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•Patients with hematology-oncology issues require platelet transfusion support that may potentiate pulmonary injury.•PRPCs decreased the probability of assisted mechanical ventilation during platelet transfusion.

Patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) often experience life-threatening relapses of the disease, and rituximab (RTX) can be used to mitigate relapse risk. However, the predictors of relapse in iTTP and the magnitude and duration of effect of RTX remain key unanswered questions. Using a multi-institutional cohort of consecutive adult patients with iTTP, we used survival analysis to compare relapse rates between patients who received RTX during the index presentation with acute iTTP and those who did not. Of 124 patients, 60 (48%) received RTX and 34 (27%) experienced relapse. Median time to relapse was 3.71 (interquartile range, 1.75-4.9) and 1.33 (interquartile range, 0.43-2.35) years for RTX-treated and untreated patients, respectively. RTX conferred protection from relapse at 1 year of follow-up (P = .01) but not at 5 years of follow-up. Extended Cox regression was then used to identify predictors of relapse and to estimate the protective effect of RTX. The following parameters were independently associated with increased risk for subsequent relapse: presenting in iTTP relapse (hazard ratio [HR], 2.97; 95% confidence interval [CI], 1.4-6.4), age younger than 25 years (HR, 2.94; 95% CI, 1.2-7.2), and non-O blood group (HR, 2.15; 95% CI, 1.06-4.39). RTX initially provided protection from relapse (HR, 0.16; 95% CI, 0.04-0.70), but this effect gradually diminished, returning to the baseline risk for untreated patients at approximately 2.6 years. Patients who are young, have non-O blood group, or present with relapsed iTTP are at increased risk for subsequent relapse. RTX appears to confer short-term protection from relapse.

Background:Immune thrombotic thrombocytopenic purpura (iTTP) is a hematologic emergency characterized by severe acquired deficiency in the von Willebrand factor-cleaving metalloprotease ADAMTS13 and thrombotic microangiopathy, resulting in end organ damage and death if untreated. Plasma exchange (PLEX) is vital to the management of iTTP and has dramatically improved survival in this disorder since the 1980s. Guidelines recommend initiation of PLEX within 4-8 hours of presentation. However, despite the importance of prompt medical intervention, determinants of the total interval between symptom onset and initiation of PLEX (S2P) and time from presentation at the pheresis center to PLEX (door to PLEX, D2P) remain unclear. Further, the impact of S2P and D2P on clinical outcomes in iTTP have not been evaluated.

This review summarizes the salient points of the symposium ‘Red Cell Genotyping 2015: Precision Medicine’ held on 10 September 2015 in the Masur Auditorium of the National Institutes of Health. The specific aims of this 6th annual symposium were to: (1) discuss how advances in molecular immunohematology are changing patient care; (2) exemplify patient care strategies by case reports (clinical vignettes); (3) review the basic molecular studies and their current implications in clinical practice; (4) identify red cell genotyping strategies to prevent alloimmunization; and (5) compare and contrast future options of red cell genotyping in precision transfusion medicine. This symposium summary captured the state of the art of red cell genotyping and its contribution to the practice of precision medicine.

Bleeding remains a significant problem for many thrombocytopenic hematology/oncology patients in spite of platelet transfusions. Factors that might contribute to bleeding were analyzed for 16 320 patient-days on or after their first platelet transfusion in 1077 adult patients enrolled in the Platelet Dose (PLADO) trial. All patients had a greatly increased risk of bleeding at platelet counts of ≤5 × 109/L (odds ratio [OR], 3.1; 95% confidence interval [CI], 2.0-4.8) compared with platelet counts ≥81 × 109/L. Platelet counts between 6 × 109/L and 80 × 109/L were also associated with a somewhat elevated bleeding risk in patients receiving allogeneic stem cell transplants (SCTs) or chemotherapy but not in those undergoing autologous SCTs. Other significant laboratory predictors of bleeding were hematocrit ≤25% (OR, 1.29; 95% CI, 1.11-1.49), activated partial thromboplastin time (aPTT) 30 to ≤50 seconds (OR, 1.40; 95% CI, 1.08-1.81; P= .01), aPTT >50 seconds (OR, 2.34; 95% CI, 1.54-3.56), international normalized ratio (INR) 1.2 to 1.5 (OR, 1.46; 95% CI, 1.17-1.83), and INR >1.5 (OR, 2.05; 95% CI, 1.43-2.95). Transfusion of either platelets or red blood cells (RBCs) on days with bleeding was often not sufficient to change bleeding outcomes on the following day. Because bleeding occurred over a wide range of platelet counts among patients undergoing allogeneic SCT or chemotherapy and because platelet transfusions may not prevent bleeding, other risk factors may be involved. These may include low hematocrit and coagulation abnormalities. This trial was registered at www.clinicaltrials.govas #NCT00128713.

Among the syndromes characterised by thrombotic microangiopathy, thrombotic thrombocytopenic purpura is distinguished by a severe deficiency in the ADAMTS13 enzyme. Patients with this disorder need urgent treatment with plasma exchange. Because ADAMTS13 activity testing typically requires prolonged turnaround times and might be unavailable in resource-poor settings, a method to rapidly assess the likelihood of severe ADAMTS13 deficiency is needed.

We have developed a tumor vaccine in which patient-derived myeloma cells are chemically fused with autologous dendritic cells (DCs) such that a broad spectrum of myeloma-associated antigens are presented in the context of DC-mediated costimulation. We have completed a phase 1 study in which patients with multiple myeloma underwent serial vaccination with the DC/multiple myeloma fusions in conjunction with granulocyte-macrophage colony-stimulating factor. DCs were generated from adherent mononuclear cells cultured with granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-α and fused with myeloma cells obtained from marrow aspirates. Vaccine generation was successful in 17 of 18 patients. Successive cohorts were treated with 1 × 106, 2 × 106, and 4 × 106 fusion cells, respectively, with 10 patients treated at the highest dose level. Vaccination was well tolerated, without evidence of dose-limiting toxicity. Vaccination resulted in the expansion of circulating CD4 and CD8 lymphocytes reactive with autologous myeloma cells in 11 of 15 evaluable patients. Humoral responses were documented by SEREX (Serologic Analysis of Recombinant cDNA Expression Libraries) analysis. A majority of patients with advanced disease demonstrated disease stabilization, with 3 patients showing ongoing stable disease at 12, 25, and 41 months, respectively. Vaccination with DC/multiple myeloma fusions was feasible and well tolerated and resulted in antitumor immune responses and disease stabilization in a majority of patients.

Background. Platelet transfusion is a critical therapy for hematology-oncology patients at risk of transfusion-transmitted infection (TTI) and pulmonary injury. Amotosalen-UVA pathogen reduction (PR) treatment of apheresis platelet components (PC) in plasma or additive solution (INTERCEPT Blood System for Platelets, Cerus, Concord, CA) is FDA approved to reduce risk of TTI and transfusion associated graft vs. host disease (TA-GVHD). PRPC meet the FDA bacteria risk reduction guidance, and approximately 50% of U.S. PC are PRPC. Amotosalen-UVA PR replaces bacteria screening, gamma irradiation, and CMV serology. PR is performed within 24 hours of collection enabling early release of PRPC with 5-day storage. We tested the hypothesis that PRPC were not inferior to conventional PC(CPC) for the incidence of pulmonary injury. Methods. An open-label sequential cohort study in platelet transfusion dependent hematology-oncology patients was conducted under routine practice conditions in 15 clinical centers. Each site enrolled a CPC cohort followed by a PRPC cohort using 4 primary therapy strata matched ± 10%: chemotherapy without hematopoietic cell transplant (HCT), HCT with myeloablation, HCT with non-myeloablative conditioning, and HCT with reduced intensity conditioning (RIC). Patients were supported with the assigned PC type for up to 21 days with 7 days of surveillance after the last PC exposure. Patients participated in only one cohort. The primary endpoint was treatment emergent assisted mechanical ventilation (TEAMV) by intubation or tight mask with positive end expiration pressure (5cm H 2O) after initiation of study PC. All endpoint patients were adjudicated by a blinded pulmonary expert panel (PEP) for diagnosis of acute respiratory distress syndrome (ARDS) by the Berlin Criteria. Secondary endpoints included: time to initiation of TEAMV, clinically significant pulmonary adverse events (CSPAE, CTCAE ≥ Grade 2), transfusion reactions, and mortality. The incidence of TEAMV by non-inferiority (margin = 2.3%), and secondary endpoints were analyzed by modified intention to treat (mITT) and per protocol (PP). Sensitivity analyses with propensity score matching for key variables were conducted for the primary endpoint. The associations between PC and categorical variables were tested by stratified Cochran-Mantel-Haenszel and continuous variables by ANOVA for two-sided significance p = 0.05. results. A total of 2291 pediatric and adult patients (1068 PRPC and 1223 CPC) were enrolled in the respective cohorts with transfusion of 5,277 PRPC and 5,491 CPC. PC assignment compliance and study completion were > 94%. For the mITT data set, the cumulative incidence of TEAMV was lower for the PRPC cohort (log rank p = 0.039) than the CPC cohort (2.9% versus 4.6%, HR = 0.633: 95% CI 0.408-0.982). PRPC by mITT were non-inferior to CPC for the incidence of TEAMV due to all indications, and for TEAMV with pulmonary dysfunction (PD) by PEP (Table). PP analyses were consistent with mITT. Relative risk (RR) of TEAMV showed significantly (p<0.05) decreased RR of PRPC respectively for baseline covariates: age < 65 (0.53), male (0.54), non-white (0.32), chemotherapy (0.40), prior pulmonary disease (0.55), and prior cardiac disease (0.58). Least squares (LS) mean days to initiation of TEAMV for patients with PD were longer for PRPC recipients. PEP adjudicated incidence of ARDS was not significantly different between cohorts (Table). Total and serious CSPAE were not different between the cohorts. There were no significant differences between cohorts in Respiratory, Thoracic, and Mediastinal Disorders, the most frequent system organ class event. Mortality was not different between cohorts. Multivariate analysis (mITT) for the probability of CSPAE or transfusion associated cardiac overload (TACO) showed PC type had no effect. The odds ratio (OR) of CSPAE or TACO during PC support was significantly increased (p< 0.05) in both cohorts for history of cardiac disease (1.35), history of pulmonary disease (2.57), diagnosis of Myelodysplasia (1.88), and diagnosis of Myelodysplasia/Myeloproliferative disease (2.27). There was a significant treatment interaction (p= 0.043) between PC type and acute myelogenous leukemia (AML), increased OR = 1.49 for CPC versus PRPC. Conclusions.PRPC did not potentiate pulmonary injury during PC support; and their use may decrease TEAMV risk with benefit of reduced TTI risk.

In a prior phase 2 study, personalized cancer vaccination with autologous dendritic cells (DCs) fused with primary MM tumor cells (DC/MM fusions) induced the expansion of circulating MM-reactive lymphocytes and was associated with conversion to complete response (CR) post-autoHCT in the absence of maintenance therapy. 1We now present a multicenter randomized phase II study that examined the efficacy of DC/MM fusion vaccination with lenalidomide maintenance therapy after autoHCT, compared with lenalidomide maintenance alone. The study offered a first-of-its-kind academic collaborative effort of personalized cell therapy using an open-source format, site-specific production, and centralized product characterization/release criteria verification.

Thrombotic thrombocytopenic purpura (TTP) is a rare, potentially fatal disease of uncertain etiology. Early diagnosis and treatment are essential to patient survival. The purpose of this report is to describe three patients with levonorgestrel implants (Norplant system) who developed TTP.

We report central venous catheter (CVC)-associated Staphylococcus aureus bacteremia detected by apheresis product culture after sterilization of standard blood cultures. A 64-year-old man with non-Hodgkin's lymphoma underwent peripheral blood stem cell (PBSC) collection. Five days after placement of a CVC, inflammation was evident at the insertion site. The CVC was removed and cephalexin was begun. Discharge at the site contained neutrophils and gram-positive cocci in pairs and clusters. Cultures of the discharge, of blood drawn via the CVC, of the CVC tip and of the apheresis product collected that day grew methicillin-susceptible S. aureus (MSSA). Cephalexin was discontinued in favor of oxacillin. Three days after removal of the CVC, PBSC collections were resumed via a contralateral CVC. Three sets of standard blood cultures drawn the day the new CVC was placed and the following day were negative, yet apheresis product cultures from each of these days grew MSSA. PBSC collections were halted, the CVC was removed, and oxacillin was continued via a peripherally inserted central catheter. Transesophageal echocardiography after two weeks of therapy revealed thickened mitral leaflets and damage to the posterior leaflet. Transthoracic echocardiography 11 weeks preceding this study had demonstrated normal mitral valve anatomy and function. Oxacillin was continued for six weeks, after which PBSC collections were resumed. Pulsed-field gel electrophoresis of the MSSA isolates revealed a clonal pattern. Cultures of apheresis product may be more sensitive to the presence of bacteremia than standard blood cultures, and they should guide clinical decisions when the bacteria isolated are potential pathogens or suggest clinical infection. J. Clin. Apheresis 18:37–39, 2003. © 2003 Wiley-Liss, Inc.

We report central venous catheter (CVC)‐associated Staphylococcus aureusbacteremia detected by apheresis product culture after sterilization of standard blood cultures. A 64‐year‐old man with non‐Hodgkin's lymphoma underwent peripheral bloodstem cell (PBSC) collection. Five days after placement of a CVC, inflammation was evident at the insertion site. The CVC was removed and cephalexin was begun. Discharge at the site contained neutrophils and gram‐positive cocci in pairs and clusters. Cultures of the discharge, of blood drawn via the CVC, of the CVC tip and of the apheresis product collected that day grew methicillin‐susceptible S. aureus(MSSA). Cephalexin was discontinued in favor of oxacillin. Three days after removal of the CVC, PBSC collections were resumed via a contralateral CVC. Three sets of standard blood cultures drawn the day the new CVC was placed and the following day were negative, yet apheresis product cultures from each of these days grew MSSA. PBSC collections were halted, the CVC was removed, and oxacillin was continued via a peripherally inserted central catheter. Transesophageal echocardiography after two weeks of therapy revealed thickened mitral leaflets and damage to the posterior leaflet. Transthoracic echocardiography 11 weeks preceding this study had demonstrated normal mitral valve anatomy and function. Oxacillin was continued for six weeks, after which PBSC collections were resumed. Pulsed‐field gel electrophoresis of the MSSA isolates revealed a clonal pattern. Cultures of apheresis product may be more sensitive to the presence of bacteremia than standard blood cultures, and they should guide clinical decisions when the bacteria isolated are potential pathogens or suggest clinical infection. J. Clin. Apheresis 18:37–39, 2003. © 2003 Wiley‐Liss, Inc.

No relevant conflicts of interest to declare.

Background:Thrombotic thrombocytopenic purpura (TTP) is a rare but deadly thrombotic microangiopathy (TMA) that is caused by a severe acquired deficiency in the ADAMTS13 enzyme. The PLASMIC clinical scoring system categorizes patients with suspected TTP as being at low (score <5), intermediate (score = 5), or high (score >5) risk of severe ADAMTS13 deficiency. Here we have modeled the potential cost savings of a management approach that incorporates the PLASMIC score to defer testing and treatment for low risk patients and compared this approach to one in which ADAMTS13 testing requests require pre-approval by the blood transfusion service.

A personalized DC/AML fusion cell vaccine promotes the expansion of leukemia-specific T cells and prolonged remission in patients.

Richardson: Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Anderson:Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership. Rowe:BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BioLineRx Ltd.: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.

Stone: Karyopharm: Consultancy; Roche/Genetech: Consultancy; Merck: Consultancy; Novartis: Research Funding; Celgene: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Amgen: Consultancy; Abbvie: Consultancy; Celator: Consultancy; AROG: Consultancy; Juno: Consultancy. Steensma:Celgene: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Onconova: Consultancy. DeAngelo:Incyte: Consultancy; Celgene: Consultancy; Agios: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Amgen: Consultancy; Pfizer: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.

No relevant conflicts of interest to declare.

Makar: Alexion Pharmaceutials: Research Funding.

We have developed a promising leukemia vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs), presenting a broad array of antigens. We are conducting a clinical trial in which AML patients who are not candidates for allogeneic transplantation undergo vaccination with DC/AML fusion cells following chemotherapy induced remission. Twenty-six patients (14 males, 12 females) underwent collection of AML cells at disease presentation for vaccine generation and immune monitoring studies. Median age of the patients is 66 years. Tumor was collected from either a bone marrow aspirate (N=16), 20 cc of peripheral blood (N=7), or leukapheresis product (N=3) at the time of presentation with newly diagnosed AML (N=25) or first relapsed AML (N=1). The mean yield of AML cells was 109 x 106cells with a mean viability of 91%. Eligible patients achieving CR following chemotherapy (N=16) underwent leukapheresis for DC generation and vaccine preparation. Adherent peripheral blood mononuclear cells were isolated, cultured in the presence of GM-CSF and IL-4 for 5-7 days, and exposed to TNFα for 48-72 hours to generate mature DCs. The mean yield of DCs was 177 x106cells with a mean viability of 89%. Fusion cells were generated by co-culture of DCs with AML cells in the presence of 50% polyethylene glycol and identified as cells co-expressing antigens that were unique to the DC and tumor population. Mean fusion efficiency and viability was 38% and 85%, respectively. As a measure of their activity as antigen presenting cells, the capacity of fusion cells to stimulate allogeneic T cell proliferation ex vivo was quantified. In contrast to the leukemia preparation (mean stimulation index (SI) 3.81), the DC and fusion cell preparation were potent stimulators (mean SI 19.61 and 13.48, respectively). Vaccination with DC/leukemia fusion cells was initiated within 12 weeks from count recovery following the final cycle of chemotherapy. 13 patients received at least two monthly vaccinations at a dose of 5x106fusion cells. 8 patients had intermediate risk cytogenetics, 3 patients had good risk cytogenetics, and 2 patients had a complex karyotype. Vaccination was well tolerated, and importantly, was not associated with clinically significant auto-immunity. Possibly related adverse events were transient and of grade 1-2 intensity, including vaccine site reactions, pruritis, arthalgias, myalgias, eosinophilia, leukopenia, thrombocytopenia. Biopsy of vaccine site reactions demonstrated a dense infiltrate of CD4 and CD8 T cells consistent with recruitment of reactive T cell populations to the vaccine bed. To date, 9 patients remain in remission (69%), with a mean follow up of 23 months. Peripheral blood samples were collected prior to each vaccination and at 1, 3, and 6 months following completion of vaccination. Vaccination resulted in the potent induction of leukemia specific immunity as measured by an increase in CD8 T cells expressing IFNγ in response to ex vivo exposure to autologous leukemia cell lysates (mean fold increase 8, n=6). Bone marrow derived T cells were isolated prior to and following vaccination in patients who are HLA2.1+. Vaccination resulted in the expansion of bone marrow infiltrating T cells recognizing MUC1 (9 fold increase), WT1 (5 fold increase), PRAME (12 fold increase) tumor antigens by tetramer analysis (n=2). In conclusion, DC/AML fusion cell vaccination results in the potent expansion of leukemia reactive T cells and durable remissions following chemotherapy. Enrollment to a second cohort is being initiated, in which patients with be treated with DC/AML fusion cell vaccination in conjunction with PD1 blockade.

Rosenblatt: CureTech Ltd.: Research Funding. Schickler:CurTech Ltd.: Employment, Research Funding. Rotem-Yehudar:CureTech Ltd: Employment, Research Funding. Avigan:CureTech Ltd: Research Funding.

Avigan: Curetech: Research Funding.

Richardson: Millenium (Research Funding and Advisory Board), Celgene, Keryx, BMS, Merck, Johnson and Johnson (All Advisory Board): Membership on an entity's Board of Directors or advisory committees, Research Funding. Anderson:Millenium (Research Funding and Advisory Board: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Keryx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Johnson and Johnson: Membership on an entity's Board of Directors or advisory committees.

Autologous stem cell transplantation results in tumor cytoreduction and improved disease outcomes in patients with multiple myeloma (MM), but patients ultimately relapse from persistent disease. A promising area of investigation is the development of cancer vaccines that educate host immunity to target and eliminate myeloma cells and can be used to eradicate residual disease following autologous stem cell transplantation. The early post-transplant period is characterized by a transient reversal of tumor mediated tolerance due to the reduction in disease bulk, the depletion of regulatory T cells. We have developed a cancer vaccine model in which DCs are fused to autologous MM cells resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. We are conducting a study in which patients with MM undergo stem cell transplantation followed by post-transplant vaccination with 3 doses of DC/MM fusions. DCs were generated from adherent mononuclear cells cultured with GM-CSF and IL-4 for 5–7 days and matured with TNFα. DCs strongly expressed costimulatory and maturation markers. Myeloma cells were isolated from bone marrow aspirates and were identified by their expression of CD38, CD138, and/or MUC1. DC and MM cells were fused with polyethylene glycol and fusion cells were quantified by determining the percentage of cells that coexpress unique DC and myeloma antigens. To date, 26 patients have been enrolled. All patients have undergone successful vaccine generation. Mean yield of the DC and myeloma preparations was 171×106 and 70×106 cells, respectively. Mean fusion efficiency was 40% and the mean cell dose generated was 4×106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 88%, 86%, and 78%, respectively. As a measure of their potency as antigen presenting cells, fusion cells potently stimulated allogeneic T cell proliferation in vitro. Mean stimulation indexes were 12, 57, 31 for T cells stimulated by myeloma cells, DCs, and fusion cell preparations at an APC: T cell ratio of 1:10. Adverse events judged to be potentially vaccine related were mild, and included injection site reactions, pruritis, myalgias, fever, chills, headache, fatigue and tachycardia. To date 14 patients have completed vaccinations and initial follow up of which 8 have achieved a complete remission and 6 a partial remission. Of note, 4 patients achieved complete remission only after undergoing post-transplant vaccination. We are examining the effect of transplant and vaccination on measures of cellular immunity, antitumor immunity and levels of activated as compared to regulatory T cells. T cell responses to PHA mitogen and tetanus toxoid were transiently depressed post-transplant. Similarly, DTH responses to candida antigen were absent post-transplant in all but 1 patient. In contrast, a significant increase was noted post-transplant in circulating tumor reactive lymphocytes as determined by T cell expression of IFNγ by CD4 and CD8 cells following ex vivo coculture with autologous myeloma cell lysate (Mean percentage of tumor reactive CD8 cells was 0.9 and 11 pre and post-transplant, respectively p=0.01; mean percentage of CD4 cells was 0.7 and 2.7; p=0.02). A further amplification of tumor reactive lymphocytes was seen with vaccination in a subset of patients (mean percentage of CD4 and CD8 tumor reactive T cells was 4.9 and 15, respectively). A decrease in the median levels of circulating regulatory T cells and a relative increase in the ratio of activated (CD4/CD25low)/regulatory (CD4/CD25high) cells was observed following transplantation. This finding suggests that although nonspecific T cell responses are muted in the early posttransplant period, there is a greater capacity to recognize tumor antigens, potentially due to the depletion of regulatory T cells and the decline in tumor mediated immune suppression. In summary, fusion cell vaccination in conjunction with stem cell transplantation was well tolerated, induced anti-tumor immunity and clinical responses in patients with MM. The post-transplant period is characterized by increased levels of activated as compared to regulatory T cells and enhanced levels of T cells with the capacity to respond to myeloma cells. The increase in tumor reactive T cells post-transplant is further amplified following exposure to the DC/MM fusion vaccine.

Autologous transplantation results in the transient reversal of tumor mediated tolerance due to the reduction in disease bulk, the depletion of regulatory T cells, and in the increased presence of tumor reactive lymphocytes during the period of lymphopoietic reconstitution. As a result, cancer vaccines are being explored as a means of targeting residual myeloma cells following stem cell transplant. We have developed a cancer vaccine in which patient derived tumor cells are fused with autologous dendritic cells (DCs). In this way multiple tumor antigens are presented in the context of DC mediated costimulation. We are conducting a study in which patients with multiple myeloma (MM) undergo stem cell transplantation followed by vaccination with 3 doses of DC/MM fusions. DCs were generated from adherent mononuclear cells cultured with GM-CSF and IL-4 for 5–7 days and matured with TNFa. DCs strongly expressed costimulatory and maturation markers. Myeloma cells were isolated from bone marrow aspirates and were identified by their expression of CD38, CD138, and/or MUC1. DC and MM cells were fused with polyethylene glycol as previously described and fusion cells were quantified by determining the percentage of cells that coexpress unique DC and myeloma antigens. To date, 19 patients have been enrolled and 18 have completed vaccine generation. Mean yield of the DC and myeloma preparations was 1.84 × 108 and 8.3 × 107 cells, respectively. Mean fusion efficiency was 40% and the mean cell dose was 4.3 × 106 fusion cells. As a measure of their potency as antigen presenting cells, fusion cells prominently stimulated allogeneic T cell proliferation in vitro. Mean stimulation indexes were 12, 57, and 31 for T cells stimulated by myeloma cells, DCs, and fusion cells, respectively. Adverse events judged to be potentially vaccine related included injection site reactions, pruritis, myalgias, fever, chills, and tachycardia. Six patients have completed the follow up period and 3 patients are currently undergoing vaccination. All patients achieved a partial response to transplant. Three patients demonstrated resolution of post-transplant paraprotein levels following vaccination. One patient with highly aggressive disease who experienced disease progression in the early post-transplant period, demonstrated initial response and then stabilization of disease with vaccination. We are examining the effect of transplant and vaccination on measures of cellular immunity, anti-tumor immunity and levels or activated as compared to regulatory T cells. T cell response to PHA mitogen was transiently depressed post-transplant. In contrast, a transient increase was noted post-transplant in mean T cell expression of IFNγ in response to autologous myeloma cell lysate. In preliminary studies, a relative increase in the ratio of activated (CD4/CD25low) to regulatory (CD4/CD25high) T cells was observed. To date, all evaluable patients demonstrated evidence of vaccine stimulated anti-tumor immunity as manifested by a rise in IFNγ expression by CD4 and/or CD8+ T cells following ex vivo exposure to autologous tumor lysate. In this ongoing study, fusion cell vaccination in conjunction with stem cell transplantation has been well tolerated, induced anti-tumor immunity and clinical responses in patients with multiple myeloma.

We have developed a cancer vaccine in which patient derived myeloma cells are fused with autologous dendritic cells (DCs). In this way, multiple tumor antigens are presented in the context of the immune stimulating machinery of the DC. We are conducting a phase I trial in which patients with multiple myeloma (MM) undergo serial vaccination with DC/myeloma fusions in conjunction with GM-CSF. Eligibility criteria included ≥20% plasma cells in the bone marrow and measurable paraprotein or light chain. To date, 16 patients have been enrolled and 15 have undergone therapy. Patients underwent leukapheresis collection and DCs were generated from adherent mononuclear cells cultured for 5 days with GM-CSF and IL-4 and matured by exposure to TNFa for 48–72 hours. The mean yield of DCs was 1.23 x108 cells with a mean viability of 88%. Patient derived myeloma cells were isolated from bone marrow aspirates. The mean yield of myeloma cells was 92.5 x 106 cells with a mean viability of 90%. Fusion cells were generated by coculture of DCs with myeloma cells in the presence of 50% polyethylene glycol. Fusion cells were quantified by determining the percentage of cells that co-expressed unique DC and myeloma antigens. Mean fusion efficiency and viability was 40% and 84%, respectively. As a measure of immunologic potency, DC/MM fusions stimulated allogeneic T cell proliferation, with mean stimulation indexes of 48, 36, and 9 for the DC, fusion and myeloma cell populations, respectively. To date, 15 patients have completed vaccination at a dose of 1–5x106 fusion cells. Adverse events potentially related to vaccine included injection site reactions, edema, rash, fever (infection), chills, fatigue, muscle aches, pruritis, and diarrhea. One patient with a history of prior deep venous thrombosis (DVT) developed a DVT and pulmonary embolus of uncertain relation to the vaccine. To date, 6/9 evaluable patients have demonstrated immunologic response, defined by at least 2 fold increase in IFNγ expression by CD4 and/or CD8 T cells in response to ex vivo exposure to autologous tumor lysate. Vaccination was associated with an increase in circulating tumor specific T cells as evidenced by an increase in CD8+ cells binding the MUC1 tetramer. The impact of vaccination on circulating levels of regulatory T cells is being examined. Humoral immune responses are being assessed by SERAX analysis. Three patients have ongoing stable disease at 3.5, 6, and 19 months following their initial vaccination. Six patients demonstrated disease stability with subsequent progression ranging from 9 weeks to 8 months following their first vaccine. Vaccination with DC/MM fusions has been well tolerated, associated with immunologic response, and disease stabilization in a majority of patients with multiple myeloma.

Multiple myeloma expresses unique antigens that potentially serve as targets for tumor specific immunotherapy. Dendritic cells (DCs) are the most potent antigen presenting cells that prominently express costimulatory molecules and are uniquely able to stimulate anti-tumor immune responses. We have developed a promising cancer vaccine in which patient derived myeloma cells are fused with autologous DC resulting in the presentation of a broad array of tumor antigens in the context of DC mediated costimulation. DC/myeloma fusions potently stimulate anti-tumor immune responses in vitro as manifested by the lysis of autologous tumor targets. We are currently conducting phase I clinical trials in which patients with myeloma undergo serial vaccination with DC/myeloma fusions alone or in conjunction with stem cell transplantation. GM-CSF (100 µg) was administered subcutaneously on the day of vaccination and for 3 days thereafter. To date, 18 patients have been enrolled (11- vaccine alone, 7 vaccine transplant). To generate mature DCs, adherent mononuclear cells were isolated from a leukapharesis collection, cultured for 5 days with GM-CSF and IL-4 and terminal maturation was induced by exposure to TNFa for 48–72 hours. DCs prominently expressed HLA class II, costimulatory and maturation markers. The mean yield and viability of the DC preparations was 1.5 x 108 cells and 88%, respectively. Patient derived myeloma cells were isolated from bone marrow aspirates and were quantified by the expression of CD38 and/or CD138. The mean yield and viability of the myeloma cell collections was 7.3 x 107 cells and 89%, respectively. Fusion cells were generated by coculture of DCs with myeloma cells at a 3:1–10:1 ratio in the presence of 50% polyethylene glycol. Fusion cells were quantified by determining the percentage of cells that co-expressed unique DC and myeloma antigens. Mean fusion efficiency and viability of the fusion cell preparation was 40% and 84%, respectively. As a measure of immunologic potency, fusion cells prominently stimulated allogeneic T cell proliferation. To date, 13 patients have completed vaccination at a dose of 1–5 x 106 fusion cells. Adverse events judged to be potentially vaccine related have included vaccine injection site reactions, edema, rash, fever (infection), chills, fatigue, muscle aches, pruritis, and diarrhea. One patient with a history of prior deep venous thrombosis (DVT) developed a DVT and pulmonary embolus of uncertain relation to the vaccine. To date, 4/6 evaluable patients have demonstrated evidence of vaccine induced anti-myeloma immunity as demonstrated by at least 2 fold increase in IFN? expression by CD4 and/or CD8 T cells in response to ex vivo exposure to autologous tumor lysate. Of patients undergoing vaccine therapy alone, 5 patients demonstrated stabilization of the myeloma paraprotein for 2–6 months following initiation of vaccination. Of 3 patients completing post-transplant vaccination, 1 patient demonstrated resolution of the persisting myeloma protein post-transplant, 1 patient exhibited stable post-transplant paraprotein levels for 6 months, and 1 patient demonstrated a transient increase followed by a progressive decline in paraprotein levels post-transplant.

Background: A Group O Policy, which issues group O red blood cells (O-RBC) to recipients whose ABO types have not been duplicated on at least 2 separate occasions, has been in place at this 550-bed hospital since 1991. Its inception was a response to the frequency of wrong blood in tube (WBIT) events identified by discrepant serial ABO typings, which had been disproportionately concentrated among critically ill patients, a population at high risk of transfusion and misdraw. This policy was thought to better address such sources of mistransfusion error than delivery-based patient identification technologies. Methods: For the period from 1/1/2004 - 12/31/2005, the policy’s impact was reviewed according to utilization of O-RBC, frequency of critical O-RBC shortage, intervals to type confirmation, the identification of protracted users of policy O-RBC, the frequency of WBIT events as revealed by ABO typing discrepancies, the frequency of ABO-incompatible transfusion errors, and the frequency with which ABO-incompatible transfusion errors were averted as a result of the policy. Results: In 2004, 479 (4.5%) of a total of 10,575 units of O-RBC were transfused due to the policy. In 2005, 689 (6.1%) of a total of 11,293 units of O-RBC were transfused due to the policy. Increase in the use of O-RBC from 2004 to 2005 was by 718 units (6.7%), while the increase in the use of group O-RBC for the policy increased disproportionately by 210 units (43.8%). Over the 2 year period, a monthly median excess of 43 units (range 24–85) of O-RBC were issued in accordance with the policy. Daily excess use of O-RBC for the policy was a median of 1 unit (range 0–19). There were 291 days when no policy-related O-RBC were used, and only 3 days when more than 10 units were used. The interval between a 1st and 2nd (confirmatory) typing, when achieved, occurred within 24 hours for 15.4% of patients, but was a median of 23 days, and ranged from minutes to 21.5 years. Confirmatory re-typing activity within less than 72h of the 1st occurred in a minority of 28%, possibly reflecting nonessential repetition and/or deliberate action to release patients from policy-related O-RBC restrictions. Patients who were protracted users of Group O policy blood (no confirmatory type and/or repeatedly indeterminate typing) were neonates (310 units [26.5%]) and recipients of ABO-incompatible hematopoietic stem cell transplants (76 units [6.5%]). The blood bank identified misdrawn blood through typing discrepancies in 6 patients over 2 years. Transfusion occurred uneventfully in 3 of these 6, as the misdraw was made apparent by comparison with prior typings on file in 2, and patient knowledge of correct type in 1. The misdrawn specimen was the 1st one in 2 of 6. Of the 3 who were not transfused, the misdraw suggested a type that would have led in all to the transfusion of clinically incompatible blood, had the knowledge of prior type, and the policy, both been absent. ABO-incompatible red cell transfusion errors did not occur, although this was a greater function of luck than the policy in the interval. Conclusions: A policy instituted to prevent ABO-incompatible transfusion errors, by providing O-RBC to those with unconfirmed blood types, appeared to be both feasible, and potentially mistransfusion-preventative, at a high-volume tertiary-care teaching hospital.

INTRODUCTION: Various guidelines define appropriateness of FFP administration based on prothrombin time (PT) greater than 1.5 times normal. However, FFP is often transfused to patients with PTs that are in the upper range of normal or mildly prolonged. There is a paucity of data on whether transfusion of FFP to such patients will change their PT. METHODS: We retrospectively reviewed all FFP transfusions that were administered to patients with a PT below 17 seconds (normal reference range 11.3–13.6 seconds) at a tertiary care hospital in 2004. Patients were included in our analysis if their PT was measured prior to and within six hours of transfusion and if they received no more than two units of FFP. We identified 143 consecutive cases that met these criteria. RESULTS: 66 patients received one unit and 77 patients received 2 units of FFP. The median decrease in PT after the infusion of one or two units of FFP was 0.4 seconds and 0.6 seconds respectively. The pretransfusion PTs in 6 patients ranged between 12.2–13 seconds (subgroup 1), in 17 patients 13–14 seconds (subgroup 2), in 46 patients 14–15 seconds (subgroup 3), in 37 patients 15–16 seconds (subgroup 4), and in 37 patients 16–17 seconds (subgroup 5). Patients in subgroups 1 and 2 were noted to have higher PT values after the administration of FFP (median prolongation of 0.6 and 0.1 seconds respectively). The PT shortened in subgroup 3 by 0.4 seconds (median) and in subgroups 4 and 5 by 0.9 seconds (median) when compared to the pretransfusion PT, which represent clinically insignificant corrections. CONCLUSION: Transfusion of blood products is not without risks; given that PT values of less than 17 seconds do not significantly change following transfusion of one to two units of FFP, this clinical practice should be avoided.

Dendritic cell (DC)-tumor fusions effectively present a broad array of tumor associated antigens in the context of DC derived costimulation. Vaccination with fusion cells induces tumor specific immunity in animal models and clinical studies. We have examined the antigen presenting characteristics of DCs fused with myeloma cells. Immature DCs were generated by culturing adherent mononuclear cells with GM-CSF and IL-4 for 1 week. Maturation was induced by exposure to TNFa for 48 hours. Compared to immature DCs, patient derived mature DC had decreased CD14 expression, increased expression of CD80 and CD83 and increased mean flourescent intensity of CD86. Using polyethyelene glycol (PEG), monocyte derived immature and mature DCs were fused to myeloma cells derived from human cell lines and patient derived bone marrow specimens. Fusion cells were isolated by flow cytometric gating of cells that co-expressed unique DC and tumor antigens. For both immature and mature DC populations, cell fusion was associated with marked upregulation of costimulatory and maturation markers. Mean expression of CD86 was 98% in both fusion cell populations and CD83 was observed in 86% and 84% of immature and mature DC/myeloma fusions, respectively. Immature and mature DC/myeloma fusions prominently express IL-12 (mean 48% and 50%, respectively) and the chemokine receptor, CCR7 (mean 33% and 46%, respectively), necessary for the migration to sites of T cell traffic in the draining lymph node. DC/myeloma fusions induce IFNγ expression by autologous T cells. In 5 serial studies, immature and mature DC/myeloma fusion cells prominently stimulate cytotoxic T lymphocyte mediated lysis of tumor targets (mean 45% and 57%, respectively). Based on these findings we have initiated a clinical trial in which successive cohorts of patients with multiple myeloma undergo vaccination with myeloma cells fused with autologous mature DCs administered in conjunction with GM-CSF. Patients with clinically stable disease who demonstrate at least 20% marrow involvement with plasma cells are potentially eligible. DCs are generated from adherent mononuclear cells collected by leukapheresis that are cultured with GM-CSF, IL-4 and TNFa. Myeloma cells are obtained from short-term culture of bone marrow aspirate specimens. DCs and myeloma cells are co-cultured in the presence of PEG and fusion cells are quantified by identifying cells that co-express DC (CD86, CD83) and myeloma (CD38, CD138) antigens. To date, 6 patients have been enrolled of which 4 have been vaccinated; 3 with 1x10(6) and 1 with 2 x 10(6) fusion cells. Mean yields of DC, tumor, and fusion cells were 7.64 x107, 1.87 x107, and 6.12 x106 cells, respectively. In contrast to myeloma cells, DC and fusion cells preparations prominently induced allogeneic T cell proliferation with mean stimulation indices of 47 and 50, respectively. Toxicities judged to be potentially vaccine related have been mild and include muscle aches/stiffness, transient fever, pruritis, rash and fatigue. Vaccination resulted in the induction of tumor specific immunity as determined by increased percentage of CD4+ and CD8+ T cells expressing IFNγ following exposure to autologous tumor lysate. The effect of vaccination on clinical markers of disease is being monitored.

The type of coagulation factors and proteins in cryoprecipitate determine the appropriate indications for its use. To determine the pattern of use at a tertiary care medical center, we performed a retrospective audit of cryoprecipitate utilization. A total of 51 patients received 88 pools of cryoprecipitate. In 39 patients, cryoprecipitate was transfused for appropriate indications: hypofibrinogenemia (n = 19), tissue plasminogen activator reversal (n = 1), management of massive transfusion (n = 7), correction of uremic bleeding (n = 2), and for making fibrin sealant (n = 10). Overall, these patients used approximately 80% of the cryoprecipitate transfused. In 12 other patients, cryoprecipitate was transfused inappropriately to attempt reversal of the anticoagulant effects of warfarin therapy (n = 6), to treat impaired surgical hemostasis in the absence of hypofibrinogenemia (n = 4), and to treat hepatic coagulopathy with multiple factor deficiencies (n = 2). The patterns of misuse, involving 24% of all cryoprecipitate orders, suggest a widespread misunderstanding and need for focused education about the coagulation factors and proteins present in cryoprecipitate and appropriate indications for its use.