bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

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

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

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

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.
Bone Health Assesment in Patients Undergoing Hematopoietic Cell Transplantation
Authors: Ramirez Pablo , Florenzano Pablo , Campusano Claudia , Jara Veronica , Bertin Pablo A , Ernst Daniel M
Published: October 2019

INTRODUCTION: The increased survival of hematological patients has required to widen the care of these patients, with emphasis in factors related to quality of life and late mortality. Among them, osteoporosis (OP) is a fundamental problem. Patients undergoing hematopoietic cell transplantation (HCT) are at great risk of OP, mainly due to prolonged exposure to chemotherapy, immunosuppresants and the hypogonadism frequently associated to these treatments. Despite this, there is lack of strong evidence on this matter and HCT guidelines are not clear on this problem.

OBJECTIVES: To evaluate parameters related to bone health in patients undergoing HCT. To measure the frequency and severity of the alterations in this group of patients.

PATIENTS AND METHODS: Observational and retrospective analysis of patients undergoing HCT at the Catholic University Hematology-Oncology Department. All patients undergoing HCT were recommended to have determination of 25-OH vitamin D, PTHi, Calcium, Phosphorus and bone densitometry (DXA). We included all patients with the evaluation before HCT (preHCT) and 1 year after HCT (postHCT).

RESULTS: We obtained data from 20 patients preHCT and 22 patients postHCT. Main diagnosis were acute myeloid leukemia (n=13; 32%), multiple myeloma (n=11; 26%), acute lymphoblastic leukemia (n=9; 21%) and Hodgkin's Lymphoma (n=5; 12%). Mean age was 40 years (range: 17-67) and 67% were males. In the preHCT group, the median 25-OH vitamin D levels were 13,6 ng/ml (range: 4,9-26,7 ng/ml) and 100% of the patients were in the insuficiency range leveles (<30 ng/ml). Median PTHi levels were 62,6 pg/ml (range: 24,4-223,7 pg/ml), and 46% of the patients had secondary hyperparathyroidism. In the postHCT group, median 25-OH vitamina D levels were 11,4 ng/ml (range: 4-29,4 ng/ml) and 100% had insuficiency levels. Median PTHi levels were 68 pg/ml (range: 37,4-135,8 pg/ml), and 56% of the patients had secondary hyperparathyroidism. 12 patients had DXA before HCT and 3 (25%) of them had DXA lumbar Z score less than -2.0. In 19 patients postHCT, 4 (21%) had decreased DXA Z score < 2.0. No difference could be established between autologus or allogeneic HCT in any meassurement.

CONCLUSIONS: HCT patients represent a high risk group of developing severe vitamin D deficiency, secondary hyperparathyrodism and decreased DXA levels. These data are a warning that this population of patients requiere early intervention to prevent long term complications. This report is the initial evaluation for the development and treatment of bone health in a prospective matter in HCT patients in our center.

Disclosures

No relevant conflicts of interest to declare.
Hematopoietic Stem Cell Transplantation for Patients with β-Thalassemia Major Underwent Pre-Transplant Splenectomy
Authors: He Yuelin , Wu Xuedong , Ruan Yongsheng , Peng Zhiyong , Feng Xiaoqin , Liao Jianyun , Liu Huaying , Xu Wenfeng , Li Na , Li Chunfu
Published: October 2019

Current study analyses the effect of splenectomy on outcomes of hematopoietic stem cell transplantation (HSCT) for patients with β-thalassemia major (TM). Twenty-two class II and III (according to Nanfang’s criteria, see Blood, 2012;120 (19): 3875-3881) patients with TM had a pre-transplant splenectomy. The outcomes of the 22 transplants were compared with 193 transplants in class II and III patients between Aug. 2008 and Dec. 2013. Patients in the splenectomy group were older (8.9±5.0 vs. 6.0±3.5 year old; p=0.001) and class III patients were more (8/22 vs. 10/193, p=0.001) than non-splenectomy. In splenectomy group 10 patients received graft from HLA matched sibling, 9 from unrelated donors and 3 from haploidentical parents plus unrelated cord blood, respectively. In non-splenectomy group 82 patients received graft from matched sibling and 121 from unrelated donors, respectively.

213 cases engrafted in 4 weeks, Splenectomized patients had a significantly shorter time to absolute neutrophil count >500/mm3 (14.5±5.2 vs 19±4.2 days p=0.001). There were no significantly different at time to platelet >2000/mm3 (16.2±5.8 vs 16.1±5.3 days, p=0.415) and hemoglobin >80g/L (15.3±4.8 vs 13.1±5.3 days p=0.135). Secondary rejection occurred in 2 patients (at 3rd month and 4th month post transplantation, respectively) in splenectomy group, but rescued successfully by second transplants from unrelated donor one year later and from haploidentical parent two years later, respectively. The patients received graft from father, one died of complications of VOD; other died of lung infection on forty-five days post transplantation. Two patients rejected their grafts in non-splenectomy group.

The mean RBC transfusion was 4u (0u-12u). The mean platelet transfusion in splenectomy group had reduced (4.4±4.5 vs 7.1±4.8 units p=0.016), with 17 patients transfused less than 6u Platelet. The incidence of acute and chronic GVHD, late infections were rare and not significantly different between the two groups. No patients died of infection due to splenectomy. Patients underwent HSCT after splenectomy had a high rate of OS (95.5% vs 92.8% p=0.001). Although TFS was lower in splenectomy group than non-splenectomy group (88.5% vs 92.3% p=0.001), this may be due to more older, class III patients and higher rate of rejection (2/22 vs 2/193).

Conclusion, our study showed pre-transplant splenectomy patients with β-thalassemia major was associated with faster engraftment and decreased require of platelet and bloodtransfusion support.

Disclosures

No relevant conflicts of interest to declare.
Encouraging Outcomes of Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation for Pediatric Patients with Acute Lymphoblastic Leukemia in Second Complete Remission
Authors: Higuchi Kohei , Sato Maho , Kondo Osamu , Ioi Aya , Mayumi Azusa , Shimizu Mariko , Sawada Akihisa , Yasui Masahiro , Inoue Masami
Published: October 2019

[Background] We have been performing reduced-intensity stem cell transplantation (RIST) to avoid preconditioning-related complications. However, the effectiveness of RIST in pediatric patients with acute lymphoblastic leukemia (ALL) remains to be clarified.

[Methods] We retrospectively reviewed 37 pediatric patients with ALL in second complete remission (CR2) who underwent first allogeneic hematopoietic stem cell transplantation (allo-SCT) between 1993 and 2012 in our institute. We compared the outcomes of RIST with those of myeloablative stem cell transplantation (MAST).

[Results] The median age at allo-SCT was 9 years (range, 1 to 18 years). There were 33 B-lineage ALL, 3 T-lineage ALL, 1 lineage unknown ALL, and none of Philadelphia chromosome-positive ALL. Sixteen patients received HLA-matched bone marrow (7 related; 9 unrelated), 12 HLA-mismatched bone marrow (11 unrelated; 1 HLA haploidentical related), 4 cord blood, and 5 CD34 positive peripheral blood stem cells (HLA haploidentical related). In all patients, the 5-year overall survival (5y-OS) rate and the 5-year event free survival (5y-EFS) rate were 75.1% and 56.5%, respectively.

Seven patients underwent RIST and 30 patients underwent MAST. The median follow-up durations of RIST and MAST groups were 3.3 years (range, 0.9 to 8.2 years) and 11.3 years (range, 0 to 21.2 years), respectively. The 5y-OS rates in RIST and MAST groups were 85.7% and 59.8%, and the 5y-EFS rates were 71.4% and 53.3%, respectively. The 5-year cumulative transplant-related mortality (TRM) rates in RIST and MAST groups were 0% and 31.0%, and the 5-year cumulative relapse rates were 28.6% and 24.3%, respectively.

[Discussion] In our series, the cumulative relapse rate in RIST group was similar with that in MAST group, and the cumulative TRM rate in RIST group was lower than that of MAST group. Therefore, both of the 5y-OS and the 5y-EFS rates in RIST group seem to be better than those in MAST group. The outcomes of RIST in our series do not seem to be poorer. Although further studies are needed because of the small size of patients and short follow-up duration, RIST can be considered as the first transplantation for pediatric patients with ALL in CR2.

Figure 1 Figure 1.

Figure 2 Figure 2.

Figure 3 Figure 3.

Disclosures

No relevant conflicts of interest to declare.
Study in the Effect of EZH2 Gene Targeting siRNA on Cell Death in HL-60 Cell Line
Authors: Luhui Lin , Huang Yiqun , Ma Xudong
Published: October 2019

Objective: To study small interfering RNA (siRNA) targeting EZH2 gene effect on cell proliferation, apoptosis and histone modulation in HL-60 cells line.

Methods: (1) Three siRNA segments targeting EZH2 gene were designed and transfected into HL-60 cells by LipofectamineTM 2000. The optimal segment was screened by RT-PCR. (2) Cell growth inhibited by EZH2 siRNA was determined by WST-1. Cell apoptosis was measured by Flow cytometry. (3) The expression of procaspase-3, procaspase-9, Bcl-2, and Bax was detected by Western blot. The levels of histone H3 lysine27 dimethylation and trimethylation and histone acetylatiom of H3 were evaluated by Western blot.

Results: (1) The efficiency of transfection with EZH2 siRNA for 6 hours was (82 ±2.97)%. The sequence of optimal segment of EZH2 siRNA was sense 5'- GAGGGAAAGUGUAUGAUAATT -3'，antisense 5'- UUAUCAUACACUUUC CCUCTT -3'。 (2) EZH2 siRNA suppressed the proliferation of HL-60 cells. (3) EZH2 siRNA downregulates the expression of procaspase-3, procaspase-9, Bcl-2 and upregulates the expression of Bax and induced cells apoptosis. (4) EZH2 siRNA suppressed the protein and mRNA of EZH2 in HL-60 cells. The levels of histone H3 lysine27 dimethylation and trimethylation was down regulated and histone acetylatiom of H3 was up regulated.

Conclusions: (1) LipofectamineTM2000 transfected siRNA for EZH2 gene into HL-60 cells has high efficiency. (2) EZH2 siRNA inhibits cell growth and induces cell apoptosis in HL-60 cell line. It might be a new therapeutic target in human leukemia. (3) EZH2 siRNA downregulates the levels of histone H3 lysine27 dimethylation and trimethylation and upregulates histone acetylation of H3. The mechanism of EZH2 effect on histone acetylation need to be further studied. RNAi technology is expected to become a new tool for targeted gene therapy of acute leukemia, and EZH2 might be one of the targets of gene therapy for leukemia.

Disclosures

No relevant conflicts of interest to declare.
Pathway Phenotyping of B-Cell Receptor Activation and Inhibition: Translation to Novel Clinical Biomarkers
Authors: Wehrman Tom S
Published: October 2019

B-cell receptor (BCR) signaling has emerged as a vital and targetable component in a number of B-cell malignancies. The recent approval and numerous ongoing clinical trials of small molecule kinase inhibitors targeting this pathway has bolstered efforts to define the signaling proteins critical to BCR activity. To this end, we first mapped BCR signaling using 93 different activation state antibodies. These results provided a high level view of BCR signaling across more than 80 intracellular proteins covering canonical and non-canonical BCR effectors. The signaling responses were then modulated with known kinase inhibitors to associate kinase activity with pathway activation. Using these data as a backdrop, three BTK inhibitors (AVL-292, CNX-774, and ibrutinib) were tested to determine which components of BCR signaling are commonly inhibited by these molecules. These studies showed that ibrutinib was the most promiscuous of the inhibitors but that all three compounds blocked activation of the S6 signal transduction pathway. Interestingly, a number of other oncogenic pathways remained intact after inhibitor treatment including the MAPK-ERK and AKT signaling pathways. After identifying the S6 pathway as a commonly blocked effector of BCR signaling, we validated the pathway readouts for their use as ex vivo pharmacodynamic assays in whole blood patient samples. Our results show that the assays yield the appropriate precision and reproducibility necessary to be used as PD markers in a clinical trial setting. Thus, the workflow of pathway phenotyping to inhibitor testing and finally to clinical biomarker development further defined the activation patterns of BCR signaling and identified novel clinical readouts for measuring BCR inhibitor activity.

Disclosures

Wehrman: Primity Bio: Employment.
MiR-146a Regulates the TRAF6/TNF-Axis in Donor T Cells during Graft-Versus-Host Disease
Authors: Stickel Natalie , Prinz Gabriele , Pfeifer Dietmar , Schmitt-Graeff Annette , Follo Marie , Finke Jürgen , Duyster Justus , Salzer Ulrich , Zeiser Robert
Published: October 2019

Introduction: Acute graft-versus-host disease (GvHD) arises from the attack of recipient tissues by donor allogeneic T cells and represents one of the major limitations of allogeneic hematopoietic cell transplantation (allo-HCT). In spite of many clinical trials, the standard immunosuppressive regimens for prevention of acute GvHD have improved little in the last two decades. Hence, a better understanding of the biology of acute GvHD may improve therapeutic options. MicroRNA-146a (miR-146a) was found to be increased in the sera of patients with GvHD. Therefore, we aimed to decipher the role of miR-146a in allogeneic donor T cells during GvHD by functional studies and in patients undergoing allo-HCT by single nucleotide polymorphism (SNP) analysis.

Methods: We used two different murine major MHC mismatch models for acute GvHD. Recipient mice were conditioned with irradiation before transplantation of bone marrow and either wildtype or miR-146a deficient T cells from allogeneic donor mice. Furthermore, genomic DNA from 289 patients that underwent allo-HCT and their respective hematopoietic stem cell donors was isolated in order to determine their miR-146a rs2910164genotype.

Results: We observed miR-146a upregulation in T cells of mice developing acute GvHD compared to untreated mice in a major MHC and a minor histocompatibility antigen mismatch model. Transfer of miR-146a deficient T cells caused increased GvHD severity, elevated TNF serum levels and reduced survival. Conversely, the phytochemical induction of miR-146a or its overexpression in donor T cells using a specific miR-146a mimic reduced GvHD severity. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was upregulated in miR-146a-/- T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased NF-κB activity and TNF production in miR-146a-/- T cells, while other pro-inflammatory cytokine levels were unaffected. The detrimental effect of miR-146a deficiency in T cells could be antagonized by TNF blockade in vivo. Moreover, in contrast to WT T cells, over expression of miR-146a in Tnf deficient T cells had no effect on their alloreactivity. In the human system, the minor genotype of the SNP rs2910164, which causes reduced miR-146a expression, was more frequent in patients developing acute GvHD grade III/IV compared to all other allo-HCT recipients (n=289).

Conclusions: Taken together we show that miR-146a functions as a negative regulator of the TRAF6/TNF-axis in allogeneic donor T cells during GvHD, leading to reduced TNF transcription. Given our observation on the predictive role of the SNP leading to decreased miR-146a expression in acute GvHD in patients and the possibility to exogenously enhance miR-146a expression, we provide a novel and targeted molecular approach to mitigate GvHD.

Disclosures

No relevant conflicts of interest to declare.
Regulation of Immune Responses during Acute GvHD Via the IL-33/ST2 Axis
Authors: Reichenbach Dawn K , Schwarze Vincent , Matta Benjamin M , Tkachev Victor , Lieberknecht Elizabeth , Liu Quan , Koehn Brent H , Pfeifer Dietmar , Taylor Patricia A , Prinz Gabriele , Dierbach Heide , Stickel Natalie , Beck Yvonne , Warncke Max , Junt Tobias , Schmitt-Graeff Annette , Nakae Susumu , Follo Marie , Wertheimer Tobias , Schwab Lukas , Devlin Jason , Watkins Simon C , Duyster Justus , Ferrara James L.M. , Turnquist Heth R. , Zeiser Robert , Blazar Bruce R.
Published: October 2019

The IL-1 superfamily member IL-33 is produced in barrier tissues. IL-33 binds to the receptor suppression of tumorigenicity 2 (ST2), expressed on stromal cells, regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), and macrophages. IL-33 has both anti-inflammatory and pro-inflammatory properties. It is not known if IL-33 plays a role in acute GvHD, and if so what properties it exerts. By immunohistochemistry staining of gut tissues, IL-33 production by non-hematopoietic cells was increased in mice post-conditioning and in patients during GvHD. To determine whether IL-33 could augment GvHD via a host signaling mechanism, we compared st2-/-to wildtype (wt) hosts and observed decreased GvHD lethality (Figure 1A). Additionally, IL-33-/- versus wt hosts had a marked decrease in GvHD lethality and reduced TNFα production. Conversely, IL-33 administration during the peak inflammatory response worsened GvHD. Previous studies have shown increased levels of the soluble form of ST2 (sST2) are a biomarker for steroid-refractory GvHD (Vander Lugt, NEJM, 2013). We hypothesized that sST2 acted not only as an indicator of tissue injury and biomarker of GvHD but also as an immune modulator during GvHD. In rodents, we found that ST2 was upregulated on alloreactive T cells and sST2 increased as GvHD progressed. St2-/-versus wt donor T cells had a marked reduction in GvHD lethality (Figure 1B) without compromise of graft-vs-leukemia responses. Comparable data was seen in 2 different strain combinations. Alloantigen-induced IL-18 receptor upregulation was significantly lower in the absence of ST2, which was linked to significantly reduced IFNγ production by st2-/- vs wt CD4 and CD8 T cells during GvHD. Similarly, sST2 transgenic hosts and wt recipients given exogenous sST2-Fc fusion protein infusions (Figure 1C) to block ST2/IL-33 interaction each had significantly reduced GVHD lethality, establishing the functional role of ST2 as a decoy receptor modulating GVHD. During the peak of the GvHD inflammatory response, IL-33 signalling of either donor or host cells promoted activation of donor T cells, while the use of exogenous sST2-Fc protein to prevent IL33/ST2 engagement ameliorates disease. Together, these studies point to targeting of the IL-33/ST2 axis as a novel and potent target for GvHD therapy.

Disclosures

Warncke: Novartis Pharma AG: Employment. Junt:Novartis Pharma AG: Employment.
Polycomb Group Member Rybp Is a Functional Tumor Suppressor Repressed By Mir-9 in MLL-Rearranged AML
Authors: Price Colles , Chen Ping , Li Shenglai , Li Zejuan , Li Yuanyuan , Jiang Xi , Huang Hao , Weng Hengyou , Ulrich Bryan , Arnovitz Stephen , Neilly Mary Beth , Chen Jianjun
Published: October 2019

MicroRNAs (miRNAs), are small non-coding RNA molecules known to be important regulators of cancer biology. Notably, we and others have shown that miRNAs play important roles in Acute Myeloid Leukemia (AML), a heterogeneous malignancies with multiple chromosomal and molecular abnormalities. Patients with chromosomal rearrangements involving mixed lineage leukemia (MLL), the mammalian homology of trithorax gene, are associated with poor survival. Previously, we have found that MLL-rearranged AML drives aberrant expression of several miRNAs, most notably microRNA-9 (miR-9). Expression of miR-9 with MLL-AF9, a common MLL-translocation, was sufficient to promote transformation normal hematopoietic progenitor cells in vitro and leukemogenesis in vivo. We previously found that miR-9 reduces expression of several genes but we did not know which genes were critical tumor suppressors. We found that the polycomb group member RING1- and YY1-Bindin Protein (RYBP) was consistently inhibited upon miR-9 expression. To assess the regulation of RYBP we used publically available data from the Cancer Genome Atlas (TCGA) and looked at genome-wide Illumina 450K methylation data. We did not find a strong correlation with methylation and RYBP expression, suggesting that expression of RYBP is likely not regulated by the DNA methylation machinery in patients. Upon looking at copy number alterations we found that a small population of AML patients contained either homozygous or heterozygous loss of RYBP, suggesting a potential role of RYBP in leukemia pathogenesis. To assess the role of RYBP we did a series of in vitro experiments. We found that expression of RYBP was sufficient to attenuate colony-forming growth driven by MLL- AF9. Furthermore, RYBP expression was able to reduce proliferation, increase apoptosis, and significantly reduce immature cell population. To determine the role of RYBP expression in vivo, we transplanted lethally irradiated mice with progenitors retrovirally transduced with MLL-AF9 compared to MLL-AF9 and RYBP. We found that expression of RYBP was sufficient to reduce leukemia burden in vivo as well as induce differentiation as shown by flow cytometry and histological analysis. Thus, this demonstrates that RYBP is a functional tumor suppressor in MLL-rearranged AML. In conclusion, we have demonstrated that chromosomal rearrangements involving MLL, the mammalian homology of trithorax, downregulates a member of the polycomb complex through upregulation of miR-9.

Disclosures

No relevant conflicts of interest to declare.
Determinants of Low Bone Mineral Density after Childhood Cancer
Authors: den Hoed Marissa , Klap Bibi C. , Te Winkel Mariël L. , Stolk Lisette , Pieters Rob , van Waas Marjolein , Neggers Sebastian J.C.M.M. , Blijdorp Karin , van Dorp Wendy , Boot A.M. , Pluijm S.M.F. , van den Heuvel-Eibrink Marry M.
Published: October 2019

Purpose: Osteopenia is a potential complication of childhood cancer treatment, but the magnitude of this problem in survivors is unknown. We examined (determinants of) bone mineral density (BMD) status in long-term survivors of adult childhood cancer (CCS).

Methods: This retrospective single-center cohort study included 346 subjects with the most common types of childhood cancer. Acute leukemia or lymphoma was diagnosed in 273 (81%) of the patients. All subjects had a median age at diagnosis of 7.0 years (range: 0.1-16.8 years) and a median follow-up time of 16.7 years (range 5.6-39.9 years). Total body BMD (BMDTB) and BMD of the lumbar spine (BMDLS) were measured by dual-X-ray absorptiometry. Osteopenia was defined as BMD standardized deviation score (SDS) below -1. Twelve candidate single nucleotide polymorphisms (SNPs) in 11 genes (COL1A1, TNFSF11, TNFRSF11, TNRFSA11B, VDR, ESR1,WLS, LRP5, MTHFR, MTRR, IL6) were investigated.

Results: Survivors had a lower BMDTB and BMDLS (mean SDS: -0.55, p<0.001; and -0.30, p<0.001, respectively) as compared to healthy peers. This was similar in cancer survivors of leukemia and lymphoma (mean SDS: -0.49, p<0.001; and -0.32, p<0.001, respectively). Osteopenia (BMDTB and/or BMDLS) was present in 45% of the survivors (46% of the leukemia and lymphoma survivors). Multivariate logistic regression analyses identified age at diagnosis <12 years, age >30 years at follow-up, male gender, underweight at follow-up, carriers of the minor allele of rs2504063 (LRP5), treatment with cranial-spinal radiotherapy and prednisone as independent prognostic factors for osteopenia. In survivors of leukemia and lymphoma, we identified low BMI at follow-up, carriers the minor allele of rs2504063 (LRP5), treatment with cranial-spinal radiotherapy and cyclophosphamide use as prognostic factors for osteopenia.

Conclusions: This large cohort of childhood cancer survivors with the long follow-up identified osteopenia in 45% of CCS. This indicates that greater awareness for low BMD is warranted, especially in survivors who are older than 30 years, male, have underweight, have a genetic predisposition, and who were treated with cranial-spinal radiotherapy and/or steroids.

Disclosures

No relevant conflicts of interest to declare.
Acute Megakaryoblastic Leukemia in Childhood: Comparison Between Patients with and without Down Syndrome
Authors: Felice Maria Sara , Rossi Jorge Gabriel , Gallego Marta , Alonso Cristina N. , Eandi Eberle Silvia , Guitter Myriam Ruth , Rubio Patricia L. , Alfaro Elizabeth M , Sanchez La Rosa Cristian , Zubizarreta Pedro
Published: October 2019

Introduction: Acute Megakaryoblastic Leukemia (AMKL) is an unusual AML subtype in childhood, being the most frequent AML in Down syndrome (DS) patients. AMKL corresponds to 19.3% of AML in our Hospital and some special characteristics have been observed.

Objective: To analyze characteristics of AMKL and to compare patients with or without DS.

Patients and Methods:From January-1990 to June-2014 AMKL was diagnosed in 77 cases: 57 in non-DS and 20 in DS children. Fivecases were secondary leukemias and 3 cases had been previouslytreated with chemotherapy due to wrong diagnosis. One patient was on induction at the time of analysis. These 9 cases were not evaluable for follow-up. Forty-eight non-DS and 20 DS caseswere evaluable. Megakayocyte lineage commitment was confirmed in all cases by fluorescence microscopy. MPO was analyzedboth by flow cytometry and cytochemistry. G-banding and RT-PCR were performed according to standard techniques. Four successive BFM-based protocols were administeredwith schedule modification for DS patients.

Results: Mean age was:2 (range: 2 mo-13 y) years of age and mean WBC count was: 27,500 (range: 1,600-250,000)/mm3. No differences were observed in age or hematological findings at diagnosis. Extramedullar compromise was detected in 10 (17.5%) of non-SD cases. Immunophenotype defined ambiguous lineage leukemia in 3 (5.0%) of the non-DS cases, with compromise of T-lineage. Interestingly, in 4 cases the megakaryoblasts also co-expressed MPO.Cytogenetic findings showed hyperdiploidy (n=7), t(1;22)(p13;q23) (n=3), t(16;21)/FUS-ERG (n=2), abnormalities in chromosome 7 (n=5) and complex karyotype (n=9). Complete remission (CR) was achieved in 51 (33 non-DS and 18 DS) (75.0%) of 68 evaluable cases;null-responses observed in7(only non-DS, 10.3%). Early deaths occurred in 10 (14.7%) during induction phase, 2 of them before treatment onset. From the 51 patients who achieved CR, 16 (14 non-DS and 2 DS) presented relapses(p: 0.04) and 9 (6 in non-DS and 3 in DS) died in CR. EFS-probability for the total population was 34 (6)%, for non-DS 21 (6)% and for DS 63 (11)%(p-value: 0.0026). LFS-probability was 45 (7)% for the total population, for non-DS 29 (8)% and for DS 70 (11)% (p-value: 0.0117). Median follow-up: 148 (range: 2-194) months.

Conclusions:

1- Extramedullar compromise and T/myeloid mixed lineage or co-expression of MPO were observed only in non-DS AMKL, in 17.5% and 12.0% respectively.

2- Null-response was observed only in non-SD patients.

3- The most frequent event of non-DS was relapse, and toxic deaths in DS.

4- The significantly better outcome of patients with DS was confirmed in our setting.

Disclosures

No relevant conflicts of interest to declare.