bluuebunny/crossref_metadata_2025 huggingface dataset:
Fatal Pulmonary Embolism and Cancer - a Post Mortem Study.
Authors: Cohen Alexander T. , Weston Gabriel J. , Tasker Andrew J. , Fellows Greg A. , Wilmott Rosalind
Published: October 2019

Introduction: During the period of 1965–1990 and 1991–2000, two separate analyses were carried out at King’s College Hospital, London, taking a retrospective review of all autopsy reports during this time. The initial aims of these reviews were to determine the number of deaths from autopsy-confirmed fatal pulmonary embolism (FPE) in hospitalised patients, and the clinical characteristics of these patients.

Methodology: Cases for inclusion were identified and data derived from manually examining copies of post-mortem reports. The data form recorded the patient gender, age, race, height, weight and surgical status. In addition, evidence of DVT, myocardial infarction (MI), stroke, chronic obstructive pulmonary disease (COPD), cardiac failure, infection or cancer was recorded from the autopsy report. Cases of fatal PE were classified as occurring in either surgical (if death occurred within 8 weeks of surgery), or non-surgical patients. The outcome of fatal pulmonary embolism was recorded as the cause of death only when the post-mortem stated that embolism was the main or contributing cause of death and identified emboli present either in the main pulmonary trunk or in the proximal right or left pulmonary arteries formed from the bifurcation of the main trunk. Emboli found in the distal pulmonary arteries after further division of the right and left pulmonary arteries were not included. Emboli derived from bone marrow, fat, tumour, or amniotic fluid were also excluded from the analysis. Ethical approval for the study was obtained from the local research ethics committee. The identity of deceased patients was protected by the use of code numbers on the data forms.

Results: Over the 35 year period there were 45,575 hospital deaths and 16,862 (37%) post-mortems. FPE was recorded as cause of death in 1,040 (6.2%) adult patients. Of these 85% (n=885) were in non-surgical patients, and 15% (n=155) in post-operative patients. Of the fatal pulmonary emboli, 347 of 1040 (33%) had either cancer (active malignancy) or a past history of cancer. Of this cancer group, 93% (n= 323) had an active malignancy, with 7% (n= 24) giving a past history of cancer. Cancer sites were gastro-intestinal in 34% (n= 119), lung 24% (n= 82), urological 10% (n= 33), gynaecological 8% (n= 29) and breast 8% (n= 28), 5% haematological (n= 17) endocrine 5% (n= 16), and dermatological 1% (n= 3). The majority of active cancers were found within the peritoneal cavity (60%, n= 194). Infection was the additional risk factor that was most prevalent in all those with FPE occurring in 32% (n=334), 76.3% (n=808) had one or more additional risk factors.

Conclusions: Most fatal pulmonary emboli occur in non-surgical patients, cancer is a very common association (33%). Active cancer is seen in 93% of all FPE deaths associated with cancer. Intra-peritoneal cavity tumours are the commonest type to be associated with FPE. Infection is a common associated risk factor.
Improved Leukemia-Free Survival after Post-Consolidation Treatment with Histamine Dihydrochloride and Interleukin-2 in AML: A Randomized Phase III Trial.
Authors: Brune Mats , Castaigne Sylvie , Catalano John , Gehlsen Kurt , Hofmann Wolf-Karsten , Ho Anthony , Hogge Donna , Nilsson Bo , Or Reuven , Rowe Jacob , Simonsson Bengt , Spearing Ruth , Stadtmauer Edward , Szer Jeff , Wallhult Elisabeth , Hellstrand Kristoffer
Published: October 2019

Background: Histamine dihydrochloride (HDC) potentiates immune-based therapies by protecting pivotal anti-neoplastic lymphocytes from phagocyte-induced suppression. Previous results in AML indicated that post-consolidation treatment with HDC and low-dose interleukin-2 (IL-2) is safe and feasible, and that the combination may prolong leukemia-free survival (LFS).

Aims: The primary objective was to determine if post-consolidation treatment with IL-2 and HDC could improve LFS for AML pts in complete remission (CR). Secondary objectives included overall survival (OS), LFS in CR1 and CR>1 subgroups, and safety.

Methods: This international, randomized, open-label, phase III study was conducted at 92 centers. From June 1998 to October 2000, 320 AML pts [148 females, 172 males, median age 57 (18–84) yrs] were enrolled in CR after completion of standard consolidation therapy. Pts were stratified by CR1 or CR>1 and randomized to either treatment (n=160) or no treatment (control, n=160) arms. The treatment was self-administered at home and included 10 cycles of low-dose IL-2 (aldesleukin, Chiron Corp) 18 000 U/kg, sc bid plus HDC (Maxim Pharmaceuticals) 0.5 mg sc bid. For cycles 1–3, each cycle comprised 3 wks of treatment and 3 wks of rest, whereas in cycles 4–10 the rest periods were 6 wks. The study arms were well balanced with respect to age, sex, karyotype-based risk, time from CR to inclusion and frequency of secondary leukemia. Pts were followed for relapse and survival using an identical assessment schedule until 3 yrs after last enrollment. All efficacy analyses were intent-to-treat.

Results: The median follow-up of living pts was 46 months. For the primary endpoint, treatment with HDC/IL-2 increased LFS in the entire study population (CR1/CR>1, n=320, p=0.026 stratified log-rank test). There was no significant difference in OS (p=0.33). In the analysis of pre-stratified subgroups, one pt was excluded. For CR1 pts (n=262), HDC/IL-2 significantly improved LFS (p=0.011, log-rank test), with 3-year Kaplan-Meier estimates of 26% (control group) and 40% (treatment group). The difference in OS did not reach significance (p=0.16). For CR>1 pts (n=57), outcome was not affected by treatment. Side effects attributable to IL-2 included fever and local inflammatory reactions. HDC treatment induced symptoms of transient vasodilatation. Serious adverse events occurred in approximately 20% of pts in both groups. There were no treatment-related deaths.

Conclusions: For AML pts in CR, post-consolidation therapy with HDC and IL-2 was safe and significantly improved LFS compared to standard of care. The benefit observed of HDC/IL-2 treatment appears to be explained by a reduction of relapses in CR1 pts.
Human Atheromatous Plaques Stimulate Thrombus Formation by Activating Platelet Glycoprotein VI.
Authors: Reininger Armin J. , Brandl Richard , Penz Sandra , Goyal Pankaj , Rabie Tamer , Rother Enno , Goetz Christine , Engelmann Bernd , Farndale Richard , Nieswandt Bernhard , Siess Wolfgang
Published: October 2019

Lipid-rich atherosclerotic plaques are vulnerable, and upon disruption trigger intraarterial thrombus formation. Tissue factor activating blood coagulation is viewed as the major prothrombotic stimulus within the plaque. We isolated lipid-rich atheromatous plaques from 50 patients with carotid artery stenosis and identified morphologically diverse collagenous structures within in the plaques. They stimulated platelet adhesion, dense granule secretion and aggregation, and triggered thrombus formation in hirudin-anticoagulated blood under arterial flow conditions. Even in fully anticoagulated flowing blood, i.e. in the absence of tissue factor-mediated coagulation, plaques were able to activate platelets. Thrombus formation was more rapid and stable when blood was anticoagulated with a low concentration of heparin, but, although fibrin was detectable within the thrombus, the initial step was always single platelet adhesion and not fibrin formation. In contrast, absence or inhibition of the platelet collagen receptor glycoprotein VI prevented platelet adhesion to atheromatous plaques and thrombus formation. We thus identified platelet glycoprotein VI as being essential and sufficient to mediate plaque-induced thrombus formation. Our study suggests a novel anti-thrombotic strategy to prevent and treat atherothrombosis in patients with vulnerable atherosclerotic plaques.

Figure Figure
Cannabinoids Inhibit the CXCL12-Induced Migration of T Lymphocytes.
Authors: Ghosh Sharmistha , Ganju Ramesh K. , Groopman Jerome E.
Published: October 2019

Cannabinoids are both exogenous compounds produced by Cannabis sativa, known for their psychoactive properties, and a family of related endogenous ligands whose functions have been recently characterized. The reported effects of cannabinoids on the immune system include inhibition of T lymphocyte proliferation, altered production of cytokines by T helper cells and decreased antibody formation by B cells. Cannabinoid receptors have been detected in several immune cells, including monocytes, macrophages, basophils and B and T lymphocytes. CB2 (Cannabinoid Receptor 2) appears to be the predominant cannabinoid receptor in the immune system, while the CB1 receptor is found primarily in the brain. We observed high levels of CB2 expression in the Jurkat T lymphocyte cell line, while others have reported expression of the CB2 receptor in CD8+ T lymphocytes. Here, we studied the ability of synthetic and endogenous cannabinoids to modulate a key function of T lymphocytes: chemotactic migration in response to the chemokine CXCL12 (also known as stromal cell-derived factor-1). We observed a 40–50% inhibition of CXCL12-induced chemotaxis in Jurkat T cells with the endogenous cannabinoid anandamide (at 15 μM concentration) and with the synthetic cannabinoids CP55,940 and Win-55,212–2 (each at 10 μM concentration). Inhibition occurred in a dose-dependent manner, indicating that it is a regulated, receptor-mediated process. Further studies showed that the inhibition of migration involved, in part, significant down-regulation of the CXCL12 receptor, CXCR4. We are currently elucidating the signaling cascade whereby endogenous cannabinoids may limit T cell migration in response to CXCL12. Thus, cannabinoids may be novel mediators of immunosuppression when clinically desired.
Stromal Cell-Derived Factor (SDF-1) Mediates Stem Cell Mobilization and Homing to the Kidney after Acute Renal Failure.
Authors: Toegel Florian E. , Isaac Jorge , Hu Zhuma , Weiss Kathy , Westenfelder Christof
Published: October 2019

The chemokine stromal cll-derived factor-1 (SDF-1) and its receptor CXCR4, expressed by hematopoietic stem cells (HSC) and other cells, represents a major mediator-system for G-CSF-induced hematopoietic stem cell (HSC) mobilization and stem cell (SC) homing to the bone marrow (BM) after BM transplantation. HSC have been shown to contribute to repair processes in kidneys damaged by ischemia and HSC as well as endothelial progenitor cells (EPC) are mobilized in response to kidney damage. The signals mediating this process are currently unknown. We tested the hypothesis that SDF-1 mediates both mobilization and homing of SCs into the kidney with acute renal failure (ARF) and may thus be crucial to SC-mediated organ repair. SDF-1 is expressed in normal kidneys and upregulated in ischemically injured (60 min bi- or unilateral renal pedicle clamp) mouse kidneys (FVB strain) as evaluated by immunohistochemistry, in-situ hybridization, quantitative RT-PCR and ELISA. Post ARF SDF-1 was upregulated in sublethally injured proximal tubular cells (PTC), peripheral blood levels of SDF-1 rose, and circulating CD34+ and Colony-forming Units of Cell numbers as well as EPCs increased significantly. In vitro chemotaxis of SC from the top of transwell inserts towards the bottom well, containing cultured PTC that exhibited augmented SDF-1 expression post partial ATP depletion, increased significantly, a response that was abolished when SC cells were preincubated with CXCR4 antibody. Injected, CFDA-labeled, BM cells were only recruited to ischemic kidneys (bi- or unilateral), a response that was also blocked after SC pre-incubation with CXCR4 antibody. Our data show that there is increased renal SDF-1 expression after ARF and secretion into the circulation, which, in turn, stimulates HSC and EPC-mobilization into the circulation and directs homing into the post-ischemic kidney. Therefore, both circulating and administered SC and other CXCR4-expressing cells can be recruited to the injured kidney, where they, as we showed previously, are able to greatly support effective tissue repair, functional recovery and animal survival following ARF.
The Use of Aprotinin to Reduce Transfusion Requirements in Children Undergoing Cardiac Surgery: A Meta-Analysis.
Authors: Arnold Donald M. , Fergusson Dean A. , Chan Anthony K. , Cook Richard J. , Fraser Graeme A. , Lim Wendy , Heddle Nancy M. , Blajchman Morris A. , Cook Deborah J.
Published: October 2019

Background. Aprotinin, an antifibrinolytic medication, reduces the requirement for allogeneic red blood cell (RBC) transfusion following elective cardiac surgery in adults. The effect of aprotinin for this indication in children is unclear.

Objective. To determine if intravenous aprotinin, administered perioperatively to patients <18 years of age undergoing cardiac surgery with cardiopulmonary bypass (CPB), reduces post-operative allogeneic blood transfusion requirements (RBCs or whole blood transfusions), compared to children who do not receive aprotinin.

Methods. Systematic review of all relevant randomized controlled trials. We searched MEDLINE, EMBASE, the Cochrane Library and published abstracts. In duplicate, 2 investigators independently selected studies based on explicit inclusion and exclusion criteria; disagreement was resolved by consensus. Using a validated scale, and blinded to outcomes, authors and journal, 2 investigators independently rated the methodological quality of each selected study. Study results were combined using the random effects model to obtain a pooled relative risk.

Results. Of 547 studies identified, 11 studies published from 1993 to 2003 were included. Four of the 11 studies were judged to be of good methodological quality. Overall, aprotinin was associated with a lower proportion of children requiring transfusion, but this was not significant (relative risk 0.73, 95% CI: 0.51–1.04). We found significant heterogeneity among studies (I2= 73.0%, p= 0.001), attributed largely to methodological quality. When we pooled the 4 studies of good methodological quality, aprotinin significantly reduced the proportion of children requiring transfusion (relative risk 0.60, 95% CI 0.38–0.95). Aprotinin had no effect on the total amount of blood transfused or on the volume of chest tube drainage.

Conclusions. Aprotinin reduced the proportion of children exposed to allogeneic blood transfusion following cardiac surgery by 40% in randomized trials of good methodological quality. Additional high quality studies are needed to confirm the potential benefit of aprotinin in this population.
Impact of HLA Mismatch on the Incidence of Acute GVHD and Rejection after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation (RICT).
Authors: Teshima Takanori , Matsuo K. , Matsue K. , Kawano F. , Taniguchi S. , Hatanaka K. , Nakao S. , Tanimoto M. , Hara M. , Eto T. , Wake A. , Abe Y. , Ohno Y. , Takemoto Y. , Harada M. , Takahashi S. , Ishida Y. , Kanda Y. , Imamura M. , Kasai M. , Takaue Y.
Published: October 2019

HLA incompatibility between the donor and recipient is the most critical factor governing the incidence of rejection and GVHD after conventional allogeneic stem cell transplantation. But the impact of HLA disparity on GVHD and graft rejection after RICT remains to be elucidated. We retrospectively analyzed the outcomes of 437 patients who underwent bone marrow (n=95) or peripheral blood stem cell RICT (n=342). The numbers of patients who received a graft from a HLA-matched (275 from siblings, 11 from family members, and 54 from unrelated donors), one-locus-mismatched, 2- or 3-loci-mismatched donor were 340, 65, and 32, respectively. The HLA-matched group included significantly higher population of patients who received cyclosporine alone for GVHD prophylaxis. The overall cumulative incidence of grade II-IV acute GVHD was 40% for all subjects. It was 38% (95% CI; 33%–43%) in recipients of HLA-matched donors, 43% (95% CI; 31%–54%) in those of one-locus-mismatched donors, and 54% (95% CI; 37%–68%) in those of 2–3-loci-mismatched donors. A Cox regression model adjusted for potential confounders including GVHD prophylaxis demonstrated that 2-3 loci-mismatch was identified as an independent risk factor of grade II-IV acute GVHD (Table). Use of antithymocyte globulin was identified as an independent better protective factor for GVHD (HR;0.66, p=.003). Cumulative incidence of rejection was significantly higher after one-locus mismatch RICT (Table) and the risk tended to increase in relation to an increase of HLA disparity. Malignant disease was identified as an independent prognostic factor for rejection. In patients with hematologic malignancies, overall survival (OS) of recipients of 2–3-loci-mismatched RICT at 1 year (38%, 95%CI; 21%–54%) was significantly worse than that after HLA-matched RICT (65%, 95%CI; 59%–70%). By contrast, there was no statistical difference in the incidence of grade II-IV acute GVHD and OS between HLA-matched RICT and one-locus-mismatched RICT. Multivariate analysis demonstrated 2–3-loci-mismatch (Table) and high-risk disease (HR; 2.3, p=.001) as independent risk factors for OS. Thus, HLA incompatibility between the donor and recipient is an important risk factor for rejection, acute GVHD and overall survival after RICT. Therefore RICT from a one-locus-mismatched donor may represent an effective alternative approach in patients lacking HLA-matched sibling donors.

multivariate analysis n acute GVHD Rejection OS HR (95%CI) p HR (95%CI) p HR (95%CI) p match 340 1.0 1.0 1.0 1-mismatch 65 1.4 (0.9–2.2) 0.20 4.5 (1.1–17.9) 0.03 1.0 (0.6–1.6) 0.88 2–3-mismatch 32 2.2 (1.2–4.1) 0.02 7.0 (0.8–64.8) 0.08 3.3 (1.8–6.2) <0.001
Outcomes Analysis of Patients Surviving ≥2 Years after Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation (AHSCT).
Authors: Sobecks Ronald , Kalaycio Matt , Pohlman Brad , Andresen Steven , Rybicki Lisa , Kosar Jennifer , Serafin Mary , Sands Karen , Lalli Christy , Brown Stacey , Kuczkowski Elizabeth , Bolwell Brian
Published: October 2019

We have previously demonstrated the value of evaluating patients with NHL undergoing autologous HSCT at 2 years post-transplant; high grade lymphoma patients in CCR 2 years post-transplant are cured, whereas patients with intermediate and low grade histologies are at ongoing risk of relapse. We elected to perform a similar analysis of AHSCT patients. We retrospectively reviewed 161 consecutive AHSCT recipients who had a minimum of 2 years follow-up and who were alive 2 years post AHSCT. Patients received their transplants from 7/1988 to 7/2002. Median age was 36 years; 75% had myeloid malignancies and 25% lymphoid; AML was the most common diagnosis (39%) followed by CML (29%), ALL (14%), NHL (10%), MDS (6%), and myeloma (2%). 98% received bone marrow alone as the hematopoietic stem cell source; 98% received a busulfan/cyclophosphamide-based preparative regimen. 129 (80%) had a matched related donor. Of those patients alive 2 years post AHSCT, 31 patients (19%) have subsequently died, with a median follow up of 6 years. Relapse was the cause of death in 29%; GVHD in 35%; secondary malignancy in 7%; infections in 16%. Patients with myeloid malignancies faired better than those transplanted with lymphoid malignancies, as shown below; (p= 0.04)

Figure Figure

When we analyzed the pts who developed their first episode of any chronic GVHD or extensive chronic GVHD there were no significant differences between lymphoid and myeloid malignacy patients. Patients receiving a matched sibling donor transplant did not have a plateau in their survival curve, and it continued to inexorably decline. Patients receiving a matched unrelated donor transplant did have a plateau of their survival curve if patients survived at least 3 years (p=0.03). We conclude that the significant majority of patients alive after AHSCT do well with extended follow-up. The graft vs. tumor effect appears to be more robust in myeloid malignancies as compared to lymphoid malignancies. Although most of these patients do well with extended follow-up, the lack of a plateau in the survival curve for patients receiving a matched sibling donor allogeneic BMT warrants additional study.
A Role for Plasma Urokinase Plasminogen Activator Receptor (uPAR) in Thrombosis in Paroxysmal Nocturnal Hemoglobinuria (PNH).
Authors: Sloand Elaine M. , Mainwaring Lori , Wong Elaine , Tang Yong , Horne McDonald K. , Young Neal S.
Published: October 2019

Venous thrombotic events are responsible for serious morbidity and the most frequent cause of death in patients with paroxysmal nocturnal hemoglobinuia (PNH). Urokinase plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-linked protein which is expressed on neutrophils and endothelial cells and mediates endogenous thrombolysis through a urokinase plasminogen activator (uPA)-dependent mechanism. On the cell surface, pro-uPA binds to uPAR via its growth factor domain, promoting the conversion of pro-uPA to active uPA. Active uPA in turn cleaves the proenzyme plasminogen, yielding active plasmin. Previous work in PNH patients has shown that the membrane form of uPAR is decreased or absent on granulocytes, and soluble uPAR levels are increased in patient’s plasma. In the current study of 45 patients with PNH and 20 normal controls, we demonstrate that plasma suPAR concentrations correlated directly with the proportion of neutrophils lacking CD16, another GPI-anchored protein (R2=0.9), and that levels are 4-10-fold greater in PNH than in controls. Patients with a history of thrombosis had higher soluble uPAR levels (in stored samples pre-dating the thrombotic event) than did patients without a history of thrombosis (mean 2.2 ng/mL compared to 0.5 ng/mL; p=0.0001). suPAR levels returned to normal in all of five patients undergoing successful bone marrow transplantation. In normal persons soluble uPAR may be generated by alternative splicing or proteolytic cleavage. In vitro we demonstrated release of suPAR from PNH hematopoietic cells: supernatants obtained from PNH CD34 cells cultured in liquid media with standard growth factor in three experiments showed suPAR levels 20 fold greater than those obtained from normal CD34 cells. Platelets also appeared to be a substantial source of suPAR: uPAR was expressed on the surface of ADP-activated CD59-positive but not CD59-negative platelets from PNH patients. By an ELISA specific for suPAR and by immunoblot, we showed that substantial quantities of suPAR were released following collagen-activation of PNH but not normal platelets. Plasma from PNH patients contained much greater quantitites of suPAR than did peripheral blood cells from those patients. Theoretically, suPAR might bind to plasma uPA, preventing its interaction with the active membrane-anchored form. In vitro, we demonstrated dose-dependent decreases in the activity of single-chain urokinase following the addition of recombinant suPAR, with loss of 25% of the activity after the addition of 0.45nM suPAR. The absence of leukocyte and platelet plasma membrane associated uPAR and high levels of soluble uPAR in the circulation may be useful in predicting thrombosis and suggest mechanisms of anticoagulation in these unusual patients.
Decreased Incidence of Clonal Evolution to Myelodysplastic Syndrome with Monosomy 7 in Children with Aplastic Anemia Following Reduced Duration G-CSF Therapy and Early Indication for BMT from an Alternative Donor.
Authors: Yagasaki Hiroshi , Kazuko Kudo , Kato Koji , Matsumoto Kimikazu , Horibe Keizo , Kojima Seiji
Published: October 2019

A serious complication of aplastic anemia (AA) is an evolution to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In a previous nationwide retrospective study, development of MDS/AML with monosomy 7 was observed in a significant proportion (22%) of AA children who were treated with cyclosporine and G-CSF. In an examination of the risk factors for such malignant transformation, our previous prospective study suggested a close relationship between use of G-CSF and secondary MDS/AML in nonresponders to immunosuppressive therapy (IST). However, a recent nationwide survey showed a prominent decreased in incidence of secondly MDS/AML. Here, we attempt to address the question as to why the incidence of malignant transformation-monosomy 7 has recently decreased in Japan. Between 1988 and 2003, 60 patients (30 male, 30 female; median age, 9 years; range, 0–17 years) at two hospitals received IST consisting of antithymocyte globulin (ATG) and cyclosporine with or without G-CSF and survived more than 1 year after diagnosis of AA. Because 13 IST nonresponders received alternative donor BMT as salvage therapy, we evaluated the incidence of malignant transformation in the 47 patients who did not received BMT. Because all patients were enrolled in the prospective multicenter study since 1993, which was the year that the Japan Marrow Donor Program was established, we compared the incidence of MDS/AML among children who were diagnosed between 1988 and 1992 (group A: n=27, with all but one receiving G-CSF), and those who were diagnosed between 1993 and 2003 (group B: n=20, with 12 receiving G-CSF). MDS with monosomy 7 was observed in 7 of the 27 patients (26%) in group A, and all of these received G-CSF, with the evolution occurring between 17 and 102 months after diagnosis, while no of the 20 patients in group B developed MDS/AML during the follow up of 1 to 11 years (median of 6 years; p=0.014). The response rate to IST did not differ significantly between group A and group B (50% vs 60%, p=). Three of 14 (21%) IST nonresponderss in group A and 10 of 12 (83%) non responders in group B received BMT from an alternative donor (p<0.01). The duration of G-CSF therapy was significantly longer in group A than in group B (186 days vs 56 days, p<0.01).
Enhanced DNA Repair Via Fanconi Anemia/BRCA Pathway Is Involved in Melphalan-Resistant Myeloma Cells.
Authors: Chen Qing , Van der Sluis Pieter C. , Hazlehurst Lori , Dalton William S.
Published: October 2019

Melphalan, a DNA crosslinker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Interstrand cross-links (ICL) are amongst the most toxic types of DNA damage; therefore, DNA cross-linking agents are important drugs in cancer treatment. Unfortunately, although most patients respond to standard and high dose melphalan therapy, eventually patients acquire drug resistance. Acquired melphalan resistance has been associated with reduced DNA crosslinks, elevated levels of glutathione and increased radiation survival. However, mechanisms associated with resistance are not well understood.

Evidence has accumulated to suggest that ICL repair contributes to the melphalan resistance. In this study, we compared the gene expression profile (GEP) of the melphalan-resistant myeloma cell line, 8226/LR5 to the 8226/S drug sensitive cell line, and found genes involved in FANC/BRCA DNA cross-link repair pathway had increased expression in drug resistant cells. The aim of our study was to determine whether FANC-BRCA pathway affects the DNA cross link repair capacity and accounts for acquired melphalan-resistance.

Using real time RT-PCR and Western Blotting, we examined the expression levels of FANC/BRCA pathway genes in two different drug sensitive and resistant cell lines: 8226/S 8226/LR5, and U266/S and U266/LR6. The results showed that increased expression of FANC/BRCA pathway genes correlated with the melphalan resistance. The formation of ICL at 5 hours after a 2 hour melphalan exposure was reduced in the LR5 compared to the drug sensitive cell 8226 using single cell comet assay. Using siRNA to knock down FANCL or FANCF in melphalan-resistant cell line LR5 reversed the drug resistance. Conversely, overexpression of FANCL or FANCF in the 8226/S drug sensitive cell line enhanced cell survival. These data show that enhanced DNA repair via Fanconi anemia/BRCA pathway is involved in melphalan-resistant myeloma cells.
A Comparison of Apheresis Cell Content Collected from the Same Donors Mobilized with Granulocyte Colony Stimulating Factor (G-CSF) Vs. a Single Injection of AMD3100.
Authors: Chakrabarti Sakti , Takahashi Y. , Read E.J. , Khuu H. , Goodwin R. , Leitman S. , Bolan C. D. , Srinivasan R. , Barrett A.J. , Calandra G. , Childs R.
Published: October 2019

AMD3100 (AMD) is a bicyclam compound that inhibits the binding of stromal cell derived factor-1 to its cognate receptor CXCR4 present on CD34+ hematopoetic progenitor cells. Recently, investigators have shown that substantial numbers of CD34+ cells are released into circulation following a single injection of AMD, making this a potentially attractive mobilization agent for both autologous and allogeneic hematopoietic cell transplantation. Mononuclear cells transplanted in G-CSF (G) mobilized allografts mediate both desirable and undesirable post-transplant events (i.e. Graft-vs-tumor and GVHD); therefore, to determine the suitability of AMD mobilized products for allografting, we assessed the cellular content of apheresis collections obtained from the same donors mobilized with AMD vs G. Between 11/03–07/04, 6 healthy donors (male=3, female=3), median age 43 years (range 18–57), underwent a 15–25 liter apheresis following G mobilization (10mcg/kg/d x 5 days); 66–143 (median 82) days later, the same donors underwent repeat apheresis 6 hours following a single subcutaneous injection of AMD (240mcg/kg); the apheresis blood volume processed after AMD mobilization was matched to the volume processed after G mobilization in 5/6 donors. Data on peripheral blood (PB) and apheresis cellular content with both mobilization agents are shown in Table-1. AMD was well tolerated (no > grade I toxicities) and effectively mobilized CD34+ cells in the majority of donors who had a previous successful G mobilization. Both drugs significantly increased PB CD34+ counts and the total WBC count, and absolute neutrophil counts (ANC), monocyte counts (AMC) and lymphocyte counts (ALC) above pre-mobilization baselines. In the PB, the increase in WBC count, ANC, and the CD34+ counts were significantly higher after G mobilization compared to AMD. In contrast, there was a trend towards a higher blood ALC increase following AMD administration compared to G. Apheresis collections mobilized with AMD contained similar numbers of mononuclear cells and CD3+ T-cells, higher numbers of CD19+ B-cells and lower numbers of monocytes and CD34+ cells compared to G mobilized collections; whether prior G mobilization negatively impacted the CD34+ cell content in AMD mobilized grafts can not be determined from this study. One patient failed mobilization with both G (CD34+ pre-count 6 /uL) and AMD (CD34+ pre-count 6 /uL); a trial investigating the efficacy of combining AMD with G in patients who fail to mobilize with G alone is currently being pursued. A detailed phenotypic analysis of lymphocyte subsets mobilized with G vs AMD3100 will be presented in a separate analysis.
Defibrotide (DF) Targets Tumor-Microenvironmental Interactions and Sensitizes Multiple Myeloma and Solid Tumor Cells to Cytotoxic Chemotherapeutics.
Authors: Mitsiades Constantine S. , Rouleau Cecile , Menon Krishna , Teicher Beverly , Iacobelli Massimo , Anderson Kenneth C. , Richardson Paul G.
Published: October 2019

Introduction: Defibrotide (DF) is a polydisperse oligonucleotide with anti-thrombotic, thrombolytic, anti-ischemic, and anti-adhesive properties, which selectively targets the microvasculature and has minimal hemorrhagic risk. DF is an effective treatment for veno-occlusive disease (VOD), an important regimen-related toxicity in stem cell transplantation characterized by endothelial cell injury. DF also augments stem cell mobilization by modulating adhesion in vivo. Because of its cytoprotective effect on the endothelium, we specifically investigated whether DF protects tumor cells from cytotoxic anti-tumor agents. Further, because of its broad anti-adhesive properties, we evaluated whether DF modulates the interaction of MM cells with bone marrow stromal cells (BMSCs), which confers growth, survival and drug resistance in the BM milieu.

Methods: In vitro studies in isogenic dexamethasone (Dex)-sensitive and resistant MM cell lines (MM-1S and MM1R, respectively) showed that DF does not attenuate the sensitivity of MM cells to Dex, the proteasome inhibitor bortezomib (PS-341), melphalan (MEL), vinca alkaloids (vincristine, vinblastine), taxanes (paclitaxel) or platinum (cisplatin), but does decrease their sensitivity to doxorubicin. These selective effects in vitro of DF in protecting tumor cells against doxorubicin and modestly sensitizing MM cells to platinum was also confirmed in solid tumor breast (MCF-7) and colon (HT-29) carcinoma cell lines. Although DF had minimal in vitro inhibitory effect on MM or solid tumor cell growth in vitro, it showed in vivo activity as a single agent and enhanced the responsiveness of MM tumors to cytotoxic chemotherapeutics, such as MEL or cyclophosphamide, in human MM xenografts in SCID/NOD mice. The in vivo single-agent activity and chemosensitizing properties of DF, coupled with its lack of major in vitro activity, suggested that DF may not directly target tumor cells, but rather modulate tumor cell interaction with BMSCs. In an ex vivo model of co-culture of primary MM tumor cells with BMSCs (which protects MM cells against conventional chemotherapy), DF alone had a only modest effect on tumor cell viability, but it significantly enhanced MM cell sensitivity to cytotoxic chemotherapy (e.g. MEL), suggesting that a major component of the biological effects of DF may be attributable not to direct targeting of tumor cells, but to modulation of the interactions that tumor cells develop with the local stromal milieu.

Conclusion: Our studies show that DF mediates in vivo anti-MM activity by abrogating interactions of MM cells with their BM milieu, thereby enhancing sensitivity and overcoming resistance to conventional chemotherapy. These data support future clinical trials of DF, in combination with both conventional and novel therapies, to improve patient outcome in MM.
No Effect of Aging on the Donor Hematopoietic Stem Cell Compartment in Allogeneic Stem Cell Transplant.
Authors: Arora Amit , Sharp John G. , Lynch J. C. , Kessinger A.
Published: October 2019

Background: Literature regarding the association of aging and stem cell numbers is conflicting. No compelling evidence exists that hematopoietic stem cell numbers decline with age. Early progenitor cells represent only a fraction of total CD34 cells. The ratio of CFU-GM/CD34 provides a relative estimate of the proportion of primitive hematopoietic stem cells in the stem cell compartment. The hypothesis is that primitive stem cell numbers decrease with age.

Methods: This study describes the relationship between the CFU-GM/CD34+ ratio (categorized as quartiles) and donor characteristics in 108 normal donors of cytokine mobilized hematopoietic stem cells for allogeneic transplant between 6/1995 and 11/2003. The relationship between the CFU-GM/CD 34+ and cell collection characteristics were also studied. Donor characteristics included: age, gender, race/ethnicity, ABO blood group, weight, height, body surface area (BSA); collection characteristics included: total number of mononuclear cells/kg (MNC), CFU-GM/kg (Colony Forming Unit- Granulocyte Monocyte), CD34+ cells/kg and the ratio of CFU-GM/CD34 collected.

Results: The median age of the donors was 42.5 years with range from 10.9 years to 71.4 years. The median CFU-GM/CD34 ratio was 0.060 with range from 0.009 to 0.248. The multivariate analysis demonstrated that higher MNC numbers were associated with a significantly higher CFU-CM/CD34 ratio (p<0.01). No significant decrease in the CFU-GM/CD34 ratio was found with increasing age indicating no general compromise in the stem cell compartment of donors with aging. A non-significant association of the CFU-GM/CD34 ratio to donor gender (p=0.071) and BSA (p=0.072) was also observed.

CONCLUSION: No significant decline in donor hematopoietic stem cell compartment was seen with aging.

Relationship of CFU-GM/CD34+ Ratio Quartiles to Donor and Collection characteristics

CFU-GM/CD34+ Ratio Quartiles First (0.009–0.034) Second (0.035–0.059) Third (0.06–0.076) Fourth (0.077–0.248) All (n=108) Abbreviation: CFU GM: Colony Forming Unit- Granulocyte Macrophage; MNC: Mononuclear cell Donor Sex (n) Male 19 19 11 18 67 Female 8 8 16 9 41 BSA sq m Median 2.04 2.02 1.81 1.96 1.99 Range (1.65–2.57) (1.59–2.35) (1.46–2.38) (1.30–2.45) (1.30–2.57) Total MNC Collected Median (10E8/kg) 5.86 5.77 6.83 8.49 6.22 Range (10E8/kg) (3.07–11.24) (2.37–13.10) (2.96–15.77) (3.31–15.48) (2.37–15.77)
Rituximab-Mediated Inhibition of the Transcription Repressor Yin-Yang 1 (YY1) in NHL B Cell Lines: Upregulation of Fas Expression and Sensitization to Fas-Induced Apoptosis.
Authors: Vega Mario I. , Huerta-Yepez Sara , Jazirehi Ali R. , Garban Hermes , Bonavida Benjamin
Published: October 2019

We have reported that rituximab triggers and inhibits anti-apoptotic gene products in NHL B-cell lines resulting in sensitization to drug-induced apoptosis (Alas et al., Clin. Cancer Res.8:836, 2001; Jazirehi et al., Mol. Cancer Therapy2:1183, 2003; Vega et al., Oncogene23:3530, 2004 ). This study investigated whether rituximab also modifies intracellular signaling pathways resulting in the sensitization of NHL cells to Fas-induced apoptosis. Treatment of the NHL cell lines (2F7, Ramos, and Raji) with rituximab (20 μg/ml) sensitized the cells to CH-11 (FasL agonist mAb) -induced apoptosis and synergy was achieved. Fas expression was up-regulated by rituximab as early as 6 h post treatment as determined by flow cytometry, RT-PCR, and Western. Rituximab inhibited both the expression and activity of the transcription repressor Yin-Yang 1 (YY1) that negatively regulates Fas transcription. Inhibition of YY1 resulted in upregulation of Fas expression and sensitization of the tumor cells to CH-11-induced apoptosis. Downregulation of YY1 expression was the result of rituximab-induced inhibition of both the p38MAPK signaling pathway and constitutive NF- κB activity. The dual roles of NF-κB and YY1 in the regulation of Fas expression were corroborated by the use of a dominant-active inhibitor of NF- κB (Ramos IκB-ER mutant) and YY1 siRNA, respectively. The role of rituximab-mediated inhibition of the p38MAPK/NF- κB/YY1 pathways, which result in both Fas upregulation and sensitization to CH11-induced apoptosis, was corroborated by the use of specific chemical inhibitors directed at various targets of these pathways. Rituximab-mediated sensitization to CH-11-induced apoptosis was executed through the Type II mitochondrial apoptotic pathway. Altogether, these findings provide a novel mechanism of rituximab-mediated signaling by inhibiting the p38MAPK/NF- κB/YY1 pathways and resulting in the sensitization of B NHL to Fas-induced apoptosis. These findings may have significant clinical implications and suggest an additional mechanism of rituximab-mediated effect in vivo in addition to CDC and ADCC.
Effects of SB559457, a Novel Small Molecule Thrombopoietin Receptor (TpoR) Agonist, on Human Hematopoietic Cell Growth and Differentiation.
Authors: Kalota Anna , Brennan Kistin , Erickson-Miller Connie L. , Danet Gwenn , Carroll Martin , Gewirtz Alan M.
Published: October 2019

Treatment of thrombocytopenia resulting from chemotherapy, radiation, or stem cell transplantation remains problematic. Hopes that TPO, the major regulator of megakaryocyte maturation, might prove clinically useful for this purpose have not been fulfilled, in part because of untoward reactions to the molecule. To avoid undesirable side effects, small molecule TpoR agonists are being developed. We have evaluated the biological effects of one such molecule, SB559457, a hydrazone compound, on hematopoietic cells. We first examined SB559457’s ability to stimulate proliferation of the recombinant human TPO (rhTPO) dependent cell line UT-7/TPO. We found a dose dependent augmentation of cell proliferation at doses between 1 and 10 μM, with maximal effects, compared to 100ng/ml of rhTPO at 10μM. We then evaluated SB559457’s growth promoting effects on normal human CD34+ cells. We again found growth stimulation to be dose dependent between 1 and 10 μM and also noted enhancement when SB559457 was combined with IL-3 and SCF. Specifically, incubation of CD34 cells in 10 μM SB559457 for 7 days lead to a 2 fold increase in cell number; incubation of cells with IL3 and SCF in addition to SB559457 lead to a 12 fold increase in cell number after 7 days. Both results were comparable to those seen with 100 ng/ml of rhTPO. Curiously, the addition of IL-6 to cultures containing IL-3 and SCF compromised the ability of SB559457, but not Tpo, to stimulate CD34+ cell growth. To determine if SB559457 stimulates megakaryocyte differentiation directly, CD34+ cells were incubated in cytokines with rhTPO or SB559457 for periods of 7–10 days, after which time cells in culture were examined for degree of polyploidization, and expression of megakaryocyte lineage markers CD41 and CD61. 14% of cells grown in SB559457 showed greater than 4N DNA content compared to 8% in rhTPO. These same cells expressed the megakaryocyte markers CD41 and CD61on 35% of cells, comparable to the results with rhTPO. Preliminary results demonstrate that incubation of CD34 cells in SB559457 maintained stem cells numbers, as assayed by NOD/SCID engraftment, for at least 4 days at a level comparable to the levels maintained by rhTPO. These results demonstrate that SB559457 is a bona fide Tpo R agonist that likely acts through activation of c-mpl, the receptor for TPO. These experiments provide a rationale for the evaluation of SB559457, or SB559457 congeners, in patients with thrombocytopenia.
Genetic Pathways in t-MDS and t-AML, a Revised Model.
Authors: Pedersen-Bjergaard Jens , Christiansen Debes H. , Andersen Mette K.
Published: October 2019

Jens Pedersen-Bjergaard, Debes H. Christiansen, Mette K. Andersen. Dept. Clinical Genetics, University Hospital Rigshospitalet, Copenhagen, Denmark. 140 unselected patients with t-MDS (n=90) or overt t-AML (n=50) were studied for chromosome aberrations, for mutations of 6 different genes and 69 patients were examined for methylation of the p15 promotor. Cytogenetically, 5q−/ −5: n=34; 7q−/ −7 but normal chromosomes 5: n=39; balanced translocations: n=23; normal karyotype: n=24; other abnormalities: n=20. Mutations of p53: n=34; AML1: n=22; FLT3: n=11; c-KIT: n=2; K- or N-RAS: n=14; BRAF: n=2; methylation of the p15 promotor: 55/69 cases.

Abnormalities of chromosomes 5 and 7 were significantly associated with t-MDS (p=0.001), whereas balanced translocations and a normal karyotype were significantly associated with overt t-AML, (p=0.0001 and p=0.006 respectively).

Mutations of p53 were significantly associated with 5q−/ −5 (p=0.001), with 17p−/−17 (p=0.0004), with a complex karyotype (p=0.001), and with MLL and AML1 amplifications. Mutations of AML1 were significantly associated with 7q−/−7 )(p=0.001) and with t-MDS progressing to t-AML (p=0.0001). Mutations of FLT3 were significantly associated with presentation as overt t-AML (p=0.0002), with a normal karyotype (p=0.004) and with previously radiotherapy only (p=0.03). RAS mutations were significantly associated with t-MDS progressing to overt t-AML (p=0.008) and borderline with a normal karyotype (p=0.07). Methylation of p15 was significantly associated with 7q−/−7 (p=0.0006).

Mutations of FLT3, RAS and c-KIT were mutually exclusive, and a synergism between AML1 and RAS mutations (22/5/14, p=0.046) and between p53 and AML1 mutations in patients with 7q−/−7 is suggested.

Based on these findings a revised model of the genetic pathways of t-MDS and t-AML (Blood2002;99: 1909–1912) is discussed.
Characteristics and Prognosis of Patients (pts) with Acute Megakaryocytic Leukemia (AMegL) Treated at M.D. Anderson Cancer Center Since 1987.
Authors: Oki Yasuhiro , Estey Elihu , Kantarjian Hagop , Cortes Jorge , Faderl Stefan , O’Brien Susan , Koller Charles , Beran Miloslav , Keating Michael J. , Pierce Sherry , Garcia-Manero Guillermo
Published: October 2019

AMegL is a rare form of acute myeloid leukemia (AML) associated with poor prognostic features. Here, we report the experience at M.D. Anderson Cancer Center from 1987 to 2003. Among 1861 pts with newly diagnosed AML (excluding APL), 38 pts (2%) had AMegL. The diagnosis of AMegL was based on (i) morphological features defined by FAB classification and (ii) immunophenotypypical positivity for CD42 and CD61 whenever available. All 38 pts received induction chemotherapy containing cytarabine and anthracyclines. Sixteen (42%) were males; median age was 54 years (range 21–78); 11 (29%) had antecedent hematologic disorder (AHD) and/or MDS, and 4 (11%) had previously received chemotherapy for other malignancies. Median white blood cell count at diagnosis was 3.5x109/L (0.5–49.9), hemoglobin 7.8 g/dl (3.2–10.9), platelet count 34.5x109/L (5–2292), and the median bone marrow blast percentage was 30% (0–80). For pts with <30% bone marrow blasts, diagnosis of AMegL was supported by peripheral blood blasts ≥30%. Significant bone marrow fibrosis was found in 24 pts (63%). Cytogenetic analysis was available in 30 pts (79%) due to insufficient material or insufficient metaphases. Twenty-seven pts (71%) showed abnormalities: deletion 7 in 15 pts (39%), deletion 5 in 14 (37%), chromosome 3 abnormality in 6 (16%) and trisomy 8 in 4 (11%). Philadelphia chromosome was detected in 2 pts (5%). Sixteen pts (42%) achieved a complete remission (CR). None of 22 pts with primary refractory disease achieved a complete remission from subsequent chemotherapies. Median overall survival of all AMegL pts (n=38) was 24 compared to 38 weeks for the control group (non AmegL AML pts, excluding APL,N=1823) (p<0.01). Median disease free survival of pts with AMegL (n=16) and other AML (n=1031) was 23 and 52 weeks, respectively (p<0.001). Two pts with primary refractory disease and three who developed relapsed disease eventually underwent allogeneic stem cell transplantation. Median survival after stem cell transplantation was 21 weeks. In summary, AMegL is a rare form of AML, which is observed in pts with AHD and/or MDS or as secondary leukemia. AMegL is highly associated with poor cytogenetic abnormalities, translating into a low CR rate and dismal outcome.