bluuebunny/crossref_metadata_2025 huggingface dataset:
Exosite Interactions Contribute to the Activated Protein C-Catalyzed Inactivation of Factor VIIIa.
Authors: Varfaj Fatbardha , Neuberg Julie , Wakabayashi Hironao , Fay Philip J.
Published: October 2019

Activated Protein C (APC) is an anticoagulant serine protease that proteolytically inactivates cofactors Va and VIIIa. Cleavage of human factor VIIIa occurs at Arg336 and Arg562 located within the A1 and A2 subunits, respectively. While cleavages are not ordered, the former site appears to represent a preferred cleavage site. Efficient catalysis requires binding of APC to a phospholipid surface and to the A3-C1-C2 subunit of factor VIIIa. The latter observation suggests that APC likely binds substrate via an exosite(s) thereby contributing to substrate specificity. A study was undertaken to evaluate contributions of substrate docking at the active site and exosite tethering to the APC catalytic mechanism. Recombinant, human factor VIII mutants where P1 Arg residues at 336 and 562 were substituted with Ala or Gln were constructed and stably expressed. Purified factor VIII was converted to factor VIIIa by thrombin and used as substrate to elucidate the mechanism of cleavages. Proteins mutated at Arg336 were also mutated at Lys338 because the latter residue may serve as an alternative APC cleavage site when residue 336 is mutated. Rates of inactivation of wild type and mutant factor VIIIa molecules and rates of cleavage at Arg336 and Arg562 by APC were monitored in the presence and absence of protein S. The R336A/K338A mutant was inactivated and cleaved at the 336 site approximately 20-fold slower than the wild type, whereas the R336Q/K338Q mutant was completely resistant to cleavage at the 336 site. These results indicate that residues other than Arg may be tolerated at the P1 site, whereas Gln yields a cleavage-resistant substrate. Indeed, the R336Q/K338Q/R562Q (triple Q) mutant was resistant to cleavage at both P1 sites. Furthermore, mutations retarding cleavage at residue 336 showed a dramatic decrease in rates of inactivation suggesting that cleavage at this site correlated with the inactivation of factor VIIIa. The importance of exosite interactions was explored by inhibition experiments examining the inactivation of wild type factor VIIIa in the presence of triple Q mutant factor VIIIa. Wild type factor VIIIa inactivation rates decreased as the proportion of triple Q factor VIIIa increased, indicating that the P1 mutant factor VIIIa effectively sequestered APC from the native substrate. Evaluation of inactivation rates suggested that APC possessed an ~8-fold greater affinity for the triple Q FVIIIa than the wild type factor VIIIa. Consistent with that observation, the Ki for triple Q factor VIIIa (29.5 ± 3.6 nM) was ~5-fold less than the Km for wild type factor VIIIa (133 ± 27 nM). Taken together, these results indicate that mutations in the P1 site that prevent cleavage may also retard dissociation of the enzyme-substrate complex. Overall, results from this study suggest that exosite interactions make a primary contribution to substrate affinity during APC-catalyzed inactivation of factor VIIIa.
Fibromodulin as a Novel Tumor-Associated Antigen (TAA) in Chronic Lymphocytic Leukemia (CLL) Which Allows Expansion of Specific CD8+ Autologous T Lymphocytes.
Authors: Mayr Christine , Bund Dagmar , Schlee Martin , Moosmann Andreas , Hallek Michael , Wendtner Clemens
Published: October 2019

BACKGROUND: Fibromodulin (FMOD), a collagen binding protein, was shown to be highly overexpressed in CLL cells compared to normal B lymphocytes by gene expression profiling. Therefore FMOD might serve as potential tumor associated antigen (TAA) in CLL, enabling expansion of FMOD-specific T cells. FMOD is physiologically expressed in articular cartilage, tendon and ligament. Furthermore, interactions of FMOD with the growth factor TGF-b were described and it may be a biologically relevant modulator of TGF-b activity.

Methods: Unpulsed native CLL cells and CD40 ligand (CD40L)-stimulated CLL cells as antigen presenting cells (APC) were used to expand autologous T cells from 13 patients.

RESULTS: In CLL samples derived from 16 patients, high expression of FMOD by real-time RT-PCR was detectable in contrast to normal B lymphocytes. The number of T cells during four weeks of in vitro culture increased 2-fold with native CLL cells as APC and 3.5-fold with CD40L-stimulated CLL cells as APC. The amount of T cells recognizing HLA-A0201 binding FMOD-derived peptides detected by HLA-A2-dimer/peptide staining increased 10-fold during in vitro culture. The T cells expanded were also able to secrete IFN-g upon recognition of the antigen demonstrated by IFN-g-ELISPOT assays. T cells not only recognized HLA-A0201 binding FMOD peptides presented by TAP-deficient T2 cells, but also FMOD overexpressing autologous CLL cells in an HLA-A0201 restricted manner. Neither HLA-A0201 negative CLL cells nor non-malignant cells, i.e. PBMC from healthy donors or tonsilar B cells, were specifically recognized by T cells. When CD40L-stimulated CLL cells were used as APC, which were pulsed with FMOD peptides prior to coincubation with the T cells, significant higher amounts of T cells specifically recognized autologous CLL cells in IFN-g-ELISPOT assays P< 0.018).

CONCLUSION: FMOD was shown for the first time to be naturally processed and ( presented as TAA in primary CLL cells. This enables the expansion of autologous tumor-specific T cells which might be applicable in clinical vaccination trials or as a tool for a CLL-specific immune monitoring in the context of vaccination approaches including CD40L-gene modified autologous leukemic cells.
Lentiviral CCR5 Intrabody Gene Delivery Provides Protection and Enrichment during CCR5-Tropic Infection.
Authors: Swan Christina H. , Buhler Bernd , Tschan Mario P. , Barbas Carlos F. , Torbett Bruce E.
Published: October 2019

The molecular mechanism of human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep mechanism. The viral envelope glycoproteins (env) binds first to CD4 and subsequently interacts with the V3 loop with a chemokine receptor, CCR5 or CXCR4, triggering the fusion event. Several findings suggest that viruses using CCR5 for entry (R5-tropic HIV-1) is the predominant species transmitted among patients. Importantly, CCR5 expression levels determine disease progression. CCR5 does not seem to be necessary for normal cell function, since individuals with a homozygous mutation (Δ32) do not appear to have clinical, immune alterations. Furthermore, these individuals are highly protected against transmission of R5-tropic HIV-1. Therefore, intervention strategies aimed at altering or blocking CCR5 expression may be beneficial for cellular protection and provide a clinical benefit against HIV-1 infection. We have constructed an HIV-1 derived vector, CAD-R5, expressing an intracellular single-chain antibody (intrabody) specific for CCR5 coupled to a KDEL endoplasmic reticulum retention signal. Intrabody expressing primary T-cells efficiently disrupted CCR5 cell surface expression with a 4.3-fold reduction in CCR5 mean fluorescence intensity (MFI) as compared to the control vector without the intrabody gene (7.9 and 33.6 MFI, respectfully). CAD-R5 transduced primary CD4 T-cells expressing the intrabody gene were resistant to R5-tropic HIV infection as shown by a 50 to 60-fold reduction in HIV-1 p24 concentration. Moreover, intrabody gene expressing cells demonstrated a selective advantage and enriched by 6.4-fold in a population of infected cells as compared to uninfected or infected cells containing vector without the intrabody gene. The SCID-human mouse model, produced by conjoining fetal human thymus and liver under the renal capsule, has become a staple for in vivo testing of T cell anti-retroviral therapy. When CAD-R5 transduced, fetal liver derived CD34+ stem cells were used for reconstitution of the human implants in SCID-hu mice (n = 2, 2 groups), 6-weeks later the human thymi displayed an average of 25% reporter gene expressing T cells. Thymocyte development was not altered by vector integration or loss of CCR5 cell surface expression as determined by the CD4+/CD8+ staining profile. Importantly, CCR5 intrabody expressing thymocytes were also highly resistant to ex vivo R5-tropic HIV-1 challenge with a 3-fold reduction in viral load. These results validate the efficacy of lentiviral delivered CCR5 intrabody mediated protection from R5-tropic HIV-1. The findings also underscore the potential advantage of intrabody gene delivery to CD34+ stem cells, which allows differentiation of protected T cell progeny.
CD34+ Cell Derived Macrophages Are Protected from HIV-1 Challenge by Intrabody-Mediated Reduction of CCR5.
Authors: Fischer Kimberlee M. , Just Margaret L. , Barbas Carlos F. , Torbett Bruce E.
Published: October 2019

The three main coreceptors that HIV-1 uses for viral entry are CD4 and the chemokine receptors CXCR4 and/or CCR5. Viral tropism dictates which chemokine receptors are used for viral entry. Naturally occurring CCR5 gene polymorphisms resulting in gene deletion (delta 32) results in loss of receptor expression and provides protection from CCR5 using viruses. There are no known deleterious effects of CCR5 loss in mice or in humans. The focus of our studies is to limit infection of myeloid cells by CCR5 using HIV-1s by decreasing CCR5 cell surface expression. We have successfully accomplished this by using HIV-1 vectors to deliver an intrabody directed against CCR5. The CCR5-specific intrabody gene, containing the endoplasmic KDEL sequence, produces a single chain antibody that sequesters CCR5 in the endoplasmic reticulum. We show that >= 93% of THP-1s, a myelomonocytic cell line, transduced with CCR5 intrabody genes stably expressed intrabodies for 6 months. Of cells expressing intrabody, CCR5 was decreased at least 80% on the cell surface. In macrophages derived from CD34+ cells, CCR5 gene expression was maintained at initial transduction levels throughout the 3-week culture period. Colony forming assays (CFU) were performed to assess the effect of intrabody expression on hematopoietic development. No differences were seen in numbers of CFU and colony types when comparing parental, control vector, and intrabody expressing cells (n = 3). To determine the level of protection afforded by CCR5 intrabody expression, macrophages were exposed to JR-FL, a CCR5 using HIV-1, at 0.3 and 0.51 multiplicity of infection. Cultures were maintained for 15 – 23 days and evaluated for p24, a measure of viral replication, at 3 – 8 day intervals. At the end of culture it was found that macrophages expressing CCR5 intrabody generated 42-, 57-, 52-fold less p24 as compared to parental and control vector containing macrophages. Thus, CCR5 intrabody expression drastically decreased viral load. Therefore, reduction of CCR5 through vector delivery of intrabody genes to CD34+ cells should reduce the likelihood of infection of myeloid cells, their function as viral reservoirs, and has promising therapeutic potential. Currently, studies are underway to evaluate this modality of treatment in humanized NOD/SCID mice.
Development of a Unique siRNA and Intrabody Combinatorial HIV-1 Vector to Knockdown CXCR4 and Protect Cells from HIV-1 Challenge.
Authors: Swan Christina H. , Tschan Mario P. , Barbas Carlos F. , Torbett Bruce E.
Published: October 2019

We overcome the problem of knocking down highly expressed gene function by combining intracellular single chain antibodies (intrabodies) and short interfering RNA (siRNA) to the same gene product. The chemokine receptor CXCR4 is expressed at high levels on T cells and is the co-receptor for pathogenic HIV-1s (termed X4-tropic). Furthermore, it has been shown that HIV-1 requires low amounts of CXCR4 for X4-tropic viral entry (Konopka and Düzgüne AIDS Res Hum Retro, 2002). In contrast to CCR5, due to the high levels of CXCR4, it has been more difficult to provide protection from HIV-1 challenge using conventional siRNA strategies. To address this issue a CXCR4 siRNA and intrabody gene combination HIV-1 vector has been generated. The CXCR4 intrabody is specific for the C-terminal domain of CXCR4 and is coupled to the KDEL endoplasmic retention signal and CXCR4 siRNA targets mRNA nucleotides 331–349 (Martinez, et al. AIDS, 2002). Using the myelomonocytic THP-1 cell line, a line that highly expresses cell surface CXCR4, it was found that CXCR4 intrabody or siRNA vectors mediated a 50% reduction in cells expressing CXCR4 as assessed by flow cytometry. In contrast, the level of CXCR4 cell knockdown increased to 82% using the CXCR4 siRNA and intrabody combinatorial vector. Moreover, the CXCR4 mean fluorescent intensity in cells containing the combination vector resembled the mean fluorescent intensity of cells analyzed with isotype control antibodies, i.e., little detectable CXCR4 expression as judged by flow cytometry. Similar CXCR4 knockdown trends were seen in primary CD4+ T-cells expressing the combinatorial vector as compared to vectors containing the intrabody gene or siRNA alone. HIV-1 X4-tropic challenge studies using a multiplicity of infection of 1 have shown that THP-1 cells expressing the combinatorial vector are resistant to infection (HIV-1 p24 production < 3 ng/ml), whereas THP-1 cells expressing the siRNA or intrabody gene vector were susceptible to infection (HIV-1 p24 > 15–35 ng/ml). These findings demonstrate that targeting both the message and protein product from the same gene is vastly superior to the targeting of either message or protein. The combinatorial vector is currently being tested in humanized mice for efficacy. Lastly, targeting a highly expressed cellular product at the message and protein level may prove beneficial for efficient gene knockdown outside the HIV-1 research and therapy field, such as for metastatic cancers associated with CXCR4/SDF-1 upregulation.
Can We Do Better? Identifying Orthopedic Surgery Patients at High Risk for Venous Thromboembolism Despite Thromboprophylaxis.
Authors: Kahn Susan R. , Schiff Renee L. , Shrier Ian , Hammouda Wahbi , Zukor David
Published: October 2019

Background

Routine administration of thromboprophylaxis is the standard of care for patients undergoing major orthopedic surgery due to the very high rate of postoperative venous thromboembolism (VTE). However, because prophylaxis is not 100% effective, a significant proportion of patients who receive thromboprophylaxis develop post-operative VTE (“breakthrough VTE”). Clinical and surgical characteristics that are associated with the development of VTE in orthopedic surgery patients who receive standard thromboprophylaxis have not been well characterized.

Objectives

To evaluate the incidence of and risk factors for breakthrough VTE in patients who underwent major orthopedic surgery at a tertiary care hospital in Montreal, Canada.

Methods

Charts from consecutive patients who underwent total hip arthroplasty (THA), total knee arthroplasty (TKA) or hip fracture surgery (HFS) (hip pinning or hemiarthroplasty) from August 1, 1999 to April 30, 2000 were abstracted using standardized case report forms. Data were collected on patient characteristics (including VTE risk factors and postoperative complications), surgical characteristics (including type of surgery, operative factors, type of anesthetic) and thromboprophylaxis regimen. The results of any tests performed for clinically suspected VTE were documented, and associations between potential risk factors and objectively confirmed VTE were examined.

Results

Over the 9 month study period, 310 patients underwent major orthopedic surgery (34% THA, 30% TKA, and 36% HFS) and received standard thromboprophylaxis with either dalteparin or enoxaparin (mean duration of prophylaxis: 7 days). Of 83 suspected VTE, 44 (7 proximal DVTs and 37 distal DVTs; 14% of study population; median onset 6 days post-op) were confirmed with objective testing (compression ultrasonography). Multivariate analyses showed that knee surgery (odds ratio [OR] 4.8, 95% confidence interval 2.3, 10.1) and type of low molecular weight heparin (enoxaparin: protective) (OR 0.39, 95% confidence interval 0.20, 0.80) independently predicted VTE. Patient characteristics such as previous VTE, malignancy, hormonal therapy or varicose veins were not associated with VTE.

Conclusion Despite receiving standard thromboprophylaxis, 14% of patients undergoing major orthopedic surgery developed symptomatic breakthrough VTE. Factors that independently predicted VTE in our population were TKR surgery and type of low molecular weight heparin. Our results suggest that TKR patients may warrant more aggressive post-operative physiotherapy and ambulation and adjunctive prophylactic measures such as pneumatic compression. In addition, due to the heterogeneity of different low molecular weight heparin compounds, direct comparison of the effectiveness of enoxaparin with dalteparin for orthopedic prophylaxis in prospective, randomized trials seems warranted.
Efficacy of Patient Self Testing (PST) for Management of the Hypercoagulable Patient.
Authors: Zucker Marcia L. , Jacobs Helena , LaDuca Frank M. , Kessler Craig
Published: October 2019

The PREVENT trial showed strong evidence of the importance of long term low intensity oral anticoagulation therapy (OAT) in patients diagnosed with hyper-coagulable disorders following an initial venous thrombo-embolic event. The current study was designed to compare patient compliance, satisfaction, safety and time in range (TIR) with OAT monitored by either PST or standard laboratory methods. All patients enrolled had a history of at least one hyper-coagulable disorder and had experienced one or more thrombotic episodes prior to enrollment. Patients randomized to the PST arm were directed to test weekly using the ProTime Microcoagulation System (ITC, Edison, NJ); those in the control arm were directed to return to the clinic monthly for Prothrombin time/International Normalized Ratio (PT/INR) testing. Additional testing was encouraged for both groups in response to changes in diet, medication or clinical condition. Of the patients screened for the trial, eight patients randomized to the control arm completed the evaluation period and twenty-three patients randomized to the PST arm have completed at least 6 months of testing. Time in range was determined by the method described by Rosendaal where the INR results between two test dates are assumed to be evenly split between the two test results. The PST patients tested on average once every 9 days (range 5 – 16 days); demonstrating a mean TIR of 73% (range 45 – 90%). The control patients tested at an average frequency of once a month (range 0.3 – 3 months); demonstrating a mean TIR of 73% (range 24 – 96%). There were no statistical differences in the TIR or time above or below range between the two groups. Satisfaction surveys completed at the time of enrollment and after 3 and 6 months of PST showed strong endorsement of PST use. This pilot study demonstrates the utility of PST in the management of OAT in patients with a number of idiopathic coagulopathies and congenital hypercoagulable states (e.g., Protein C deficiency, hyperhomocysteinemia) as an alternative to routine clinical management without compromise to the patient’s well-being.
Sequential Therapy with Cytarabine and Bismuth-213 (213Bi)-Labeled-HuM195 (Anti-CD33) for Acute Myeloid Leukemia (AML).
Authors: Mulford Deborah A. , Pandit-Taskar Neeta , McDevitt Michael R. , Finn Ronald D. , Weiss Mark A. , Apostolidis Christos , Morgenstern Alfred , Divgi Chaitanya R. , Larson Steven M. , Scheinberg David A. , Jurcic Joseph G.
Published: October 2019

HuM195, a humanized anti-CD33 monoclonal antibody, targets myeloid leukemia cells and has activity against minimal residual disease. To enhance the potency of native HuM195 and avoid nonspecific cytotoxicity seen with β-emitting radioimmunoconjugates, the α-emitting radiometal 213Bi was conjugated to HuM195. The feasibility, safety, and antileukemic activity of therapy with 213Bi-HuM195 were shown in a phase I trial (Jurcic et al. Blood 2002). Because of the short-range (50–80 μm) and high linear energy transfer (8400 keV) of α particles, radioimmunotherapy with 213Bi is ideally suited for the treatment of residual disease. To determine the effects of 213Bi-HuM195 after partial cytoreduction with chemotherapy, we treated 25 patients (median age, 67 years; range, 49–80) with cytarabine 200 mg/m2/day for 5 days followed by 213Bi-HuM195 in a phase I/II trial. Fourteen patients had relapsed or primary refractory AML; 5 patients had previously untreated de novo AML, and 6 patients had untreated secondary AML. Sixteen patients had intermediate-risk cytogenetics, and 9 had poor-risk cytogenetics. During the phase I portion of the study, cohorts of 3–6 patients were treated with 0.5, 0.75, 1, and 1.25 mCi/kg. At the 1.25 mCi/kg dose level, 2 of the 4 patients had dose-limiting myelosuppression (grade 4 leukopenia lasting ≥ 35 days) and 1 patient died of progressive pneumonia. Therefore, the maximum tolerated dose was determined to be 1 mCi/kg. No responses were seen at the first two dose levels. Seven of the 19 patients (37%) who received 1 mCi/kg (n=15) or 1.25 mCi/kg (n=4) responded. There were 2 CRs lasting 9 and 12 months; 3 CRp (CR with incomplete platelet recovery) lasting 1, 2, and 6 months and 2 PRs lasting 3 and 8 months. The median time from initiation of chemotherapy to recovery of leukocyte counts was 34 days (range, 21–59 days). Delayed count recovery was attributed to persistent leukemia in 6 patients. Neutropenic fever occurred in all patients, and 19 patients had documented infections. At the phase II dose level, two of the 15 patients died of progressive infections, and one had grade 4 hyperbilirubinemia. The most common extramedullary toxicities were transient, low-grade elevations in liver function tests (n=19) and serum creatinine (n=11). Thirteen patients had infusion-related reactions following the first injection of 213Bi-HuM195, typically characterized by reversible grade 1 or 2 fever and/or chills. One patient had orthostatic hypotension and syncope after the first antibody infusion associated with concomitant bacteremia. Sequential administration of cytarabine and 213Bi-HuM195 is tolerable and can produce complete remissions in patients with AML.
Addition of Cladribine to the Standard AML Treatment Improves Long-Term Outcome in High Tumor Burden and Older Than 40 Years Acute Myeloid Leukemia Patients. Five-Year Follow-Up of the Polish Adult Leukemia Group (PALG 1999 DAC vs. DA Study).
Authors: Holowiecki Jerzy , Grosicki Sebastian , Robak Tadeusz , Krzemien Slawomira , Giebel Sebastian , Hellmann Andrzej , Skotnicki Aleksander , Jedrzejczak Wieslaw W. , Konopka Lech , Kuliczkowski Kazimierz , Zdziarska Barbara , Dmoszynska Anna
Published: October 2019

The goal of the study was to evaluate long-term outcome of acute myeloid leukemia (AML) patients treated within the PALG 1999 DAC vs, DA Study.

Between 1999–2002, 445 patients, aged 18–60 years, were randomized to the induction DAC-7: daunorubicin 60 mg/m2/d iv 1–3; cytarabine 200 mg/m2/d ci 1–7; cladribine 5 mg/m2 2h inf. iv d 1–5 or standard DA-7 regimen (the same regimen without cladribine). Patients achieving CR received two courses of subsequent intensive consolidation: 1) HAM (HD AraC, mitoxantrone) 2) HD AraC with or without cladribine in the DAC-7 or DA-7 arm, respectively. In case of PR after the first induction course the same regimen was repeated, NR patients received CLAG (2-CDA, HD-AraC, G-CSF), regardless the randomization arm. Post-consolidation therapy was in both arms comparable.

As previously reported, a single course of DAC-7 induction resulted in 17% higher CR rate compared to the DA-7 treatment. The difference was particularly pronounced in a population of patients >40 years and for those with initial WBC >100x109/L. In the latter subgroup also the overall CR rate (achieved after >=1 induction course) was higher in the DAC-7 arm (71% vs. 43%). [Leukemia. 2004 May;18(5):989–97]

In the present report we analyzed long-term outcome (median follow-up 3.2 years) in the whole study group and in pre-defined subgroups taking into account initial tumor burden, age, cytogenetics, FAB subtype, and preceding myelodysplasia. At five years the overall survival (OS) rate equaled 31% for DAC-7 and 25% for DA-7 arm (p=0.42) and leukemia free survivall (LFS) 32% vs. 29% (p=0.38), respectively.

The subgroup analysis revealed higher probability of the OS in patients with initial WBC ≥100 G/l assigned to DAC-7 compared to DA-7 arm (39% vs. 11%, p=0.02). The LFS rate and the probability of relapse equaled 50% and 32% (p=NS) and 36% and 68% (p=0.0497), respectively.

In patients aged >40 years, the therapy containing cladribine was associated with improved LFS (30% for DAC-7 vs. 20% for DA-7, p=0.01), and a tendency to improved OS (28% vs. 18%, p=0.09). In this subgroup DAC-7 therapy resulted in reduced relapse incidence (60% vs. 69%, p=0.04).We conclude that addition of cladribine to induction/consolidation therapy of AML may improve long-term outcome in higher age ranges patients and in those with high tumor burden. The improvement results mainly from reduced risk of relapse, and, in patients with high initial WBC, from higher CR rate.
Effects of the FLT3 Inhibitor PKC 412 in Combination with Cytostatic Drugs In Vitro in Acute Myeloid Leukemia.
Authors: Möllgård Lars , Bengtzen Sofia , Fioretos Thoas , Andersson Anna , Paul Christer , Lehmann Sören
Published: October 2019

FLT3 is a member of the class III tyrosine kinase receptor family. Interaction with its ligand results in reactions that involve different signalling pathways in the cell. A majority of acute myeloid leukemias (AML) express FLT3. FLT3 is mutated and activated in about 30% of all patients with AML. The new compound PKC412 was found to specifically an potently inhibit the growth of leukemic cell lines with a FLT3 mutation.

In the first part of this study the primary objective was to evaluate the in vitro effect of the combination of PKC412 and ara-C focusing on the timing of the two drugs. Samples from 21 patients with AML have been included. IC50 curves for PKC 412 curves were obtained to find appropriate concentrations for the study. Two different in vitro models were used. In the 4+3 model the incubation time for the first drug was 4 days and the second drug was added after 1 day. In the 2+2 model the cells were incubated for 2 days with the first drug, then centrifuged and resuspended and incubated with the second drug for another 2 days. The FLT3 status was analysed with PCR and the ATP bioluminiscens method was used to determine viable cells after the incubations.

The preliminary results show that most of the possible combinations of the 2 drugs had an additive effect. In the 2+2 model 2 days with ara-C 0,5 m M followed by 2 days with PKC 0,2 m M resulted in a synergistic effect in the AML patient cells with FLT3-ITD. We also found a synergistic effect in the FLT3 mutated cells when PKC412 0,2 m M and daunorubicin 0,15 m M was combined in a 4 days incubation

Our results suggest that the timing of PKC412 in combination with cytostatic drugs can influence the effect. The timing of the drugs may be of importance in the designing of future clinical trials of PKC412 in AML patients.
Radiotherapy-Based Conditioning Is Effective Immunosuppression for Patients Undergoing Transplantation with T-Cell Depleted Stem Cell Grafts for Severe Aplasia.
Authors: Novitzky Nicolas , Thomas Valda , Stubbings Helen , Hale Geoff , Waldmann Herman
Published: October 2019

The combination of cyclophosphamide and ALG is highly effective in inducing engraftment in patients with aplastic anaemia undergoing allogeneic stem cell transplantation. However, the incidence of graft vs. host disease remains substantial. We studied the outcome of individuals with aplastic anaemia conditioned with a radiation containing regimen followed by the infusion of stem cell grafts that had been depleted of lymphocytes with CAMPATH-1H. The conditioning regime consisted of fractionated (f) total body irradiation 8 Gy followed by f total nodal irradiation 6 Gy. Additionally, patients received cyclophosphamide 60 mg/kg on 2 consecutive days. Cytokine mobilised peripheral blood grafts from HLA identical siblings were T-cell depleted with CAMPATH-1H “in the bag”. Cyclosporin was commenced on day -1 and continued until day +90. Seventeen heavily transfused patients with aplastic anaemia with a median age of 18 years (range 14–56) were studied. Median time from diagnosis to transplantation was 172 days (range 34–443). The median CD34+ cell number infused was 3.47 x106/kg (range 1.03–18.4). All patients engrafted. Recovery was fast and patients reached 0.5 x 109/L polymorphs by median day 11 (range 9–17) days. Toxicity from the conditioning included grade 4 haematological toxicity in all. Other major toxicity was gastrointestinal mucosal damage, which exceed grade 2 in only 2 instances. 1 patient developed thrombotic thrombocytopenic purpura which responded to substitution of cyclosporin with tacrolimus and plasmapheresis. Another, who had normal blood counts died of infection on day 241. Chimerism studies at 6 months post transplantation confirmed donor origin of haematopoiesis in all 7 patients tested. None of the patients developed acute or chronic graft vs. host disease. There was no delayed graft failure and 94% of patients survive disease free, at a median of 1303.5 days (range 216–2615) from graft infusion. In this cohort of multiply transfused patients, the radiation containing schedules described in this study led to universal engraftment with limited toxicity despite T-cell depletion. No patient developed graft vs. host disease or late graft failure. Lower doses of radiation containing conditioning should be explored further.
Fludarabine Is Superior to Cladribine When Added to Busulfan and Low Dose TBI as Reduced Intensity Conditioning for Allogeneic Hematopoietic Cell Transplantation (HCT): A Prospective Randomized Trial.
Authors: Markova M. , Barker Juliet N. , Wagner John E. , Miller Jeffrey S. , Arora Mukta , Burns Linda J. , MacMillan Margaret L. , Repka Tanya , Blazar Bruce R. , Weisdorf Daniel J.
Published: October 2019

Purine analogues have been combined with alkylator/irradiation as immunosuppressive and anti-tumor conditioning prior to allogeneic hematopoietic stem cell transplantation (HCT) though differing purine analogues have not been compared. We prospectively tested Fludarabine (F) 40 mg/m2/d x 5d vs. Cladribine (C) 10 mg/m2/d x 5d plus Busulfan (Bu) (2mg/kg q12h x 2d) and total body irradiation (T) 200cGy followed by cyclosporine and mycophenylate mofetil in 19 recipients of matched sibling peripheral blood stem cell and 13 unrelated donor (URD) marrow HCT. Patients in each randomly assigned cohort [FBuT vs. CbuT] were similar in age (median 52 years in both groups), diagnosis (leukemia/MDS 38 vs. 31%; lymphoid malignancy 57 vs 69%), extensive pre-HCT therapy (56 vs. 63%), high risk disease status (81 vs. 93%) and Karnofsky (median 90 in each)[all p= NS] though fewer FBuT were URD recipients 25% vs. CBuT 56%, p=0.07. Engraftment was prompt in both groups (median 11 vs. 12 days), but the cumulative incidence of neutrophil engraftment was 75% (95% C.I. 54–96%) using CBuT vs. 100% with FBuT (p<0.01) and randomization was halted. Platelet recovery was prompt (median FBuT 18 vs CBuT 24 days) and after FBuT 75% (95% C.I. 49–100) vs. CBuT 69% (43–95) recovered platelets > 50,000/μL by day +180, p=0.19. The cumulative incidence of GVHD after FBuT vs. CbuT was similar (acute grade II/IV 56 vs. 69%, p=0.26) and (chronic 50 vs. 31%, p=0.27). Transplant related mortality at day +180 was also similar [FBuT 25% (4–46) vs. CbuT 38% (14–61), p=0.47]. Survival was equivalent: at 1 year 50% in each group; at 3 years FBuT 25% vs. CBuT 38%, p=0.55. Multivariate analyses adjusted for age, donor type, diagnosis and stage as well as conditioning regimen showed lower relative risk (RR) of engraftment with CBuT (RR 0.6 (95% C.I. 0.2–1.3) p=0.16) and with URD RR 0.4 (0.2–1.0) p=0.04). RR of Platelet recovery was equivalent with FBuT (RR 0.7 (0.3–1.7) p=0.45) but inferior with URD (RR .16 (.05–.5) p<0.01). RR of GHVD II/IV similar with FBuT RR 1.1, p=0.95, but more frequent with URD RR 2.0, p=.2 and high risk status patients (RR 4.5, 1.5–13.5, =<0.01). Prevalence of remission (CR or PR) at 18 months was high and was similar in both groups (FBuT 100% vs CBuT 86%, p=NS). These data suggest that older patients with advanced hematologic malignancies can achieve satisfactory post-transplant outcomes using either of these combination/reduced intensity conditioning regimens. Fludarabine may be superior to cladribine as a component of pre-HCT conditioning with Bu/TBI due to reduced risks of graft failure. Further modifications of the regimen may confirm universal engraftment with even lower peri-transplant morbidity.
High Relapse Rate Following Alemtuzamab Use in Allogeneic Transplants for Myeloid Hematologic Malignancies.
Authors: Shea Thomas C. , Serody Jonathan , Gabriel Don , Comeau Terrance , Harvey Donald , Irons Robert , Morris David , Sharf Andrew , Krasnov Carol , Dunphy Cherie , Banderenko Nicholas , Brecher Mark
Published: October 2019

Chakravarti, (Blood, 2002, p1071) and others have described the use of alemtuzamab, a monoclonal antibody that binds to CD52 on T cells, B cells, monocytes and dendritic cells in vivo for control of graft vs host disease in reduced intensity or fully-ablative matched related and unrelated transplant regimens for hematologic malignancies. Control of GVHD has been good, but high relapse rates and infectious complications have been seen. We report our experience with 42 pts who underwent matched related (27) or unrelated (15) bone marrow (8), blood stem cell (33), or both (1) transplants for hematologic malignancies. There were 16 women and 26 men, median age 48, and median f/u is 9 months. All AML pts had high-risk disease defined as beyond CR1 (11), CR1 with high risk cytogenetics (5), AML with tri-lineage dysplasia (6), or failed prior autologous transplant (1) Three had >10% marrow blasts at time of transplant. All other pts had advanced and multiply recurrent disease. All pts received alemtuzamab either at 20 mg/m2/d x 5 (17), or 30 mg/m2/d x 3 (25) between days -8 through -4. The conditioning regimens also included 12.8 mg/kg IV busulfan, (27), 6.4 mg/kg IV busulfan (8), melphalan 140 mg/m2 (6), or Cy/TBI (1). All non-TBI patients received 30 mg/m2/d x 5 of fludaribine. Median recovery of ANC to >500/ul and platelets > 20,000/ul w/o transfusion were 13 and 14 days, respectively. TRM included 1 pt each with intracranial hemorrhage at day 8, multi-organ failure/sepsis at day 28, and IPS/ARDS at day 119. Two of the 3 deaths were in MRD transplants; no serious episodes of VOD were observed. Grade 2–4 GVHD was seen in 4/14 evaluable MUD pts and 1 (sex mismatched) of 26 evaluable MRD pts. CMV reactivation has been seen in nearly all CMV sero+ pts, and bk reactivation was seen in 25 pts with hemorrhagic cystitis in 10. One case of PTLD, 3 graft failures, and one severe case of disseminated adenovirus infection resulting in nephritis, cystitis and transient cardiomyopathy were also seen. Relapse/# evaluable for different diseases are as follows: AML 12/22, Gleevec-resistant CML 3/3, MDS 0/4, MM 3/3, NHL 0/3, ALL 0/3, CLL 0/2. Relapse rates in evaluable MRD pts, 15/26 (58%), were statistically higher compared to MUD pts, 3/14 (21%) (p = 0.05). RR in marrow, 2/8, vs BSC, 16/32, were comparable. Alemtuzamab use has been effective at controlling GVHD and infectious complications can be reduced with early and pre-emptive use of ganciclovir. Nevertheless, high relapse rates remain a significant problem and preclude routine use of this approach in MRD patients with advanced myeloid diseases. Current use of this agent has been individualized for donor (MRD vs MUD) and disease (myeloid vs other) status.〉
Radiation Dose Determines Degree of Donor Chimerism after Nonmyeloablative Hematopoietic Cell Transplantation.
Authors: Kahl Christoph , Mielcarek Marco , Iwata Mineo , Harkey Michael , Storer Barry , Torok-Storb Beverly
Published: October 2019

Efforts to replace total body irradiation (TBI) used for transplant conditioning with agents that have less acute and long-term toxicities require a better understanding of the biological effects of low dose TBI. We therefore retrospectively analyzed the role of radiation dose, stem cell source, and type of immunosuppression on both the stability and degree of donor chimerism in canine recipients of matched littermate hematopoietic cell transplants. Recipients were prepared with 200 cGy (n=26), 100 cGy (n=76) or 50 cGy (n=19) total body irradiation (TBI) at 7 cGy/min. Stem cell sources included bone marrow (BM) alone (n=58), BM plus G-CSF mobilized peripheral blood mononuclear cells (G-PBMC) (n=42), BM and CD14-depleted G-PBMC (n=13), or BM and T-cell-depleted G-PBMC (n=8). Posttransplant immunosuppression consisted of cyclosporin (CSP) only (n=53), CSP plus mycophenolate mofetil (MMF) (n=23), CSP and rapamycin (n=12), CSP, MMF and rapamycin (n=5); or CSP and MMF in combination with pretransplant immunosuppression (n=28). The percentage of donor granulocytes in the peripheral blood, as determined by PCR amplification of variable numbers of tandem repeats (VNTR), served as a marker for engraftment. TBI dose and stem cell source were both significantly associated with long-term (>26 weeks) stable engraftment in multivariate analysis (p=0.0001 and p=0.004, respectively). Among the 39 dogs with stable engraftment, however, TBI dose was the only factor examined that was associated with the degree of donor chimerism (mean % of donor granulocytes after 200 cGy, 100 cGy and 50 cGy of TBI: 65%, 52%, and 24%, respectively; p=0.008). To determine whether low-dose irradiation directly affected recipient stem/progenitor cell numbers and thereby conferred a competitive disadvantage to donor cells, CD34+ cells were isolated from two normal human donors. One preparation of CD34 cells was ex vivo irradiated (=200 cGy) and then injected into NOD/SCID beta2m-/- mice in combination with an equal number of unirradiated CD34 cells from the second donor. The contributions of each donor to human engraftment were assessed at 10 weeks by VNTR. After 200 cGy, the irradiated population contributed 74% less than expected, 24% less after 100 cGy, but only 6% less after 50 cGy. Flow analysis of Caspase-3 activation indicated that a significant percentage of cells irradiated with 200 cGy were apoptotic, and that this was associated with the loss of L-selectin and P-selectin glycoprotein ligand-1. In conclusion, our findings suggest that TBI, in addition to its well-characterized immunosuppressive effects, determines the degree of donor cell engraftment by directly compromising recipient stem cells, thereby providing a competitive advantage to donor stem cells.
Engraftment and Survival Following Non-Myeloablative Allogeneic Peripheral Blood Hematopoietic Cell Transplantation for Malignant Diseases Is Affected by CD8+ T-Cell Dose.
Authors: Cao Thai M. , Shizuru Judith A. , Wong Ruby M. , Sheehan Kevin , Laport Ginna G. , Stockerl-Goldstein Keith E. , Johnston Laura J. , Stuart Monic J. , Brown Janic M.Y. , Grumet F. C. , Negrin Robert S. , Lowsky Robert
Published: October 2019

The influence of graft composition on clinical outcomes after non-myeloablative allogeneic hematopoietic transplantation is not well characterized. In this report we evaluated the influence of CD34+, CD3+, CD4+, and CD8+ cell doses on donor engraftment, graft-versus-host disease (GVHD), freedom from progression (FFP), and overall survival (OS). Patients (n=63) were given total body irradiation 200 cGy (n=8) or total body irradiation 200 cGy plus fludarabine (n=55) followed by allografting with granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell (G-PBMC). The median age was 53 years, and donors were HLA-identical siblings (n=38) or HLA-matched unrelated individuals (n=25). Almost all patient were treated for a hematologic malignancy (n=60), with 30 patients (48%) characterized as having advanced diseases. G-PBMC cell doses were prospectively enumerated by FACS analysis at the time of collection. Median donor G-PBMC cell doses were 7.4x106 CD34+/kg (range: 1.9–17.7), 3.2x108 CD3+/kg (range: 0.8–6.4), 2.1x108 CD4+/kg (range: 0.4–4.1), and 1.1x108 CD8+/kg (range: 0.2–3.4). By univariate analysis only G-PBMC CD8+ T-cell dose ≥ the 50th percentile favorably correlated with attainment of full donor blood T-cell chimerism (P = .03). The incidences of grade 2–4 acute GVHD was 16.3% (95% CI: 6.9–25.8) and extensive chronic GVHD was 42.9% (95% CI: 27.8–57.9). No G-PBMC cell dose significantly influenced grade 2–4 acute or extensive chronic GVHD. With a median follow-up of 13 months (range: 5–45), estimates for FFP was 53.7% (95% CI: 37.7–69.7) and OS was 33.4% (95% CI: 17.4–49.4). G-PBMC CD8+ T-cell dose ≥ the 50th percentile T-cell dose was favorably prognostic for both FFP (P = .001) and OS (P = .01). Survival curves for FFP and OS are shown in Figure 1. Extensive chronic GVHD also predicted better FFP (P = .02), but in multivariate analysis only G-PBMC CD8 + T-cell dose (P = .004; RR = 0.2, 95% CI = 0.1–0.6) was associated with improved FFP. Infusion of low G-PBMC CD8+ T-cell dose for non-myeloablative allografting with total body irradiation 200 cGy plus fludarabine may adversely affect donor T-cell engraftment, disease progression and survival outcomes.
Invasive Pulmonary Aspergillosis Is Not a Contraindication to Reduced-Intensity Conditioned Allogeneic Haematopoietic Stem Cell Transplantation (RIC allo-HSCT).
Authors: Ho Aloysius Y.L. , Desai Sujal R. , Stanton Ian D.P. , Copley Susan J. , Wells Athol U. , Pagliuca Antonio , Devereux Stephen , Mufti Ghulam J.
Published: October 2019

Ten patients with focal abnormalities on CT prior to RIC allo-ASCT, in whom follow-up CT studies were available, were studied. All 10 had received prior chemotherapy for AML/MDS and had received treatment for high probability invasive pulmonary aspergillosis (IPA). The RIC protocol comprised “FBC”- fludarabine 30 mg/m2 intravenously from Day -9 to Day -5; busulphan 4mg/kg/day orally from Day -3 to Day -2 and alemtuzumab (CAMPATH-1H) 20mg intravenously from Day -8 to Day -4. Unselected peripheral blood stem cells (PBSCs) or bone marrow (BM) was infused on Day 0. Immunosuppression was achieved with cyclosporine 1.5 mg/kg intravenously 12 hourly from Day -1. Neoralâ was substituted when a good oral intake was tolerated and tapered from Day +56 in the absence of GvHD. Filgrastim 300 mcg iv/sc was administered from Day +7 to neutrophil engraftment (neutrophils ≥ 0.5x109/l). All patients received standard nursing and supportive care protocols for neutropenic patients. In addition these patients received Ambisome 3mg/kg intravenously daily until full neutrophil regeneration had occurred, then prophylactic Ambisome (2mg/kg thrice a week) till immunosuppression was stopped. Two thoracic radiologists (blinded to the timing of examinations) independently reviewed the CT studies pre- and post-RIC allo-HSCT. The site and size of individual lesions was evaluated. The likelihood (recorded as probable, possible, indeterminate or unlikely; denoting probabilites of ≥75%, ≥50%, ≥25% and <25% [respectively]), that individual lesions represented foci of angioinvasive fungal infection was noted. Serial changes in size, the appearance of new lesions (if any), and an overall impression of change in each patient (somewhat better or worse, significantly better or worse, no change) at follow-up CT, were also recorded. There were 48 lesions (probable, n=16; possible, n=18; indeterminate, n=13 and unlikely, n=1) on CT prior to RIC allo-HSCT. At follow-up CT, 12/16 (75%) probable and 10/18 (56%) possible lesions had either regressed or were smaller than prior to RIC allo-HSCT. Only 1/18 (6%) lesions graded as possible had increased in size following RIC allo-HSCT. Five new lesions (probable, n=2; possible, n=2; indeterminate, n=1) were demonstrated in five patients. The overall impression on follow-up was that CT studies were somewhat better (n=3) or significantly better (n=6) in 9/10 (90%) patients. In patients with pre-existing lesions on CT prior to RIC allo-HSCT, there was no evidence of progression in the majority of patients, and no patient died of IPA in the follow-up period. Documented IPA may not be a contraindication to RIC allo-HSCT in patients with haematological malignancy, which can be safely performed with our conditioning and supportive care protocol.
Prediction and Prevention of Transplant Related Mortality from Pulmonary Causes Following Total Body Irradiation and Allogeneic Stem Cell Transplantation.
Authors: Savani Bipin , Montero Aldemar , Gochiuo Bernadette , Nlonda Nene , Childs Richard , Solomon Scott , Barrett John
Published: October 2019

Between 7/1997 and 8/2004, 146 consecutive patients with hematological malignancies received a T cell depleted peripheral blood stem cell transplant (PBSCT) from an HLA identical sibling using three successive conditioning regimens: (A): 13.6Gy total body irradiation (TBI) + cyclophosphamide 120mg/kg (Cy) (n=85) , (B) 12.0Gy TBI with lung shielding to 9.0 Gy + Cy + fludarabine 125mg/m2 (Flu) , n= 35, (C) 12.0Gy TBI with lung shielding to 6.0 Gy + Cy + Flu, n= 26. Ninety-four (65.4%) had standard risk disease (transplant in first complete remission of acute leukemia, CML in chronic phase, and MDS-RA); the remainder had more advanced disease or unfavorable diagnoses. Actuarial transplant related mortality (TRM) was 16.4 ± 3%, at a median time of 103 days (range 23–238). Of the 21 transplant related deaths 14 (67%) were from pulmonary causes (6, idiopathic interstitial pneumonia (IP), 4 acute respiratory distress syndrome (ARDS), and pneumonia from CMV (2), RSV (1) and bacterial origin (1). Median time to death from IP and ARDS was 71 and 66 days post-transplant respectively. Kaplan-Meier analysis was used to study factors affecting pulmonary TRM. Pre-transplant characteristics predictive for pulmonary-related TRM are shown in the table. Patients with high risk disease and CML patients who had received busulfan for more than one month were at significantly greater risk of developing both pulmonary TRM and IP. Patients who smoked were significantly more at risk to develop ARDS and fatal pulmonary infection. Pre-transplant pulmonary function tests were highly predictive for pulmonary TRM. Diffusion capacity of the lung for carbon monoxide (DLCO), vital capacity and FEV-1 were highly correlated, but the most predictive parameter was DLCO: Of 48 patients with <85% normal vs. 98 with >85% of normal diffusion capacity, 6 vs 0 had IP, p<0.001, 4 vs 0 had ARDS, p=0.01 and 11 vs 3 had pulmonary TRM, p=0.0003. The death from all pulmonary causes was highest in protocol A (no lung shielding) and significantly less in conditioning regimens B+C (lung shielding) (12/85 vs 2/61 deaths, p = 0.05). These results indicate that after TBI and PBSCT, pulmonary causes contribute significantly to TRM, but can be predicted by patient characteristics and pulmonary function tests and may be reduced by lung shielding.