bluuebunny/crossref_metadata_2025 huggingface dataset:
Dasatinib-Induced Platelet Dysfunction.
Authors: Quintas-Cardama Alfonso , Han Xin , Kantarjian Hagop , Cortes Jorge
Published: October 2019

Dasatinib is efficacious and safe in patients (pts) with chronic myelogenous leukemia (CML) after imatinib failure. However, dasatinib therapy has been linked to bleeding in some pts, typically affecting mucosal surfaces. We analyzed the effect of dasatinib on basic coagulation tests and platelet aggregation function in 55 pts with CML in chronic phase at diagnosis (1), >8 weeks after tyrosine kinase inhibitor (TKI) discontinuation (3), or during therapy with the ABL/SRC inhibitors dasatinib (n=27) and bosutinib (n=12), or the aminopyrimidine derivatives imatinib (n=8) and nilotinib (n=4). Median age was 54 yrs (range, 22–80), WBC 5.5 ×109/L (range, 2.1–58.5), hemoglobin 12.2 g/dL (range, 9.7–15.1), and platelets 230 ×109/L (range, 80–746). Only 2 pts (both on imatinib and warfarin) had abnormal basic coagulation parameters (prolonged prothrombin time [PT]). In the remainder 53 pts, the median PT time was 11.6 sec (9.8–13), activated partial thromboplastin time (aPTT) 28.5 sec (range, 21–33.9), and fibrinogen level 495 mg/dL (range, 336–700), suggesting an intact secondary hemostasis. To evaluate the primary hemostasis status, platelet aggregation was measured by turbidometry in 1 mL of platelet-rich plasma upon stimulation with 5 μM of ADP, 1 μg/ml of collagen, 1 μM of epinephrine, 1 mM arachidonic acid, or 8 μL of ristocetin by using a Whole Blood Lumi-Aggregometer (Chrono-Log Corporation, Havertown, PA). Aggregation was expressed as the maximal percent change in light transmittance from baseline using platelet-poor plasma as a reference. Five (9%) patients (2 bosutinib, 1 nilotinib, and 2 imatinib) were on aspirin and showed an aspirin-like effect (decreased platelet aggregation with arachidonic acid [AA] and epinephrine [Epi]). Patients off-TKI or untreated had a normal platelet aggregation test. Results on the remaining 46 assessable patients demonstrate that dasatinib is strongly associated platelet aggregation defects, particularly an aspirin-like effect, when compared with other TKIs.

Platelet function analysis by PFA-100 showed that addition of dasatinib (400 nM) to fresh whole blood obtained from a healthy volunteer rapidly induced platelet dysfunction. Closure times were prolonged from 100 sec (baseline) to >300 sec in response to Epi (normal <160 sec) but not in response to ADP (from 72 to 84 sec, normal <120 sec) after 30 min incubation. In summary, our data indicate that dasatinib does not interfere with secondary hemostasis. Rather, it inhibits platelet function in an aspirin-like manner. This effect, while independent, can be additive to dasatinib-induced thrombocytopenia and may account for the bleeding diathesis observed in pts with CML receiving dasatinib.

Table 1. Effect of BCR-ABL kinase inhibitors on platelet aggregation Dasatinib Bosutinib Nilotinib or Imatinib Platelet aggregation test n=27 n=10 n=9 p value Normal (%) 4 (15) 8 (80) 6 (67) < 0.01 Abnormal (%) 23 (85) 2 (20) 3 (33) < 0.006 Only with Epi 7 1 0 With both Epi and AA 14 1 3 With all agonists 2 0 0
DNA-Based Measurement of BCR-ABL in Chronic Myeloid Leukemia (CML).
Authors: Morley Alexander , Bartley Paul , Martin-Harris Henry , Latham Sue , Ross David , Hughes Timothy
Published: October 2019

Aim To develop a DNA-based method for measurement of minimal residual disease (MRD) in CML

Background Currrent RNA-based methods for quantification of the BCR-ABL translocation have a number of disadvantages, including the potential for RNA degradation, requirement for a reverse transcription step, uncertain sensitivity and specificity, and only an indirect relationship between assay result and cell number.

Methods Tagged PCR primers were designed to span the common 3kb breakpoint region of BCR and corresponding 140kb breakpoint region of ABL. Primers were spaced to bind at approximately 500bp intervals in order to limit the size of the target sequence and ensure efficient amplification. PCR was performed with pools of 1-6 BCR forward primers and pools of 24–282 ABL reverse primers. The first round products were diluted and amplified in a second round using similarly sized pools of nested primers. The resulting products were sequenced, the sites of breakpoint identified and 3 pairs of PCR primers were synthesised for each patient. A 3 round nested quantitative PCR was established, in which amplification remained exponential until the third round, which was a quantitative real-time PCR using a Taqman probe or EvaGreen to measure fluorescence. Dilutions of the diagnosis DNA were used to establish a standard curve. Test samples, corresponding to different levels of MRD, were artificial mixtures made by diluting diagnosis DNA in up to 25 mcg of normal peripheral blood DNA. Specificity was determined by quantifying BCR-ABL sequences in 25 mcg of normal peripheral blood DNA.

Results Samples from 23 patients are being studied and the translocation breakpoint has thus far been isolated and sequenced in 15. Study of the remaining patients is in progress. No favoured breakpoint site or sequence has as yet become evident, although the numbers are small. The results of assays of mixtures of DNA from 9 patients are shown in Fig 1.

There results follow a 1:1 linear relationship (line shown) between expected and measured MRD levels down to approximately 10−6. Use of a Taqman probe or EvaGreen for quantification gave similar results DNA samples from 15 normal individuals were tested against each of the set of BCR-ABL primers from 3 patients. No BCR-ABL translocations were detected.

Conclusions

Isolation and sequencing of the BCR-ABL translocation breakpoint is feasible in the majority of patients with CML. Short-range multiplex PCR is a promising strategy for doing this. Sensitive and specific DNA-based measurement of BCR-ABL sequences is possible for CML patients in whom the BCR-ABL breakpoint sequence can be determined. The clinical utility of this approach to measurement of MRD in CML remains to be determined.

Figure Figure
A 3,239 -Patient Combined Eastern Cooperative Oncology Group (ECOG), M.D. Anderson Cancer Center (MDA) Analysis of the Effect of CR vs. Responses < CR on Long-Term Survival in Newly-Diagnosed AML Treated with Ara-C-Containing Regimens: Implications for Targeted Drug Development.
Authors: Estey Elihu , Sun Zhuoxin , Rowe Jacob , Faderl Stefan , Cassileth Peter , Sartiano George , Tartaglia Anthony , Garcia-Manero Guillermo , Cripe Larry , Bennett John , Kantarjian Hagop , Tallman Martin
Published: October 2019

New definitions of response for patients with AML have been suggested, e.g. CRp, marrow CR, hematologic improvement. Although the criteria for these are less demanding than those for CR, some have proposed that their attainment can convert AML to a “chronic disease” associated with long-term survival. To test this hypothesis, we used ECOG and MDA data to assess the effect of the achievement of CR rather than responses < CR following initial Rx on probabilities of survival 3 and 5 years after diagnosis. 2,061 pts treated on 6 ECOG trials 1976–1999 and 1,651 pts given various ara-C-containing regimens at M.D. Anderson 1980–1999 formed our database. We excluded from the survival analyses the 473 pts (11% ECOG,16% MDA)who died within 4 weeks of beginning treatment. CR rates were 62% and 60% for ECOG and MDA pts, respectively. 11% of ECOG and 54% of MDA pts were age ≥ 60. CR rates in these pts were 46% ECOG and 49% MDA. Survival probabilities according to site and response to initial Rx are as follows:

Group Site/Response to Initial Rx Patients Probability Survival at 3 Years (%+/−SE) Probability Survival at 5 Years (%+/− SE) All Pts ECOG/CR 1277 38+/− 1 33+/−1 MDA/CR 996 30+/−1 24+/−1 ECOG/<CR 567 11+/1 9+/−1 MDA/<CR 399 4+/−1 2+/−0 Age ≥ 60 ECOG/CR 106 25+/4 15+/−3 MDA/CR 374 19+/2 12+/−2 ECOG/<CR 62 2+/2 0+/−0 MDA/<CR 221 2+/−1 0+/−0 Age < 60 ECOG/CR 1171 39+/−1 34+/−1 MDA/CR 622 38+/−2 31+/2 ECOG/<CR 505 12+/−1 10+/−1 MDA/<CR 178 6 +/−2 4+/−2

We are assessing whether pts who survived 3 or 5 years despite not achieving CR with initial Rx attained CR with salvage Rx (e.g. allogeneic transplant) or had CRp or marrow CR rather than complete resistance after initial Rx. Nonetheless, our data indicate that in older pts given ara-C-containing regimens, long-term survival is essentially impossible without achievement of CR. Similarly, very few younger pts not achieving CR survive at 3–5 years. Therefore, it appears likely that newer “targeted agents” will have to contribute more than producing responses < CR and must fundamentally change the biology of the disease to improve long-term survival in AML.
Recovery of the NK Cells and Its Intending Clinical Roles after Unmanipulated Haploidentical Blood and Marrow Transplantation.
Authors: Huang Xiao Jun , Zhao Xiang-yu , Chang Ying-jun
Published: October 2019

Objectives: The goal of this study was to investigate the association of natural killer (NK) cell recovery with clinical outcomes after unmanipulated haploidentical blood and marrow transplantation.

Methods: We sequentially monitored the reconstitution kinetics of circulating NK cells, CD56bright and CD56dim, in 43 patients by flow cytometry, and the functionality recovery of cytokine or cytotoxicity of NK cells by flow cytometry or LDH release assay after transplantation.

Results: Reconstitution of NK cells was rapid but accompanied by skewing of cell subsets mainly in CD56bright, which had a greater growing speed. Linear regression analysis demonstrated that dose of CD34+ cells in the allografts was inversely correlated with the ratio of T/NK cells (β=−0.506, P=0.003) and CD56dim/CD56bright cell (β=−0.403, P=0.018) by day 14 after HSCT, and dose of CD3+ T cells in the allografts was also inversely correlated with the ratio CD56dim/CD56bright cells by day 14 after HSCT (β=−0.474, P=0.005). Compared with non-acute graft-versus-host disease (aGVHD) patients, patients with aGVHD had a higher level of NK subsets during week 2 post-transplantation. Cox regression analysis revealed that the patients with more CD56bright NK cells in the recovery stage had a higher survival rate (hazard risk [HR], 0.353; p=0.019) and the patients with higher ratio of T/NK (>1.0) had a higher chance of getting aGVHD (HR, 3.889; p=0.034) and cGVHD (HR, 3.925; p=0.028).

Conclusions: Our results suggest that the recovery of NK cells is and can be used as an indicator to predicate the clinical outcomes after unmanipulated haploidentical transplantation.

Figure 1. High number of circulating CD56bright NK cells correlated with better overall survival, and less TRM. Patients were subgrouped into ‘High’, ‘Middle’, and ‘Low’, group based on 33% and 67% percentage of circulating CD56bright NK cell count by day 14 after transplantation. Kaplan-Meier survival estimates OS (Figure 1A) in these three groups and cumulative incidence estimates TRM (Figure 1B). Figure 1. High number of circulating CD56bright NK cells correlated with better overall survival, and less TRM. Patients were subgrouped into ‘High’, ‘Middle’, and ‘Low’, group based on 33% and 67% percentage of circulating CD56bright NK cell count by day 14 after transplantation. Kaplan-Meier survival estimates OS (Figure 1A) in these three groups and cumulative incidence estimates TRM (Figure 1B).
IL23R Arg381Gln Variant in the Donor Confers Strong Protection Against Acute Graft-Versus Host Disease after Hematopoietic Stem Cell Transplantation in Children with Hematological Malignancies.
Authors: Gruhn Bernd , Intek Juliane , Pfaffendorf Nadine , Wittig Susan , Wolff Ilona , Schiller Isabella , Voigt Astrid , Häfer Ralf , Fuchs Dietlinde , Zintl Felix
Published: October 2019

The IL23R gene on chromosome 1p31 encodes a subunit of the receptor for the proinflammatory cytokine interleukin-23. Recently, IL23R could be identified as a novel inflammatory bowel disease susceptibility gene. The exchange of arginine with glutamine at position 381 of IL23R causes a blockade of the IL-23 signaling pathway which is associated with a reduced risk of both Crohn’s disease and ulcerative colitis. IL-23 is a pivotal cytokine in the differentiation of T cells into inflammatory, IL-17-producing T cells. Because of the requirement for IL-23 in autoimmune disease we hypothesized that IL23R Arg381Gln reduces the risk of graft-versus host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). 141 donors and 141 children (median age, 12 years) with acute lymphoblastic leukemia (n=63), acute myeloid leukemia (n=40), myelodysplastic syndrome (n=26) or chronic myeloid leukemia (n=12) who underwent allogeneic bone marrow (n=93) or peripheral blood stem cell transplantation (n=48; T-cell depleted: n=22) in a single center were genotyped of IL23R for rs11209026 (c.1142G>A, p.Arg381Gln) using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 60% of transplants and HLA-identical related in 30% of transplants. Conditioning regimen was myeloablative in all cases. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 65% of transplants and cyclosporine A alone in 24% of transplants. The IL23R Arg381Gln variant was present in 16 of the 141 donors (11.3%) and in 15 of the 141 patients (10.6%). Interestingly, we found a significantly reduced incidence of acute GVHD grade II–IV in patients who were transplanted from a donor with this specific variant (6.2% versus 33.6%; p=0.025). Furthermore, there was no severe acute GVHD grade III–IV if the variant occurred in the donor (0% versus 14.4%). In three of the donor-patient pairs the variant was present in both individuals and no acute GVHD was observed. The occurrence of the IL23R variant, in either donors or recipients, had no significant impact on chronic GVHD, relapse rate, treatment related mortality, and overall survival. In conclusion, IL23R Arg381Gln variant in the donor confers strong protection against acute GVHD after HSCT in children with hematological malignancies. Therefore, the IL23 signaling pathway seems to play an important role in the pathogenesis of acute GVHD. Blockade of the IL23 receptor by a monoclonal antibody could be a rational therapeutic strategy for acute GVHD.
Phase I/II Study of the P-Glycoprotein (Pgp) Inhibitor Zosuquidar Administered by Continuous Infusion (CIV) with Daunorubicin (DNR) and Cytarabine (ARA-C) as Primary Therapy in Older Patients with Pgp-Positive Acute Myeloid Leukemia (AML).
Authors: Lancet Jeffrey E. , Gotlib Jason , Wetzler Meir , Luger Selina , Cripe Larry D. , Tallman Martin S. , Gojo Ivana , Mulford Deborah , Baer Maria R. , List Alan F. , Multani Pratik S. , Sikic Branimir I.
Published: October 2019

Background: Pgp expression in AML increases with age and correlates with inferior therapeutic response and survival. Zosuquidar is a potent and highly specific Pgp inhibitor with minimal pharmacokinetic (PK) interaction with conventional xenobiotic antineoplastics. Prolonged Pgp blockade is believed necessary to optimize antineoplastic sensitization in resistant cells in vitro. We previously reported that 72-hour CIV Zosuquidar at 700 mg/d resulted in rapid and sustained Pgp inhibition during the entire period of anthracycline administration in Pgp+ AML (Lancet, et al. ASH 2006).

Methods: We report interim results from a Phase I/II trial of 72-hr CIV zosuquidar in older adults with newly diagnosed Pgp+ AML. Primary objective: to determine the response (CR + CRp) rate in Pgp+ patients. Eligibility included ages 55–75, ECOG PS 0-2, adequate end-organ function, and Pgp+ by functional assay. Zosuquidar was initiated 4 hrs prior to the first doses of DNR (45 mg/m2/d x 3d) and ARA-C (100 mg/m2/d CIV x 7d) and continued for 72 hrs. Reinduction with the same dosing schedule was permitted in patients with significant cytoreduction without aplasia. Patients who achieved a CR/CRp received up to 2 cycles of consolidation with the same agents using an abbreviated schedule.

Results: 67 patients with Pgp+ AML were enrolled on the study; 80 patients were included in the safety analysis. Sixty-two of the 67 Pgp+ patients received Zosuquidar at 700 mg/day, while 5 received 800 mg/day (during phase I). Median age was 66; M/F was 46/21; cytogenetics: adverse (19), intermediate (29), and unknown/not done (19); de novo/secondary AML: 36/31. Mean percentage of planned Zosuquidar actually administered was 94%. CR/CRp was achieved in 32 of 66 (48%) evaluable Pgp+ patients (CR=25, CRp=7). Fifteen of 31 (48%) patients with secondary AML, and 6 of 19 (32%) with adverse-risk cytogenetics achieved CR/CRp. Eight of 14 (57%) patients age ≥ 70 responded. Induction-related death (< 30 days) occurred in 10% of patients. Other common toxicities included infection/febrile neutropenia (89%), tremor (42%), hallucinations (11%), nausea (52%), and diarrhea (51%). Median times to neutrophil (≥ 1000/μL) and platelet (≥ 100,000/μL) recovery were 34 and 33 days, respectively. With a median followup of 7.5 months, the median overall survival was 8.9 months and the median relapse-free survival was 9.3 months.

Conclusions: CIV Zosuquidar 700 mg/d with DNR and ARA-C is well tolerated, with signs of clinical benefit in poor-risk older patients with Pgp+ AML, warranting continued study of this combination. Accrual to the current trial is ongoing.
Pre-Transplant Test Dose vs. PK Studies during Conditioning Regimen To Target iv Busulfan in Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Chunduri Sandeep , Beri Rakesh , Dobogai Lisa C. , Hurter Elizabeth , Mactal-Haaf Christina , Sweiss Karen I. , Shord Stacy , Peace David , Rondelli Damiano
Published: October 2019

In this study we tested the efficacy of a test dose of iv busulfan in targeting blood levels of this drug during the conditioning regimen prior to an allogeneic hematopoietic stem cell transplant. We analyzed blood samples of 22 patients undergoing allogeneic hematopoietic stem cell transplantation with a busulfan-based conditioning regimen. Patients received a test dose of busulfan at 0.8 mg/kg as a 60 minute intravenous infusion. Serial blood samples were drawn at eight time points: 15 minutes before dose, at end of infusion, 15 minutes after completion, 30 minutes after completion, 60 minutes after completion, 2 hours after completion, 4 hours after completion, and 6 hours after completion. Pharmacokinetics (PK) studies were then performed at the Seattle Cancer Care pharmacokinetics laboratory. The AUC was determined using WinNonlin Professional software. The conditioning dose of busulfan was calculated by multiplying the test dose in mg/AUC × 4800. After the first dose of busulfan was administered, the same protocol was used to test busulfan PK. If the Busulfan AUC was therapeutic (between 4800 μM*min and 5200 μM*min) then the same dose was continued. If the Busulfan AUC was low or high then the third and fourth doses of busulfan were adjusted. The test dose of 0.8 mg/kg intravenous did not have any hematological side effects. The mean historic dose (solely based on weight) was 3.2 ± 0.1 mg/kg and the mean dose based on the test dose was 3.5 ± 0.5 mg/kg (p=0.02). In 12 patients where we also analyzed PK after the first day of conditioning regimen, AUC values of busulfan obtained during test dose and after day 1 dose were not different (p=0.7). The mean dose of busulfan based on test dose was 3.5 ± 0.6 mg/kg while the final dose based on day 1 busulfan PK was 3.6 ± 0.7 mg/kg (p=0.9). Nevertheless, in 2 CML patients who were on treatment with dasatinib or nilotinib at the time of the test dose, a higher AUC was observed (AUC 6065 and 6200, respectively). A pre-transplant busulfan test dose can be safely performed anytime prior to transplant and allows targeting the dose of busulfan efficiently, thus avoiding the requirement of PK studies during the conditioning regimen.
Allogeneic Stem Cell Transplantation in Angioimmunoblastic T-Cell Lymphoma (AITL): A Retrospective Survey from the Lymphoma Working Party (LWP) of the European Group for Blood and Marrow Transplantation (EBMT).
Authors: Kyriakou Charalampia , Canals C. , Taghipour G. , Finke J. , Kolb H. , Goldstone A. , Sureda A. , Schmitz N.
Published: October 2019

AITL is a rare peripheral T-cell lymphoma characterised by an aggressive behaviour, which primarily affects the elderly. Chemotherapy regimens fail to alter the high relapse rate and overall survival hardly exceeds 25% at 5 years. To date, there is no information on the potential role of allogeneic stem cell transplantation (allo-SCT) in the management of AITL. We report the outcome of 39 patients with a median age of 47 years (24–68), who underwent an allo-SCT between 1995 and 2004 for AITL, and were reported to the EBMT registry. The median time from diagnosis to transplant was 10 months (4–72). Thirty-four patients (87%) had previously received two or more treatment lines, and 16 patients (41%) a previous autologous SCT. Fifteen patients (38%) had a primary refractory disease, 13 (33%) were transplanted in partial remission and the remaining patients were in complete remission (CR) (mostly in 2nd and 3rd CR). Twenty-four patients were transplanted from an HLA-identical sibling and 15 from a matched unrelated donor. A myeloablative conditioning regimen (MAC) was used in 21 patients (cyclophosphamide + total body irradiation in 14), while 18 patients received fludarabine-based reduced intensity conditionings (RIC). Peripheral blood was the source of stem cells in 35 patients (90%). Three patients failed to engraft (one patient in the RIC group). Twenty-one patients (54%) developed acute graft versus host disease (grade I-II, n=16; grade III-IV, n=5). Twenty-eight patients (72%) achieved a CR after the allogeneic procedure. Nine patients died from transplant related mortality (TRM) and 5 patients from disease progression. The cumulative incidence of TRM at 12 months was 19% for the MAC and 26% for the RIC group. After a median follow-up for the surviving patients of 20 months (6–74), 25 patients are alive. Relapse rates at 1 and 3 years were estimated at 10% and 18% for the MAC and 16 and 20% for the RIC patients. Progression free survival rates at 3 years were 67% and 50% and the overall survival at the same time 71% and 56% for the MAC and RIC group of patients, respectively. Although follow up is rather short, these data suggest that allo-SCT results in good overall response and is associated with a low relapse rate in this group of poor risk heavily pre-treated and rather elderly group of AITL patients. Allo-SCT could be considered a therapeutic option for eligible high-risk AITL patients. Nevertheless, the impact of this approach should be further explored in prospective collaborative studies.
Rivaroxaban - An Oral, Direct Factor Xa Inhibitor - for Thromboprophylaxis after Total Knee Arthoplasty: The RECORD3 Trial.
Authors: Lassen Michael R. , Turpie Alexander G.G. , Rosencher Nadia , Borris Lars C. , Ageno Walter , Lieberman Jay R. , Bandel Tiemo J. , Misselwitz Frank
Published: October 2019

Introduction Rivaroxaban is a novel, oral, direct Factor Xa inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. In RECORD3, a phase III trial, the efficacy and safety of once-daily (od) rivaroxaban was compared with od enoxaparin for the prevention of venous thromboembolism (VTE) in patients undergoing total knee arthroplasty (TKA).

MethodsIn this multicenter, double-blind, double-dummy trial, patients undergoing TKA were randomized to receive either oral rivaroxaban 10 mg od or subcutaneous enoxaparin 40 mg od. Enoxaparin was initiated 12 hours before surgery, and rivaroxaban 6–8 hours after surgery; both were continued for 10–14 days. The primary outcome was the composite of deep vein thrombosis (DVT), pulmonary embolism (PE), and all-cause mortality. Secondary efficacy outcomes included major VTE (the composite of proximal DVT, PE and VTE-related death) and symptomatic VTE. The main safety outcome was major bleeding, and secondary outcomes included non-major bleeding and adverse events.

Results A total of 2531 patients were randomized; 2459 were eligible for inclusion in the safety population and 1702 for the modified intention-to-treat population. The primary efficacy outcome occurred in 9.6% of patients receiving rivaroxaban compared with 18.9% of those receiving enoxaparin (relative risk reduction 49%; p<0.001; Table). Rivaroxaban was also significantly more effective than enoxaparin at reducing major VTE, with a relative risk reduction of 62%. Symptomatic VTE was also lower with rivaroxaban than with enoxaparin. The incidences of major and non-major bleeding were similar in both the rivaroxaban and enoxaparin groups, as was the incidence of other adverse events. During the treatment period, there were no deaths or PEs in the rivaroxaban group, and two deaths and four PEs occurred in the enoxaparin group.

Conclusions Rivaroxaban was superior to enoxaparin for the prevention of VTE after TKA in this study, with a similar, low rate of bleeding. This is the first study to demonstrate the safety and efficacy of a fixed, unmonitored regimen of an oral, direct Factor Xa inhibitor - rivaroxaban - for the prevention of VTE after major orthopaedic surgery.

Rivaroxaban 10 mg od % (n/N) Enoxaparin 40 mg od % (n/N) Relative risk reduction (%) p -value for difference aModified intention-to-treat-population; bModified intention-to-treat population valid for major VTE analysis; cSafety population who underwent surgery; dSafety population; *Calculated for the absolute risk difference DVT, non-fatal PE, and all-cause mortalitya 9.6% (79/824) 18.9% (166/878) 49% p <0.001 Major VTEb 1.0% (9/908) 2.6% (24/925) 62% p =0.016 Symptomatic VTEc 0.7% (8/1201) 2.0% (24/1217) 66% p =0.005 Major bleedingd 0.6% (7/1220) 0.5% (6/1239) - p =0.774* Non-major bleedingd 4.3% (53/1220) 4.4% (54/1239) - p =0.990*
Once Daily YM150, an Oral Direct Factor Xa Inhibitor, for Prevention of Venous Thromboembolism in Patients Undergoing Elective Primary Hip Replacement.
Authors: Eriksson Bengt I. , Turpie Alexander G.G. , Lassen Michael R. , Prins Martin H. , Agnelli Giancarlo , Kälebo Peter , Wetherill Graham , Meems Lambertus
Published: October 2019

YM150 is an orally active direct FXa inhibitor in clinical development. Proof of concept of YM150 was previously obtained in a dose escalation study, demonstrating a statistically significant dose-related trend in VTE prevention in patients undergoing total hip replacement (ONYX; J Thromb Haemost2007;5:1660–5). The present study with YM150 is a double blind, parallel, multi-center dose finding study in patients undergoing elective primary hip replacement surgery, with open label enoxaparin as control. Patients were randomly treated with 5, 10, 30, 60 or 120 mg YM150 once daily orally or 40 mg enoxaparin once daily subcutaneously. Patients were treated in hospital for approximately 1–2 weeks followed by home treatment for another 4 weeks, for in total 33–38 treatment days (prolonged treatment for hip replacements according to guidelines of Am. College of Chest Physicians (Geerts et al. Chest2004:126;338–400)). The administration of YM150 started 6–10 h after surgery, while treatment with enoxaparin started 12 ± 2 h before surgery. Mandatory bilateral venography was performed after 7–10 days of treatment. Follow up visits were performed at 2 and 4 weeks during home treatment, and 2, 4, and 8 weeks after end of study treatment. The sample size was estimated to be 960 randomized patients in order to yield 110 patients per treatment group with an evaluable venogram. The primary efficacy endpoint was defined as deep vein thrombosis, and/or symptomatic VTE and/or death, and primary safety endpoint as the incidence of major bleeding during the first 7–10 days of study treatment. Secondary efficacy endpoints were proximal and distal DVT (during the first 7–10 days) and symptomatic VTE during home treatment, and follow up. Secondary safety endpoints were the incidences of major, clinically relevant non-major (CRNM) and minor bleeding during the three study periods. Independent Adjudication Committees reviewed all venograms, bleeding reports, symptomatic VTEs, and deaths unaware of treatment allocation. An independent DSMB monitored the data to ensure the safety of the patients. 1017 patients were randomized from 81 sites in 17 European countries. Preliminary data showed that DVT was detected in 149 out of 727 evaluable venograms, one patient had symptomatic VTE, and one patient died (myocardial infarction) during the hospital treatment period. This results in a total primary efficacy outcome rate of 20.7% (151/729) in the evaluable study population, with a corresponding rate of major bleeding of 0.6% (6/1017). The breakdown of the results according to treatment group will be presented.
Subcutaneous Alemtuzumab (MabCampath) in Fludarabine-Refractory CLL (CLL2H Trial of the GCLLSG).
Authors: Stilgenbauer Stephan , Winkler Dirk , Bühler Andreas , Zenz Thorsten , Groner Silja , Busch Raymonde , Hensel Manfred , Dührsen Ulrich , Finke Jürgen , Dreger Peter , Jäger Ulrich , Lengfelder Eva , Trümper Lorenz , Söling Ulrike , Schlag Rudolf , Hallek Michael , Döhner Hartmut
Published: October 2019

Fludarabine-refractory CLL has a poor prognosis with a median overall survival time of less than 12 months despite salvage chemotherapy and intravenous alemtuzumab (Campath-1H) is the approved treatment based on a remission rate of 33% and a median survival time of 16 months (Keating et al., Blood 2002). The CLL2H trial of the GCLLSG was initiated to evaluate the subcutaneous application of 3 × 30 mg alemtuzumab weekly in fludarabine refractory CLL. The current analysis is based on 109 consecutive patients enrolled until completion of the trial in April 2006. Median age was 63 (36–81) years, 71% were male. A median number of 3 (1–9) prior lines of therapy had been given. Subcutaneous treatment was performed on an outpatient basis in all cases and had to be temporarily interrupted in 68 patients due to neutropenia (43%), anemia (6%), thrombocytopenia (3%), infections (40%, CMV reactivations 30%), and was stopped early in 63 cases due to insufficient response (44%), hematotoxicity (16%), infection (17%), and CMV reactivation (13%). The median alemtuzumab dose given was 722 (3–2203) mg. Toxicity was mostly grade I/II apart from hematotoxicity (grade III/IV anemia: 42%, thrombocytopenia: 52%, neutropenia: 54%) and grade III/IV infections (25%). After a median follow up time of 21.4 months, 56 deaths have occurred (due to progression 52%, infections 39%, not CLL related 9%). The overall response rate was 33% (CR 4%, PR 27%), the median progression free survival time was 7.7 months, and median overall survival time was 19.1 months. Genetic high-risk factors were present in the vast majority of cases (unmutated VH 66%, 17p–29%, 11q–19%, TP53 mutation 39%). Responses (CR or PR) were observed in 22% of VH unmutated, 24% of 11q-, 39% of 17p-, and 33% of TP53 mutated cases. Progression free survival and overall survival were not significantly different when comparing the genetic subgroups, particularly TP53 mutated, 11q-, and 17p- (see figure). In conclusion, subcutaneous alemtuzumab is feasible in an outpatient setting in a high-risk population of fludarabine-refractory CLL and appears to be of similar efficacy as by intravenous administration. Most importantly, genetic high risk subgroups with unmutated VH, 11q- or 17p- appear to respond to alemtuzumab.

Figure Figure
A Phase II Study of Dasatinib in Relapsed and Refractory Chronic Lymphocytic Leukemia (CLL/SLL).
Authors: Amrein Philip C. , Attar Eyal C. , Takvorian Tak , Hochberg Ephraim P. , Ballen Karen , Zahrieh David , Brown Jennifer R.
Published: October 2019

Preclinical studies have shown that proliferation and survival of CLL cells are associated with overexpression of the Lyn kinase protein, and in vitro inhibition of Lyn kinase leads to apoptosis of the CLL cells (Contri, J Clin Invest 2005). Because dasatinib inhibits Lyn kinase in CML cells at doses easily achievable in patients, we undertook this phase II study of dasatinib in patients with previously treated CLL/SLL. Patients were required to be over 18 years of age, have a diagnosis of CLL/SLL by flow cytometry/immunostains, and have failed at least 1 course of treatment with a fludarabine-containing regimen or at least 2 courses of non-fludarabine containing regimens. The starting dose of dasatinib was 140 mg daily by mouth. This dose could be reduced to 100 mg or 80 mg daily for toxicity. At baseline all patients had bone marrow biopsies and CT scans, and these were repeated at 2 months. Sequential blood and bone marrow samples were tested for Lyn kinase activity. The design of the study provided for 2 phases such that if 3 responses were seen among the first 15 patients, the trial would expand to enroll another 20 patients. Among the first 9 patients enrolled there were 4 male and 5 female subjects with a median age of 59 years (40–78 years). ECOG performance status was 0 in 4 subjects, 1 in 3, and 2 in 2 subjects. All patients had previously received fludarabine: 1 subject had 1 prior treatment, 3 had 2 prior treatments, 3 had 3 prior treatments, and 2 had 4 prior treatments. By cytogenetic/FISH analysis there were 2 patients with del(17p) and another 4 patients with del(11q). All patients required treatment by NCI Working Group criteria. The major toxicity encountered was myelosuppression: grade 3 + 4 neutropenia in 7 subjects, grade 3 + 4 thrombocytopenia in 5 subjects. Gastrointestinal toxicity was minor with only 1 subject experiencing grade 3 diarrhea. One subject developed a grade 2 pleural effusion. There was 1 patient with an electrolyte imbalance consisting of a transient serum K=6.8, and in 1 patient there was a transiently prolonged QTc of 516 ms. There were no fatal events, and all toxicities were reversible. The median duration of treatment on study was 9 weeks with a range of 4 to 23 weeks. The clinical response data of the first 15 patients will be presented along with the correlative studies of Lyn kinase inhibition by dasatinib.
Cytotoxicity of the Type 4 Phosphodiesterase Inhibitor CD160130 for Freshly Isolated Human CLL Cells In Vitro.
Authors: Weinberg J. Brice , Jiang Ning , Volkheimer Alicia D. , Chen Youwei , Bond Karen M. , Moore Joseph O. , Gockerman Jon P. , Diehl Louis F. , de Castro Carlos M. , Rizzieri David A. , Levesque Marc C. , Mugridge Kenneth , DeAngelo Joe
Published: October 2019

B cell chronic lymphocytic leukemia (CLL), the most common subtype of leukemia in the United States of America and in Europe, is treatable but incurable. New drugs are needed for its management. Phosphodiesterase inhibitors (PDEi) block catabolism of cyclic nucleotides resulting in accumulation of cellular cAMP and cGMP. These PDEi also decrease production of inflammatory cytokines such as tumor necrosis factor. Furthermore, they inhibit expression of inducible nitric oxide synthase (NOS2) mRNA and NO production. Researchers have previously noted that nonspecific PDE inhibitors such as theophylline as well as a relatively specific PDE4 inhibitor (rolipram) cause CLL cell death in vitro, while relatively sparing normal peripheral blood mononuclear cells (PBMC). The purpose of this study was to evaluate the effectiveness of CD160130 in the killing of freshly isolated CLL cells in vitro. CD160130 is a novel, orally available heterocyclic pyrimido-indole derivative that is relatively PDE4-specific. Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CLL cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. Samples from 10 CLL patients (6 male and 4 female) and 10 normal controls were examined. Nine of 10 patients were stage 0 at presentation, and 1 was stage 2. They had been followed 6.1 yr (median; range 0.7–19.1 yr). One of 10 was CD38 positive, and 6 of 10 were Zap-70 positive. Of nine analyzed, one had unmutated IgVH gene, and 9 were mutated. Six patients had not been treated, and 4 had been treated with chlorambucil, fludarabine, and/or rituximab. Four had normal cytogenetics by FISH analysis; one had trisomy 12; three had 13q14 del; and one had 17p del. Of seven determined, two had elevated CLL cell lipoprotein lipase mRNA elevated. CD160130 induced cell death in a dose-dependent fashion in all patients’ samples, with a mean cytotoxicity of 96% at 12.5 uM. The mean ED50 for killing was 233 nM in media with FBS and 314 nM in serum-free medium, while that for PBMC was higher at 7500 nM with FBS and 5210 nM with serum-free medium. The agent was 32.2 fold more potent for killing of CLL cells compared to PBMC in medium with FBS and 16.6 fold more potent for CLL cells in serum-free medium. In summary, the PDE4 inhibitor CD160130 potently kills freshly isolated CLL cells in vitro in the presence or absence of serum. The killing is relatively selective for CLL cells compared to normal PBMC. In vivo trials of CD160130 in patients with CLL should help determine the toxicity and efficacy in patients.
Neutralization of Tumor-Derived Soluble Glucocorticoid-Induced TNF-Related Protein (GITR) Ligand Present in Cancer Patient Sera Increases Anti-Tumor Reactivity of NK Cells.
Authors: Baltz Katrin M. , Krusch Matthias , Baessler Tina , Bringmann Anita , Kanz Lothar , Brossart Peter , Salih Helmut R.
Published: October 2019

Glucocorticoid-induced TNF-related protein (GITR) and its ligand (GITRL) are members of the TNF/TNF receptor (TNFR) superfamily, which mediates multiple cellular functions including proliferation, differentiation, and cell death. Recently we reported that NK cells express GITR while tumor cells express GITRL, and GITR-GITRL interaction downregulates NK cell-mediated anti-tumor immunity (Baltz et al., FASEB J 2007). Many TNF family members are released as soluble forms, which affects cell-cell interactions by reduction of ligand density and distally modulates effector cells bearing the respective receptor. Here we report that human tumor cells spontaneously release a soluble form of GITRL (sGITRL), which can be detected in tumor cell culture supernatants by ELISA (detection limit 0.01ng/ml). We demonstrated that NK cell cytotoxicity and IFN-γ production in cocultures with the tumor cell lines SK-Mel (Melanoma), PC-3 (prostate), HCT116 (colon), and LX-1 (lung) were significantly (both p<0.01, Mann-Whitney U-test) and concentration dependently reduced (up to 50%) by tumor-derived sGITRL, and NK cell effector functions could be restored by neutralization of sGITRL using a GITR-Fc fusion protein. While tumor-derived GITRL did not induce apoptosis in NK cells, it diminished nuclear localized RelB indicating that sGITRL negatively modulates NK cell NF-κB activity. Furthermore, we demonstrate that significantly elevated sGITRL levels (mean 0.4ng/ml, range from 0.01 to 3.5ng/ml) were contained in 40 out of 50 sera of patients with various cancers (colon, lung and germ line), while sera of healthy volunteers (n=8) contained no detectable levels of sGITRL. Addition of sGITRL containing patient sera to cocultures of NK cells and GITRL-negative tumor cells significantly reduced NK cell cytotoxicity and IFN-γ production about 30% and 45%, respectively (both p<0.05, Mann-Whitney U-test). Again the inhibitory effects of sGITRL on NK cell effector functions could be completely restored by neutralization of sGITRL with GITR-Fc. The strong correlation of tumor incidence and elevated sGITRL levels clearly suggests that sGITRL is released at significant amounts from malignant cells in vivo and may reduce immune surveillance of human tumors. Our data indicate that determination of sGITRL levels may be implemented as an immunological diagnostic marker in tumor patients, and GITRL-neutralization may be employed in therapeutic strategies like adoptive NK cell transfer.
Recombinant Thrombin Has a Superior Immunogenicity Profile Compared with Bovine Thrombin in a Well-Controlled, Randomized, Double-Blind, Phase 3 Study of Topically Applied Thrombins in Four Surgical Settings.
Authors: Zuckerman Linda A. , Pederson Susan , Boster Dan R. , Appesland Lisa , Jackson Dana M. , Giste Erika , Newman Jonathan D. , Alexander Allan
Published: October 2019

Topical hemostatic agents have been used for more than 50 years. Currently, bovine thrombin is isolated from bovine plasma and has an inherent risk of transmitting blood-borne pathogens. Approximately 20% or more patients who receive various bovine thrombin preparations have been reported to develop antibodies to bovine thrombin product and impurities. In rare cases, these antibodies can cross-react with human endogenous coagulation proteins and may lead to alterations in coagulation laboratory parameters, hypersensitivity reactions, or severe bleeding or thrombosis. ZymoGenetics, Inc. has developed recombinant Thrombin (rThrombin) as an alternative to bovine and human plasma-derived thrombin preparations. The potential immunogenicity of rThrombin (n=198) and bThrombin (n=200) were compared in a well-controlled, randomized, double-blind Phase 3 study. Two enzyme-linked immunosorbent assays (ELISAs) were developed and validated under the principles of FDA Guidance Documents (FDA Guidance for Industry: BioAnalytical Method Validation, May 2001) to detect antibodies binding to either rThrombin or bThrombin product. A time-to-clot based assay was developed and validated to characterize whether antibodies to rThrombin had neutralizing activity for plasma-derived thrombin. Study results demonstrated rThrombin’s superior immunogenicity profile compared with bThrombin (1.5% versus 22%, respectively, p < 0.0001; antibodies developed to the products, as defined by seroconversion or significant change in titer units). Characterization of the binding antibodies demonstrated none of the anti-rThrombin antibodies neutralized human plasma thrombin. No subjects in either group had evidence of antibody-mediated coagulopathy. These results demonstrate that rThrombin has a superior immunogenicity profile when compared with bThrombin.
A Point Mutation in the Juxtamembrane Wedge of CD45 Impacts Myeloid Development and Generates a Novel Model for Hemophagocytic Disorders in the Context of a MHC I-Restricted Transgene.
Authors: Hermiston Michelle L. , de Graaf Fleur R. , Weiss Art
Published: October 2019

The receptor like-protein tyrosine phosphatase CD45 is highly expressed on all nucleated hematopoietic cells and functions by modulating the activity of Src family kinases. We previously generated mice containing a point mutation in the juxtamembrane wedge of CD45 that leads to constitutive phosphatase activity. Demonstrating the critical negative regulatory function of the wedge, the CD45E613R mutation led to a lymphoproliferative disorder, lupus-like autoimmune syndrome, and premature death at 9–12 months of age. To address the intrinsic effects of the wedge mutation on the CD8 T cell lineage, we introduced a CD8 restricted T-cell receptor (TCR) transgene, OT1, specific for the antigen ovalbumin, into CD45E613R mice. Surprisingly, 100% of the CD45E613R/OT1+ mice succumb by 6 weeks of age to a rapidly progressive disease characterized by severe cachexia, hemophagocytosis, and markedly elevated interferon gamma levels. This phenotype shares similarity to the human hemophagocytic syndromes hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS). The aims of this study are to identify the mechanistic basis and the cell type(s) responsible for this phenotype. Using a genetic approach, we demonstrate that the phenotype is maintained in CD45E613R/OT1+mice on a Rag1−/− background, thus implicating myeloid and/or CD8 T cells in disease pathogenesis. Analysis of myeloid cell development, phagocytic function, and signaling indicate that the wedge mutation operates in this lineage. Despite the intrinsic hyperresponsiveness of CD45E613R macrophages, adoptive transfer experiments demonstrate they are not essential for disease. However, adoptive transfer of purified CD45E613R/OT1+ CD8 T cells into either CD45 mutant or CD45 wildtype (wt) is sufficient to initiate disease. Interestingly, CD45E613R CD8 OT+ T cells are not dominant over CD45wt CD8+ cells. In addition we show that transfer of CD45wt CD8+ T cells into ill mice can delay disease progression. We hypothesize that, in the setting of the wedge mutation, the restricted repertoire of the CD8 T cells in CD45E613R/OT1+ mice might not be able to limit macrophage activation. We believe these mice may represent a useful model for understanding the molecular pathogenesis of hemophagocytic disorders, devastating and often fatal diseases in humans.
Gene Expression Analysis of B-Cell Post Transplant Lymphoproliferative Disorders Provides Insights into Disease Biology.
Authors: Vakiani Efsevia , Basso Katia , Klein Ulf , Dalla-Favera Riccardo , Pasqualucci Laura , Bhagat Govind
Published: October 2019

Background: B-cell post transplant lymphoproliferative disorders (PTLDs) encompass a diverse group of entities, the majority representing EBV-driven lymphoproliferations, which arise in the setting of iatrogenic immunosuppression. Their pathogenesis, especially the genetic relationship of PTLD with other types of B-cell non-Hodgkin lymphomas (B-NHLs), is not well understood and the association of polymorphic PTLD (P-PTLD) with monomorphic PTLD (M-PTLD) is presently unclear. Thus, in order to address these issues we analyzed gene expression profiles of PTLD and compared them with those derived from other types of B-NHL, normal B-cell subsets, and B-cell derived cell lines.

Material and methods: Paraffin embedded and H&E stained sections were used for morphologic analysis and PTLD were classified according to the WHO classification. Phenotypic characterization was performed using a comprehensive panel of antibodies and in situ hybridization for EBER. Frozen tissue from PTLD, different B-NHL, purified normal B-cell subsets, EBV-transformed lymphoblastoid cell lines (LCLs) and multiple myeloma cell lines, was used to generate complementary RNA (cRNA) that was hybridized to HGU95 arrays (Affymetrix). Gene expression data were analyzed using unsupervised and supervised clustering algorithms and classifiers were generated to determine the relationship between PTLD and cells corresponding to different stages of mature B-cell development.

Results: The 12 PTLD analyzed represented an infectious mononucleosis-like (IM-like) lesion, 5 P-PTLD, and 6 M-PTLD. All P-PTLD and 5 M-PTLD had late-germinal center (GC)/post-GC phenotypes and 8 PTLD, including the IM-like lesion, were EBV+, while 4 PTLD were EBV−. The expression profiles of late GC/post GC PTLD appeared homogeneous and distinct from other B-NHL, however no segregation of P-PTLD and M-PTLD or EBV+ and EBV− PTLD was noted. Surprisingly, on supervised analysis, only 4 genes were expressed at higher levels in PTLD compared to B-NHL and normal B-cell subsets (BLIMP-1, XBP-1, IFITM3, PCMT1) and only 2 genes were differentially upregulated in EBV+ PTLD (CD27 ligand, MTDH). The expression profiles of PTLD appeared most similar to LCLs.

Conclusions: B-cell PTLDs of late-GC/post-GC phenotype represent a distinct type of B-NHL, which are possibly derived from activated B-cells. Despite morphologic distinctions, P-PTLD and M-PTLD have overlapping genetic profiles suggesting disease heterogeneity within different categories.