bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Telomere Length in Dyskeratosis Congenita Lymphocytes with Increased Sensitivity to Radiation and Anti-Mitotic Agents.
Authors: Beasley Michael A. , Singhal Vibha , Klingelhutz Aloysius J. , Akabogu Ike , Goldman Frederick D.
Published: April 2020

Dyskeratosis congenita (DC) is a premature aging syndrome characterized by progressive bone marrow failure, abnormal skin pigmentation and nail dystrophy. We have recently described an autosomal dominant form of DC (AD DC) in a large three-generation kindred that is due to a mutation in the gene encoding human telomerase RNA (hTR). Importantly, we have noted progressive shortening of telomeres in lymphocytes from the most recent generation, correlating with earlier onset of severe cytopenias in some of these patients. While telomere shortening is a normal consequence of the aging process, DC patients display accelerated telomere shortening in many somatic cell types. Allogeneic hematopoietic stem cell transplant (HSCT) remains the only curative therapy for marrow failure in DC. However, HSCT in DC is generally poorly tolerated and associated with significant morbidity, perhaps as a consequence of increased sensitivity of dividing cells to cytotoxic agents. To test this hypothesis, we characterized lymphocytes from nine AD DC patients and age matched controls that had been placed in long term culture following in vitro exposure to irradiation (137Cs) and varying doses of Taxol. Cell proliferation and viability were quantitated by direct visual counting on a hemocytometer, and flow cytometry was employed to assess apoptosis and cell surface expression of senescent markers. CD57 and CD95, markers of cellular senescence and apoptosis, were significantly upregulated on DC T lymphocytes after two weeks in culture relative to controls. In addition to DC lymphocytes having a decreased proliferative capacity, an increased sensitivity to Taxol was noted, with an average decrease of 21% in cell growth relative to similarly treated control cells. This effect was also noted in irradiated DC cells. Finally, DC lymphocytes displayed an increased apoptotic index in the presence of varying doses of Taxol. These results suggest that telomere shortening may be an important factor in determining cellular tolerance to cytotoxic therapy and support the concept of reduced intensity HSCT regimens in both aged individuals and DC patients.
Increased TCF-4 Expression Correlates with Reduced Caspase-3 Induction and Confers Resistance to Bortezomib.
Authors: Shringarpure Reshma , Bhole Deepak , Burger Renate , Catley Laurence , Podar Klaus , Tai Yu-Tzu , Hideshima Teru , Mitsiades Constantine , Munshi Nikhil C. , Chauhan Dharminder , Anderson Kenneth C.
Published: April 2020

Bortezomib (PS-341 / Velcade®) has achieved responses in the setting of relapsed refractory multiple myeloma, yet some patients either fail to respond or become refractory after an initial response. Moreover, despite the essential role of proteasomes in cell proliferation, not all tumor cell types respond to Bortezomib treatment. To define mechanisms for differential sensitivity to Bortezomib, we used gene expression profiling to compare global gene expression patterns in SUDHL-4 and SUDHL-6 B-cell lymphoma cell lines, which respond differentially to Bortezomib treatment. Of genes that were differentially expressed in the two cell lines, we chose to further examine differential expression of the transcription factor ‘T-cell factor 4′ (TCF-4), a downstream effector in Wnt signaling. TCF-4 expression was 15 fold higher in resistant SUDHL-4 cells compared to sensitive SUDHL-6 cells by microarray analysis, which was confirmed by real-time PCR. TCF-4 was constitutively associated with ß-catenin, its transcriptional co-activator, only in resistant SUDHL-4 cells, but not in sensitive SUDHL-6 cells. TCF-4 overexpression was associated with increased transcription of TCF-4 target genes cyclin D1 (3.48 fold) and c-myc (1.7 fold). Since all TCF-4 target genes are not defined, we also confirmed increased transcription by the TCF-4 / ß-catenin complex using TCF-4 / ß-catenin driven reporter genes. Increased TCF-4 expression in resistant SUDHL-4 cells was associated with lower induction of caspase-3 expression and activity in response to Bortezomib. Importantly, siRNA-mediated downregulation of TCF-4 sensitized cells to Bortezomib treatment, suggesting that blocking TCF-4 can restore sensitivity to Bortezomib-induced apoptosis. Our studies provide the rationale to use small molecule antagonists of TCF-4 / ß-catenin interaction to overcome Bortezomib resistance and improve patient outcome.
The CXCR4 Antagonist, AMD3100, Inhibits AML Transmigratory Activity but Does Not Alter Blast Proliferation or Survival.
Authors: Liesveld Jane L. , Lancet Jeffrey E. , Rosell Karen E. , Bechelli Jeremy , Abboud Camille N. , Phillips Gordon L.
Published: April 2020

Stromal cell derived factor-1 (SDF-1α) and its receptor, CXCR4 play a role in the trafficking of CD34+ cells. AMD3100, a selective CXCR4 antagonist, can mobilize hematopoietic progenitors from marrow to peripheral blood in healthy human volunteers and in patients with multiple myeloma and non-Hodgkin’s lymphoma (Flomenberg et al, Blood 102, 39a, 2003). Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation and NOD/SCID repopulating capacity (Kahn et al. Blood 103:2942, 2004). Since CXCR4 has been found to regulate the migration and development of AML stem cells in NOD/SCID mice, we studied the effect of AMD3100 on AML cells from the standpoint of proliferation and in vitro transendothelial transmigration utilizing a transwell system. AMD3100 (from AnorMED, Inc.), at concentrations from 0.1 to 1.0 ng/ml did not affect the viability or porliferation of purified AML blasts (n=4). AMD3100 did not influence the adherence of AML blasts to endothelial monolayers. In the presence of 0.1 to 1 ng/ml AMD-3100, the transmigration of normal CD34+ cells stimulated by 100 ng/ml SDF-1α through a human umbilical vein endothelial cell (HUVEC) monolayer was completely inhibited. Likewise, the transmigration of AML blasts through HUVECs was not altered by AMD3100 exposure, but the SDF-1α mediated transmigration was inhibited by AMD3100 from 0.1 to 1 ng/ml. The same effect was noted with AML transmigration through marrow stromal layers. The increase in transmigration through endothelial cells stimulated with G-CSF was not inhibited by AMD3100 whereas the transmigration stimulated by interleukin-8 was inhibited. When AMD3100 was placed in the bottom of the migration chamber, no independent effects on AML transmigration were noted. Co-culture of AML blasts with stromal monolayers protected blasts from apoptosis. This protection was not altered by SDF-1α, AMD3100, nor by the combination. These in vitro results demonstrate that AMD3100 can influence the migratory capacity of AML cells but has no direct effects on their proliferation or survival. Further in vitro and in vivo studies will be required to elucidate the role that this unique chemokine antagonist has in the mobilization potential of AML blasts or progenitors or in the interactions of AML cells with their microenvironment. Such studies have implications for AML autografting and AML blast interactions with extramedullary endothelial cells.
Williams-Beuren Syndrome Critical Region-5/Non-T Cell Activation Linker: A Novel Target Gene of AML1/ETO.
Authors: Lübbert Michael , Stock Michael , Berg Tobias , Fliegauf Manfred
Published: April 2020

The chromosomal translocation (8;21) fuses the AML1 gene on chromosome 21 and the ETO gene on chromosome 8 in human acute myeloid leukemias, resulting in expression of the chimeric transcription factor AML1/ETO. AML1/ETO-mediated dysregulation of target genes critical for hematopoietic differentiation and proliferation is thought to contribute to the leukemic phenotype. Several mechanisms, including recruitment of histone deacetylases (HDACs) to AML1 target genes, may be responsible for altered gene expression. We used an ecdysone-inducible expression system in the human monoblastic U-937 cell line to isolate genes that were differentially expressed upon induction of AML1/ETO expression. By representational difference analysis (cDNA-RDA), we identified 26 genes whose expression levels were significantly modulated following AML1/ETO induction for 48 hours. None of these genes has previously been described as a target of AML1, ETO or AML1/ETO. One gene down-regulated by AML1/ETO in vitro, Williams Beuren Syndrome critical region 5 (WBSCR5), was expressed in primary t(8;21) negative AML blasts but not in primary t(8;21) positive AML blasts, strongly implying a role of this gene in the phenotype of t(8;21) positive AML. WBSCR5 is part of the critical region located on chromosome 7q11.23 that is deleted in the Williams Beuren syndrome (OMIM 194050), an autosomal dominant disorder comprising vascular, neurological, behavioral and skeletal abnormalities. WBSCR5 has recently been shown to have a role in the activation and differentiation of B cells (Brdicka et al., J. Exp. Med. 196:1617, 2002) and thus was also termed Non-T cell activation linker.. WBSCR5 as well as seven other regulated genes were further studied using all-trans-retinoic acid (ATRA), an inducer of differentiation of U-937 cells, and Trichostatin A (TSA), an HDAC inhibitor. WBSCR5 and two other out of these eight genes were regulated during ATRA-induced monocytic differentiation of U-937 cells, however none of them antagonistically, upon both ATRA-treatment and AML1/ETO-induction. Since repression of WBSCR5 might be mediated by recruitment of HDACs through the fusion gene, cells were treated with TSA prior to transgene induction. However, the AML1/ETO-associated dysregulation of WBSCR5 gene expression (as well as that of the other seven genes studied) was not mediated by a TSA-sensitive mechanism. The identified genes provide a useful model to study the mechanism by which the AML1/ETO fusion protein exerts its function in transcriptional dysregulation in acute myeloid leukemia. The role of WBSCR5 in malignant hematopoietic cells warrants further study.
Expression of Erythropoietin and Erythropoietin Receptor in Squamous Cell Carcinomas of the Head and Neck: Levels of Erythropoietin Expression Correlate with Tumor Hypoxia.
Authors: Arcasoy Murat O. , Amin Khalid , Chou Shu-Chuan , Haroon Zishan A. , Varia Mahesh , Raleigh James A.
Published: April 2020

Erythropoietin (EPO), an oxygen-regulated glycoprotein hormone, is a hematopoietic cytokine that stimulates erythropoiesis by binding to its cellular receptor EPOR. The recombinant form of human EPO is widely used in clinical practice for the prevention or treatment of anemia associated with cancer and chemo-radiation therapy. However, in a recent randomized, placebo-controlled trial involving patients receiving curative radiotherapy for squamous cell carcinoma of the head and neck, EPO treatment was associated with poorer loco-regional progression-free survival. The purpose of this study was to determine whether EPOR and its ligand EPO are expressed in primary squamous cell carcinomas of the head and neck. We also investigated the hypothesis that EPO expression in malignant cells may be associated with the presence of tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness and poor prognosis. Twenty-one patients with squamous cell carcinoma of the head and neck were enrolled in a tumor hypoxia study under a research protocol approved by the Institutional Review Board at the University of North Carolina Hospitals. All patients provided signed informed consent. The patients received an intravenous infusion of the hypoxia marker pimonidazole hydrochloride (Hypoxyprobe-1™) prior to multiple tumor biopsies. Two or more biopsies were available from all except one primary tumor. The tissue specimen from one patient with laryngeal carcinoma was excluded because of availability of only a single, small and fragmented biopsy. Contiguous sections from 74 biopsies were analyzed by immunohistochemistry for expression of EPOR and EPO as well as pimonidazole binding. We found EPOR expression in tumor cells in 97% of the biopsies. The pattern of EPOR immunoreactivity was predominantly cytoplasmic but was found to be localized to the membrane in some sections. Co-expression of EPO was observed in 90% of biopsies. Qualitative and semi-quantitative analyes for EPO staining and tumor hypoxia on a section-by-section basis revealed that EPO and pimonidazole adduct staining did not always co-localize within tumors but there was a significant positive correlation between levels of micro-regional EPO expression and pimonidazole binding (r = 0.736, P < 0.001, n=20 by two-tailed Spearman’s rank correlation analysis). These data demonstrate the co-expression of EPOR and its ligand EPO in squamous carcinoma cells suggesting that EPO may play a novel role as a potential autocrine or paracrine growth factor in head and neck cancer. Furthermore, EPO expression in tumor cells may be modulated, at least in part, by tumor hypoxia. The expression of EPOR needs to be taken into consideration in the design of future clinical trials investigating the role of recombinant human EPO in head and neck cancer.
Time Interval from Last Chemotherapy to Stem Cell Collection Correlates with Peripheral Blood Absolute Lymphocyte Count at Apheresis and Survival Post-Autologous Stem Cell Transplantation in Non-Hodgkin’s Lymphoma.
Authors: Holtan Shernan G. , Porrata Luis F. , Inwards David J. , Ansell Stephen A. , Micallef Ivana N.M. , Litzow Mark R. , Gertz Morie A. , Lacy Martha Q. , Dispenzieri Angela , Gastineau Dennis A. , Tefferi Ayalew , Elliott Michelle , Hogan William J. , Hayman Suzanne , Markovic Svetomir N.
Published: April 2020

The infused autograft absolute lymphocyte count (A-ALC) is an independent prognostic factor for survival after autologous hematopoietic stem cell transplant (AHSCT) in non-Hodgkin’s lymphoma (NHL). Previous studies have shown that A-ALC directly correlates with the peripheral blood absolute lymphocyte count (PC-ALC) at the time of apheresis collection. However, factors affecting the PC-ALC at apheresis remain undefined. We hypothesized that one possible factor impacting PC-ALC may be the time interval from last chemotherapy to stem cell collection (TILC). Data from 160 patients who underwent AHSCT for treatment of relapsed NHL at Mayo Clinic between 1993 and 2001 were collected and analyzed. The primary end point of this study was correlation between TILC and PC-ALC, and our analysis revealed a strong correlation (r = 0.67, p < 0.0001). Further analysis revealed higher PC-ALC numbers in patients with TILC ≥ 60 days versus TILC < 60 days (median of 7.44 x 109/L in the ≥ 60 day group versus 3.87 x 109/L in the < 60 day group, p < 0.0001). Both the median overall survival (figure below) and the progression-free survival were longer in the TILC ≥ 60 days group versus the TILC < 60 days group (76 versus 21 months, p < 0.0037; 76 versus 11 months, p < 0.018, respectively). These findings were independent of other prognostic indicators for relapsed NHL patients undergoing APHSCT. The data supports our hypothesis that TILC affects PC-ALC and survival post-AHSCT in NHL.

Figure Figure
More Than One Mutation Is Required for Leukemia Development Following Retroviral Vector-Mediated Gene Transfer.
Authors: Kustikova Olga , Schmidt Manfred , Modlich Ute , Li Zhixiong , Schmidt Sonja , Deichmann Anette , Rudolph Cornelia , von Neuhoff Nils , Schlegelberger Brigitte , Fehse Boris , Kalle Christof , Baum Christopher
Published: April 2020

As recent case reports have suggested that proto-oncogene activation by replication-deficient retroviral vectors may trigger leukemia in mice and men, there is an urgent need for models allowing predictive investigations into the pathomechanisms of this complication. Previously, we have shown that dose-escalated replication-defective retrovirus vectors (such as HaMDR1 or SF91dsRedpre) may trigger combinatorial leukemia-initiating events in single cells, with a probability that allows prospective studies in simple mouse models (C57Bl/6). Meanwhile, we have performed molecular analyses for 5 different leukemias, harboring 6 to 12 vector insertions according to Southern blot. 34 insertions recovered from these clones by LM-PCR and LAM-PCR showed that 4 leukemias had combinatorial hits in at least one established proto-oncogene (class 1 genes: Hivep1, Mllt3, Fli1, HoxA7, Brd2, Evi1, Csfr3, Bcl11a, Ski) with at least one hit in other signaling genes (class 2 genes). One leukemia for which 2 insertions remain to be identified showed at least 3 class 2 hits, so far without a class 1 event. All leukemias presented with hits in genes involved in cytoplasmic signal cascades along with genes contributing to nuclear regulatory networks. Some of these leukemias had a normal karyotype. A comparison with 62 insertion sites from healthy control animals (determined in peripheral blood DNA harvested 7 months after transplantation) revealed a significant enrichment of class 1 hits in leukemias (14-fold over expected and 3-fold over control). Healthy animals still showed 6 hits (~10%) in class 1 genes, obviously not being sufficient for leukemia induction. Three of these occurred upstream of Evi1, one upstream of Nfic, one upstream of Pdclg2, and one in Bcl11a. Interestingly, upstream vector insertion was preferred in class 1 gene hits. Overall, insertions recovered from both normal and leukemic samples revealed an overrepresentation of hits within (or upstream of) known or predicted genes (89 and 90%, respectively). In line with the data of a related murine marking study (Kustikova, Fehse et al., ASH 2004), our study strongly suggests that single hits in class 1 or class 2 genes are not sufficient to induce leukemia in mice. However, certain combinations of such events in single cells may well be leukemogenic with a given latency. Our findings establish a powerful approach for both oncogene discovery and safety validation in gene therapy.
BCR/ABL→STAT5 Pathway Stimulates Helix-Loop-Helix Id-1 Protein To Enhance the MMP9 - Dependent Invasiveness of Leukemia Cells.
Authors: Nieborowska-Skorska Margaret , Hoser Grazyna , Malecki Maciej , Wasik Mariusz , Skorski Tomasz
Published: April 2020

BCR/ABL oncogenic tyrosine kinase activates STAT5, which plays as important role in leukemogenesis (Nieborowska-Skorska et al., J.Exp.Med., 189:1229,1999). We reported before that the bcl-2 family member A1 and the proto-oncogene pim-1, the two downstream effectors of STAT5, collaborate in BCR/ABL-positive cells to induce protection from apoptosis and growth factor independence (Nieborowska-Skorska et al., Blood, 99:4531,2002). We show here that expression of the helix-loop-helix protein Id1 is enhanced by BCR/ABL in the STAT5-dependent manner. This effect requires activation of STAT5 by the signaling from SH3+SH2 domains of BCR/ABL. Enhanced expression of Id1 played a key role in the BCR/ABL-mediated cell invasion in vitro, but not in protection from apoptosis in the absence of growth factors and in promotion of growth factor-independent proliferation. Leukemogenesis in mice was prolonged after injection of BCR/ABL-positive cells, in which Id-1 was downregulated by the antisence cDNA. In addition, infiltration of central nervous system (CNS) by leukemia cells was also reduced if Id-1 was inhibited. Id1 was responsible for BCR/ABL-induced production of MMP9 metalloproteinase. Abrogation of MMP9 catalytic activity by a selective inhibitor or blocking antibody decreased the invasive capability of leukemia cells. These data indicate that BCR/ABL→STAT5→Id1→MMP9 pathway may play a critical role in the BCR/ABL-dependent leukemogenesis by regulation of the invasive capability of leukemia cells. Moreover, this pathway may protect CML cells from imatinib mesylate (IM) by facilitation of their localization in CNS, a sanctuary for leukemia cells during IM therapy.
Tumor-Derived Chaperone-Rich Cell Lysate (CRCL) Can Be Effectively Combined with Syngeneic Bone Marrow Transplantation to Treat Bcr-abl+ Leukemia in a Murine Model.
Authors: Chen Xinchun , Zeng Yi , Li Gang , Thompson Sylvia , Sepassi Marjan , Romanoski M. Angela , Graner Michael W. , Katsanis Emmanuel
Published: April 2020

We have previously reported that purified tumor derived chaperone-rich cell lysate (CRCL) containing multiple heat shock proteins (HSPs) is a promising cancer vaccine, capable of generating tumor-specific T cell responses and protective immunity in different animal tumor models. In this study, we have explored the therapeutic applicability of CRCL in the context of syngeneic bone marrow transplantation (BMT) to treat preexisting leukemia. BALB/c mice received a 10-fold LD100 dose of bcr-abl+ leukemia cells (12B1) subcutaneously (s.c.) on day -7. On day -1, 900cGy of total body irradiation (TBI) was given followed by syngeneic bone marrow transplantation (BMT) on day 0. Transplanted mice received 20X106 bone marrow cells along with 50X106 splenocytes (SPC) intravenously (i.v.). We have demonstrated that BMT/SPC prolonged survival of mice by a median time of 7 days (median survival time [MST] from tumor inoculation of no BMT versus BMT/SPC =19 vs 26 days), however, all mice eventually died of disseminated leukemia. When recipients received BMT and SPC from syngeneic donors that had been previously immunized with 12B1-derived CRCL (immune BMT/SPC) the MST was increased to 29 days with 26.1% of transplanted mice surviving without tumor (p<0.01 immune BMT/SPC vs naive BMT/SPC). Vaccination of immune BMT/SPC recipients with 20 mg 12B1-derived CRCL in the early post-transplant period (day +1 and day +6) increased MST to 32 days (p<0.01 vs naïve BMT/SPC control) but did not significantly improve overall survival (26.3%) when compared to immune BMT/SPC mice not receiving post transplant 12B1-CRCL vaccine. Eleven to 17 weeks later, mice with no evidence of disease were re-challenged with 12B1 cells in one groin and A20 B cell leukemia cells in the opposite groin. Eighty percent of the mice demonstrated long term tumor specific immunity by rejecting the 12B1 rechallenge while 100% of the mice developed A20 tumors. Our results indicate that CRCL is a promising vaccine that can be used to generate specific anti-tumor immunity that can be effectively transferred to a host via BMT.
Mitochondrial Mutations as Molecular Markers for Minimal Reidual Disease (MRD) in Acute Myeloid Leukemia.
Authors: Morley Alexander A. , Grist Scott A. , Lu Xiao J. , Patsouris Katrina
Published: April 2020

Measurement of minimal residual disease (MRD) is being increasingly used in haematological malignancies in order to assess prognosis and decide on treatment. However for some patients, including many patients with acute myeloid leukemia (AML), molecular techniques for MRD measurement cannot be performed owing to lack of a molecular marker. We have detected mitochondrial mutations (MM) in the D loop of the mitochondrial genome of the leukemic cells in approximately 40% of patients with AML and have investigated 2 techniques - denaturing gradient gel electrophoresis (DGGE) and single nucleotide primer extension (SNUPE) - to detect and quantify them. Mixing experiments showed that DGGE had a sensitivity of approximately 0.5% for detection of MM, and it was able to detect leukemia in remission marrow in 5 of 6 patients with AML. When present in a patient, MM are usually multiple. We therefore tested a 2-step strategy, as shown in the figure, which involved initial enrichment by PCR using primers directed at 1 or 2 flanking mutations followed by DGGE to detect an inner mutation. In 2 mixing experiments this 2-step strategy increased sensitivity of detection down to 0.0001% (this level of detection was evident in the original gel photo).

Figure Figure

SNUPE was more sensitive than DGGE and in 3 mixing experiments it showed a sensitivity of 0.02–0.05%. Four patients with AML have now been studied by SNUPE and the levels of MRD in the marrow documenting morphological remission at the end of induction therapy were 2.8%,16.1%,40.8% and 15.7%. The relatively high levels of MRD as measured by PCR in 3 patients suggest that some of the leukaemic cells at the end of induction may be more mature than blasts and thus not identifiable by morphology. While questions remain to be answered, mitochondrial mutations are promising molecular markers for quantifying MRD in AML and possibly other haematological disorders
Mutations in the Receptor Tyrosine Kinase Pathway Are Associated with Clinical Outcome in Patients with Acute Myeloblastic Leukemia Harboring t(8;21)(q22;q22).
Authors: Nanri Tomoko , Matsuno Naofumi , Kawakita Toshiro , Suzushima Hitoshi , Kawano Fumio , Mitsuya Hiroaki , Asou Norio
Published: April 2020

AML1-MTG8 generated by t(8;21)(q22;q22) contributes to leukemic transformation but additional events are required for full leukemogenesis. We examined whether mutations in the receptor tyrosine kinase (RTK) pathway could be the genetic events that cause acute myeloblastic leukemia (AML) harboring t(8;21). Mutations in the second tyrosine kinase domain, juxtamembrane domain and exon 8 of the C-KIT gene were observed in 7, 1 and 3 of 37 AML patients with t(8;21), respectively. Three patients showed an internal tandem duplication in the juxtamembrane domain of the FLT3 gene. One patient had a mutation in the K-Ras gene at codon 12. As the occurrence of these mutations was mutually exclusive, a total of 15 (41%) patients showed mutations in the RTK pathway. These results suggest that AML1-MTG8 predisposes cells to the acquisition of activating mutations in the RTK pathway as an additional event leading to the development of AML. Ten of 15 patients with mutations in the RTK pathway relapsed, compared with only 3 of 19 patients lacking such mutations (p=0.0042). Furthermore, the 6-year disease-free survival (DFS) in patients with mutations was 12% compared to 55% in those without mutations (p=0.0344). When patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT) were censored at the date of the HSCT, patients with mutations had a 6-year DFS of 0% versus 60% for the patients without mutations (p=0.0096) These observations indicate that RTK mutations are associated with the clinical outcome in t(8;21) AML.
DNA Immunization Against Melanoma Antigens Enhances Tumor Immunity in Mouse Models of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).
Authors: Diab Adi , Perales Miguel-Angel , Cohen Adam , Hubbard Vanessa M. , Eng Jeff , Huggins Deonka , Engelhorn Manuel , Guevarra Jose A. , Alpdogan Onder , Wolchok Jedd D. , Kochman Adam , Houghton Alan N. , van den Brink Marcel
Published: April 2020

Allogeneic HSCT is an important therapy with curative potential for a variety of malignant diseases, including leukemias, lymphomas and some solid tumors. Despite significant progress in reducing treatment-related mortality, malignant relapse remains a major problem. We are developing DNA vaccines that encode gene products closely related to self-antigens, including xenogeneic DNA and mutated DNA, and have initiated clinical trials of DNA vaccines in patients with advanced melanoma or prostate cancer. Using the B16 mouse melanoma model, we have shown that immunization with human TRP-2 DNA (xenogeneic melanoma differentiation antigen - MDA) or Opt-Tyrp1 DNA (a mutated MDA related to TRP-2, which we have optimized for CD8 epitopes), can induce tumor protection, including against established tumors. We hypothesized that immunization of allogeneic HSCT recipients (or their donors) against specific tumor antigens could decrease the risk of relapse without enhancing graft-versus-host disease (GVHD). In an MHC-matched minor antigen-mismatched mouse HSCT model (LP into B6), we found that: (1) by day 28 after transplant, recipients of an allogeneic T-cell depleted (TCD)-HSCT have considerable numbers of splenic T cells, including de novo generated donor T cells, which suggests that vaccination aimed at T cells might be feasible; (2) post-HSCT DNA immunization against a single tumor antigen can provide protection from a tumor challenge that is comparable to that observed with a whole cell vaccine (B16-GM-CSF) and significantly greater than HSCT alone; (3) DNA immunization post-HSCT can induce tumor-specific CD8+ T cells of donor origin (detected by ELISPOT or intracellular cytokine flow cytometry assay); (4) the combination of donor leukocyte infusion (DLI) and post-HSCT DNA immunization further enhances tumor-free survival (Figure); (5) there is no evidence of GVHD in multiple experiments using a clinical GVHD score to monitor recipients; and (6) the effects of post-HSCT DNA immunization on both tumor-free survival and CD8+ T cell responses have been validated for two different DNA vaccine strategies (hTRP-2 + GM-CSF DNA, or Opt-Tyrp1 DNA). These results demonstrate that DNA immunization after allogeneic TCD-HSCT can induce potent anti-tumor effects without the induction of GVHD. This and similar investigations provide a strong rationale for the development of novel therapeutic strategies that combine allogeneic HSCT, post-transplant tumor vaccination and adoptive cell therapy in human clinical trials.

Figure Figure
DNA Immunization Against Melanoma Antigens Enhances Tumor Immunity in Mice Following Sub-Lethal Irradiation and Immune Reconstitution.
Authors: Diab Adi , Hubbard Vanessa M. , Cohen Adam , Huggins Deonka , Kochman Adam , Guevarra Jose A. , Engelhorn Manuel , Wolchok Jedd D. , van den Brink Marcel , Houghton Alan N. , Perales Miguel-Angel
Published: April 2020

The development of successful cancer vaccines is contingent on the ability to induce effective anti-tumor immunity against self-antigens that do not typically elicit immunity. We are working on strategies to overcome immunologic tolerance/ignorance to cancer through the use of gene products closely related to self-antigens, including xenogeneic DNA and mutated DNA, and have initiated clinical trials of DNA vaccines in patients with advanced melanoma or prostate cancer. Recent studies have demonstrated that homeostasis-driven T-cell proliferation in the reconstituted lymphodepleted host could enhance the efficacy of whole-cell tumor vaccines. We hypothesized that immunization of sub-lethally irradiated and immune reconstituted mice against specific tumor antigens could increase anti-tumor immunity. B6 mice were irradiated with 600 cGy and immediately reconstituted with 30 x 106 syngeneic splenocytes. Weekly DNA immunization using either human tyrosinase-related protein (TRP)-2 DNA (xenogeneic melanoma differentiation antigen (MDA) or Opt-Tyrp1 DNA (a mutated MDA, which has been optimized for CD8 epitopes), was begun on day 1. We have found that: (1) by day 14 after irradiation and reconstitution, recipients have considerable numbers of splenic T cells, including de novo generated donor T cells, which suggests that vaccination aimed at T cells might be feasible; (2) DNA immunization against a single tumor antigen can provide protection from a tumor challenge that is significantly greater than that observed in immunized non-irradiated hosts (Figure); (3) DNA immunization induces higher levels of tumor-specific CD8+ T cells in the irradiated and reconstituted recipients (detected by intracellular cytokine flow cytometry assay); and (4) the effects of DNA immunization after lymphodepletion with immune reconstitution on both tumor-free survival and CD8+ T cell responses have been validated for two different DNA vaccine strategies (TRP-2 DNA and Opt-Tyrp1 DNA). These results demonstrate that DNA immunization following sub-lethal irradiation and immune reconstitution can induce potent anti-tumor effects. Furthermore, they provide a strong rationale for the development of novel therapeutic strategies that combine lymphodepletion with immune reconstitution and DNA immunization in human clinical trials.
The Variable Success of Managing Bleeding Disorder Related Menorrhagia: A Multicenter Study within the Hemophilia Treatment Center Network.
Authors: Kouides Peter A. , Cohen Alice J. , DiMichele Donna , Petruzzi Mary Jane , Saidi Parvin
Published: April 2020

The growing awareness to screen women with menorrhagia for an underlying bleeding disorder has led to an increase in enrollment of identified cases in Hemophilia Treatment Centers (HTCs). However, the outcome of menorrhagia management in these women has not been well-described.

Objective : To determine the efficacy/outcome of the management of bleeding disorder-related menorrhagia in patients enrolled within the HTC network.

Patients and Methods : Registry study of initial data collection of retrospective obstetrical and gynecological events with prospective yearly follow-up for 5 years for new obstetrical and gynecological events.

Results : 99 women from five HTCs in New York-New Jersey Region II have been registered to date, mean age 38±16.3, median age 40. Diagnoses are: von Willebrand disease (VWD) in 71%; thrombocytopathy in 6%; factor deficiency in 23%. Menstrual status and outcomes presently:

⇒32/99 (32%) are not menstruating due to hysterectomy or post/peri-menopausal status; 13/99 have undergone a hysterectomy (n=10) or endometrial ablation (n=3) for menorrhagia control; in 7/10 of the women undergoing a hysterectomy for menorrhagia control, the diagnosis of VWD was previously established; 6/7of these women attempted medical treatment prior to hysterectomy

⇒68/99 (68%) are presently menstruating. Of those menstruating, menstrual status is as follows:

Normal menses: 23.5% (16/68) Menorrhagia under control: 23.5% (16/68); of these patients; 11/16 have achieved control with oral contraceptive(OC), the remaining 5 patients have achieved control with intranasal DDAVP or Amicar Menorrhagia presently: 53% (36/68); 22/36 (61%) have undergone a course of ineffective menorrhagia treatment including intranasal DDAVP in 17/22

Conclusions : The majority of women with menorrhagia enrolled in HTCs have persistent menorrhagia despite evaluation and management within the HTC network. Only a small proportion have menorrhagia control due to intranasal DDAVP use and a proportion still necessitate hysterectomy for menorrhagia control. The value in United States HTCs of hemostatic measures alone such as intranasal DDAVP in the management of bleeding disorder related menorrhagia is uncertain at this time. Further study of combined therapy (intranasal DDAVP and Amicar ± OC) and novel approaches (levonorgestrel-intrauterine device) is warranted as are efforts to procure tranexamic acid for use in the U.S.
Variability in Bleeding Pattern of Severe Hemophilia.
Authors: van Dijk Karin , Fischer Kathelijn , van der Bom Johanna G. , Mauser-Bunschoten Eveline P. , Roosendaal Goris , van den Berg H. Marijke
Published: April 2020

Introduction

The aim of this study was to describe variability in bleeding pattern in a single center cohort of severe hemophilia patients treated with prophylaxis.

Methods

At the Van Creveldkliniek, all patients with severe hemophilia born between 1944 and 2002 were followed from 1972 onwards. Data on bleeding characteristics and treatment were collected yearly and Pettersson scores were performed with five-year intervals. Prophylactic dose was adjusted always according to bleeding pattern. Since the number of accepted bleeds has decreased over the years, each treatment dependent indicator was described stratified for age. Age specific quartiles for the different indicators were used as arbitrary cut-off values.

Results

Data on prophylaxis were available for 247 patients, and a total of 2760 follow up years on prophylaxis were collected. Variability in treatment characteristics is shown in Table 1.

Treatment characteristics and clinical manifestations per age group

Year of birth 1985–2002 1968–1985 1944–1968 Values are means (interquartile range) n 66 87 94 Follow-up per patient (yr) 6.6 18.0 22.6 Age at first joint bleed (yr) 1.8 (0.7–3.7) - Annual clotting factor use (IU/kg/yr) 2790 (2282–3321) 1989 (1654–2350) 1458 (1119–1790) Joint bleeds per year 2.6 (1.0–3.7) 2.9 (1.3–5.1) 4.5 (2.2–9.9) Pettersson score (max 78 points) 0.0 (0.0–2.5) 15.1 (6.0–22.0) 44.8 (35.5–56.2)

The variation in annual clotting factor use was used as a marker of bleeding pattern. Age at first joint bleed was inversely related to annual clotting factor use in the youngest age group. Using cut off levels of the 25th and 75th percentiles (P25 and P75) of age at first joint bleed and annual clotting factor use, 10% of patients were identified as patients with a milder bleeding pattern (i.e. age at first joint bleed above P75 and annual clotting factor use below P25) and 9.3% as patients with a more severe bleeding pattern. Using annual clotting factor use and joint bleed frequency in the middle group, 8.0% of patients were identified as patients with a milder phenotype and 10.3% as patients with a more severe bleeding pattern. In the oldest group, Pettersson scores were positively associated with annual clotting factor use. Using these parameters, 14% of the patients were identified as patients with a milder bleeding pattern. Due to the ceiling effect of the Pettersson score, these parameters could not be used to identify patients with a more severe bleeding pattern.

Conclusion

There is considerable variation in bleeding pattern of patients with severe hemophilia. Combinations of bleeding and treatment characteristics could be used as indicators of phenotype identifying 8 to 14% patients with a milder bleeding pattern and 9 to 10% of patients with a more severe bleeding pattern.
The Impact of Latent Epstein-Barr Virus Infection on Outcome in Children and Adolescents with Hodgkin’s Lymphoma.
Authors: Claviez Alexander , Tiemann Markus , Lueders Heike , Parwaresch Reza , Schellong Guenther , Doerffel Wolfgang
Published: April 2020

The prognostic significance of latent Epstein-Barr virus (EBV) infection in Hodgkin’s lymphoma (HL) is debated controversially. Especially in the pediatric age group, no conclusive data are available due to small series.

842 children and adolescents (55% male) with a median age of 13.7 years (range, 2–20) from consecutive pediatric DAL/GPOH multicenter treatment studies HD-90 and HD-95 were studied for the presence of latent EBV infection in Hodgkin and Reed-Sternberg cells by immunostaining against latent membrane protein 1 (LMP-1). Histology subtypes were as follows: nodular sclerosis (NSHL) 549, mixed cellularity (MCHL) 190, lymphocyte predominance (NLPHL) 90, lymphocyte depletion (LDHL) 6, lymphocyte-rich classical HL (LRCHL) 5, not specified 2. 88 patients had stage I, 470 had stage II, 172 had stage III and 112 had stage IV. B symptoms were present in 274 patients (33%). LMP status was compared with clinical parameters and established risk factors.

A total of 263 patients (32%) were LMP positive. EBV infection correlated with gender (male 39% vs. female 23%; p<.001), histological subtype (MCHL 69% vs. NSHL 22% vs. NLPHL 6%; p<.001) and age (<10 years 67% vs ≥10 years 28%, p<.001. With a median follow-up of 4.9 years (0.3–12) 820 patients (97%) are alive. Probability of overall survival at 10 years (±SD) for EBV negative and positive patients was 98±1% and 95±1%, respectively (p=.017 by log-rank test). Probability of failure-free survival (FFS) in LMP positive and negative patients was 89±2% and 84±4%, respectively (p=.86). With respect to LMP status, a negative effect of latent EBV infection on overall survival became evident only for patients treated for advanced stages (p=.003), those with nodular sclerosis subtype Bennett II (p=.02) and B symptoms (p=.05). In a multivariate regression analysis, allocation to treatment group (RR=3.7) and LMP positivity (RR=3.01) were independent factors for overall survival and presence of B symptoms (RR=2.4) for FFS.

Under current highly effective polychemotherapy with or without involved field radiotherapy in pediatric HL, latent EBV infection has no influence on FFS in univariate and multivariate analysis. LMP positivity, however, seems to be associated with an inferior overall survival in some subgroups.
Effects of Biochemically-Confirmed Smoking Cessation on Periperhal Leukocyte Count.
Authors: Abel Gregory A. , Hays J. Taylor , Decker Paul , Croghan Gary , Kuter David J. , Rigotti Nancy A.
Published: April 2020

BACKGROUND: Previous work has demonstrated that smokers have higher white blood cell counts than nonsmokers, but the effect of biochemically-confirmed smoking cessation on peripheral leukocyte counts has not been demonstrated in a large prospective study.

METHODS: Subjects were healthy smokers enrolled in a trial of sustained-release bupropion for relapse prevention after smoking cessation. White blood cell (WBC) counts and absolute neutrophil counts (ANC) were measured at baseline, week 7 and week 52. Smoking status was assessed at weeks 7 and 52 by self-report and biochemically confirmed with expired air carbon monoxide levels. Multivariate analyses compared changes in WBC count and ANC between smokers who did and did not stop smoking, adjusting for treatment group, age, sex, and body mass index.

RESULTS: Of 784 smokers enrolled, 461 had biochemically-confirmed tobacco abstinence after 7 weeks of bupropion; 429 were randomly assigned to receive continued bupropion or placebo until week 52. At baseline, the mean WBC count was 8.4 x 109/L (SD ±2.3) and the mean ANC was 5.3 x 109/L (SD ±1.9). The baseline WBC count and ANC were higher in smokers with greater tobacco exposure, measured by daily cigarette consumption and serum cotinine level. Between baseline and week 7, there was a significantly larger decrease in WBC count in continuously abstinent subjects compared with continuing smokers (adjusted p =.031). At 52 weeks, biochemically-confirmed continuously abstinent subjects, as compared with continuing smokers, had a greater decline from baseline in WBC count (1.2 x 109/L versus 0.1 x 109/L, p<0.001) and ANC (0.1 x 109/L versus 0.2 x 109/L, p<0.001).

CONCLUSION: The WBC count and ANC of healthy smokers increase with increasing tobacco exposure. Biochemically-confirmed tobacco abstinence leads to a rapid and sustained decrease in WBC count and ANC, possibly reflecting a decrease in an underlying state of tobacco-induced inflammation.
A Novel Non-Cleavable Cell Surface Mutant of CD40-Ligand Induces Anti-Leukemic Immune Response and Prevent Systemic Inflammatory Reaction.
Authors: Kato Kazunori , Masuta Yukari , Tomihara Kei , Sasaki Katsunori , Hamada Hirofumi
Published: April 2020

CD40-ligand (CD40L), a member of the TNF family, is expressed transiently on activated CD4-positive T cells and mediates cognate interaction between T cell and antigen-presenting cell (APC) such as dendritic cells. We and other investigators have reported previously that transduction of human leukemia cells with adenovirus encoding full-length CD40-ligand resulted in upregulation of immune costimulatory molecules, enhance APC activity and generation of CTL to leukemia B cells. However, CD40L is cleaved to a soluble form (sCD40L) by metalloproteases and high levels of sCD40L may contribute to the systemic inflammatory diseases including systemic lupus erythematosus and rheumatoid arthritis, suggesting a potentially deleterious side effect of CD40L gene therapy. In this study we generated a non-cleavable mutant of CD40L to develop a potentially less toxic molecule for CD40L gene therapy. Four mutants of human CD40L (termed CD40Lm1, m2, m3 and m4) with point mutation of amino acids from E112 to P120 (suggested cleavage site) were created by RT-PCR and cloned into retrovirus and adenovirus vectors. These four mutants of CD40L were transduced into tumor cells and assessed sCD40L production by ELISA, demonstrating that all four mutants resulted in a fully non-cleavable mutant of CD40L. We also confirmed that CD40L mutants could stimulate CD40-positive B and dendritic cells and induce phenotypic alterations and IL-12 production. In order to examine systemic side effect of CD40L, we transplanted tumor cells expressing wild-type (CD40Lwt) or non-cleavable mutant of CD40L (CD40Lm3) in nude mice and have observed for one month period. Two weeks after transplantation, mice with tumors expressing CD40Lwt exhibited arthritis, systemic edema and slight diarrhea, but CD40Lm3 did not induce any systemic inflammatory effect. We also found increased plasma levels of sCD40L (>800 pg/ml) in mice transplanted with CD40Lwt transfectant but not in CD40Lm3 transplanted mice. Additionally, mice with CD40Lwt resulted in increased number of infiltrating mononuclear cells in the liver and kidney, whereas no inflammatory cells were observed in the liver of mice with CD40Lm3. Overall, non-cleavable mutant of CD40L is fully capable of inducing immune response with less toxic molecule and useful tool for CD40L gene therapy of leukemia and lymphoma.
A Novel p53-PP2A Signaling Pathway Is Responsible for Regulation of Hematopoiesis Induced by TGFß.
Authors: Takimoto Rishu , Kato Junji , Takada Koichi , Takahari Daisuke , Niitsu Yoshiro
Published: April 2020

TGFβ has been reported to play an important role in regulating the proliferation of hematopoietic cells. Dysfunction of signal transduction pathway of TGFβ may result in leukemogenesis. It has been generally accepted that Smads mediate the TGFβ signaling to inhibit cell proliferation, while several Smad4-defective cell lines have been shown to retain certain levels of responsiveness to TGFβ, suggesting an additional TGFβ signaling pathway for cell cycle arrest. We previously demonstrated the evidence for a novel signal pathway for G1 arrest linking TGFβ and p53/Protein phosphatase 2A (PP2A) in CD4+ T lymphocyte and megakaryocytes (ASH, San Diego, 2003). We also showed that, upon TGFβ stimulation, PP2A was transiently recruited by heteromeric complex of TGFβ receptors (TβRs), released into cytosol, and activated. Subsequently, PP2A dephosphorylated p53 to import it into nucleus by interacting with importin-α/β, followed by p21Waf1 induction. In this study, we extended our observation to explore TGFβ-induced growth suppression is required for increment of PP2A activity by using SiRNA against PP2A. When PP2A of CD4+ lymphocytes was knocked-down by specific SiRNA for PP2A, the recruitment of PP2A by TβRs and TGFβ-induced growth suppression as well as nuclear import of p53 were inhibited. As a result, TGFβ stimulation induces PP2A activation and subsequently dephosphorylated-p53 was translocated into nucleus. Thus these results further define a novel pathway of TGFβ-induced growth suppression linking p53-PP2A, and suggest that dysfunction of p53 may result in the escape from growth inhibitory signal of TGFβ in hematological malignancies.