ALBERT

Description: INTRODUCTION. There is substantial pre-clinical data demonstrating single agent and synergistic activity of the proteasome inhibitor (PI) bortezomib and deacetylase inhibitors (DACi) in multiple myeloma (MM). This is the first clinical trial combining these two classes of drugs. METHODS. This is an ongoing open label single-centre single-arm phase I/II dose escalation trial of bortezomib, dexamethasone and romidepsin (depsipeptide) in patients with relapsed or refractory MM. All patients received bortezomib (1.3 mg/m2; d1,4,8,11) with dexamethasone (20mg d1,2,4,5,8,9,11,12). The dose escalation of romidepsin commenced at a dose of 8 mg/m2 IV d1, 8, 15 every 28d and involved an initial accelerated dose escalation phase, with intra-patient dose escalation of romidepsin. Response rates were assessed according to M-protein response criteria, with complete responses documented by EMBT criteria. RESULTS. To date, 8 patients have entered the study with 7 evaluable for response and toxicity. Median number of prior therapies = 2 (range: 2–5). Most patients had previously taken potential neurotoxic medications; vincristine-containing (3), thalidomide (4), bortezomib (1). No dose-limiting toxicities (NCI-CTC v3: defined as platelets 4 week suspension of treatment due to toxicity) were demonstrated at romidepsin doses of 8mg (n=1) or 10mg (n=3). At doses of 12mg, 3 episodes of Grade 4 thrombocytopenia occurred and one episode of febrile neutropenia. Of note, 2 of the patients with Grade 4 thrombocytopaenia had platelets below 100 × 109/L prior to commencing the combination (at inclusion, patients must have had a platelet count 〉50 × 109/L). Other drug-related toxicities observed include: Grade 3: fatigue (n=1), neutropaenia (n=1), sepsis (n=1); Grade 2: fatigue (n=1), peripheral neuropathy (n=2), nausea (n=1), diarrhoea (n=1). Two patients required dose reduction of bortezomib due to peripheral neuropathy (these patients were co-administered 12 mg/m2 romidepsin). As of August 2007, the median number of cycles delivered was 3; 3 patients have progressed (after Cycle 1 (n=2), and Cycle 4 (n=1). There has been 1 immunofixation negative Complete Response, 3 Partial Responses and 1 Minor Response with an overall response rate of 5/7 (71%). Most patients remain on the combination therapy (n=5/8). The trial continues accruing patients at the doses of romidepsin (10 mg/m2) with bortezomib (1.3 mg/m2). CONCLUSION. These early results demonstrate efficacy with a promising response rate and some durable responses with acceptable toxicity of a proteasome inhibitor-DACi combination. There are planned regimen scheduling modifications to address the current dose limiting toxicity of transient thrombocytopenia.

Description: In patients (pts) with Hodgkin lymphoma (HL) receiving first-line chemotherapy, interim restaging with 18F-FDG-PET scan (FDG-PET) after 1 or 2 cycles has been shown to predict event free survival (EFS) with high sensitivity and specificity overriding the clinical International Prognostic Score (IPS). The predictive value of FDG-PET in patients with relapsed or refractory HL undergoing high dose chemotherapy and autologous stem cell transplantation (ASCT) is less well established. Previous studies have either included functional imaging with both gallium and FDG-PET scans or performed analyses of Hodgkin and non-Hodgkin lymphoma as a combined cohort. Our aim was to determine the predictive value of FDG-PET in pts with HL planned to receive ASCT. A retrospective analysis was undertaken of 52 consecutive pts treated at three centres. Pts with primary refractory (n=25) or relapsed HL (n=27) underwent FDG-PET scanning after salvage chemotherapy and before ASCT. Remission status by FDG-PET post salvage, treatment details, including salvage type and peri-transplant involved field radiotherapy (IFRT), and clinical characteristics were recorded and EFS and overall survival (OS) post ASCT were evaluated. Survival analyses were performed using Kaplan-Meier estimates and cohorts were compared using the Log-rank (Mantel-Cox) and the Gehan-Breslow-Wilcoxon Test. The contingency of data between different groups was analysed using Fisher’s exact test. The median age of pts at the time of ASCT was 38 [18–61] years, 27/52 (52%) were male. The majority of pts received salvage chemotherapy with VIC (etoposide 100mg/m2 day (d)1-3, ifosfamide 5g/m2 d2, carboplatin AUC 5), n=24) or MADEC (methotrexate 400mg/m2 d1, cytosine arabinoside 75mg/m2 d1-5, dexamethasone 40mg d1-4, etoposide 75mg/m2 d1-5, cyclophosphamide 750mg/m2 d2, n=13), other chemotherapy regimens used were BEACOPP, n=3, IGEV, n=3, FGIV, n=2, DHAC, n=2 or others, n=1 each. After salvage, 23/52 (44%) of pts were FDG-PET-negative and 29/52 (56%) were positive. With a median follow-up of 30 [4–115] months in surviving pts, the 6-year actuarial rates for EFS and OS for FDG-PET negative versus FDG-PET positive pts were 73% and 34% (p=0.03), and 95% and 64%, respectively (p=0.06). Overall, the addition of peri-transplant IFRT did not impact on EFS or OS. However, in 13 pts with post salvage FDG-PET-avid disease which was limited to an area entirely encompassed by IFRT, actuarial rates of 6-year EFS and OS were 51% and 66% and therefore not significantly different from those obtained in FDG-PET-negative pts (p=0.47 and 0.39, respectively). Female gender was the only factor predictive for obtaining a complete FDG-PET-remission post salvage therapy (p=0.011). Female gender and duration of first remission of ≥ 12 months also independently predicted for superior EFS (p=0.017 and 0.039), respectively. Other characteristics including the presence of B-symptoms, extra-nodal disease prior to salvage, age ≥ 38 years, type of salvage or conditioning regimen used, or transplant centre did not influence EFS or OS. Our data show that FDG-PET-status after salvage chemotherapy for relapsed or refractory HL is a powerful predictor of EFS after ASCT demonstrating an excellent outcome for FDG-PET-negative pts. In pts with limited FDG-PET-avid disease following salvage chemotherapy, the addition of IFRT may reduce the poor prognostic impact of residual FDG-PET positivity in a subset of patients.

Description: Adoptive transfer (AT) of autologous T cells genetically-redirected against tumor antigens has considerable potential as cancer immunotherapy [Kershaw, Nat Rev Immunol. 2005]. However, the in vivo persistence of AT T cells is critical for tumor control and requires the development (in vitro or in vivo) of a memory T cell subset. We investigated the generation of memory T cell subsets in a novel chimeric T cell receptor-expressing T cell product prior to, and after exposure to cognate antigen. Gene-modified T cells (LeY-T) express a chimeric receptor comprising a single chain variable fragment (scFv) specific for Lewis Y (LeY) antigen coupled to the intracellular signaling domains of CD3 zeta and CD28, capable of inducing T cell effector granule release and target killing [Westwood PNAS 2005]. To produce LeY-T cells, PBMC from healthy donors (n=20) or multiple myeloma patients (n=2) were cultured with anti-OKT3 (30ng/ml) and IL-2 (600IU/ml) for three days, followed by two rounds of transduction with retroviral supernatant. Subsequently, T cells were expanded in high dose IL-2 (600IU/ml) from day 5 onwards. T cells were harvested for this study on culture days 10–12, CD8+ and CD4+ T cells expressed the chimeric protein (50–60)%. LeY CD8+ T cell subsets were assessed as naïve (N), central memory (CM), effector memory (EM) or effector (E) based on three features:- phenotype (CD45RA, CCR7, CD28, CD27 and perforin); homeostatic cytokine (IL-15/IL-7) proliferation; response to Lewis antigen contact including cell proliferation and cytokine secretion. We repeatedly observed that CD8+ LeY-T cells analyzed directly from the initial expansion culture demonstrate an effector memory (EM) phenotype (CD45RA−/CCR7−/CD28+/perforinhi and variable CD27 expression) (Figure 1A). Furthermore in vitro expanded LeY CD8 T cells express IL- 15R beta (CD122) and the common gamma chain (CD132), they proliferate in response to IL-15 (86% cell division, division index 1.82), but less with IL-7 (30% cell division, division index 0.56). Baseline expanded CD8+ LeY-T cells respond to the presence of LeY antigen by proliferating and secreting IFN-gamma (4–8% of CD8 T cells) but not IL-2. Importantly, no IFN-gamma secretion was seen in control T cells transduced with empty vector (Figure 1B, OVCAR cells). Furthermore, no IFN-gamma was secreted by the control or the CD8+ LeY-T cells in response to the Lewis antigen negative cell line (Figure 1C, HCT116 cells). To explore the memory component further, we examined the functional status of the CD8+ LeY-T cells seven and 30 days following a 48-hour exposure to LeY antigen (OVCAR cells), and compared this to CD8+ LeY-T cell functional status at baseline. Thus, direct from transduction, expansion culture LeY CD8+ T cells were largely EM phenotype (95%) a small population of cells (1–5)% had a CM phenotype (CD45RA−/CCR7+/CD28+/perforinlo). In contrast, seven days after Lewis antigen contact the EM cells had decreased to (76–88)% and CM increased to (10–21)%; this distribution was retained up to day 30 post-antigen exposure. In addition, seven days after Lewis antigen exposure, CD8+ LeY-T cells retain the capacity to proliferate in response to Lewis antigen and to secrete IFN-gamma, at no stage do these cells secrete IL-2. In conclusion, the CD8+ LeY-T cells produced by in vitro transduction and expansion culture have an EM functional status direct from in vitro culture indicating that they are an appropriate starting population for in vivo adoptive transfer. After exposure to LeY expressed on tumor cell lines in vitro, CD8+ LeY T cells show further polarization to either EM or CM cells. These results suggest that the LeY-chimeric T cells have the potential to form long-term memory populations in vivo after adoptive transfer. Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116. Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116.

Description: Treatment with single agent rituximab (R), alkylating agents or purine analogues is safe and moderately effective as first-line treatment for patients (pts) with Waldenström Macroglobulinemia (WM). However, efficacy in relapsed/refractory WM is unsatisfactory. We analysed if combinations of fludarabine (F) with alkylators and/or R were safe and more effective. From 12/94 - 08/08, 25 courses of at least 2 cycles of intravenous F-combination therapy were administered to 22 pts with WM: FC (F 25mg/m2 d1–3, cyclophosphamide (C) 250mg/m2 d1–3; n=7); FCR (FC+R 375mg/m2 d1; n=14); FM (F+mitoxantrone (M) 10mg/m2 d1; n=3); FR, n=1). The median age of pts was 57yrs [range; 36–89], 18/22 (84%) male, 6 pts received F-combination as their primary treatment (24% of courses). The 19 pre-treated pts had a median number of 2 [1–7] prior treatments, prior F-exposure in 5 cases (20%), 9 pts (36%) were alkylator-refractory. The median time from diagnosis to F-based treatment was 23 Mo [0–153], baseline paraprotein (PP) 30g/L [7–64]. A total of 99 cycles were administered, median 4 [2–6] per pt. Treatment was generally well tolerated with 5% infusion reactions grade (G) 2, gastro-intestinal toxicity was mostly limited to G 1 or 2 (35% of cycles) with only one G3 episode, no renal toxicity or hepatic side effects 〉 G 1. G 3 thrombocytopenia occurred in 5%, G ≥ 3 neutropenia and infections complicated 20% and 3% of cycles, respectively, none of which were life-threatening. However, 3 heavily pre-treated pts subsequently developed secondary AML/MDS (1 fatal) at 52, 61 and 99 Mo post-treatment. The overall response rate (RR) was 21/25 (84%) with a median PP reduction of 90% [30–100%]. One pt achieved a complete remission, 17/25 (68%) had a partial response, resulting in an objective RR of 72%, 3 responses (12%) were minor (PP-reduction of 25–60 years or increased baseline beta2 microglobulin impacted on EFS or OS. Anemia (Hb

Description: Introduction: Although there have been progress in treatment and outcomes of acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) in younger patients, treatment of elderly patients, who are not eligible for intensive treatment is still challenging. The demethylating agents azacitidine (AZA) or decitabine recently became a standard of care for first line treatment of elderly or unfit patients with high-risk MDS or AML. Despite the improvement in overall survival with epigenetic therapies, almost all patients eventually develop disease progression, for which no standard therapeutic option is available. The aim of this retrospective analysis was to evaluate the outcome of low-dose cytarabine (LDARAC) treatment compared to best supportive care (BSC) as second-line palliative treatment options after failure of AZA. Patients and Methods: From 2009 to 2014 we treated 75 consecutive patients with newly diagnosed high-risk MDS or de novo AML, with AZA (75mg/m2 sc. d1-7, qd29). Patients who responded to AZA after at least four courses and had a subsequent relapse, as defined by worsening of peripheral blood counts, increase of blasts or increase in transfusion frequency, received a second-line treatment with subcutaneous LDARAC (2x 10 mg/m2/d sc., d1-10, qd29) until progression or were followed by best supportive care, including symptomatic treatment, transfusions or treatment of clinical infections- but without chemotherapy. Results: After first-line AZA 48/75 (64%) achieved a partial hematologic response (n=23), stable disease (n=14) or clinical benefit (n=7). The median overall survival (OS) of all 48 patients with response or clinical benefit to AZA was 27 months. Median duration of response was 13 (4-56) months. Cytogenetic risk at diagnosis was a significant prognostic factor for OS (P

Description: Abstract 4180 High risk acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) may be amenable to immunotherapy with adoptive transfer of chimeric antigen receptor (CAR) T cells. However, persistence and generation of central memory (CM) are essential for ongoing clinical responses (Morgan et al Science 2006). Our prior work showed that the carbohydrate antigen LewisY (LeY) is expressed in 46% of AML patients (n=33) to varying degrees, making it a suitable and novel target for CAR therapy. In our phase I study, we have assessed the trafficking, persistence and functional capacity of CAR-T cells transduced with an anti-LeY chimeric receptor gene in high-risk patients with LeY positive AML or MDS. METHOD: The CAR comprised extracellular humanized scFv recognizing the LeY Ag, linked to an extracellular CD8 hinge region, a transmembrane and cytoplasmic CD28 and CD3 zeta signalling domain. The humanized anti- LeY scFv-CD28-ζ receptor vector was produced as described previously (Westwood J et al PNAS 2005). T cells, harvested from the patient, were transduced with LeY scFv-CD28-ζ pSAMEN retroviral vector. Bulk transduced T cells (LeY-T) were re-infused into the patient after lymphodepleting fludarabine chemotherapy. AML immunophenotyping and cytogenetics were used to monitor minimal residual disease (MRD). PB and BM samples were collected prior to and post CAR-T adoptive transfer. An optimized PCR assay (sensitivity 1:1e5) for the presence of the LeY transgene (TG) was performed on genomic DNA extracted from each sample. To measure both CAR-T cell functional polarization and persistence of CAR expression, PB or BM cells were co-cultured with a cell line expressing LeY (OVCAR-3), in the presence or absence of MHC class I or II blocking antibodies. Culture supernatant was collected at 24 hours and assessed by Luminex assay for Th1 (IFN-g, IL-2), Th2 (IL-4, IL-10), pro-inflammatory (IL-6, IL-17 and TNF) cytokines and TGF-b. RESULTS: Five AML patients have been enrolled to date. Four patients have had sufficient CAR-T cells generated to provide doses of 0.5 × 109 −1.3 × 109T cells (transduced T cells: 8 – 30% of total T-cells]) with a viability of 96.1– 98.5%. 2 patients (01 and 04) had progressive disease 1 and 2 months after infusion. 2 patients (02 and 05) remain in morphologic remission with stable cytogenetic MRD, 3 and 14 months post infusion. Patients 01, 02 and 04 all had LeY TG in PB and BM samples at all time points measured, up to day 49 in patient 01 (Figure 1) and up to 10 months in patient 02. In patient 04 LeY TG was also detected in a skin biopsy (day +6) of leukemic skin infiltrate. Co-culture supernatant cytokine analysis showed LeY-T cells from patient 01, 02 and 04 secreted high levels of IFN-g (805.5 ± 198.9 pg/ml) and low levels of IL-2 (3.14 ± 0.65 pg/ml) prior to adoptive transfer. In contrast, post adoptive transfer, co-culture with OVCAR-3 cells, and MHC class I and II blocking antibody, induced these PB LeY-T cells to secrete IL-4 (24.13 ± 12.47pg/ml) and IL-10 (9.35 ± 0.44 pg/ml); no IFN-g or IL-2 was detected. This Th2 polarization of CAR-T cells was shown in PB samples taken at days 5 to 28 (patient 01), day 28 to 8 months (patient 02) and day 1 to day 28 (patient 04) post-adoptive transfer (Figure 2). In addition, when Th1/Th2 cytokines were measured in patient 02 PB and BM plasma, Th1 cytokines were detected immediately post-adoptive transfer (IFN-g=39 pg/ml, IL-2=8.6 pg/ml at peak); however these decreased to basal levels by day 3 (IFN-g=1.8 pg/ml) and day 2 (IL-2=2.6 pg/ml). The same trend in serum cytokines was observed for patient 01. Therefore, our studies reveal that, after adoptive transfer, LeY CAR-T cells persist in vivo, maintain their expression of the CAR, but show rapid polarization from a Th1 to a Th2 phenotype. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: 18FDG-PET early during first-line chemotherapy of Hodgkin Lymphoma (HL) has a high positive and negative predictive value. The predictive value of functional imaging (FI) in refractory or relapsed HL after salvage treatment and prior to high-dose (HD) chemotherapy and autologous stem cell transplantation (AUSCT) is less well established. Methods: A retrospective analysis was undertaken of the clinical characteristics, FI results, event free survival (EFS) and over all survival (OS) data for 66 consecutive patients (pts) with refractory or relapsed HL treated with salvage chemotherapy and AUSCT between 1997 and 2007. Results: The median follow-up was 52 months (range 0 to 125 months). Following salvage chemotherapy, and prior to AUSCT, 63 of the 66 evaluable pts (96%) had response assessment with a conventional CT scan, 53 pts also had FI with a gallium scan (n=18), 18FDG-PET scan (n=31) or both (n=4). Of the 53 pts with FI prior to AUSCT, 21 (40%) were in metabolic complete remission (FI-negative) and 32 pts (60%) had residual disease (FI-positive). The median EFS and OS for the whole cohort were 29 and 52 months, respectively. Median EFS was 31 and 13 months (p=0.096), and median OS was 60 and 39 months (p=0.35) for those with negative and positive FI, respectively. Significantly more pts with negative FI achieved CR with conventional imaging compared to those with positive FI (9/19 = 47% vs. 2/32 = 6%, p=0.001). Male pts were disproportionately represented in the cohort with positive FI (20/32) compared to the pts with negative FI (7/21, p=0.038). There was no association between FI-positivity and the clinical features of age, short duration (

Description: AML and MM are sensitive to immune control as evidenced by T cell mediated allogeneic graft versus leukemia/myeloma effect. Adoptive immunotherapy with gene modified T cells has shown clinical activity in some solid tumors and B cell non Hodgkin lymphomas. The carbohydrate antigen Lewis Y (LeY) is a tumor associated antigen expressed on numerous epithelial cancers. Our aim was to generate gene-modified clinical-grade T cells directed against LeY positive hematological malignancies. Moreover, we aimed to produce cells that possessed T cell memory, essential for in vivo T cell persistence and long-term control of tumor cell targets. MM and AML cell lines were found to express differing levels of the LeY antigen ranging from negative (median fluorescence intensity (MFI) equal to mature lymphocytes (lymph) as internal control) to strongly positive (up to10×MFI lymph). Furthermore, 25/46 (54%) and 15/29 (52)% of primary MM and AML bone marrow samples were LeY-positive (≥5×MFI lymph), respectively. Lewis Y expression did not correlate with patient age, gender, clinical risk status, cytogenetic abnormalities, extent of previous chemotherapy, degree of bone marrow infiltrate, disease subtype, cytopenias in peripheral blood (MM and AML), LDH, WBC 〉 or ≤ 30×109/L (AML), beta2 microglobulin, albumin or pretreatment with bortezomib, thalidomide or lenalidomide (MM). We manufactured a novel retroviral vector construct enabling efficient transduction of PBMC-derived T cells with resultant high expression (up to 65%) of a single-chain anti-LeY chimeric T cell receptor comprising T cell activation via CD3zeta and CD28 signaling domains. Under GMP conditions, we achieved 〉100-fold expansion of T cells using a 12 day culture protocol. Anti-LeY T cells lysed LeY-positive tumor cells in vitro while sparing LeY-negative control tumor cells and LeY expressing neutrophils (moderate LeY expression, 〉3×MFI lymph). Similar transduction rates were achieved in CD4 and CD8 T cell subsets. Twelve-day culture T cells showed low expression levels of CD45RA and CCR7, moderate levels of the co-stimulatory molecules CD27 and CD28, and active proliferation in response to IL-2 and IL-15, suggesting an effector memory phenotype. On re-exposure to LeY expressing tumor cells, anti-LeY T cells displayed active proliferation and IFN-gamma production. In vivo efficacy was demonstrated in three independent experiments of a MM xenograft mouse model showing improved disease free survival of mice receiving anti LeY T cells compared to control mice treated with untransduced T cells. Transplantation of syngeneic murine anti LeY T cells into sublethally irradiated Balb/C mice subsequently monitored for up to two years was found to be safe without generation of lymphoproliferative disorders arising from the anti LeY T cells and no impact on OS compared to irradiated control mice not receiving adoptive T cell transfer. Consequently, we have developed a first in human phase I trial of autologous anti LeY T cells for patients with LeY-positive MM or AML. LeY is a promising and immunologically relevant target for T cell immunotherapy and our product is likely to lead to persistence of anti-LeY T cells in patients, an outcome which will be specifically addressed in our upcoming study.