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Background & Aims Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disorder resulting from genetic and environmental factors. Hyperferritinemia has been associated with increased hepatic iron stores and worse outcomes in patients with NAFLD. The aim of this study was to evaluate the prevalence of variants of iron-related genes and their association with hyperferritinemia, hepatic iron stores and liver disease severity in patients with NAFLD. Methods From a cohort of 328 individuals with histological NAFLD, 23 patients with ferritin >750 ng/ml and positive iron staining, and 25 controls with normal ferritin and negative iron staining, were selected. Patients with increased transferrin saturation, anemia, inflammation, β-thalassemia trait, HFE genotype at risk of iron overload and ferroportin mutations were excluded. A panel of 32 iron genes was re-sequenced. Literature and in silico predictions were employed for prioritization of pathogenic mutations. Results Patients with hyperferritinemia had a higher prevalence of potentially pathogenic rare variants (73.9% vs. 20%, p = 0.0002) associated with higher iron stores and more severe liver fibrosis (p Conclusions Variants in non-HFE iron genes, particularly ceruloplasmin, are associated with hyperferritinemia and increased hepatic iron stores in patients with NAFLD. Carriers of such variants have more severe liver fibrosis, suggesting that genetic predisposition to hepatic iron deposition may translate into liver disease. Lay summary Non-alcoholic fatty liver disease (NAFLD) is a common disease which can progress to cirrhosis and liver cancer. Increased levels of serum ferritin are often detected in patients with NAFLD and have been associated with altered iron metabolism and worse patient outcomes. We found that variants of genes related to iron metabolism, particularly ceruloplasmin, are associated with high ferritin levels, hepatic iron deposition and more severe liver disease in an Italian cohort of patients with NAFLD.

The standard of care for breast cancer has gradually evolved from empirical treatments based on clinical-pathological characteristics to the use of targeted approaches based on the molecular profile of the tumor. Consequently, an increasing number of molecularly targeted drugs have been developed. These drugs target specific alterations, called driver mutations, which confer a survival advantage to cancer cells. To date, the main challenge remains the identification of predictive biomarkers for the selection of the optimal treatment. On this basis, we evaluated a panel of 25 genes involved in the mechanisms of targeted treatment resistance, in 16 primary breast cancers and their matched recurrences, developed during treatment. Overall, we found a detection rate of mutations higher than that described in the literature. In particular, the most frequently mutated genes were ERBB2 and those involved in the PI3K/AKT/mTOR and the MAPK signaling pathways. The study revealed substantial discordances between primary tumors and metastases, stressing the need for analysis of metastatic tissues at recurrence. We observed that 85.7% of patients with an early-stage or locally advanced primary tumor showed at least one mutation in the primary tumor. This finding could explain the subsequent relapse and might therefore justify more targeted adjuvant treatments. Finally, the mutations detected in 50% of relapsed tissues could have guided subsequent treatment choices in a different way. This study demonstrates that mutation events may be present at diagnosis or arise during cancer treatment. As a result, profiling primary and metastatic tumor tissues may be a major step in defining optimal treatments.

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681-686, 2016. © 2016 Wiley Periodicals, Inc.

PubMedID: 26612772 The autosomal co-dominant disorder familial hypobetalipoproteinemia (FHBL) may be due to mutations in the APOB gene encoding apolipoprotein B (apoB), the main constituent peptide of chylomicrons, very low and low density lipoproteins. We describe an 11month-old child with failure to thrive, intestinal lipid malabsorption, hepatic steatosis and severe hypobetalipoproteinemia, suggesting the diagnosis of homozygous FHBL, abetalipoproteinemia (ABL) or chylomicron retention disease (CMRD). The analysis of candidate genes showed that patient was homozygous for a variant (c.1594 C>T) in the APOB gene causing arginine to tryptophan conversion at position 505 of mature apoB (Arg505Trp). No mutations were found in a panel of other potential candidate genes for hypobetalipoproteinemia. In vitro studies showed a reduced secretion of mutant apoB-48 with respect to the wild-type apoB-48 in transfected McA-RH7777 cells. The Arg505Trp substitution is located in the ß?1 domain of apoB involved in the lipidation of apoB mediated by microsomal triglyceride transfer protein (MTP), the first step in VLDL and chylomicron formation. The patient's condition improved in response to a low fat diet supplemented with fat-soluble vitamins. Homozygosity for a rare missense mutation in the ß?1 domain of apoB may be the cause of both severe hypobetalipoproteinemia and intestinal lipid malabsorption. © 2015 Elsevier B.V. 2010C4JJWB-002 Ministero dell’Istruzione, dell’Università e della Ricerca This work was supported by a grant from MIUR (Italian Ministry of University and Research) No. 2010C4JJWB-002 to Patrizia Tarugi.

Background Ruxolitinib (RUX) is a JAK1 & JAK2 inhibitor that resulted in rapid and durable reductions in splenomegaly and improved disease-related symptoms and quality of life in patients (pts) with myelofibrosis (MF) compared with either placebo (COMFORT-I) or best available therapy (BAT; COMFORT-II). In addition, RUX-treated pts had longer overall survival (OS) compared with placebo and BAT. We recently reported that, among 879 primary MF pts receiving conventional BAT, those harboring a mutation in any one of EZH2, ASXL1, IDH1/2 and SRSF2 constituted an IPSS- and DIPSS-plus prognostic score–independent “high molecular risk” (HMR+) category associated with shorter OS and greater risk of leukemia compared with pts with no mutations (“low molecular risk”; LMR) (Vannucchi AM, et al. Leukemia. 2013). The aim of this study was to analyze the impact of mutational status on spleen volume reduction, anemia development, and OS in pts receiving RUX in the COMFORT II trial. Patients and methods In COMFORT-II, pts with primary or post–polycythemia vera/–essential thrombocythemia MF were randomized to receive RUX (n=146) or BAT (n=73). Mutations in 12 genes (JAK2, MPL, EZH2, ASXL1, TET2, IDH1/2, CBL, SRSF2, SOCS1, SOCS2, SOCS3, and SH2B3) were genotyped, in DNA derived from whole blood, at baseline in 166 pts (RUX n=120, BAT n=46). Analysis was performed by next-generation sequencing with the Ion Torrent PGM or Roche 454 platform. Sequencing data were analyzed with Nextgene software or Roche 454 Analysis software v2.6 with variant frequency cutoff adjusted to 5%. All mutations were confirmed at least twice. Missense, nonsense, and frameshift mutations only were considered; in the case of novel mutations, SNPs were excluded by database searching and by germline DNA genotyping when available. Development of anemia was defined as a drop of hemoglobin level by more than 1 g/dL from baseline to a value Results The frequency of mutations was: JAK2V617F 75.47%; MPLW515 7.74%; ASXL1 32.53%; TET2 10.69%; EZH2 7.24%; CBL 4.4%; SRSF2 3.01%; SH2B3 1.3%; IDH1-2 0.7%; SOCS1 0.65%; SOCS2 0.65%; SOCS3 0.0%, with no difference between RUX and BAT. Forty-six (38.3%) and 20 (43.5%) pts in the RUX and BAT groups, respectively, were classified as HMR+. We first determined whether an HMR+ status impacted the achievement of a ≥35% spleen volume reduction (primary study endpoint). The percentage of RUX treated pts achieving ≥35% spleen volume reduction was 36.3% (16/44) and 33.8% (21/62) at 24 wk and 30.7% (12/39) and 36.3% (20/55) at 48 wk in the HMR+ and LMR categories, respectively. Mean spleen volume reduction was also similar: -29.0% and -23.5% in HMR+ vs -29.9% and -30.6% in LMR pts at 24 and 48 wk. None of the other mutations analyzed correlated with spleen volume reduction in pts receiving RUX. We also found that an HMR+ status did not predict for the development of anemia associated with RUX administration: the percentage of anemic pts was 74% in the HMR+ group vs 72% in the LMR group. This was also independent of the presence of mutation in any one of the genes associated with JAK2/STAT signaling (JAK2, MPL, SH3B2, CBL, and SOCSs): anemic pts were 74% in mutated vs 72% in wild-type ones. The survival estimate at 114 wk of follow-up in BAT pts was 0.58 and 0.71 in HMR+ and LMR pts, confirming the negative impact of the mutational risk category. In the RUX arm, the survival estimate was 0.79 and 0.85 for HMR+ and LMR pts, indicating a benefit of RUX treatment in both groups. In the multivariate Cox model, a risk of death with RUX compared with BAT was reduced by 43% (HR=0.57, 95% CI: 0.30-1.08) and LMR patients had a reduction in risk compared with HMR+ by 38% (HR=0.62, 95% CI: 0.33-1.16; Figure). Conclusions The prognostic value of detrimental mutations comprising an HMR+ category in MF was maintained within this subset of COMFORT II patients. The results suggest that the negative impact of HMR+ status on survival in MF pts in COMFORT-II is halved by treatment with RUX compared with BAT and that the effect of RUX is similar in both the HMR+ and LMR groups; in addition, HMR+ status did not affect the likelihood of obtaining a ≥35% spleen volume reduction nor the risk of developing anemia under treatment. Disclosures: Stalbovskaya: Novartis Pharma AG: Employment, Equity Ownership. Squires:Novartis Pharma AG: Employment. Harrison:NOVARTIS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; YM Bioscience: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Honoraria; Shire: Speakers Bureau; Sbio: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Vannucchi:NOVARTIS: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Background: We reported that mutations in ASXL1, EZH2, IDH1/2 and SRSF2 are negative prognostic variables for survival in primary myelofibrosis (PMF). Patients harboring any one of these mutations comprise IPSS and DIPSS-plus independent high molecular risk (HMR) category. Conversely, prognostic variables in secondary myelofibrosis (sMF), including post-polycythemia vera (PPV) and post-essential thrombocythemia (PET) MF are not defined. Aims: The aim was to evaluate the correlations of HMR mutational status with hematologic characteristics and clinical presentation, and the role for outcome predition, in PPV- and PET-MF. Methods: PPV- and PET-MF were diagnosed by IWG-MRT criteria; all pts provided informed consent. Previously published methods were used to screen mutations involving JAK2,MPL, CALR, EZH2, ASXL1, IDH1/2 and SRSF2. The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression. Results: A series of 200 sMF pts from 3 Italian centres was collected: 108 were PPV-MF (54%), 92 PET-MF (46%). PPV-MF cohort: Median age was 65y. Median follow up from PPV-MF diagnosis 4.7y (0.2-25.9y) and median time from PV to PPV-MF 10y (1.08-30.7y). Death occurred in 34 pts (31.8%), 7 pts (6.5%) developed leukemia. Median OS from PPV-MF diagnosis was 9.5y (7.1-11.9y). Frequency of mutations was: JAK2V617F 100% (median allele burden 77%, range 23-100), ASXL1 17.8%, EZH2 3.7%, IDH 5.6%, SRSF2 1%; 29 patients (26.9%) were classified as HMR cases. Hematologic characteristics: median leucocytes 13.0x109/L, hemoglobin 13.3g/dL, platelets 328x109/L, blasts ≥1% 23.4%. Constitutional symptoms in 45.8%, splenomegaly 94.1% (43.3% >10cm from LCM), pruritus 49%; median percentage of BM cellularity was 90% (25-100%) and grade 3 fibrosis in 16.8%. Overall 73 patients were evaluable for karyotype and abnormalities were detected in 42.5%. We did not find significant correlations between individual mutations or HMR category and hematologic and clinical characteristics. PET-MF cohort: Median age was 63.6y. Median follow up from the PET diagnosis 3.3y (0.2-14.5y), median time from ET to PET-MF 11.8y (0.9-30.6y). Death occurred in 30 pts (32.6%), 11 pts (12.0%) developed leukemia. Median OS from PET-MF diagnosis was 9.2y (4.9-13.6y). Frequency of mutations was: JAK2V617F 48.9% (median allele burden 49%, range 0-100), CALR 40.2%, MPL 4.3% and triple negativity (TN) 6.5%; ASXL1 29.3%, EZH2 6.5%, IDH 1.1%, SRSF2 3.3%; 33 pts (35.9%) were HMR. Hematologic characteristics: median leucokytes 8.0x109/L, hemoglobin 10.9 g/dL, platelets 375x109/L, blasts ≥1% 26.4%. Constitutional symptoms 35.4% of pts, splenomegaly 82% (18.2% >10cm from LCM), pruritus i31%; median percentage of BM cellularity was 70% (15-90%) and grade 3 fibrosis was found in 31.7%. Overall 56 patients were evaluable for karyotype and abnormalities were detected in 25.0%. By correlating hematologic and clinical characteristics with unique mutations and HMR category, we found that HMR mutated pts presented greater leukocytosis (P=0.04) and higher JAK2V617F allele burden (P=0.017) than pts in the low-molecular risk –LMR- category (ie, wild-type for ASXL1, EZH2, SRSF2, IDH1/2). Comparison of PPV-MF and PET-MF: a comparison of hematologic and clinical characteristics according to the diagnosis sMF disclosed that PPV-MF pts presented more frequently splenomegaly (P=0.008; for >10cm from LCM P Univariate analysis disclosed significant correlations between shortened survival and mutated ASXL1 (P=0.02, HR 2.2 95% CI, 1.02-4.8) or EZH2 (P=0.05, HR 5.0 95% CI, 1.0-40.7) in PPV-MF. A HMR category was associated with reduced survival in PPV-MF: median survival 6.1y versus 9.5yr LMR (HR 1.07, 95%CI 1.0-4.4; P=0.04). In PET-MF survival was 4.8y in HMR versus 10.9y in LMR (HR1.6, 95%CI 0.8-3.4; P=0.2). Conclusion: We conclude that a HMR status is associated with shorter survival in sMF, but the overal impact is narrower than in primary MF, even though the rate of mutations is similar (Table1). Disclosures No relevant conflicts of interest to declare.

BACKGROUND. In primary myelofibrosis (PMF), survival from time of diagnosis is predicted by the international prognostic scoring system (IPSS), and from time of referral, by the dynamic IPSS (DIPSS) or DIPSS-plus. JAK2/CALR/MPL mutational status and presence and number of other prognostically-relevant mutations (ASXL1, SRSF2, EZH2, IDH1/2) provide IPSS/DIPSS-plus independent prognostic information. The objective in the current study was to revise IPSS, by including mutation-relevant prognostic information. METHODS. The study included 986 PMF patients divided into learning (n=588) and validation (n=398) cohorts. Previously published methods were used to screen for mutations involving JAK2,MPL, CALR, EZH2, ASXL1, IDH1/2 and SRSF2. The clinical variables assessed were those previously identified as being prognostically-relevant by IPSS or DIPSS-plus. The prognostic model (MIPSS) was developed through a stepwise selection process, based on a z-test of the regression coefficients, and its relative quality was measured by means of the Akaike information criterion. RESULTS. In the Italian learning cohort (n=588),median follow-up was 3.6years (95% CI, 0.03-30.8) for alive patients and 196 (33.3%) deaths and 67 leukemic transformations were documented. IPSS risk distribution was low in 26%, intermediate-1 in 30%, intermediate-2 in 24% and high in 20%. Mutational frequencies were approximately 63% for JAK2, 20% CALR, 6% MPL, 7% EZH2, 22% ASXL1, 2.5% IDH1/2 and 9% SRSF2. Constitutional symptoms were recorded in 28.6% of patients, splenomegaly 75% (18% >10cm from LCM) and grade 3 fibrosis 21%. Overall 252 patients were evaluable for karyotype and abnormalities were detected in 35%, including 9.5% unfavorable. Univariate analysis identified the following risk factors for inferior survival: age >60 years, constitutional symptoms, hemoglobin 25x109/L, platelets 10cm, grade 3 fibrosis, unfavorable karyotype, triple-negativity for JAK2, MPL and CALR (TN), JAK2 or MPL mutation, and mutations in ASXL1, SRSF2, EZH2 or IDH1/2. In multivariable analysis, age >60 years, constitutional symptoms, hemoglobin In the Mayo Clinic validation cohort (n=398), median age 63 years; males 64%,median follow-up for alive patients was 7.5 years and 271 (68%) deaths and 51 (13%) leukemic transformations were documented. DIPSS-plus risk distribution was low in 13%, intermediate-1 18%, intermediate-2 37% and high 32%. Mutational frequencies were approximately 51% for JAK2, 30% CALR, 7.5% MPL, 5% EZH2, 36% ASXL1, 5.6% IDH1/2 and 13% SRSF2. MIPSS performed equally well in this cohort, as illustrated in figure 1B, which includes number of patients in each risk category with their median survival and HR and 95% CI values. Leukemia-free survival in the Mayo cohort was also predicted by high (p CONCLUSIONS. MIPSS addresses the mutation-relevant prognostic information gap in IPSS and performs equally well in the setting of patients seen at time of diagnosis or referral. Karyotype carries MIPSS-independent prognostic value that is recognized in an alternative GIPSS prognostic model (see accompanying ASH 2014 abstract), which complements MIPSS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.