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PDF file - 1065KB, Supplemental Data for Figure 4.

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Purpose: Previously, we showed that adoptive transfer of in vivo vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at day +12 after transplant increased CD4 and CD8 T-cell counts at day +42 and augmented vaccine-specific immune responses in patients with myeloma. Here, we investigated the safety and kinetics of T-cell recovery after infusing ex-T at day +2 after transplant.Experimental Design: In this phase I/II two-arm clinical trial, 50 patients with myeloma received autografts after high-dose melphalan followed by infusions of ex-T at day +2 after transplant. Patients also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine only (arm B; n = 24) or the pneumococcal conjugate vaccine plus an HLA-A2–restricted multipeptide vaccine for HLA-A2+ patients (arm A; n = 26).Results: The mean number of T cells infused was 4.26 × 1010 (range, 1.59-5.0). At day 14 after transplant, the median CD3, CD4, and CD8 counts were 4,198, 1,545, and 2,858 cells/μL, respectively. Interleukin (IL)-6 and IL-15 levels increased early after transplant and IL-15 levels correlated significantly to day 14 T-cell counts. Robust vaccine-specific B- and T-cell responses were generated. T-cell infusions were well tolerated with no effect on hematopoietic recovery. Eight patients (16%) developed a T-cell “engraftment syndrome” characterized by diarrhea and fever that was clinically and histopathologically indistinguishable from grade 1 to 3 acute graft-versus-host disease (GVHD) of the gastrointestinal tract (seven patients) and/or grade 1 to 2 cutaneous GVHD (four patients).Conclusions: Adoptive T-cell transfers achieve robust T-cell recovery early after transplant and induce moderate-to-severe autologous GVHD in a subset of patients.

PDF file - 54KB, Adverse Events Table.

PDF file - 198KB, MAGE-A3 Trojan Peptide Vaccine Reactogenicity.

PDF file - 40KB, ELISA Antibody Responses for the PCV (Prevnar-13(registered trademark)).

Supplementary Data from Rapid Immune Recovery and Graft-versus-Host Disease–like Engraftment Syndrome following Adoptive Transfer of Costimulated Autologous T Cells

Purpose: Myeloma-directed cellular immune responses after autologous stem cell transplantation (ASCT) may reduce relapse rates. We studied whether coinjecting the TLR-3 agonist and vaccine adjuvant Poly-ICLC with a MAGE-A3 peptide vaccine was safe and would elicit a high frequency of vaccine-directed immune responses when combined with vaccine-primed and costimulated autologous T cells.Experimental Design: In a phase II clinical trial (NCT01245673), we evaluated the safety and activity of ex vivo expanded autologous T cells primed in vivo using a MAGE-A3 multipeptide vaccine (compound GL-0817) combined with Poly-ICLC (Hiltonol), granulocyte macrophage colony-stimulating factor (GM-CSF) ± montanide. Twenty-seven patients with active and/or high-risk myeloma received autografts followed by anti-CD3/anti-CD28–costimulated autologous T cells, accompanied by MAGE-A3 peptide immunizations before T-cell collection and five times after ASCT. Immune responses to the vaccine were evaluated by cytokine production (all patients), dextramer binding to CD8+ T cells, and ELISA performed serially after transplant.Results: T-cell infusions were well tolerated, whereas vaccine injection site reactions occurred in >90% of patients. Two of nine patients who received montanide developed sterile abscesses; however, this did not occur in the 18 patients who did not receive montanide. Dextramer staining demonstrated MAGE-A3–specific CD8 T cells in 7 of 8 evaluable HLA-A2+ patients (88%), whereas vaccine-specific cytokine-producing T cells were generated in 19 of 25 patients (76%). Antibody responses developed in 7 of 9 patients (78%) who received montanide and only weakly in 2 of 18 patients (11%) who did not. The 2-year overall survival was 74% [95% confidence interval (CI), 54%–100%] and 2-year event-free survival was 56% (95% CI, 37%–85%).Conclusions: A high frequency of vaccine-specific T-cell responses were generated after transplant by combining costimulated autologous T cells with a Poly-ICLC/GM-CSF–primed MAGE-A3 vaccine. Clin Cancer Res; 20(5); 1355–65. ©2013 AACR.

Background Racial disparity in the incidence of multiple myeloma is well established; however, to the authors' knowledge, little is known regarding the impact of racial differences on disease characteristics, response to therapy, and clinical outcome. Methods The authors studied 453 patients (174 of whom were black and 279 of whom were white) who underwent transplant between 2000 and 2013. The median follow-up was 4.4 years. Results Black patients were significantly younger than white patients (median age, 54 years vs 59 years; P

We retrospectively evaluated the tolerability and efficacy of fractionated total body irradiation (TBI) (1,200 cGy) and melphalan (MEL) (100–110 mg/m2) myeloablative conditioning in 48 patients with nonremission AML (n = 14), ALL (n = 10), NHL (n = 18), and other refractory hematologic malignancies (n = 6) who received allogeneic stem cell transplantation (SCT) between 2002 and 2011. Median age was 48 years (22 to 68); 14 out of 26 leukemia patients (54 %) had circulating blasts at transplant, 20 (50 %) evaluable patients had poor-risk cytogenetics, 12 (25 %) had prior SCT, and 10 (21 %) received stem cells from a mismatch donor. All patients received tacrolimus with or without methotrexate for GVHD prophylaxis. At the time of analysis, 13 patients (27 %) were alive and disease free. Engraftment was complete in all patients. The median time to ANC recovery (>500) was 12 days (range, 6–28). The most common grade III and IV toxicities were mucositis and infections. Eighteen patients (43 %) developed grade II–IV acute GVHD, and eight (26 %) had extensive chronic GVHD. Of 44 evaluable patients for response, 28 (64 %) achieved a complete remission (CR), and seven (15 %) had a partial remission after the transplant. With a median follow-up of 30 months (4 to 124 months) for surviving patients, the cumulative incidence of relapse was 45 % at 1 year, and the probability of overall survival (OS) at 5 years was 22.5 %. Multivariate analysis showed that platelet count (500 IU/L) at SCT were associated with relapse. Age less than 53 years and CR after SCT were associated with better OS. Our data suggest that TBI-MEL can result in CR in two thirds, durable remission in one third, and 5-year survival in about one quarter of patients with nonremission hematologic malignancies. Further studies with TBI-MEL in standard risk transplant patients are warranted.

Major clinical response to nivolumab in relapsed/refractory Hodgkin lymphoma after allogeneic stem cell transplantation

Splenectomy as a measure to treat prolonged post-transplant cytopenia associated with hypersplenism

The primary cause of death (COD) provides important information in many studies of hematopoietic stem cell transplantation (HSCT). A panel of experts critically assessed the CODs submitted by 15 transplantation centers for 281 patients who died in a randomized multicenter trial of unrelated HSCT. The panel reviewed the CODs reported by the transplantation centers, which used the Center for International Blood and Marrow Transplant Research and National Marrow Donor Program COD reporting form. The panel determined that the existing criteria for primary and contributing CODs lacked sufficient stringency for uniform interpretation. A hierarchy was developed and applied to the T cell depletion project. Using its scheme, the panel reclassified 157 CODs (56%) reported by the transplantation centers. The changes resulted in increased recognition of graft-versus-host disease as the primary COD and a concomitant decrease in attribution of the primary COD to infection. This algorithm promotes consistent assignment of primary and contributing CODs for patients with leukemia or lymphoma who expire after myeloablative allogeneic HSCT.

Serious infections are a major obstacle limiting the usefulness of unrelated donor marrow transplantation. Graft-versus-host disease (GVHD) and its therapy are associated with a high risk of opportunistic infection. In this study, patients were randomized to receive 1 of 2 GVHD prophylaxis strategies, marrow T cell depletion, and cyclosporine (TCD) or methotrexate/cyclosporine (M/C) after transplantation. The patients underwent transplantation between March 1995 and October 2000 as part of a multicenter randomized trial. As a secondary analysis, we analyzed infections in this study cohort. Among the 404 patients who underwent transplantation, a total of 1598 infections were reported. The rates of serious and fatal infections did not differ between the TCD and M/C groups. Bacterial infections accounted for 1/3 of serious infections in each treatment arm. A significantly higher incidence of severe cytomegalovirus (CMV) and life-threatening or fatal aspergillus infections was observed in the patients receiving TCD (CMV, 28% vs 17% [P = .02]; aspergillosis, 16% vs 7% [P < .01]). The only independent risk factor for serious infection was the development of grade III-IV acute GVHD (aGVHD; hazard ratio = 1.41; 95% confidence interval = 1.03-1.91). Strategies to speed immune recovery, even in the absence of GVHD, are needed to overcome the risk of infection after unrelated donor transplantation.

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Herein we report results of a phase I/II trial to evaluate the safety and activity of autologous T-cells engineered to express an affinity-enhanced T-cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4×109 engineered T cells two days after autologous stem cell transplant (ASCT). Infusions were well-tolerated without clinically apparent cytokine release syndrome, despite high IL-6 levels. Engineered T-cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, consistent with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression free survival of 19.1 months. NY-ESO-1/LAGE-1 TCR-engineered T-cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.

Donor-derived T cells have been proposed to play a role in pathogenesis of chronic graft-versus-host disease (cGVHD). The impact of ex vivo T-cell depletion (TCD) on cGVHD was analyzed in a randomized multicenter trial involving unrelated donor marrow transplants. A total of 404 patients diagnosed with hematologic malignancies received a total body irradiation-based myeloablative conditioning regimen. GVHD prophylaxis included TCD plus cyclosporine (CSA) or unmodified grafts with CSA plus methotrexate (M/C). Median recipient age was 31.2 years (range, 0.5-55.6 years); median follow-up time since randomization was 4.2 years. The mean number of T cells infused was 1 log lower on the TCD arm. The incidence of cGVHD at 2 years was similar between the TCD and M/C arms, 29% versus 34% (P = .27), respectively. Survival at 3 years from diagnosis of cGVHD was also similar, (TCD 51% versus M/C 58%; P = .29). The proportion of patients with cGVHD who discontinued immunosuppression at 5 years was not different (TCD 72% versus M/C 63%; P = .27), and incidence of serious infections and leukemia relapse were similar on both treatment arms. In spite of a significant reduction of acute GVHD, TCD did not reduce the incidence of cGVHD or improve survival in patients who developed cGVHD.

The objective of this study was to investigate the safety, tolerability, and pharmacokinetics of oral voriconazole in subjects at high risk of developingfungal infections. This was a multicenter, randomized, double-blind, double-dummy, parallel-group, dose escalation study with a fluconazole active control. Twenty-four subjects with hematological malignancies, solid tumors, or autologous bone marrow transplants were randomized to receive voriconazole 200 mg q 12 h (n = 9), voriconazole 300 mg q 12 h (n = 9), or fluconazole 400 mg OD (n = 6)for a period of 14 days. Blood samples were taken for the assessment of voriconazole pharmacokinetics in plasma on Days 1 and 14. Using a 200 mg q 12 h dosing regimen, geometric mean voriconazole peak plasma concentrations (Cmax) were 904 ng/ml on Day 1 and 2996 ng/ml on Day 14. Geometric mean voriconazole exposure, as measured by the area under the curve within a dosing interval (AUCtau), was 4044 and 20308 ng x h/ml on Days 1 and 14, respectively. On Day 1, geometric mean Cmax and AUC were 1.80- and 1.94-fold higher in subjects receiving voriconazole 300 mg q 12 h than in those receiving 200 mg q 12 h. Similarly, on Day 14, geometric mean Cmax and AUC were 1.56- and 1.80-fold greater in the high-dose group. Although the confidence intervals are large, this trend suggests nonlinearity in pharmacokinetics with respect to dose as seen in healthy volunteers. The absorption of orally administered voriconazole was relatively rapid, with t(max) achieved in 1.7 to 3.0 hours. There was a mean 5.4- and 5.0-fold accumulation of voriconazole over the 14-day study period in the 200 mg and 300 mg q 12 h dose groups, respectively. Voriconazole was generally safe and well tolerated. Mild, reversible visual disturbances were the most commonly reported adverse event but were not associated with treatment discontinuation. No patient developed a breakthrough fungal infection. It was concluded that in this group of patients at risk of fungal infection, voriconazole pharmacokinetics was consistent with that reported in healthy volunteers.

Racial disparity in the incidence of multiple myeloma is well established; however, to the authors' knowledge, little is known regarding the impact of racial differences on disease characteristics, response to therapy, and clinical outcome.The authors studied 453 patients (174 of whom were black and 279 of whom were white) who underwent transplant between 2000 and 2013. The median follow-up was 4.4 years.Black patients were significantly younger than white patients (median age, 54 years vs 59 years; P.0001), more frequently presented with anemia (P = .04), had more of the immunoglobulin G isotype (P.001), and had a borderline favorable cytogenetic risk (P = .06). Overall response to induction was similar, but deeper responses were observed in more white patients compared with black patients receiving immunomodulatory drug-based induction (P = .02). Referral for transplant was significantly delayed in black individuals (median, 1.3 years vs 0.9 years; P = .003). Overall survival from the time of transplant was similar for black and white patients, with medians of 6.2 years and 5.7 years, respectively, but survival from the time of diagnosis was significantly longer among black individuals (median, 7.7 years vs 6.1 years; P = .03). Maintenance therapy was found to positively impact progression-free survival but not overall survival, irrespective of race.The results of the current study confirm ethnic differences in age, referral patterns, response to therapy, and overall survival. Future validation of these disparities is urgently needed.

Purpose: Myeloma-directed cellular immune responses after autologous stem cell transplantation (ASCT) may reduce relapse rates. We studied whether coinjecting the TLR-3 agonist and vaccine adjuvant Poly-ICLC with a MAGE-A3 peptide vaccine was safe and would elicit a high frequency of vaccine-directed immune responses when combined with vaccine-primed and costimulated autologous T cells. Experimental Design: In a phase II clinical trial (NCT01245673), we evaluated the safety and activity of ex vivo expanded autologous T cells primed in vivo using a MAGE-A3 multipeptide vaccine (compound GL-0817) combined with Poly-ICLC (Hiltonol), granulocyte macrophage colony-stimulating factor (GM-CSF) ± montanide. Twenty-seven patients with active and/or high-risk myeloma received autografts followed by anti-CD3/anti-CD28–costimulated autologous T cells, accompanied by MAGE-A3 peptide immunizations before T-cell collection and five times after ASCT. Immune responses to the vaccine were evaluated by cytokine production (all patients), dextramer binding to CD8+ T cells, and ELISA performed serially after transplant. Results: T-cell infusions were well tolerated, whereas vaccine injection site reactions occurred in >90% of patients. Two of nine patients who received montanide developed sterile abscesses; however, this did not occur in the 18 patients who did not receive montanide. Dextramer staining demonstrated MAGE-A3–specific CD8 T cells in 7 of 8 evaluable HLA-A2+ patients (88%), whereas vaccine-specific cytokine-producing T cells were generated in 19 of 25 patients (76%). Antibody responses developed in 7 of 9 patients (78%) who received montanide and only weakly in 2 of 18 patients (11%) who did not. The 2-year overall survival was 74% [95% confidence interval (CI), 54%–100%] and 2-year event-free survival was 56% (95% CI, 37%–85%). Conclusions: A high frequency of vaccine-specific T-cell responses were generated after transplant by combining costimulated autologous T cells with a Poly-ICLC/GM-CSF–primed MAGE-A3 vaccine. Clin Cancer Res; 20(5); 1355–65. ©2013 AACR.

Background: P is used in combination with G-CSF to improve the mobilization of peripheral blood hematopoietic stem cells in poor mobilizers. Limited data are available in regard to effects of P on post-transplant outcomes in comparison with non-P chemomobilized patients. Methods: In this retrospective study, we compare the engraftment and the outcomes of 34 patients mobilized with P + G-CSF or just-in-time rescue P in combination with chemotherapy and G-CSF to 143 patients (control group) mobilized with G-CSF with or without chemotherapy in lymphoma and myeloma patients who underwent ASCT between February 2012 and April 2014 at the University of Maryland Greenebaum Cancer Center. Post-transplantation outcomes including infections, hematologic recovery, relapse, progression and survival were recorded. Results: The median number of collected of CD34+ cells/Kg was 5.9 x 10(6) in the P group and 12.3 x 10(6) in the control group (p=0.0002). Median time to neutrophil engraftment (>0.5 × 10(9) /L) was comparable between the 2 groups: 12 days for the P group and 11 for the control group (p=0.28). There was a trend toward a shorter time to platelet engraftment (>20 × 10(9) /L) in the control (12 days) compared to the P group (14 days) (p=0.056). Progression-free survival at 1 year after (ASCT) was 88.2% in the P group and 81.8% in the control group. There was no difference in the overall survival of both groups (p=0.62) Conclusions: Short and long-term engraftment and outcomes after ASCT seem to be comparable in lymphoma and myeloma patients receiving plerixafor compared to chemomobilized patients without plerixafor. This observation support the use of plerixafor + G-CSF or just-in-time rescue P in patients who mobilize poorly without P. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

In a phase 1/2 two-arm trial, 54 patients with myeloma received autografts followed by ex vivo anti-CD3/anti-CD28 costimulated autologous T cells at day 2 after transplantation. Study patients positive for human leukocyte antigen A2 (arm A, n = 28) also received pneumococcal conjugate vaccine immunizations before and after transplantation and a multipeptide tumor antigen vaccine derived from the human telomerase reverse transcriptase and the antiapoptotic protein survivin. Patients negative for human leukocyte antigen A2 (arm B, n = 26) received the pneumococcal conjugate vaccine only. Patients exhibited robust T-cell recoveries by day 14 with supraphysiologic T-cell counts accompanied by a sustained reduction in regulatory T cells. The median event-free survival (EFS) for all patients is 20 months (95% confidence interval, 14.6-24.7 months); the projected 3-year overall survival is 83%. A subset of patients in arm A (36%) developed immune responses to the tumor antigen vaccine by tetramer assays, but this cohort did not exhibit better EFS. Higher posttransplantation CD4+ T-cell counts and a lower percentage of FOXP3+ T cells were associated with improved EFS. Patients exhibited accelerated polyclonal immunoglobulin recovery compared with patients without T-cell transfers. Adoptive transfer of tumor antigen vaccine-primed and costimulated T cells leads to augmented and accelerated cellular and humoral immune reconstitution, including antitumor immunity, after autologous stem cell transplantation for myeloma. This study was registered at www.clinicaltrials.gov as NCT00499577.

A carcinogen-transformed rat hepatoma cell line (Reuber H-35) was utilized as a model system for investigation of the biochemical factors which may limit the effectiveness of chemotherapy in intrinsically resistant tumors such as hepatocellular carcinoma. Northern blotting demonstrated expression of mRNA coding for the P-170 membrane-glycoprotein associated with the multi-drug resistance phenotype, while Western blotting identified the P-170 glycoprotein in the hepatoma cell membrane. Consistent with these observations, tumor cell sensitivity to the vinca alkaloids, vincristine and vinblastine, to the anthracycline antibiotics, Adriamycin ® and daunorubicin, and to the demethyl-epipodophyllotoxin derivative, VM-26, was enhanced by continuous incubation in the presence of the calcium channel antagonist, verapamil. Verapamil produced a minimal change in cell sensitivity to the demethylepipodophyllotoxin derivative, VP-16, and to the aminoacridine, m -AMSA. Relatively high detoxification potential via the glutathione metabolic pathway was also observed in the hepatoma cell. The capacity of topoisomerase II in nuclear extracts from the hepatoma cell to mediate cleavable complex formation stimulated by VM-26, VP-16 and m -AMSA appeared to be at least comparable to, if not greater than that from drug-sensitive HL-60 cells, suggesting that drug resistance may not occur at the level of this enzyme. Consistent with findings in a number of tumor cell lines resistant to antineoplastic drugs, the antiproliferative activity of the topoisomerase II inhibitors VM-26, VP-16 and m -AMSA appeared to be dissociable from the induction of DNA strand breaks, suggesting that such lesions in DNA may fail to fully account for the antiproliferative activity of these agents in the hepatoma cell.

Purpose: Previously, we showed that adoptive transfer of in vivo vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at day +12 after transplant increased CD4 and CD8 T-cell counts at day +42 and augmented vaccine-specific immune responses in patients with myeloma. Here, we investigated the safety and kinetics of T-cell recovery after infusing ex-T at day +2 after transplant.Experimental Design: In this phase I/II two-arm clinical trial, 50 patients with myeloma received autografts after high-dose melphalan followed by infusions of ex-T at day +2 after transplant. Patients also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine only (arm B; n = 24) or the pneumococcal conjugate vaccine plus an HLA-A2–restricted multipeptide vaccine for HLA-A2+ patients (arm A; n = 26).Results: The mean number of T cells infused was 4.26 × 1010 (range, 1.59-5.0). At day 14 after transplant, the median CD3, CD4, and CD8 counts were 4,198, 1,545, and 2,858 cells/μL, respectively. Interleukin (IL)-6 and IL-15 levels increased early after transplant and IL-15 levels correlated significantly to day 14 T-cell counts. Robust vaccine-specific B- and T-cell responses were generated. T-cell infusions were well tolerated with no effect on hematopoietic recovery. Eight patients (16%) developed a T-cell “engraftment syndrome” characterized by diarrhea and fever that was clinically and histopathologically indistinguishable from grade 1 to 3 acute graft-versus-host disease (GVHD) of the gastrointestinal tract (seven patients) and/or grade 1 to 2 cutaneous GVHD (four patients).Conclusions: Adoptive T-cell transfers achieve robust T-cell recovery early after transplant and induce moderate-to-severe autologous GVHD in a subset of patients.

This report illustrates the use of cardiac magnetic resonance imaging (MRI) to quantify the initial extent of a cardiac rhabdomyosarcoma and, more importantly, its response to chemotherapy. Image slices spanning the heart and adjacent structures were analyzed using Simpson's rule applied to the image slices to estimate the tumor volume initially, then after 5 weeks, and again after 4 months of chemotherapy. A substantial, progressive reduction in tumor volume during chemotherapy was shown. After chemotherapy was discontinued, an increase in tumor volume was shown. It is suggested that, in addition to being useful in patient care, the technique may be useful in clinical investigations by providing an objective, quantitative measure of tumor response to therapy.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Adoptive immunotherapy for cancer has been limited by a lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We hypothesized that infusion of genetically modified tumor-specific T cells following autologous stem cell transplant (ASCT) may overcome these barriers for multiple myeloma (MM). To test this, we conducted a phase I/II clinical trial ([NCT01352286][1]) in which T cells engineered with an HLA-A*0201 restricted, affinity-enhanced TCR recognizing NY-ESO-1 / LAGE-1 peptides (NY-ESOc259-T), were infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy given with ASCT. HLA-A*0201 MM patients eligible for ASCT, with antigen positive tumor were enrolled. NY-ESOc259-T was manufactured in a 10 day process using anti-CD3/CD28 microbeads and lentiviral vector, and was administered two days following ASCT. IMWG criteria were used to assess response at day 100 with the addition of a near complete response category (nCR) due to the common occurrence of oligoclonal banding observed following rapid post-ASCT immune reconstitution. Blood and marrow samples were taken at multiple timepoints for serum cytokine analysis, NY-ESOc259-T persistence and trafficking, multiparameter flow analysis to examine the phenotype and function of NY-ESOc259-T, and tumor biomarker analysis. 25 of 29 enrolled patients were infused. A mean of 2.8 × 109 engineered cells were administered (range 8.3 × 108-4.2 × 109), and the average transduction efficiency was 33% (range 30%-45%). Patients tended to have advanced disease (64% chromosomal abnormalities, and 24% prior ASCT). At 3 months, 67% (16/24) and 58% (14/24) of patients were in VGPR and nCR or better, respectively. Infusions were well-tolerated and no cytokine release syndrome was reported. NY-ESOc259-T persisted at 6 months in all but one patient, and in a subset of patients at 2 years; marrow infiltration was consistently observed from day 7 through day 180. NY-ESOc259-T initially displayed a dominant activated effector phenotype which converted towards a dominant effector memory phenotype by 1 year post infusion, in a pattern that mirrored clinical responses. Persisting cells demonstrated a polyfunctional response (IFN-γ and TNF-α) with a cytotoxic (CD107a and granzyme B) signature without overexpression of exhaustion markers (PD-1, LAG-3, and TIM-3). Tumor biomarker analysis is ongoing. MM relapse occurred in 13/25 patients. This data show that NY-ESOc259-T cells exhibit robust trafficking and expansion, durable persistence without exhaustion, and follow a natural immune expansion and contraction pattern consistent with an antigen-driven mechanism of action. Relapse correlated with a loss of persistence or tumor antigen escape, suggesting that targeting multiple antigens and maintenance infusions may increase durable remissions. Citation Format: Aaron Rapoport, Edward Stadtmauer, Luca Melchiori, Ryan Wong, Eduardo Davila, Gwendolyn Binder-Scholl, Tom Holdich, Dan Vogl, Brendan Weiss, Jeffrey Finkelstein, Simon Lacey, Sarah Bond, Marylene Fortin, Yoav Peretz, Joanna Brewer, Alan Bennett, Andrew Gerry, Nick Pumphrey, Helen Tayton-Martin, Lilliam Ribeiro, Ashraf Badros, Saul Yanovich, Nancy Hardy, Jean Yared, Naseem Kerr, Sunita Philip, Sandra Wesphal, Bruce L. Levine, Carl June, Michael Kalos, Bent Jakobsen. NY-ESO T cells administered post ASCT for MM exhibit extended functionality without exhaustion in a natural pattern of effector and memory programming. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2015-4701 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01352286&atom=%2Fcanres%2F75%2F15_Supplement%2F4701.atom

The primary objective of this study was to compare health-related quality of life (HRQL) in adult patients undergoing either ex vivo T cell–depleted bone marrow transplantation or conventional marrow transplantation. Data on patients' HRQL were gathered as part of a multicenter randomized trial comparing the effect of ex vivo T-cell depletion versus methotrexate and cyclosporine immunosuppression on disease-free survival. HRQL assessments were conducted at baseline, day +100, 6 months, 1 year, and 3 years. There were no treatment arm differences 1 year after transplantation on the Functional Assessment of Cancer Therapy, Bone Marrow Transplantation, the Medical Outcomes Study Short-Form 36, and the Centers for Epidemiological Studies of Depression. The lack of treatment differences was robust across types of data analyses that took baseline functioning into account and that recognized the sensitivity of outcome measures to assumptions concerning missing data. The trajectory of recovery revealed an initial decrease in function and then a recovery to pretreatment levels that were similar for both treatment arms. Furthermore, the patients in both treatment groups returned to a functional level that approximated general US population norms. Even though the incidence of acute graft-versus-host disease was slightly higher in the conventional treatment arm, T-cell depletion did not differentially affect HRQL at 1 year after transplantation.

BACKGROUND Oral mucositis is a complication of intensive chemotherapy and radiotherapy with no effective treatment. We tested the ability ofpalifermin (recombinant human keratinocyte growth factor) to decrease oral mucosal injury induced by cytotoxic therapy. METHODS This double-blind study compared the effect ofpalifermin with that of a placebo on the development of oral mucositis in 212 patients with hematologic cancers; 106 patients received palifermin (60 μg per kilogram ofbody weight per day) and 106 received a placebo intravenously for three consecutive days immediately before the initiation of conditioning therapy (fractionated total-body irradiation plus high-dose chemotherapy) and after autologous hematopoietic stem-cell transplantation. Oral mucositis was evaluated daily for 28 days after transplantation. RESULTS The incidence oforal mucositis of World Health Organization (WHO) grade 3 or 4 was 63 percent in the palifermin group and 98 percent in the placebo group (P

Patients with neutropenia and persistent fever are often treated empirically with amphotericin B or liposomal amphotericin B to prevent invasive fungal infections. Antifungal triazoles offer a potentially safer and effective alternative.In a randomized, international, multicenter trial, we compared voriconazole, a new second-generation triazole, with liposomal amphotericin B for empirical antifungal therapy.A total of 837 patients (415 assigned to voriconazole and 422 to liposomal amphotericin B) were evaluated for success of treatment. The overall success rates were 26.0 percent with voriconazole and 30.6 percent with liposomal amphotericin B (95 percent confidence interval for the difference, -10.6 to 1.6 percentage points); these rates were independent of the administration of antifungal prophylaxis or the use of colony-stimulating factors. There were fewer documented breakthrough fungal infections in patients treated with voriconazole than in those treated with liposomal amphotericin B (8 [1.9 percent] vs. 21 [5.0 percent], P=0.02). The voriconazole group had fewer cases of severe infusion-related reactions (P0.01) and of nephrotoxicity (P0.001). The incidence of hepatotoxicity was similar in the two groups. Patients receiving voriconazole had more episodes of transient visual changes than those receiving liposomal amphotericin B (22 percent vs. 1 percent, P0.001) and more hallucinations (4.3 percent vs. 0.5 percent, P0.001). Parenteral voriconazole was changed to the oral formulation in 22 percent of the voriconazole group, with a reduction in the mean duration of hospitalization by one day in all patients (P=0.17) but by two days in patients at high risk (P=0.03).Voriconazole is a suitable alternative to amphotericin B preparations for empirical antifungal therapy in patients with neutropenia and persistent fever.

Background Despite recent therapeutic advances, multiple myeloma (MM) remains primarily an incurable cancer. Patients experiencing rapid recovery of T cells post autologous stem cell transplant (auto-SCT) may have improved outcomes, and spontaneous cellular responses to tumor can occur, suggesting immune mediated control of tumor is possible. We and others have investigated therapeutic cancer vaccines that have shown promise in pilot studies, in particular following post-transplant infusion of activated autologous T cells. However, efficacy of these approaches may be limited by thymic selection which restricts the repertoire of T cell receptors (TCRs) to low affinity TCRs that cannot recognize the low level of antigen present on most tumor cells. We hypothesized that incorporation of affinity-enhanced tumor antigen-specific TCRs into autologous T cells infused post-transplant would overcome this limitation and improve response rates in the post auto-SCT setting. Methods We report interim results of a Phase II clinical trial (NCT01352286) to evaluate the safety and activity of autologous T cells genetically engineered to express an affinity-enhanced TCR that recognizes the NY-ESO-1/LAGE-1 peptide complex HLA‐A*0201‐SLLMWITQC; these cells are infused in the setting of profound lymphodepletion that accompanies high dose chemotherapy administered during auto-SCT. Patients with high risk or relapsed MM, who are HLA‐A*0201 positive, and whose tumor is positive for NY-ESO-1 and/or LAGE-1 by RT-PCR are eligible. CD25 depleted CD4 and CD8 T cells are activated and expanded using anti-CD3/CD28 antibody conjugated microbeads, and genetically modified with a lentiviral vector containing the TCR construct at a multiplicity of infection of 1. Engineered T cells are administered four days after high dose melphalan and two days following auto-SCT, at a dose range of 1-10 billion total cells with a minimum gene modification requirement of 10%. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months post infusion. At 3 months, patients start lenalidomide maintenance. The initial 6 patient phase is complete and a 20 patient extension phase is ongoing. Results Prior to enrollment on study, patients had received a median of 3 prior therapies including 6 with prior transplant. 50% of tumors contained high risk chromosomal abnormalities, and NY-ESO-1 expression is correlated with adverse prognosis. 20 patients (average age of 57) have been infused with an average of 2.3 X 109 engineered T cells (range 4.5 X 108-3.9 X 109); this reflects an average clinical scale transduction efficiency of 34% (range 18% – 49%). Infusions have been well tolerated, and the majority of adverse events were related to the high dose melphalan. Possibly related SAEs were neutropenia and thrombocytopenia, and GI and metabolism disorders including diarrhea, colitis, hyponatremia and hypomagnesemia. 10, 4, 2, and 2 patients have reached the 1 year, 9 month, 6 month and 3 month assessment timepoints, respectively, and 17/20 patients are alive. Best response by day 100 is sCR/CR in 2/15 (13%), nCR in 10/15 (67%), and PR in 3/15% (20%), which compares favorably to historic responses in patients undergoing first or second transplant. Engineered T cells expanded and persisted in blood and marrow at 180 days by Q-PCR and flow-cytometry in all but one case (Figure). 7 patients progressed after day 100, which was accompanied either by loss of engineered T cells or loss of tumor antigen. Detailed phenotyping and functional analysis of engineered T cells, and correlates with clinical responses, is underway. Summary This is the first clinical evaluation of engineered T cells in the MM setting. Infusions are safe, well tolerated, and are associated with encouraging responses in a high risk myeloma population. A study evaluating the engineered T cells in a non-transplant study is underway. Disclosures: Stadtmauer: Celgene: Consultancy. Binder-Scholl:Adaptimmune: Employment. Smethurst:Adaptimmune: Employment. Brewer:Adaptimmune: Employment. Bennett:Adaptimmune: Employment. Gerry:Adaptimmune: Employment. Pumphrey:Adaptimmune: Employment. Tayton-Martin:Adaptimmune: Employment. Ribeiro:Adaptimmune: Employment. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Jakobsen:Adaptimmune: Employment. Kalos:Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. June:Novartis: Patents & Royalties, Research Funding.

Background African Americans (AAs) have a higher incidence of MM, about twice as much as Caucasians, and present at a younger age. Several studies have shown that presentation and outcomes of AA are similar to Caucasians treated with standard and high dose chemotherapy. The impact of induction with novel agents (IMIDs and proteosome inhibitors) and prognostic markers such as cytogenetics on different ethnic groups has not been fully evaluated. Methods We analyzed MM patients (AA=174 and Caucasians= 279) who underwent auto-SCT over fifteen years (1998-2013). Baseline characteristics are summarized in table below. Patients received induction therapy with standard chemotherapy (n=33) or dexamathesone (n=43) before 2001 or IMIDs or protesome inhibitors in addition to either chemotherapy (n=72) or dexamethasone (n=203) when treated after 2001. All patients received high-dose melphalan followed by auto-SCT. Response criteria were assessed according to the International Uniform Response criteria. Results AA were significantly younger but with no differences in disease stage or CRAB criteria at MM diagnosis with the exception of anemia. More AA women were referred and underwent auto-SCT relative to AA men. The subtype distribution was significantly different between AA and Caucasians (P Conclusion MM is heterogeneous disease. AA disease characteristics are different from Caucasians (subtype and cytogenetics). AA have inferior responses to novel agents, despite comparable and probably better survival to Caucasians, suggesting a favorable disease biology. Future studies focusing on AA patients are urgently needed to validate these observations prospectively. Disclosures: No relevant conflicts of interest to declare.

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic‐acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic‐acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co‐amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96‐well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3‐log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic‐acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay. J. Clin. Lab. Anal. 14:32–37, 2000. © 2000 Wiley‐Liss, Inc.

Advanced atherosclerotic lesions are characterized by lipid accumulation, chronic inflammation, and defective efferocytosis; an ideal therapy should address all aspects of this multi-factorial disease. Ixmyelocel-T, an expanded autologous multicellular therapy showing clinical promise in the treatment of severe diseases associated with atherosclerosis, includes a novel population of M2-like macrophages. We examined the properties of these macrophages to determine their potential mechanisms of action in atherosclerotic states. Flow cytometry revealed that ixmyelocel-T macrophages express surface receptors of alternative activation (CD206 and CD163) and scavenger receptors involved in lipid and apoptotic cell uptake (CD36, LDLR, CD91, MSR1, and MerTK). ELISA analysis revealed ixmyelocel-T macrophages secrete substantial levels of IL-10 (1060 278 vs. 1978 313 pg/ml, p < 0.05) and IL-1ra (12673 1242 vs. 37576 3650 pg/ml, p < 0.001) before and after overnight LPS stimulation. Baseline secretion of the pro-inflammatory cytokines IL-1b, TNFa, and IL-12 is minimal and remains low after LPS stimulation. Fluorescent microscopy and flow cytometry revealed that ixmyelocel-T readily phagocytosed apoptotic cells. Apoptosis analyzed by a caspase 3/7 assay demonstrated that ixmyelocel-T isn’t sensitive to the lipotoxic effects of modified cholesterol (87 10.78 vs. 100 14.58% of control). Real time PCR revealed that the cholesterol transporter genes ABAC1 (1.49 0.32 vs. 0.42 0.11 fold change, p < 0.01) and ABCG1 (0.41 0.13 vs. 0.07 0.03 fold change, p < 0.05) are both significantly upregulated when ixmyelocel-T macrophages are loaded with cholesterol. Ixmyelocel-T also exhibits enhanced ApoAI mediated cholesterol efflux (13.91 1.90 vs. 3.42 0.31%, p < 0.001 compared to THP-1). In addition, ixmyelocel-T effluxed cholesterol in vivo in using a mouse model for reverse cholesterol transport. These biological properties of ixmyelocel-T macrophages suggest that this cellular therapy may exert beneficial effects on atherosclerotic disease by providing macrophages that potentially limit atheroma development through secretion of anti-inflammatory cytokines, removal of apoptotic cells, and promotion of cholesterol efflux.

Background: Adoptive immunotherapy for cancer has been limited by lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We are conducting an early phase clinical trial (NCT01352286) attempting to overcome these barriers using T cells engineered with an HLA-A0201 restricted, affinity-enhanced TCR that recognizes an epitope expressed by the NY-ESO-1 and LAGE-1 cancer testis antigens; these cells are infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy with autologous stem cell transplant (aSCT) for patients with high risk or relapsed multiple myeloma (MM). Methods: Inclusion criteria include: 1) eligibility for aSCT, 2) PS of 0-2, 3) high risk MM or relapse after prior therapy, 4) HLA-A0201 positive, and 5) NY-ESO-1 and/or LAGE-1 positive tumor by PCR. CD25 depleted T cells are activated and expanded using anti-CD3/28 antibody conjugated microbeads, and genetically modified with a lentiviral vector. T cells are administered four days after high dose melphalan and two days following auto-SCT. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months. At 3 months, patients with adequate marrow function start lenalidomide maintenance. Blood and marrow are monitored for persistence of engineered cells by qPCR and by surface expression of the NY-ESO-1 / LAGE-1 TCR using dextramerTM reagents. NY-ESO-1 and LAGE-1 antigen expression in marrow was assessed by qRT-PCR at baseline and post infusion. Results: As of November 2012, 21 patients have been enrolled, 15 have been infused; 4 were taken off study prior to infusion due to disease progression. An average of 2.7 x 109 engineered T cells were administered per patient (range 8.3 x 108-4.2 x 109), and the average transduction efficiency was 33% (range 30%-45%). More than 50% (8/15) of patients have high risk chromosomal abnormalities, and 3 (20%) have received prior aSCT. At 3 months post aSCT, 73% of patients were in a very good partial response (VGPR) or better. Gastrointestinal toxicity resulting from autologous GVHD (aGVHD) occurred in a subset of patients at a higher rate than reported following aSCT alone or aSCT and T cell infusion, and was resolved in all cases. Infused T cells typically showed peak expansion in blood at day 14, followed by durable persistence in blood and marrow at 6-12 months in all but one patient. Disease progression is typically accompanied by very low levels or loss of engineered T cell persistence or loss of target antigen on tumor. Conclusions: We report for the first time that possible correlates of clinical response in this study include persistence of engineered T cells and loss of antigen, suggesting specific activity of the infused cells. Infusions are well tolerated with a possible risk of manageable aGVHD. Citation Format: Aaron P. Rapoport, Edward A. Stadtmauer, Dan T. Vogl, Brendan Weiss, Gwendolyn K. Binder-Scholl, Dominic P. Smethurst, Jeffrey Finkelstein, Irina Kulikovskaya, Minnal Gupta, Erica Suppa, Tatiana Mikheeva, Joanna E. Brewer, Alan D. Bennett, Andrew B. Gerry, Nick J. Pumphrey, Helen K. Tayton-Martin, Lilliam Ribeiro, Elizabeth Veloso, Zhaohui Zheng, Ashraf Z. Bados, Saul Yanovich, Gorgun Akpek, Karen Dengel, Naseem Kerr, Sunita Philip, Kelly-Marie Betts, Sandra Westphal, Bruce L. Levine, Bent K. Jakobsen, Carl H. June, Michael Kalos. Correlates of clinical response following autologous stem cell transplant and adoptive immunotherapy with engineered T cells expressing an affinity-enhanced T cell receptor in patients with mutliple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2013-4575

Abstract 755 Background: Adoptive transfer of T cells genetically engineered to express affinity-enhanced T cell receptors (TcRs) specific for self-antigens is one approach to generate potent anti-tumor immunity, and this approach is being evaluated in a number of clinical trials. Although persistence of engineered T cells is positively correlated with response rates, a recurring observation to-date has been poor T cell persistence beyond the initial treatment window. We have recently initiated a Phase II clinical trial (NCT01352286) in patients with recurrent multiple myeloma (MM), to evaluate the safety and activity of autologous T cells genetically engineered to express an HLA-A201 restricted, affinity-enhanced TCR that targets an epitope shared by the NY-ESO-1 and LAGE-1 cancer testis antigens commonly expressed by MM. Clinical study details and outcomes are reported elsewhere in this meeting (Rapoport, A, Stadtmauer, E. et al.). Here we report the in vivo expansion, bioactivity, trafficking, durable persistence, and anti-tumor activity of TcR modified cells in this trial. Methods: Subjects received high-dose melphalan, infusion of autologous hematopoietic stem cells on day 0, and infusion of gene engineered cells (GEC) on day +2. Persistence of GEC was assessed by quantitative ABI-Taqman based PCR and a qualified assay that detects the lentivirus backbone sequence. Surface expression of the introduced affinity-enhanced TcR α/β receptor was detected by multiparametric flow cytometry and TcR-specific dextramer™reagents. NY-ESO-1 and LAGE-1 antigen expression in marrow samples was assessed by quantitative ABI-taqman based PCR and inventoried primer/probe sets. Results: As of August 2012, we have screened marrow from 46 patients for NY-ESO- and LAGE-1 expression. Of these, 29 (63%) and 17 (37%) expressed LAGE-1 and NY-ESO-1, respectively by PCR. LAGE-1 co-expression was observed in all cases with NY-ESO-1. To date 12 patients have been infused with GEC. Robust engraftment was observed, with peak levels of gene-marked cells in peripheral blood and marrow ranging from 1 × 102-1 × 103 cells/μL and peak levels detected within the first 21 days post infusion. By day +100 the absolute number of GEC showed patient-specific and variable contraction, with a subset of patients maintaining GEC at 1 × 102-1 × 103 cells/μL, and a second subset where GEC contracted to 1 × 101-1 × 102cells/μL. Trafficking to and persistence of GEC in marrow was observed. At both early (up to day +30) and, in a subset of patients, late timepoints (post day+42), circulating CD3+ T cells could be detected with surface expression of the affinity enhanced TcR. Engineered cells are detected in all patients at their latest timepoint by Q-PCR. To date we see no evidence for TCR mispairing with endogenous TCR. Tumor-antigen and tumor-specific targeting by GEC was evaluated using Q-PCR and quantifying transcript levels of NY-ESO-1, LAGE-1, and CD138 (as a plasma cell marker) in marrow biopsies at enrollment, and days +42, +100, and +180. In the initial cohort of 5 patients, selective reappearance of CD138 transcript is observed in 4 cases (2 sCR, 1 VGPR, 1 PR), with non-detectable or very weak expression of LAGE-1/NY-ESO-1 transcripts, suggesting specific targeting of tumor cells that express LAGE-1/NY-ESO-1. In the fifth patient, who has progressive disease and low engineered cell persistence, CD138 and LAGE-1/NY-ESO-1 transcripts are both present above pre-treatment levels. A second infusion was recently given to this patient. Summary: Our studies show for the first time that adoptive transfer of affinity-enhanced TCR engineered T cells in MM patients results in prolonged T cell persistence, homing to marrow, and anti-tumor activity. Disclosures: Binder-Scholl: Adaptimmune: Employment. Smethurst:Adaptimmune Ltd: Employment. Brewer:Adaptimmune Ltd: Employment. Bennett:Adaptimmune Ltd: Employment. Gerry:Adaptimmune Ltd: Employment. Pumphrey:Adaptimmune Ltd: Employment. Tayton-Martin:Adaptimmune Ltd: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. Jakobsen:Adaptimmune Ltc: Employment. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

Abstract 472 Background: Despite recent therapeutic advances, multiple myeloma (MM) remains primarily an incurable cancer. Patients experiencing rapid recovery of T cells post auto-SCT may have improved outcomes, and spontaneous cellular responses to tumor can occur, suggesting immune mediated control of tumor is possible. We and others have investigated therapeutic cancer vaccines that have shown promise in pilot studies. However, vaccine efficacy may be limited by thymic selection which restricts the repertoire of T cell receptors (TCRs) to low affinity TCRs that cannot recognize the reduced level of antigen present on most tumor cells. We hypothesized that incorporation of affinity-enhanced tumor antigen specific TCRs into autologous T cells would overcome this limitation. Methods: We report interim results of a Phase II clinical trial (NCT01352286) to evaluate the safety and activity of autologous T cells genetically engineered to express an HLA-A201 restricted, affinity-enhanced TCR targeting an epitope shared by the NY-ESO-1 and LAGE-1 tumor antigens (gene engineered cells, or GEC). Inclusion criteria include: 1) eligibility for auto-SCT, 2) PS of 0–2, 3) high risk MM or relapse after prior therapy (prior allo-SCT excluded), 4) HLA-A201 positive, and 5) NY-ESO-1 and/or LAGE-1 positive tumor. GEC are manufactured from monocyte and T regulatory cell depleted apheresis, followed by lentivector gene transfer and T cell expansion by bead-based artificial antigen presenting cells. GEC are administered four days after high dose melphalan and two days following auto-SCT, at a dose range of 1–10 billion cells. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months post infusion. At 3 months, patients start lenalidomide maintenance. The initial 6 patient phase is complete and a 20 patient extension phase is ongoing. Results: 19 patients have been enrolled, 12 have been infused, 3 are pending infusion, and 4 were taken off study prior to infusion due to disease progression. The average TCR transfer efficiency was 31.9% (range 18.2%-45%). 6, 4, and 1 patients have reached the 6 month, 3 month, and 6 week post infusion MM assessment time points, respectively. All treated patients are alive. 3/11 (27%) and 7/11 (64%) patients have a stringent CR or best response of VGPR or better, respectively; 3/11 (27%) have stable or partial responses, and 1/11 (9%) had progressive disease. In patients with VGPR, oligoclonal banding was common, marrow showed no expansion of clonal plasma cells, and all had normalized light chain ratios, suggesting at most minimal residual disease. These results are encouraging, especially considering that 3/11 patients had prior ASCT and 8/11 had adverse cytogenetics. GEC infusions have been well tolerated. The majority of adverse events are related to the high dose melphalan. Several patients had a transient skin rash with lymphocytosis and others had a diarrheal syndrome that occurred later than expected for melphalan-induced mucositis. In all cases GI toxicity has been self-limited or treatable with a short dose of corticosteroids. Possibly related SAEs were cytopenias such as neutropenia and thrombocytopenia, and GI and metabolism disorders such as diarrhea, colitis, hyponatremia and hypomagnesemia. GEC have been detected for up to 1 year at 1% in the circulation and bone marrow. Detailed correlative analysis is reported elsewhere in this meeting (Kalos, M. et al). Summary: This is the first test of GEC in the MM setting. Infusion of GEC administered post auto-SCT is safe, well tolerated, and leads to high response rate in a high risk myeloma population. Phase II accrual continues and a study evaluating GEC outside of auto-SCT is planned. Disclosures: Stadtmauer: celgene: Consultancy; millenium: Consultancy. Binder-Scholl:Adaptimmune : Employment. Brewer:Adaptimmune Ltd: Employment. Bennett:Adaptimmune Ltd: Employment. Gerry:Adaptimmune Ltd: Employment. Pumphrey:Adaptimmune Ltd: Employment. Smethurst:Adaptimmune Ltd: Employment. Tayton-Martin:Adaptimmune Ltd: Employment. Lilliam:Adaptimmune: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. Jakobsen:Adaptimmune Ltc: Employment. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

Abstract 352 Background: Autologous stem cell transplantation (ASCT) for MM leads to complete responses in ∼20–40% of patients but rare cures. Patients with rapid recovery of T cells post ASCT may have improved outcomes suggesting possible immune mediated tumor control. We have shown that adoptive T-cell transfers after ASCT for MM using vaccine-primed and ex-vivo costimulated autologous T-cells in combination with pre- and post-transplant immunizations using a tumor antigen vaccine (+ GM-CSF and montanide) led to vaccine-directed T-cell responses in about 1/3 of patients and enhanced recovery of polyclonal T and B cell counts and function. We hypothesized that addition of Poly-ICLC (Hiltonol®) – a TLR-3 agonistic vaccine adjuvant could increase tumor antigen vaccine responses through better priming and boosting. Methods: We report interim results of a Phase II clinical trial (NCT01245673) evaluating safety and activity of autologous T cells primed in-vivo with a MAGE-A3 multipeptide vaccine (Orphan Drug Designation: GL-0817) mixed with GM-CSF and Poly-ICLC (Hiltonol®) +/− montanide. Inclusion criteria included measurable disease or high-risk cytogenetics. MAGE-A3 is expressed in ∼50% MM cases and more frequently in relapsed/extramedullary/proliferative disease. The MAGE-A3 vaccine has 2 HLA-A2-restricted class I peptides and 1 promiscuous class II peptide linked to an HIV-1-TAT membrane translocation sequence (Trojan peptide) to enhance peptide presentation. Vaccine-primed T cells were collected by leukapheresis, costimulated and expanded ex-vivo using anti-CD3/anti-CD28 mAb conjugated beads. T-cells were infused at day +2 after ASCT followed by booster immunizations at days 14, 42, 90, 120 and 150 post-transplant. Lenalidomide maintenance was started at day +100. Patients were evaluated for MM responses in accordance with IMWG criteria at days 60, 100, 180 post-transplant and Q3 months thereafter. T-cell and B-cell responses to the vaccine were evaluated by IFN-g or IL-2 cytokine production (all patients), dextramer binding to CD8 cells (HLA-A2 positive patients) and ELISA antibody assays at days 14, 60, 100 and 180 post-transplant. Results: 25 patients were transplanted on study. At a median followup of 6 months (range 2–18 months), 24 patients are surviving while 1 patient relapsed at about day +60 and died. Four additional patients have relapsed at 7,9,18 and 18 months post-transplant, yielding a 1-year Kaplan-Meier EFS of 77%. Of the 16 patients evaluable for response at day 100, 7/16 (44%) had CR/nCR using the study enrollment (post-induction) myeloma markers as a baseline while at day 180, 7/13 (53%) had CR/nCR. T-cell infusions were well-tolerated with no probable/definite grade 3 or higher toxicities. Vaccinations were associated with > 50 mm injection site reactions (redness, induration or both) after 1 or more immunizations in the majority of patients. Two patients developed large and prolonged inflammatory reactions which evolved into sterile abscesses. These resolved over 2–3 months with conservative management but as a result montanide was eliminated from the vaccine formulation for patients 11–25. Thereafter vaccine reactogenicity was decreased with no additional sterile abscesses. Of 16 patients tested for immune responses to date, 2 patients were unevaluable due to poor sample viability. Dextramer staining demonstrated MAGE-A3-specific CD8 T-cells in 4/4 (100%) of evaluable HLA-A2+ patients. Cytokine production in response to MAGE-A3 stimulation was seen in 11/14 (79%) patients; responses usually peaked at day 100 or day 180. Robust MAGE-A3 antibody responses were detected in 7/9 patients who received montanide in the vaccine formulation but in 0/7 patients who did not. Conclusions: Combination immunotherapy using a MAGE-A3 multipeptide tumor antigen vaccine plus vaccine-primed and costimulated autologous T-cells after ASCT for MM is well-tolerated and associated with encouraging early clinical responses. The addition of Poly-ICLC (Hiltonol®) to the vaccine formulation was associated with a high frequency of post-ASCT T-cell functional responses. The combination of Poly-ICLC +GM-CSF + Montanide led to robust injection site reactions that were occasionally severe and prolonged. Elimination of the montanide reduced injection site reactogenicity but may also have compromised the B-cell responses to the tumor antigen vaccine. Disclosures: Rapoport: Gliknik, Inc: Research Funding. Stadtmauer:Celgene: Consultancy, Speakers Bureau; Millenium: Consultancy, Speakers Bureau. Vogl:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Consultancy, Research Funding; Otsuka: Consultancy; Acetylon: Research Funding. Strome:Gliknik, Inc: Equity Ownership, Patents & Royalties, Research Funding. Salazar:Oncovir, Inc: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

e19565 Background: OM is a common complication of high-dose melphalan in MM patients (pts). Proposed risk factors for OM in SCT include: low albumin and high serum creatinine (Cr) levels, both were evaluated in MM patients undergoing Mel/ASCT. (Grazziutti, ML, Bone Marrow Transplant 2006). Methods: This is a single center retrospective chart review of 214 sequentially treated MM pts who received Mel 200mg/m2 conditioning prior to SCT between January 2005-September 2011. Data collected included: demographics, Hgb, Cr, C-reactive protein and albumin on the day of SCT, length of hospital stay. OM assessment was graded as follows: Grade 1, no OM; Grade 2, mild OM; the pts maintained adequate oral intake; Grade 3, decreased oral intake and/or use of oral narcotics; Grade 4, severe OM needing intravenous narcotics. Results: The table below describes pt characteristics grouped by OM grade. Overall, 56 pts (27%) had grade 3/4 OM. Multivariate analysis of variance revealed no statistically significant correlation between OM grade and Hgb, Cr, albumin, CRP; the overall test’s p value = 0.55. There were no racial or gender differences with regard to grade of mucositis, the p-values range are 0.75 and 0.31, respectively (likelihood ratio chi-square test). Most interestingly, OM did not impact length of hospital stay. Conclusions: We did not establish any predictive risk factors for OM as previously described. Analysis of the impact of OM on MM response and event and overall survival will be presented. Studies of Mel pharmacogenetics may provide insight to patients' predisposition to OM. [Table: see text]

Abstract 784 BACKGROUND: Previously, we showed that adoptive transfer of in vivo vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at day +12 after transplant increased CD4 & CD8 levels at day +42 and augmented vaccine-specific immune responses in pts with myeloma (MM)[Nat Med 2005; 11: 1230-7]. DESIGN: We conducted a phase I/II 2-arm clinical trial in which 54 pts with advanced MM received autografts after high-dose melphalan followed by infusions of ex-T at day +2 after transplant. Pts also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine (PCV) only (ARM B; n = 26) or the PCV plus an HLA-A2 restricted multipeptide tumor antigen vaccine (3 hTERT peptides + 1 survivin peptide + 1 CMV peptide) for HLA-A2+ patients (ARM A; n = 28). After adoptive transfer of 4.26 × 10e10 costimulated T cells (range 1.59-5.0), the median CD3, CD4 and CD8 counts were 4198, 1545 and 2858 cells per microliter at day +14 post-transplant and 16% of pts developed significant autologous GVHD [Clin Cancer Res 2009;15: 4499 – 507]. Here we first report clinical outcomes from this trial and describe several novel observations about immune reconstitution after day +2 T-cell transfers. CLINICAL RESULTS: For the 54 total transplant pts, the median age was 55 (range 37-68), 28 (52%) were male and 21 (39%) were AA while 32 (59%) were Caucasian and 1 (2%) was Asian. 66% had IgG and 28% had IgA paraproteins while 6% excreted light chains only; 41% had abnormal cytogenetics. With a median followup of 1 year (0.25 - 2.5 years) 50 pts (93%) are alive and 31 (57%) remain event-free (EFS). The projected median EFS is 15 mos with no difference between ARMS A and B. Based on the Cox regression analysis, EFS did not correlate significantly to gender, race nor absolute lymphocyte counts (ALC). However, age correlated inversely to EFS with older pts exhibiting a lower EFS (coefficient = -0.0667, p-value = 0.0398). MM responses at day +60 and day +100 did not correlate to EFS, but the response at day +180 did correlate to EFS (p IMMUNOLOGICAL RESULTS: Preliminary results indicated that 6 of 15 pts (40%) in ARM A had positive tetramer responses to hTERT and/or survivin peptides at one or more timepoints after immunization, defined as tetramer staining by flow cytometry > 0.1% (and > 3-fold increase). Based on analysis of the first 36 patients, the mean sum of the antibody responses to 4 of the 7 serotypes of the pneumococcal conjugate vaccine (PCV; serotypes 6B, 14, 19F, 23F) were 83.55 mcg/ml at day +100 and 74.13 mcg/ml at day +180, highly protective levels, compared to 5.17 mcg/ml at enrollment. We also observed several novel features of immune system recovery after early T-cell transfers. Compared to an historical cohort of 103 MM pts who were autografted without ex-T, post-transplant quantitative IgG, IgA and IgM levels at day +180 (and days +60/+100 for IgG and IgA) were significantly higher in the pts who received day +2 T-cell transfers (IgG: 1525 mg/dl vs 1137 mg/dl, p= 0.014; IgA: 264 mg/dl vs 76 mg/dl, p = 0.0029; IgM: 58 mg/dl vs 39 mg/dl, p = 0.029). Furthermore, when IgG recovery was examined for the IgA MM pts only, the IgG level at day +180 was significantly higher in the ex-T trial (mean IgG = 810 mg/dl vs 611 mg/dl, p = 0.0478). Conversely, when IgA recovery was examined for the IgG MM pts only, the IgA level was significantly higher in the ex-T trial at days +60 (mean IgA = 129.82 mg/dl vs 71.70 mg/dl, p = 0.0036), day +100 (mean IgA = 114.43 mg/dl vs 59.44 mg/dl, p = 0.0075) and day +180 (mean IgA = 98.38 mg/dl vs 48.74 mg/dl, p = 0.0037). Remarkably, a pattern of CD8 T-cell “reprogramming” was also observed after day +2 T-cell transfers such that the % of CD8+/CD45RA+ T-cells at day +60 post-transplant was 75% vs 36% in a cohort of MM transplant pts who did not receive ex-T (p< 0.0001). In addition, the log ratio of Teffector/Tregulatory cells was significantly higher among the patients who received early T-cell transfers (2.57 vs. 1.93, p = 0.0066). CONCLUSIONS: Early adoptive transfers of anti-CD3/anti-CD28 costimulated autologous T-cells significantly enhanced post-transplant humoral and cellular immune reconstitution, a pattern which may be associated with better myeloma control and protection from infection. These data may have important implications for efforts to induce clinically significant immune responses to cancer vaccines in the transplant setting. Disclosures: Vonderheide: University of Pennsylvania and Dana Farber Cancer Institute: Patents & Royalties. June:University of Pennsylvania: Patents & Royalties.

The incidence of MM in African-Americans is more than double that in Caucasians. Historically AAs have had a higher mortality rate than Caucasians; but over the past 10 yrs, the age-adjusted mortality rate has been on the decline for AAs while it has been stable for Caucasians as a result of ASCT and novel agents. Previous studies (n=74 AA patients) suggested that response to ASCT is similar, if not better, for AA patients (Verma et al 2008, Saraf et al 2006). We retrospectively analyzed the clinical presentation of a large cohort of AA patients (n=103) who underwent ASCT at our center between 1998 and 2008 and compared their outcome to that of Caucasians patients (n=183) transplanted in the same time period. AA patients were significantly younger than Caucasian patients at diagnosis with median age 53 (range: 32–75) vs 59 (range: 27–80), respectively (p 3.5 mg/L and > 5.5 mg/L, respectively. Initial cytogenetic data were not available for the majority of patients. Median time from diagnosis to ASCT was significantly longer for AA than for Caucasian patients at 0.8 yrs (range: 0.23–9.2) vs 0.5 yrs (range: 0.1– 7.0), respectively (p

Bortezomib has demonstrated activity in heavily pretreated MM patients. The dose limiting toxicity is peripheral neuropathy (PN) that affects up to 35% of the patients (Richardson et al JCO 2006). We retrospectively reviewed the incidence and severity of PN in 78 patients who received bortezomib at our institution. The median age was 57 years (range: 33–80), 62% were men, and 37% were African Americans (AA). Risk factors for PN included prior use of thalidomide in 53 patients (68%) and vincristine in 31 patients (40%). Seventeen patients (22%) had diabetes mellitus. Before bortezomib treatment 29 patients (37%) reported subjective grade 1–2 PN. Patients received bortezomib alone (n=10) or in combination with dexamethasone (n=36) and thalidomide (n=20) or chemotherapy (n=12). Responses included complete, near complete and partial responses in 5%, 10% and 27%, respectively. Grade 2 and higher PN occurred in 52% of the patients including Grade grades 3 and 4 PN in 15% and 7%, respectively. Twelve patients stopped bortezomib due to side effects that included PN (n=8), diarrhea (n=2) and CMV pneumonia (n=1), and 11 patients had dose reductions because of PN and fatigue. Grade 4 PN affected 6 patients (sensory n=4, and motor/sensory, n=2), 5 of whom were AA and 4 of whom had DM. The onset of grade 4 PN was acute rather than cumulative as noted with lower grades and took a median of 8 months (range: 4–16+) to improve compared to 3–4 months for lower grade PN. Neuropathy grade was not associated with age, sex, race or renal function (10 patients had creatinine > 2 mg/dl, including 2 on dialysis). Factors predictive of PN were baseline neuropathy (p=0.002) in addition to prior thalidomide use (p=0.03) and presence of DM (p=0.03). Responses were independent of neuropathy grade and whether bortezomib was combined with chemotherapy or thalidomide. The duration of therapy in responding patients was longer in patients receiving bortezomib in combination with thalidomide with a median of 5 cycles (range: 2–36) versus those who received other bortezomib combinations 3 cycles (range 1–19). Two of four patients with grade 4 sensory PN had their best response ever after 3 cycles of bortezomib; both had failed transplant and thalidomide based therapies; one remains in continuous complete remission for 24 months. PN therapy was mostly supportive including combinations of analgesics duloxetine, gabapentin, and pregabalin. Interestingly, 6 of 9 patients with PN who received lenalidomide as salvage therapy after progression on bortezomib had significant improvement in their symptoms; 3 of them stopped analgesics. In conclusion, the highest risk and grade of bortezomib neurotoxicity was seen in patients with baseline PN secondary to prior thalidomide use and DM. On the other hand, responding patients who received bortezomib in combination with thalidomide remained longer on therapy with minimal dose reductions. Although this may represent a section bias it is possible that thalidomide’s known anti-inflammatory and anti-angiogenesis properties protect against bortezomib-induced PN. Similar effects may explain the unexpected symptomatic improvement of PN on lenalidomide.

ONJ is a serious complication in MM patients; incidence is time-dependent with highest risk seen after long-term use of BP in older MM patients, often after dental surgical procedure (Badros, et al JCO 2006). Therapy has focused on debridement of necrotic bone, infection management, pain control and discontinuation of BP. The association between BP and ONJ remains provisional; theoretically and anecdotally, however there is evidence that BP withdrawal may allow for recovery of normal osteoclast function and reversal of antiangiogenic effects of the drug on the periosteum allowing for bone healing. We have observed an increased risk of skeletal complications in MM patients after long interruptions of BP therapy (6–12+months), even though patients had been on BP therapy for a median of 3 yrs (range: 2–10+yrs). Table below summarizes the complications that occurred off BP therapy in MM patients with ONJ (n=28) and without ONJ who stopped BP ahead of dental procedures (n=20). Skeletal complications included avascular necrosis of the hip (AVN), fractures, new lytic lesions on skeletal surveys and bone pain; all occurred in relapsed patients with active MM. These events highlighted the importance of BP cornerstone for treatment of skeletal complications of MM; of the 28 ONJ patients, 8 died from progressive MM and 3 patients had non-healing oral lesions and did not resume BP. Twelve of 17 ONJ patients were restarted on BP after complete healing of the oral lesions; all of whom had relapsed MM and bone disease. They received pamidronate every 3 months. So far 3 of 12 patients (25%) had recurrent ONJ and none had skeletal complications. Of the 20 patients who stopped BP before dental procedures, 3 (15%) went on to develop ONJ. Fifteen had restarted BP 2–3 months after the procedure site was well-healed; one patient developed ONJ at the healed extraction site 6 months after restarting BP therapy. This patient was in remission on maintenance thalidomide but with extensive myeloma bone disease. In conclusion, interruption of BP in MM patients with ONJ as well as before and after dental procedures had inconsistent results with regards to both healing and prevention of ONJ. On the other hand, the damage of holding BP therapy is evident with increased frequency of skeletal complications. Until further studies define the optimal frequency and duration of BP therapy, we propose continuous administration of BP therapy probably less frequently after 2 years of monthly infusions with careful assessment of the risk status of each individual patient. Skeletal complications off bisphosphonate therapy FracturesΔ AVN New lytic lesions Bone pain Δ Fractures affecting long bones or ribs. λ Two had bone disease as first site of relapse. π One patient had a femoral Infarction and severe pain. ONJ; n=28 5 5 2 4 Dental Procedure; n=20 3 1 3λ 1π

Busulfan (BU) and Fludarabine (F) has been reported by de Lima et al. to be a well-tolerated myeloablative conditioning regimen with 3% incidence of hemorrhagic cystitis (Blood2004;104:857). Between 5/2005 and 5/2006, we performed allogeneic stem cell transplantation (SCT) in 29 patients following BU+F (n=17) and other (n=12) myeloablative regimens. Nineteen (66%) patients were in remission and 10 (34%) had active disease at BMT. Twenty patients had AML, 4 ALL and 5 had other malignancies. Busulfan was given at 130mg/M2 over 3 hours on 4 successive days, along with Fludarabine 40mg/M2 on 4 successive days. Eighteen (72%) had matched-related 7 (24%) had matched-unrelated donor (MUD) transplant. GVHD prophylaxis consisted of Tacrolimus and low-dose Methotrexate. Thymoglobulin (n=4) and CAMPATH (n=3) were given along with conditioning in MUD/one-antigen mismatch transplant recipients (5 BU+F and 2 other). As of August 1st, 2006, 19 (65%) patients are alive. Of 26 evaluable patients, the incidence of bacterial (59% vs. 89%), fungal (29% vs. 11%) infections were similar between Bu+F and other group, respectively. CMV reactivation was observed in 78% in other group as compared to 35% in BU+F recipients (p=0.09). Hemorrhagic cystitis (HC) occurred in 6 patients (35%) in BU+F group and in 1 (11%) patient in other group (p=0.35). BK virus was detected by quantitative PCR in all patients with HC. Two BK(+) cases were in BU+F+CAMPATH group. Considering 18 AML patients who received transplant in remission and had at least 2 months of follow-up, 5 patients (38%) in BU+F group relapsed within 6 months of transplant. Two out of 5 patients relapsed in the other group. Our findings suggest that BU+F myeloablative conditioning appears to be associated with high incidence of BK-related hemorrhagic cystitis and may have long-term consequences. Further studies are warranted.

Translocation of the hydrocarbon fluorescent probe diphenylhexatriene (DPH) between membranes was studied by fluorescence polarization ( P ) analysis. First, using a model system, the high P value (0.324) of DPH-labeled cholesterol/phosphatidylcholine liposomes and the low P value (0.157) of DPH-labeled phosphatidylcholine liposomes allowed detection of DPH translocation between interacting liposomes. This was monitored by the change in P in either direction. Early events during cell-virus interactions were similarly studied by monitoring DPH translocation. The P value of DPH-labeled Epstein-Barr Virus (EBV) was significantly higher (0.350-0.392) than the P value of DPH-labeled lymphoid cells (0.238-0.289). Hence, DPH translocation could be detected by changes in P following incubation of DPH-labeled EBV and nonlabeled cells. A marked decrease in P was observed after incubation of DPH-labeled EBV with either nonlabeled lymphoblastoid Raji cells or fresh human B lymphocytes. However, only a slight decrease in P was obtained when DPH-labeled EBV was incubated with either nonlabeled fresh human T lymphocytes or fresh T or B rabbit lymphocytes. Moreover, incubation of fresh human B lymphocytes with the purified C3 component of complement (a putative inhibitor for the EBV receptor) prior to the addition of DPH-labeled EBV abolished the observed decrease in the P value. Most of these experiments were carried out with both the P3HR-1 and the B95-8 strains of EBV. DPH translocation, as determined by fluorescence polarization analysis, is, therefore, measuring some early event during interaction of this enveloped virus and mammalian cells. The potential applicability of this technique to other viruses is illustrated by an experiment with Semliki Forest virus.

Surface marker studies were performed on "hairy cells" from 7 patients with hairy cell leukemia (HCL). Using sensitive analytic techniques including specific antisera and Fluorescence Activated Cell Sorter (FACS-1), further definition of the abnormal cell was achieved. Four different antisera were used in infestigating the cell surface characteristics of these patients: anti-p23,30, an antiserum reactive with B cells and a subset of monocytes, anti-311, which reacts only with T cells, pepsin digested anti-F(ab')2 which reacts with B cells only and pepsin digested anti-lysozyme reactive with monocytes and myeloid cells, but not with B or T cells. In all cases strong reactivity was observed with anti-p23,30 and anti-F(ab')2, but no reactivity with anti-311. Five out of the seven cases were reactive with anti-lysozyme in a pattern similar to normal monocytes. Furthermore, when cells were separated according to binding to anti-p23,30, anti-F(ab')2 and anti-lysozyme and in two cases, according to cell size, the majority of reactivity and large cells were "hairy" when examined under microscopy. In contrast, the small and nonreactive (dull cells) appeared as normal mature lymphocytes. Thus, our data supports the view that HCL cells bear in most cases B cell and monocytic membrane markers.

Purified populations of T and B cells were obtained from individuals with chronic lymphocytic leukemia (CLL) by the use of anti-F(ab′) 2 affinity column chromatography techniques in which twice the amount of column material used for normal separations was used for the separation of Ig − and Ig + cells from CLL patients. Furthermore, the flow rate was reduced in order to decrease the possibility that some B cells from CLL patients, containing a low density of surface Ig, would pass through without binding to the coupled Sephadex G-200. Examination of surface Ig, P23,30 antigens, and T-cell antigens in the separated populations indicated that both the Ig − and Ig + populations were highly enriched. These T cells were also tested for their response to mitogenic stimulation as well as their ability to cooperate with leukemic B cells, normal B cells, or B cells from a transformed B-cell line. T cells from leukemic patients were found to be significantly deficient in both the response to mitogens and ability to cooperate with B cells in the synthesis and secretion of immunoglobulin.

The incubation of linoleic acid with cells causes profound effects on membrane asociated phenomenon. Using the fluorescent probe diphenyl hexatriene (DPH) to monitor lipid changes in the microenvironment of the cell surface, we find that linoleic acid reduces the polarization values (P) in mouse lymphocytes and BHK cells. Measurements on lipids extracted from the cells grown in linoleic acid produce similar results. We also find in the mouse lymphocyte that capping of Ig is inhibited and con A stimulated mitogenesis is unaffected. In contrast to the latter effect, LPS and PHA stimulated mitogenesis is inhibited and in the rat lymph node, con A stimulated mitogenesis, greatly enhanced. We also show that linoleic acid alters the binding of antibodies to the cell surface of EL-4 lymphoma cells. These observations suggest that linoleic acid alters cellular function by interfering with protein/lipid interactions within the surface membrane.