PV Patients Research Articles

Ropeginterferon alpha-2b targets JAK2V617F-positive polycythemia vera cells in vitro and in vivo

Polycythemia vera is characterized by the acquisition of the JAK2V617F mutation. Recommended treatments include hydroxyurea and interferon-alpha. Several groups have reported a reduction in the JAK2 mutant allele burden in interferon-treated patients, but significance of this observation is questioned. We characterized the activity of ropeginterferon alpha-2b, a novel form of interferon-alpha recently shown to be safe and efficacious in polycythemia vera. Ropeginterferon was able to inhibit the proliferation of the HEL, UKE-1, and UT-7 JAK2-mutant cell lines while sparing JAK2-wild-type UT-7 and normal CD34+ cells growth. In vitro treatment of erythroid progenitors derived from PV patients showed that ropeginterferon could considerably inhibit the growth of endogenous erythroid colonies, a hallmark of polycythemia vera. Finally, we could study in sequential samples the clonal architecture of erythroid progenitors derived from patients included in a randomized study comparing hydroxyurea to ropeginterferon. After 1 year of treatment with ropeginterferon, the ratio of JAK2-mutated to wild-type colonies grown from bone marrow progenitors was reduced by 64%, compared to 25% in patients receiving hydroxyurea. This study shows that ropeginterferon has a potent targeted activity against JAK2-mutant cells and is able to drastically reduce the proportion of malignant progenitors in patients treated with this drug.

Outcomes of living donor liver transplantation for patients with preoperative portal vein problems

Introduction: Portal vein thrombosis (PVT) is a common complication for patients with end-stage liver disease. The presence of PVT used to be a contraindication to LDLT. This is related to technical difficulties of PV reconstruction, increased blood loss, and the risk for postoperative PV complications. Methods: We reviewed the data of LDLT patients at Liver Transplantation Unit, Mansoura University, Egypt during the period between May 2004 till March 2017. Patients were divided into three groups. Group I: patients without PVT, Group II: attenuated PV patients (PV diameter <8 mm), and Group III PVT patients. Results: During the study period, 500 cases underwent LDLT. Group I included 446 patients (89.2%), Group II included 26 patients (5.2%), and Group III included 28 patients (5.6%). Higher incidence of hematemesis and encephalopathy was detected in Group III. Longer anhepatic phase duration was found in Group III. There were no significant differences regarding operation time, blood loss, and transfusion requirements. Higher incidence of postoperative vascular complications was found in Group III. The median OS was 33 months (4-169). The 1-, 3-, and 5-years OS survival rates of Group I were 80.5%, 77.7%, and 75%, while for Group II were 84.6%, 79.6%, and 73.5%, and for Group III were 88.3%, 64.4%, and 64.4% respectively. There was no significant difference between the groups regarding OS rates (Log-Rank: 0.793). Conclusion: Preoperative PVT increases the complexity of LDLT operation and the operative trauma to the patient, but it does not reduce the OS rates.

Involvement of Nail Apparatus in Pemphigus Vulgaris in Ethnic Poles Is Infrequent.

Pemphigus vulgaris lesions have a tendency to localize around natural body orifices. The aim here was to analyze the involvement of nail apparatus in pemphigus vulgaris. Sixty seven ethnic Poles suffering from pemphigus vulgaris on photographic files archiving initial presentation were retrospectively evaluated. Pemphigus vulgaris was diagnosed using combination of clinical data, H+E histology, direct immunofluorescence of plucked scalp hair and/or perilesional tissue also for IgG1 and IgG4 deposits evaluation, indirect immunofluorescence on mosaic substrate and/or monkey esophagus, mono-analyte ELISA with desmoglein 1/3 or multi-analyte ELISA. The nail apparatus involvement was found in 9 of 67 patients (13.4%; 3 females and 6 males). Periungual fingernail lesions were found in 6 patients (2 females, 4 males), whereas periungual toenail lesions in just 3 patients (1 female, 2 males). Our patients nail apparatus changes included, by order of frequency, paronychia, nail discoloration, onychorrhexis, Beau lines, periungual hemorrhages, onychomadesis, cross-ridging, onycholysis, and trachyonychia. The average time between the onset, as recalled by patients, and the diagnosis of pemphigus vulgaris with direct immunofluorescence was not statistically different in PV patients with and without nail apparatus lesions. In this article the molecular and immunological rationale for of periungual involvement is discussed. Our single-center study suggests that nail apparatus involvement is infrequent in pemphigus vulgaris in ethnic Poles. Due to the fact that nail apparatus lesions in pemphigus vulgaris may clinically resemble onychomycosis, giving the proper diagnosis can be difficult particularly when other lesions are overlooked or misinterpreted.

Splanchnic vein thrombosis in myeloproliferative neoplasms: treatment algorithm 2018

Myeloproliferative neoplasms (MPNs) are a leading cause of splanchnic vein thrombosis (SVT). SVT is observed in all MPNs and frequently affects young patients. Therapy should be addressed to three main goals: preventing thrombosis recurrence, managing the underlying MPN, and supporting liver dysfunction. Life-long oral anticoagulation with vitamin K antagonists is the cornerstone of the antithrombotic treatment. However, recurrences of SVT or other thrombosis may occur in 15–20% of patients. Direct oral anticoagulants can represent an alternative and preliminary data encourage comparative studies. Survival of patients with SVT in MPN is primarily influenced by the natural history of the underlying neoplasms, rather than the SVT event. An aggressive management is recommended and a treatment algorithm based on the different MPN subtypes is proposed. Hydroxyurea is the cytoreductive drug of choice in polycythemia vera and essential thrombocythemia, whereas ruxolitinib is indicated in intermediate and high-risk patients with myelofibrosis and in PV patients resistant or intolerant to hydroxyurea. The management of SVT in MPNs requires a multidisciplinary approach that may include a hematologist, a gastroenterologist, an interventional radiologist, and a surgeon. In the case of clinical deterioration despite pharmacological therapy, patients with SVT should be considered for invasive procedures or liver transplantation.

80-W GreenLight Laser Vaporization Versus Transurethral Resection of the Prostate for Treatment of Benign Prostatic Obstruction: 5-Year Outcomes of a Single-center Prospective Randomized Trial

To assess long term functional and safety follow-up data after 80-W GreenLight photoselective vaporization (GL PV) of the prostate and transurethral resection of the prostate (TURP). Prospective randomized trial at a single tertiary referral center (Geneva, Switzerland). Patients were recruited in the outpatient clinic if they met the criteria for surgical treatment of benign prostatic obstruction. At baseline, 238 patients were treated either with the 80-W GL PV or monopolar TURP. After 5 years, data were available from 105 patients: 44 GL PV patients and 61 TURP patients. The primary outcome measure was the International Prostate Symptom Score (IPSS). Secondary outcome measures included maximum urinary flow rate (Qmax), postvoidal residual (PVR) and reoperation rate. Statistical analyses were performed using Stata 14 (StataCorp). After 5 years of follow-up, mean improvements in International Prostate Symptom Score, postvoidal residual and maximum urinary flow rate were similar in both groups. The re-treatment rate was 14.3% in the GL PV group vs 11.9% in the TURP group (P = .9). Noninferiority of the GL PV to TURP was confirmed in all functional and safety outcomes at 5-year follow-up. GL-PV could be a safe surgical alternative for patients suffering from benign prostatic obstruction.

Lymphoproliferative disorders in patients with chronic myeloproliferative neoplasms: A systematic review.

Patients with a Ph-negative myeloproliferative neoplasm (MPN) may harbor or develop lymphoproliferative disorders (LPD), however, the clinical and molecular determinants of MPN and LPD co-occurrence are still uncertain. To systematically pool the available knowledge, we conducted a scoping review of literature published since January 2005 and analyzed single-patient clinical data from 50 papers reporting 214 individuals harboring both MPN and LPD. Patients had been diagnosed essential thrombocythemia (44%), polycythemia vera (29%), or myelofibrosis (23%) at a median age of 67 years (26-94): half of them incurred a LPD after a median of 72 months from MPN diagnosis, while in 20% the LPD diagnosis was antecedent or synchronous. Patients mainly incurred indolent LPD, particularly chronic lymphocytic leukemia (CLL), while aggressive lymphomas and multiple myeloma were a relevant portion of the LPDs occurring in the follow-up of MPN. CLL was preferentially diagnosed in PV patients and was associated with a very high male-to-female ratio, as well as an older age at MPN diagnosis. On converse, multiple myeloma was rarely reported in PV patients and was preferentially diagnosed in female patients not harboring the JAK2 V617F mutation. Based on the 46 cases reporting follow-up data, median survival after MPN diagnosis was 96 months. This thorough review of published evidence confirms that LPD are relevant clinical events in the history of MPN patients. Controlled studies are needed to better refine individuals at higher risk of developing LPD, to support surveillance programs and to avoid therapies possibly favoring LPD.

Association of Cytotoxic T Lymphocyte Antigen-4 Gene Polymorphisms with Psoriasis Vulgaris: A Case-Control Study in Turkish Population.

Psoriasis is a common, chronic, and autoimmune skin disease in which dysregulation of immune cells, particularly T cells, is thought to play an important role in the pathogenesis. Cytotoxic T lymphocyte antigen-4 (CTLA-4) expressed only on activated T cells is an immunoregulatory molecule and plays a role in the pathogenesis of autoimmune disorders. We aimed to determine whether CTLA-4 gene polymorphisms are associated with development and/or clinical features of psoriasis vulgaris (Pv). Genotyping of SNPs (−318C>T, +49A>G, and CT60A>G) in CTLA-4 gene was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 103 Pv patients and 102 controls. No statistically significant associations were detected in any of the investigated genetic models for the −318C>T polymorphism. The genotype distributions of +49A>G and CT60A>G were associated with Pv development. In haplotype analysis, while frequency of CAA haplotype was significantly higher in the control group, frequencies of CGG and CAG haplotype were significantly higher among the patients. However, all of CTLA-4 polymorphisms and haplotypes do not have an effect on severity and onset age of Pv. In conclusion, the +49A>G and CT60A>G polymorphisms may be risk factors for Pv development. Furthermore, CGG and CAG haplotypes may contribute to Pv development, while CAA haplotype may be protective against Pv.

Hungarian Philadelphia negative chronic myeloproliferative neoplasia registry. Evaluation of the Polycythemia vera patients

Data of 351 JAK2V617F and exon 12 mutation positive polycythemia vera patients were collected online from 15 haematology centres reporting epidemiologic, clinical characteristics, diagnostic tools, therapeutic interventions, thromboembolic complications, disease transformations. Vascular events prior to and after diagnosis were evaluated upon the Landolfi risk assessment scale. 116 thromboembolic events were reported in 106 PV patients prior to diagnosis and 152 occasions in 102 patients during follow-up. The frequency of major arterial events were significantly reduced (p<0.0001) and the minor venous events were significantly elevated (p<0.0001) after the diagnosis. Major hemorrhagic complications were found in 25 and transformation in 26 cases. Our registry allows to collect and evaluate the features of patients with polycythemia vera. The Landolfi risk stratification was proven to be useful. Based on evaluated data, accuracy of diagnostic criteria and compliance to risk-adapted therapeutic guidelines are needed. Orv Hetil. 2017; 158(23): 901-909.

Whole-exome sequencing identifies novel candidate predisposition genes for familial polycythemia vera

BackgroundPolycythemia vera (PV), characterized by massive production of erythrocytes, is one of the myeloproliferative neoplasms. Most patients carry a somatic gain-of-function mutation in JAK2, c.1849G > T (p.Val617Phe), leading to constitutive activation of JAK-STAT signaling pathway. Familial clustering is also observed occasionally, but high-penetrance predisposition genes to PV have remained unidentified.ResultsWe studied the predisposition to PV by exome sequencing (three cases) in a Finnish PV family with four patients. The 12 shared variants (maximum allowed minor allele frequency <0.001 in Finnish population in ExAC database) predicted damaging in silico and absent in an additional control set of over 500 Finns were further validated by Sanger sequencing in a fourth affected family member. Three novel predisposition candidate variants were identified: c.1254C > G (p.Phe418Leu) in ZXDC, c.1931C > G (p.Pro644Arg) in ATN1, and c.701G > A (p.Arg234Gln) in LRRC3. We also observed a rare, predicted benign germline variant c.2912C > G (p.Ala971Gly) in BCORL1 in all four patients. Somatic mutations in BCORL1 have been reported in myeloid malignancies. We further screened the variants in eight PV patients in six other Finnish families, but no other carriers were found.ConclusionsExome sequencing provides a powerful tool for the identification of novel variants, and understanding the familial predisposition of diseases. This is the first report on Finnish familial PV cases, and we identified three novel candidate variants that may predispose to the disease.

Clinical significance of JAK2、CALR and MPL gene mutations in 1 648 Philadelphia chromosome negative myeloproliferative neoplasms patients from a single center

目的研究Ph染色体阴性的慢性骨髓增殖性肿瘤（MPN）患者JAK2、CALR及MPL基因突变情况，比较不同类型基因突变及突变阴性患者部分临床参数的差异。方法采用基因测序法分别检测1 648例Ph阴性MPN患者JAK2V617F、JAK2基因12号外显子、CALR基因9号外显子及MPL基因10号外显子突变情况。结果①JAK2V617F突变总检出率为82.8%（1 364/1 648），其中真性红细胞增多症（PV）为92.7%（471/508），原发性血小板增多症（ET）为78.1%（819/1 049），原发性骨髓纤维化（PMF）为81.3%（74/91）；JAK2基因12号外显子突变总检出率为0.5%（9/1 648），均为PV患者[1.7%（9/508）]；CALR基因突变总检出率为8.7%（143/1 648）,其中ET为12.6%（132/1 049），PMF为12.1%（11/91）；MPL基因突变总检出率为0.6%（10/1 648）,其中ET为0.9%（9/1 049），PMF为1.1%（1/91）。未发现两种及以上基因突变共存病例。②JAK2V617F突变组中位年龄[61（15~95）岁]高于JAK2 12号外显子突变组[49（33~62）岁]及突变阴性组[42（3~78）岁]（P值均<0.001），与CALR突变组[57（17~89）岁]、MPL突变组[59（22~71）岁]比较差异无统计学意义（P>0.05）。JAK2V617F突变阳性组白细胞计数、血红蛋白浓度均高于CALR突变阳性及阴性组（P<0.05），而与MPL突变阳性组比较仅表现为高白细胞计数（P=0.013）；CALR突变组血小板计数高于JAK2V617F突变组[966（400~2 069）×109/L对800（198~3 730）×109/L，P<0.001]。③共1 160例患者进行了有效染色体核型分析，CALR基因突变阳性、阴性患者异常核型检出率差异无统计学意义[9.8%（8/82）对7.4%（80/1 078），P=0.441]；JAK2V617F突变阳性、阴性患者异常核型检出率差异无统计学意义[7.7%（75/971）对6.9%（13/189），P=0.688]。CALR突变阳性组与JAK2V617F突变阳性组的异常核型检出率差异无统计学意义[9.8%（8/82）对7.7%（75/971），P=0.512]。7例JAK2基因12号外显子突变阳性及6例MPL基因突变阳性患者均未发现异常核型。结论基因检测使MPN的诊断及预后判断有了更可靠的依据，不同MPN亚型基因突变的发生频率、分布情况不同，不同基因突变导致独特的临床表型。

Monocytosis in Polycythemia Vera: Clinical and Molecular Correlates

BackgroundPolycythemia Vera (PV) constitutes one of the three BCR-ABL1-negative myeloproliferative neoplasms and is characterized by clonal erythrocytosis and the almost invariable presence of JAK2 mutation. An absolute monocyte count (AMC) of ≥1 x 10(9)/L defines chronic myelomonocytic leukemia (CMML) but can also be seen in other myeloid disorders including myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). The presence or development of monocytosis has previously been shown to confer poor prognosis in both primary myelofibrosis (PMF), which is one of the three BCR-ABL1-negative MPN (Leukemia Research 2007;31:1503, Mod Pathol. 2013 Feb;26(2):204) and MDS (Haematologica. 1997;82:25). In the current study, we examined the clinical, prognostic and molecular correlates of monocytosis in PV.MethodsStudy patients were selected from our institutional database of MPN and fulfilled the 2008 World Health Organization (WHO) criteria for the diagnosis of PV (Blood. 2009;114:937). Cytogenetic and mutational analyses were performed according to conventional methods (Leukemia. 2014;28:2206) any assignment as unfavorable karyotype was per PMF criteria (Leukemia. 2011;25:82). Mutation screening included TET2, ASXL1 and SRSF2 because of their known association with CMML (Leukemia. 2014;28:2206) Statistical analyses considered clinical and laboratory parameters obtained at time of diagnosis.ResultsPatient characteristics:Analysis was conducted on 587 patients (median age 60 years; 48% males) who met WHO criteria for diagnosis of PV. Amongst them, accurate documentation of AMC was available in 237 patients, cytogenetic information in 239, and ASXL1, TET2 and SRSF2 mutational status in 133 patients. Median (range) values were for AMC 0.6 x 10(9)/L (0-4.7) and leukocytes 11.6 x 10(9)/L (3.8-171.6). 31% of 506 informative patients had palpable splenomegaly, 34% of 551 had microcirculatory symptoms, 30% of 566 had pruritus, 8% of 504 had erythromelalgia, 42% of 581 had hypertension, 9% of 584 had diabetes and 11% of 575 were active tobacco users. 25% of the patients presented with history of thrombosis and 22% developed thrombosis after diagnosis. Cytogenetic findings were abnormal in 19%, of whom 20% were unfavorable. TET2, ASXL1 and SRSF2 mutations were documented in 18%, 11% and 3%, respectively. During follow-up, 224 (38%) patients died and median follow-up for living patients was 109 months. Median survival was 16 years and leukemic or fibrotic transformations were documented in 4% and 14%, respectively.Comparison of patients with and without monocytosis:Among 237 informative patients, 32 (14%) displayed monocytosis (AMC ≥1 x 10(9)/L) at time of diagnosis. PV patients with monocytosis were older (p=0.006) and displayed higher leukocyte count (p<0.0001) and higher incidences of leukocytosis (p=0.024) and unfavorable cytogenetic abnormalities (p=0.02). There was no association between monocytosis and mutations for TET2 (p=0.1), ASXL1 (p=0.7) and SRSF2 (p=0.3) or thrombosis before (p=0.9) or after (p=0.5) diagnosis (p=0.5), palpable splenomegaly (p=0.6), pruritus (p=0.7) or microcirculatory symptoms (p=0.1).Among the 237 PV patients in whom information regarding AMC was available, 70 (30%) died during follow-up and 49 (21%), 23 (10%), 9 (4%) developed thrombosis, leukemic transformation or fibrotic progression, respectively. In univariate analysis, overall (p=0.009; HR 2.0, 95% CI 1.2-3.4) but not leukemia-free (p=0.79), myelofibrosis-free (p=0.13) or thrombosis-free (p=0.48) survivals were different between patients with or without monocytosis. Furthermore, the significant difference in survival was no longer apparent when analysis was adjusted for age (p=0.13), unfavorable karyotype (0.17) or leukocytosis (p=0.06).ConclusionsMonocytosis (AMC ≥1 x 10(9)/L) is not infrequent in PV (14%). However, the presence of monocytosis does not appear to represent a significantly different phenotype in terms of molecular characteristics although it is associated with older age, leukocytosis and unfavorable karyotype. The latter associations account for the inferior survival seen in patients with monocytosis. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Bone Marrowmmorphology in Addition to JAK2 Allele Burden Is Essential for Assessing Disease Status in Recombinant Interferon Alpha-Treated Polycythemia Vera Patients

IntroductionPolycythemia vera (PV) is a myeloproliferative neoplasm (MPN) associated with the JAK2 V617F mutation. Recombinant interferon alfa-2b (rIFNα-2b) or pegylated rIFN2α (pegrIFNα-2a) treatment in PV is effective in inducing hematologic and molecular responses. Reduction in JAK2V617Fallele burden (%V617F) has been used increasingly as a surrogate marker of disease response to treatment and as a unique criterion for discontinuation of therapy. To date, no studies have evaluated the relationship between changes in %V617F and established indicators of disease activity such as bone marrow cellularity and degree of fibrosis. Having observed persistent marrow hypercellularity or progressive fibrosis in PV patients despite a significant reduction in %V617F, we therefore systematically examined the correlation of %V617F with marrow morphology. We also assessed immunohistochemical expression of pSTAT5 as a marker of JAK2 activation.MethodsThe diagnosis of PV was based on the WHO criteria which include presence of erythrocytosis and/or an increased Cr51 red cell mass, JAK2V617F mutation and typical bone marrow findings such as hypercellularity due to panmyelosis and megakaryocyte morphology consistent with PV. Patients with two marrow examinations and quantitative JAK2 levels measured at a median of 1.2 months from the biopsy date were eligible for inclusion. The JAK2V617Fallele burden in peripheral blood samples was determined by pyrosequencing. Clinical, hematologic and molecular responses were graded according to ELN criteria. Marrow fibrosis was scored according to the WHO three-tiered semi quantitative grading system. Immunohistochemical staining for p-STAT5 was performed on clot sections in a subset of patients.ResultsWe identified 15 patients for inclusion. The median %V617F at the time of the initial biopsy was 62.2% (18.2-100%), which decreased to a median of 33% (0-93.7%) on subsequent evaluation (p=0.02). Patients were initially treated with peg rIFNα-2a 45-90 mcgm weekly (n=13) or rIFNα-2b (n=2), 1x106units thrice weekly, and gradually increased based upon response and tolerance. The median duration of treatment between biopsy was 4.2 years (0.8 - 6.6). All patients achieved either a complete (CHR) (n=8) or a partial (PHR) (n=7) hematologic response. Of these 15 patients, 3 achieved a complete molecular response (CMR), 4 a partial molecular response (PMR), and 8 patients no molecular response (NMR). We observed no correlation between clinical and molecular response: of the 8 patients who achieved CHR, 2 achieved a CMR and 6 did not. There was no correlation between clinical response and fibrosis or cellularity; of 8 patients who achieved CHR, 5 showed persistent/increased hypercellularity and 6 had persistent/increased fibrosis.We also found no correlation between marrow morphologic changes and molecular response. Of the 3 patients with CMR, all had persistent hypercellularity (range: 60-90%) and increased/persistent fibrosis. Among the 4 patients who achieved a PMR, 2 had increased cellularity and 2 decreased cellularity. All had increased or persistent fibrosis. Among the 8 patients with NMR, 2 had an increase in marrow cellularity, 2 no change, and 4 an insignificant decrease. Fibrosis increased in 2, was unchanged in 3, and decreased in 3. Thus, there were no significant correlations between changes in %V617F and cellularity (κ=0.02) and fibrosis (κ=0.04). Immunohistochemical staining for p-STAT5 expression in megakaryocyte nuclei showed no correlation between degree of positivity and %V617F.ConclusionsAlthough we observed a statistically significant decrease in %V617F after treatment with rIFNα, there were no parallel changes in marrow morphology. In all patients with CMR, we observed persistent or worsening marrow disease, suggesting to us a need for continued therapy. In patients who achieved either CHR or PHR, we observed persistent hypercellularity and persistent/progressive fibrosis. Our results question the use of JAK2V617F allele burden as the sole criterion for discontinuation of rIFNα therapy. The prognostic significance of failing to attain a morphologic response in patients who have achieved a hematologic and/or molecular response remains unresolved. Resolution of these issues with longer follow-up is important, as premature discontinuation of rIFNα may allow the pathogenic mechanisms of the disease to progress unfettered. DisclosuresOrazi:Novartis: Honoraria.

An ED paradox: patients who arrive by ambulance and then leave without consulting an ED provider

BackgroundScientists have called for strategies to identify ED patients with unmet needs. We identify the unique profile of ED patients who arrive by ambulance and subsequently leave without consulting a...

A statistical analysis plan for the efficiency and safety of Chinese herbal medicine used concurrently with topical therapy for psoriasis vulgaris.

BackgroundPsoriasis vulgaris (PV) has been causing increasing concern due to its highly prevalent, harmful and therapy-resistant characteristics. The YXBCM01 (Chinese herbal medicine) for PV trial evaluates the effects of YXBCM01 on relapse rate in patients suffering from PV. As an update to the published design and method for the trial, this paper presents the statistical plan for the main publication to avoid the risk of outcome reporting bias, selective reporting, and data-driven results.Methods/designThis trial is a multicenter, randomized, double-blind, placebo-controlled, parallel-group clinical trial. A total of 600 PV patients (300 in each group) will be randomized to one of two arms: participants in the experimental group will receive the YXBCM01 granule 5.5 g twice daily for 12 weeks. Placebo granules are given to patients in the control group at a dose of 5.5 g twice daily for 12 weeks. The sequential topical therapy is administrated simultaneously to all eligible patients by using calcipotriol betamethasone ointment once daily (a treatment area of up to 30 % body surface area (BSA), fingertip unit is recommended) in the first 4 weeks (maximum of 100 g weekly), followed by calcipotriol betamethasone ointment once daily for the remaining 8 weeks (maximum of 100 g weekly). The primary outcome measure is relapse rate in the treatment period and follow-up period. The secondary outcome measures include time to relapse, time to onset, rebound rate, cumulative consumption of topical medicine, visual analog scale (VAS), BSA, the Dermatology Life Quality Index (DLQI) and the Medical Outcomes Study (MOS) 36-item short form health survey (SF-36).ConclusionsApplication of this statistical analysis plan to the YXBCM01 for PV trial will facilitate unbiased evaluation of these important clinical data. This study will provide evidence regarding the value of YXBCM01 as an intervention for PV patients.Trial registrationChinese Clinical Trial Registry: ChiCTR-TRC-13003233, registered on 26 May 2013.Electronic supplementary materialThe online version of this article (doi:10.1186/s13063-016-1610-z) contains supplementary material, which is available to authorized users.

Homozygous calreticulin mutations in patients with myelofibrosis lead to acquired myeloperoxidase deficiency

AbstractThe pathogenesis of acquired myeloperoxidase (MPO) deficiency, a rare phenomenon observed in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), is unknown. MPO is a glycoprotein (GP) chaperoned by calreticulin (CALR) in the endoplasmic reticulum. Mutations in CALR are frequently found in patients with myelofibrosis (MF) and essential thrombocythemia (ET) with nonmutated Janus kinase 2 (JAK2). We hypothesized that acquired MPO deficiency in MPN could be associated with the presence of CALR mutations. A cohort of 317 patients with MPN (142 polycythemia vera [PV], 94 ET, and 81 MF) was screened for MPO deficiency. MPO deficiency was observed in 6/81 MF patients (7.4%), but not in PV or ET patients. Susceptibility to infections had been documented in 2/6 (33%) MPO-deficient patients. Five out of 6 patients with MPO deficiency carried a homozygous CALR mutation and were also deficient in eosinophilic peroxidase (EPX). In contrast, 1 patient with MF, a JAK2-V617F mutation, and MPO deficiency, carried 2 previously reported MPO mutations and showed normal EPX activity. Patients with homozygous CALR mutations had reduced MPO protein, but normal MPO messenger RNA (mRNA) levels supporting a posttranscriptional defect in MPO production. Finally, we demonstrate in vitro that in the absence of CALR, immature MPO protein precursors are degraded in the proteasome. Therefore, 4 decades after the first description of acquired MPO deficiency in MPN, we provide the molecular correlate associated with this phenomenon and evidence that CALR mutations can affect the biosynthesis of GPs.

Anti-Desmoglein 1 and 3 Autoantibody Levels in Endemic Pemphigus Foliaceus and Pemphigus Vulgaris from Brazil.

Pemphigus is a group of autoimmune blistering diseases of which the major forms are pemphigus foliaceus (PF) and vulgaris (PV). In Brazil, PF occurs in an endemic form also known as fogo selvagem. The main autoantibody in PF is against desmoglein 1 (DSG1), while in PV the main antibody is anti-desmoglein 3 (DSG3), but often anti-DSG1 is also present. The aim of the present study was to analyze the levels of anti-DSG1 and antiDSG3 autoantibodies in Brazilian PF and PV patients, considering different stages of the disease for PF patients and comparing these levels to those of healthy individuals living in and outside the endemic regions. Levels of anti-DSG1 and anti-DSG3 were measured in the sera of Brazilian PF (n = 68) and PV (n = 20) patients as well as in clinically healthy (control) individuals (n = 48) by Enzyme Linked Immunosorbent Assay (ELISA). Comparisons were made using Kruskal-Wallis and Mann-Whitney tests. As expected, anti-DSG1 was more prevalent among PF patients (84% against 43% in PV), while antiDSG3 was more prevalent in PV patients (50% against 4% in PF). Levels of anti-DSG1 in PF patients in remission differed from those in patients undergoing active disease (p = 0.003), and patients in long-term remission (more than two years without presenting new lesions) were similar to control individuals living in the endemic region and surrounding area (p = 0.09). Moreover, patients with a more severe form of the disease had higher levels of anti-DSG1 (at least 134 U/mL, mean of 233 U/mL) than patients with a less severe form (fewer lesions) (mean of 193 U/mL, including two negative individuals). Despite the importance of these antibodies for diagnosis and management purposes, their presence in a healthy individual and in patients under remission indicates that caution should be taken when using anti-DSG for diagnosis, especially in endemic areas.

Successful Treatment of HU-Refractory Polycythemia Vera with Atorvastatin and Low Dose Hydroxyurea. Results from a Pilot Study in Bolivia

BackgroundPolycythemia Vera (PV) is a clonal myeloproliferative neoplasm, characterized by the JAK2V617F mutation.The main goal of current therapies for PV is to prevent thrombotic events and delay transformation to Myelofibrosis (MF) or Acute Myeloid Leukemia (AML).Treatment for PV to keep an hematocrit (Hct) level <45 %, has been associated with a reduction in cardiovascular deaths and thrombotic events (Marchioli, R et al. NEJM 2013). Currently, low-risk PV patients (<60 years and no previous thrombotic events) are treated with aspirin and phlebotomy while high-risk patients require additional cytoreductive therapy, usually with Hydroxyurea (HU). Resistance to HU is associated with an increased risk of transformation and reduced survival. This is why for HU-refractory patients, second line treatments with interferon alpha, anagrelide or even ruxolitinib are recommended. In Latin America, because of high cost and drugs availability, this last group reflects difficulties to be treated. Because statins have been reported to modulate the erythroid clonogenic activity of normal BM erythroid colonies we performed a pilot study to investigate in vitro and in vivo the biologic and clinical activity of atorvastatin in PV patientsPatients and MethodsTen high risk PV patients with a median age of 64.3 years (range 58-73) entered into this study. The diagnosis of PV was done according to the 2008 World Health Organization diagnostic criteria and patients were stratified according to an algorithm proposal provided by Griesshammer et al. (Ann Hematol, 2015). The definition of HU resistance (Barosi, G et al.: BJH 2009) was applicable to five patients (median age 63.9 years) failing to achieve a satisfactory hematologic response upon treatment with more than 2 g of HU, 100 mg of Aspirin and phlebotomies. The assessment of the JAK2V617F mutation was performed as previously described (Guerini et al.: Leukemia 2009). Colony assay, proliferation and apoptosis tests were performed with or without Simvastatin (3.5 uM), as previously described (Amaru, A, Experimental Hematology 2012), on cell lines (UKE1 and K562) and bone marrow mononuclear cells obtained from PV patients and healthy donors. Patients with HU refractory PV (n=5) and high risk PV with hypercholesterolemia (n=5) were eligible to receive Atorvastatin (20 mg/day) added on the top of the ongoing treatment with phlebotomies, Aspirin (100 mg/day) and cytoreductive HU therapy (500 mg/day). All treated patients were high altitude residents (> 3.600 m.a.s.l.) of La Paz (Bolivia) where the normal Hct level of healthy subjects is 48-57% for men and 44-54% for women. This pilot study was approved by the Review Board of the Hospital and the University of San Andres, La Paz.ResultsIn a preliminary set of in vitro proliferation cell assays, simvastatin (3.5 uM), added for 5 days, induced a 33% inhibition of cell proliferation of UKE-1 (JAK2V617F mutated) as compared to 5 % of K562 (BCR/ABL positive). A comparable result was obtained in a 7-day clonogenic cell assay where the colony inhibition was 50 % for UKE-1 and 10 % for K562. On the basis of these results similar experiments were also performed using BM mononuclear cells derived from PV patients and healthy donors. In these experiments performed with the addition of simvastatin, it induced a 41% of inhibition in BFU-E colonies of PV patients and a 25% of inhibition in healthy donors. Furthermore, BFU-E colonies inhibited by simvastatin presented a decrease in hemoglobinization and the size of colonies.HU refractory PV patients and High-risk PV patients with hypercholesterolemia treated with the addition of Atorvastatin, Aspirin, cytoreductive HU and phlebotomies; after a follow-up of 2.6 years (1-7 years), induced a decrease of WBC from 16.500 to 9.270/ul, Hct 61.1 to 52.3% and PLT 457.900,000 to 324.7000/ul. The number of required phlebotomies is reduced in comparison to the required at starting treatment. None of the patients presented thrombotic or cardiopulmonary event. One patient died within two years of starting treatment, due to complications of diabetes mellitus.ConclusionsIn vitro and in vivo, statins showed some evidence of inhibitory activity of the hematopoiesis of PV patients. These preliminary results might indicate the opportunity to further investigate the potential clinical value of these molecules in the treatment of PV. DisclosuresOff Label Use: Atorvastatin was used for its antiproliferative activity on myeloid progenitor cells shown by in vitro experiments.

The Impact of Interferon-alpha2 on HLA-Genes in Patients with Polycythemia Vera and Related Neoplasms

IntroductionGene expression profiling studies in the Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have unraveled significant deregulation of several immune and inflammation genes of potential importance for clonal evolution (Skov V et al., Eur J Haematol 2011; Leuk Res 2012; Exp Hematol 2012). Other mechanisms might be downregulation of major histocompatibility (MHC) class I and II genes, which are used by tumor cells to escape antitumor T-cell-mediated immune responses. In a previous study, several genes encoding human leukocyte antigen (HLA) class I and II molecules, beta2microglobulin and members of the antigen processing machinery of HLA class I molecules (LMP2, LMP7, TAP1, TAP2 and tapasin) have been shown to be significantly downregulated (Skov V, Leuk Lymphoma 2012). Upregulation of HLA-genes is considered one of the mechanisms of action of interferon-alpha2 (IFN-alpha2) but regulation of these genes during IFN-alpha2 treatment of patients with MPNs has never been studied. The purpose of this study was to assess the regulation of several HLA genes before and during treatment with IFN-alpha2, the hypothesis being that IFN-alpha2 - as in other cancers - may restore downregulation of HLA genes in MPNs as well.Patients and MethodsUsing Affymetrix HG-U133 2.0 Plus microarrays, gene expression profiling have been performed on whole blood from patients with ET (n =8), PV (n = 21), and PMF (n = 4) before and after 3 months of treatment with IFN-alpha2. Background correction, normalization, and gene expression index calculation were performed with the robust multi-array (rma) method. The regularized t-test limma for pairwise data was used to calculate differences in gene expression between patients before and after treatment with IFN-alpha2. A p-value < 0.05 was considered significant.ResultsStatistical analysis of all 54,675 probe sets on the microarray revealed 6261, 10,008, 2828, and 12,390 probe sets to be significantly differentially expressed in ET, PV, PMF, and MPNs as a whole, respectively, in response to treatment with IFN-alpha2 (P-value <0.05). To examine if treatment with IFN-alpha2 restores preexisting abnormalities of HLA genes in MPNs, pretreatment vs. posttreatment gene expression changes of MPNs were analyzed. Several HLA genes were significantly deregulated. Of 23 HLA major histocompatibility complex (MHC) class I and II genes and 26 HLA associated and complex group genes, 15, 24, 6 and 24 genes were significantly deregulated in ET, PV, PMF, and MPNs as a whole, respectively (P<0.05). In ET patients, BAT4, HCG26, HCP5, HLA-B, HLA-C, HLA-F, HLA-G, TAP1, and PSMB9 were significantly upregulated and BAT5, CIITA, HCG11, HLA-DOA, HLA-DQA1, and HLA-DRB4 were significantly downregulated. In PV patients, the significantly upregulated genes included CD74, HCG26, HCG8, HCP5, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DOB, HLA-DPA1, HLA-DRB1, HLA-DRB5, HLA-DRB6, MR1, TAP1, TAP2, TAPBPL, PSMB8, and PSMB9 and the significantly downregulated genes included CITTA, HLA-DOA, and HLA-DPB2. In PMF, HCP5, HLA-DOA, and HLA-DQA1 were significantly upregulated and HCP5, TAP1, and PSMB9 significantly downregulated. In MPNs as a whole, BAT4, HCG26, HCG8, HCP5, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DOB, HLA-DPA1, HLA-DRB1, HLA-DRB5, HLA-DRB6, MR1, TAP1, TAP2, TAPBPL, PSMB8, and PSMB9 were significantly upregulated and CIITA, HLA-DOA, and HLA-DPB2 were significantly downregulated (all P<0.05).Conclusions: Whole blood transcriptional profiling has unraveled that IFN-alpha2 potently up-regulates several HLA-genes of importance for tumor immune surveillance. Our findings may be crucial for the efficacy of IFN-alpha2 in the treatment of MPNs, since the potential of cytotoxic immune cells for tumor killing is likely by this mechanism significantly enhanced and the path towards induction of minimal residual disease accordingly improved as well. DisclosuresBjerrum:Bristoll Myers Squibb, Novartis and Pfizer: Other: educational activities. Hasselbalch:Novartis: Research Funding.

Assessment and Validation of the EQ-5D Among a Population of Myeloproliferative Neoplasm Patients

IntroductionIndividuals with the Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) have previously been shown to experience symptoms in excess to age and comorbidity matched peers. Although numerous assessments of symptom burden and quality of life have been utilized among MPN populations, including the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30), Patient Reported Outcomes Measurement Information System (PROMIS¨) fatigue scale, and Functional Assessment of Cancer Therapy-Lymphoma (FACT-Lym) and the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-10), there are currently no validated assessments of overall health. Developed by the EuroQol Group to assess heath status, the EQ-5D is an internationally validated self-assessment which may be useful in the assessment of health state among MPN patients (Annals of Medicine, 2001. 33:337-43).MethodsThe EQ-5D evaluates five dimensions of mobility, self-care, usual activities, pain/discomfort and anxiety/depression, on a scale from no problems to extreme problems and assesses patient reported health state via a numerical visual analogue scale (VAS) ranging from 0 to 100. The health state score is a composite score of these items ranging from 0 to 1.0, with higher scores indicating an improved health state. EQ-5D survey participants were essential thrombocythemia (ET) and polycythemia (PV) patients participating in the MAJIC United Kingdom (UK) trial. The MAJIC trial is a randomized, phase II study of best available therapy versus JAK inhibition in individuals with PV or ET who have been resistant or intolerant of cytoreduction with hydroxyurea /hydroxycarbamide. This study was one of the first Trials Acceleration Programme (TAP) trials and is funded by Leukaemia Lymphoma Research, with drug supplied by Novartis. Only baseline EQ-5D assessments taken at study enrollment were included in this analysis. Comparative analysis was preformed using the MPN-10 (J Clin Oncol.2012. 30:4098-103).Results:Participant Demographics: Sixty six patients with ET and 55 patients with PV completed the EQ-5D questionnaire. Median respondent age was 65 years old and half of respondents were female (54%). Average MPN duration was 10 years for ET and 9.2 years for PV.EQ-5D Scoring: Severe to extreme difficulties in the items of mobility, self-care, usual activities, pain/discomfort and anxiety/depression were common (Table 1).Patients with PV tended to have higher proportion of patients with severe to extreme difficulties in individual disease dimensions, higher VAS score, and worsened health state compared to their essential thrombocythemia counterparts, although these effects were not significant. Average VAS health score was 70.4 (+21.2) for ET and 66.5 (+19.6). When evaluating the health state composite score, the mean ET score was 0.74 (+0.32) and mean PV score was 0.66 (+0.28).EQ-5D Validation: The EQ-5D had excellent internal consistency (CronbachÕs alpha=0.87) and strong internal correlation. When evaluating external validity, strong correlations existed between the EQ-5D and the MPN-SAF TSS scales for dimensions of mobility, usual activities, pain and overall health state (many items with r >0.5 and p<0.001).Conclusions:The EQ-5D is a valid and accurate instrument for the brief assessment of health state among MPN patients. This instrument demonstrates excellent clinical utility in a large clinical study cohort and can be used in both clinical practice and trial settings. When comparing EQ-5D health state scores, ET and PV patients had worsened health than the general UK population age 60 to 69 (0.774; Med Decis Making. 2011.31(6)800-4), but similar health to individuals with other malignancies including breast cancer (0.760), prostate cancer (0.743), and lung cancer (0.689). [Display omitted] DisclosuresMesa:NS Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Incyte Corporation: Research Funding; Pfizer: Research Funding; Promedior: Research Funding; CTI Biopharma: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Pfizer: Research Funding. Harrison:CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Shire: Speakers Bureau; Gilead: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.

No Development of Neutralizing Antibodies Against Recombinant Interferon-Alpha in Ph-Negative Myeloproliferative Neoplasms - a Prospective Study

BackgroundTreatment of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPNs) with recombinant pegylated interferon alpha2a/b (rIFN-alpha) has proven effective. It is well known that prolonged therapy with recombinant type 1 interferons (IFN-alpha and IFN-beta) may induce neutralizing antibodies (nAbs) against the drug leading to treatment failure. Most data on type 1 IFN immunogenicity are available from studies of patients with multiple sclerosis treated with rIFN-beta, and patients with hepatitis C treated with rIFN-alpha. A few reports have demonstrated nAbs in MPN patients not responding adequately to rIFN-alpha treatment, but the phenomenon has not been thoroughly investigated in MPNs.Patients and MethodsNewly diagnosed patients with MPNs enrolled in the Danish multicenter trial - DALIAH (Low-dose rIFN-alpha versus Hydroxyurea in The Treatment of Ph-Negative MPNs) were included. Patients were randomized to treatment with either rIFN-alpha 2a or 2b at a starting dose of 45 and 35 mikrograms once weekly, respectively. The occurrence of neutralizing Abs (nAbs) against rIFN-alpha was investigated at baseline, month 12 and month 24 by reporter gene assays (iLiteTM alphabeta and iLiteTM antialpha, Biomonitor A/S, Copenhagen, Denmark). JAK2 V617F quantitative mutation analyses were performed as previously described (Larsen TS, BJH 2007). Statistical analyses were performed using STATA version 9.0.ResultsNinety-two patients on sustained treatment with rIFN-alpha2a (n=48) and rIFN-alpha2b (n=44) for 12 months were analyzed for this study. Forty-five patients had ET, 39 patients had PV and 8 patients had proliferative PMF. Thirty-six out of 39 (92%) PV patients, 22 out of 45 (49%) ET patients and 4 out of 8 (50%) PMF patients were JAK2V617F mutated. Hematological responses at 12 months: ET: 67% CR, 29 % PR; PV: 64% CR, 31% PR (ELN 2009 criteria); PMF: 50% had at least a minor response (EUMNET).The median serum concentration of bioactive IFN-alpha at 12 months was 12,4 (range <2,4-86,4) and 2,6 (range <2,4-12,8) IU/mL serum, for patients treated with rIFN-alpha 2a and -2b respectively. No significant association between hematological or molecular response and serum IFN-alpha activity was found. Serum from 92 patients was analyzed at 12 months and 33 patients were analyzed at both 12 and 24 months and no occurrence of nAbs was seen during treatment. Twenty-four patients had pre-treatment levels of IFN nAbs measured. Notably, one patient was tested positive for the presence of nAbs before rIFN-alpha exposure. This autoAb-positive patient remained positive throughout the study and has shown low IFN serum activity (< 2,4 IU/mL) and only partial hematological and molecular response after 24 months of treatment.ConclusionsDevelopment of nAbs in MPN patients completing treatment for 12 months with rIFN-alpha seems exceedingly rare as no patients, neither complete responders nor patients not meeting criteria for complete hematological remission developed nAbs after 12 (24) months of therapy. Its apparent rarity does not justify a routine investigation of nAbs in patients not responding to rIFN-alpha treatment. There was no significant correlation between serum concentration of rIFN-alpha 2a and -2b and clinical or molecular responses. The intriguing finding that one of 24 patients had pre-existing cross reacting nAbs against rIFN-alpha 2a and 2b before commencing rIFN-alpha treatment is interesting and was associated with an insufficient response. DisclosuresOff Label Use: Recombinant interferon-alpha 2a and -2b in the treatment of chronic Philadelphia-negative myeloproliferative neoplasms.. El Fassi:Novartis Denmark: Honoraria, Other: Have conducted an educational session for Novartis Denmark, regarding MPNs and ruxolitinib, for this a honorarium was received.. Bjerrum:Bristoll Myers Squibb, Novartis and Pfizer: Other: educational activities. Hasselbalch:Novartis: Research Funding. Bendtzen:Pfizer: Honoraria; Eurodiagnostica AB: Equity Ownership; Novo-Nordisk: Equity Ownership.