Giovanna De Matteis

Hepcidin is not useful as a biomarker for iron needs in haemodialysis patients on maintenance erythropoiesis-stimulating agents

BACKGROUND It has been suggested that hepcidin may be useful as a tool for managing iron therapy in haemodialysis (HD) patients on erythropoiesis-stimulating agents (ESA). METHODS We used SELDI-TOF mass spectrometry assay to measure serum hepcidin-25 (Hep-25) and hepcidin-20 (Hep-20) in 56 adult HD patients on maintenance ESA to assess their ability to predict haemoglobin (Hb) response after 1 g intravenous iron (62.5 mg ferric gluconate at 16 consecutive dialysis sessions) and their relationship with markers of iron status, inflammation and erythropoietic activity. RESULTS At multivariate analysis (in a model that also included Hb, reticulocyte, ESA dose, HFE genotype, soluble transferrin receptor [sTfR] and C-reactive protein), Hep-25 independently correlated with ferritin (β = 0.03, P = 0.01) and the percentage of hypochromic red blood cells [%Hypo] (β = 1.84, P = 0.01), suggesting that Hep-25 may be a useful biomarker for iron stores and bone marrow iron availability. Hep-20 correlated independently with Hep-25 (β = 0.159, P < 0.001) and ferritin (β = 0.006, P = 0.05), suggesting that it may be a useful additional biomarker for iron stores. On receiver operating characteristics curve analysis, neither Hep-25 nor Hep-20 significantly predicted who will increase their Hb after iron loading (AUC = 0.52 ± 0.09 and 0.54 ± 0.08, P = 0.612), and the same applied to ferritin and transferrin saturation (AUC = 0.55 ± 0.08 and 0.59 ± 0.08, P = 0.250), whereas %Hypo and reticulocyte Hb content were significant predictors (AUC = 0.84 ± 0.05 and 0.70 ± 0.08, P < 0.01). At multivariate logistic regression analysis, %Hypo was the only biomarker independently associated with iron responsiveness. CONCLUSIONS Although our study suggests an important role for hepcidin in regulating iron homeostasis in HD patients on ESA, our findings do not support its utility as a predictor of iron needs, offering no advantage over established markers of iron status.

Analysis of B- and T-cell clonality and HLA class II alleles in patients with idiopathic thrombocytopenic purpura: correlation with Helicobacter pylori infection and response to eradication treatment.

Many authors have recently found a positive correlation between Helicobacter pylori infection and idiopathic thrombocytopenic purpura (ITP), the most common autoimmune hematological disorder. In order to clarify the pathogenic mechanism of H. pylori-associated ITP, we have investigated 52 consecutive ITP adult patients for Helicobacter pylori infection, B- and T-cell clonality and HLA class II alleles. Thirty-four ITP patients (65.4%) were infected by H. pylori and bacterium eradication was accompanied by a long-term platelet response in 17 (53.1%) of them. A B-cell clonality was found in three patients (5.8%, two patients H. pylori-negative and one patient H. pylori-positive). The ITP patients with H. pylori infection showed a HLA–DRB1*11, *14 and –DQB1*03 frequencies significantly higher and a –DRB1*03 frequency significantly lower than in H. pylori-negative patients. Moreover, an HLA–DQB1*03 pattern was associated with a higher probability of platelet response to eradication treatment. If our study documents the efficacy of eradication treatment in H. pylori-infected ITP patients, it may also help to identify different subgroups of ITP patients with probably different pathogeneses of thrombocytopenia and, finally, different responses to eradication treatment.

Clonal mast cell disorders in patients with severe Hymenoptera venom allergy and normal serum tryptase levels

BACKGROUND Systemic mastocytosis is a clonal mast cell (MC) disease that can lead to potentially fatal anaphylactic reactions caused by excessive MC mediator release. The prevalence of mastocytosis in patients with Hymenoptera venom allergy is high, and thus the disease should be suspected in patients with severe reactions caused by Hymenoptera stings and increased serum basal tryptase (SBT) levels. OBJECTIVE We sought to evaluate the presence of clonal MC disorders in patients seen at our mastocytosis center with Hymenoptera sting-induced anaphylaxis, documented hypotension, absence of urticaria pigmentosa, and normal SBT levels. METHODS Twenty-two patients with Hymenoptera sting-induced anaphylaxis, without skin lesions, and with tryptase levels of less than 11.4 ng/mL underwent bone marrow evaluation. Bone mineral density was assessed in those patients with ascertained mastocytosis. RESULTS In 16 of 22 patients, a diagnosis of indolent mastocytosis could be established, and 1 patient had a monoclonal MC activation syndrome. Patients with mastocytosis had higher SBT levels (P = .03) but only rarely had angioedema/urticaria associated with hypotension (P = .004). CONCLUSIONS The absence of urticaria or angioedema in severe reactions to Hymenoptera stings with hypotension might represent the most relevant factor in identifying patients with mastocytosis, regardless of their serum tryptase levels.

Real-time polymerase chain reaction quantification of free DNA in serum of patients with polyps and colorectal cancers

Abstract Background: Colorectal cancer (CRC) is one of the most frequent causes of cancer related deaths worldwide. Recently, the use of cell-free DNA as diagnostic tools to identify cancer has been investigated. The aim of this work was to assess whether circulating DNA could be considered a useful marker for detection of early stage CRC and polyps. Methods: A total of 118 patients with CRC were included in the study along with 49 patients with colorectal polyps and 26 control subjects. Cell-free DNA was quantified using a real-time TaqMan-polymerase chain reaction assay. Non-parametric tests (Mann-Whitney test and Spearman correlation) were utilized for statistical analysis. Results: Serum DNA concentrations were significantly higher in CRC patients and patients with polyps (median value 105.0 ng/mL and 40.0 ng/mL) compared with controls (median value 14.0 ng/mL; p<0.05). Although carcinoembryonic antigen was above the cut-off in only 13/66 (19.7%) patients with early stage CRC, serum free DNA showed values above the threshold identified using receiver operator characteristic (ROC) curve analysis in 53/66 (80.3%) patients. Conclusions: Our data confirm that serum DNA concentrations are significantly increased in CRC patients with early stage disease and in patients with polyps. This marker might be useful for identifying high-risk individuals. Clin Chem Lab Med 2010;48:1665–8.

Somatic D816V KIT mutation in a case of adult-onset familial mastocytosis

Rank # Genes Highest OR (L95-U95) Smallest P-value # Genes Highest OR (L95-U95) Smallest P-value # Genes Highest OR (L95-U95) Smallest P-value 1 IRAK1 0.70 (0.55-0.90) 0.006 IRAK1 0.73 (0.54-0.99) 0.042 IRAK1 0.50 (0.28-0.86) 0.013 2 MKK3 1.28 (1.10-1.50) 0.002 IRAKM 0.53 (0.31-0.90) 0.019 SIGIRR 0.54 (0.35-0.85) 0.008 3 ERK2 1.22 (1.05-1.42) 0.010 RIP1 1.49 (1.04-2.12) 0.012 TRAF6 0.65 (0.46-0.90) 0.009 4 IKBKE 0.68 (0.51-0.91) 0.010 JNK2 0.78 (0.65-0.94) 0.010 MKK6 2.29 (1.23-4.25) 0.009 5 Mek1 1.33 (1.00-1.76) 0.025 MKK6 0.55 (0.31-0.95) 0.029 TAB2 0.63 (0.46-0.86) 0.004 6 TOLLIP 0.82 (0.70-0.96) 0.009 MKK3 1.27 (1.05-1.53) 0.014 IRAK4 0.42 (0.20-0.89) 0.024 7 JNK2 1.42 (1.07-1.87) 0.014 IL1RL1 0.79 (0.65-0.96) 0.015 MyD88 2.07 (1.10-3.90) 0.024 8 SIGIRR 0.77 (0.62-0.95) 0.014 IRF7 0.74 (0.56-0.98) 0.029 IKBKE 1.47 (1.08-1.98) 0.013 9 TAB2 0.82 (0.70-0.95) 0.009 NFKB1 0.61 (0.39-0.97) 0.034 RIP1 1.85 (1.05-3.25) 0.034 10 TAB1 0.83 (0.71-0.97) 0.022 TIRAP 1.65 (1.01-2.69) 0.045 TRAM1 1.75 (1.02-3.01) 0.043 11 AKT1 0.77 (0.61-0.98) 0.030 IRF3 0.76 (0.59-0.98) 0.031 P38 1.67 (1.03-2.72) 0.039 12 IRF3 0.79 (0.64-0.97) 0.026 Mek1 1.25 (1.02-1.53) 0.032 JNK1 1.60 (1.03-2.48) 0.038 13 IRAKM 1.24 (1.04-1.47) 0.015 AKT1 1.25 (1.01-1.55) 0.037 JNK2 1.51 (1.04-2.20) 0.030 14 RIP1 1.38 (1.03-1.84) 0.029 AP1 0.68 (0.46-1.00) 0.049 15 TAK1 1.30 (1.03-1.64) 0.026 16 TRAM1 1.37 (1.01-1.86) 0.045 17 NFKB1 0.68 (0.47-1.00) 0.048 18 IL1RL1 0.84 (0.72-0.99) 0.031 19 MKK4 1.25 (1.00-1.55) 0.047

Efficacy and safety of phlebotomy to reduce transfusional iron overload in adult, long‐term survivors of acute leukemia

BACKGROUND:  Transfusional iron overload is a frequent finding in long‐term survivors of acute leukemia (AL). Only a few studies have reported the results of iron depletion therapy in this category of patients.

The impact of sensitive KIT D816V detection on recognition of Indolent Systemic Mastocytosis

Patients with Systemic Mastocytosis (SM) need a highly sensitive diagnostic test for D816V detection of the KIT receptor gene. Along with histology/cytology and flow cytometry evaluation, bone marrow (BM) from 110 consecutive adult patients referred with a suspicion of SM to Multidisciplinary Outpatient Clinic for Mastocytosis in Verona were tested both by Amplification Refractory Mutation System Reverse Transcriptase quantitative real time Polymerase Chain Reaction (ARMS-RT-qPCR) and RT-PCR+Restriction Fragment Length Polymorphism (RFLP) followed by Denaturing-High Performance Liquid Chromatography (D-HPLC) and Sanger sequencing. ARMS-RT-qPCR identified D816V mutation in 77 patients, corresponding to 100% of cases showing CD25(+) mast cells (MCs) whereas RT-PCR+RFLP/D-HPLC+sequencing revealed D816V mutations in 47 patients. According to the 2008 WHO criteria 75 SM, 1 Cutaneous Mastocytosis (CM), 1 monoclonal MC activation syndrome (MMAS), and 1 SM Associated with Haematologic Non-Mast Cell Disorder (SM-AHNMD) were diagnosed. Seventeen out 75 SM patients (23%) would have not satisfied sufficient WHO criteria on the basis of the sole RT-PCR+RFLP: these patients had significantly lower serum tryptase levels and amount of CD25(+) MCs. Therefore, ARMS-RT-qPCR might result particularly useful, in patients that do not fulfil major BM histological criterion, for the recognition of indolent SM with a very low MC burden.

Identification of novel mutations in hemochromatosis genes by targeted next generation sequencing in Italian patients with unexplained iron overload

Hereditary hemochromatosis, one of the commonest genetic disorder in Caucasians, is mainly associated to homozygosity for the C282Y mutation in the HFE gene, which is highly prevalent (allele frequency up to near 10% in Northern Europe) and easily detectable through a widely available “first level” molecular test. However, in certain geographical regions like the Mediterranean area, up to 30% of patients with a HH phenotype has a negative or non‐diagnostic (i.e. simple heterozygosity) test, because of a known heterogeneity involving at least four other genes (HAMP, HJV, TFR2, and SLC40A1). Mutations in such genes are generally rare/private, making the diagnosis of atypical HH essentially a matter of exclusion in clinical practice (from here the term of “non‐HFE” HH), unless cumbersome traditional sequencing is applied. We developed a Next Generation Sequencing (NGS)‐based test targeting the five HH genes, and applied it to patients with clinically relevant iron overload (IO) and a non‐diagnostic first level genetic test. We identified several mutations, some of which were novel (i.e. HFE W163X, HAMP R59X, and TFR2 D555N) and allowed molecular reclassification of “non‐HFE” HH clinical diagnosis, particularly in some highly selected IO patients without concurring acquired risk factors. This NGS‐based “second level” genetic test may represent a useful tool for molecular diagnosis of HH in patients in whom HH phenotype remains unexplained after the search of common HFE mutations. Am. J. Hematol. 91:420–425, 2016.

Reduced DNA methylation and hydroxymethylation in patients with systemic mastocytosis

As disruption of epigenetic control is a frequent event in solid tumors and leukemia, we investigated changes in DNA methylation (5mC) and hydroxymethylation (5hmC) in patients with systemic mastocytosis (SM), a rare myeloproliferative disease with a wide spectrum of severity, characterized by the accumulation of mast cells in various organs.

Baseline factors associated with response to ruxolitinib: an independent study on 408 patients with myelofibrosis

In patients with Myelofibrosis (MF) treated with ruxolitinib (RUX), the response is unpredictable at therapy start. We retrospectively evaluated the impact of clinical/laboratory factors on responses in 408 patients treated with RUX according to prescribing obligations in 18 Italian Hematology Centers. At 6 months, 114 out of 327 (34.9%) evaluable patients achieved a spleen response. By multivariable Cox proportional hazard regression model, pre-treatment factors negatively correlating with spleen response were: high/intermediate-2 IPSS risk (p=0.024), large splenomegaly (p=0.017), transfusion dependency (p=0.022), platelet count <200×109/l (p=0.028), and a time-interval between MF diagnosis and RUX start >2 years (p=0.048). Also, patients treated with higher (≥10 mg BID) average RUX doses in the first 12 weeks achieved higher response rates (p=0.019). After adjustment for IPSS risk, patients in spleen response at 6 months showed only a trend for better survival compared to non-responders. At 6 months, symptoms response was achieved by 85.5% of 344 evaluable patients; only a higher (>20) Total Symptom Score significantly correlated with lower probability of response (p<0.001). Increased disease severity, a delay in RUX start and titrated doses <10 mg BID were associated with patients achievinglower response rates. An early treatment and higher RUX doses may achieve better therapeutic results.In patients with Myelofibrosis (MF) treated with ruxolitinib (RUX), the response is unpredictable at therapy start. We retrospectively evaluated the impact of clinical/laboratory factors on responses in 408 patients treated with RUX according to prescribing obligations in 18 Italian Hematology Centers. At 6 months, 114 out of 327 (34.9%) evaluable patients achieved a spleen response. By multivariable Cox proportional hazard regression model, pre-treatment factors negatively correlating with spleen response were: high/intermediate-2 IPSS risk (p=0.024), large splenomegaly (p=0.017), transfusion dependency (p=0.022), platelet count <200x109/l (p=0.028), and a time-interval between MF diagnosis and RUX start >2 years (p=0.048). Also, patients treated with higher (≥10 mg BID) average RUX doses in the first 12 weeks achieved higher response rates (p=0.019). After adjustment for IPSS risk, patients in spleen response at 6 months showed only a trend for better survival compared to non-responders. At 6 months, symptoms response was achieved by 85.5% of 344 evaluable patients; only a higher (>20) Total Symptom Score significantly correlated with lower probability of response (p<0.001). Increased disease severity, a delay in RUX start and titrated doses <10 mg BID were associated with patients achievinglower response rates. An early treatment and higher RUX doses may achieve better therapeutic results.