WB) confirmed the expression of the truncated SETD2 isoform resulting from the nonsense mutation. The SETD2 gene encodes a histone methyltransferase responsible for trimethylation of Lysine 36 of histone H3 (H3K36Me3), a key hystone mark associated not only with active chromatin but also with transcriptional elongation, alternative splicing, DNA replication and repair. Loss of the highly conserved WW and SRI domains was predicted to impair SETD2 binding with RNA polymeraseII and hNRNP L, as confirmed by co-immunoprecipitation. Accordingly, RNA-seq showed evidence of spurious transcripts initiated from cryptic promoter-like sequences within genes as well as non-canonical splice isoforms. More importantly, WB confirmed that H3K36Me3 was completely abrogated. In line with the role of SETD2-dependent H3K36Me3 in DNA repair and genome stability, Cytoscan HD arrays and WES showed that several losses at many chromosomal loci, together with more than 70 additional point mutations, undetectable at diagnosis, were acquired at the time of progression. Absence of SETD2 protein expression and/or reduced H3K36Me3 were detected in 3/3 additional MCLs and in 6/8 aggressive SMs so far screened. Our data point to epigenetic regulation and/or DNA repair as candidate pathways deserving further investigation in SM - in an attempt to elucidate the mechanisms underlying enhanced clinical aggressiveness and to identify more effective treatment modalities. Molecular characterization of 10 additional MCLs and aggressive SMs is ongoing. Supported by FP7 NGS-PTL project and Progetto Regione-Università 2010-12(L. Bolondi) Citation Format: Simona Soverini, Caterina De Benedittis, Michela Rondoni, Manuela Mancini, Cristina Papayannidis, Luca Zazzeroni, Viviana Guadagnuolo, Elisa Zago, Francesca Griggio, Alberto Ferrarini, Marianna Garonzi, Massimo Delledonne, Giorgina Specchia, Roberta Zanotti, Omar Perbellini, Livio Pagano, Michele Cavo, Peter Valent, Giovanni Martinelli. Integrated molecular characterization of mast cell leukemia reveals recurrent inactivation of the SETD2 tumor suppressor gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3957. doi:10.1158/1538-7445.AM2015-3957

Background and Aims: Next generation amplicon-based deep sequencing (DS) on the Roche, Illumina or Ion Torrent instruments is becoming accessible to a wider and wider number of diagnostic laboratories. Although conventional sequencing is still the gold standard, DS has been hailed by many as the future of diagnostic BCR-ABL1 kinase domain (KD) mutation screening. BCR-ABL1 KD mutations are infrequent in newly diagnosed chronic myeloid leukemia (CML) patients (pts) receiving 1st-line TKI therapy, but remain a challenge in relapsed pts, who usually display a greater genetic instability. Indeed, pts already harboring BCR-ABL1 KD mutations have a higher likelihood of developing additional, dasatinib (DAS)- or nilotinib (NIL)-resistant mutations – which is defined as a ‘failure’ by the 2013 European LeukemiaNet (ELN) recommendations. Taking advantage of a next-generation amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. Methods: among the imatinib (IM)-resistant CML pts who switched to 2nd-line TKI therapy and were referred to our laboratory for routine BCR-ABL1 transcript level monitoring and KD mutation screening by conventional sequencing, 51 acquired DAS- or NIL-resistant mutations after a median of 9 months (range, 3-27 months) of therapy and had leftover cDNA available at previous timepoints. To reconstruct the dynamics of mutation emergence, resequencing on a Roche GS Junior instrument was performed from the time of failure and mutation detection by conventional sequencing backwards. Runs were designed to achieve high sequencing depth, allowing reliable detection of variants down to 1% abundance. BCR-ABL1/ABL1%IS transcript levels and/or cytogenetic response, whichever available, were used to define whether the patient had an ‘optimal response’, ‘warning’ or ‘failure’ at the time of first mutation detection by DS. Results: baseline mutation status, as assessed by conventional sequencing, was available for all the 51 CML pts included in this retrospective study; 29/51 pts were positive for BCR-ABL1 KD mutations, with switch to NIL or DAS selected accordingly. Twenty-six pts were later found to have acquired DAS-resistant mutations (T315I, n=13; F317L/V, n=10; V299L, n=3) and 25 pts were later found to have acquired NIL-resistant mutations (T315I, n=4; F359V/I/C, n=7; Y253H, n=6; E255K, n=9; one patient acquired two mutations). DS was able to backtrack the DAS- or NIL-resistant mutations to the previous sample(s) in 23/51 (45%) pts. Median mutation burden at the time of first detection by DS was 5% (range, 1-17%); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 3-9 months). In 5 cases, the mutations were traceable at baseline; in the remaining cases, correlation with response at the time mutations were first detected by DS revealed a ‘warning’ according to the 2013 ELN definitions of response to 2nd-line therapy in 13 cases; an ‘optimal response’ in one case; a ‘failure’ in 4 cases. As a control, we used DS to explore BCR-ABL1 KD mutation status in 10 randomly selected pts with ‘warning’ at various timepoints, that later turned into optimal responses; no DAS- or NIL-resistant mutations were detected. Conclusions: the 2011 ELN recommendations for mutation analysis suggest BCR-ABL1 KD to be screened by conventional sequencing in case of ‘failure’ of 2nd-line TKI therapy – according to the provisional definitions available at the time. Earlier detection of emerging BCR-ABL1 KD mutations allows a greater leeway in tackling drug resistance and enhancing therapeutic efficacy. Data presented herein indicate that: 1) DS may reliably pick TKI-resistant mutations earlier than conventional sequencing in a proportion of pts, and that 2) the recently introduced definitions of ‘warning’ may provide a rational trigger, besides ‘failure’, for DS-based BCR-ABL1 KD mutation screening in CML pts on 2nd-line TKI therapy. A prospective cost-benefits evaluation of using DS in this and other settings is warranted, and will contribute useful information to the revision of the ELN recommendations for BCR-ABL1 KD mutation analysis. Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Soverini: Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Ariad: Consultancy. Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Bonifacio:Amgen Inc.: Consultancy. Rosti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.