Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Does the nasogastric tube has a role in elective colo-rectal surgery?

INTRODUCTION: Routine use of nasogastric tubes (NGT) after abdominal operations is intended to hasten the return of bowel function, diminish the risk of anastomotic leakage and prevent pulmonary complications. The aim of our study was to prospectively assess the tolerability and the safety of the non use of NGT after elective colorectal open operations. PATIENTS AND METHODS: Between March 2009 and December 2010, 110 consecutive patients underwent colo-rectal elective open surgery for neoplasm without nasogastric decompression. We analyzed the incidence of nausea and vomiting, the pulmonary complications, the return of bowel function the deep wound breakdown (fascial dehiscence) and the anastomotic leakage. RESULTS: Only 15 patients (13,6%) reported nausea without vomiting immediately after surgery and 9 cases of vomiting were observed (8%), requiring the insertion of the NGT (nasogastric tube) in 5 (4,5%). A total of 105 patients (96,3%) were NGT free. No deep wound dehiscence was observed and only one real pneumonia occurred. Anastomotic dehiscence occurred in 4 patients (3,6%) and a second surgical procedure was needed in three cases. The return of bowel function, except in the last four patients, occurred in 3,8 days average (range 2-7 days). CONCLUSION: We confirm the uselessness of the NGT in the framework of fast track program adopted in elective open colo-rectal surgery.

Prospective identification of high-risk polycythemia vera patients based on JAK2(V617F) allele burden.

The aim of this study was to determine whether the burden of JAK2(V617F) allele correlated with major clinical outcomes in patients with polycythemia vera (PV). To this end, we determined JAK2 mutant allele levels in granulocytes of 173 PV patients at diagnosis. The mean (+/-s.d.) mutant allele burden was 52% (+/-29); 32 patients (18%) had greater than 75% mutant allele. The burden of JAK2(V617F) allele correlated with measurements of stimulated erythropoiesis (higher hematocrit, lower mean cell volume, serum ferritin and erythropoietin levels) and myelopoiesis (higher white cell count, neutrophil count and serum lactate dehydrogenase) and with markers of neutrophil activation (elevated leukocyte alkaline phosphatase and PRV-1 expression). As compared to those with less than 25% mutant allele, patients harboring greater than 75% JAK2(V617F) allele were at higher relative risk (RR) of presenting larger spleen (RR 4.7; P<0.001) or suffering from pruritus (RR 3.1; P<0.001). In these patients, the risk of requiring chemotherapy (RR 1.8; P=0.001) or developing major cardiovascular events (RR 7.1; P=0.003) during follow up were significantly increased. We conclude that a burden of JAK2(V617F) allele greater than 75% at diagnosis points to PV patients with high-risk disease.

Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

A quantitative assay for JAK2(V617F) mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis.

A point mutation in the Janus tyrosine kinase 2 (JAK2) gene has been described in patients with chronic myeloproliferative disorders (MPD), but the clinical significance of JAK2(V617F), which may be harbored in either the heterozygote or homozyote status, is still largely undefined. There are indirect suggestions that clinical phenotype and also some biological characteristics are dependent on the mutated allele levels. We have designed and validated in 179 MPD patients an amplification-refractory mutation sequencing PCR assay that allows the relative quantitation of mutated and normal JAK2 mRNAs using dye-labelled mutation-specific primers and capillary electrophoresis. Direct sequencing confirmed the specificity of the assay, which has a detection limit congruent with1% and allowed to identify 9% more JAK2-mutated patients as compared to conventional allele-specific PCR. The mutated mRNA ratio ranged from 5 to 51% in the JAK2(V617F) heterozygote and from 45 to 100% in the homozygote patients. Expression levels of both PRV-1 and NF-E2 gene, previously found to be overexpressed in MPD patients, were significantly correlated to the amount of mutated JAK2 mRNA. We propose that this method might complement current technologies based on genomic DNA analysis, and lead prospectively to a better clinically oriented assessment of the impact of JAK2(V617F) mutation in MPD.

A clinical-molecular prognostic model to predict survival in patients with post polycythemia vera and post essential thrombocythemia myelofibrosis.

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms with variable risk of evolution into post-PV and post-ET myelofibrosis, from now on referred to as secondary myelofibrosis (SMF). No specific tools have been defined for risk stratification in SMF. To develop a prognostic model for predicting survival, we studied 685 JAK2, CALR, and MPL annotated patients with SMF. Median survival of the whole cohort was 9.3 years (95% CI: 8-not reached-NR-). Through penalized Cox regressions we identified negative predictors of survival and according to beta risk coefficients we assigned 2 points to hemoglobin level <11 g/dl, to circulating blasts ⩾3%, and to CALR-unmutated genotype, 1 point to platelet count <150 × 109/l and to constitutional symptoms, and 0.15 points to any year of age. Myelofibrosis Secondary to PV and ET-Prognostic Model (MYSEC-PM) allocated SMF patients into four risk categories with different survival (P<0.0001): low (median survival NR; 133 patients), intermediate-1 (9.3 years, 95% CI: 8.1-NR; 245 patients), intermediate-2 (4.4 years, 95% CI: 3.2-7.9; 126 patients), and high risk (2 years, 95% CI: 1.7-3.9; 75 patients). Finally, we found that the MYSEC-PM represents the most appropriate tool for SMF decision-making to be used in clinical and trial settings.

A data-driven network model of primary myelofibrosis: transcriptional and post-transcriptional alterations in CD34+ cells.

microRNAs (miRNAs) are relevant in the pathogenesis of primary myelofibrosis (PMF) but our understanding is limited to specific target genes and the overall systemic scenario islacking. By both knowledge-based and ab initio approaches for comparative analysis of CD34+ cells of PMF patients and healthy controls, we identified the deregulated pathways involving miRNAs and genes and new transcriptional and post-transcriptional regulatory circuits in PMF cells. These converge in a unique and integrated cellular process, in which the role of specific miRNAs is to wire, co-regulate and allow a fine crosstalk between the involved processes. The PMF pathway includes Akt signaling, linked to Rho GTPases, CDC42, PLD2, PTEN crosstalk with the hypoxia response and Calcium-linked cellular processes connected to cyclic AMP signaling. Nested on the depicted transcriptional scenario, predicted circuits are reported, opening new hypotheses. Links between miRNAs (miR-106a-5p, miR-20b-5p, miR-20a-5p, miR-17-5p, miR-19b-3p and let-7d-5p) and key transcription factors (MYCN, ATF, CEBPA, REL, IRF and FOXJ2) and their common target genes tantalizingly suggest new path to approach the disease. The study provides a global overview of transcriptional and post-transcriptional deregulations in PMF, and, unifying consolidated and predicted data, could be helpful to identify new combinatorial therapeutic strategy. Interactive PMF network model: http://compgen.bio.unipd.it/pmf-net/.

Splanchnic vein thrombosis in myeloproliferative neoplasms: risk factors for recurrences in a cohort of 181 patients.

We retrospectively studied 181 patients with polycythaemia vera (n=67), essential thrombocythaemia (n=67) or primary myelofibrosis (n=47), who presented a first episode of splanchnic vein thrombosis (SVT). Budd-Chiari syndrome (BCS) and portal vein thrombosis were diagnosed in 31 (17.1%) and 109 (60.3%) patients, respectively; isolated thrombosis of the mesenteric or splenic veins was detected in 18 and 23 cases, respectively. After this index event, the patients were followed for 735 patient years (pt-years) and experienced 31 recurrences corresponding to an incidence rate of 4.2 per 100 pt-years. Factors associated with a significantly higher risk of recurrence were BCS (hazard ratio (HR): 3.03), history of previous thrombosis (HR: 3.62), splenomegaly (HR: 2.66) and leukocytosis (HR: 2.8). Vitamin K-antagonists (VKA) were prescribed in 85% of patients and the recurrence rate was 3.9 per 100 pt-years, whereas in the small fraction (15%) not receiving VKA more recurrences (7.2 per 100 pt-years) were reported. Intracranial and extracranial major bleeding was recorded mainly in patients on VKA and the corresponding rate was 2.0 per 100 pt-years. In conclusion, despite anticoagulation treatment, the recurrence rate after SVT in myeloproliferative neoplasms is high and suggests the exploration of new avenues of secondary prophylaxis with new antithrombotic drugs and JAK-2 inhibitors.

Evaluation of plitidepsin in patients with primary myelofibrosis and post polycythemia vera/essential thrombocythemia myelofibrosis: results of preclinical studies and a phase II clinical trial.

Previous data established that plitidepsin, a cyclic depsipeptide, exerted activity in a mouse model of myelofibrosis (MF). New preclinical experiments reported herein found that low nanomolar plitidepsin concentrations potently inhibited the proliferation of JAK2V617F-mutated cell lines and reduced colony formation by CD34+ cells of individuals with MF, at least in part through modulation of p27 levels. Cells of MF patients had significantly reduced p27 content, that were modestly increased upon plitidepsin exposure. On these premise, an exploratory phase II trial evaluated plitidepsin 5 mg/m(2) 3-h intravenous infusion administered on days 1 and 15 every 4 weeks (q4wk). Response rate (RR) according to the International Working Group for Myelofibrosis Research and Treatment consensus criteria was 9.1% (95% CI, 0.2-41.3%) in 11 evaluable patients during the first trial stage. The single responder achieved a red cell transfusion independence and stable disease was reported in nine additional patients (81.8%). Eight patients underwent a short-lasting improvement of splenomegaly. In conclusion, plitidepsin 5 mg/m(2) 3-h infusion q4wk was well tolerated but had a modest activity in patients with primary, post-polycythaemia vera or post-essential thrombocythaemia MF. Therefore, this trial was prematurely terminated and we concluded that further clinical trials with plitidepsin as single agent in MF are not warranted.

Profound parental bias associated with chromosome 14 acquired uniparental disomy indicates targeting of an imprinted locus.

Acquired uniparental disomy (aUPD) is a common finding in myeloid malignancies and typically acts to convert a somatically acquired heterozygous mutation to homozygosity. We sought to identify the target of chromosome 14 aUPD (aUPD14), a recurrent abnormality in myeloid neoplasms and population cohorts of elderly individuals. We identified 29 cases with aUPD14q that defined a minimal affected region (MAR) of 11.2 Mb running from 14q32.12 to the telomere. Exome sequencing (n=7) did not identify recurrently mutated genes, but methylation-specific PCR at the imprinted MEG3-DLK1 locus located within the MAR demonstrated loss of maternal chromosome 14 and gain of paternal chromosome 14 (P<0.0001), with the degree of methylation imbalance correlating with the level of aUPD (r=0.76; P=0.0001). The absence of driver gene mutations in the exomes of three individuals with aUPD14q but no known haematological disorder suggests that aUPD14q may be sufficient to drive clonal haemopoiesis. Analysis of cases with both aUPD14q and JAK2 V617F (n=11) indicated that aUPD14q may be an early event in some cases but a late event in others. We conclude that aUPD14q is a recurrent abnormality that targets an imprinted locus and may promote clonal haemopoiesis either as an initiating event or as a secondary change.

[Caustic lesions of the upper digestive tract. Our experience]. / Le lesioni da caustici del tubo digestivo superiore. Nostra esperienza.

The authors take into consideration 21 cases of caustic burns of the superior digestive tract. In 6 cases (29%) the extent of lesions has impelled emergency surgery. In case of total exeresis of the esophagogastric tract, according to their experience, the preferred operation is at closed thorax following the esophageal stripping technique.

Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value.

Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.

CALR and ASXL1 mutations-based molecular prognostication in primary myelofibrosis: an international study of 570 patients.

Current prognostication in primary myelofibrosis (PMF) is based on the dynamic international prognostic scoring system (DIPSS)-plus, which employs clinical and cytogenetic variables. We recently reported DIPSS-plus independent prognostic significance for calreticulin (CALR) (favorable) and ASXL1 (unfavorable) mutations. In the current study, 570 PMF patients were recruited for derivation (n=277) and validation (n=293) of a molecular prognostic model based on these two mutations. Survival was the longest in CALR(+)ASXL1(-) (median 10.4 years) and shortest in CALR(-)ASXL1(+) patients (median, 2.3 years; hazard ratio (HR), 5.9; 95% confidence interval (CI), 3.5-10.0). CALR(+)ASXL1(+) and CALR(-)ASXL1(-) patients had similar survival and were grouped together in an intermediate-risk category (median survival, 5.8 years; HR, 2.5; 95% CI, 1.5-4.0). The CALR/ASXL1 mutations-based prognostic model was DIPSS-plus independent (P<0.0001) and effective in identifying low-/intermediate-1-risk patients with shorter (median, 4 years) or longer (median 20 years) survival and high-/intermediate-2-risk patients with shorter (median, 2.3 years) survival. Multivariable analysis distinguished CALR(-)ASXL1(+) mutational status as the most significant risk factor for survival: HR 3.7 vs 2.8 for age >65 years vs 2.7 for unfavorable karyotype. These observations signify immediate clinical relevance and warrant i) CALR and ASXL1 mutation determination in all patients with PMF and ii) molecular revision of DIPSS-plus.

Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms.

With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.

The number of prognostically detrimental mutations and prognosis in primary myelofibrosis: an international study of 797 patients.

We recently defined a high-molecular risk category (HMR) in primary myelofibrosis (PMF), based on the presence of at least one of the five 'prognostically detrimental' mutated genes (ASXL1, EZH2, SRSF2 and IDH1/2). Herein, we evaluate the additional prognostic value of the 'number' of mutated genes. A total of 797 patients were recruited from Europe (n=537) and the Mayo Clinic (n=260). In the European cohort, 167 (31%) patients were HMR: 127 (23.6%) had one and 40 (7.4%) had two or more mutated genes. The presence of two or more mutations predicted the worst survival: median 2.6 years (hazard ratio (HR) 3.8, 95% confidence interval (CI) 2.6-5.7) vs. 7.0 years (HR 1.9, 95% CI 1.4-2.6) for one mutation vs 12.3 years for no mutations. The results were validated in the Mayo cohort and prognostic significance in both cohorts was independent of International Prognostic Scoring System (IPSS; HR 2.4, 95% CI 1.6-3.6) and dynamic IPSS (DIPSS)-plus (HR 1.9, 95% CI 1.2-3.1), respectively. Two or more mutations were also associated with shortened leukemia-free survival (HR 6.2, 95% CI 3.5-10.7), also Mayo validated. Calreticulin mutations favorably affected survival, independently of both number of mutations and IPSS/DIPSS-plus. We conclude that the 'number' of prognostically detrimental mutations provides added value in the combined molecular and clinical prognostication of PMF.

Mutations and prognosis in primary myelofibrosis.

Patient outcome in primary myelofibrosis (PMF) is significantly influenced by karyotype. We studied 879 PMF patients to determine the individual and combinatorial prognostic relevance of somatic mutations. Analysis was performed in 483 European patients and the seminal observations were validated in 396 Mayo Clinic patients. Samples from the European cohort, collected at time of diagnosis, were analyzed for mutations in ASXL1, SRSF2, EZH2, TET2, DNMT3A, CBL, IDH1, IDH2, MPL and JAK2. Of these, ASXL1, SRSF2 and EZH2 mutations inter-independently predicted shortened survival. However, only ASXL1 mutations (HR: 2.02; P<0.001) remained significant in the context of the International Prognostic Scoring System (IPSS). These observations were validated in the Mayo Clinic cohort where mutation and survival analyses were performed from time of referral. ASXL1, SRSF2 and EZH2 mutations were independently associated with poor survival, but only ASXL1 mutations held their prognostic relevance (HR: 1.4; P=0.04) independent of the Dynamic IPSS (DIPSS)-plus model, which incorporates cytogenetic risk. In the European cohort, leukemia-free survival was negatively affected by IDH1/2, SRSF2 and ASXL1 mutations and in the Mayo cohort by IDH1 and SRSF2 mutations. Mutational profiling for ASXL1, EZH2, SRSF2 and IDH identifies PMF patients who are at risk for premature death or leukemic transformation.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.