Peptide/MHC tetramer-based sorting of CD8⁺ T cells to a leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire.

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8⁺ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer⁺ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor ß (TCRß) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCRß clonotypes in a patient with a UNC-CDK4-1 tetramer⁺ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRß repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCRß clonotypes represented >17% of the entire TCRß repertoire-far in excess of the UNC-CDK4-1 tetramer⁺ frequency-indicating that the recurrent TCRß clonotypes identified from UNC-CDK-4-1 tetramer⁺ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1-driven clonal T-cell expansion. Mapping recurrent TCRß clonotype sequences onto TCRß repertoires can help confirm or refute antigen-specific T-cell expansion in vivo.

Effectiveness of an algorithm-based approach to the utilization of plerixafor in patients undergoing chemotherapy-based stem cell mobilization.

Autologous stem cell transplantation remains a mainstay of therapy for diseases such as multiple myeloma and relapsed lymphoma. The use of plerixafor has been shown to augment the ability to collect adequate stem cells, but the optimal use of this agent when used with chemotherapy is not yet clear. We utilized an algorithm-based approach with the addition of plerixafor to 54 patients undergoing chemomobilization with reduced-dose etoposide who had a less than optimal preapheresis CD34(+) cell count. We used a CD34(+) precount of 20 cells/µL as a threshold to initiate stem cell apheresis. Ninety-four percent of patients were successfully collected and proceeded to transplantation. Fourteen of 51 (28%) patients who successfully collected required plerixafor to augment stem cell yield. Of the patients who successfully collected, 94% (89% of the entire population) were able to collect in 2 or fewer days. Compared with previous data from our institution, the rate of patients collecting > 4 × 10(6) CD34(+) cells/kg in a single collection was increased from 39% to 69%. The safety profile of this approach was acceptable. The use of this algorithm-based method to determine when and whether to add plerixafor to chemomobilization was shown to be a successful and cost-effective approach to stem cell collection.

Role of procoagulant microparticles in mediating complications and outcome of acute liver injury/acute liver failure.

UNLABELLED: Microparticles (MPs), membrane fragments of 0.1-1.0 µm, are derived from many cell types in response to systemic inflammation. Acute liver failure (ALF) is a prototypical syndrome of systemic inflammatory response syndrome (SIRS) associated with a procoagulant state. We hypothesized that patients with ALF develop increased procoagulant MPs in proportion to the severity of systemic complications and adverse outcome. Fifty patients with acute liver injury (ALI), 78% of whom also had hepatic encephalopathy (HE; ALF), were followed until day 21 after admission. MPs were characterized by Invitrox Sizing, Antigen Detection and Enumeration, a light-scattering technology that can enumerate MPs as small as 0.15 µm, and by flow cytometry. Procoagulant activity was assessed by a functional MP-tissue factor (MP-TF) assay. Sixteen patients (32%) died and 27 (54%) recovered without liver transplantation (LT). Total MPs (0.15-1.0 µm) were present in nearly 19-fold higher concentrations in ALI/ALF patients, compared to healthy controls (P < 0.0001). MP-TF assays revealed high procoagulant activity (9.05 ± 8.82 versus 0.24 ± 0.14 pg/mL in controls; P = 0.0008). MP concentrations (0.28-0.64 µm) were higher in patients with the SIRS and high-grade HE, and MPs in the 0.36-0.64-µm size range increased in direct proportion to SIRS severity (P < 0.001) and grade of HE (P < 0.002). Day 1 MPs (0.28-0.64 µm) correlated with laboratory predictors of death/LT (higher phosphate and creatinine; lower bicarbonate), and day 1 and 3 MPs were higher in patients who died or underwent LT, compared to spontaneous survivors (P ≤ 0.01). By flow cytometry, 87% of patients had circulating CD41(+) MPs, indicating platelet origin. CONCLUSION: Highly procoagulant MPs of specific size ranges are associated with the SIRS, systemic complications, and adverse outcome of ALI/ALF. MPs may contribute to the multiorgan system failure and high mortality of ALF.

B cells from patients with chronic GVHD are activated and primed for survival via BAFF-mediated pathways.

Recent data reveal an important role for B cells in the pathogenesis of chronic GVHD (cGVHD). Patients with cGVHD have delayed B-cell reconstitution and elevated BAFF to B-cell ratios compared to patients without cGVHD. The mechanisms promoting and sustaining B-cell activation in this disease, however, remain unknown. As BAFF increases murine B-cell metabolism and survival and maintains autoreactive B-cell clones, we performed ex vivo analyses of peripheral B cells from 51 patients who either had or did not have active cGVHD and were greater than 1 year from the time of allogeneic hematopoietic stem cell transplantation. We found that B cells from patients with active cGVHD were in a heightened metabolic state and were resistant to apoptosis. Exogenous BAFF treatment amplified cell size and survival in B cells from these patients. We found significantly increased signaling through ERK and AKT that associated with decreased levels of proapoptotic Bim, suggesting a mechanistic link between elevated BAFF levels and aberrant B-cell survival. Thus, we identify a role for BAFF in the pathogenesis of cGVHD and define B-cell activation and survival pathways suitable for novel therapeutic development in cGVHD.

Lenalidomide after stem-cell transplantation for multiple myeloma.

BACKGROUND: Data are lacking on whether lenalidomide maintenance therapy prolongs the time to disease progression after autologous hematopoietic stem-cell transplantation in patients with multiple myeloma. METHODS: Between April 2005 and July 2009, we randomly assigned 460 patients who were younger than 71 years of age and had stable disease or a marginal, partial, or complete response 100 days after undergoing stem-cell transplantation to lenalidomide or placebo, which was administered until disease progression. The starting dose of lenalidomide was 10 mg per day (range, 5 to 15). RESULTS: The study-drug assignments were unblinded in 2009, when a planned interim analysis showed a significantly longer time to disease progression in the lenalidomide group. At unblinding, 20% of patients who received lenalidomide and 44% of patients who received placebo had progressive disease or had died (P<0.001); of the remaining 128 patients who received placebo and who did not have progressive disease, 86 crossed over to lenalidomide. At a median follow-up of 34 months, 86 of 231 patients who received lenalidomide (37%) and 132 of 229 patients who received placebo (58%) had disease progression or had died. The median time to progression was 46 months in the lenalidomide group and 27 months in the placebo group (P<0.001). A total of 35 patients who received lenalidomide (15%) and 53 patients who received placebo (23%) died (P=0.03). More grade 3 or 4 hematologic adverse events and grade 3 nonhematologic adverse events occurred in patients who received lenalidomide (P<0.001 for both comparisons). Second primary cancers occurred in 18 patients who received lenalidomide (8%) and 6 patients who received placebo (3%). CONCLUSIONS: Lenalidomide maintenance therapy, initiated at day 100 after hematopoietic stem-cell transplantation, was associated with more toxicity and second cancers but a significantly longer time to disease progression and significantly improved overall survival among patients with myeloma. (Funded by the National Cancer Institute; ClinicalTrials.gov number, NCT00114101.).

Infusional mitoxantrone plus bolus melphalan as a stem cell transplant conditioning regimen for multiple myeloma.

This study combined infusional mitoxantrone with bolus melphalan as a transplant preparative regimen for multiple myeloma. Mitoxantrone was infused over 6 hr on days 6 and 5. Melphalan was given as a 15 min bolus on day 1 followed by autologous transplant on day 0. Thirty-five patients were enrolled; 57% of enrollees had received ≥ 2 prior treatments. The median overall survival was 5 years and 8 months, with 37% of the subjects alive >7 years posttransplantation. Myelosuppression and mucositis were the most frequent adverse events. This regimen is well tolerated and the survival compares well to other transplant trials.

Microparticle sizing and counting using light scattering methods.

New light scattering methods offer many advantages to particle size distribution characterization. In addition to ease of operation, speed, and accuracy, the particle size, particle surface characteristics, interaction of the surface with specific ligands, and hydrodynamic volume of the particle are easily obtained. Extensions of these methods also permit the assessment of surface reactions in real time and without reporter group conjugation to the reactant. These methods offer the ability to examine binding constants and kinetics of binding without chemical modification and offer true advantages in product development and clinical diagnostics and therapeutic monitoring.

Role of hematopoietic stem cell transplant in the management of follicular lymphoma.

Despite decades of published data regarding the application of autologous and allogeneic stem cell transplant in patients with follicular lymphoma, there remain no uniform indications for its use in this disease. Autologous transplant has been shown to lead to longer progression-free survival times in randomized trials when compared with postremission interferon-based chemoimmunotherapy. However, the development of rituximab and its use in frontline, salvage, and maintenance therapy complicates the decision to pursue autologous transplant, a modality developed prior to the advent of anti-CD20 monoclonal antibodies. Allogeneic transplant offers the advantages of lymphoma-free grafts and the immunologic graft-versus-lymphoma effect. These factors may confer the possibility of long-term remission, though historically they have been accompanied by high rates of upfront morbidity and mortality, especially in heavily pretreated patients with a poor performance status or chemotherapy-refractory disease. Advances in patient selection, human leukocyte antigen (HLA) matching, conditioning regimens, and supportive care have reduced transplant-related mortality and the incidence of graft-versus-host disease. Recently published data focus on the incorporation of rituximab and radioimmunoconjugates prior to, during, and following autologous transplant. Furthermore, reduced-intensity allogeneic stem cell transplantation has increasingly been used for relapsed follicular lymphoma patients with comorbidities or advanced age. Several recent reports suggest that reduced-intensity regimens may provide a high likelihood of long-term disease-free survival for patients up to 70 years of age with a good performance status, chemotherapy-sensitive disease, and HLA-matched sibling donors. Such patients with relapsed disease should be referred to a transplant center that can enroll them in one of the forthcoming clinical trials that aim to confirm these outcomes.

Sequential high-dose ifosfamide, carboplatin and etoposide with rituximab for relapsed Hodgkin and large B-cell non-Hodgkin lymphoma: increased toxicity without improvement in progression-free survival.

Non-cross resistant drugs given at high-dose intensity may maximise tumor cell kill leading to improved patient outcomes. We investigated the feasibility and efficacy of administering ifosfamide, carboplatin and etoposide +/- rituximab as sequential high-dose single agents. Twenty-two patients with relapsed/refractory Hodgkin lymphoma (n = 9) or non-Hodgkin (n = 13) lymphoma (NHL) were included. Therapy included: cycle 1 ifosfamide (15 g/m(2)), cycle 2 etoposide (900 mg/m(2)) and cycle 3 carboplatin (area under the curve 15). Patients with NHL received rituximab (375 mg/m(2)) with cycles 1 and 2. Blood stem cell collection was performed after etoposide. Primary endpoints were overall response (complete response (CR) + PR) and ability to mobilise stem cells after etoposide. Secondary endpoints were to assess the toxicity of the regimen and to evaluate the ability of patients to proceed to stem cell transplant (SCT). Overall response rate was 54% with CR in 4/22 (18%) subjects and PR in 8/22 (36%). Median progression-free survival was 15 months and overall survival has not been reached at 40 months. Thirteen participants proceeded to SCT. Grade 3/4 thrombocytopenia and neutropenia occurred in 58% of cycles and 91% of subjects respectively. Forty-five percent of patients required hospitalisation for toxicity and two patients died from complications of therapy. Sequential dose intense ifosfamide, etoposide, carboplatin +/- rituximab was more toxic and no more effective than the same drugs given in a conventional fashion.

Antiplatelet therapies and the role of antiplatelet resistance in acute coronary syndrome.

Acute coronary syndrome is the number one killer in the industrialized world and, as such, continues to be one of the most well-studied disease states in all of medicine. Advancements in antiplatelet therapies for use in patients undergoing percutaneous coronary intervention have improved outcomes dramatically. However, a proportion of patients on long-term antiplatelet therapy continue to have cardiovascular events. Resistance to antiplatelet drugs may explain some of these events and this topic has become one of major interest and rapid evolution. This review describes the pathogenesis of acute coronary syndromes, outlines the evidence behind the use of the available antiplatelet agents, and examines the current data surrounding antiplatelet resistance.

Coagulopathy of acute liver failure.

Coagulopathy is an essential component of the acute liver failure (ALF) syndrome and reflects the central role of liver function in hemostasis. ALF is a syndrome characterized by the development of hepatic encephalopathy and coagulopathy within 24 weeks of the onset of acute liver disease. Coagulopathy in this setting is a useful prognostic tool in ALF and a dynamic indicator of the hepatic function. If severe, it can be associated with bleeding and is commonly a major obstacle to the performance of invasive procedures in patients with ALF. This review focuses on the epidemiology, pathophysiology, presentation, evaluation, and management of coagulopathy in ALF.

Unfractionated heparin reduces the anti-platelet effects of abciximab but not eptifibatide during PCI.

In 29 patients undergoing percutaneous coronary intervention (PCI), we obtained blood samples at baseline, 10 minutes after standard weight-based abciximab (n=15) or double-bolus eptifibatide (n=14) and 5 minutes after unfractionated heparin (UFH; 70 U/kg bolus). The median percent inhibition was significantly higher in the eptifibatide group compared with the abciximab group both before (96.5% [94-100] vs. 85% [77-89.5] [adenosine diphosphate; ADP]; 89.5% [84-95] vs. 59% [37.5-76.5] [thrombin receptor agonist peptide; TRAP], p<0.001 for both) and after UFH (95% [93-100] vs. 79% [68.8-87.5] [ADP]; 82% [77-93] vs. 51% [34.5-71.3] [TRAP], p<0.001 for both). Addition of UFH significantly reduced platelet inhibition in the abciximab group (85% [77-89.5] vs. 79% [68.8-87.5] [ADP]; 59% [37.5-76.5] vs. 51% [34.5-71.3] [TRAP], p<0.05 for both) but not in the eptifibatide group (96.5% [94-100] vs. 95% [93-100] [ADP]; 89.5% [84-95] vs. 82% [77-93] [TRAP], p=ns for both). Eptifibatide achieved superior platelet inhibition before but especially after UFH compared with abciximab.

Pharmacokinetics of ganciclovir after oral valganciclovir versus intravenous ganciclovir in allogeneic stem cell transplant patients with graft-versus-host disease of the gastrointestinal tract.

The pharmacokinetics of ganciclovir after oral valganciclovir versus intravenous ganciclovir were compared in allogeneic stem cell transplant recipients with stable graft-versus-host disease of the gastrointestinal tract. Twenty-two evaluable adult patients were randomized to receive a single dose of open-label study drug (900 mg of oral valganciclovir or 5 mg/kg of intravenous ganciclovir). After a washout period of 2 to 7 days, patients were crossed over to receive the alternate study drug. Ganciclovir and valganciclovir concentrations in plasma were measured over 24 hours after dosing. Noninferiority of 900 mg of valganciclovir relative to intravenous ganciclovir was concluded if the lower limit of the 1-sided 95% confidence interval of the ratio of least-square means of the ganciclovir area under the curve (AUC) for the 2 study drugs was >80%. Valganciclovir was found to be rapidly absorbed and converted into ganciclovir. The ganciclovir exposure after 900 mg of valganciclovir noninferior to that of intravenous ganciclovir (AUC0-infinity, 52.1 and 53.8 microg.h/mL, respectively; 95% confidence interval of the ratio of least square means of AUC0-infinity, 82.48%-118.02%). Oral valganciclovir could be a useful alternative to intravenous ganciclovir in certain stable stem cell transplant patients who require prophylaxis or preemptive therapy for cytomegalovirus infection.

Cytoprotection by amifostine during autologous stem cell transplantation for advanced refractory hematologic malignancies.

This study evaluated whether amifostine protects against mucositis and other toxicities in patients with advanced, refractory, or recurrent hematologic malignancies undergoing high-dose chemotherapy and total body irradiation. Thirty-five patients (20 with non-Hodgkin lymphoma, 12 with Hodgkin disease, and 3 with acute myelogenous leukemia) who underwent autologous stem cell transplantation were conditioned with total body irradiation 2 Gy twice daily on days -8 through -6; cyclophosphamide 6 g/m(2), etoposide 1.8 g/m(2), and carboplatin 1 g/m(2) on days -5 through -3; and amifostine 500 mg/m(2) on days -8 through -2. Prior institutional experience in patients treated without amifostine was used as a historical comparison (no-amifostine group). Severe mucositis occurred in 14 (40%) of 35 patients in the amifostine group, compared with 33 (94%) of 35 in the no-amifostine group (P < .0001). Total parenteral nutrition was used by 4 (11%) of 35 amifostine-treated patients and 34 (97%) of 35 no-amifostine patients (P < .0001). The median duration of narcotic use decreased from 15.5 days with no amifostine to 11 days with amifostine (P = .002). Granulocyte and platelet engraftment times were similar. Prospective trials with innovative designs and clearly defined stopping rules are warranted to confirm whether amifostine reduces the toxicities of a myelosuppressive conditioning regimen before autologous stem cell transplantation without compromising therapeutic response.

Rare cause of ureteral obstruction.

This report presents bilateral ureteral obstruction due to possible ureteritis in a bone marrow transplant patient with resurgence of BK virus after hemorrhagic cystitis. This is believed to be the first description of this type of ureteral obstruction. The presented case includes the management plan.

Phase 1 trial of the proteasome inhibitor bortezomib and pegylated liposomal doxorubicin in patients with advanced hematologic malignancies.

Proteasome inhibitors, a novel class of chemotherapeutic agents, enhance the antitumor efficacy of anthracyclines in vitro and in vivo. We therefore sought to determine the maximum tolerated dose (MTD) and dose-limiting toxicities of bortezomib and pegylated liposomal doxorubicin (PegLD). Bortezomib was given on days 1, 4, 8, and 11 from 0.90 to 1.50 mg/m2 and PegLD on day 4 at 30 mg/m2 to 42 patients with advanced hematologic malignancies. Grade 3 or 4 toxicities in at least 10% of patients included thrombocytopenia, lymphopenia, neutropenia, fatigue, pneumonia, peripheral neuropathy, febrile neutropenia, and diarrhea. The MTD based on cycle 1 was 1.50 and 30 mg/m2 of bortezomib and PegLD, respectively. However, due to frequent dose reductions and delays at this level, 1.30 and 30 mg/m2 are recommended for further study. Pharmacokinetic and pharmacodynamic studies did not find significant drug interactions between these agents. Antitumor activity was seen against multiple myeloma, with 8 of 22 evaluable patients having a complete response (CR) or near-CR, including several with anthracycline-refractory disease, and another 8 having partial responses (PRs). One patient with relapsed/refractory T-cell non-Hodgkin lymphoma (NHL) achieved a CR, whereas 2 patients each with acute myeloid leukemia and B-cell NHL had PRs. Bortezomib/PegLD was safely administered in this study with promising antitumor activity, supporting further testing of this regimen.

Monitoring coagulation and the clinical effects of recombinant factor VIIa.

Accurately monitoring hemostasis in patients with coagulation disorders is vital in order to allow physicians to optimize anticoagulant therapy and deliver effective patient management. Traditional assays, based on the measurement of the time to first evidence of clot formation, fail to describe the kinetics of clot production that ultimately define the quality of the final clot. This review describes the technologies that have been developed to accurately describe and monitor the kinetics of clot formation. Using clinical examples, the utility of these technologies is assessed and conclusions drawn about the most useful diagnostic measures for monitoring hemostasis.

Simultaneous testing of the heparin effect on the soluble phase and platelet component of hemostasis.

BACKGROUND: This study assessed the anticoagulant effect of heparin on both platelet activity and soluble phase coagulation. METHODS AND RESULTS: Blood samples were collected from 32 patients undergoing cardiac catheterization before and 5 minutes after a heparin injection (2000 U). Activated clotting time (ACT), activated partial thromboplastin time (aPTT), and whole blood platelet aggregation [adenosine diphosphate (ADP) and collagen] were compared with the flow device variables platelet hemostasis time (PHT) and collagen-induced thrombus formation (CITF). Before heparin, all patients had a normal aPTT and all but 1 had a normal ACT. After heparin, all patients showed a prolonged aPTT and ACT. In contrast, the flow device showed considerable variability after heparin. Only 47% of patients increased both PHT and CITF above the upper limit of normal, and 13% did not prolong either. After heparin, enhanced platelet aggregation to ADP and collagen occurred in 53% and 63% of patients, respectively. CONCLUSIONS: Although patients seem to have an anticoagulant effect after heparin based on ACT and aPTT results, the flow device identified a lack of any hemostatic impairment in 25 to 41% of patients. These findings probably reflect the variable effect of heparin on platelet function and may explain the poor heparin effect or, alternatively, the excessive bleeding after heparin administration that occurs in some patients.