bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of Real-Time PCR and Droplet Digital PCR for the Determination of JAK2V617F Mutation in Ph'-Negative Myeloproliferative Neoplasms
Authors: Fontanelli Giulia , Baratè Claudia , Ciabatti Elena , Guerrini Francesca , Morganti Riccardo , Arici Roberta , Barsotti Sara , Petrini Iacopo , Del Re Marzia , Petrini Mario , Galimberti Sara 
Published: October 2019

Abstract
Myeloproliferative neoplasms (MPNs) are a group of clonal diseases associated with JAK2 mutation (V617F) in a percentage varying from 50% (Essential Thrombocytemia ET, and Primary Myelofibrosis PMF) to 95% (Polycitemia Vera, PV). The standard method for determining the V617F mutation is today represented by the Real Time PCR (RT-PCR). However, in recent years some studies have focused attention on a new method, the Droplet Digital PCR (DD-PCR), that allows an absolute quantitation of the mutated allele without any reference curve.
The main objective of this study was the comparison of two PCR methods (RT-PCR and DD-PCR) for the analysis of the JAK2V617F mutation in order to assess the sensitivity, specificity and feasibility of the two techniques.
In the study 225 patients with MPNs JAK2V617F-positive were enrolled; in 99 cases, DNA was still available for further assays and so DD-PCR tests were performed and compared with the results coming from RT-PCR (MutaQuant kit, Ipsogen, Luminy Biotech, Marseille, France).
The results show a good sensitivity for both methods; nevertheless, after appropriate dilution tests, the DD-PCR was able to detect the JAK2V617F mutation in the 5x10-4 dilution versus 5x10-3 for the RT-PCR, in front of a fully comparable specificity. A linear regression analysis showed an absolute correlation between the 2 methods (R2=0.91), and the median mutated allele burden was not different when measured by the DD-PCR or the RT-PCR (p=0.67), with the highest median allele burden observed in secondary myelofibrosis (86% by RT-PCR and 91% by DD-PCR) and the lowest one in ET (23% by RT-PCR and 25% by DD-PCR) (see Figure 1 and Figure 2), as expected.
Figure 1 Figure 1.
Figure 2 Figure 2.
Our study demonstrates that a new molecular technique, the DD-PCR, may represent a new frontier in the world of molecular techniques for the detection of JAK2 mutation with a high sensitivity and specificity. In addition the method allows obtain two types of information, qualitative and quantitative, with one analysis, saving time and at a lower cost than the standard method.

Disclosures
No relevant conflicts of interest to declare.
Heterozygosity for JAK2V617F Is a Risk Factor of Developing Post-Polycythemia Vera Myeloofibrosis?
Authors: Cacciola Rossella , Torre Maria , Cacciola Emma 
Published: October 2019

Abstract
Animal model studies report that heterozygous JAK2 V617F polycythemia vera (PV) has a low risk of progression to myelofibrosis. This clinical characteristic in humans is controversial. Therefore, we evaluated JAK2 V617F allele burden and myelofibrotic indicators such as reticulin level, bone marrow (BM) cellularity, CD34 levels and spleen size in 75 patients with heterozygous JAK2 V617F PV (45 males, 30 females, mean age 68 years) diagnosed according WHO criteria. Their mean duration of disease was 11 years (range, 1-26 years). All patients were on aspirin. The percentage of granulocyte mutant alleles was evaluated using a quantitative real-time polymerase chain reaction-based allelic discrimination assay. Reticulin level and BM cellularity were evaluated by ad hoc panel of 4 hematologists and 2 pathologists. CD34 levels were measured by flow cytometric assay. The spleen size was on ultrasound scan by measuring the longitudinal diameter (centimeters). Of 75 patients, 42 had allele burden > 10% (34±5%) (group 1) and 33 had allele burden < 10% (6±1%) (group 2). All patients had minor reticulin fibrosis (0.4±0.8), normal BM cellularity (76±8%), normal CD34 level (0.08±0.09%) whereas 24/42 (57%) of the group 1 and 3/33 (9%) of the group 2 had palpable splenomegaly (14±2.6 cm and 18 cm, respectively). These results suggest that heterozygosity > 10% might indicate a higher risk into the evolution of the so-called spent phase of the PV.

Disclosures
No relevant conflicts of interest to declare.
Long Term Follow-up of 107 Patients with Essential Thrombocythemia: Role of Cardiovascular Events
Authors: Arboscello Eleonora , Molinari Elisa , Bellodi Andrea , Del Corso Lisette , Favorini Serena , Sicbaldi Vera , Agnelli Giacchello Jacopo , Salvetti Chiara , Balleari Enrico , Ghio Riccardo , Van Lint Maria Teresa 
Published: October 2019

Abstract
BACKGROUND Essential Thrombocythemia (ET) is a Philadelphia-negative chronic myeloproliferative neoplasm characterized by haemorrhagic and thrombotic complications. Haemorrhagic events and arterial and venous thrombosis, including microcirculation transient occlusions, are the major causes of morbidity in ET patients. The control of these events represents the goal of standard therapeutic approaches.
MATERIALS AND METHODS We retrospectively analysed data about 107 ET patients who received diagnosis between January 1980 and June 2014. Median follow-up was 80 months,16 patients were lost during follow-up. The medium age at diagnosis was 60 years, with a prevalence of female (66 patients).We recorded adverse cardiovascular events at diagnosis and during follow-up, assessing whether cytoreductive ad antiplatelet therapy could reduce such events and improve quality of life. Finally, we evaluated the impact of additional cardiovascular risk factors.
OBJECTIVES to observe incidence and kind of thrombotic events in patients affected by ET at diagnosis and during follow-up.
RESULTS 30 patients (27.7%) had a history of thrombosis at diagnosis (8 transient cerebral ischemia, 7 myocardial infarction/unstable angina, among them 7 patients experienced a rethrombosis during follow-up. 16 patients (15%) developed a first thrombotic event, all patients were under cytoreductive treatment. 21 patients with a history of thrombosis had more than 60 years at diagnosis, 19 patients (63%) had at least one additional cardiovascular risk factor among arterial hypertension, dyslipidemia, diabetes, obesity, hyperuricemia and smoking. Median platelet count was 813000/mm3, leukocyte count greater was more than 10000/mm3 in half of patients. Evolution to acute leukemia/myelofibrosis occurred in 3 (2,7%) and 7 (6,5%) patients of total.
CONCLUSIONS The occurrence of thrombotic events even in patients with good hematologic response of disease and during antiplatelet and cytoreductive therapy, indicates the presence of a residual risk of thrombosis. This risk is not yet fully clarified by retrospective studies published until now. Prospective studies will be useful to evaluate the role and the importance of comorbidity in these patients with long-prognosis, in order to optimize therapy, reduce cardiovascular events and improve quality of life

Disclosures
No relevant conflicts of interest to declare.
Calreticulin Mutations in JAK2-Negative Myeloproliferative Neoplasms. Experience in Our Center
Authors: Penalva Moreno Maria Jose , Martinez-Laperche Carolina , Osorio Prendes Santiago , Buces Gonzalez Elena , Diez-Martin Jose Luis , Buño Borde Ismael 
Published: October 2019

Abstract
Introduction: Calreticulin (CALR) is a multifunctional protein regulated by calcium that is located in the endoplasmic reticulum. Recently, mutations in the calreticulin gene have been described in patients with the diagnosis of essential thrombocytemia (ET) and primary myelofibrosis (PMF), mainly in JAK2-negative cases. CALR mutations are localized to exon 9 and generate deletions or insertions that lead to a frameshift change resulting in a mutant protein. The detection of these mutations helps in the actual diagnosis of JAK2 mieloproliferative syndromes (MPN). Our aim is to assess the utility of the determination of these mutations in the management of patients with diagnosis of MPN in our center.
Patients and methods: This study includes 94 patients with diagnosis of JAK2-negative MPN retrospectively selected following clinical and analytical criteria between 2008 and 2014 in our center (Table 1, 2). CALR mutations were performed with the use of fluorescent PCR following the methods described by Klampf et al. (NEJM, 2013).
Results: 94 patients were analyzed, 77 of them had the diagnosis of TE, 8 of PMF and 9 of others disorders of myelodisplastic/mieloproliferative. 22% of the cases of ET had mutations in CALR (Table 1). In these mutations, a total of 53% were type I mutations (52-bp deletion) and 47% were type II mutations (5-bp insertion). Only one mutation was infrequent, a 46-pb deletion. We have found statistical correlation in the number of platelets depending on the presence of the mutation and in the largest number of platelets in type II mutations. 33% of the cases of PMF had mutations in CALR, all of them type I. Among other diseases not included in MPN, one of them had a type I mutation (data not shown).
Conclusions: Our results are close to recently published results regarding the frequency of mutation and as the largest number of platelets in type II mutations with respect to mutation type I. This study confirms the importance of CALR mutations determination in the diagnosis of JAK2-negative ET and PMF with high clinical suspicion.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Up-Regulation of Prok-2 in Granulocytes Is Present BOTH in MGUS and MM
Authors: Romano Alessandra , Simeon Vittorio , La Cava Piera , Parrinello Nunziatina Laura , La Fauci Alessia , Vetro Calogero , Tibullo Daniele , Giallongo Cesarina , Cavalli Maide , Chiarenza Annalisa , Conticello Concetta , Musto Pellegrino , Di Raimondo Francesco 
Published: October 2019

Abstract
Introduction
Our previous work showed that in Multiple Myeloma (MM) the immune function is impaired, including immunosuppressive properties of granulocytes due to their increased amount of arginase-1 and reduced phagocytic activity (Parrinello, manuscript in preparation). It is currently unknown if granulocyte dysfunction occurs in progression from MGUS to MM.
Aim
Providing a gene expression profile of mature granulocytes isolated from peripheral blood at the steady-statein MGUS and MM.
Methods
Using oligonucleotide microarrays we first evaluated the gene expression profile of granulocytes at the steady state in 5 MM, 3 MGUS and 3 healthy subjects matched for sex and age.
Then, we validated the first up-regulated gene PROK-2, obtained from preliminary findings in granulocytes from peripheral blood in 85 consecutive newly diagnosed MGUS (N=45), MM (N=40) and 15 healthy subjects, in RT-PCR (validation set).
Results
We found 708 genes differentially expressed (467 up- and 241 down regulated) in MGUS versus healthy granulocytes at the steady state. The set of annotated, differentially expressed genes could befunctionally organized by “gene ontology” (http://www.geneontology.org/) into the following major categories:
i) receptors and signal transduction (including up-regulation of CD14, Toll-like receptor 5 (TLR-5), IL-7 Receptor (CD127), IL-11 receptor, TGF-beta receptor 2, hematopoietic cells kinase (HCK), IFNAR1);
ii) negative regulation of adaptive immune response (including up regulation of CD127, STAT6, IFNAR1, OSCAR, PROK-2 and down regulation of p50, p65,NFKBIA, IL8, ELK-1, HIF-1 alpha, CEBP-beta, CEBP-zeta).
In MM samples we confirmed a statistically significant up-regulation of PROK-2 (a key molecule of VEGF-independent angiogenesis), CD14 (mediator hypersensitive innate immune response to lipopolysaccharide) and HCK (the hematopoietic cell kinase, involved in neutrophil migration and degranulation).
In the validation set, PROK-2 expression was two times higher in MGUS than healthy subjects (p=.02) and up to ten times higher in MM (p=.001). In MM patients, increased levels of PROK-2 were positively associated with advanced bone disease and unfavourable cytogenetics.
Conclusion
Granulocytic impairment is present in MGUS and worsened in MM patients due to increased expression of genes that negatively regulate adaptive immune response. PROK-2 is a key molecule involved in the granulocyte dysfunction and could be involved in the progression from MGUS to MM.

Disclosures
Musto: Celgene: Honoraria; Janssen: Honoraria.
Targeting HMGB1 Increased the Sensitivity of MM Cells to Chemotherapy
Authors: Guo Xing , He Donghua , Chen Jing , Yang Li , Li Yi , Chen Qingxiao , He Jingsong , Yang Yang , Zhang Enfan , Wu Wenjun , Han Xiaoyan , Zheng Gaofeng , Zhao Yi , Huang He , Cai Zhen 
Published: October 2019

Abstract
Background ：Multiple myeloma (MM) is an malignant disease of clonal plasma cell hyperplasia, which incidence is increasing in recent years. Chemotherapy is the main method to treat MM, but because of drug resistance, MM is still an incrurable disease. High-mobility group box 1 protein (HMGB1) is a chromatin associated nuclear protein, which participated in DNA damage repair , rearrangement, and it also as an extracellular damage associated molecular pattern molecule(DAMP) involved in inflammation, neoplastic progression, angiogenesis, invasion and metastasiss and drug resistance of kinds of tumor cells. There’s no research about the role of HMGB1 in multiple myeloma before.
Methods: First, We detected HMGB1 mRNA and protein expression level in MM cell lines and primary MM cells by semiquantitative real time-polymerase chain (qRT-PCR)and western blot respectively. The concentration of HMGB1 in the culture supernatants of MM cell lines and primary MM cells was detected by ELISA. Second, we constructed Lentivirus vector with HMGB1 shRNA, infected the lentivirus to MM cells to knockdown HMGB1 expression. Then Chemo-drugs (ADM, quercetin and bortezomib) were added to MM cells, flow cytometry was used for the detection of cell apoptosis.
Results:MM cell line and primary MM cell all express HMGB1 with high level. The concentration of HMGB1 in the MM cell supernatants was significantly elevated after chemotherapy treatment compared with untreated cells(p<0.05), and HMGB1 in MM patients’ bone marrow supernatants was very high. Knockdown of HMGB1 increased 8226 and ARP-1 cell apoptosis induced by ADM and quercetin (NC shRNA VS HMGB1 shRNA, p<0.05), but there’s no significant difference induced by bortezomib.
Conclusions: Our study showed that the concentration of HMGB1 in the MM cell supernatants was significantly elevated after chemotherapy treatment. Further more, knockdown of HMGB1 by shRNA increased the sensitivity of MM cells to chemotherapy.

Disclosures
No relevant conflicts of interest to declare.
Statin Use and Its Effects on Skeletal Related Events in Patients with Multiple Myeloma
Authors: Vargas Madueno Fernando M , Benyounes Amin , King Gentry , Gary Huang Kuan-Hsiang , Varadi Gabor 
Published: October 2019

Abstract
Introduction: Skeletal related events (SRE) in multiple myeloma (MM) patients are associated with significant morbidity and mortality. Studies assessing the anti myeloma effect of statins in conjunction with chemotherapy have shown conflicting results with regards to overall survival or disease response. To our knowledge there have been no studies evaluating the effect of statins on SRE. Here we sought to assess the relationship between statin use and the presence of SRE.
Methods: We retrospectively reviewed 101 patients seen at our institution between the years 2007-2012 who had a diagnosis of MM separating them in 2 groups, patients on statin therapy (n=50) and those without statin therapy (n=51). Statin use was considered if present prior to the occurrence of a SRE. SRE were defined as pathologic fractures, necessity for orthopedic intervention, radiation therapy or spinal cord compression. MM stage as per the International Staging System (ISS), as well as history of osteoporosis, malignancy, hypothyroidism, Paget's disease of the bone, alcohol abuse, smoking status, calcium-vitamin D, and bisphosphonate use were also recorded.
Results: In our cohorts the prevalence of SRE was significantly lower in patients on statin medication when compared to statin naive patients, (36% vs. 58.8% respectively, p = 0.029, Fisher's exact test, Figure 1). No significant differences were noted between statin treated group and statin naive group in the following variable subgroups (Fisher's exact test): the history of smoking or alcohol abuse, the documented diagnosis of osteoporosis, coexistent malignancy, hypothyroidism, or Paget's disease of the bone, and the use of other medications including bisphosphonates or calcium - vitamin D supplementation. There were no significant differences in distribution of cancer staging (according to ISS) when we compared between statin use status (chi-square test for trend).
Conclusion: The use of statins was associated with a decrease in the prevalence of SRE in patients with MM. This was found independent of disease stage, history of osteoporosis or second malignancy, and bisphosphonate use.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Host Natural Killer T Cells Regulate Allogeneic Donor CD8+ T Cell Expression of Homing and Chemokine Receptors and Tissue Migration
Authors: Li Nainong , Lou Jingwei , Racine Jeremy , Forman Stephen J. , Zeng Defu 
Published: October 2019

Abstract
Natural killer T (NKT) cells recognize CD1d, and include invariant Jα18+ iNKT and non-invariant subpopulations. NKT prevent graft versus host disease (GVHD) via IL-4, but the mechanisms remain unclear. We have reported that donor CD8+ T cells are confined to host lympho-hematological tissues in anti-CD3-conditioned recipients, and the confinement is associated with presence of high percentage of residual host NKT. But it is unknown whether NKT can regulate T cell migration. In the current studies, using NKT deficient CD1d-/-, Rag-2-/-, and iNKT deficient Jα18-/- host-type mice, and by add-back of iNKT cells from wild-type or IL-4-/- host-type mice to in vivo transplantation and/or in vitro co-culture experiments, we observed that 1)host NKT down-regulate donor CD8+ T expression of α4β7, CCR5, CCR6, CXCR3 but upregulate expression of CCR3, as well as down-regulate GVHD target tissue expression of ccl3-5 and Cxcl9-11 chemokines in an IL-4 dependent manner. 2) iNKT and non-invariant NKT have redundant role in regulating donor T expression of chemokine receptors; but iNKT are required for down-regulating tissue release of chemokines. 3) iNKT down-regulate donor CD8+ T expression of α4β7 without reduction of IFN-g production. These indicate that NKT preventing GVHD can be via reducing donor T tissue migration capacity. (This work was supported by Nesvig Lymphoma foundation)

Disclosures
No relevant conflicts of interest to declare.
Mobilisation of PBSC for Allogenic Transplantation By the Use of G-CSF Biosimilar Zarzio® in Healthy Donors
Authors: Anguita Javier , Balsalobre Pascual , González-Arias Elena , Redondo Sara , Pérez-Corral Ana , Pascual Cristina , Serrano David , Kwon Mi , Bastos Mariana , MuÃ±oz Cristina , Gayoso Jorge , Diez-Martin Jose Luis 
Published: October 2019

Abstract
Introduction
The human recombinant filgrastim G-CSF has been widely used for mobilisation of CD34+ cells of healthy donors (HD) since 90´s. In 2008, G-CSF biosimilars were approved by the European Medicines Agency for the same indications as those authorized for original filgrastim (Neupogen®). These new drugs were authorized on the basis of extensive physicochemical and biological protein characterization and pharmacodynamics data as well as clinical comparative studies in the particular field of chemotherapy–induced neutropenia in patients with solid tumors. Since then, the reported experience on the use of G-CSF biosimilars for PBSC mobilisation has been very limited particularly, in the setting of HD mobilization for allogeneic transplant. We report our single center experience on the use of filgrastim biosimilar Zarzio® (Sandoz Biopharmaceuticals®) for mobilization of HD, compared with a cohort of donors mobilized with the original filgrastim.
Methods
Retrospective comparative analysis of all consecutive HD undergoing a 1st attempt of PBSC mobilisation using 10 mcg/Kg/24h x4 days G-CSF (Filgrastim biosimilar Zarzio® [case cohort] or Filgrastim, Neupogen® [control cohort]) and collected with the same cellular separator (Cobe Spectra, Terumo®). Apheresis was initiated on day 5 and was daily repeated, if needed, in order to achive at least 3.5 x106/Kg patient body weight (PBW). SAEs were also analysed in both cohorts. For the porpoise of the post-transplant engraftment analysis both cohorts were independently compared according to the grade of HLA compatibility (HLA identical vs Haploidentical). Volunteer HD were excluded from the engraftment analysis. Non-parametric test was used for 2 cohorts comparison.
Results
Forty five HD cases and 41 controls undergoing G-CSF mobilization between June-09 and April-14 were eligible for the analysis. Donor features were comparable for both cohorts (table 1). Doses of G-CSF required, pre-apheresis analytical parameters, leukapheresis procedures, processed volume and collected graft parameters were found similar in both groups (table 1). All donors collected more than 2 x106/Kg CD34+ cells. Cummulative incidence of engraftment at day 30 was 100% for both cohorts, the HLA identical setting (p=0.53) and the haplo sub-group (p=0.25). AES were not observed for both cohorts.
Conclusions
In our experience, efficacy of G-CSF biosimilar Zarzio® was similar to reference filgrastim (Neupogen®) in terms of dose administered, CD34+ cells collected, stem cell functionality and engraftment. No SAEs were found for both cohorts. These preliminary results should be confirmed with a longer follow up within a prospective randomized trial.
Figure 1 Figure 1.
Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.
Multiplex PCR for Virus Infections after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Inazawa Natsuko , Hori Tsukasa , Yamamoto Masaki , Igarashi Keita , Tsutsumi Hiroyuki , Nojima Masanori , Suzuki Nobuhiro , Shimizu Norio , Hatakeyama Naoki 
Published: October 2019

Abstract

Background: Viral infections following hematopoietic stem cell transplantation (HSCT) are thought to occur due to breakdown of both cell-mediated and humoral immunity. We used a multiplex polymerase chain reaction (PCR) method to detect 13 different viral infections from before to six weeks after transplantation.
Methods: One hundred and five patients who underwent allogeneic HSCT between September 2010 and December 2012 were included. Weekly blood samples were collected from prior to transplantation to 42 days after transplantation. We used whole blood for comprehensive analysis for the following 13 viruses; herpes simplex virus 1 (HSV-1); HSV-2 varicella-zoster virus (VZV); Epstein-Barr virus (EBV); cytomegalovirus (CMV); human herpesvirus 6 (HHV-6); HHV-7; HHV-8; Adeno virus (ADV); BK virus (BKV); JC virus (JCV); parvovirus; and hepatitis B virus (HBV).
Results: Viruses were detected in 75 (71.4%) patients. Before transplantation, four types of viruses were detected in 12 patients. The detection rate gradually increased following transplantation; the number of patients having at least one type of virus peaked at 45 (45.5%) on post-transplant day 21. The most frequently detected virus during the HSCT courses was HHV6 (n=63; 60.0%), followed by EBV and CMV (n=11 each; 10.5%). No other viruses were detected. Viral infections were significantly more common among patients undergoing CBT, those with HLA mismatched HSCT, those treated with either steroids, and those with GVHD. EBV infection tended to be more common in children than in adults. No sex- or pretreatment-related differences were observed in the viral infection rate.
Conclusion: These results suggest that in the presence of these factors, we should pay attention to the increased risk of viral infection.

Disclosures
No relevant conflicts of interest to declare.