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Increased microvessel density contributes to abnormal BM and spleen microenvironment in myelofibrosis (MF). Taking advantage of the JAK2V617F mutation as a marker of malignancy, in the present study, we investigated whether splenic endothelial cells (ECs) obtained from capillaries by laser microdissection or from fresh spleen tissue by cell culture or cell sorting harbored such mutation in patients bearing the mutation in their granulocytes and undergoing splenectomy for therapeutical reasons. To extend the analysis to the ECs of large vessels, endothelial tissue from the splenic vein was also studied. We found JAK2V617F+ ECs in 12 of 18 patients also bearing the mutation in their granulocytes. In 3 patients, the mutation was found in at least 2 different EC samples obtained by laser microdissection, cell culture, or cell sorting. The mutation was detected in the splenic vein ECs of 1 of 6 patients investigated. In conclusion, we provide evidence that some ECs from the spleen and splenic veins of patients with MF bear the JAK2V617F mutation. We suggest that splenic ECs are involved in the process of malignant transformation in MF.

PurposeIn the WHO diagnostic classification, prefibrotic myelofibrosis (pre-MF) is included in the category of primary myelofibrosis (PMF). However, strong evidence for this position is lacking.Patients and methodsWe investigated whether pre-MF may be aligned along a clinical and biological continuum in 683 consecutive patients who received a WHO diagnosis of PMF.ResultsAs compared with PMF-fibrotic type, pre-MF (132 cases) showed female dominance, younger age, higher hemoglobin, higher platelet count, lower white blood cell count, smaller spleen index and higher incidence of splanchnic vein thrombosis. Female to male ratio and hemoglobin steadily decreased, while age increased from pre-MF to PMF- fibrotic type with early and to advanced bone marrow (BM) fibrosis. Likely, circulating CD34+ cells, LDH levels, and frequency of chromosomal abnormalities increased, while CXCR4 expression on CD34+ cells and serum cholesterol decreased along the continuum of BM fibrosis. Median survival of the entire cohort of PMF cases was 21 years. Ninety-eight, eighty-one and fifty-six percent of patients with pre-MF, PMF-fibrotic type with early and with advanced BM fibrosis, respectively, were alive at 10 years from diagnosis.ConclusionPre-MF is a presentation mode of PMF with a very indolent phenotype. The major consequences of this contention is a new clinical vision of PMF, and the need to improve prognosis prediction of the disease.

Rationale: Catecholaminergic polymorphic ventricular tachycardia is an inherited disease that predisposes to cardiac arrest and sudden death. The disease is associated with mutations in the genes encoding for the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2). CASQ2 mutations lead to a major loss of CASQ2 monomers, possibly because of enhanced degradation of the mutant protein. The decrease of CASQ2 is associated with a reduction in the levels of Triadin (TrD) and Junctin (JnC), two proteins that form, with CASQ2 and RyR2, a macromolecular complex devoted to control of calcium release from the sarcoplasmic reticulum. Objective: We intended to evaluate whether viral gene transfer of wild-type CASQ2 may rescue the broad spectrum of abnormalities caused by mutant CASQ2. Methods and Results: We used an adeno-associated serotype 9 viral vector to express a green fluorescent protein-tagged CASQ2 construct. Twenty weeks after intraperitoneal injection of the vector in neonate CASQ2 KO mice, we observed normalization of the levels of calsequestrin, triadin, and junctin, rescue of electrophysiological and ultrastructural abnormalities caused by CASQ2 ablation, and lack of life-threatening arrhythmias. Conclusions: We have proven the concept that induction of CASQ2 expression in knockout mice reverts the molecular, structural, and electric abnormalities and prevents life-threatening arrhythmias in CASQ2-defective catecholaminergic polymorphic ventricular tachycardia mice. These data support the view that development of CASQ2 viral gene transfer could have clinical application.

Background. Primary myelofibrosis (PMF) is a clonal, neoplastic disorder of the hematopoietic stem cells, characterized by an increased number of circulating CD34+ cells. Available evidence indicates that enhanced activation of JAK-STAT pathway is the main molecular mechanism of myeloproliferation due to somatic mutations in JAK2, CALR or MPL genes. A subset of patients display immune-related abnormalities that suggest an (auto)immune pathogenesis. In addition, it has been reported a significant association between personal history of a broad span of autoimmune diseases and subsequent risk of myeloproliferative neoplasm (Kristinsson SY Haematologica 2010; 95:1216-1220). CD4+ T cells maintain cancer surveillance and immune tolerance. Chronic inflammation has been proposed as a driver of clonal evolution in myeloproliferative neoplasms (MPN) (Hasselbalch HC et al. Leuk Res 2013;37:214-20), suggesting that T cells play an important role in their pathogenesis. A recent paper by Keohane C et al (BJH 2015; doi 10.1111/bjh 13519) shows that peripheral blood (PB) T regulatory cells (Tregs) are reduced in MPN patients compared to healthy subjects (CTRLs) and that this decrease is even more pronounced following JAKi therapy (Massa M et al Leukemia 2014;28:449-51). Unpublished data from our group further indicate a significant (p Study design. To investigate the possible factors favouring a decrease of Tregs, we examined in patients with PMF and Results. 1) The percentages of PB CD4+, CD4+CD25+, or CD4+CD25++CD127low/- cells expressing membrane Annexin V were comparable in patients with PMF and CTRLs (data not shown). 2) The phosphorilation of Stat1, Stat3, Stat5, Stat6, and p38-MAPK (expressed as mean fluorescence intensity: MFI) on CD4+, CD4+CD25-, CD4+CD25+, and CD4+CD25++FOXP3+ cells before and after stimulation with IFNα, IL6, IL4, or PMA was comparable in patients and CTRLs. The Erk1,2 phosphorilation was comparable in patients and CTRLs at baseline condition, while in patients with PMF Erk1,2 phosphorilation following stimulation with PMA was significantly lower in both CD4+CD25+ cells (p=0.039; median MFI 5.4, range 0.3-40) and CD4+CD25++FOXP3+ cells (p=0.01; median MFI 4.3, range 1.4-9.3) than in CTRLs (CD4+CD25+ cells median MFI 8.1, range 4.9-20; CD4+CD25++FOXP3+ cells median MFI 7.3, 3.7-10.8). 3) sIL2rα plasma levels were higher (p=0.01) in patients with PMF (1658 pg/ml, range 476-8968) than in CTRLs (1040 pg/ml, 482-1719); the sIL2rα plasma levels were significantly (R=-0.35, p=0.039) inversely correlated with the percentages of circulating Tregs. No significant correlation was found with the presence (n=13)/absence (n=14) of JAK2V617F mutation. Conclusions. These results indicate that in patients with PMF Tregs present an untuned Erk signaling; moreover, sIL2rα plasma levels are higher in patients than in CTRLs, and significantly inversely correlated with Treg percentages. These findings may partly explain the reduced frequency of Tregs in PMF since Erk signaling is pivotal in T cell differentiation, regulation of T cell homeostasis, and defects in Erk expression have been associated with autoimmunity (Chiung-Fang Chang J Immunol 2012; 189:721-731). Moreover, increased levels of sIL2rα may impact on IL-2-mediated functions and alterate the optimal activation of Erk1/2 that is IL-2 dependent (Lequn Li, Blood. 2005;106:3068-3073), Disclosures No relevant conflicts of interest to declare.

A contribution of Helicobacter pylori gastritis to the pathogenesis of non-ulcer dyspepsia (NUD) remains uncertain.Administration of an appropriate clinical questionnaire followed by endoscopy allowed us to select, among 139 outpatients with dyspepsia, 87 non-ulcer dyspepsia patients with more severe and group-distinctive symptoms, 35 of whom were classified as having ulcer-like (ULD). 38 as dysmotility-like (DLD), and 14 as reflux-like dyspepsia (RLD). Biopsy specimens were evaluated for H. pylori gastritis in accordance with the Sydney system. The 70 H. pylori-positive cases were treated with omeprazole, 20 mg twice daily, and amoxycillin, 1 g three times daily for 2 weeks.Higher rates of H. pylori colonization were found histologically in the gastric mucosa of ULD (91%) and RLD (86%) than in that of DLD (68%) or asymptomatic (42%) patients. ULD differed from RLD patients in their higher score of antritis activity. Three and 6 months after H. pylori eradication ULD (but not DLD) showed significant regression of dypspetic symptoms scores.It seems likely that H. pylori gastritis, with special reference to active antritis, is among causative factors of ULD. Its role in the pathogenesis of RLD and DLD needs further investigation.

To clarify the mechanisms of the gastric mucosal immune—inflammatory response to Helicobacter pylori infection, surgical and biopsy specimens from asymptomatic uninfected, gastritis-free individuals and from H. pylori-positive ulcer patients with chronic gastritis were investigated using light and electron microscopy. Activation of the antigen-transporting endocytic-endosomal system, enhanced expression of the antigen-processing enzyme cathepsin E and de novo expression of antigen-presenting human leukocyte antigen (HLA)-DR molecules have been detected in H. pylori-colonized gastric epithelium. These findings may be crucial in the production of a mucosal immune-inflammatory response to H. pylori infection. Cytoplasmic swelling and vacuolation, micropapillary change, mucin loss, erosion of the juxtaluminal cytoplasm and cell desquamation were the main effects of bacterial cytotoxicity on gastric surface-foveolar epithelium. Activated macrophages and granulocytes (partly linked to the mucosal IgG immune res...

The incidence and mean score of Helicobacter pylori-related, active antroduodenitis, lesions of superficial antral epithelium and duodenal gastric-type metaplasia were higher in endoscopic biopsies from a large series of patients with duodenal ulcer, when compared with asymptomatic patients or patients with non-ulcer dyspepsia. In 65 out of 73 patients with duodenal ulcer who could be followed up, H. pylori was eradicated using a combination of amoxycillin, 3 g daily, metronidazole, 1 g daily, and omeprazole, 20 mg daily. Rapid and permanent (6-month follow-up) abolition of both gastroduodenitis activity and lesions of the gastric surface epithelium was observed in these 65 patients. There was also a progressive decrease in total immune-inflammatory cells but without a substantial change in duodenal gastric-type metaplasia. Similar, but transient and quantitatively less prominent, improvements were observed in the antroduodenal mucosa, which had been temporarily cleared of H. pylori by treatment with omeprazole alone. Conversely, increased gastritis activity, epithelial lesions and immune-inflammatory cell scores were found in the short term in the corpus mucosa, which was not cleared of H. pylori after omeprazole treatment. It is concluded that, of the various H. pylori-related mucosal changes, antroduodenitis activity and antral epithelial lesions most closely reflect the severity of mucosal damage and are probably the most important factors in duodenal ulcerogenesis. Their complete and rapid suppression after bacterial eradication may be a key factor in preventing ulcer relapse.

Sorafenib is the only therapy approved for advanced hepatocellular carcinoma no longer eligible for transcatheter arterial chemoembolization. Hepatic intra-arterial chemotherapy has been shown to be an effective and safe therapy for advanced hepatocellular carcinoma. Cetuximab has been administered intravenously to patients with advanced hepatocellular carcinoma, showing encouraging results in terms of its safety and toxicity profile.Our purpose was to evaluate the safety and feasibility of hepatic arterial chemotherapy with cetuximab, cisplatin and 5-fluoruracil for patients with advanced hepatocellular carcinoma, not responsive or not eligible for sorafenib therapy.From January 2010 to January 2011, 12 patients received a 2-day course of chemotherapy consisting of repeated daily hepatic arterial administration of 20 mg of cisplatin as 2-h infusion, 5-fluorouracil at 500 mg/m(2) as 5-h infusion and cetuximab 500 mg/m(2) as 12-h infusion. Cycles were repeated every 14 days.After a mean of four months of therapy, computed tomography revealed five partial responses, five cases of stable disease and two of progressive disease. The toxicity profile was favourable, with no G4 gastrointestinal, hematologic or skin side-effects, or severe deterioration of liver function.Hepatic intra-arterial chemotherapy with cetuximab is a safe and feasible treatment for advanced hepatocellular carcinoma, with promising results in patients with initial poor prognosis.

ECL cells are argyrophilic endocrine cells of the stomach. Their distribution is species specific, however they are consistently located in the oxyntic mucosa and, in particular, in very close contact with the adenomeres of acidopeptic glands. ECL cells store histamine and are considered a key element in the mechanisms of gastric acid secretion as controlled by gastrin stimulus. Their peculiar anatomical disposition and secretory properties strongly suggest that ECL cells exert their function by a paracrine mechanism, i.e. by releasing histamine in the extracellular spaces surrounding acid-producing parietal cells. ECL cell activity is strongly stimulated by gastrin, which, once applied as a long-standing stimulus, also exerts a potent proliferating effect. Long-lasting hypergastrinaemia has been demonstrated to elicit ECL cell proliferation in laboratory animals, inducing ECL cell hyperplasia, dysplasia and ECL cell tumours, i.e. argyrophilic gastric carcinoids. However, in experimental rodents it is believed that hypergastrinaemia is not per se a stimulus capable of inducing ECL cell transformation, a predisposing genetic background being required for tumour development in endocrine organs. In man, long-standing hypergastrinaemia exerts the same proliferative pressure on ECL cells and is associated with hyperplasia with or without dysplastic changes and carcinoid development. Clinical evidence suggests that other factors, both genetic and environmental, are required to induce ECL cell transformation and carcinoid development. For this reason human gastric argyrophilic ECL carcinoids are subdivided into three main groups depending on their clinical background: (1) gastric carcinoids in patients with chronic atrophic gastritis; (2) gastric carcinoids in patients with Zollinger-Ellison and multiple endocrine neoplasia type 1 syndrome (MEN-ZES); and (3) solitary, sporadic gastric carcinoids. The clinical assessment of carcinoid-bearing patients is strongly recommended for better diagnosis and management of patients.

TGFβ1 is secreted as latent protein that requires activation to become biologically active. It negatively regulates the progenitor cell growth, and favours the deposition of extra-cellular matrix in different tissues. We have studied TGFβ1 levels in Philadelphia-negative (Ph-) myeloproliferative diseases, evaluating patients with primary myelofibrosis (PMF) that is characterized by increased numbers of circulating progenitor cells and bone marrow (BM) fibrosis, and patients with polycythemia vera (PV) or essential thrombocythemia (ET) that do not present BM fibrosis. We found that patients with PMF, PV or ET have higher peripheral blood (PB) plasma levels of both bioactive and total TGFβ1 than healthy controls, with a balance bioactive/total TGFβ1 in favour of the latter. The balance between bioactive/total TGFβ1 in the BM plasma of patients mirrored that of PB, with most of TGFβ1 in the latent form; on the contrary, in the BM plasma of healthy controls most of the TGFβ1 was in the bioactive form. In conclusion, increased plasma levels of TGFβ1 and an altered ratio bioactive/total TGFβ1 in BM are not peculiar of patients with PMF suggesting that, whether altered levels of TGFβ1 have a role in myelofibrosis, this may not be related to the induction of BM fibrosis.

Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit–endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.

Intrahepatic cholangiocarcinoma (ICC) is a rare life-threatening disease, whose only treatment with potential for cure is surgical resection. However, only 27% of patients at most are suitable for surgery when first diagnosed. For patients with unresectable disease, therapeutic options are chemotherapy or chemoradiation. We evaluated the feasibilty and safety of oxaliplatin-eluting microspheres transarterial chemoembolization (OEM-TACE) associated with chemotherapy (ChT) in patients affected by unresectable ICC. Between December 2005 and May 2008 we treated nine patients (six female and three male) with unresectable ICC. All patients had undergone OEM-TACE associated with chemotherapy with oxaliplatin and gemcitabine. A retrospective comparison was carried out with a historical group of 11 patients treated with ChT only, estimating the prevalence of adverse effects and the median survival of the two groups. A total of 30 TACEs were performed during the observational time (ranging from one to seven procedures per patient). OEM-TACEs were followed by few adverse effects (AEs), without G4 AEs, according to CTACAE 3.0. According to RECIST criteria, 44% (4/9) of patients achieved partial responses and 56% (5/9) stabililization of disease. Overall survival analysis in the two groups showed a significantly increased survival in patients treated with ChT and OEM-TACE, with respect to those treated with ChT (30 vs. 12.7 months; p = 0.004). In conclusion, in our experience OEM-TACE associated with ChT in the treatment of advanced unresectable ICC is a safe and feasible treatment causing no major adverse events. Although RECIST criteria can underestimate the rate of responses in patients treated with locoregional therapies, we achieved very encouraging results. A randomized multicentric trial is warranted to assess the actual superiority of OEM-TACE associated with ChT compared to conventional chemotherapy.

Antral gastrin cell hyperfunction is a rare condition, often associated with severe duodenal ulcer disease. In children, clinical and functional characteristics of this syndrome are poorly known. Two cases are described here: one child had melena and the other had moderate abdominal pain, both without peptic ulceration. Basal and postprandial increase of gastrin levels showed a response over the upper normal range, indicating gastrin cell hyperfunction. Acid hypersecretion, both basal and after pentagastrin stimulation, was also found in the two children, confirming the biological effect of their sustained hypergastrinemia. Gastrin cell counts were within the normal range, while the number of somatostatin D cells was significantly reduced. This report stresses the importance of diagnosing antral gastrin cell hyperfunction in children because this unrecognized condition may manifest with serious complications (bleeding) or nonspecific abdominal symptoms.

The spectrum of endocrine cell changes occurring in 80 cases of body-fundus chronic atrophic gastritis (CAG), mostly type A or multifocal, including various types of hyperplasia, precarcinoid lesions (20 cases), and neoplasia (carcinoid, 24 cases) have been analyzed histologically, histochemically, and ultrastructurally. Changes associated with gland atrophy, pyloric- or intestinal-type metaplasia, regenerative hyperplasia, and hypergastrinemia have been identified and their neoplastic potential evaluated in the light of their proliferative capacity (bromodeoxyuridine incorporation) and clinicopathologic behavior. A close link between disseminated precarcinoid lesions of non-tumor mucosa and multiple carcinoids (carcinoidosis) arising in hypergastrinemic type-A CAG is suggested. Hyperplastic changes, including endocrine cell clusters, seem to have no or only minimal neoplastic potential.

Intrahepatic cholangiocarcinoma account for 13% of annual cancer-related deaths worldwide and for 3% in the USA. Patient with unresectable disease can benefit from palliative therapies such as systemic chemotherapy. However, the only curative treatment for intrahepatic cholangiocarcinoma is complete surgical resection with histologically negative resection margins.

Among 994 patients with essential thrombocythemia (ET) who were genotyped for the MPLW515L/K mutation, 30 patients carrying the mutation were identified (3.0%), 8 of whom also displayed the JAK2V671F mutation. MPLW515L/K patients presented lower hemoglobin levels and higher platelet counts than did wild type (wt) MPL; these differences were highly significant compared with MPLwt/JAK2V617F–positive patients. Reduced hemoglobin and increased platelet levels were preferentially associated with the W515L and W515K alleles, respectively. MPL mutation was a significant risk factor for microvessel disturbances, suggesting platelet hyperreactivity associated with constitutively active MPL; arterial thromboses were increased only in comparison to MPLwt/JAK2wt patients. MPLW515L/K patients presented reduced total and erythroid bone marrow cellularity, whereas the numbers of megakaryocytes, megakaryocytic clusters, and small-sized megakaryocytes were all significantly increased. These data indicate that MPLW515L/K mutations do not define a distinct phenotype in ET, although some differences depended on the JAK2V617F mutational status of the counterpart.

To the editor: The Budd-Chiari syndrome (BCS) and extrahepatic portal vein obstruction (EHPVO) are splanchnic vein thromboses (SVT) that may occur as presenting complications of undiagnosed chronic myeloproliferative disorders (CMPD). Diagnosis of the underlying CMPD may be difficult because

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disorder characterized by life threatening arrhythmias elicited by physical and emotional stress in young individuals. The recessive form of CPVT is associated with mutation in the cardiac calsequestrin gene ( CASQ2 ). We engineered and characterized a homozygous CASQ2 R33Q/R33Q mouse model that closely mimics the clinical phenotype of CPVT patients. CASQ2 R33Q/R33Q mice develop bidirectional VT on exposure to environmental stress whereas CASQ2 R33Q/R33Q myocytes show reduction of the sarcoplasmic reticulum (SR) calcium content, adrenergically mediated delayed (DADs) and early (EADs) afterdepolarizations leading to triggered activity. Furthermore triadin, junctin, and CASQ2-R33Q proteins are significantly decreased in knock-in mice despite normal levels of mRNA, whereas the ryanodine receptor (RyR2), calreticulin, phospholamban, and SERCA2a-ATPase are not changed. Trypsin digestion studies show increased susceptibility to proteolysis of mutant CASQ2. Despite normal histology, CASQ2 R33Q/R33Q hearts display ultrastructural changes such as disarray of junctional electron-dense material, referable to CASQ2 polymers, dilatation of junctional SR, yet normal total SR volume. Based on the foregoings, we propose that the phenotype of the CASQ2 R33Q/R33Q CPVT mouse model is portrayed by an unexpected set of abnormalities including (1) reduced CASQ2 content, possibly attributable to increased degradation of CASQ2-R33Q, (2) reduction of SR calcium content, (3) dilatation of junctional SR, and (4) impaired clustering of mutant CASQ2.

Twenty-three children with Helicobacter (Campylobacter) pylori-associated chronic gastritis are reported. Family history of peptic disease, previous digestive procedures, and nonspecific epigastric pain were the most frequently encountered clinical features. Antral nodularity at endoscopy and histologic evidence of follicular gastritis were characteristic morphological aspects. Rapid urease tests suggested the diagnosis in 90% of patients. Significant increases of serum IgG and IgA against Helicobacter pylori allowed the identification of infected children with 95% cumulative sensitivity. Treatment with amoxicillin and bismuth subcitrate eradicated the infection and improved gastritis in 13 of 19 children. These findings provide further evidence for the etiologic role of Helicobacter pylori in chronic antral gastritis in children.

M1, a mucin antigen, and cathepsin E, an aspartic proteinase, are both expressed in normal gastric superficial-foveolar epithelial cells. In this study, we determined by immunohistochemical staining the prevalence of these antigens in 316 gastric cancers representative of the main histologic types and stages of the disease. M1 was expressed in 201 cases (64%) and cathepsin E was expressed in 235 cases (75%) of the 313 cases investigated. Both antigens were expressed more commonly in diffuse and mixed cancers than in glandular tumors. M1 was found in 64 of 83 (77%) diffuse cancers and in 48 of 59 (81%) mixed cancers, but in only 74 of 146 (51%) glandular cancers. For cathepsin E, the prevalence was 93% in diffuse cancer, 81% in mixed cancer, and 71% in the 143 glandular cancers examined. Among 25 mucoid tumors, 15 (60%) expressed M1 but only eight (32%) expressed cathepsin E. Overall, 262 (84%) of the tumors expressed at least one of these antigens and of these, 173 (66%) expressed both antigens. No significant difference in the prevalence of M1 or cathepsin E was found between early and advanced cancer or between metastatic and nonmetastatic cancer. The two markers differed in their intracellular localization. In superficial-foveolar cells, M1 immunostaining was concentrated in secretory granules, Golgi complex, and luminal mucous, whereas cathepsin E was found in the endoplasmic reticulum. Moreover, cathepsin E, but not M1, was found in the enterocytes of duodenal villi and, occasionally, in mucopeptic cells. Parallel histochemical and ultrastructural investigations confirmed the occurrence in gastric cancer of foveolar-type cells, manifested by periodic acid-Schiff- and/or alcian blue-reactive mucous granules having a punctate substructure. We conclude that superficial-foveolar cell differentiation is common in gastric cancer and is a major component of this type of tumor. However, pure foveolar cell differentiation is rare. Rather, most gastric cancers consist of cells exhibiting features of foveolar, intestinal, and mucopeptic cell lines.

The clinical and haematological phenotype of patients with myelofibrosis harbouring MPL(W515L/K) mutation has not been thoroughly investigated. Of 217 myelofibrosis subjects, 18 (8.2%) had an MPL mutation, four of which (22%) co-existed with JAK2(V617F) mutation. When compared with MPL wild-type patients, irrespective of JAK2(V617F) status, those with MPL(W515L/K), were more frequently female, were older (61 years vs. 57 years; P = 0.02), presented with more severe anaemia (haemoglobin, 101 g/l vs. 121 g/l; P = 0.002) and were more likely to require regular transfusional support (P = 0.012). These data indicate that MPL mutation in myelofibrosis characterises patients with more severe anaemic phenotype.

Background Primary myelofibrosis (PMF) is an acquired clonal disease of the hematopoietic stem cell compartment, characterized by bone marrow fibrosis, anemia, splenomegaly and extramedullary hematopoiesis. About 60% of patients with PMF harbor a somatic mutation of the JAK2 gene (JAK2-V617F) in their hematopoietic lineage. Recently, a splicing isoform of JAK2, lacking exon 14 (JAK2Δ14) was described in patients affected by myeloproliferative diseases. Materials and Methods By using a specific RT-qPCR method, we measured the ratio between the splicing isoform and the JAK2 full-length transcript (JAK2+14) in granulocytes, isolated from peripheral blood, of forty-four patients with PMF and nine healthy donors. Results We found that JAK2Δ14 was only slightly increased in patients and, at variance with published data, the splicing isoform was also detectable in healthy controls. We also found that, in patients bearing the JAK2-V617F mutation, the percentage of mutated alleles correlated with the observed increase in JAK2Δ14. Homozygosity for the mutation was also associated with a higher level of JAK2+14. Bioinformatic analysis indicates the possibility that the G>T transversion may interfere with the correct splicing of exon 14 by modifying a splicing regulatory sequence. Conclusions Increased levels of JAK2 full-length transcript and a small but significant increase in JAK2 exon 14 skipping, are associated with the JAK2-V617F allele burden in PMF granulocytes. Our data do not confirm a previous claim that the production of the JAK2Δ14 isoform is related to the pathogenesis of PMF.

Splenic extramedullary hematopoiesis is a major clinico-pathological feature of patients with myelofibrosis. As in the bone marrow (BM), hematopoiesis in the spleen occurs thank to the interplay of hematopoietic progenitor cells with the microenvironment, which provides the regulatory mechanism for their differentiation, proliferation and trafficking. Among other components, such as vessels and extra-cellular matrix proteins, this microenvironment encompasses different types of accessory cells, including mesenchymal stromal cells (MSCs). We have recently reported that MSCs from the BM of patients with myelofibrosis harbor genetics abnormalities and display an altered functional activity, suggesting that a primary MSC defect may either lead to or favor the pathogenesis of the disease. Here, we describe the phenotypical, functional, and genetic profile of MSCs isolated from the spleen of 23 patients with myelofibrosis, who underwent splenectomy for anemia and/or for excessive size of the spleen, and compare them to splenic mesenchymal stromal cells (s-MSCs) from 7 healthy subjects (HSs) who were splenectomized following traumatic lesion. The study was approved by the institutional review board of IRCSS Policlinico San Matteo Foundation; patients and HSs gave written informed consent for participating to the study. Mononuclear cells (MNCs) were obtained by dissociation of small spleen fragments by means of the GentleMacs Dissociator device (Miltenyi Biotech, Germany), and s-MSCs were isolated and expanded according to the standard procedures used for BM-MSCs. S-MSCs were obtained in 9/23 patients and in 3/7 HSs and displayed no significant differences for morphology and differentiation ability into adipocytic and osteoblastic lineages. However, the clonogenic efficiency of s-MSCs from patients with myelofibrosis was statistically higher than that of HSs (0.07 colonies/106 MNCs, range 0.03-0.01, vs 0.03/106 MNCs, range 0.03-0.04, respectively; p=0.048), whereas doubling time and time to senescence were not statistically different. Flow cytometric assessment of standard surface antigens (CD13, CD14, CD34, CD45, CD73, CD90, CD105) confirmed the mesenchymal nature of the cells grown in the cultures, and was similar between patients’ and HSs’ s-MSCs. When nestin expression was determined, no significant differences in the frequency of MSCs expressing this antigen was observed; however, nestin Mean Fluorescence Intensity (MFI) of patients’ s-MSCs was significantly lower than that of s-MSCs from HSs (22, range 6-45, vs 97, range 65-100, respectively; p=0.035). Patients’ s-MSCs also displayed a reduced capacity to sustain long term hematopoiesis in vitro in a classical Long Term Culture-Initiating Cell assay. However, when normal cord blood-derived CD34+ cells were co-cultured onto patients’ s-MSCs in a transwell system for 13 days, the output of CD41+ megakaryocytic cells increased with respect to culture where CD34+ cells were plated onto HSs' s-MSCs [21,5% vs 14,2% w/o recombinant human thrombopoietin (rhTPO), respectively, p=0,043; 60,2% vs 33,6% with rhTPO, respectively, p=0,01] at detriment of CD33+ cells (41,5% vs 48,6% w/o rhTPO, respectively, p=0,049; 10,4% vs 29,4% with rhTPO, respectively, p=0,012]. Finally, an abnormal karyotype [46XXt(5;17)(4-12)] was detected in 1 out of 18 metaphases of 1 out of 3 patient s-MSCs, while a normal karyotype was always observed in 2 out of 2 HSs’ s-MSC. This extensive characterization of s-MSCs shows that s-MSCs of patients with myelofibrosis display functional and genetic abnormalities compared to those isolated from HSs. The low level of nestin expression suggests that the hematopoietic niche of the spleen of patients with myelofibrosis can be defective and responsible for the increased trafficking of CD34+ cells that is observed in these patients, whereas the increased differentiation into the megakaryocytic lineage indicates a role of the splenic niche in leading hematopoiesis toward a pathological profile. All together, our data suggest that s-MSCs play a role in the pathogenesis of myelofibrosis and could be, therefore, a potential target for the treatment of the disease. Disclosures No relevant conflicts of interest to declare.

Data from COMFORT-I and COMFORT-II trials indicate that the JAK1/JAK2 inhibitor ruxolitinib is effective in reducing splenomegaly and reversing symptoms in patients with MPN-associated MF (Verstovsek S. et al, NEJM 2012;366:799; Harrison C. et al. NEJM 2012;366:787). However, not all subjects in these studies needed therapy for splenomegaly according newly-reported criteria (Barosi et al, Leuk Res 2014;38:155). Moreover, splenomegaly response was defined as 35% reduction in spleen volume by magnetic resonance imaging, which does not always correspond to a clinically relevant response as defined by the IWG-MRT/ELN revised response criteria (Tefferi et al. Blood 2012;122:1395). We evaluated response in 57 subjects with a spleen > 10 cm from the costal margin or with a progressive splenomegaly. Patients had been enrolled in clinical trials, or in individual patient care. The majority of patients were males (32/57, 56.1%), and the median patient age was 57 years (range: 30–81 years). The disease conditions included primary MF in 45 patients (78.9%), post-ET or post- PV MF (12 patients, 21.1%). Genotype was JAK2V617F in 37, 26 with > 50% (45.6%) and 11 (19.3%) with 50% or less allele burden. Fifteen (26.3%) had a CALR mutation, 4 a MPL mutation (7%), and 1 no detectable mutation. Starting dose of ruxolitinib given twice daily was: 20 mg in 32, 15 mg in 22, and 10 mg in 3. Doses were adjusted as appropriate. Median actuarial follow-up was 23.3 months. Thirteen subjects (37%) had a dose-reduction at a median time of 34 days (range 8-510 days). Twenty-eight (49.1%) discontinued therapy at a median of 17. months: 3-month and 1-,2-, and 3-year discontinuation rates were 22%, 40%, 55%, and 67%, respectively. Reasons for therapy-discontinuation were lack of response/progression (N=14), or toxicity/adverse events (N=14). Nineteen subjects (33%) responded according to the IWG-MRT/ELN response criteria at a median time of 3.9 months (range, 12 days - 21 months). However, 7 subsequently lost their response after a median time of 2.3 months (range, 1-35 months). 3-year continuous response rate was 24%. We next estimated failure-free survival (FFS), defined as continuing on ruxolitinib, no start of another therapy for splenomegaly, and/or no disease progression. This composite endpoint is more relevant than the individual components, but its validity after ruxolitinib in MF has not been evaluated. Actuarial FFS was 84% at 3 months, 71% at 6 months, 61% at 12 months, 44% at 2 years, and 36% at 3 years. Subjects with a JAK2V617F allele burden >50%, or with a liver 50% as independently associated with FFS. Relative failure risk of subjects with >50% JAK2V617F allele burden was 0.28 (95% CI = 0.12 to 0.66), P=0.003 relative to all other genotypes (Figure). There was no significant association between FFS and age, gender, type of MF, hemoglobin concentration, white blood cells, platelet count, reason for starting therapy, spleen size, CD34+ levels, starting dose, or dose reductions. In conclusion, ruxolitinib produced a 3-year IWG-MRT/ELN response rate of 24% in persons needing therapy for splenomegaly. However, actuarial 3-year FFS was 36%. Patients with JAK2V617F and more than 50% allele burden had significantly better FFS than other genotypes, perhaps reflecting a greater sensitivity to inhibition of JAK1/JAK2 by ruxolitinib. These results may help define which persons with MPN-associated MF and splenomegaly needing therapy are most likely to benefit from ruxolitinib. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Post-transplant lymphoproliferative disorder (PTLD) is a complication of solid organ and allogeneic hematopoietic stem cell transplantation (HSCT); following autologous HSCT only rare cases of PTLD have been reported. Here, a case of Hodgkin's disease (HD), as unusual presentation of PTLD after autologous HSCT for malignant glioma is described. Case presentation 60-years old man affected by cerebral anaplastic astrocytoma underwent subtotal neurosurgical excision and subsequent high-dose chemotherapy followed by autologous HSCT. During the post HSCT course, cranial irradiation and corticosteroids were administered as completion of therapeutic program. At day +105 after HSCT, the patient developed HD, nodular sclerosis type, with polymorphic HD-like skin infiltration. Conclusion The clinical and pathological findings were consistent with the diagnosis of PTLD.

While examining some histological sections of a clinically suspected neoplastic nodule in a woman's breast, sections of Dirofilaria repens were noted in the same nodule along with sections of a different nematode. The latter appeared to be a specimen possibly belonging to the genus Anatrichosoma (family Trichosomoididae), a parasitic group of helminths rarely reported in humans. In view of the diagnostic interest in an exceptional event, such as a double parasitic infection in the same nodule, we report the details of the case and the morphological findings.

Several studies support a role of 18q21 LOH, involving the DCC locus, in colorectal cancer progression; however, its contribution to the natural history of gastric cancer is less clear. Recently, a number of cancer-related genes have been mapped in the 18q21 region, either centromeric or telomeric to DCC. This study searched for 18q21 LOH in 161 gastric cancers representative of all tumour stages and main histological types. To this purpose, seven highly polymorphic markers were used flanking the 18q21 band and spanning the entire region. Thirty-four out of 147 (23.1%) informative cases showed LOH. In 27 of 34 cases (79%), LOH involved all the informative loci. The remaining seven cases showed LOH at more telomeric sites and retained heterozygosity at more centromeric markers, mostly those proximal to the DCC gene. A strong correlation between 18q21 LOH and level of gastric wall invasion, lymph node metastases, or stage was found in cohesive (glandular+solid) and mixed tumours, but not in diffuse cancers. Cox univariate and multivariate analysis showed that invasion level, lymph node metastases, distant metastases, TNM stage, and histology were effective predictors of survival, whereas 18q21 LOH did not show predictive power. The simultaneous deletion of a variety of cancer-related genes with different and even opposite roles might explain why, apparently, 18q21 LOH does not per se contribute significantly to the natural history of gastric cancer, despite strong correlation with stage.

Background and aims: The usefulness of histological diagnosis of gastroesophageal reflux disease (GERD) is limited by poor specificity or sensitivity of available diagnostic tools. Recently, ultrastructural morphometry showed interstitial space dilation (ISD) to be a reliable sign of reflux disease. Aims of this study were to (a) search for a light microscopy equivalent of ISD, (b) test its diagnostic value, and (c) look for a possible role of intercellular glycoconjugates in its genesis. Methods: Esophageal grasp biopsies were taken during endoscopy, 2–3 cm and 6–7 cm above the squamo- columnar junction, from patients under investigation for GERD symptoms. The biopsies were fixed in aldehyde solutions and embedded in resin for electron microscopy or in paraffin for routine histology, and the glycoconjugates underwent immunohistochemistry using 3-fucosyl-N-acetylactosamine antibodies. Results: Irregular intercellular space dilation was detected in the basal and prickle layers using both light and electron microscopy. Hematoxylin–eosin preparations showed ISD in 20 of 22 (90%) erosive esophagitis cases, 30 of 44 (68%) endoscopy negative GERD cases, and 1 of 12 (8%) controls, with good interobserver (K=0.75) and bioptic site reproducibility. ISD correlated with loss or rearrangement of intercellular glycoconjugates of the overlying layers and with granulocyte (eosinophil and/or neutrophil) infiltration. Conclusions: Light microscopy ISD is a suitable index of GERD. Alterations of intercellular glycoconjugates are likely to have a role in the genesis of ISD and GERD.

Intestinal metaplasia (IM) is by far the most frequent metaplastic change occurring in the gastric mucosa. True pyloric type metaplasia of oxyntic glands and pancreatic metaplasia of the cardia and oxyntic glands is relatively infrequent and focally restricted. So-called ‘pseudopyloric metaplasia’ of oxyntic glands, a prominent component of diffuse corpus-fundus (type A) atrophic gastritis, is better interpreted as mucous-neck cell hyperplasia with lack of acid-peptic differentiation, rather than as true metaplasia1. Three types of IM are usually considered: (1) type I or complete or small intestine type, composed of columnar crypt/absorptive enterocytes with sparse globet cells, with or without Paneth cells and intestinal type endocrine cells; (2) type II or incomplete sulphomucin-negative type, consisting of goblet cells scattered among gastric foveolar and neck cells; and (3) type III or incomplete sulphomucin-positive type, showing goblet cells scattered among sulphomucin-producing columnar cells. The latter cells show gastric or hybrid gastric/intestinal cell patterns under histochemical and ultrastructural investigation and overlie a relatively immature proliferative component, often sulphomucin-negative but CAR-5 (an intestinal oncofetal antigen) positive, whose expansion apparently leads to dysplasia2. Cytokeratin 7-positive glands or microcysts, mainly non-proliferative, are sometimes found at the bottom of type III or mixed complete/incomplete types of metaplasia, often combined with Brunner-type glands.

Early observations on inflammation of mucosa at the gastric cardia in patients with reflux symptoms1–3, confirmed by more recent studies4,5, raised speculation as to a possible involvement of cardia gastritis (carditis) in the genesis of reflux symptoms or in some mechanism favouring reflux disease5–7. On the other hand, investigation of the cardia mucosa of patients with Helicobacter pylori gastritis showed frequent involvement of the cardia7,8, thus raising the question as to whether carditis is merely to be interpreted as a cardia extension of H. pylori gastritis or, at least in patients with reflux disease, may represent an independent pathological entity related to acid reflux4,5. The possibility that precursor lesions to cancer at the cardia may develop in chronically inflamed and/or acid-damaged cardia mucosa, and may contribute to the increasing incidence of such cancer, is of special interest.

Prefibrotic myelofibrosis (pre-MF) is a prodromic variant of primary myelofibrosis (PMF) characterized by prevalence of female sex, young age, high frequency of splanchnic vein thrombosis and indolent disease course (Barosi et al, PLoS One. 2012;7(4):e35631). Here we report the molecular characteristics at diagnosis of 177 consecutive pre-MF patients included in the institutional data-base of PMF patients at the Center for the Study of Myelofibrosis in Pavia from January 2001 through July 2013. As compared with patients with fibrotic type-MF (bone marrow (BM) fibrosis= 1 to 3), the frequency of JAK2V617F mutation in pre-MF patients (BM fibrosis=0) was higher (69.2% vs. 60.9%; P=0.02) but the JAK2V617F allele burden was lower (39% vs. 50.6%; P= Disclosures: No relevant conflicts of interest to declare.

Ruxolitinib, a JAK1/JAK2 inhibitor, is considered effective and safe in the relief of splenomegaly and symptoms in Myelofibrosis (MF), and it is now approved for this use in US and EU. Since 2009, we have treated 47 patients with MF enrolled in two consecutive clinical trials with ruxolitinib (Clinicaltrials.gov Identifiers: [NCT00463385][1] and [NCT00669578][2]) or under an individual patient therapeutic use. Among these patients, one developed tuberculosis, two herpes zoster infections, three herpex simplex reactivation, and one non-bacterial pneumonia of suspected viral origin. This incidence of infectious adverse effects is in agreement with the Novartis reported high incidence of urinary tract infections (12.3%), herpes zoster (4.3%), and tuberculosis (1%) (JAKAVI, Summary of product characteristics. Basel, CH: Novartis Pharma AG; 2012). Accordingly, a case of Cryptococcus neoformans pneumonia in a patient receiving ruxolitinib has been recently described (Wysham NG et al, Chest 2013;143(5):1478-9). Considering that a protective role for regulatory T-cells (Tregs) against specific viral pathogens, some parasites, and fungal pathogens has been documented (Rowe JH et al, Immunology 2012;136:1-10), we have investigated the possible role of ruxolitinib in modulating immune surveillance, by asseessing the frequency of circulating Tregs in patients affected by MF receiving ruxolitinib. In particular, we evaluated by cytofluorimetric analysis both total peripheral blood (PB) CD4+ T cells and the CD25+CD127low/-FOXP3+ Tregs subset in 11 patients (JAK2V617F: n=9) with MF (disease duration: median 60 months, range 6-360) who never received JAK2 inhibitors and were out of chemotherapy since at least three months. The evaluations were performed before they received ruxolitinib (full approved dose) (T0) and at defined time points for 1 year. Results are expressed as % of CD4+ cells. We found that the PB CD4+ T cell frequency was stable during the study (data not shown), whereas a significant reduction of the Treg frequency was evident in all patients since 30 days after the first dose of ruxolitinib ([Figure][3], panel A) as well as at the subsequent time points, with values that were below our lower limit of normality (dotted line). One of these patients experienced ruxolitinib withdrawal after 120 days of therapy: the frequency of Tregs on day 150 increased up to normal levels, speaking in favour of a direct effect of the drug on Treg ferquency ([Figure][3], panel B). Based on our results, we argue that the severe reduction in circulating Tregs may count as a major cause of immunosurveillance disruption during ruxolitinib therapy. This observation is in agreement with previous evidence that inhibition of the JAK2 pathways reduces in vitro FOXP3 expression blocking STAT5 signalling (Murawski MR et al Ann.N.Y.Acad.Sci 2006; Barrio S et al Br J Hematol 2013). ![Figure][4] Disclosures: No relevant conflicts of interest to declare. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00463385&atom=%2Fbloodjournal%2F122%2F21%2F4057.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00669578&atom=%2Fbloodjournal%2F122%2F21%2F4057.atom [3]: #F1 [4]: pending:yes

Abstract 2829 Background: The somatically acquired Janus Kinase 2 (JAK2) mutation (V617F) is borne by approximately 60% of patients with primary myelofibrosis (PMF). The mutation increases JAK2 kinase activity, with the potential to affect phenotype and clinical outcome of mutated subjects. Accordingly, PMF V617F mutants result associated with higher white-blood cell counts, higher propensity to develop large splenomegaly, and are less likely to require transfusion during follow-up. At variance, different results have been obtained as far as the influence of JAK2 V617F mutation on the major disease outcomes, such as blast transformation (BT) and overall survival (OS). In this work we considered our cohort of patients with PMF-fibrotic type consecutively registered from 1990 and prospectively followed, who reached a median follow-up of more than 4 years and we used statistical analysis to account for competing risks. With this cohort of patients we strove to provide a more informative population and appropriate analysis for studying the influence of JAK2 V617F mutation on BT and OS in PMF. Patients and Methods: In 462 PMF –fibrotic type patients (bone marrow [BM] fibrosis grade >0) we computed the incidence of BT and death in the framework of Cox regression analysis and of Fine and Gray competing risks analysis for BT. Results: At the Cox regression analysis, having either a wild-type (wt) or a homozygous JAK2 V617F genotype were factors for BT (HR, 1.98 and 2.04, respectively, with respect to the heterozygous genotype), but not for OS. At the competing risks regression analysis, the risk for BT in wt and homozygous V617F patients increased with respect to Cox analysis, giving a sHR of 2.17 and 2.12, respectively. Correcting the results for the variables that could have influence on BT, JAK2 V617F wt and homozygous genotypes remained independently associated with BT. In a validation cohort of 133 independent cases with PMF-prefibrotic type (BM fibrosis grade =0), the BT predictive model including JAK2 V617F genotype, older age, and lower hemoglobin retained high discriminant capacity (C statistics, 0.70; 95% CI, 0.47 to 0.92). A total of 147 patients were genotyped with a quantitative assay for JAK2 V617F mutation in granulocyte DNA collected within 6 months from diagnosis, provided that no treatment had been delivered in the meantime. A total of 97 patients were JAK2 V617F mutated, giving an overall frequency of 65.9%. Comparing the four groups, the time to BT, death for any cause, and death for BT among the 4 quartiles were not significantly different (P=0.5). However, there was a shorter time to BT in the upper quartile compared with lower quartiles (P= 0.04). Conclusion: The accumulation of mutated alleles in the JAK2 V617F clone or the selective acquisition of a proliferative advantage in the wt clone are two relevant routes to BT in PMF. The influence of these results on treatment decisions with anti-JAK2 agents should be tested. Disclosures: No relevant conflicts of interest to declare.

Investigation of extensively sampled nontumor gastric mucosa from 205 early gastric cancers showed Helicobacter pylori colonization in 85% of cases, including 100% of diffuse and 78% (83% in 97 cases with Swiss rolls) of glandular or mixed cancers. Intestinal metaplasia, including its type III variant, was prominent in the mucosa associated with glandular and mixed (but not diffuse) early cancers. Both glandular (usually called "intestinal") and diffuse-type cancers showed admixtures of intestinal and gastric tumor cell phenotypes. Both p53 gene mutations and p53 protein immunostaining were essentially restricted to glandular or mixed cancers and associated dysplastic lesions. Their appearance in the advanced stage of diffuse cancer was partly due to a change of the histologic pattern from glandular to diffuse during progression of some tumors. Loss of laminin, beta I integrin, or zonula adherens junctions was a common finding in both early and advanced diffuse cancer. It is concluded that two main pathways operate in gastric carcinogenesis, both starting from H. pylori gastritis and both leading to phenotypically variable, often mixed gastric/intestinal tumor growth. However, only one of the two pathways involves intestinal metaplasia, its type III variant, p53 gene alteration, and dysplasia to end in glandular cancer. In the other pathway, diffuse cancer apparently arises directly from hyperplastic, sometimes atypical necks of mostly nonmetaplastic gastric glands, through primary involvement of genes affecting cell-cell and cell-matrix junctional proteins.

Still unexplored is the correlation betweenplasmaD-dimer levels and axillary node involvement, as detected following SNB investigation, in operable breast cancer.1 This prospective study attempts to verify the correlation between D dimer level and lymph node involvement, including patients with positive Sentinel Node. Between January 2007 and October 2010, preoperative D-dimer levels were evaluated in 142 consecutive operable breast cancer, receiving axillary lymph node dissection, either preoperatively planned (41) or after sentinel node biopsy (SNB) (101). Forty-one (41) patients were candidate to quadrantectomy or mastectomy plus axillary dissection, not satisfying eligibility criteria for SNB procedure. In second group,101 patients were candidate for conservative treatment following SNB procedure. Patientswith positive SN (22) received standard axillary dissection. In order to determine a threshold in D dimer levels distinguishing between subjects with involved lymph-nodes and subjects with no lymph-nodal involvement, a ROC curve analysis was performed on both the whole sample and subgroups identified according to surgical procedure. No difference was found between patients with negative or positive lymph nodes, either in case of axillary dissection or after SNB procedure. The hypothesis that D-dimer level might be related to breast cancer clinical stage is not supported.

Helicobacter pylori infection has been described in children with peptic ulcer disease and chronic type B gastritis. In all reported pediatric series the most frequent symptom associated with the infection was recurrent epigastric or abdominal pain. Combination of amoxycilhn with colloidal bismuth subcitrate [2] or with tinidazole [4] eradicated the infection and improved symptoms and gastritis in approximately 70% of infected children. Follow-up of these patients was generally limited to a few months after discontinuation of therapy. To study the outcome of the disease after successful treatment we prospectively followed- up children and adolescents with H. pylori-associated nonulcer dyspepsia for a mean period of 3 years after eradication of the infection.

Recent studies suggest that Helicobacter pylori (HP) gastritis is a major risk factor for both peptic ulcer disease [36] and cancer [16, 31, 32]. However, the histologic factors and mechanisms leading from gastritis to ulcer or cancer are poorly known. Gastritis activity and HP-linked surface epithelium lesions have been suggested to play some role in ulcerogenesis [7, 9, 12, 13, 15, 34] while intestinal metaplasia (IM) has long been indicated as a factor in cancerogenesis [6, 21, 22].

Abstract 1743 Background: WHO diagnostic criteria of myeloproliferative neoplasms (MPNs) identify a preMF variant including patients having absence or early grade fibrosis, dual myeloid and megakaryocytic dominance, and clusters of atypical, ectopic, variable in size megakaryocytes in the bone marrow (BM). Many issues on the variant are critical, such as its distinction from essential thrombocythemia, recognisability, biological identity and true place among MPNs. Here we describe the clinical presentation of patients with preMF collected among a large cohort of patients diagnosed with PMF according WHO and for whom long-term follow-up data were available. The aim is to investigate whether there is an underlying structure of the patients' characteristics that allows to assign them to a distinct variant of MPNs or to align them along a continuum in the realm of PMF. Methods: We included in this study patients with PMF who were referred to the Center for the Study of Myelofibrosis of IRCCS Policlinico S. Matteo Foundation, Italy from 1990 to 2011. In all patients the BM biopsy taken at diagnosis was reviewed (pathologist: UM) and the diagnosis of preMF was established adopting the stringent criterion that, besides typical BM cellularity and megakaryocyte morphology, BM had less than grade 1 reticulin fibrosis (EUMNET criteria). Results: Of the 659 patients with PMF, 126 fulfilled the criteria we established for the diagnosis of preMF, thus accounting for 19.1% of the cohort. Female sex accounted for 59.5% of the preMF cohort while it accounted for 33.8% of PMF fibrotic type (P In order to assign patients with PMF to groups with different degrees of BM fibrosis, we further categorized MF fibrotic type into early MF (BM fibrosis grade 1) and advanced MF (BM fibrosis grade 2 or 3). Female frequency steadily varied from 59.5% to 43.2% and 25.6% in the 3 categories of BM fibrosis, respectively. Age varied from 39.2, 50.4, 57.0 years, respectively. Hemoglobin and platelet count steadily decreased (Hb=14.0, 13.3, 11.2 g/dL, respectively; platelet count=647, 554, 346 x109/L, respectively). Spleen size steadily increased along with increasing of BM fibrosis (spleen index=120, 152, 200). All these parameters exhibited a statistically significant trend along the continuum of BM fibrosis grade (ANOVA, P Conclusions: The BM picture of preMF identifies a group of patients with PMF associated with a very indolent hematologic phenotype, female dominance and very young age at presentation characterized by high incidence of splanchnic vein thrombosis. Disclosures: Barosi: Novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract 3844 In recent years the diagnosis and treatment of patients with myeloproliferative disorders has focused on the JAK2 gene. Recently, an alternative splicing of JAK2 transcript lacking the entire exon 14 (88bp), has been described (Ma et al. (2010) PLoS ONE, 5, e12165). This mRNA has a stop codon in exon 15 and is translated into a truncated protein that has a deletion within the JH2 pseudokinase domain and complete deletion of the kinase domain (JH1). The alternative transcript was detected in plasma of patients with myeloproliferative neoplasms (MPNs) (58% of patients positive for the V617F mutation and 33% of negative) in proportions ranging from 2% to 26% compared to the normal transcript, but never in healthy subjects. However, quantification of the transcript was performed by quantitative fragment length analysis, which is not the most appropriate method for such determination especially if, as in this case, the PCR is not limited to the exponential phase (Cirigliano, V. et al. (2001) Mol. Hum. Reprod., 7, 1001–1006). We investigate the mRNA levels of JAK2 and its splicing variant lacking exon 14, in granulocytes isolated from peripheral blood of 9 healthy individuals and 41 patients diagnosed with primary myelofibrosis (19 negative and 22 positive for the JAK2 V617F mutation). To overcome the limitations of quantitative fragment length analysis, the levels of the splice variant were assessed by qPCR, using a boundary-spanning primer designed on the junction between exons 13 and 15 (Vandenbroucke II, et al. (2001) Nucleic Acids Res., 29:E68). The JAK2 V617F allele burden was measured as previously described (Lippert, E. et al. (2006) Blood, 108, 1865–1867). Low levels of the alternative-spliced product was found in all RNA samples from patients and from all healthy individuals, in proportions ranging from 0.3 % to 3.5% compared to the normal transcript. The wild-type patients (95% C.I.: 0.3–0.8%) did not differ from controls (95% C.I.: 0.4–0.7%) while, in patients with the JAK2 V617F mutation, the levels correlates with allele burden (R^2= 0.513; p= 0.0002) (Figure 1).Figure 1Figure 1. To explain this observation we performed a bioinformatic analysis of the exon 14 sequence, using the software Human Splice Finder 2.4.1 (Desmet, F.O. et al. (2009) Nucleic Acids Res37 (e67) and ESE finder 3.0 (Cartegni, L. et al. (2003) Nucleic Acids Res31 (13)). The G/T transversion causing the aminoacidic substitution V617F also breaks an high-score, putative, Exon Splicing Enhancer (ESE) sequence that potentially binds the splicing regulator protein SRP55 (TGTGTC; positions 70 to 75 of exon 14). The higher level of the splice variant found in patients with the mutation could be due to an alteration of the binding of molecular factors that determine the inclusion of exon 14 in mature transcripts. The low levels of alternative transcripts observed, could be due to the presence of a stop codon in exon 15 as a result of the frameshift caused by the exclusion of exon 14. We know that a stop codon at more than 55 bases upstream of the last exon junction (in this case between exons 24 and 25) determines the activation of the Nonsense Mediated mRNA Decay (NMD) system and the consequent destruction of the messenger. In order to knowhow a small percentage of mRNA, subjected to degradation by NMD system, can produce the predicted 70 KDa protein that has been detected after immunoprecipitation and western blot analysis using JAK2 N-terminal antibodies, we are characterizing the 3' end of the alternative transcript. We show that the somatic mutation in exon 14 of JAK2 gene, in addition to the effects caused by the V617F amino acid change, has a moderate influence on splicing, increasing the levels of a splicing variant that is produced in small amounts also in healthy subjects. Our study demonstrates the efficacy of qPCR technique and the predictive power of bioinformatics tools used here. Further studies are needed to understand how this isoform is processed at the post-transcriptional level and whether there are significant effects on clinical phenotype. Disclosures: Barosi: Novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract 2811 Among chronic myeloproliferative neoplasms (MPN) myelofibrosis (MF) has the worst prognosis. The International Prognostic Score System-IPSS reliably discriminates four categories of patients (pts) with different survival (OS); however, the possibility to identify pts with very high-risk disease, based on intrinsic characteristics of the underlying disease, would be particularly useful for therapeutic choices that include early stem cell transplantation. Several studies have addressed the prognostic value of molecular abnormalities harbored by MF pts, but a part from a negative role of low JAK2V617F allele burden on survival independently reported by the Mayo Clinic and our own group, none of them impacted significantly on survival. Here we have genotyped at diagnosis 370 pts with primary MF (PMF) and 148 with post-polycythemia vera/essential thrombocythemia MF (PPV/PET-MF) for mutations of EZH2 and ASXL1. EZH2 is the catalytic component of polycomb repressive complex 2 involved in trimethylation of H3 lysine 27, while ASXL1 is a member of enhancer of trithorax and polycomb group required for repression of HOX genes through deubiquitination of histone H2A. All 20 EZH2 exons were screened by high-resolution melting followed by direct sequencing whereas direct sequencing was used for ASXL1 exon 12 genotype. Pts median FU was 39 mo (1–340); 128 pts died (25.9%), 81 (18.3%) because of leukemia (AL). Among PMF pts survival differed significantly according to the IPSS category and was also predicted by the lowest V617F quartile. EZH2 mutation. We found 22 PMF (6%), 1 PPV-MF (1.2%) and 6 PET-MF (9.4%) mutated pts, harboring 25 different mutations, 20 exonic (heterozygous in 83%), 5 intronic. Concurrent JAK2 V617F, MPL W515L/K, IDH1/2, TET2, CBL and ASXL1 mutations were found in 41.4%, 0%, 0%, 6.9%, 3.4% and 20.7% of EZH2 -mutated pts. In multivariate analysis EZH2 mutation was associated with leukocytosis (12.3±13.2 vs 18.7±11.5×10e9/L) in PMF pts, but no additional clinical/hematological correlation was discovered. EZH2 mut pts clustered preferentially in the IPSS high-risk category (52.6%; P=.002). More EZH2 mut than wt PMF pts died (51.9% vs 24.4%; P ASXL1 mutation. In a total number of 334 evaluated patients we found 38 different mutations in 49 pts (14.7%), 35 PMF (15%), 5 PPV-MF (8.6 %) and 9 PET-MF (21.4%). Mutations were frameshift, non sense and missense mutations, heterozygous in 46 of 49 cases. Co-expression of JAK2 V617F, MPL W515L/K and EZH2 mutation was found in 55.1%, 4.1% and 12.2%, respectively. Co-espression of ASXL1 and IDH1/2, TET2 or CBL was observed in two, one and one of the evaluable subjects, respectively. ASXL1 mutation was associated with leucocytosis (P=.006), a blast count >1% (43.8% vs 16.6%, P According to EZH2 and ASXL1 mutational status, median OS was significantly shorter in double EZH2/ASXL1 mutated PMF pts compared with single EZH2 or ASXL1 mutated or WT patients (34 vs 116 vs 198 vs 272 mo, respectively. P No impact of EZH2 or ASXL1 mutations on OS or LFS was seen in PPV/PET-MF. Overall, these results showed that mutations in genes involved in epigenetic gene regulation such as EZH2 and ASXL1 negatively impact on OS and LFS in pts with PMF, suggesting that screening for these mutations might be useful for risk stratification and treatment decisions. Disclosures: Barosi: Novartis: Membership on an entity's Board of Directors or advisory committees. Vannucchi:Novartis: Honoraria.

In children Helicobacter pylori infection is strikingly associated with peptic disease [1]. Family history of peptic disease, previous digestive procedures, and nonspecific epigastric pain are the most frequently encountered clinical features [2]. Isolated vomiting and more severe symptoms such as hematemesis are rarely described. At endoscopy antral nodularity, and gastric and duodenal ulcers are the typical lesions [2]. The infection is almost invariably associated with histologic evidence of chronic gastritis [2, 3]. Serum immune response against the bacterium with production of specific immunoglobulin G (IgG) and IgA is found in the majority of infected children [4, 5]. An increase in the level of serum pepsinogen I usually occurs [5].

Helicobacter pylori colonization and the incidence, severity, activity and topography of gastritis were investigated systematically in antrum and corpus mucosal biopsies of 1177 subjects undergoing endoscopy in the absence of gastric complaints (asymptomatic, 49) or for non-ulcer dyspepsia (NUD; 631 patients, 72 of whom had gastric and/or duodenal erosions), active gastric ulcer (GU, 76 patients), active duodenal ulcer (DU, 138 patients), and healed gastric (HGU, 39 cases) or duodenal ulcer (HDU, 230 cases). In the antrum, H. pylori colonization and the incidence, severity and activity of gastritis increased progressively in the sequence asymptomatic, erosion-free NUD, erosive NUD, healed ulcer and active ulcer. The same trend was observed in the corpus as regards H. pylori and gastritis incidence, whereas the severity and activity of gastritis were lower in active DU and erosive NUD and higher in active, proximal GU than in the remaining patients. Active DU and erosive NUD showed the highest incidence of non-atrophic gastritis and lowest type-A or AB atrophic gastritis, while active GU had lowest normal mucosa or type-A gastritis and highest type-B atrophic gastritis. In conclusion, H. pylori colonization and gastritis incidence, severity and, especially, activity of the antrum might all contribute to mucosal erosion and ulceration, whereas the same factors, at least in part and with the exception of proximal GU, seem to have a preventive role when affecting corpus mucosa.

Gastritis-associated epithelial changes are known to have a major role in the pathogenesis of gastric diseases such as peptic ulcer and exocrine or endocrine cell neoplasia [1, 26, 27, 31, 33]. Thus, a careful cytologic characterization of these epithelial changes at the light and electron microscopy level should prove helpful in understanding the cause and evolution of such diseases.

Bone marrow and spleen neoangiogenesis is a relevant feature of patients with myelofibrosis (MF). We have previously reported that patients with MF have an increased percentage of circulating endothelial progenitor cells (EPC) assessed as CD34+CD133+VEGFR2+ cells compared with patients with other Ph-negative myeloproliferative disorders (polycythemia vera, PV, and essential thrombocytemia, ET) and healthy subjects. However, neither the functional activity of these putative EPC nor their belonging to the malignant clone have been yet fully characterized. In order to address these issues we have grown in vitro EPC-derived colonies from the peripheral blood (PB) of 36 patients with MF, 9 patients with PV or ET and 10 healthy subjects. Seventeen MF patients harbored a V617F JAK-2 mutation (8 heterozygous and 9 homozygous) whereas 2 patients showed a W515L MPL mutation (both heterozygous). Eight out of 9 PV/ET patients had a V617F JAK-2 mutation (5 heterozygous and 3 homozygous). Mononuclear cells were cultured in collagen coated 6 well plates in the presence of EBM-2MV medium according to Ingram et al (Blood104:2752; 2004). The endothelial origin of the colonies was ascertained by assessment of the expression of CD105, CD146, CD144, CD31, vWf, VEGFR-2, CD14 and CD45 antigens. V617F JAK-2 and W515L MPL mutations were assessed by PCR, followed by enzymatic digestion, of endothelial cells after tripsinization of the EPC-derived colonies. The median frequency (number of colonies per 107 mononuclear cells plated) of EPC-derived colonies was statistically higher in MF patients (0.25, range 0–8.1) compared to healthy subjects (0.05, 0–0.3; P=0.037), but not different form that of PV/ET patients (0, 0–4.4; P=NS). Immunophenotyping confirmed that the cells expressed the endothelial antigens CD105, CD146, CD144, CD31, vWf, and VEGFR-2 but not the hematopoietic specific antigens CD45 and CD14. The capacity of colony-derived endothelial cells of MF patients to form capillary-like structures in the Matrigel assay was not different from that of healthy subjects. No correlation was found between the number of colonies and the mutational status of either JAK-2 or MPL. In 11 MF patients harboring either a JAK-2 (n=9) or a MPL (n=2) mutation, colony growth was observed and PCR was performed on EPC-derived colonies. In 0/9 and 0/2 cases neither JAK-2 nor MPL mutations were found, respectively. In addition, no V617F JAK-2 mutation was found in the EPC-derived colonies of 8 PV/ET patients who carried the mutation in their granulocytes. Taken together, our data show that patients with MF have an increased frequency of EPC in their PB compared to healthy subjects and that these mobilized EPC are not clonally-related to the JAK-2 or MPL mutated clone. Whether or not circulating EPC derive from an earlier progenitor cell compared to the one in which the JAK-2/MPL mutations arise remains to be determined.