bluuebunny/crossref_metadata_2025 huggingface dataset:
Telomere Elongation in Dyskeratosis Congenita Induced Pluripotent Stem Cells.
Authors: Agarwal Suneet , Loh Yuin-Han , McLoughlin Erin M , Huang Junjiu , Park In-Hyun , Miller Justine D , Huo Hongguang , Okuka Maja , Reis Rosana Maria dos , Loewer Sabine , Keefe David , Goldman Frederick D , Klingelhutz Aloysius , Liu Lin , Daley George Q.
Published: October 2019

Abstract 497

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer premature degeneration of multiple tissues. Bone marrow failure is the principal cause of mortality, and allogeneic stem cell transplantation is limited by increased treatment-related mortality. Somatic cells can be reprogrammed using defined genetic and chemical factors, yielding “induced pluripotent stem” (iPS) cell lines which have the capacity to differentiate into any tissue. Patient-specific iPS cells therefore hold promise as therapeutic agents and disease models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, and we have found that telomere length is increased in human iPS cells relative to the normal primary somatic cells from which they are derived. Here we investigated whether defects in telomerase function would limit derivation or self-renewal of iPS cells from patients with DC. We reprogrammed primary fibroblasts from patients with X-linked and autosomal dominant DC, caused by mutations in the genes encoding dyskerin and telomerase RNA component (TERC), respectively. We were able to establish multiple DC-specific iPS lines showing all hallmarks of pluripotency, including the formation of hematopoietic progenitors in vitro. Unexpectedly, DC-specific iPS cells were able to sustain continual proliferation in vitro, in contrast to the premature senescence displayed by the DC fibroblasts. Although early passage DC iPS cells had shorter telomeres than donor fibroblasts, we found that telomere length in DC iPS cells increased with continued passage in culture. To explain this finding, we discovered that steady state levels of TERC, which are critically limiting in several forms of DC, are upregulated in normal and DC iPS cells. We found that TERC upregulation is a feature of the pluripotent state, that the TERC locus is a target of pluripotency-associated transcription factors, and that transcriptional silencing accompanies a 3' deletion at the TERC locus in autosomal dominant DC. Our results demonstrate that reprogramming restores self-renewal capacity in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to enhance endogenous TERC expression may be feasible and therapeutically beneficial in DC patients. The studies demonstrate the value of patient-specific iPS cells for basic and translational discovery, and further the rationale for autologous iPS based cellular therapy of genetic hematologic disorders.

Disclosures:

Daley: MPM Capital: Consultancy; Solasia: Consultancy; Epizyme: Consultancy; iPierian: Consultancy, Equity Ownership.
Is JAK2 Inducible by Cytotoxic Chemotherapy?
Authors: Bendaly Edmond A. , Rahman Saud S. , Zafar Samiah , Haglof Karen , Ibrahim Sherif , Raphael Bruce G.
Published: October 2019

Abstract 4979

Introduction

The JAK2V617F mutation accounts for most cases of myeloproliferative neoplasms (MPN). Only a few case reports of MPN following cytotoxic chemotherapy have been reported, and all of them were published prior to the discovery of the JAK2V617F mutation. We report a series of 6 patients who developed a JAK2V617F positive MPN following cytotoxic chemotherapy.

Patients

From 2006 to 2009, 6 patients with a history of a hematologic or an oncologic malignancy who developed an MPN were identified and their medical records retrospectively reviewed. One patient had acute lymphoblastic leukemia, 1 had Hodgkin lymphoma, 1 had squamous cell carcinoma of the head and neck, 1 had cervical cancer, and 2 had breast cancer. All patients were in remission from their primary malignancies at the time the MPN was diagnosed. Five were females. The median age at diagnosis was 72 years. Median time to development of the myeloproliferative neoplasm was 14 years. Type of chemotherapy exposure, MPN diagnosis and time to MPN in each case is shown in the table below. The JAK2V617F mutation was detected either in the peripheral blood or the bone marrow of all patients. There was no predominance of any specific MPN diagnosis. Patients who received platinum-based chemotherapy developed the MPN sooner than those who received alkylators (6 vs 17.5 years respectively). Treatment consisted of phlebotomy, hydroxyurea, anagrelide, aspirin or a combination as deemed appropriate by the treating hematologist.

Conclusion

These findings lead us to hypothesize whether the development of JAK2V617F positive MPN may be related to prior exposure to cytotoxic chemotherapy. Exposure to platinum-based chemotherapy may cause the disorder to appear sooner compared with exposure to alkylators. Recently, JAK2V617F positive MPN was found to be strongly associated with a specific constitutional haplotype, 46/11 suggesting increased susceptibility to this mutation. Chromosomal analyses are planned to show whether any of the reported patients exhibit this haplotype.

Ref: 1.Jones et al, Nat Genet. 2009 Apr;41(4):446-9. 2009 Mar 15.

The authors have no relevant disclosure.

Disclosures

No relevant conflicts of interest to declare.
Jak2 Mutation in Myeloproliferative Neoplasms: Is It Just An Exon Episode?.
Authors: Sun Xuemei , Xu Zuqiong , Lai Rengsheng , Zhang Wenxi , Zhu Xuejun , Jiang Pengjun , Xie Ling
Published: October 2019

Abstract 4982

The discovery of the V617F point mutation of JAK2 has revolutionized the understanding of the pathogenesis of BCR-ABL-negative myeloproliferative neoplasms (MPNs). Further discoveries of other JAK2 exon 12-15 and MPL mutations have extended the scope of the research in this field. However, a very small proportion of patients with polycythemia vera (PV) and 50% of patients with essential thrombocythemia (ET), as well as primary myelofibrosis (PMF), are negative for JAK2 or MPL mutations. This indicates that other forms of the mutations may exist and needs further evaluation

Recently, we treated a patient with ET who did not carry JAK2 V617F or JAK2 exon 12 mutations. The genomic DNA of mononuclear cells from heparin-treated bone marrow as well as he gastric tissue from paraffin embedding samples obtained from the patient was extracted using a DNA extraction kit, according to the manufacturer's instruction (Promega, Madison, WI, USA), and stored at -20°C. For mutation analysis of the JAK2 exon 12 and exon 14, genomic DNA was amplified respectively by polymerase chain reaction. And the PCR products either 358bp or 308bp were generated by forward primers within the exon 12 or exon 14 and forward primers within the intron 12 or intron 14(primer sequences and PCR conditions are provided on request). The direct sequencing on ABI-3100-Avant was used for mutation detection.

No mutation was found within JAK2 exon 12 or exon 14. However, a C/T mutation was detected in intron 14 at the position 14 downstream of the exon 14 in bone marrow cells. Informatively, this mutation was not found in genomic DNA of the gastric tissue. Further more, this novel mutation was not found in all samples from 59 MPN patients in our institute, among which 23 were diagnosed with PV, 22 ET, 12 PMF and 2 MPN-U. No such mutation was demonstrated in bone marrow mononuclear cells from 10 normal subjects. Preliminary results by flow cytometric analysis showed that hematopoietic cells including granulocytes, erythrocytes and megakaryocytes all showed elevated tyrosine kinase activity.

We concluded that mutations in JAK2 mRNA splicing region might also contribute to the aberrant signal transduction in hematopoiesis. And direct sequencing for JAK2 whole genomic DNA might be a suitable method for finding novel JAK2 novel mutation in JAK2 negative patients. Further functional study for evaluation of the effect of nucleotide changes within mRNA splicing sequences in wild type and mutated JAK2 cell lines as well as animal models would provide more convincing evidence of the relationship between JAK2 intron mutation and MPNs..

Disclosures

No relevant conflicts of interest to declare.
Progression Free Survival Is Predicated by Categorical Response, and Supports the Attainment of VGPR/CR as An Objective for Long Term Disease Control in Waldenstrom's Macroglobulinemia.
Authors: Treon Steven , Ioakimidis Thea , Hanzis Christina , Manning Robert , Patterson Christopher J , Hunter Zachary , Sheehy Patricia , Turnbull Barry
Published: October 2019

Abstract 4993

The attainment of higher response rates, as well as better categorical responses has occurred with the introduction of novel biological agents, including rituximab in the treatment of WM. However, there is a paucity of knowledge as to whether the attainment of better categorical responses is associated with improved outcomes. We therefore analyzed the impact of categorical responses on progression free survival (PFS) in 159 WM patients naïve to rituximab therapy and who received a rituximab based regimen: cyclophosphamide (n=58), nucleoside analogue (n=43), immunomodulatory agent (n=35), or bortezomib (n=23). The median follow-up for all patients was 83.5 months. Baseline characteristics were as follows: median age 62 years; serum IgM 3670 mg/dL; hematocrit 31.7%. Eighty-two patients had a B2M ≥3.0 g/L, and the IPSS score was I (n=64); II (n=57) and III (n=38). One hundred eight (68%) patients were previously untreated. Categorical responses were as follows: Complete Response (CR) defined as absence of clinically evident bone marrow or extramedullary disease, normal range serum IgM and negative immunofixation study (n=14); Very Good Partial Response (VGPR) defined as ≥90% reduction in serum IgM (n=21); Partial Response (PR) defined as ≥ 50% to <90% reduction in serum IgM (n=79); Minor Response (MR) defined as ≥ 25% to <50% in serum IgM (n=30); No Response (NR; n=14). No association between categorical response attainment and baseline serum IgM, B2M, Hematocrit or IPSS score was observed when employing a proportional hazards model. Achievement of better categorical responses was incrementally associated with improved PFS (p<0.0001 by log rank analysis), including attainment of a minor response, in both previously untreated and treated patients. No separation of curves was observed between VGPR and CR (Figure 1). Median TTP for patients achieving CR or VGPR was 71.8 months versus 38.6 months for those with PR or MR (Figure 2; p=0.008). Attainment of at least a VGPR served as an independent marker for improved PFS by univariate analysis. The results suggest that categorical responses, including MR and VGPR have prognostic value in WM, and that efforts aimed at improving categorical responses may lead to improved treatment outcomes in patients with WM.

Disclosures

No relevant conflicts of interest to declare.
Clinical Characteristics and Outcomes of 102 Patients with Adult T-Cell Leukemia/Lymphoma in Peru.
Authors: Beltran Brady , Morales Domingo , Quinones Pilar , Castillo Jorge
Published: October 2019

Abstract 5001

Background

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive disease associated with the human T-lymphotropic virus type-I (HTLV-I). ATLL has heterogeneous clinical presentations and outcomes. Shimoyama's ATLL classification includes acute, lymphomatous, chronic and smoldering subtypes. The objective of this study is to define prognostic factors for survival in patients with ATLL.

Methods

102 ATLL cases were collected in our center between January 1997 and September 2008. A diagnosis of ATLL was made according to the following criteria: a clinical history consistent with ATLL, a positive HTLV-1 antibody by ELISA and Western Blot or evidence of HTLV-1 proviral integration by PCR, and histological findings compatible with ATLL. Descriptive statistics were used to assess categorical and continuous variables. Survival curves for OS were estimated using the Kaplan-Meier method and compared using the log-rank test. For the multivariate analysis, the Cox Proportional-hazard regression test was used.

Results

The median age at diagnosis was 60.5 years (range 23–92 years), with a female-to-male ratio of 1.2:1. Forty nine cases (48%) had a performance status ≥ 2. Clinical types were acute (n=45), lymphomatous (n=43), smoldering (n=3), cutaneous (n=10) and chronic (n=1). Median hemoglobin was 12.0 g/dl (range 5.2–17.4 g/dl), median albumin was 3.3 g/dl (range 1.8–4.6 g/dl), median beta-2 microglobulin was 4.4 g/dl (range 1.1–16.9 g/dl), and LDH was 808 UI/ml (range 298–13000 IU/ml). Twenty nine patients with acute ATLL were treated with chemotherapy obtaining an overall response rate (ORR) of 14% (4/29), 3 complete responses (CR) and 1 partial response (PR), while in 38 patients with lymphomatous ATLL, an ORR of 32% (12/38) was obtained, 8 CR and 4 PR. Smoldering and cutaneous ATL types received mainly topic treatments. Median overall survival (OS) was 2.4 months for the acute type, 11.4 months for the lymphomatous type, 17.2 months for the smoldering type and 39.4 months for the cutaneous type (p<0.00001 for trend). In the univariate analysis, presence of B symptoms (median OS 5.4 vs. 20.2 months; p=0.005), performance status ≥ 2 (median OS 3.5 vs. 15.6 months; p<0.005), clinical stage > 2 (median OS 5.7 vs. 39.4 months; p<0.005), high LDH levels (median OS 4.3 vs. 15.4 months; p<0.005), and bone marrow involvement (median OS 3.3 vs. 15.6 months; p<0.005) were associated with worse prognosis. In the multivariate analysis, bone marrow involvement and elevated LDH remained as adverse prognostic factors for survival.

Conclusions

ATLL is a heterogeneous disease with poor outcomes. Each ATLL subtype has distinct clinical features, including response rates and survival times. In this cohort of 102 cases, bone marrow involvement and elevated LDH levels were independent prognostic factors. Prospective studies are needed to further validate these observations.

Disclosures

No relevant conflicts of interest to declare.
A Review of HIV Related Burkitt's Lymphoma/Leukemia in Sub Saharan Africa.
Authors: Novitzky Nicolas , Singh Jaimendra
Published: October 2019

Abstract 5016

Introduction

Burkitt's malignancies have increased substantially in frequency In HIV infected patients regardless of CD4 count. There is limited information about the clinical presentation and outcome of adult HIV+ patients with Burkitt's malignancies (BM) outside the endemic areas of Africa.

Methods

Patients with histologically confirmed BM were treated with antiretroviral medication and intense, anthracycline containing combinations (HyperC-VAD, COP-ADM). A retrospective review of the clinical characteristics and prognostic factors relevant for outcome was conducted comparing to an HIV –ve cohort.

Results

Between 1999 and 2009, 32 HIV positive patients and 9 HIV negative individuals with Burkitt's malignancy were referred to a single centre. The median age was 33 (13-61) years, 19 were female; 18 patients had leukemia while 23 patients had Burkitt's lymphoma. Extranodal disease was seen in 16 patients, the presentation serum median LDH was 1902 (292-45100) i.u., CNS disease was present in 7. In the HIV+ population, the median presentation CD4 count was 193 (10-967)/uL, 18% were on HAART prior to diagnosis but in the majority the malignancy led to the initial presentation. Of the 32 HIV+ve subjects who received intense cytotoxic therapy, at median follow up of 261 (146-2564) days, 14 (44%) survive, 12 longer than 1 year. The main cause of death was disease progression, relapse (particularly in CNS) and opportunistic infections. Male gender (p= 0.02), nodal presentation (p= 0.01), higher Hb (p= 0.001) and platelets (p= 0.03) predicted for better outcome. Cox analysis confirmed that male gender (p= 0.03), lower LDH (p= 0.02), and nodal presentation (p=0.03) were independent factors for survival. Non parametric statistics showed that HIV-ve Burkitt's patients were predominantly male (p= 0.01), Caucasian (p= 0.02), had significantly higher presentation serum albumin (p= 0.01) and lower creatinine (p= 0.02). In the HIV-ve group 78% survive longer than 1 year (log Rank p= 0.01).

Conclusions

In South Africa there are distinct clinical differences between HIV+ve and negative patients who present with Burkitt's malignancies. While outcome in the HIV –ve population is favourable, it is significantly worse in the HIV+ve cohort. For this group, a different strategy is required to improve outcome.

Disclosures

No relevant conflicts of interest to declare.
Upregulation of Krüppel-Like Factor 4 (KLF-4) Expression as a Potential Tumor Progression Gene Product in Non-Hodgkin's Lymphoma.
Authors: Vega Mario I. , vazquez-Islas Juan , Martinez-Paniagua Melisa , Hernandez-Atenogenes Miriam , martinez-Miguel Bernardo , Maldonado-Valenzuela Altagracia , Gonzalez-Bonilla Cesar , Huerta-Yepez Sara , Martinez-Maza Otoniel , Bonavida Benjamin
Published: October 2019

Abstract 5022

Krüppel-like factor 4 (KLF-4) is highly expressed in epithelial tissues such as the gut and skin. Several studies based on clinical evidence suggest that KLF-4 functions as a tumor suppressor in cancers of the colon, bladder, stomach and in leukemia. In contrast, KLF-4 expression is increased in primary breast ductal carcinomas and in oral dermal squamous cell carcinomas, suggesting that KLF-4 is important in tumor development and progression. However, KLF-4 expression in lymphomas has not been investigated. Our preliminary studies have examined KLF-4 expression in lymphoma cell lines and a TMA containing fresh tissues derived from patients with several types of lymphoma. There was a significantly higher expression of KLF-4 in Burkitt's lymphoma compared with other lymphomas such as follicular or DLBCL. These findings suggest that KLF-4 may be considered as a new biomarker in Non-Hodgkin's lymphoma (NHL). Further analyses based on the clinical outcome revealed that KLF-4 protein expression was significantly associated with poor patient's survival. The increased KLF-4 expression was associated with an inferior survival duration (P= 0.002). The survival for 12 patients who had a tumor with weak KLF-4 expression and 13 patients with negative KLF-4 expression was significantly longer than that for the 30 patients with strong KLF-4 expression (P< 0.001). Other variables that affected survival in univariate analyses included stages and completeness of resection were shown to have a statistically significant effect on survival (P=0.001), however age or sex did not have a statistically significant effect on survival. These data provide the first clinical and causal evidences that alterations of KLF-4 expression can play a critical role in the development and progression of NHL.

Disclosures

No relevant conflicts of interest to declare.
Investigation of the Safety and Usefulness of Computed Tomography-Guided Percutaneous Needle Biopsy of Paraaortic Lymph Nodes.
Authors: Aramaki Takeshi , Ikeda Takashi , Moriguchi Michihisa , Tsushima Takahiro , Endo Masahiro , Asakura Koiku , Sawada Akihiro , Kimura Fumihiko
Published: October 2019

Abstract 5027

Background

Various factors such as malignant lymphoma cause abdominal lymph node enlargement. A histological diagnosis of malignant lymphoma is important in planning therapy, and although superficial lymph node biopsies are often obtained, target lesions occasionally arise only around the abdominal aorta. These require surgical, or more recently, endoscopic biopsies. To intraoperatively determine whether target lymph nodes are correctly resected during a surgical biopsy can be difficult, and therapy could be delayed due to postoperative complications.

Objective

To assess the safety and effectiveness of percutaneous biopsy of paraaortic lymph nodes under computed tomography (CT) fluoroscopic guidance.

Subjects and Methods

We retrospectively investigated puncture success rates based on findings of diagnostic imaging, size of target lymph nodes, rate of confirmed diagnosis, frequency of changes in therapeutic plans and incidence of complications among 36 patients after percutaneous needle biopsy using an 18G cutting needle between September 2002 and March 2009.

Results

The pre-biopsy diagnoses for the 36 subjects who underwent percutaneous needle biopsy included: malignant lymphoma (n = 26), lymph node metastasis of esophageal cancer (n = 3), lymph node metastasis of lung cancer (n = 3), lymph node metastasis of pancreatic cancer (n = 3) and unknown (n = 3). The median size of the target lymph nodes was 25 mm (range: 9–102 mm). The results of diagnostic imaging indicated that all punctures were successful. The diagnoses were confirmed in 33 of the 36 patients (91.7%). Histopathological findings confirmed malignant lymphoma (n = 21; Follicular Lymphoma in 11 patients, Diffuse Large B cell Lymphoma in 8, T-Cell Lymphoma in 1, Hodgkin Disease in 1), squamous cell carcinoma (n = 2), poorly differentiated adenoma (n = 2), other malignancies (n = 5), benign lesions (n = 3) and unknown in 3. Therapeutic strategies were altered based on the results of CT-guided percutaneous needle biopsies in 12 of the 36 patients (33.3%). Three patients developed 4 adverse events comprising grade 1 radiation dermatitis (n = 1), grade 1 hematoma (n = 2) and grade 1 pneumothorax (n = 1).

Conclusions

Target paraaortic lymph nodes can be accurately punctured using CT-guided percutaneous needle biopsy, and the specimens were too small to examine the flowcytometry or chromosome analysis, however it was enough to confirmed the diagnosis according to WHO classification of malignant lymphoma. Percutaneous biopsy of paraaortic lymph nodes under CT fluoroscopic guidance is useful for confirming diagnoses with a low risk of serious deep organ damage.

Disclosures

No relevant conflicts of interest to declare.
Acquisition of Isochromosome 7 Is a Late Change in the Pathogenesis of Hepatosplenic Lymphoma, Documented On a Case of Adolescent Girl 5 Years After Renal Transplant with Preceding TCR Gamma-Delta Positive LGL Leukemia.
Authors: Mejstrikova Ester , Kabickova Edita , Sumerauer David , Seeman Tomas , Fronkova Eva , Zemanova Zuzana , Lizcova Libuse , Sedláček Petr , Smisek Petr , Muzikova Katerina , Pospisilova Klara , Janda Ales , Kodet Roman , Capkova Linda , Stary Jan , Hrusak Ondrej
Published: October 2019

Abstract 5031

Background

Post-transplant lymphoproliferative disorders (PTLD) occur in 1-20% of recipients receiving solid organ transplantation. We describe a patient who suffered from hepatosplenic T-cell lymphoma occurring after previous PTLD in a renal transplant recipient.

Patient/Methods

An 11-yr-old girl underwent kidney transplantation for end-stage Fanconi's nephronophthisis in 2002. In October 2006 significant neutropenia (<200/uL) was firstly detected, without any abnormality in bone marrow (BM) aspirate and without hepato- or splenomegaly. Episodes of neutropenia resolved spontaneously or after enhanced immunosuppression and G-CSF. In January 2007 new episodes of neutropenia and newly significant “monocytosis” were detected in peripheral blood (PB) and BM. Percentage of “monocytes” corresponded with immunophenotypically atypical TCR gamma/delta positive T cells (CD7weakposCD5negCD3bright) in PB and BM. Clonal TCR gamma and delta rearrangements were identified which enabled qPCR minimal residual disease (MRD) assessment. No lymphadenopathy was present, slight hepatosplenomegaly was identified by sonography. Conventional and molecular cytogenetic analyses didn't reveal any chromosomal aberration in PB and BM including changes on chromosome 7. No increased levels of EBV and CMV load by PCR were found. Partial increase of granulocytes and slight decrease of atypical TCR gamma/delta T cells were detected after administration of corticosteroid bolus and mercaptopurin. Three months later she presented with fever, rapidly progressive hepatosplenomegaly and pancytopenia, clinically corresponding with hepatosplenic lymphoma. At this time, newly acquired isochromosome 7q was detected by FISH.

Results

Initial therapy with campath and fludarabine was ineffective. She didn't respond to the 2nd line treatment (prednisone, vincristine, daunorubicine,asparaginase) and died 2 weeks later from lymphoma progression. Autopsy identified severe hemophagocytosis in the liver. Retrospectively, we identified identical clonal TCR rearrangements in the PB samples from March 2006 (∼0.03% of lymphoma PB MRD level), when neither changes in PB count nor clinical symptoms were found.

Conclusion

We detected a “pre-lymphoma” phase with clonal expansion of atypical TCR gamma/delta T cells more than 1 year before lymphoma manifestation. The presence of isochromosome 7q was a late change during this lymphoma genesis.

Grant support

IGA NS/9997-4; IGA NR/9531-3, IGA NS 10480-3, Research Projects MZCR 000064203, MSM0021620813

Disclosures

No relevant conflicts of interest to declare.
The Src Kinases Play a Crucial Role in the Growth of Hematopoietic Cells Transformed with Constitutively Activated Stat5 Mutants.
Authors: Nyga Remy , Gouilleux Fabrice , Cartier Flora , Pecquet Christian , Regnier Aline , Marolleau Jean-Pierre , Rochette Jacques , Moriggl Richard , Bouhlal Hicham , Lassoued Kaiss
Published: October 2019

Abstract 5041

Stat5A and Stat5B transcription factors (TF) play an important role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological malignancies and solid tumours. Studies conducted with constitutively active (ca) Stat5 mutants (Stat51*6 and cS5F) have shown that deregulated Stat5 activity promotes leukemogenesis. In order to evaluate the role of Src kinases in the transforming properties of Stat5 TF, caStat5 expressing Ba/F3 cells and primary murine bone marrow cells were treated with the PP1 Src kinase inhibitor and with its inactive analogue PP3 as a control. We found that PP1 but not PP3 strongly inhibited Stat5A1*6- and Stat5B1*6- expressing Ba/F3 cell growth and survival while no changes were observed in IL-3 stimulated-parental Ba/F3 cells when treated with PP1. Similarly, we were able to demonstrate specific requirement of Src kinases in cS5F-induced primary bone marrow cell growth. We then examined the contribution of Src kinases to the phosphorylation of various signaling molecules involved in cell growth and survival like Stat5, PI3-K/Akt, Ras/Mapk and NF-kB. The treatment of caStat5 (Stat51*6 or cS5F)-expressing Ba/F3 cells with PP1 resulted in a strong decrease of Erk1/2 phosphorylation, but not of Stat5, Akt and IKK species. In addition, caStat5-expressing Ba/F3 cells were found to express a constitutive molecular complex comprising Stat5, Shc, Grb2, Sos, Erk, Gab2, p85, Lyn and Hck. Finally we found that the Lyn Src kinase was overexpressed in the caStat5-expressing cells. Our data suggest a direct implication of Src kinases in the proliferation of caStat5- Ba/F3 transformed cells through the Shc/Erk1/2 signaling pathway and that Src family members are required for caStat5 (Stat51*6 and cS5F)-induced hematopoietic cell growth.

Disclosures

No relevant conflicts of interest to declare.
Mdm2 and Mdmx Are Not the Major Negative Regulators of the p53 Pathway in Myeloid Leukemias.
Authors: Abramowitz Julia , Neuman Tzahi , Samman Rania , Perlman Riki , Ben-Yehuda Dina
Published: October 2019

Abstract 5046

Mutations in the p53 tumor suppressor gene are rare in human hematological malignancies, suggesting that aberrant p53 function may be due to alterations in its regulatory pathways. p53 is negatively regulated by MDM2 through ubiquitin-dependent degradation and by Mdmx through inhibition of transcriptional function. There is little information on the expression of Mdm2 and Mdmx in most myeloid leukemias. We determined the gene expression and protein levels of Mdm2 and Mdmx in bone marrow samples of leukemia patients. We first performed quantitative evaluation of Mdm2 and Mdmx gene expression in bone marrow samples of 144 leukemic patients at the time of diagnosis: 29 de novo AML patients (AML, M0-M7, M3 excluded), 30 de novo AML M3 patients (APL), 39 therapy-related AML patients (t-AML, M0-M7, M3 included) and 46 CML chronic phase (CML-CP) patients in comparison to 35 normal bone marrow samples from Hodgkin's disease patients. Quantitative Real-Time PCR analysis showed no global statistically significant over expression of Mdm2 or Mdmx in any of the tested leukemias. However, a number of patients in both de novo and therapy-related AML had elevated levels of Mdm2 or Mdmx. Significant down regulation of Mdm2, Mdmx and a splicing variant of Mdmx lacking exon 6 (Mdmx-S) was observed in CML-CP. We next performed IHC staining, evaluated by semi-quantitative score, to examine the levels of Mdm2 and Mdmx protein expression in 151 leukemic patients at the time of diagnosis: 40 AML patients, 23 APL, 47 t-AML and 41 CML-CP patients in comparison to 58 normal bone marrow samples from Hodgkin's disease patients. Protein expression analysis also showed no global statistically significant over expression of Mdm2 in any of the tested leukemias. Nonetheless, a number of patients in both de novo and therapy-related AML had elevated levels of Mdm2 protein. Specifically, APL patients segregated into 2 groups: while half of the patients did not express the Mdm2 protein, the other half over-expressed Mdm2. A significant down-regulation of Mdmx was observed in APL. In summary, while in some leukemic patients Mdm2 and Mdmx might play a role in the regulation of p53, our quantitative analysis indicates that these negative regulators do not seem to provoke the inactivation of the p53 pathway in most myeloid leukemias patients. These findings have implications on the possible use of Mdm2 antagonists like nutlin-3 in myeloid leukemias. To the best of our knowledge this is the largest group of myeloid leukemia patients studied for Mdm2 and Mdmx expression.

Disclosures

No relevant conflicts of interest to declare.
High Plasma Levels of Factor VIII and Factor IX Increased the Incidence of Recurrent VTE.
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: October 2019

Abstract 5070

The aim of this study was to evaluate the diagnostic accurancy of the measurement of procoagulant factors FVIII and FIX levels and the risk of recurrence of venous thromboembolism (VTE). We have studied 56 patients (14 male, 42 female) with an objectively diagnosis of first deep vein thrombosis enrolled from February 2003 to February 2008, all patients were <60 years, without known malignancies, heart failure, chronic lung disease, infection, recent surgery, or genetic defects as Factor V Leiden (FV-Arg 506 Glu), prothrombin G20210A, deficiencies of Protein S or C or ATIII, hyperhomocysteinemia, dysfibrinogenemias; with increased levels of FVIII (>218 IU/dl), and FIX (>136 UI/dl). FVIII and FIX antigen levels were measured by ELISA. All patients received subcutaneous low molecular-weight eparin as thromboprophylactic treatment for the first 3 month after primary VTE, color Doppler ultrasonography (DUS) was performed periodically after primary VTE, the venous hemodynamic parameters studied included: blood flow velocity (cm/sec), vein cross sectional area (cm/2) blood volume flow (ml/sec), and microvascular injury was evaluated. Our studies have confirmed that a markedly elevated plasma levels of FVIII >256 IU/dl (range 218-294), and FIX >164IU/dl (range 136-192) among the patients with first episode of VTE are independent predictors of early recurrence about 52-74%: these patients have increased fibrin formation, D-Dimer levels (234.6ng/ml), and blood rheologic variables such as plasma viscosity are still present in all patients: plasma viscosity mPa/s shear rate >1.98 and blood viscosity mPa/s shear rate >3.48 especially under low shear conditions. These factors show to be associated with VTE and above all with the recurrence (54 patients), and emerged as relatively independent of other thrombophilic factors. The higt risk of VTE recurrence in these patients requires strategies for its prevention: secondary prophylaxis with the administration of standard oral anticoagulant at moment the use of coumarins (INR: 2.0-3.0) and possibly the need for prolonged, probably life-long treatment.

Disclosures

No relevant conflicts of interest to declare.
A Prospective Study of Platelets Function During the Process of Stem Cells Mobilisation in Healthy, Allogeneic Blood Stem Cell Donors Using the Method of Impedance Aggregometry.
Authors: Najda Jacek , Wojnar Jerzy , Kyrcz-Krzemien Slawomira
Published: October 2019

Abstract 5079

Potential haemostasis disturbances accompanying the process of peripheral stem cells harvest are still underestimated. Although the last two decades have witnessed an important increase in the use of peripheral blood stem cells (PBSC) for allogeneic transplantations, we are still lacking a satisfactory analysis of mobilization related risk of thrombosis or an unexpected bleeding. It was previously proved, that G-CSF administration may induce a hypercoagulable state by increasing levels of FVIII;C. On the other hand, platelet aggregation is reduced after cytokine application. Numerous, remaining unsolved questions in this matter prompted us to study prospectively 26 PBSC healthy donors. All donors were medically evaluated initially to eliminate any identifiable pathology in their medical history, especially concerning cardiovascular system. Any concomitant medication was excluded. All donors studied were excluded if they had an abnormal platelet count, activated partial thromboplastin time (APTT) or prothrombin time(PT) prior to cytokine administration.

All donors received G-CSF (Neupogen, Amgen) at 5ug/kg of body weight subcutaneously twice daily for nine consecutive doses. We were analyzing the group of donors in the context of hemostasis alteration using the method of impedance aggregometry. The basic device used was “MULTIPLATE' aggregometer produced by DYNABYTE (Munich). We analyzed the primary hemostasis measuring the reaction of platelets to collagen (COL-TEST), ADP (ADP-test) and TRAP –test.

We performed platelets aggregation analysis before G-CSF administration, and subsequently on the first day of stem cells mobilization, three times. The first sample was obtained just before the beginning of separation, the second in the half time point of the procedure and the last sample was drawn immediately after the cessation of stem cell collection procedure. We found reduced platelet aggregation after G-CSF administration, comparing the data before cytokine stimulation and preceding the leukapheresis (p<0,05).

In contrast to this finding, there was a considerable increase in platelet activation parameters in the course of apheresis procedure, concerning both COL-test (p<0,04) nad TRAP-test (p<0,05).

The clinical implications of these findings confirm important and significant coagulation system stimulation during separation procedure.

Disclosures

No relevant conflicts of interest to declare.
Poor Potential of Proliferation and Differentiation in Bone Marrow Mesenchymal Stem Cells Derived From Children with Severe Aplastic Anemia.
Authors: Peng Ching-Tien
Published: October 2019

Abstract 5081

Introduction

Idiopathic severe aplastic anemia (SAA), characterized by failure of hematopoiesis, is rare and potentially life-threatening to children. However, the pathogenesis has not been completely understood, and insufficiency in the hematopoietic microenvironment can be an important factor. Mesenchymal stem cells (MSCs) play an important role in maintaining bone marrow microenvironment. Therefore, we aimed at the intrinsic defects of bone marrow MSCs derived from SAA children.

Materials and Methods

Bone marrow MSCs were obtained from 5 SAA children and 5 controls. The morphology, immunophenotyping, proliferative capacity and differentiation potential of MSCs from SAA children were determined and compared with those of MSCs from controls.

Results

In vitro, MSCs of SAA and control group shared a similar spindle-shaped morphology. Both revealed a consistent immunophenotypic profile which was negative for CD45, CD14 and CD34, and positive for CD105, CD73, and CD44. However, SAA MSCs had slower expansion rate and smaller cumulative population doubling from passage 4 to 6 (1.83± 1.21 vs 3.36± 0.87; p = 0.046), indicating lower proliferative capacity. Besides, only 3 of 5 cultures of SAA group retained the ability to continue expansion till 80%-90% confluent cell layer beyond passage 6, suggesting earlier senescence of SAA MSCs. After osteogenic induction, SAA MSCs showed lower alkaline phosphatase activity (1.46± 0.04 vs 2.27± 0.32; p = 0.013), less intense von Kossa staining and lower gene expression of core binding factora1 (0.0015± 0.0005 vs 0.0056± 0.0017; p = 0.013). Following adipogenic induction, SAA MSCs showed less intense Oil red O staining (0.86± 0.22 vs 1.73± 0.42; p = 0.013) and lower lipoproteinlipase expression (0.0105± 0.0074 vs 0.0527± 0.0254; p = 0.013).The results of real time-PCR analysis for the assessment of lineage-specific genes were consistent with the findings of histochemical stains, and both indicated that SAA MSCs had poor osteogenic and adipogenic potential.

Conclusions

In this study, we demonstrated that bone marrow MSCs from children with SAA had poor potential of proliferation and differentiation. These alterations in MSCs may contribute to the failure of hematopoiesis, and lead to the development of the disease. Further studies are needed to elucidate the relationship between MSCs and SAA.

Disclosures

No relevant conflicts of interest to declare.
Novel Homozygous Mutation (R329X) in the Hemojuvelin Gene Is Associated with Hypogonadotrophic Hypogonadism, Diabetes Mellitus and Heart Failure Duo to Juvenile Hemochromatosis.
Authors: Zhou Rong-Fu , Ouyang Jian , Guo Xue-mei , Chen Bing , Xu Jing-yan , Shao Xiao-Yan , Zhang Qi-Guo
Published: October 2019

Abstract 5102

Juvenile hemochromatosis (JH) is a rare autosomal recessive disorder characterized by the early onset of severe iron overload. A large variety of mutations within the genes encoding hepcidin (HAMP) and hemojuvelin (HJV) have been identified in patients with JH. But in the Chinese population, the prevalence of JH is quite low. No HJV mutation has been reported so far.

Methods and results

The proband was a 25-year-old young man of Asia decent presented with hypogonadotrophic hypogonadism, diabetes mellitus and heart failure but no family history of iron disorders. His serum iron level was 34μmol/L, with a transferrin concentration of 8.5g/L, serum ferritin concentration was 8140 μg/L. Echocardiography revealed that he had generalized cardiac enlargement, cardiac dysfunction, and severe mitral and tricuspidal insufficiency, pulmonary hypertension. Ultra-sonography showed diffuse hepatomegaly and splenomegaly, seroperitoneum and right hydrothorax. Liver biopsy showed severe diffuse hepatocellular siderosis and cirrhosis,hemosiderin pigmentation. To search for possible variants in the HJV gene, we performed PCR and direct sequencing in proband and his family. A homozygous nonsense mutation in exon 4 of HJV (R329X) was identified in the JH patient and heterozygous mutation of R329X in his father and mother. No mutations were found in other genes associated with adult or juvenile hemochromatosis including HFE, transferrin receptor-2 (TFR2), ferroportin (SLC40A1), hepcidin (HAMP).

Conclusions

A novel nonsense mutation (R329X) has been identified in the HJV gene for the first time in China. This mutation elevates ferritin levels and leads to JH associated with hypogonadotrophic hypogonadism, diabetes mellitus and severe cardiomyopathy.

Disclosures

No relevant conflicts of interest to declare.
Hematologic Manifestations and Changes of Cytokines or Hematopoietic Growth Factors in Mice with Iron Overlaod.
Authors: Jeong Dae-Chul , Hwang Hui Seung , Chung Nack Gyun , Cho Bin , Shin Hyun Jung , Kim Hack Ki , Kim Yoo Jin , Han Chi Wha
Published: October 2019

Abstract 5103

Background

Iron overload by repeated transfusions induced organ toxicity including liver, heart. We investigated hematologic manifestations and cytokines or hematopoietic growth factors in murine secondary hemochromatosis.

Materials and methods

We established murine secondary hemochromatosis model using 6 week-old male C57/BL6 (H-2b) with iron dextran. Mice (n=10∼12) were intraperitoneally injected with 10 mg of iron dextran for 2 or 4 weeks. We divided five groups: control (PBS injection), iron 100mg, iron 200mg, iron 200mg with deferasirox (DFX) 300mg, and only DFX 300mg. We examined hematocrit, platelet counts and plasma iron concentration (PIC) in peripheral blood, and liver iron contents (LIC) by atomic absorption spectrophotometer. We evaluated colony forming capacity from bone marrow according to experimental group. For cytokines and hematopoietic growth factors, we performed real-time PCR for IL-1b, iNOS, IFN-g, TNF-a, TGF-b, SCF, TPO, GM-CSF, and IL-11 in bone marrow. We compared each values of relative ratio with b-actin.

Results

There was no difference of hematocrit among experimental groups. The platelet counts were significantly decreased in iron 200mg among groups (P<0.05), and showed increased trends after administration of DFX. The levels of LIC and PIC were dependent on cumulative dose of iron loaded, and decreased by DFX (P<0.01). This findings showed positive correlation between PIC and LIC (P<0.01, R2=0.726). The CFU-GEMM and CFU-GM decreased in iron 200mg, iron 200mg+DFX300mg, and DFX300mg compared with control and iron 100mg (P<0.01). Most colonies in DFX300mg were not observed except CFU-GM. In cytokines, there was shown no difference for IL-1b, iNOS, IFN-g, TNF-a, TGF-b according to experiments (P>0.05). However, SCF was shown diminished expressions for treated mice compared with control (P=0.02). The levels of TPO were increased in hemochromatosis, and decreased after administration of DFX (P=0.05). The GM-CSF was observed significantly lower in iron 200mg, iron 200mg plus DFX, DFX than control and iron 100mg (P<0.01).

Conclusions

Our results suggested that iron overload might affect hematopiesis and these findings were due to effects of hematopoietic growth factors including SCF, TPO, GM-CSF, not inhibitory cytokines. Also, we need further study for DFX in hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Effort to Protect Totos, the Smallest Tribe in the World.
Authors: Basak Jayasri , Majumdar Deboshree , Pal Nabamita , Paul Sanghita , Mukhopadhyay Soma , Mukhopadhyay Ashis
Published: October 2019

Abstract 5113

Background

Toto is a primitive Indo-Bhutanese tribe residing in a small enclave called Totopara in the Jalpaiguri district of West Bengal, India. Toto culture and language is totally unique to the tribe, and is clearly distinguished from the neighboring Rajbongshis, Koch or the Bhutanese Sharchop tribes. Totos use to do consanguineous marriage and stay in the same locality even after marriage. Totos are becoming extinct since 1950. Total population of Totos are 1377 (smallest tribe of the world) and it is declining day by day. We noticed that their life span is very short (35-40 years). With a motive to investigate the reason behind it we started our work and got the answers of our queries. Almost 50% of them are carrier of beta Thalassemia, which is a genetic disease and are caused by the mutation of beta globin gene. The objectives of our study are,

Materials & Methods

After awareness program, 3-4 ml of blood sample was collected from each Toto along with their written consent. On the spot inexpensive NESTROF (Naked Eye Single Tube Red Cell Osmotic Fragility) test was done. After returning to institute we use to do CBC (Complete Blood Count) followed by HPLC (High Performance Liquid Chromatography). DNA was isolated from those samples detected as carrier or patients using the protocol standardized in our lab. Mutation was detected by ARMS (Amplification Refractory Mutation System)–PCR.

Conclusion

Total individual already screened in the Totopara under the age limit 10- 35 years is 688. Number of Totos and Non-Totos are 628 and 60 respectively. From NESTROFT and CBC result it was revealed that different blood parameters e.g., MCV, MCH, RBC, RDW, Hb% etc of 71% Totos do not match with the normal value and found to be significantly altered. HPLC results show that 51% Totos are carrier of HbE and 20% of them are HbE homozygous. Whereas 97% non-Totos are found to be normal. DNA analysis by ARMS-PCR confirmed the result of HPLC. The percentage of HbE carrier and HbE homozygous is very high among Totos and this is the real cause of their early death. To protect this primitive tribe we have to restrict their marriage between two carriers. For Totos thalassemia carrier detection should be mandatory before marriage.

Disclosures

No relevant conflicts of interest to declare.
Platelet Adhesion Plaques: Anchors for Contraction of the Hemostatic Plug.
Authors: White James G. , Burris Steven M. , Escolar Gines
Published: October 2019

Abstract 5128

Exposure of GPIIb-IIIa and other receptors on the surface of activated platelets, binding of fibrinogen, molding of shape-changed cells into tight aggregates, internal assembly of actin molecules into filaments and movement of talin, an actin-binding protein, to the inner membrane surface provides the framework for clot retraction. However, the direction of contractile force towards the center of large aggregates or clots would lift the hemostatic plug away from the edges of vascular injury. Another mechanism must be present to facilitate the direction of contractile force toward the damaged vessel wall. This may be accomplished by development of adhesion plaques as platelets spread out on the injured vessel. The present study has used scanning (SEM) and transmission electron microscopy (TEM), confocal and immunofluorescence microscopy to detect adhesion plaques developing at sites of contact as platelets spread on surfaces. Rhodamine-phalloidin was used to detect actin filaments, and an anti-talin antibody identified by protein-A gold or Alexa Fluor 488 labeled rabbit anti mouse IgG to demonstrate talin. Normal human platelets were spread on clean glass slides or plastic chambers for intervals of up to 90 min, extracted with Triton X100 or fixed intact then labeled for talin and actin, and prepared for study by the several microscopic techniques. Triton-extracted spread platelets revealed attachment plaques well stained for talin and actin when examined by SEM or TEM. Inmunofluorescence studies of spread platelets stained with rhodamine-phalloidin and antibodies also revealed co-participation of actin filaments and talin in formation of the adhesion plaques. The association of actin and talin remained intact at all intervals for up to 90 min. Clearly adhesion plaques serve as the anchors for contraction and sealing of hemostatic plug to damaged vascular surfaces.

Disclosures

No relevant conflicts of interest to declare.