bluuebunny/crossref_metadata_2025 huggingface dataset:
Leukemia-Associated Antigen Specific T-Cell Responses Following Combined PR1 and WT1 Peptide Vaccination in Patients with Myeloid Malignancies.
Authors: Rezvani Katayoun , Yong Agnes S.M. , Mielke Stephan , Savani Bipin N. , Musse Laura , Superata Jeanine , Jafarpour Behnam , Boss Carol , Barrett A. John
Published: October 2019

The graft-versus-leukemia (GVL) effect following allogeneic stem cell transplantation (SCT) is evidence that T lymphocytes can eradicate leukemia. The successful identification of a range of leukemia-associated antigens such as proteinase 3 (PR3) and Wilms tumour-1 (WT1) has stimulated efforts to induce leukemia-specific T-cell responses to these antigens using peptide vaccines. Here we describe the safety and immunogenicity of a combined vaccine of two leukemia-associated antigenic peptides, PR1 and WT1. Eight HLA-A*0201 positive patients with myeloid malignancies (2 myelodysplasia, 5 acute myeloid leukemia and 1 chronic myeloid leukemia) received one subcutaneous dose each of PR1 and WT1 vaccines in Montanide adjuvant, with granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients completed 4 weeks follow-up to monitor toxicity and immunological responses. Toxicity was limited to grade 1–2. All remain alive at a median of 252 days (range 105–523). We analyzed the immunological response to vaccination using PR1/HLA-A*0201 and WT1/HLA-A*0201 tetrameric complexes and flow cytometry for intracellular interferon-gamma (IFN-γ) in samples obtained pre- and weekly post-vaccination. A significant CD8+ T-cell response to the vaccine was defined as the emergence of PR1 or WT1-specific CD8+ T-cells when the pre-study analysis was negative or a twofold increase in frequencies when responses were present pre-vaccination. Following vaccination, a significant CD8+ T-cell response to PR1 was seen in 7/8 patients (median 0.34%, range 0.04–0.48%), to WT1 in 5/8 patients (median 0.29%, range 0–0.42%) and to one or both antigens in 8/8 patients. Vaccine-induced CD8+ T-cells were seen as early as 1 week post-vaccination, produced IFN-γ and were preferentially expanded in the effector compartment (CD45RO+/-CD27−). Post-vaccination, there was a strong correlation between the emergence of PR1 or WT1+CD8+ T-cells and a reduction in WT1 mRNA expression, a marker of minimal residual disease, suggesting a vaccine-driven anti-leukemia effect. Loss of response was associated with reappearance of WT1 transcripts (P<0.01). Two patients with detectable CD8+ T-cell responses to PR1 who failed to have a reduction in MRD relapsed 3–6 months following completion of vaccination. This is the first demonstration that a combined PR1 and WT1 vaccine is immunogenic. Based on these results we have initiated a phase 2 study of repeated vaccination with PR1 and WT1 peptides in patients with myeloid malignancies.
Role of High-Dose Chemotherapy with Autologous Stem Cell Transplantation in Primary Systemic Amyloidosis: A Systematic Review and Meta-Analysis.
Authors: Behera Madhusmita , Kumar Ambuj , Kharfan-Dabaja Mohamed A. , Djulbegovic Benjamin
Published: October 2019

Background: Primary systemic amyloidosis (AL) is a rare plasma cell clonal disorder(8/million) characterized by extracellular deposits of material composed mainly of fragments of light chain immunoglobulin throughout a body. Standard chemotherapy (e.g. melphalan and prednisone) is associated with poor outcomes (typical median survival is between 12–18 months with less than 5% survive 10 years). Autologous stem cell transplant (ASCT) has been increasingly advocated for treatment of AL. However, it is uncertain whether ASCT is better than standard chemotherapy. To address this uncertainty, we undertook a systematic review/meta-analysis to evaluate the efficacy of high-dose chemotherapy and autologous stem-cell transplant (HSCT) versus conventional chemotherapy in patients with AL.

Methods: Data search of published studies included Medline [all randomized controlled trials (RCTs)], Cochrane library and hand search of references. Studies were included if they were comparison trials of HSCT versus conventional chemotherapy, regardless if they were RCTs, prospective studies with historical control, or single arm studies. The studies were eligible if patients had biopsy proven AL with at least one major organ involved. Data were extracted on benefits as well as harms (overall survival, event-free survival, response, treatment related mortality, treatment-related morbidity).

Results: Out of 34 identified studies only 13 met the inclusion criteria for the current systematic review (2 RCTs, 2 prospective non-randomized trials involving historical control, and 9 single arm trials). Altogether these trials enrolled 1056 patients. Pooled data from 4 trials with controls (RCT and non-RCT) found similar overall survival for ASCT and conventional therapy arms [hazard ratio (HR) of 1.10 (95% CI 0.88, 1.36, p=0.4); p= 0.6]. Analysis of data according to trial design also did not find any difference in survival [HR for RCTs was 1.10 (95% CI 0.88, 1.37) and for non RCTs HR was 0.98 (95% CI 0.29, 3.35)]. The complete hematological response was also similar in both arms in RCTs (Odds ratio [OR]=1.38, 95%CI 0.67, 2.85; p=0.4) and non RCTs (OR=1.78, 95%CI 0.22, 14.65; p=0.32). The pooled proportion of treatment-related deaths in the single arm studies for AHCT was 0.119 (95% CI = 0.09 to 0.14)].

Conclusion: The results from the meta-analysis indicate that there is no statistically significant difference between the treatment effects from high-dose chemotherapy with ASCT and conventional chemotherapy. Hence, the efficacy of ASCT in improving overall survival and complete hematological response remains to be proven.
ADAMTS-13 Levels Do Not Predict Outcome in Thrombotic Thrombocytopenic Purpura: Protein S Levels Are Maintained during Plasma Exchange with Solvent Detergent Treated Plasma.
Authors: Rock Gail A. , Anderson David , Benny Barrett , Barth David , Clark William , Foley Ronan , Sutton David , Leblond Pierre
Published: October 2019

Introduction The ultimate therapy for TTP is not yet defined. While approximately 85% of patients respond to plasma exchange with FFP, the use of alternative solutions such as cryosupernatant plasma, which is deficient in the high molecular weight forms of vWF. has not been proven by appropriate randomized prospective sample trials. However, it is intellectually appealing. Additionally, the use of the ADAMTS 13 level either to predict disease or outcome has not been resolved.

Methods Cryosupernatant plasma (CSP) was compared to solvent detergent treated plasma (SDP) over one cycle (nine days) of plasma exchange (PE) and subsequent procedures as required. Throughout treatment, ADAMTS 13 and protein S levels were followed.

Results At the primary end point of one month, 3 of 35 of the CSP and 1 of 27 of the SDP patients died. The average platelet count was 184 × 109 per litre for CSP and 209 × 109 per litre for SDP. While vWF and FVIII were elevated in all patients at entry, no unusually large vWF multimers were seen at any time. At entry 9 patients had ≤ 0–10% ADAMTS 13 and 14 of 62 had normal levels with the rest in between. At presentation only 9 patients had 100% inhibitor activity with no inhibitor in 18 patients. Of the four patients who died, two who received CSP, had 100% enzyme activity at presentation whereas one had no ADAMTS-13 and another had 10%. All 4 patients died within 10 days. Fifteen patients with less than 10% enzyme activity had >1.5 vWF. However, elevated (>3u/mL) vWF was seen in the presence of normal ADAMTS-13 levels. Six months after remission, and in the presence of a normal platelet count, ADAMTS13 remained low in 5 patients and normal in 15.Protein S levels in 41/62 patients studied showed little variation during therapy with values on day 3 and 5, similar to those seen on entry.

Conclusion In idiopathic TTP, ADAMTS 13 levels do not correlate with disease, and are not predictive of outcome. While protein S levels in the SDP were lower than those found in FFP, this did not appear to have clinical significance.
Inactivation of Human White Blood Cells in Red Blood Cell Products Using the MIRASOL® System for Whole Blood.
Authors: Fast Loren D. , Marschner Susanne , DiLeone Gilbert , Doane Suzann , Fitzpatrick Christy , Ray Goodrich
Published: October 2019

Transfusion of blood products containing white blood cells (WBC) can result in the induction of immune responses that can negatively impact the recipient. An approach that would mitigate these consequences would be beneficial. Previous studies had shown that exposure of platelet concentrates to light in the presence of riboflavin was able to inhibit immune responses mediated by WBC. To make this protocol more widely applicable the effect of treating whole blood units with riboflavin and varying amounts of light was tested. Human peripheral blood mononuclear cells were purified by Ficoll-Hypaque discontinuous centrifugation from aliquots of nonleukoreduced whole blood units that were untreated or exposed to Mirasol treatment using varying light dosages. Viability and phenotype of treated cells was unchanged compared to untreated controls. The results showed that exposure of whole blood units to 33J/mL red blood cells (RBC) UV light in the presence of riboflavin completely inhibited proliferation of WBC in response to polyclonal stimulators such as phytohemagglutinin, and anti-CD3/CD28 or to allogeneic stimulator cells in a mixed lymphocyte culture (MLC). Additional assays showed that treated WBC were unable to induce proliferation of normal responder cells in an MLC. Treated cells did not produce inflammatory or TH1/TH2 cytokines when stimulated with lipopolysaccharide for 24 hours or anti-CD3/CD28 for 72 hours. In addition, treatment was found to inhibit T cell activation as evidenced by the lack of CD69 expression in treated compared to untreated control cells when incubated with phorbol 12-myristate 13-acetate. These treatment conditions did not induce crossmatch incompatibility. Methemoglobin levels and hemolysis in RBC units stayed below 1% during storage for 42 days in AS-3. Platelet and plasma units separated from whole blood after treatment showed acceptable cell and protein quality over 5 days in storage or as fresh frozen plasma, respectively. In summary, Mirasol treatment was able to functionally inactivate WBC in whole blood products without adversely affecting the quality of the RBC, platelets and plasma. This technique offers potential means to achieve inactivation of WBC in whole blood units that can subsequently be separated into RBC, platelet and plasma components.
The Intracellular ABC Transporter A3 Contributes to Imatinib Resistance of CML Cells by Subcellular Sequestration.
Authors: Chapuy Bjoern , Albrecht Melanie , Radunski Ulf , Koch Raphel , Haase Detlef , Truemper Lorenz , Wulf Gerald G.
Published: October 2019

Inhibition of bcr-abl tyrosine kinase activity has evolved as a mainstay of therapy for patients with chronic myeloid leukaemia (CML). However, a fraction of leukaemia cells persists under targeted therapy and can lead to disease progression on cessation of treatment. Analysing leukemic progenitor cells with the side population phenotype we found that the bcr-abl positive leukemic clones contributed to this compartment in CML patients, and that the intracellular ABC Transporter A3 is strongly expressed in such leukemic SP cells. Subsequently we found ABCA3 expressed in all of 39 whole bone marrow and peripheral blood samples from 39 CML patients, with transcripts numbers substantially exceeding those of patients with AML or normal volunteers. Similarly, the CML cell lines K562, Lama-84 and BV17.3 showed high endogenous ABCA3 expression levels, allowing specific downregulation by siRNA. As measured in viability and colony forming assays, the functional knock down of ABCA3 induced a significantly increased susceptibility to imatinib. Furthermore, we applied subcellular fractionation techniques to investigate the subcelluar distribution of imatinib, and found the majority of [14C]-labelled imatinib to accumulate in the lyosomal fraction, in K562 and Lama84 leukemic cells as well as the HEK293 model system. In conclusion, the intracellular ABC transporter A3 is expressed consistently and at high levels expressed in CML cells, and may contribute to imatinib resistance via lysosomal sequestration.
PD-1 Signaling on Chronic Myeloid Leukemia-Specific T Cells Results in T Cell Exhaustion and Disease Progression.
Authors: Mumprecht Sabine , Schwaller Juerg , Solenthaler Max , Ochsenbein Adrian F.
Published: October 2019

Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease of hematopoietic stem cells with a characteristic chronic phase of several years before progression to acute myeloid leukemia. The immune system may contribute to disease control at this stage. Here we analyzed leukemia-specific immune responses in a murine retroviral bone marrow transduction and transplantation model using the glycoprotein of lymphocytic choriomeningitis virus as a model leukemia antigen. We found that CML-specific cytotoxic T cells (CTLs) became exhausted after initial activation and expansion. Only a small fraction of CML-specific CTLs persisted longterm. They maintained some limited cytotoxic activity but did not produce IFNγ or TNFα or expand after restimulation. CML-specific CTLs were characterized by high expression of programmed death 1 (PD-1), whereas CML cells expressed PD-ligand 1 (PD-L1). Blocking PD-1 signaling in CML mice by transferring BCR/ABL-NUP98/HOXA9 transduced bone marrow cells to PD-1 deficient recipient mice resulted in improved CML disease control and prolonged survival. In addition, we extended our findings from the preclinical CML model to human patients. PD-1 was expressed at significantly higher levels on CD8+ T cells of CML patients when compared to healthy donors. These data identify PD-1 as a potential target to restore the function of exhausted CML-specific CTLs and therefore to treat CML.
Altered Expression of PP2A Regulatory Subunits in Chronic Myelogenous Leukemia: Identifying Targets for Improved Therapies.
Authors: Roberts Kathryn G. , Ashman Leonie K. , Sim Alistair T.R. , Verrills Nicole M. ,   (Intr. by Danilo Perrotti)
Published: October 2019

Over 90% of chronic myelogenous leukemia (CML) cases are associated with constitutive activation of the BCR/ABL oncoprotein, which induces transformation of hematopoietic stem cells. Inhibition of BCR/ABL with imatinib has shown remarkable success in treating CML patients. However, the emergence of resistance is becoming a major clinical problem. To direct the development of improved therapies, signaling pathways activated by BCR/ABL must be identified. The aim of this project is to determine whether the activity and expression of the tumor suppressor protein phosphatase 2A (PP2A) is altered in the presence of BCR/ABL. PP2A is a serine/threonine phosphatase that is composed of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B), of which there are three different families. Each family of PP2A-B subunits has a number of isoforms that determine the substrate specificity, subcellular localization and catalytic activity of the enzyme. In this study we show that transfection of BCR/ABL into FDC-P1, a mouse factor-dependent myeloid cell line, increased tyrosine phosphorylation on the PP2A-C subunit, and this correlated with a reduction in enzymatic activity. Imatinib treatment rescued PP2A activity in wild type BCR/ABL+ cells, but not the imatinib-resistant Y253F BCR/ABL+ cells. Importantly, pharmacological enhancement of PP2A by forskolin and FTY720 resulted in reduced proliferation in both cell lines. Furthermore, we have demonstrated for the first time that BCR/ABL increases the expression of three specific PP2A-B subunit isoforms - B55α, B56α, and B56δ. It is predicted that enhanced levels of these subunits cause displacement of alternate PP2A-B isoforms to alter enzyme composition and hence the specificity of PP2A. To determine the functional role of each individual B subunit, shRNA studies are being performed. These findings confirm that functional inactivation of PP2A is an essential component of BCR/ABL-induced leukemogenesis in CML, and provide novel insight into the mechanisms by which this may occur. Elucidating the importance of each regulatory subunit may identify novel targets to develop improved therapies for CML patients.
Deletions Adjacent to BCR and ABL Breakpoints Occur in a Substantial Minority of Chronic Myeloid Leukaemia Patients with Masked Philadelphia Rearrangements.
Authors: De Melo Valeria A. , Kotzampaltiris Paul , Nacheva Elisabeth , Marin David , Apperley Jane F. , Reid Alistair G.
Published: October 2019

Chronic myeloid leukaemia (CML) results from the malignant transformation of haematopoietic stem cells by the BCR-ABL fusion gene, generated by a reciprocal translocation between chromosomes 9 and 22, the Philadelphia (Ph) translocation, or variations thereof. Common permutations of the Ph include translocations involving one or more additional chromosomes to 9 and 22 (“variant” Ph), and rearrangements resulting in cryptic insertion of chromosome 9 into chromosome 22, or vice versa, leaving apparently normal chromosomes 9 and 22 (“masked” Ph). Recently the development of new FISH techniques led to the identification of unexpected deletions at the Ph translocation breakpoints in approximately 15% of CML patients. The deletions, encompassing sequences 3′ of the BCR breakpoint and/or 5′ of the ABL breakpoint, were associated with a shorter duration of chronic phase and shorter survival in patients treated with interferon therapy, although their impact in patients treated with tyrosine kinase inhibitors is currently unclear. Interestingly, the incidence of deletions has been shown to vary for different cytogenetic subgroups of CML, with a significantly higher incidence of deletion in patients with a variant Ph translocation. The frequency of such events in patients with masked Ph rearrangements, however, has not yet been explored because of limitations inherent to the widely adopted dual-colour BCR-ABL FISH approach. We report the evaluation of 14 patients with masked Ph-positive CML for the presence of deletions extending 3′ from BCR and 5′ from ABL using two 3-colour BCR-ABL probes. Deletions were identified in 3 patients in total (21%), encompassing sequences 5′ to ABL in two of these and sequences 3′ to BCR in the remaining patient, thus demonstrating that the phenomenon is a significant feature of the masked Ph CML subgroup. Furthermore, our findings are consistent with the notion that loss of genomic material is a potential side effect of any DNA breakage event at the 9q34.1 and 22q11.2 chromosomal regions, regardless of the subsequent mechanism of chromosomal rearrangement.
HyperCKemia in CML Patients on Imatinib Mesylate.
Authors: Hewitt Joanne D. , Rusnak Deborah A. , Cox-Kennett Nanette , Larratt Loree M. , Turner Robert
Published: October 2019

Imatinib mesylate (IM) is the standard therapy for Philadelphia chromosome positive chronic myelogenous leukemia (CML). It is generally well tolerated with most non-hematologic toxicities being mild to moderate in severity. Muscle cramps and/or myalgia may occur in up to half of patients (pts) treated, particularly with doses of IM > 400 mg/day. Prompted by a case of rhabdomyolysis, we began to monitor creatine kinase (CK) levels in CML patients on IM in 2005. 71 patients with chronic phase CML (diagnosed between 1994 and 2007 and treated with IM between 2000 and 2007) had CK levels done every 3 months. Elevated CK occurred in 33/71 (47%) of patients (30% grade 1; 11% grade 2; 4% grade 3 and 2% grade 4); the majority (91%) (including those with ≥ grade 3 toxicity) were taking ≤ 400 mg/day of IM. Of these 33 patients, 21 had a normal baseline CK. The mean and median duration of IM exposure at the time of first documented CK elevation in these 21 patients was 82.5 weeks and 53 weeks respectively (range 4 – 300 weeks). 36% (12/33) of the patients with elevated CK levels reported associated symptoms (muscle cramps, muscle spasms and/or myalgia): the majority of the adverse events were ≤ grade 2 in severity, with some pts reporting more than 1 symptom. CK elevation was documented once in 8 patients, continuously in 9 patients and intermittently in 16 patients. 7 patients required dose modification; all subsequently had a reduction in CK levels to ≤ grade 1. Of the 38/71 who had normal CK levels, 24% (9/38) reported either grade 1 or 2 muscle cramps and/or myalgia: only 1 of these pts required dose reduction due to muscle related toxicity. Possible factors contributing to CK elevation included hypothyroidism (9 pts), vigorous physical activity (7 pts), antipsychotics (7 pts), cholesterol lowering agents (6 pts), alcohol use (2 pts), beta blockers and infection (1 pt each).

Conclusions: hyperCKemia is common in patients on IM for CML (1);hyperCKemia is not correlated with myalgia or muscle cramps;many patients had multiple potential etiologies for increased serum CK;hyperCKemia responds to IM dose modification.
Absence of Residual Vein Thrombosis after an Episode of Idiopathic Deep Vein Thrombosis: Short-Term Anticoagulation Is Safe. The “Extended Dacus Study”.
Authors: Siragusa Sergio M. , Malato Alessandra , Bellisi Mario , Attanzio Maria Teresa , Cigna Valeria , Caramazza Domenica , Lo Coco Lucio , Milio Glauco , Bajardi Guido
Published: October 2019

Background. The optimal duration of Oral Anticoagulant Therapy (OAT) for Deep Vein Thrombosis (DVT) can be tailored by Residual Vein Thrombosis (RVT) (Siragusa S et al. Blood2003;102(11):OC183), a marker able to assess the individual risk for recurrent thrombosis. However, in patients with idiopathic DVT the safety of early interruption of OAT, because of absence of RVT, is still debated.

Objective of the study. In the present study, we evaluated the safety of withholding OAT, in patients with idiopathic DVT and without RVT, three months after the index thrombotic episode.

Study design. Prospective controlled study with two groups: patients without RVT stopped OAT after 3 months while those with RVT continued for additional 3 months.

Materials and Methods. Consecutive patients with a first episode of idiopathic DVT of the lower limbs; patients with cancer or known thrombophilia were excluded. At the third months of OAT, RVT was assessed as previously described; briefly, RVT was considered absent when a clot occupying less than 40% of the vein lumen was detected by compression ultrasonography. Events, classified as recurrent DVT and/or Pulmonary Embolism (PE) and/or major and minor bleeding were evaluated; all patients were followed-up for at least 12 months after OAT discontinuation.

Results. During the period 1999–2006, 518 patients were included in the study. In 206 (39.7%) RVT was considered absent (RVT negative group) and they stopped OAT; the remaining 312 patients continued anticoagulants for additional 3 months (RVT positive group). Total duration of follow-up (FU) was 184.7 years for RVT negative group (with a mean FU of 3.0 ± 0.83 years) and 191.3 years for RVT positive group (with a mean FU of 3.1 ± 0.89 years). The rate and type of events during FU is reported in table and figure.

Conclusions. This investigation shows that in patients without RVT, three months of OAT are safe even after an episode of idiopathic DVT. This hold for at least 30% of the entire DVT population and has an important clinical impact; in fact, it is possible to select a group of patients with a very low risk for recurrences over a period of 3 years. This approach carries also a negligible risk for bleeding.

Events between RVT Negative and Positive Groups Outcomes RVT Neg. group (206) RVT Pos. group (312) "p" value *After OAT discontinuation, **During OAT Recurrences, n/total (%)* 2/206 (0.9) 63/312 (20.2) <0.0005 Recurrences, n/100 person-year (%)* 2/184.7 (1.1) 63/191.3 (32.9) <0.0005 Type of recurrent VTE DVT 1 43 DVT + PE 0 6 Isolated PE 0 3 Controlateral 1 11 Major bleeding, n/total (%)** 0/206 3/312 (0.9) Major bleeding, n/100 person-Yr (%)** 0/184.7 3/191.3 (1.5) Figure Figure
Outpatient Management of Both Myeloablative and Reduced-Intensity Allogeneic HCT Results in Low Non-Relapse Mortality Irrespective of HCT-CI.
Authors: Bashey Asad , Manion Karen , Morris Lawrence E. , Holland Kent , Conaghan Gerard , Matthews Rebekkah , Dooley Amy , Sizemore Connie , Kerns Leslie , Solomon Scott R.
Published: October 2019

Myeloablative allogeneic hematopoietic transplantation continues to be performed as an inpatient procedure from the start of the preparative regimen until neutrophil recovery in most centers. Advances in supportive care and improved remission status pre-transplant may enable predominantly outpatient management of such patients during their transplant. We have analyzed transplant outcomes for consecutive unselected allogeneic transplants performed at our center between February 2005 and June 2006. Patients were scheduled to be admitted to the inpatient unit for less than one day for the stem cell infusion. The remainder of the transplant procedure including the preparative regimen and the period of pancytopenia prior to hematopoietic recovery were managed in the outpatient setting. Patients were admitted to the inpatient BMT unit for the management of complications only. Outpatient care was performed in a dedicated, fully HEPA-filtered facility with detailed infection control measures. If necessary, patients were admitted directly to the inpatient unit and use of the emergency department of the hospital was avoided. Patients were assessed daily for a minimum of 28d post HCT. Fifty-nine patients underwent first allogeneic HCT using non-cord blood donors during the period (median age 41 (21–63); 41 M, 18F; diagnoses - AML=17, ALL=11, NHL=8, MDS/MPS=7, MM=6, HD=2, OTH=7; Myeloablative=35, RIC =24; Related donors=38, MUD=21; ASBMT disease risk score - high=39, intermediate=4, low=16. Median inpatient stay from start of preparative regimen to d100 was 18d (1–63d). With a median follow-up of 565d (383–873d) for all patients, estimated probability of overall survival (OS) at 100d, 12m and 24m are 95%, 80% and 58% respectively. Corresponding probabilities of non-relapse mortality (NRM) are 3.5%, 14% and 23%. For patients undergoing myeloabative HCT, OS and NRM probabilities were 96%, 78%, 58% and 5%, 16%, 22% respectively. The HCT-comorbidity index (HCT-CI, Sorror et al Blood106:2912 [2005]) was used to assess co-existing morbidities. Probabilities of OS and NRM by HCT-CI risk group are shown in the Table. These data suggest that planned outpatient management of allogeneic HCT including myeloblative HCT can be performed without compromising OS and NRM if the appropriate facilities are provided.

HCT-CI 0 1–2 ≥3 Overall Survival d100 100% 97% 83% p=NS 12m 87% 81% 67% Non-Relapse Mortality d100 0% 3% 9% p=NS 12m 10% 14% 20%
A Non-Myeloablative Regimen of Fludarabine and Melphalan Is Safe and Well Tolerated for Allogeneic Transplantation in Multiple Myeloma.
Authors: Qazilbash Muzaffar H. , Saliba Rima M. , Parikh Gaurav C. , Hosing Chitra , Mendoza Floralyn , Weber Donna M. , Wang Michael , Thomas Sheeba K. , Flosser Thuy S. , Kebriaei Partow , Popat Uday , Alousi Amin M. , de Lima Marcos , Champlin Richard E. , Giralt Sergio A.
Published: October 2019

Background: Allogeneic transplantation is a potentially curative approach in patients with myeloma. The potential benefit is offset by a higher risk of transplant-related mortality (TRM), graft-vs.host disease (GVHD) and opportunistic infections. The success of reduced intensity conditioning approaches has renewed interest in allogeneic transplantation. In this randomized phase II trial we evaluated whether lowering the dose of melphalan in a nonablative preparative regimen can lower the toxicity and TRM. Two reduced intensity regimens:

fludarabine + melphalan 140 mg/m2 (FM 140) and fludarabine + melphalan 100 mg/m2 (FM 100) were compared in patients undergoing allogeneic stem cell transplantation.

Methods: We enrolled 22 patients, 11 in each arm, who were 70 years of age or younger and had an HLA-identical sibling donor. Patients underwent allogeneic transplantation between April 2002 and January 2007. Median age was 52.5 years (Range: 32–63). GVHD prophylaxis consisted of tacrolimus and methotrexate 5mg/m2 on days 1, 3, 6 and 11. Median interval between diagnosis and transplant was 28 months (9–232). Only 6 patients (27%) received transplant for the consolidation of first remission, the rest had relapsed or refractory disease (73%). Twenty-one patients had a prior autotransplant; 3 of these had 2 prior autotransplants. Median number of prior treatment regimens was 5 (1–10). Cytogenetic abnormalities were observed in 8/22 patients (36%).

Results: The 2 groups were comparable in terms of age, disease status, prior therapy and other prognostic factors. Median time to neutrophil engraftment in both arms was 12 days. TRM in the first 100 days was zero in both arms. Response rate (CR 18% + PR 64%) was 82%, with no significant difference between the 2 arms. After a median follow up of 25 months (26 months in FM 100, 21 months in FM 140), Kaplan-Meier estimates of 2-year PFS in FM 100 vs. FM 140 were 41% vs. 42%, respectively. Kaplan-Meier estimates of 2-year OS in FM 100 vs. FM 140 were 60 vs. 36%, (p=0.4), respectively. There was no significant difference in the incidence of grade II-IV acute GVHD (27 vs. 36%) or chronic GVHD (50 vs. 43%) between the 2 arms. There was a decrease in overall grade II-IV toxicity in the FM 100 arm (45% vs. 73%) that did not reach statistical significance (p=0.2) Patients transplanted in relapse had shorter PFS (0.09).

Conclusions: The dose of melphalan in preparative regimen can be safely reduced without adversely impacting the engraftment in this heavily pretreated patient population. There was a trend towards lower grade II-IV toxicity and prolonged OS in the FM 100 arm. This approach may be more beneficial when used early in the course of treatment.
Dalteparin Thromboprophylaxis in Critically Ill Patients with Severe Renal Insufficiency: The Direct Study.
Authors: Douketis James , Cook Deborah , Zytaruks Nicole , Heels-Ansdell Diane , Crowther Mark
Published: October 2019

Background: Critically ill patients with renal insufficiency are at high risk for deep vein thrombosis (DVT). Low-molecular-weight heparins (LMWHs) effectively prevent DVT but are avoided in patients with renal insufficiency because of potential bioaccumulation and potential bleeding risk. However, evidence is lacking that LMWHs bioaccumulate in such patients.

Objectives: The objectives of DIRECT were, in critically ill patients with severe renal insufficiency, to determine if dalteparin prophylaxis leads to bioaccumulation and bleeding from excessive anticoagulation and to determine the pharmacodynamic profile of dalteparin.

Methods: Multi-center, open-label, prospective cohort study of critically ill patients with a creatinine clearance <30 mL/min who receive dalteparin, 5000 IU once-daily, subcutaneously for up to 30 days. Dalteparin bioaccumulation in a patient was defined by a trough anti-Xa level >0.40 IU/mL, measured twice-weekly 20 hours after the prior dalteparin dose. The pharmacodynamic profile of dalteparin was assessed by anti-Xa levels measured at 0, 1, 2, 4, 8, 12, 20, and 24 hours after the prior dose on days 3, 10, and 17 of treatment.

Results: We enrolled 156 patients with a mean (SD) creatinine clearance of 18.9 (6.5) mL/min; of these, 18 were excluded because they died or were discharged before testing (n = 3) or had prevalent DVT (n = 14) or pulmonary embolism (n = 1) within 48 hours of enrollment. Of 138 patients included, the median (inter-quartile range [IQR]) duration of dalteparin treatment was 7 days (4, 12). In 120 pa tients who had ≥1 trough anti-Xa measured (427 total), none had dalteparin bioaccumulation; the median (IQR) trough anti-Xa level was <0.1 IU/mL (<0.1, <0.1). In 138 patients who received ≥1 dose of dalteparin,10 patients (7.2%; 95% CI: 4.0, 12.8) had a major bleed, all with trough anti-Xa levels ≤0.18 IU/mL. The pharmacodynamic profile of dalteparin, shown in the Figure, was typical for drugs that do not bioaccumulate, with a typical peak and decline after 3, 10, and 17 days of treatment.

Conclusions: In critically ill patients with severe renal insufficiency, thromboprophylaxis with dalteparin did not bioaccumulate. Dalteparin, 5000 IU once-daily, appears to be a reasonable option for thromboprophylaxis of critically ill patients with severe renal insufficiency.

Figure Figure
Sirolimus as Primary Immunoprophyllaxis for Alternative Donor Allotranspplant after Non-Myeloablative Conditioning.
Authors: Karch Jon M. , Ehmann W.C. , Rybka Witold B. , Chase Gary A. , Claxton David F.
Published: October 2019

We have explored the immunosuppressive agent Sirolimus for immunoprophyllaxis after allogeneic hematopoietic transplant in order to exploit its favorable immunotolerance and antineoplastic properties. Where possible, co-administered tacrolimus has been tapered early so as to minimize its restraint of graft versus tumor activity. Between May 2001 and February 2007, 68 patients with matched unrelated or 5/6 antigen-matched related donors received non-ablative hematopoietic cell transplantation. Conditioning consisted of fludarabine 25mg/m2 (d-7, -6, -5, -4, -3) and cyclophosphamide 1mg/m2 (d-7, -6) followed by 5mg/m2 methotrexate days 1, 3, 6 and combination sirolimus and tacrolimus each dosed at 6–10 ng/ml levels. Median age was 55.2 (range 23.3 to 72.2). Disease diagnoses were ALL - 4, AML - 30, CLL - 7, CML - 3, HD - 1, MDS - 3, MM - 2, MPD - 4, NHL - 14. Only 22/68 patients were in remission (CR1 or CR>1) at transplant. 62/67 patients (5/6 BM and 57/61 PBSC) engrafted stably with donor cells by day 30. Donor blood chimerism was >90 percent in 35/58 by day 100 and was correlated with CD34 cells infused (R=0.263, p=0.033), but not degree of HLA match. In 19/47 patients without GVHD by day 30, tacrolimus was tapered and stopped between days 40 and 100. Sirolimus was continued in the absence of a contraindication for 8 months at least. In 6/19 patients GVHD developed within 2 months of discontinuation of tacrolimus. 2/6 patients developed grade ≥3 GVHD, and in 1/6 GVHD was the cause of death. In 4/6 grade 1–2 GVHD was controlled with steroids and/or reinstitution of tacrolimus. Toxicity was manageable. Post transplant creatinine levels >3.0mg/dl were noted at least briefly in 16/68 patients. Only 4 patients required dialysis. Tacrolimus was discontinued because of renal toxicity in 8/16. In the other 8/16 patients tacrolimus was resumed after improvement in renal function. Thrombotic microangiopathy occurred in only 1 patient. Transplant related mortality (TRM) occurred in 6/68 (9%) by 100 days, and 12/59 (20%) by year 2. The overall survival (OS) was 85% (58/68) at 100 days, and 42% (25/59) at 2 years. In 62 patients engrafting stably, median survival has not been achieved for 21/62 in CR, and was 483 days for 41/62 not in CR at transplant (see figure below.) This data suggests that non-ablative hematopoietic cell transplantation with combination sirolimus and tacrolimus therapy can be accomplished with manageable nephrotoxicity, and sirolimus-based immunosuppression provides long term survival in patients with advanced hematopoietic malignancies. In many patients without GVHD tacrolimus may be discontinued early, with immunosuppression then provided by single agent sirolimus for an extended period.

Figure Figure
Haploidentical Allogeneic Hematopoietic Cell Transplantation in Adults with CD3/CD19-Depleted Grafts after Reduced-Intensity Conditioning: A Phase I/II Study.
Authors: Bethge Wolfgang A. , Bornhaeuser Martin , Beelen Dietrich W. , Stuhler Gernot , Stelljes Matthias , Schwerdtfeger Rainer , Faul Christoph , Vogel Wichard , Handgretinger Rupert , Kanz Lothar
Published: October 2019

Introduction: Haploidentical hematopoietic cell transplantation (HHCT) after high dose conditioning with a megadose of CD34-selected stem cells has been complicated by regimen related toxicities, slow engraftment and delayed immune reconstitution leading to a high treatment related mortality (TRM). A new regimen using graft CD3/CD19 depletion and reduced intensity conditioning (RIC) may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, dendritic-, natural killer- and other graft-facilitating cells which may allow stable engraftment even without the use of a megadose of CD34+ cells.

Methods: A multicenter phase I/II study of HHCT using RIC with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), OKT-3 (5 mg/day, day −5 to +14) and graft CD3/CD19 depletion with anti-CD3 and anti-CD19 coated microbeads on a CliniMACS device has been initiated. No post grafting immunosuppression was applied if the graft contained <5×104 CD3+ cells/kg.

Results: To date, 36 patients (median age=46 [range, 21–65] years) have been enrolled in this study. Diagnosis were AML (n=20), ALL (n=7), NHL (n=3), MM (n=2), CML (n=2) and MCL (n=2). Patients were high risk with refractory disease or relapse after preceding HCT (auto=6, allo=12). The CD3/CD19 depleted haploidentical grafts contained a median of 7.5 × 106 (range, 3.4–17×106) CD34+cells/kg, 2.8×104(range, 0.4–44×104) CD3+T-cells/kg and 3.8×107 (range, 0.02–37.3 x107) CD56+cells/kg. Donor-recipient KIR-ligand-mismatch was found in 20 of 36 patients. The regimen was well tolerated with maximum acute toxicity being grade 2–3 mucositis. Five cases of reversible peripheral neuropathy and 3 cases of progressive multifocal leukencephalopathy (PML) occurred posttransplant in heavily pretreated patients. Engraftment was rapid with median time to >500 granulocytes/μL of 12 (range, 9–21) days, >20000 platelets/μL of 11 (range, 7–30) days and full donor chimerism after 2–4 weeks in all but one patient. Incidence of grade II-IV GVHD was 36% with grade II=9, III=2 and IV=2. TRM in the first 100 days was 10/36 (27%). Overall survival is 16/36 patients (44%) with deaths due to relapse (n=9), infection (n=9), GVHD (n=1) and PML (n=1) with a median follow-up of 194 days (range, 14–1271), resulting in a Kaplan-Meier 1 year survival estimate of 41%. We did not observe a statistical significant survival advantage for patient transplanted from a KIR-mismatched donor.

Conclusion: This regimen is promising in high risk patients lacking a suitable donor, and a prospective phase I/II study is ongoing.
Transcription Factor NF-kB as a Biomarker for Disease Progression and Target for Treatment in Chronic Lymphocytic Leukaemia.
Authors: Hewamana Saman , Clement Mathew , Lin Thet Thet , Jenkins Chris , Fegan Chris , Rowntree Clare , Brennan Paul , Pepper Chris
Published: October 2019

NF-kB is part of a multi-component signalling pathway that regulates the expression of hundreds of genes involved in the control of cell proliferation, cell survival, stress responses, innate immunity and inflammation. The principal constituents of mammalian NF-kB are p65, p50, p52, c-Rel and Rel-B and in their inactive state they are bound in the cytoplasm to the inhibitor protein IKB. Phosphorylation of IKB by IKB kinase has been shown to result in the translocation of NF-kB into the nucleus where it can bind to kB responsive elements. It has been reported that NF-kB is constitutively activated in chronic lymphocytic leukaemia (CLL) cells. However, no previous studies have prospectively quantified the subunits of NF-kB in primary CLL cells or examined their relative expression between patient samples. Therefore, the aim of our study was to perform quantitative subunit analysis of NF-κB in freshly isolated CLL cells and examine their relationship with some of the well established clinical and molecular prognostic markers. NF-κB was evaluated using EMSA (n= 48), supershift assays and p65 subunit-specific ELISA (n=100). Apoptosis was measured at 24h and 48h using annexin V/propidium iodide labelling. We demonstrated a wide variation in NF-κB expression in CLL samples and p50, p65 and c-rel were consistently shown to be the predominant NF-kB subunits. Importantly, we showed a negative correlation between in vitro apoptosis and nuclear p65 NF-kB expression (r2 = −0.22) suggesting that high levels of p65 confer a cytoprotective effect on primary CLL cells. Furthermore, we found that p65 has a positive correlation (r2 = 0.3) with total white cell count (WBC) and CLL samples with a lymphocyte doubling time below 12 months have significantly higher p65 DNA binding (p= 0.02) implicating this NF-κB subunit in disease progression. Finally we have also demonstrated that the NF-κB inhibitor, LC-1, has potent in vitro apoptosis-inducing effects on primary CLL cells. Together, this work supports the use of specific inhibitors of NF-kB, particularly targeting p65, for treatment of CLL.
Early Fludarabine Treatment of TCL-1 Transgenic Mice Promotes Drug Resistance through a Mechanism Not Involving p53 Mutations: Implications for Patients with Chronic Lymphocytic Leukemia (CLL).
Authors: De Lay Michael D. , Johnson Amy J. , Lucas David M. , Durr Darius T. , Sass Ellen M. , Goettl Virginia M. , Lehman Amy , Jarjoura David , Grever Michael R. , Byrd John C.
Published: October 2019

Initial therapy for Chronic Lymphocytic Leukemia (CLL) typically includes nucleoside analogs such as fludarabine. Mutations of p53 are uncommon in CLL patients at diagnosis, but are found at increased frequency in patients with prior treatment and are often associated with resistance to standard therapies including fludarabine. The causality of fludarabine treatment to p53 mutation has not been clearly defined. In previous work with the TCL-1 model of CLL, we demonstrated that mice with active leukemia do not have p53 mutations and respond to fludarabine therapy with a modest survival advantage (Johnson et al, Blood108:1334, 2006). We therefore initiated a randomized trial of fludarabine treatment (34mg/kg daily intraperitoneal injection for five days every 28 days) versus observation (treatment with volume-matched vehicle controls on the same dosing schedule) in TCL-1 transgenic mice prior to the onset of leukemia. The goals of this study were to determine if early treatment with fludarabine would 1) delay disease onset; 2) eventually yield a fludarabine-resistant phenotype; or 3) promote development of p53 mutations in mice developing progressive leukemia. Transgenic TCL-1 mice between the ages of eight and 12 weeks were continually treated with fludarabine (n=32) or saline (n=45) until death while being screened monthly for disease progression through changes in WBC count by peripheral blood differentiation smear, lymphocyte subsets by flow cytometry, and nodal and spleen exams. Despite continued treatment with fludarabine, drug resistance as manifested by leukemia and eventual death occurred. Median time to leukemia onset was 308 days (95% CI: 224, ∞) in the fludarabine group and 338 days (95% CI: 252, ∞) in the saline group. Thus, the risk of developing leukemia was not significantly different between the two groups. Of interest, p53 mutations in exons 2–11 were not found in either group (n=10 each) at the time of death. Microarray studies are currently underway to discover other genes differentially expressed between treated and untreated mice that might contribute to fludarabine resistance and will be presented. In conclusion, our data demonstrate that early fludarabine treatment in the TCL-1 transgenic model of CLL does not prevent onset of leukemia, but instead results in disease resistant to fludarabine. The source of fludarabine resistance in this mouse model was not through acquisition of p53 mutations, but rather an alternative unidentified mechanism(s).
The Proliferative Response to CpG ODN Stimulation Predicts Disease Progression in B-Cell Chronic Lymphocytic Leukemia.
Authors: Laurenti Luca , Longo Pablo G. , Tarnani Michela , Gobessi Stefania , Sica Simona , Leone Giuseppe , Efremov Dimitar G.
Published: October 2019

Chronic lymphocytic leukemia (CLL) B-cells from certain patients exhibit marked proliferation following stimulation with unmethylated CpG-oligodeoxynucleotides (CpG-ODN). In a recent study we observed that CpG-ODN induced proliferation occurs primarily in leukemic B-cells from patients with progressive disease and unmutated immunoglobulin (Ig) VH genes. To further investigate whether the proliferative response following CpG-ODN stimulation can be used as a prognostic marker in CLL, we evaluated progression-free survival (PFS) and treatment-free survival (TFS) in a larger series of well characterized patients that had been followed for an extended period. In addition, we correlated the proliferative response with other prognostic factors, including Ig VH gene mutation status, ZAP-70 expression and the presence of “high risk” cytogenetic abnormalities. Analysis of 3H thymidine incorporation in purified CLL B-cells stimulated for 72 hours with CpG-ODN 2006 revealed a proliferative response in 23 (40%) of the 58 evaluated cases. Eighteen (78%) of the proliferating cases had unmutated and 5 (22%) had mutated (M) Ig VH genes, whereas among the 34 non-proliferating cases with available Ig VH gene data 8 (24%) had unmutated and 26 (76%) mutated Ig VH genes (P=<0.001 by chi-square test). ZAP-70 was expressed by 16 of the proliferating (70%) and 14 of the non-proliferating cases (40%), but this difference was not statistically significant (P = 0.053). The high-risk risk cytogenetic abnormalities del(17p13.1) and del(11q22.3) were detected in 6 of the 21 evaluated proliferating (29%) and in 3 of the 30 evaluated non-proliferating cases (10%) (P = 0.136). Median PFS was 25 months in the proliferating and 72 months in the non-proliferating subset (P=0.001). Similarly, median TFS was significantly shorter in the proliferating (30 months) than in the non-proliferating subset (114 months, P=0.001). In summary, these data confirm that the proliferative response to CpG-ODN stimulation is strongly correlated with Ig VH gene mutation status, but does not depend on ZAP-70 expression. Although high-risk cytogenetic abnormalities were more frequent in the proliferating subset, the difference was not significant. Cases with a positive proliferative response had significantly shorter PFS and TFS, indicating that the proliferative capacity of the leukemic cells is an important determinant of the clinical course in CLL. Elucidation of the mechanisms that underlie the different proliferative capacity of CLL B-cells should provide additional insight in the pathogenesis of CLL and may lead to the identification of novel and clinically useful prognostic markers.