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Introduction: Brentuximab vedotin (BV) is an antibody-drug conjugate that delivers monomethyl auristatin E (MMAE) to CD30+ cells and is safe and effective in relapsed/refractory (r/r) Hodgkin lymphoma (HL). Although most patients respond to BV, only a minority will obtain a complete response (CR), and almost all patients eventually progress. Ibrutinib is a Bruton's tyrosine kinase (BTK) inhibitor highly active in multiple subtypes of non-Hodgkin lymphoma; limited data exist regarding its use in HL. It irreversibly inhibits interleukin-2-inducible kinase (ITK) with Th1 based immune responses. As we previously observed preclinical synergy between ibrutinib and BV, we hypothesized ibrutinib may enhance the antitumor activity of BV in HL. We designed and conducted a phase II trial of ibrutinib plus BV in patients with R/R HL, and herein report the final primary analysis of safety and efficacy., Methods: This was a multicenter phase II trial with a lead-in cohort in patients with r/r HL. Eligibility criteria included age ≥ 15 years with r/r HL after at least one prior line of therapy. Treatment consisted of 1.8 mg/kg BV intravenously every 3 weeks and ibrutinib 560 mg PO daily (420 mg PO daily in the lead-in cohort). Prior BV was allowed if patients were not refractory. The primary endpoint was the CR rate according to Lugano 2014. Secondary endpoints included toxicities, overall response rate (ORR), and duration of response (DOR)., Results: The 39 patients were enrolled onto the study, of which 67% were male; the median age was 33 (range: 17-71). 38% had extranodal disease at baseline, 51% had advanced stage disease, 51% were refractory to the prior therapy, and 21% had prior BV. Of 36 patients who were evaluable for response, the CR rate was 33% and ORR 64%; median DOR was 25.5 months. Thirteen patients proceeded to autologous transplant and 3 patients proceeded to allogeneic transplant for consolidation after response. The most common adverse events were nausea (67%), peripheral neuropathy (62%), diarrhea (59%), fatigue (46%), thrombocytopenia (46%), headache (41%), rash (41%), elevated ALT (38%), anemia (36%), vomiting (36%), abdominal pain (33%), fever (33%), and hypertension (33%). Six patients experienced unacceptable toxicity, defined as Gr 3/4 non-hematologic toxicity or non-resolving Gr 3/4 hematologic toxicity including one patient who died of multiorgan failure from suspected COVID-19 infection during cycle 1., Discussion: The combination of BV and ibrutinib was active in r/r HL; however, given significant toxicity, it cannot be recommended for future development., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This phase 1 study evaluated the addition of vorinostat to pembrolizumab in patients with relapsed/refractory (RR) classical Hodgkin lymphoma (cHL), diffuse large B-cell lymphoma, and follicular lymphoma. We report the results in cases of cHL. Adult patients with RR cHL who had received ≥1 prior lines of therapy and were ineligible for transplantation were treated in a dose-escalation cohort with 2 dose levels (DLs) and then on an expansion cohort at the recommended phase 2 dose (RP2D) in 21-day cycles. Vorinostat 100 mg twice a day (DL1) and 200 mg twice a day (DL2) was administered orally from days 1 to 5 and 8 to 12; all patients received pembrolizumab 200 mg IV every 3 weeks. The primary end point was safety and determination of RP2D. In total, 32 patients with cHL were enrolled, including 30 at DL2 (RP2D); 78% had received prior anti-programmed cell death 1 (anti-PD-1) therapy, and 56% were PD-1 refractory. Grade ≥3 adverse events (AEs) included hypertension (9%), neutropenia (9%), hypophosphatemia (9%), thrombocytopenia (6%), and lymphopenia (6%). Immune-related AEs included grade 1 or 2 thyroiditis (13%), grade 1 rash (6%), and grade 3 esophagitis/duodenitis (3%). The overall response rate (ORR) was 72% and complete response (CR) rate was 34%. Patients refractory to prior PD-1 blockade (n = 18) had ORR and CR rates of 56% and 11%, respectively. Pembrolizumab and vorinostat was well tolerated with a high ORR rate in RR cHL including in anti-PD-1-refractory disease. This trial was registered at www.clinicaltrials.gov as #NCT03150329., (© 2023 by The American Society of Hematology.)

Background: Allogeneic hematopoietic stem cell transplantation (alloHCT) is potentially curative for relapsed/refractory (r/r) B-cell non-Hodgkin's lymphoma (B-cell NHL). However, relapse remains a major cause of treatment failure, especially in patients with either positron emission tomography (PET)-positive and/or chemoresistant disease prior to alloHCT. 90 Y-ibritumomab tiuxetan (Zevalin) is a radiolabeled anti-CD20 antibody which is a safe and effective therapy in multiple histologic subtypes of B-cell NHL and has also been incorporated in both autologous HCT (autoHCT) and alloHCT conditioning regimens., Objectives: The purpose of this study was to evaluate the efficacy and confirm the safety of the radiolabeled anti-CD20 antibody ibritumomab tiuxetan (Zevalin) combined with the reduced intensity conditioning (RIC) regimen of fludarabine and melphalan (Flu/Mel) in patients with high-risk B-cell NHL., Study Design: We conducted a phase II trial (NCT00577278) of Zevalin with Flu/Mel in patients with high-risk B-cell NHL. We enrolled 41 patients from October 2007 to April 2014, all of whom had either a fully matched sibling or 8/8 or 7/8 matched unrelated donor (MUD). Patients received 111 In-Zevalin (5.0 mCi) on day -21 pre-HCT, followed by 90 Y-Zevalin (0.4 mCi/kg) on day -14. Fludarabine (25 mg/m 2 daily) was given from days -9 to -5 and melphalan (140 mg/m 2 ) was administered on day -4. All patients received rituximab 250 mg/m2 on day +8 and an additional dose on either day +1 or -21 depending on the baseline rituximab level. Patients with a low rituximab level were given rituximab on days -21 and -15. All patients received tacrolimus/sirolimus (T/S) with or without methotrexate (MTX) for graft-versus-host disease (GVHD) prophylaxis starting on day -3, and stem cells were infused on day 0., Results: The 2-year overall survival (OS) and progression-free survival (PFS) for all patients were 63% and 61%, respectively. The incidence of relapse at 2 years was 20%. Nonrelapse mortality (NRM) at day +100 and 1 year were 5% and 12%, respectively. The overall cumulative incidence of grade II-IV and III-IV acute GVHD (aGVHD) were 44% and 15%, respectively. Extensive chronic GVHD (cGVHD) occurred in 44% of patients. On univariate analysis, histology (diffuse large B cell lymphoma (DLBCL) vs. others) was negatively predictive for OS (P = .0013) and PFS (P = .0004), while histology (DLBCL vs. others, P = .0128) predicted for relapse. PET positivity pre-HCT did not correlate with any of the efficacy endpoints., Conclusion: Addition of Zevalin to Flu/Mel is safe and effective in high-risk NHL and met the prespecific endpoint. Results were suboptimal in patients with DLBCL., Competing Interests: Disclosure Declarations of interest: MM reports consultancies with Novartis, SeaGen, Janssen, EUSA, CTI, participation in speakers’ bureaus with SeaGen and Morphosys, and research funding from BMS, BeiGene, and Morphosys. AFH reports consultancies with BMS, Genentech, Merck, SeaGen, AstraZeneca, Karyopharm, ADC Therapeutics, Takeda, Tubulis, Regeneron, Genmab, Pfizer, Caribou, Adicet Bio, Abbvie, Allogene, and research funding from Genentech, Merck, Seagen, KiTE Pharma, Gilead Sciences, AstraZeneca, ADC Therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: Assessing the risk of common, complex diseases requires consideration of clinical risk factors as well as monogenic and polygenic risks, which in turn may be reflected in family history. Returning risks to individuals and providers may influence preventive care or use of prophylactic therapies for those individuals at high genetic risk., Methods: To enable integrated genetic risk assessment, the eMERGE (electronic MEdical Records and GEnomics) network is enrolling 25,000 diverse individuals in a prospective cohort study across 10 sites. The network developed methods to return cross-ancestry polygenic risk scores, monogenic risks, family history, and clinical risk assessments via a genome-informed risk assessment (GIRA) report and will assess uptake of care recommendations after return of results., Results: GIRAs include summary care recommendations for 11 conditions, education pages, and clinical laboratory reports. The return of high-risk GIRA to individuals and providers includes guidelines for care and lifestyle recommendations. Assembling the GIRA required infrastructure and workflows for ingesting and presenting content from multiple sources. Recruitment began in February 2022., Conclusion: Return of a novel report for communicating monogenic, polygenic, and family history-based risk factors will inform the benefits of integrated genetic risk assessment for routine health care., Competing Interests: Conflict of Interest N.S.A.-H. is an employee and equity holder of 23andMe; serves as a scientific advisory board member for Allelica, Inc; received personal fees from Genentech Inc, Allelica Inc, and 23andMe; received research funding from Akcea Therapeutics; and was previously employed by Regeneron Pharmaceuticals. T.W. has grant funding from Gilead Sciences, Inc. L.O. and T.R.-B are founders of a company developing MeTree. T.S., E.D.E., and E.H. are employees and stockholders of Invitae Corporation. E.M.M. has been a consultant for Avidity Bioscience, Amgen Inc, AstraZeneca, Cytokinetics, Invitae Corporation, Janssen Pharmaceuticals, Pfizer Inc, PepGen Inc, Tenaya Therapeutics, and Stealth BioTherapeutics Inc; she is also the founder of Ikaika Therapeutics. E.E.K. received personal fees from Illumina Inc, 23andMe, and Regeneron Pharmaceuticals and serves as a scientific advisory board member for Encompass Bioscience, Foresite Labs, and Galateo Bio. B.K. is an advisory board member and stockholder of Genome Medical. M.S. is a member of the Institutional Review Board of the All of Us Research Program. E.F.P. is a paid consultant for Allecia Inc. J.F.P. is a paid consultant for Natera Inc. All other authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The efficacy of repetitive transcranial magnetic stimulation (rTMS) for depression may vary depending on the subregion stimulated within the dorsolateral prefrontal cortex (DLPFC). Clinical TMS typically uses scalp-based landmarks for DLPFC targeting, rather than individualized MRI guidance., Objective: In rTMS patients, determine the brain systems targeted by multiple DLPFC stimulation rules by computing several surrogate measures: underlying brain targets labeled with connectivity-based atlases, subgenual cingulate anticorrelation strength, and functionally connected networks., Methods: Forty-nine patients in a randomized controlled trial of rTMS therapy for treatment resistant major depression underwent structural and functional MRI. DLPFC rules were applied virtually using MR-image guidance. Underlying cortical regions were labeled, and connectivity with the subgenual cingulate and whole-brain computed., Results: Scalp-targeting rules applied post hoc to these MRIs that adjusted for head size, including Beam F3, were comparably precise, successful in directly targeting classical DLPFC and frontal networks, and anticorrelated with the subgenual cingulate. In contrast, all rules involving fixed distances introduced variability in regions and networks targeted. The 5 cm rule targeted a transitional DLPFC region with a different connectivity profile from the adjusted rules. Seed-based connectivity analyses identified multiple regions, such as posterior cingulate and inferior parietal lobe, that warrant further study in order to understand their potential contribution to clinical response., Conclusion: EEG-based rules consistently targeted DLPFC brain regions with resting-state fMRI features known to be associated with depression response. These results provide a bridge from lab to clinic by enabling clinicians to relate scalp-targeting rules to functionally connected brain systems., Competing Interests: Declaration of competing interest Dr. Rosen, Ms. Bhat, Dr. Cardenas, Dr. Ehrlich, Ms. Horwege, Mr. Lavacot, Mr. Roach, Dr. Glover, Dr. Forman, Dr. Yurgelun-Todd, and Dr. Yesavage have no conflicts to disclose. Dr. Mathalon is a consultant for Boehringer Ingelheim and Caden Therapeutics. Dr. Badran owns a minority stake in Bodhi NeuroTech Inc., which manufactures meditation enhancing devices and holds patents in this area assigned to the Medical University of South Carolina. Dr. George holds research grants through the Medical University of South Carolina with Brainsway and LivaNova. He is on the scientific advisory board, uncompensated, for Magstim, Brainsway. He has equipment loaned from MECTA and Nexstim. He is on the scientific advisory board for Neuralief (compensated). He has no ownership of any brain stimulation device company. He is the editor in chief of Brain Stimulation, published by Elsevier. Dr. Thase reports no conflicts of interest with this research. Dr. Thase reports the following other relationships over the past three years: Advisory/Consultant—Acadia, Alkermes, Allergan (Forest, Naurex), Axsome, Clexio, Johnson & Johnson (Janssen, Ortho-McNeil), Lundbeck, Merck, Novartis, Otsuka, Pfizer, Shire, Sunovion, Takeda; Grant Support—Acadia, Allergan (Forest, Naurex), Alkermes, Axsome, Clexio, Myriad (AssureRx), Axsome, Intracellular, Janssen, National Institute of Mental Health, Otsuka, Patient Centered Outcomes Research Institute, Takeda; Royalties—American Psychiatric Press, Guilford Publications, Herald House, W.W. Norton & Company, Inc. Dr. Thases's spouse, Diane Sloan, PharmD, is a Senior Medical Director for Peloton Advantage, which does business with a number of pharmaceutical companies., (Copyright © 2021. Published by Elsevier Inc.)

Background: Precise targeting of brain functional networks is believed critical for treatment efficacy of rTMS (repetitive pulse transcranial magnetic stimulation) in treatment resistant major depression., Objective: To use imaging data from a "failed" clinical trial of rTMS in Veterans to test whether treatment response was associated with rTMS coil location in active but not sham stimulation, and compare fMRI functional connectivity between those stimulation locations., Methods: An imaging substudy of 49 Veterans (mean age, 56 years; range, 27-78 years; 39 male) from a randomized, sham-controlled, double-blinded clinical trial of rTMS treatment, grouping participants by clinical response, followed by group comparisons of treatment locations identified by individualized fiducial markers on structural MRI and resting state fMRI derived networks., Results: The average stimulation location for responders versus nonresponders differed in the active but not in the sham condition (P = .02). The average responder location derived from the active condition showed significant negative functional connectivity with the subgenual cingulate (P < .001) while the nonresponder location did not (P = .17), a finding replicated in independent cohorts of 84 depressed and 35 neurotypical participants. The responder and nonresponder stimulation locations evoked different seed based networks (FDR corrected clusters, all P < .03), revealing additional brain regions related to rTMS treatment outcome., Conclusion: These results provide evidence from a randomized controlled trial that clinical response to rTMS is related to accuracy in targeting the region within DLPFC that is negatively correlated with subgenual cingulate. These results support the validity of a neuro-functionally informed rTMS therapy target in Veterans., Competing Interests: Declaration of competing interest Dr. Rosen, Ms. Bhat, Dr. Cardenas, Dr. Ehrlich, Ms. Horwege, Mr. Roach, Dr. Glover, Dr. Forman, Dr. Yurgelun-Todd, Dr. Scott, Dr. Tian, and Dr. Yesavage have no conflicts to disclose. Dr. Mathalon is a consultant for Boehringer Ingelheim and Caden Therapeutics. Dr. Badran owns a minority stake in Bodhi NeuroTech Inc., which manufactures meditation enhancing devices and holds patents in this area assigned to the Medical University of South Carolina. Dr. George holds research grants through the Medical University of South Carolina with Brainsway and LivaNova. He is on the scientific advisory board, uncompensated, for Magstim, Brainsway. He has equipment loaned from MECTA and Nexstim. He is on the scientific advisory board for Neuralief (compensated). He has no ownership of any brain stimulation device company. He is the editor in chief of Brain Stimulation, published by Elsevier. Dr. Thase reports no conflicts of interest with this research. Dr. Thase reports the following other relationships over the past three years: Advisory/Consultant—Acadia, Alkermes, Allergan (Forest, Naurex), Axsome, Clexio, Johnson & Johnson (Janssen, Ortho-McNeil), Lundbeck, Merck, Novartis, Otsuka, Pfizer, Shire, Sunovion, Takeda; Grant Support—Acadia, Allergan (Forest, Naurex), Alkermes, Axsome, Clexio, Myriad (AssureRx), Axsome, Intracellular, Janssen, National Institute of Mental Health, Otsuka, Patient Centered Outcomes Research Institute, Takeda; Royalties—American Psychiatric Press, Guilford Publications, Herald House, W.W. Norton & Company, Inc. Dr. Thases’s spouse, Diane Sloan, PharmD, is a Senior Medical Director for Peloton Advantage, which does business with a number of pharmaceutical companies. Dr. Doyen and Dr. Sughrue are directors of Onminscient Neurotechnology Pty. and hold company shares. Dr. Doyen, Dr. Sughrue, and Mr. Nicholas are employees of Omniscient Neurotechnology Pty. Mr. Nicholas holds company employee options in Omniscient Neurotechnology Pty., (Copyright © 2021. Published by Elsevier Inc.)

The objective of this study was to evaluate the safety and efficacy of bermekimab, an IL-1α inhibitor, in the treatment of hidradenitis suppurativa (HS). This study was a phase II, multicenter, open-label study of two dose cohorts of bermekimab in patients with moderate-to-severe HS who are naïve to or have failed prior anti-TNF therapy. Patients with HS (n = 42) were divided into groups A and B based on whether or not they had previously failed an anti-TNF therapy. In group A (n = 24), bermekimab was administered subcutaneously at a dose of 400 mg weekly (13 doses) in patients who had previously failed anti-TNF therapy; in group B (n = 18), bermekimab was administered subcutaneously at a dose of 400 mg weekly (13 doses) in patients who were anti-TNF naïve. Bermekimab, previously found to be effective in treating HS, was evaluated using a subcutaneous formulation in patients with HS naïve to or having failed anti-TNF therapy. There were no bermekimab-related adverse events with the exception of injection site reactions. Bermekimab was effective despite treatment history, with 61% and 63% of patients naïve to and having failed anti-TNF therapy, respectively, achieving HS clinical response after 12 weeks of treatment. A significant reduction in abscesses and inflammatory nodules of 60% (P < 0.004) and 46% (P < 0.001) was seen in anti-TNF naïve and anti-TNF failure groups, respectively. Clinically and statistically significant reduction was seen in patients experiencing pain, with the Visual Analogue Scale pain score reducing by 64% (P < 0.001) and 54% (P < 0.001) in the anti-TNF naïve and anti-TNF failure groups, respectively. IL-1α is emerging as an important clinical target for skin disease, and bermekimab may represent a new therapeutic option for treating moderate-to-severe HS., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Abrocitinib, an oral selective Janus kinase 1 inhibitor, was effective and well tolerated in adults with moderate-to-severe atopic dermatitis in a phase 2b trial. We aimed to assess the efficacy and safety of abrocitinib monotherapy in adolescents and adults with moderate-to-severe atopic dermatitis., Methods: In this multicentre, double-blind, randomised phase 3 trial (JADE MONO-1), patients (aged ≥12 years) with moderate-to-severe atopic dermatitis (Investigator Global Assessment score ≥3, Eczema Area and Severity Index [EASI] score ≥16, percentage of body surface area affected ≥10%, and Peak Pruritus Numerical Rating Scale score ≥4) with a bodyweight of 40 kg or more, were enrolled at 69 sites in Australia, Canada, Europe, and the USA. Patients were randomly assigned (2:2:1) to oral abrocitinib 100 mg, abrocitinib 200 mg, or placebo once daily for 12 weeks. Randomisation was done using an interactive response technology system, stratified by baseline disease severity and age. Patients, investigators, and the funder of the study were masked to study treatment. The coprimary endpoints were the proportion of patients who had achieved an Investigator Global Assessment response (score of 0 [clear] or 1 [almost clear] with a ≥2-grade improvement from baseline), and the proportion of patients who achieved at least a 75% improvement in EASI score from baseline (EASI-75) score, both assessed at week 12. Efficacy was assessed in the full analysis set, which included all randomised patients who received at least one dose of study medication. Safety was assessed in all randomised patients. This study is registered with ClinicalTrials.gov, NCT03349060., Findings: Between Dec 7, 2017, and March 26, 2019, 387 patients were enrolled: 156 were assigned to abrocitinib 100 mg, 154 to abrocitinib 200 mg, and 77 to placebo. All enrolled patients received at least one dose of study treatment and thus were evaluable for 12-week efficacy. Of the patients with available data for the coprimary endpoints at week 12, the proportion of patients who had achieved an Investigator Global Assessment response was significantly higher in the abrocitinib 100 mg group than in the placebo group (37 [24%] of 156 patients vs six [8%] of 76 patients; p=0·0037) and in the abrocitinib 200 mg group compared with the placebo group (67 [44%] of 153 patients vs six [8%] of 76 patients; p<0·0001). Of the patients with available data for the coprimary endpoints at week 12, compared with the placebo group, the proportion of patients who had achieved an EASI-75 response was significantly higher in the abrocitinib 100 mg group (62 [40%] of 156 patients vs nine [12%] of 76 patients; p<0·0001) and abrocitinib 200 mg group (96 [63%] of 153 patients vs nine [12%] of 76 patients; p<0·0001). Adverse events were reported in 108 (69%) of 156 patients in the abrocitinib 100 mg group, 120 (78%) of 154 patients in the abrocitinib 200 mg group, and 44 (57%) of 77 patients in the placebo group. Serious adverse events were reported in five (3%) of 156 patients in the abrocitinib 100 mg group, five (3%) of 154 patients in the abrocitinib 200 mg group, and three (4%) of 77 patients in the placebo group. No treatment-related deaths were reported., Interpretation: Monotherapy with oral abrocitinib once daily was effective and well tolerated in adolescents and adults with moderate-to-severe atopic dermatitis., Funding: Pfizer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: We previously demonstrated that brentuximab vedotin (BV) used as second-line therapy in patients with Hodgkin lymphoma is a tolerable and effective bridge to autologous hematopoietic cell transplantation (AHCT). Here, we report the post-AHCT outcomes of patients treated with second-line standard/fixed-dose BV and an additional cohort of patients where positron-emission tomography adapted dose-escalation of second-line BV was utilized., Patients and Methods: Patients on the dose-escalation cohort received 1.8 mg/kg of BV intravenously every 3 weeks for two cycles. Patients in complete remission (CR) after two cycles received two additional cycles of BV at 1.8 mg/kg, while patients with stable disease or partial response were escalated to 2.4 mg/kg for two cycles. All patients, regardless of treatment cohort, proceeded directly to AHCT or received additional pre-AHCT therapy at the discretion of the treating physician based on remission status after second-line BV., Results: Of the 20 patients enrolled to the BV dose-escalation cohort, 8 patients underwent BV dose-escalation. BV escalation was well-tolerated, but no patients who were escalated converted to CR. Of 56 evaluable patients treated across cohorts, the overall response rate (ORR) to second-line BV was 75% with 43% CR. Twenty-eight (50%) patients proceeded directly to AHCT without post-BV chemotherapy, and a total of 50 patients proceeded to AHCT. Thirteen patients received consolidative post-AHCT therapy with either radiation, BV, or a PD-1 inhibitor. After AHCT, the 2-year progression-free survival (PFS) and overall survival were 67% and 93%, respectively. The 2-year PFS among patients in CR at the time of AHCT (n = 37) was 71% compared with 54% in patients not in CR (p = 0.12). The 2-year PFS in patients who proceeded to AHCT directly after receiving BV alone was 77%., Conclusions: Second-line BV is an effective bridge to AHCT that produces responses of sufficient depth to provide durable remission in conjunction with AHCT (clinicaltrials.gov: NCT01393717).

Background Aims: Light chain amyloidosis (AL) is a protein deposition disorder that is a result of a plasma cell dyscrasia, similar to multiple myeloma (MM). Immunotherapy is an attractive approach because of the low burden of disease, but the optimal target for AL is unclear. CS1 and B-cell maturation antigen (BCMA) are two potential targets because they are expressed on normal plasma cells and MM cells., Methods: We performed a prospective study evaluating bone marrow specimens of 20 patients with plasma cell diseases, 10 with AL and 10 with MM. We evaluated the clonal population of plasma cells for BCMA and CS1 expression. We designed a second-generation CS1 chimeric antigen receptor (CAR) construct, comprising a CS1 antigen-specific scFv, shortened hinge region and CD28 costimulatory domain. Purified central memory T cells were activated and transduced with a lentiviral vector encoding the CS1 CAR. Cytotoxicity was evaluated using 51 Cr release assays. Five days after tumor inoculation, NSG mice were injected intravenously with CS1 CAR T cells., Results: Whereas CS1 is present on the plasma cells of AL patients, we found BCMA expression in AL to be markedly low. CS1 CAR T cells were cytotoxic against CS1 positive tumor cells and induced durable tumor regressions in mice., Discussion: Our work represents a novel application of CS1-directed CAR T cells while revealing that BCMA would not be a suitable target. We expect AL to be particularly susceptible to CAR T-cell therapy because of the low tumor burden in the bone marrow., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Cutaneous sclerosis is one of the most common clinical manifestations of chronic graft-versus-host disease (cGVHD). Donor CD4(+) T and B cells play important roles in cGVHD pathogenesis, but the role of antibodies from donor B cells remains unclear. In the current studies, we generated immunoglobulin (Ig)H(µγ1) DBA/2 mice whose B cells have normal antigen-presentation and regulatory functions but cannot secrete antibodies. With a murine cGVHD model using DBA/2 donors and BALB/c recipients, we have shown that wild-type (WT) grafts induce persistent cGVHD with damage in the thymus, peripheral lymphoid organs, and skin, as well as cutaneous T helper 17 cell (Th17) infiltration. In contrast, IgH(µγ1) grafts induced only transient cGVHD with little damage in the thymus or peripheral lymph organs or with little cutaneous Th17 infiltration. Injections of IgG-containing sera from cGVHD recipients given WT grafts but not IgG-deficient sera from recipients given IgH(µγ1) grafts led to deposition of IgG in the thymus and skin, with resulting damage in the thymus and peripheral lymph organs, cutaneous Th17 infiltration, and perpetuation of cGVHD in recipients given IgH(µγ1) grafts. These results indicate that donor B-cell antibodies augment cutaneous cGVHD in part by damaging the thymus and increasing tissue infiltration of pathogenic Th17 cells., (© 2016 by The American Society of Hematology.)

Targeting oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) in acute myeloid leukemia (AML) can reduce blast survival and tumor immune evasion. Decoy oligodeoxynucleotides (dODNs), which comprise STAT3-specific DNA sequences are competitive inhibition of STAT3 transcriptional activity. To deliver STAT3dODN specifically to myeloid cells, we linked STAT3dODN to the Toll-like receptor 9 (TLR9) ligand, cytosine guanine dinucleotide (CpG). The CpG-STAT3dODN conjugates are quickly internalized by human and mouse TLR9(+)immune cells (dendritic cells, B cells) and the majority of patients' derived AML blasts, including leukemia stem/progenitor cells. Following uptake, CpG-STAT3dODNs are released from endosomes, and bind and sequester cytoplasmic STAT3, thereby inhibiting downstream gene expression in target cells. STAT3 inhibition in patients' AML cells limits their immunosuppressive potential by reduced arginase expression, thereby partly restoring T-cell proliferation. Partly chemically modified CpG-STAT3dODNs have >60 hours serum half-life which allows for IV administration to leukemia-bearing mice (50% effective dose ∼ 2.5 mg/kg). Repeated administration of CpG-STAT3dODN resulted in regression of human MV4-11 AML in mice. The antitumor efficacy of this strategy is further enhanced in immunocompetent mice by combining direct leukemia-specific cytotoxicity with immunogenic effects of STAT3 blocking/TLR9 triggering. CpG-STAT3dODN effectively reducedCbfb/MYH11/MplAML burden in various organs and eliminated leukemia stem/progenitor cells, mainly through CD8/CD4 T-cell-mediated immune responses. In contrast, small-molecule Janus kinase 2/STAT3 inhibitor failed to reproduce therapeutic effects of cell-selective CpG-STAT3dODN strategy. These results demonstrate therapeutic potential of CpG-STAT3dODN inhibitors with broad implications for treatment of AML and potentially other hematologic malignancies., (© 2016 by The American Society of Hematology.)

Acute myeloid leukemia (AML) is sustained by small populations of leukemia stem cells (LSCs) that can resist available treatments and represent important barriers to cure. Although previous studies have shown increased signal transducer and activator of transcription (STAT)3 and STAT5 phosphorylation in AML leukemic blasts, the role of Janus kinase (JAK) signaling in primary AML compared with normal stem cells has not been directly evaluated. We show here that JAK/STAT signaling is increased in LSCs, particularly from high-risk AML. JAK2 inhibition using small molecule inhibitors or interference RNA reduced growth of AML LSCs while sparing normal stem cells both in vitro and in vivo. Increased JAK/STAT activity was associated with increased expression and altered signaling through growth factor receptors in AML LSCs, including receptor tyrosine kinase c-KIT and FMS-related tyrosine kinase 3 (FLT3). Inhibition of c-KIT and FLT3 expression significantly inhibited JAK/STAT signaling in AML LSCs, and JAK inhibitors effectively inhibited FLT3-mutated AML LSCs. Our results indicate that JAK/STAT signaling represents an important signaling mechanism supporting AML LSC growth and survival. These studies support continued evaluation of strategies for JAK/STAT inhibition for therapeutic targeting of AML LSCs.

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and immune checkpoint commonly activated in cancer cells and in tumor-associated immune cells. We previously developed an immunostimulatory strategy based on targeted Stat3 silencing in Toll-like receptor 9 (TLR9)-positive hematopoietic cells using CpG-small interfering RNA (siRNA) conjugates. Here, we assessed the therapeutic effect of systemic STAT3 blocking/TLR9 triggering in disseminated acute myeloid leukemia (AML). We used mouse Cbfb-MYH11/Mpl-induced leukemia model, which mimics human inv(16) AML. Our results demonstrate that intravenously delivered CpG-Stat3 siRNA, but not control oligonucleotides, can eradicate established AML and impair leukemia-initiating potential. These antitumor effects require host's effector T cells but not TLR9-positive antigen-presenting cells. Instead, CpG-Stat3 siRNA has direct immunogenic effect on AML cells in vivo upregulating major histocompatibility complex class-II, costimulatory and proinflammatory mediators, such as interleukin-12, while downregulating coinhibitory PD-L1 molecule. Systemic injections of CpG-Stat3 siRNA generate potent tumor antigen-specific immune responses, increase the ratio of tumor-infiltrating CD8(+) T cells to regulatory T cells in various organs, and result in CD8(+) T-cell-dependent regression of leukemia. Our findings underscore the potential of using targeted STAT3 inhibition/TLR9 triggering to break tumor tolerance and induce immunity against AML and potentially other TLR9-positive blood cancers.

Purpose: To determine the distribution of eating disorders (ED) in children and adolescents comparing the fourth edition of the Diagnostic and Statistical Manual (DSM) to the proposed fifth edition DSM criteria., Methods: A total of 215 consecutive patients (15.4 ± 3.3 years) presenting for initial ED evaluation to adolescent medicine physicians from six institutions were assigned ED diagnoses using current DSM-IV criteria as well as proposed DSM-5 criteria., Results: Diagnoses of anorexia nervosa and bulimia nervosa increased using the proposed DSM-5 criteria (from 30.0% to 40.0% and from 7.3% to 11.8%, p < .001). Approximately 14% of patients received the presumptive DSM-5 diagnosis of avoidant/restrictive food intake disorder. Cases of ED not otherwise specified decreased from 62.3% to 32.6% (p < .001)., Conclusions: Proposed DSM-5 criteria substantially decreased the frequency of ED not otherwise specified diagnoses and increased the number of cases of anorexia nervosa and bulimia nervosa in a population of young patients presenting for ED treatment. Avoidant/restrictive food intake disorder appears to be a significant diagnosis., (Copyright © 2013 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)

STAT3 operates in both cancer cells and tumor-associated immune cells to promote cancer progression. As a transcription factor, it is a highly desirable but difficult target for pharmacologic inhibition. We have recently shown that the TLR9 agonists CpG oligonucleotides can be used for targeted siRNA delivery to mouse immune cells. In the present study, we demonstrate that a similar strategy allows for targeted gene silencing in both normal and malignant human TLR9(+) hematopoietic cells in vivo. We have developed new human cell-specific CpG(A)-STAT3 siRNA conjugates capable of inducing TLR9-dependent gene silencing and activation of primary immune cells such as myeloid dendritic cells, plasmacytoid dendritic cells, and B cells in vitro. TLR9 is also expressed by several human hematologic malignancies, including B-cell lymphoma, multiple myeloma, and acute myeloid leukemia. We further demonstrate that oncogenic proteins such as STAT3 or BCL-X(L) are effectively knocked down by specific CpG(A)-siRNAs in TLR9(+) hematologic tumor cells in vivo. Targeting survival signaling using CpG(A)-siRNAs inhibits the growth of several xenotransplanted multiple myeloma and acute myeloid leukemia tumors. CpG(A)-STAT3 siRNA is immunostimulatory and nontoxic for normal human leukocytes in vitro. The results of the present study show the potential of using tumoricidal/immunostimulatory CpG-siRNA oligonucleotides as a novel 2-pronged therapeutic strategy for hematologic malignancies.

STAT3 plays a crucial role in promoting progression of human cancers, including several types of B-cell lymphoma. However, as a transcription factor lacking its own enzymatic activity, STAT3 remains difficult to target with small-molecule drugs in the clinic. Here we demonstrate that persistent activated STAT3 colocalizes with elevated expression of S1PR1, a G-protein-coupled receptor for sphingosine-1-phosphate (S1P), in the tumor cells of the activated B cell-like subtype of diffuse large B-cell lymphoma patient specimens. Inhibition of S1PR1 expression by shRNA in the lymphoma cells validates that blocking S1PR1 affects expression of STAT3 downstream genes critically involved in tumor cell survival, proliferation, tumor invasion, and/or immunosuppression. Using S1PR1 shRNA, or FTY720, an antagonist of S1P that is in the clinic for other indications, we show that inhibiting S1PR1 expression down-regulates STAT3 activity and causes growth inhibition of the lymphoma tumor cells in vitro and in vivo. Our results suggest that targeting S1P/S1PR1 using a clinically relevant and available drug or other approaches is potentially an effective new therapeutic modality for treating the activated B cell-like subtype of diffuse large B-cell lymphoma, a subset of lymphoma that is less responsive to current available therapies.

This phase 1/2 study assessed the augmentation of reduced-intensity conditioning (RIC) with total marrow and lymph node irradiation (TMLI), for peripheral blood stem cell transplantation, in patients with advanced hematologic disease. The regimen consisted of fludarabine 25 mg/m(2) per day for 5 days, melphalan 140 mg/m(2) for one day, and TMLI radiation at 150 cGy/fraction in 8 fractions over 4 days. Eligible patients were over 50 years old and/or had compromised organ function. Median age of the 33 evaluable patients was 55.2 years. Eighteen events of nonhematologic grade III or higher toxicities occurred in 9 patients. Day 30 and day 100 mortalities were 3% and 15%, respectively. Patients achieved myeloid and platelet engraftment at a median of 14 days after transplantation. Long-term toxicities occurred in 2 patients: hypokalemia and tremor, both grade III, on days 370 and 361 after transplantation. Fourteen patients died, 7 of relapse-related causes and 7 of non-relapse-related causes. With a median follow-up for living patients of 14.7 months, 1-year overall survival, event-free survival, and non-relapse-related mortality were 75%, 65%, and 19%, respectively. Addition of TMLI to RIC is feasible and safe and could be offered to patients with advanced hematologic malignancies who might not otherwise be candidates for RIC.

Background: Although secondary acute leukemias and myelodysplasia are the known complications of adjuvant chemotherapy for breast cancer, the treatment outcome of these secondary malignancies is presently unclear. We examined the clinical and pathological features as well as the treatment results of a series of patients with acute leukemia/myelodysplasia arising after adjuvant chemotherapy for breast cancer., Patients and Methods: Patients referred to our institution during a 5-year period for treatment of acute leukemia/myelodysplasia and who had received adjuvant chemotherapy for breast cancer are included. Leukemia-free survival for the whole group and for patients who underwent hematopoietic stem cell transplantation (HSCT) was estimated., Results: Fifteen women (14 with acute leukemia and one with myelodysplasia) were identified. Seven of 15 patients had received an anthracycline, cyclophosphamide and a taxane. Ten patients developed acute leukemia/myelodysplasia with a latency period of 2 years or less from initiation of chemotherapy. Although mixed-lineage leukemia (MLL) rearrangement was the commonest chromosomal abnormality (8 of 15 patients), various other chromosomal abnormalities were also detected. Twelve of 15 patients underwent HSCT (11 allogeneic and one autologous). Eleven of these 12 patients who underwent HSCT were in remission at a median follow-up of 20.4 months (range 4.4-53.3 months)., Conclusion: Durable remissions can be achieved in patients who develop acute leukemia/myelodysplasia secondary to adjuvant chemotherapy for breast cancer and are able to undergo allogeneic HSCT. Our results indicate that HSCT should be an early consideration in the management of such patients who are suitable candidates for the procedure.

In acute graft-versus-host disease (GVHD), naive donor CD4(+) T cells recognize alloantigens on host antigen-presenting cells and differentiate into T helper (Th) subsets (Th1, Th2, and Th17 cells), but the role of Th subsets in GVHD pathogenesis is incompletely characterized. Here we report that, in an MHC-mismatched model of C57BL/6 donor to BALB/c recipient, WT donor CD4(+) T cells predominantly differentiated into Th1 cells and preferentially mediated GVHD tissue damage in gut and liver. However, absence of interferon-gamma (IFN-gamma) in CD4(+) T cells resulted in augmented Th2 and Th17 differentiation and exacerbated tissue damage in lung and skin; absence of both IL-4 and IFN-gamma resulted in augmented Th17 differentiation and preferential, although not exclusive, tissue damage in skin; and absence of both IFN-gamma and IL-17 led to further augmentation of Th2 differentiation and idiopathic pneumonia. The tissue-specific GVHD mediated by Th1, Th2, and Th17 cells was in part associated with their tissue-specific migration mediated by differential expression of chemokine receptors. Furthermore, lack of tissue expression of the IFN-gamma-inducible B7-H1 played a critical role in augmenting the Th2-mediated idiopathic pneumonia. These results indicate donor CD4(+) T cells can reciprocally differentiate into Th1, Th2, and Th17 cells that mediate organ-specific GVHD.

Host dendritic cells (DCs) play a critical role in initiating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL), and separation of GVL from GVHD remains a major challenge in the treatment of hematologic malignancies by allogeneic hematopoietic cell transplantation (HCT). Here, we show that preconditioning with anti-CD3 monoclonal antibody before conditioning with total body irradiation (TBI) prevents GVHD but retains GVL in a HCT model of major histocompatibility complex (MHC)-mismatched C57BL/6 donor to BALB/c host. Prevention of GVHD is associated with inhibition of donor T-cell expression of homing and chemokine receptors, and inhibition of GVHD target tissue expression of chemokines. Furthermore, inhibition of donor T-cell expression of gut homing alpha4beta7 and chemokine receptor (CCR)9 by anti-CD3 preconditioning results from a reduction of CD103(+) DCs in draining mesenteric lymph nodes (LNs), which is associated with down-regulation of DC expression of CCR7, a receptor required for tissue DC migration to draining LNs. These results indicate that anti-CD3 preconditioning reduces not only tissue release of chemokines but also prevents tissue DC migration to draining LNs and subsequently reduces the capacity of DCs of draining LNs to imprint donor T-cell tissue tropism. Therefore, modulation of host DCs by anti-CD3 preconditioning before HCT represents a new approach for separating GVL from GVHD.

Th17 is a newly identified T-cell lineage that secretes proinflammatory cytokine IL-17. Th17 cells have been shown to play a critical role in mediating autoimmune diseases such as EAE, colitis, and arthritis, but their role in the pathogenesis of graft-versus-host disease (GVHD) is still unknown. Here we showed that, in an acute GVHD model of C57BL/6 (H-2(b)) donor to BALB/c (H-2(d)) recipient, IL-17(-/-) donor T cells manifested an augmented Th1 differentiation and IFN-gamma production and induced exacerbated acute GVHD. Severe tissue damage mediated by IL-17(-/-) donor T cells was associated with increased Th1 infiltration, up-regulation of chemokine receptors by donor T cells, and enhanced tissue expression of inflammatory chemokines. Administration of recombinant IL-17 and neutralizing IFN-gamma in the recipients given IL-17(-/-) donor cells ameliorated the acute GVHD. Furthermore, the regulation of Th1 differentiation by IL-17 or Th17 may be through its influence on host DCs. Our results indicate that donor Th17 cells can down-regulate Th1 differentiation and ameliorate acute GVHD in allogeneic recipients, and that treatments neutralizing proinflammatory cytokine IL-17 may augment acute GVHD as well as other inflammatory autoimmune diseases.

CD103 (alphaEbeta7) has been shown to be an excellent marker for identifying in vivo-activated FoxP3(+)CD4(+) regulatory T (Treg) cells. It is unknown whether reinfusion of in vivo-activated donor-type CD103(+) Treg cells from recipient can ameliorate ongoing chronic graft-versus-host disease (GVHD). Here, we showed that, in a chronic GVHD model of DBA/2 (H-2(d)) donor to BALB/c (H-2(d)) recipient, donor-type CD103(+) Treg cells from recipients were much more potent than CD25(hi) natural Treg cells from donors in reversing clinical signs of GVHD and tissue damage. Furthermore, in contrast to CD25(hi) natural Treg cells, CD103(+) Treg cells expressed high levels of CCR5 but low levels of CD62L and directly migrated to GVHD target tissues. In addition, the CD103(+) Treg cells strongly suppressed donor CD4(+) T-cell proliferation; they also induced apoptosis of in vivo-activated CD4(+) T and B cells and significantly reduced pathogenic T and B cells in GVHD target tissues. These results indicate that CD103(+) Treg cells from chronic GVHD recipients are functional, and reinfusion of the CD103(+) Treg cells can shift the balance between Treg cells and pathogenic T cells in chronic GVHD recipients and ameliorate ongoing disease.

We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph(-)) acute lymphoblastic leukemia (ALL). Two model human Ph(-) ALL cell lines (T-cell MOLT-4 and pre-B-cell Reh) were treated with LBH589 and evaluated for biologic and gene expression responses. Low nanomolar concentrations (IC(50): 5-20 nM) of LBH589 induced cell-cycle arrest, apoptosis, and histone (H3K9 and H4K8) hyperacetylation. LBH589 treatment increased mRNA levels of proapoptosis, growth arrest, and DNA damage repair genes including FANCG, FOXO3A, GADD45A, GADD45B, and GADD45G. The most dramatically expressed gene (up to 45-fold induction) observed after treatment with LBH589 is GADD45G. LBH589 treatment was associated with increased histone acetylation at the GADD45G promoter and phosphorylation of histone H2A.X. Furthermore, treatment with LBH589 was active against cultured primary Ph(-) ALL cells, including those from a relapsed patient, inducing loss of cell viability (up to 70%) and induction of GADD45G mRNA expression (up to 35-fold). Thus, LBH589 possesses potent growth inhibitory activity against including Ph(-) ALL cells associated with up-regulation of genes critical for DNA damage response and growth arrest. These findings provide a rationale for exploring the clinical activity of LBH589 in the treatment of patients with Ph(-) ALL.

Purpose: Using data from an eating disorders screening initiative conducted in high schools across the United States, we examined the relationship between vomiting frequency and irregular menses in a nonclinical sample of adolescent females., Methods: A self-report questionnaire was administered to students from U.S. high schools participating in the National Eating Disorders Screening Program in 2000. The questionnaire included items on frequency of vomiting for weight control in the past 3 months, other eating disorder symptoms, frequency of menses, height, and weight. Multivariable regression analyses were conducted using data from 2791 girls to estimate the risk of irregular menses (defined as menses less often than monthly) associated with vomiting frequency, adjusting for other eating disorder symptoms, weight status, age, race/ethnicity, and school clusters., Results: Girls who vomited to control their weight one to three times per month were one and a half times more likely (risk ratio [RR] = 1.6; 95% confidence interval [CI] = 1.2-2.2), and girls who vomited once per week or more often were more than three times more likely (RR = 3.2; 95% CI = 2.3-4.4), to experience irregular menses than were girls who did not report vomiting for weight control. Vomiting for weight control remained a strong predictor of irregular menses even when overweight and underweight participants were excluded., Conclusions: Our study adds to the evidence that vomiting may have a direct effect on hormonal function in adolescent girls, and that vomiting for weight control may be a particularly deleterious component of eating disorders.

We ascertained the prevalence of self-reported late occurrence of diabetes, hypertension, and cardiovascular (CV) disease in 1089 hematopoietic cell transplantation (HCT) survivors who underwent HCT between 1974 and 1998, survived at least 2 years, and were not currently taking immunosuppressant agents and compared them with 383 sibling controls. All subjects completed a 255-item health questionnaire. The mean age at survey completion was 39.3 years for survivors and 38.6 years for siblings; mean follow-up was 8.6 years. Adjusting for age, sex, race, and body mass index (BMI), survivors of allogeneic HCT were 3.65 times (95% confidence interval [CI], 1.82-7.32) more likely to report diabetes than siblings and 2.06 times (95% CI, 1.39-3.04) more likely to report hypertension compared with siblings but did not report other CV outcomes with any greater frequency. Recipients of autologous HCTs were no more likely than siblings to report any of the outcomes studied. Allogeneic HCT survivors were also more likely to develop hypertension (odds ratio [OR]=2.31; 95% CI, 1.45-3.67) than autologous recipients. Total body irradiation (TBI) exposure was associated with an increased risk of diabetes (OR=3.42; 95% CI, 1.55-7.52). Thus, HCT survivors have a higher age- and BMI-adjusted risk of diabetes and hypertension, potentially leading to a higher than expected risk of CV events with age.

The aim of this study was to understand the impact of chronic graft-versus-host disease (cGVHD) on the overall health status of hematopoietic cell transplantation (HCT) survivors. Subjects included 584 individuals who had undergone allogeneic HCT between 1976 and 1999, survived 2 or more years, and completed a 255-item health questionnaire. Global assessment of health status was facilitated by measurement of 6 health status domains: general health, mental health, functional impairment, activity limitation, pain, and anxiety/fear. Information regarding diagnosis of cGVHD was abstracted from medical records, and presence of active cGVHD in the preceding 12 months was self-reported. The incidence of cGVHD in participants was 54%, of whom 46% reported active cGVHD. In multivariable analyses, subjects with active cGVHD were more likely to report adverse general health, mental health, functional impairments, activity limitation, and pain than were those with no history of cGVHD. However, health status did not differ between those with resolved cGVHD and those who never had cGVHD. We conclude that active cGVHD has a significant impact on many aspects of the overall health status of HCT survivors and that, most importantly, those successfully treated for cGVHD do not appear to have long-term impairments.

Chronic graft-vs-host disease (GVHD) is a major cause of morbidity and mortality of long-term survivors of allogeneic hemato-poietic cell transplantation (HCT). Chronic GVHD can have features of an autoimmune collagen vascular disease with clinical manifestations similar to autoimmune scleroderma and systemic lupus erythematosus (SLE). However, the pathogenesis of chronic GVHD is poorly understood. It is unclear how autoreactive T and B cells are generated in chronic GVHD recipients. We have recently developed a new chronic GVHD model by transplantation of donor DBA/2 (H-2d) spleen cells into major histocompatibility complex (MHC)-matched but minor antigen-mismatched sublethally irradiated BALB/c (H-2d) recipients as well as athymic BALB/c(nu/nu) and adult-thymectomized BALB/c recipients. Both euthymic and athymic BALB/c recipients developed high levels of serum IgG autoantibodies, sclerodermatous skin damage, and glomerulonephritis. Disease induction required both donor CD25-CD4+ T and B cells in transplants. In contrast, donor CD25+CD4+ T regulatory (Treg) cells prevented the disease induction. These results indicate that host thymus is not required for induction of chronic GVHD and that quiescent autoreactive T and B cells in transplants from nonautoimmune donors may be activated and expanded to cause chronic GVHD with autoimmune manifestations in allogeneic recipients, and donor Treg cells can suppress this process.

Background: The production of therapeutic T-cell populations for adoptive immunotherapy of cancer requires extensive ex vivo cell processing, including the isolation or creation of Ag-specific T cells and their subsequent propagation to clinically relevant numbers. These procedures must be performed according to the principles of current good manufacturing practices (cGMP) for phase I clinical trials to ensure the identity, purity potency and safety of the cellular product. In this report we describe our approach to manufacturing and characterizing bulk populations of gene-modified autologous T cells for use in treating follicular lymphoma., Methods: PBMC from healthy donors, obtained after informed consent, were stimulated in vitro with Ab to CD3epsilon (OKT3) and recombinant human IL-2 and then electroporated with plasmid DNA containing a human CD19-specific chimeric Ag receptor (CAR) gene and HSV-1 thymidine kinase (TK) gene. Stably transfected cells were selected in cytocidal concentrations of hygromycin B over multiple 14-day stimulation culture cycles and then cryopreserved. Vials of cryopreserved/selected T cells were used to initiate T-cell expansion cultures to produce cell products for clinical infusion. These cultures were characterized for phenotype, function and suitability for use in adoptive immunotherapy studies., Results: Our results demonstrate that bulk populations of gene-modified T cells derived from peripheral blood of healthy donors express CD19+ chimeric Ag receptor at low levels and can specifically lyse CD19+ target cells in vitro. These cells display a differentiated T-effector phenotype, are sensitive to ganciclovir-mediated killing and display a non-transformed phenotype. TCR Vbeta usage indicated that all populations tested were polyclonal. Ex vivo cell expansion from cryopreserved cell banks is sufficient to produce doses of between 5 x 10(9) and 1 x 10(10) cells/run. One of three transductions resulted in a population of cells that was not suitable for infusion but was identified during release testing. No populations displayed any evidence of bacterial, fungal or mycoplasma contamination., Discussion: We have established a manufacturing strategy that is being used to produce T cells for a phase I clinical trial for follicular lymphoma. Genetically modified T cells have been characterized by cell-surface marker phenotype, functional activity against CD19+ targets and requisite safety testing. These pre-clinical data confirm the feasibility of this approach to manufacturing T-cell products.

We conducted a phase 1/2 trial of high-dose 90Y-ibritumomab tiuxetan in combination with high-dose etoposide (VP-16) 40 to 60 mg/kg (day -4) and cyclophosphamide 100 mg/kg (day -2) followed by autologous stem cell transplantation (ASCT) in 31 patients with CD20+ non-Hodgkin lymphoma (NHL). Patients underwent dosimetry (day -21) with 5 mCi (185 MBq) 111In-ibritumomab tiuxetan following 250 mg/m2 rituximab, followed a week later by 90Y-ibritumomab tiuxetan to deliver a target dose of 1000 cGy to highest normal organ. Bone marrow biopsy was done on day -7 to estimate radiation dose and stem cells were reinfused when the radiation dose was estimated to be less than 5 cGy. The median 90Y-ibritumomab tiuxetan dose was 71.6 mCi (2649.2 MBq; range, 36.6-105 mCi; range, 1354.2-3885 MBq). Histology included follicular lymphoma (n = 12), diffuse large B-cell (n = 14), and mantle cell (n = 5). The median number of prior chemo-therapy treatments was 2. The treatment was well tolerated. The median times to reach an absolute neutrophil count greater than 500/microL and platelet count more than 20,000/microL were 10 days and 12 days, respectively. There were 2 deaths and 5 relapses. At a median follow-up of 22 months, the 2-year estimated overall survival and relapse-free survival rates are 92% and 78%, respectively. We conclude that high-dose 90Y-ibritumomab tiuxetan can be combined safely with high-dose etoposide and cyclophosphamide without an increase in transplant-related toxicity or delayed engraftment.

We have engineered an anti-CD20-interleukin 2 (IL-2) immunocytokine (ICK) based on the Leu16 anti-CD20 antibody and have deimmunized both the variable (V) regions as well as the junction between the heavy (H) chain constant region and IL-2. Mutations were made to remove potential T-cell epitopes identified by in silico binding to major histocompatibility complex (MHC) class II molecules. The resulting immunocytokine, DI-Leu16-IL-2, retained full anti-CD20 activity as assessed by fluorescence-activated cell-sorting (FACS) analysis, and had enhanced antibody-dependent cellular cytotoxicity (ADCC) effector function relative to the DI-Leu16 antibody or control anti-CD20 antibody (rituximab). In a severe combined immunodeficient (SCID) mouse model of disseminated, residual lymphoma, anti-CD20-IL-2 immunocytokines based on Leu16 were far more effective at a dose of 0.25 mg/kg than anti-CD20 antibody given at 25/mg/kg, despite a shorter half-life of the ICK. Anti-CD20-IL-2 was also far more effective than a control ICK targeted to an antigen with greatly reduced expression on Daudi tumor cells, or various combinations of anti-CD20 antibodies and IL-2. Antitumor activity of DI-Leu16-IL-2 was shown to partially but not entirely depend on Fc receptor (R) binding, suggesting that ADCC and targeting of IL-2 both play roles in the mechanism of tumor clearance. Based on these animal models, DI-Leu16-IL-2 could offer therapeutic potential for patients with CD20 positive lymphoma. Clinical trials are currently under development.

Prevention of autoimmune diabetes and induction of islet transplantation tolerance in nonobese diabetic (NOD) mice can be reached by induction of mixed chimerism via bone marrow transplantation (BMT), but this procedure requires total body irradiation (TBI) conditioning of the recipients. The toxicity of radiation and potential for graft-versus-host disease (GVHD) prevents its clinical application. Donor CD8+ T cells play a critical role in facilitation of engraftment but also contribute to induction of GVHD in TBI-conditioned recipients. Here, we showed that high doses of donor CD8+ T cells in combination with bone marrow (BM) cells induced mixed chimerism without GVHD in NOD recipients conditioned with anti-CD3 monoclonal antibody (mAb). The prevention of GVHD in those recipients was associated with low-level production of inflammatory cytokines (ie, tumor necrosis factor alpha [TNF-alpha]), high-level production of anti-inflammatory cytokines (ie, interleukin 4 [IL-4] and IL-10), and confining of the donor CD8+ T-cell expansion to lymphohematopoietic tissues. The chimeric NOD recipients showed donor-specific tolerance and reversal of insulitis. These results demonstrate that donor CD8+ T-cell-mediated facilitation of engraftment can be separated from GVHD in nonirradiated recipients. This regimen may have potential application in the treatment of autoimmune disorders as well as induction of transplantation tolerance.

6-O-(4,4,5,5,6,6,7,7,7-Nonafluoro-2-hydroxyheptyl)-, 6-O-(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluoro-2-hydroxynonyl)-, and 6-O-(4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoro-2-hydroxyundecyl)-d-galactopyranose (9, 10, and 11, resp.) were prepared by a two-step synthesis including the reaction of 1,2:3,4-di-O-isopropylidene-alpha-d-galactopyranose with 2-[(perfluoroalkyl)methyl]oxiranes under catalysis with BF(3).Et(2)O. Similarly, 1-O-(4,4,5,5,6,6,7,7,7-nonafluoro-2-hydroxyheptyl)-, 1-O-(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluoro-2-hydroxynonyl)-, 1-O-(4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoro-2-hydroxyundecyl)-dl-xylitol (18, 19, and 20, resp.) were prepared by a two-step synthesis from the corresponding 1,2:3,4-di-O-isopropylidene-dl-xylitol. Most of the both types of fluoroalkylated carbohydrate derivatives 9-11 and 18-20 generally displayed very low level of hemolytic activity and excellent co-emulsifying properties on testing on perfluorodecalin-Pluronic F-68 microemulsions.

Background: Immunotherapy for B-cell lymphomas has evolved significantly with the advent of CD20-targeted Ab-based therapeutics. Strategies to invoke or augment cellular anti-lymphoma immune responses may also have considerable therapeutic potential and serve to further augment the clinical efficacy of MAbs., Methods: We report here the aquisition by priming human cytotoxic T lymphocyte (CTL) effectors of re-directed CD20 specificity by their genetic modification to express a chimeric immunoreceptor consisting of an anti-CD20 single chain Ab extracellular domain molecularly fused to the T-cell receptor complex CD3-zeta cytoplasmic tail (scFvFczeta). Peripheral blood-derived human T-cells were transduced with naked DNA plasmid vector by electoporation then selected for G418 resistance., Results: Following cloning in limiting dilution and ex vivo expansion to large numbers scFvFczeta+ TCRalpha/beta+ CD4- CD8+ CTL display re-directed HLA-unresricted CD20-specific lymphoma cell cytolysis proportional to the cell-surface density of the chimeric immunoreceptor. Engineered CTL clones are also activated through the chimeric immunoreceptor to produce Tc1 cytokines (IFN-gamma) upon co-culture with CD20+ lymphoma stimulator cells. Additionally, CD20-specific CTL proliferate in the presence of lymphoma stimulators and IL-2 (5 U/mL)., Discussion: These studies provide the rationale for exploring the clinical utility of adoptive therapy with CD20-specific CTL as a component of immunotherapeutic targeting of CD20+ malignancy.

Is peripheral stem cell mobilization followed by autologous stem cell transplantation (ASCT) feasible in patients with human immunodeficiency virus (HIV)- associated lymphoma (HIV-L)? Studies have demonstrated that, in the HIV- negative (HIV(-)) setting, ASCT may improve lymphoma-free survival in high-risk non-Hodgkin lymphoma (NHL) or relapsed Hodgkin disease (HD) and NHL. Given the poor prognosis of HIV-L with conventional chemotherapy, this dose-intensive approach was explored. Nine patients with HIV-HD or NHL mobilized a median of 10.6 x 10(6) CD34(+) cells/kg and engrafted after ASCT. CD4 counts recovered to pretransplantation levels and HIV viral loads were controlled in patients compliant with antiretroviral therapy. Seven of 9 patients remain in remission from their lymphoma at a median of 19 months after transplantation. Thus, patients with HIV-L on antiretroviral therapy can engraft following ASCT. Prolonged lymphoma remissions, without significant compromise of immune function, can be seen, suggesting that ASCT can be used in selected patients with HIV-L.

It was recently reported that transplantation of clonally derived murine neurosphere cells into sublethally irradiated allogeneic hosts leads to a donor-derived hematopoietic reconstitution. The confirmation of the existence of a common neurohematopoietic stem cell in the human brain will have a significant effect on stem cell research and on clinical transplantation. Here, it is demonstrated that the human fetal brain contains separate but overlapping epidermal growth factor (EGF)-responsive and basic fibroblast growth factor (FGF-2)-responsive neural stem cells. The majority (> 85%) of cells within these EGF- and/or FGF-2-generated neurospheres express characteristic neural stem/progenitor cell markers including nestin, EGF receptor, and FGF-2 receptor. These neural stem cells can be continuously passaged in vitro, and demonstrate a constant 20-fold expansion in every passage for up to the fifth passage (the longest period that has been carried out in the authors' laboratory). These neural stem cells are multipotential for neurons, astrocytes, and oligodendrocytes. After transplantation into SCID-hu mice, all neural stem cells, regardless of passages, culture conditions, and donors, are able to establish long-term hematopoietic reconstitution in the presence of an intact human bone marrow microenvironment.

The associations of cytogenetics with complete remission (CR) rates, overall survival (OS), and outcomes after CR were studied in 609 previously untreated AML patients younger than 56 years old in a clinical trial comparing 3 intensive postremission therapies: intensive chemotherapy, autologous transplantation (ABMT), or allogeneic bone marrow transplantation (alloBMT) from matched related donors. Patients were categorized into favorable, intermediate, unfavorable, and unknown cytogenetic risk groups based on pretreatment karyotypes. CR rates varied significantly (P <.0001) among the 4 groups: favorable, 84% (95% confidence interval [CI], 77%-90%); intermediate, 76% (CI, 71%-81%); unfavorable, 55% (CI, 48%-63%); and unknown, 54% (CI, 33%-74%). There was similar significant heterogeneity of OS (P <.0001), with the estimated relative risk of death from any cause being 1.50 (CI, 1.10-2.05), 3. 33 (CI, 2.43-4.55), and 2.66 (CI, 1.59-4.45) for the intermediate, unfavorable, and unknown risk groups, respectively, compared with the favorable group. In multivariate analyses, the effects of cytogenetic risk status on CR rate and OS could not be explained by other patient or disease characteristics. Among postremission patients, survival from CR varied significantly among favorable, intermediate, and unfavorable groups (P =.0003), with significant evidence of interaction (P =.017) between the effects of treatment and cytogenetic risk status on survival. Patients with favorable cytogenetics did significantly better following ABMT and alloBMT than with chemotherapy alone, whereas patients with unfavorable cytogenetics did better with alloBMT. Cytogenetic risk status is a significant factor in predicting response of AML patients to therapy; however, to tighten treatment correlates within genetically defined AML subsets, a significantly larger leukemia cytogenetic database is warranted.

The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic stem cells (HSCs) is vital to stem cell research. Establishment of such culture systems will have significant impact on ex vivo manipulation and expansion of transplantable stem cells in clinical applications such as gene therapy, tumor cell purging, and stem cell transplantation. We have recently developed a stromal-based culture system that facilitates ex vivo expansion of transplantable human HSCs. In this stromal-based culture system, 2 major contributors to the ex vivo stem cell expansion are the addition of leukemia inhibitory factor (LIF) and the AC6.21 stromal cells. Because the action of LIF is indirect and mediated by stromal cells, we hypothesized that LIF binds to the LIF receptor on AC6.21 stromal cells, leading to up-regulated production of stem cell expansion promoting factor (SCEPF) and/or down-regulated production of stem cell expansion inhibitory factor (SCEIF). Here we demonstrate a secreted SCEPF activity in the conditioned media of LIF-treated AC6.21 stromal cell cultures (SCM-LIF). The magnitude of ex vivo stem cell expansion depends on the concentration of the secreted SCEPF activity in the SCM-LIF. Furthermore, we have ruled out the contribution of 6 known early-acting cytokines, including interleukin-3, interleukin-6, granulocyte macrophage colony-stimulating factor, stem cell factor, flt3 ligand, and thrombopoietin, to this SCEPF activity. Although further studies are required to characterize this secreted SCEPF activity and to determine whether this secreted SCEPF activity is mediated by a single factor or by multiple growth factors, our results demonstrate that stromal cells are not required for this secreted SCEPF activity to facilitate ex vivo stem cell expansion. (Blood. 2000;95:1957-1966)

We analyzed data on 612 patients who had undergone high-dose chemoradiotherapy (HDT) with autologous stem cell rescue for Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) at the City of Hope National Medical Center, to evaluate the incidence of therapy-related myelodysplasia (t-MDS) or therapy-related acute myeloid leukemia (t-AML) and associated risk factors. A retrospective cohort and a nested case-control study design were used to evaluate the role of pretransplant therapeutic exposures and transplant conditioning regimens. Twenty-two patients developed morphologic evidence of t-MDS/t-AML. The estimated cumulative probability of developing morphologic t-MDS/t-AML was 8.6% +/- 2.1% at 6 years. Multivariate analysis of the entire cohort revealed stem cell priming with VP-16 (RR = 7.7, P = 0.002) to be independently associated with an increased risk of t-MDS/t-AML. The influence of pretransplant therapy on subsequent t-MDS/t-AML risk was determined by a case-control study. Multivariate analysis revealed an association between pretransplant radiation and the risk of t-MDS/t-AML, but failed to reveal any association with pretransplant chemotherapy or conditioning regimens. However, patients who had been primed with VP-16 for stem cell mobilization were at a 12. 3-fold increased risk of developing t-AML with 11q23/21q22 abnormalities (P = 0.006). Patients undergoing HDT with stem cell rescue are at an increased risk of t-MDS/t-AML, especially those receiving priming with VP-16 for peripheral stem cell collection. (Blood. 2000;95:1588-1593)

The reported results (The Post Coronary Artery Bypass Graft Trial Investigators. The effect of aggressive lowering of low-density lipoprotein cholesterol levels and low-dose anticoagulation on obstructive changes in saphenous-vein coronary-artery bypass grafts. New Engl J Med 1997;336:153-162) of the Post Coronary Artery Bypass Graft (Post CABG) trial have shown that aggressive lowering was more effective than moderate lowering of low density lipoprotein (LDL) cholesterol in reducing the progression of atherosclerosis in saphenous-vein grafts (27 vs. 39%; P < 0.001); low dose warfarin had no effect on the progression of atherosclerosis. The present report describes the effect of long-term (an average of 4.3 years) aggressive treatment with high (40-80 mg/day) and moderate treatment with low (2.5-5 mg/day) doses of lovastatin on lipids, apolipoproteins (apo) and apoA- and apoB-containing lipoprotein families. To achieve the target LDL-cholesterol levels (60-85 mg/dl for aggressive group and 134-140 mg/dl for moderate group), cholestyramine (8 g/day) was given to 25% of subjects on aggressive and 5% of subjects on moderate treatment. Although with both treatment strategies there were significant decreases (P<0.001) in the levels of total cholesterol, LDL-cholesterol, apoB, LDL-apoB and cholesterol-rich Lp-B family, percent changes in the levels of these variables were greater in the aggressive- than in the moderate-treatment groups. These treatments had only marginal effects in increasing the levels of high density lipoprotein cholesterol, apoA-I and Lp-A-I and Lp-A-I:A-II families. The long-term aggressive treatment exerted no effect on the concentrations of triglycerides, apoC-IlI, apoC-III in VLDL + LDL and triglyceride-rich Lp-Bc families. Neither treatment affected the levels of Lp(a). The potentially modifying influence of warfarin and apoE phenotypes on lovastatin-induced changes in lipoprotein variables was found to be of little significance. It is likely that the beneficial effect of lovastatin in reducing the progression of atherosclerosis in grafts is mediated through its specific lowering effect on cholesterol-rich Lp-B particles.

We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34(+) thy-1(+) cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34(+) thy-1(+) cells on AC6.21 stroma, a murine bone marrow-derived stromal cell line, caused expansion of cells with CD34(+) thy-1(+) phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34(+) thy-1(+) phenotype. The ex vivo-expanded CD34(+) thy-1(+) cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34(+) thy-1(+) cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34(+) CD38(-) cells, shows a similar pattern of proliferative response. This suggests that ex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34(+) thy-1(+) cells.

Objectives: We sought to examine possible differences in medical status at presentation in 1996, compared to 1991, of adolescents with eating disorders (EDs) at a hospital-based multidisciplinary care program to reflect the increasing market penetration of managed care., Design: Charts were reviewed for all new patients scheduled in a hospital-based outpatient ED program in 1996 and 1991. The 92-item standardized data extraction form included information on demographics, indicators of illness severity at the first visit, and subsequent hospitalization. The need for primary care referral was verified using billing records. Data were analyzed with Student's t-test, Chi-square, Fisher's exact, and Mann-Whitney U tests using SPSS 7.5., Results: Of the 153 total patients, 133 kept their intake appointment and 130 (98%) of these had charts available for review. The age, racial/ethnic characteristics, and average length of disordered eating behaviors were not significantly different over the 5-year period. Referral from a primary care clinician was more commonly required in 1996 than 1991 (59% vs. 11%; p < .0001). Eighteen percent of the patients seen in 1996 were admitted from the initial appointment for medical stabilization, compared to 1.5% in 1991 (p = .002). Comparing 1996 to 1991, a similar number of patients had symptoms consistent with anorexia nervosa, whereas fewer patients in 1996 gave a history of bingeing and purging (22% vs. 40%; p = .027). There were no significant differences in indicators of illness severity, treatment by primary care clinician prior to referral, or hospitalization rates for those patients with and without managed care., Conclusions: Patients in 1996 were more likely to require referrals, were less likely to have symptoms consistent with bulimia nervosa, and were more likely to be admitted for medical stabilization. There were no differences in patient presentation characteristics or initial hospitalization rates based on their managed care status. Further research is needed to investigate the changes in illness severity at presentation and to assess the role that managed care plays in the treatment of patients with eating disorders.

Leukemic blast cells express the CD33 antigen in most patients with acute myeloid leukemia (AML), but this antigen is not expressed by hematopoietic stem cells. We conducted a study to determine whether normal hematopoiesis could be restored in patients with AML by selective ablation of cells expressing the CD33 antigen. In a dose escalation study, 40 patients with relapsed or refractory CD33(+) AML were treated with an immunoconjugate (CMA-676) consisting of humanized anti-CD33 antibody linked to the potent antitumor antibiotic calicheamicin. The capacity of leukemic cells to efflux 3, 3'-diethyloxacarbocyanine iodide (DiOC2) was used to estimate pretreatment functional drug resistance. Leukemia was eliminated from the blood and marrow of 8 (20%) of the 40 patients; blood counts returned to normal in three (8%) patients. A high rate of clinical response was observed in leukemias characterized by low dye efflux in vitro. Infusions of CMA-676 were generally well tolerated, and a postinfusion syndrome of fever and chills was the most common toxic effect. Two patients who were treated at the highest dose level (9 mg/m2) were neutropenic >5 weeks after the last dose of CMA-676. These results show that an immunoconjugate targeted to CD33 can selectively ablate malignant hematopoiesis in some patients with AML.

Lineage-specific growth factors mobilize peripheral blood progenitor cells (PBPC) and accelerate hematopoietic recovery after high-dose chemotherapy. Recombinant human thrombopoietin (rhTPO) may further increase the progenitor-cell content and regenerating potential of PBPC products. We evaluated the safety and activity of rhTPO as a PBPC mobilizer in combination with granulocyte colony-stimulating factor (G-CSF) in 29 breast cancer patients treated with high-dose chemotherapy followed by PBPC reinfusion. Initially, patients received escalating single doses of rhTPO intravenously (IV) at 0.6, 1.2, or 2.4 micrograms/kg, on day 1. Subsequent patients received rhTPO 0.6 or 0.3 micrograms/kg on days -3, -1, and 1, or 0.6 micrograms/kg on days -1 and 1. G-CSF, 5 micrograms/kg IV or subcutaneously (SC) twice daily, was started on day 3 and continued through aphereses. Twenty comparable, concurrently and identically treated patients (who were eligible and would have been treated on protocol but for the lack of study opening) mobilized with G-CSF alone served as comparisons. CD34(+) cell yields were substantially higher with the first apheresis following rhTPO and G-CSF versus G-CSF alone: 4.1 x 10(6)/kg (range, 1.3 to 17.6) versus 0.8 x 10(6)/ kg (range, 0.3 to 4.2), P =.0003. The targeted minimum yield of 3 x 10(6) CD34(+) cells/kg was procured following a single apheresis procedure in 61% of the rhTPO and G-CSF-mobilized group versus 10% of G-CSF-mobilized patients (P =.001). In rhTPO and G-CSF mobilized patients, granulocyte (day 8 v 9, P =.0001) and platelet recovery (day 9 v 10, P =.07) were accelerated, and fewer erythrocyte (3 v 4, P =.02) and platelet (4 v 5, P =.02) transfusions were needed compared with G-CSF-mobilized patients. Peripheral blood platelet counts, following rhTPO and G-CSF, were increased by greater than 100% and the platelet content of PBPC products by 60% to 110% on the first and second days of aphereses (P <.0001) with the greatest effect seen with repeated dosing of rhTPO at 0.6 microgram/kg. rhTPO is safe and well tolerated as a mobilizing agent before PBPC collection. Mobilization with rhTPO and G-CSF, in comparison to a comparable, nonrandomized G-CSF-mobilized group of patients, decreases the number of apheresis procedures required, may accelerate hematopoietic recovery, and may reduce the number of transfusions required following high-dose chemotherapy for breast cancer.

We evaluated the capacity of adeno-associated virus (AAV) vectors to transduce primitive human myeloid progenitor cells derived from marrow and cord blood in long-term cultures and long-term culture-initiating cell (LTC-IC) assays. Single-colony analyses showed that AAV vectors transduced CD34(+) and CD34(+)38(-) clonogenic cells in long-term culture. Gene transfer was readily observed in LTC-ICs derived from 5-, 8-, and 10-week cultures. Recombinant AAV (rAAV) transduction was observed in every donor analyzed, although a wide range of gene transfer frequencies (5% to 100%) was noted. AAV transduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing comparable or better transduction relative to week-5 LTC-ICs. Fluorescence in situ hybridization (FISH) analyses performed to determine the fate of AAV vectors in transduced cells showed that 9% to 28% of CD34(+) and CD34(+)38(-) cells showed stable vector integration as evidenced by chromosome-associated signals in metaphase spreads. Comparisons of interphase and metaphase FISH suggested that a fraction of cells also contained episomal vector at early time points after transduction. Despite the apparent loss of the episomal forms with continued culture, the number of metaphases containing integrated vector genomes remained stable long term. Transgene transcription and placental alkaline phosphatase (PLAP) expression was observed in CD34(+), CD34(+)38(-) LTC-ICs in the absence of selective pressure. These results suggest that primitive myeloid progenitors are amenable to genetic modification with AAV vectors.

(1) We review evidence that anesthetics inhibit peripheral nAChR cation translocation by binding directly to a protein site in the transmembrane pore. (2) This site is near the middle of the pore-forming M2 domains on alpha and beta subunits, but further from the homologous portions of gamma and delta subunits. (3) Interactions between both anesthetics and nonanesthetics with the nAChR pore site are determined primarily by hydrophobic forces rather than steric factors. (4) Anesthetics and nonanesthetics display different state-dependent accessibility to this site, suggesting a mechanism for the different in vivo actions of these two classes of drugs.

We have conducted a pilot study to investigate the role of high-dose therapy and autologous bone marrow/stem cell transplantation (ASCT) during first complete or partial remission in 52 patients with poor-risk aggressive lymphoma. There were 42 patients with intermediate-grade or immunoblastic lymphoma who were considered to be high (60%) and high-intermediate risk (40%) groups at diagnosis based on the age-adjusted International Prognostic Index (IPI) and 10 patients with high-grade, SNCCL (small non-cleaved cell, Burkitt's, and non-Burkitt's), who at presentation had poor-risk features defined as elevated serum lactate dehydrogenase level, stage IV, and bulky mass >/=10 cm. The median age was 34 years (range, 16 to 56 years). Thirty-nine were transplanted in first complete remission and 13 in first partial remission after conventional therapy. Conditioning regimens consisted of total body irradiation (TBI) administered as a single fraction 750 cGy in 3 patients and in fractionated doses for a total of 1,200 cGy in 44 patients, in combination with 60 mg/kg etoposide and 100 mg/kg cyclophosphamide. Five patients with prior radiotherapy received 450 mg/m2 carmustine instead of TBI. Stem cell sources were either bone marrow and/or peripheral blood. No in vitro purging was used. All patients engrafted. Two SNCCL patients died of venoocclusive disease at 25 days and acute leukemia at 27 months posttransplantation. There were six relapses at 1.5 to 12.8 months posttransplantation. At a median follow-up of 44 months (range, 1 to 113 months), the estimated 3-year overall survival (OS) and disease-free survival (DFS) for all patients was 84% (95% confidence interval [CI], 70% to 92%) and 82% (95% CI, 68% to 91%), respectively. In the subset of patients with intermediate-grade and immunoblastic lymphoma, the 3-year DFS was 89% (95% CI, 74% to 96%) for all patients, 87% (95% CI, 67% to 96%) for high-risk patients, and 92% (95 CI, 61% to 99%) for high-intermediate risk patients. The 3-year OS and DFS for SNCCL patients were identical at 60% (95% CI, 30% to 84%). These results suggest that high-dose therapy and ASCT during first remission may improve the survival and prognosis of patients with poor-risk intermediate- and high-grade lymphoma. A prospective randomized study comparing high-dose therapy and ASCT with conventional chemotherapy in IPI high-risk patients with aggressive non-Hodgkin's lymphoma should be undertaken.

The development of a protective cellular immune response against human cytomegalovirus (HCMV) is the most important determinant of recovery from HCMV infection after allogeneic bone marrow transplantation (BMT). The ultimate aim of our study is to develop an antigen-specific and peptide-based vaccine strategy against HCMV in the setting of BMT. Toward this end we have studied the cellular immune response against the immunodominant matrix protein pp65 of HCMV. Using an HLA A*0201-restricted T-cell clone reactive against pp65 from peripheral blood from a seropositive individual, we have mapped the position of the cytolytic T lymphocyte (CTL) epitope from HCMV pp65 to an 84-amino acid segment. Of the four peptides which best fit the HLA A*0201 motif in that region, one nonamer sensitized an autologous Epstein-Barr virus immortalized lymphocyte cell line for lysis. In vitro immunization of PBMC from HLA A*0201 and HCMV seropositive volunteers using the defined nonamer peptide stimulated significant recognition of HCMV infected or peptide-sensitized fibroblasts. Similarly, HLA A*0201 transgenic mice immunized with the nonamer peptide developed CTL that recognize both the immunizing peptide and endogenously processed pp65 in an HLA A*0201 restricted manner. Lipid modification of the amino terminus of the nonamer peptide resulted in its ability to stimulate immune responses without the use of adjuvant. This demonstration of a vaccine function of the nonamer peptide without adjuvant suggests its potential for use in an immunization trial of BMT donors to induce protective CTLs in patients undergoing allogeneic BMT.