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Background Berzosertib (formerly M6620, VX-970) is a highly potent and selective, first-in-class inhibitor of ataxia telangiectasia and Rad3-related protein kinase (ATR). We assessed multiple ascending doses of berzosertib + gemcitabine ± cisplatin in patients with resistant/refractory advanced solid tumours. Methods We evaluated the safety, tolerability, pharmacokinetics (PK) and preliminary efficacy of intravenous berzosertib + gemcitabine ± cisplatin using a standard 3 + 3 dose-escalation design. The starting doses were berzosertib 18 mg/m2, gemcitabine 875 mg/m2 and cisplatin 60 mg/m2. Results Fifty-two patients received berzosertib + gemcitabine and eight received berzosertib + gemcitabine + cisplatin. Four patients receiving berzosertib + gemcitabine had a total of seven dose-limiting toxicities (DLTs) and three receiving berzosertib + gemcitabine + cisplatin had a total of three DLTs. Berzosertib 210 mg/m2 (days 2 and 9) + gemcitabine 1000 mg/m2 (days 1 and 8) Q3W was established as the recommended Phase 2 dose (RP2D); no RP2D was determined for berzosertib + gemcitabine + cisplatin. Neither gemcitabine nor cisplatin affected berzosertib PK. Most patients in both arms achieved a best response of either partial response or stable disease. Conclusions Berzosertib + gemcitabine was well tolerated in patients with advanced solid tumours and showed preliminary efficacy signs. Clinical trial identifier NCT02157792.

Purpose Berzosertib (formerly M6620) is the first-in-class inhibitor of ataxia–telangiectasia and Rad3-related protein, a key component of the DNA damage response, and being developed in combination with chemotherapy for the treatment of patients with advanced cancers. The objectives of this analysis were to characterize the pharmacokinetics (PK) of berzosertib across multiple studies and parts, estimate inter-individual variability, and identify covariates that could explain such variability. Methods A population PK analysis was performed using the combined dataset from two phase I clinical studies (NCT02157792, EudraCT 2013-005100-34) in patients with advanced cancers receiving an intravenous infusion of berzosertib alone or in combination with chemotherapy. The analysis included data from 240 patients across 11 dose levels (18–480 mg/m2). Plasma concentration data were modeled with a non-linear mixed-effect approach and clinical covariates were evaluated. Results PK data were best described by a two-compartment linear model. For a typical patient, the estimated clearance (CL) and intercompartmental CL were 65 L/h and 295 L/h, respectively, with central and peripheral volumes estimated to be 118 L and 1030 L, respectively. Several intrinsic factors were found to influence berzosertib PK, but none were considered clinically meaningful due to a very limited effect. Model simulations indicated that concentrations of berzosertib exceeded p-Chk1 (proximal pharmacodynamic biomarker) IC50 at recommended phase II doses in combination with carboplatin, cisplatin, and gemcitabine. Conclusions There was no evidence of a clinically significant PK interaction between berzosertib and evaluated chemo-combinations. The covariate analysis did not highlight any need for dosing adjustments in the population studied to date. Clinical Trial information NCT02157792, EudraCT 2013-005100-34

BackgroundBerzosertib (formerly M6620, VX-970) is a highly potent and selective, first-in-class ataxia telangiectasia-mutated and Rad3-related protein kinase (ATR) inhibitor. We assessed the safety, tolerability, pharmacokinetics, and preliminary efficacy of berzosertib plus cisplatin.MethodsAdult patients with advanced solid tumours refractory or resistant to standard of care therapies received ascending doses of cisplatin (day 1) and berzosertib (days 2 and 9) every 3 weeks (Q3W).ResultsThirty-one patients received berzosertib (90–210 mg/m2) and cisplatin (40–75 mg/m2) across seven dose levels. The most common grade ≥3 treatment-emergent adverse events were neutropenia (20.0%) and anaemia (16.7%). There were two dose-limiting toxicities: a grade 3 hypersensitivity reaction and a grade 3 increase in alanine aminotransferase. Berzosertib 140 mg/m2(days 2 and 9) and cisplatin 75 mg/m2(day 1) Q3W was determined as the recommended Phase 2 dose. Cisplatin had no apparent effect on berzosertib pharmacokinetics. Of the 31 patients, four achieved a partial response (two confirmed and two unconfirmed) despite having previously experienced disease progression following platinum-based chemotherapy.ConclusionsBerzosertib plus cisplatin is well tolerated and shows preliminary clinical activity in patients with advanced solid tumours, warranting further evaluation in a Phase 2 setting.Clinical Trials IdentifierNCT02157792.

BackgroundCV8102 is a non-coding, non-capped RNA complexed with a carrier peptide activating the innate (via TLR7/8, RIG-I) and adaptive immune system.1 2 An ongoing phase I trial is investigating the intratumoral (i.t.) administration of CV8102 in patients with advanced cutaneous melanoma (cMEL), squamous cell carcinoma of the skin (cSCC) or head and neck (hnSCC) and adenoid cystic carcinoma (ACC), either as a single agent or in combination with systemic anti-PD-1 antibodies. Preliminary immune profiling results will be reported.MethodsAn open-label, cohort-based, dose escalation and expansion study in patients with advanced cMEL, cSCC, hnSCC or ACC is ongoing investigating CV8102 i.t. as single agent and in combination with anti-PD-1 antibodies. Eight i.t. injections of CV8102 were administered over a 12 week period with optional continuing treatment in case of clinical benefit. In the initial dose escalation part, the recommended phase II dose for subsequent cohort expansion was defined. Blood samples for immune cell phenotyping, RNA sequencing (RNAseq) and serum cytokine/chemokine analysis were collected at baseline and multiple time points during the treatment period. For characterization of the tumor microenvironment (TME), optional core needle biopsies of injected and/or non-injected lesions were taken before, during and after treatment. Changes on various tumor-infiltrating immune cells were assessed by multiplex immunofluorescence (MultiOmyx < sup >TM ) and immune-related gene expression profiling using nCounter® Pan Cancer IO360 < sup >TM panel (NanoString).ResultsDuring the dose escalation part, 33 patients received CV8102 (dose range of 25–900 µg) as single agent and 25 patients received CV8102 in combination with an anti-PD-1 antibody. A dose of 600 µg was selected as recommended phase II dose. Serum cytokine/chemokine and blood RNAseq analysis showed transient increases in several markers like interferons alpha and gamma after the first dose. First analyses of paired biopsies showed changes in the TME of injected and non-injected lesions. Complete results of cytokine and chemokine analysis in serum and blood RNAseq for the dose escalation cohorts will be presented. Multiplex immunofluorescence and gene expression profiling from paired biopsies from individual patients will be also included.ConclusionsIntratumoral injection of CV8102 activated several cytokine/chemokine pathways in the peripheral blood and showed immunological changes in the tumor microenvironment of injected and non-injected lesions.Trial RegistrationNCT03291002ReferencesZiegler A, Soldner C, Lienenklaus S, Spanier J, Trittel S, Riese P, Kramps T, Weiss S, Heidenreich R, Jasny E, Guzmán CA, Kallen KJ, Fotin-Mleczek M, Kalinke U. A New RNA-Based Adjuvant Enhances Virus-Specific Vaccine Responses by Locally Triggering TLR- and RLH-Dependent Effects. J Immunol 2017;198(4):1595–1605. doi: 10.4049/jimmunol.1601129.Heidenreich R, Jasny E, Kowalczyk A, Lutz J, Probst J, Baumhof P, Scheel B, Voss S, Kallen KJ, Fotin-Mleczek M. A novel RNA-based adjuvant combines strong immunostimulatory capacities with a favorable safety profile. Int J Cancer 2015 Jul 15;137(2):372–84. doi: 10.1002/ijc.29402.Ethics ApprovalThe study was approved by the Central Ethics Committees in Tuebingen, Germany under 785/2016AMG1, in France by the COMITE DE PROTECTION DES PERSONNES SUD-EST I under 2019–49, approval dated 17-May-2019, in Barcelona, Spain by the CEC COMITÉ DE ÉTICA DE INVESTIGACIÓN CLÍNICA CON MEDICAMENTOS del Hospital Universitari Vall d’Hebron, approval date 28-Nov-2019 under the EUdraCT number, in Austria by the Central Ethics Committee in Graz under 31–426 ex 18/19 approved on 19-Sep-2019.ConsentWritten informed consent from the patient was obtained for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Ataxia-telangiectasia mutated and Rad3-related kinase (ATR) is one of the key players in the DNA damage response (DDR) to replication stress. M6620 is a potent and selective ATR inhibitor which has been shown to sensitize cancer cells to the lethal effects of DNA-damaging chemotherapeutic agents. We previously demonstrated that M6620 significantly enhances the anti-tumor efficacy of dual combinations of platinum-based chemotherapy and avelumab, an FDA-approved anti-PD-L1 monoclonal antibody, in syngeneic tumor models in vivo. The purpose of this study was to further elucidate the potential mechanisms of action that drive the therapeutic benefit of this triplet combination therapy. Treatment with certain chemotherapeutic agents has been reported to promote immunogenic cell death (ICD) in tumor cells. We utilized in vitro assays to test the expression of markers of immunogenic cell death in murine MC38 colorectal carcinoma cells. After 72 hours of treatment, carboplatin and M6620 dual combination treatment significantly increased the levels of Calreticulin, HMGB1, and ATP relative to treatment with either single agent. In addition, gene expression profiling was used to determine pathways differentially induced by the drug treatments. RNA-sequencing (RNA-seq) analysis was performed on MC38 cells treated in vitro with carboplatin, M6620, or dual combination therapy. Comparison of gene expression between treatment groups at 48 hours post-treatment revealed that the dual combination therapy significantly increased expression of genes regulated by type I/II interferons (IFN), including CCl5, CXCL10, CXCL11 and ISG15. RNA-seq gene expression analysis was also performed on MC38 tumor samples from mice treated with carboplatin, M6620, avelumab, carboplatin/avelumab dual therapy, or carboplatin/M6620/avelumab triple combination therapy. Triple combination therapy induced a more robust upregulation of IFN-inducible genes at day 3 compared with monotherapies or carboplatin/avelumab dual combination, consistent with in vitro observations that M6620 and carboplatin dual combination therapy enhanced expression of genes regulated by IFNs. Taken together, these data suggest that potentiation of immunogenic cell death and induction of pro-inflammatory responses by M6620, in the context of chemotherapy-induced DNA damage, may contribute to the enhanced anti-tumor efficacy observed with carboplatin, M6620, and avelumab triple combination therapy. Citation Format: Marat Alimzhanov, Ashwin George, Parantu Shah, Astrid Zimmerman, Andreas Schroeder, Martin Falk, Christiane Amendt, Joern-Peter Halle, Andree Blaukat, Frank Zenke, Patricia Soulard. Induction of immunogenic cell death and interferon signaling by carboplatin and the ATR inhibitor M6620 may contribute to anti-tumor activity of M6620-carboplatin-avelumab triplet combination in MC38 tumor model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 964.

Background Tecemotide is a MUC1-antigen-specific cancer immunotherapy. The phase III START study did not meet its primary end point but reported notable survival benefit with tecemotide versus placebo in an exploratory analysis of the predefined patient subgroup treated with concurrent chemoradiotherapy. Here, we attempted to gain further insight into the effects of tecemotide in START. Patients and methods START recruited patients who did not progress following frontline chemoradiotherapy for unresectable stage III non-small-cell lung cancer. We present updated overall survival (OS) data and exploratory analyses of OS for baseline biomarkers: soluble MUC1 (sMUC1), antinuclear antibodies (ANA), neutrophil/lymphocyte ratio (NLR), lymphocyte count, and HLA type. Results Updated OS data are consistent with the primary analysis: median 25.8 months (tecemotide) versus 22.4 months (placebo) (HR 0.89, 95% CI 0.77–1.03, P = 0.111), with ∼20 months additional median follow-up time compared with the primary analysis. Exploratory analysis of the predefined subgroup treated with concurrent chemoradiotherapy revealed clinically relevant prolonged OS with tecemotide versus placebo (29.4 versus 20.8 months; HR 0.81, 95% CI 0.68–0.98, P = 0.026). No improvement was seen with sequential chemoradiotherapy. High sMUC1 and ANA correlated with a possible survival benefit with tecemotide (interaction P = 0.0085 and 0.0022) and might have future value as biomarkers. Interactions between lymphocyte count, NLR, or prespecified HLA alleles and treatment effect were not observed. Conclusion Updated OS data support potential treatment benefit with tecemotide in patients treated with concurrent chemoradiotherapy. Exploratory biomarker analyses suggest that elevated sMUC1 or ANA levels correlate with tecemotide benefit. ClinicalTrials.gov number NCT00409188.

e21048Background: Ataxia telangiectasia and Rad3-related protein (ATR) is an essential regulator of the DNA damage response and is required for the survival of proliferating cells. DNA-damaging age...

Background: There is increasing interest in using combination immunotherapy in the neoadjuvant setting in prostate cancer, however, endpoints for such studies remain elusive. We have conducted a clinical trial evaluating immunotherapy with ADT in patients with high risk prostate cancer. Patients were assessed for immune responses and changes in endorectal (er) MRI which can be used to assess intraprostatic tumors. Methods: Treatment-naïve high-risk (Gleason 8-10, PSA>20, or stage T3) prostate cancer patients (pts) were randomized to standard ADT+Radiation + an immunotherapy targeting MUC1 (tecemotide, aka L-BLP25) in this trial (NCT01496131). ADT consisted of gonadotropin-releasing hormone therapy. Immunotherapy included low dose (300 mg/m2, maximum 600 mg) pre-treatment cyclophosphamide for regulatory T-cell depletion. erMRI was done at baseline and after 2 months of immunotherapy including multiparametric MRI evaluation of apparent diffusion coefficient (ADC) maps from diffusion-weighted MRI. Monthly peripheral blood assessments analyzed immune cell subsets using flow cytometry and intracellular cytokine (ICC) staining for MUC-1 specific responses. Results: 28 pts with high risk prostate cancer were enrolled (n=14/arm). As expected, PSA declined in all pts 2 months after ADT. erMRI after 2 months of treatment suggested greater improvements in ADC values in pts receiving immunotherapy+ADT vs. ADT alone. Improved ADC on MRI indicates increased intratumoral diffusion and has been associated with decreased tumor density. The improvements in ADC were seen when one dominant tumor per patient was evaluated (p=0.17) but were more pronounced when up to 3 lesions were evaluated per pt (n=44 lesions; p=0.031). Compared to baseline, there were trends to increases in CTLA4+ CD8+ T-cells consistent with immune activation and decreases in myeloid derived suppressor cells (MDSCs) in pts receiving immunotherapy+ADT coinciding with the erMRI changes. These immune findings were not seen in the ADT alone group. 3 of 14 pts had MUC1 specific immune response by ICC. 2 of these patients had the greatest changes in ADC noted on erMRI over a 2-year period. Conclusions: Based on assessments by erMRI, pts who received ADT+immunotherapy had greater improvements in ADC than pts receiving ADT alone. Given that ADC improvements are associated with decreased tumor density, this suggests a possible greater anti-tumor effect of the ADT-immunotherapy combination vs. ADT alone. These findings were associated with trends to increased activated CD8+ T-cells and decreased MDSCs in pts receiving immunotherapy+ADT, with 3/14 pts having MUC1 specific immune responses. Further studies are required to confirm the potential to use ADC on erMRI as a potential (bio)marker of anti-tumor effect of immune combinations including ADT. Citation Format: Ravi A. Madan, Baris Turkbey, Lauren M. Lepone, Renee N. Donahue, Italia Grenga, Samuel Samuel Borofsky, Peter A. Pinto, Deborah Citrin, Aradhana Kaushal, Andra Krauze, Sheri McMahon, Myrna Rauchhorst, Anna Couvillon, Martin H. Falk, S Peter Eggleton, Stephen C. Greco, Peter L. Choyke, William L. Dahut, Jeffrey Schlom, James L. Gulley. Neoadjuvant immunotherapy with androgen deprivation therapy (ADT) prior to radiation in prostate cancer: Impact on multiparametric prostate MRI and immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-059. doi:10.1158/1538-7445.AM2017-LB-059

Many cell lines are sensitive to growth at low cell density and undergo apoptosis induced by oxidative stress if the cell density is decreased below a critical threshold. In stable transfection experiments this cell density-dependent growth may be the limiting factor, since during drug selection the cell density falls below the critical threshold, precluding outgrowth of transfected clones. We describe here a simple protocol for the establishment of stably transfected human B cell lines making use of the protective action of antioxidants. The protocol includes: (i) seeding the cells in medium supplemented with sodium pyruvate, alpha-thioglycerol and bathocuproine disulfonate; (ii) delaying the onset of dominant marker selection to improve recovery of the cells after electroporation. Stably transfected clones have thus been obtained from Burkitt's lymphoma lines, which have been regarded as untransfectable. Using this protocol the stable transfection efficiency with episomal plasmids approaches the transient transfection efficiency, indicating that virtually every transfected cell can be established as a stably transfected clone. This protocol should also prove useful for other cell lines, e.g. neuronal cells, having similar sensitivities to oxidative stress.

Burkitt lymphoma (BL) cells are highly sensitive to suboptimal growth conditions and undergo apoptosis when seeded at reduced serum concentration or low cell density. Irradiated fibroblasts can protect BL cells from apoptosis induced by lowering the serum concentration or cell density through secretion of a survival- and proliferation-promoting activity which is soluble and labile. Murine B cells have a restricted uptake capacity for cystine and require cysteine for proliferation, which can be supplied efficiently by feeder cells. Therefore, we have studied the role of cysteine and other compounds with free thiol groups for survival and proliferation of BL cells. Cysteine, when added alone, exerted strong toxicity on BL cells. This toxicity could be counteracted by the addition of catalase, pyruvate or bathocuproine disulfonate (BCS), all of which interfere with the production of hydrogen peroxide. Inhibition of the toxicity of cysteine was necessary to unravel the survival- and growth-promoting activity of cysteine at low cell density. Alpha-thioglycerol, beta-mercaptoethanol and dithiothreitol had similar toxic activity in the absence of catalase, pyruvate and BCS and, through stimulation of cysteine uptake and glutathione synthesis, displayed a similar survival- and growth-promoting activity in the presence of the protective agents. The survival- and proliferation-inducing activity of thiol compounds in the presence of catalase, pyruvate and BCS was not associated with induction of BCL-2 or BAX. Cysteine/cystine uptake and the intra/cellular glutathione level are thus important parameters, determining the susceptibility vs. resistance of BL cells to apoptosis.

30 Background: Endorectal(er) MRI is an emerging tool in assessing intraprostatic tumors. Immunotherapy development in prostate cancer has been limited due to the lack of intermediate (bio)markers of response. Methods: Untreatedpts with high-risk prostate cancer were randomized in a trial (NCT01496131) of standard ADT+Radiation + an immunotherapy targeting MUC1 (tecemotide, aka L-BLP25). Pts had erMRI at baseline and after 2 months of ADT+/- biweekly immunotherapy. Low dose (300 mg/m2, maximum 600 mg) cyclophosphamide for regulatory T-cell depletion preceded first immunotherapy. Multiparametric MRI included evaluation of apparent diffusion coefficient (ADC) maps from diffusion-weighted MRI. Monthly peripheral blood assessments utilized flow cytometry to evaluate immune cell subsets. This analysis focuses on the 2 month neoadjuvant period of ADT+/-immunotherapy before radiation. Results: 28 pts (n = 14/arm) with high risk prostate cancer (Gleason 8-10, PSA > 20, or stage T3) were enrolled. PSA declined in all pts 2 months after ADT. erMRI findings at 2 months indicated greater improvements in ADC values in pts receiving immunotherapy+ADT vs. ADT alone. Improved ADC on MRI suggests improvements in intratumoral diffusion and has been associated with decreased tumor density. This relative improvement between the groups occurred both per patient (p = 0.16) and per lesion (p = 0.031). Relative to baseline, pts receiving immunotherapy+ADT had increases in CTLA4+ CD8+ T-cells consistent with immune activation (p = 0.0134) and decreases in myeloid derived suppressor cells (MDSCs; p = 0.0353) during the neoadjuvant period corresponding to the erMRI changes. These immune findings were not seen in the ADT alone group. Conclusions: Pts who received immunotherapy+ADT for 2 months had greater improvements in ADC values on erMRI, consistent with decreased tumor density, relative to pts receiving ADT alone. Corresponding increases in activated CD8+ T-cells and decreases in MDSCs were seen in pts receiving vaccine+ADT. These preliminary findings suggest that ADC on MRI may be useful in assessing immunologic impact. Further study is warranted. Clinical trial information: NCT01496131.

To present an updated survival analysis of an open-label, parallel-group, phase IIB trial of BLP25 liposome vaccine (L-BLP25) in patients with stage IIIB or IV non-small-cell lung cancer (NSCLC). Patients were randomized to either L-BLP25 plus best supportive care (BSC) or BSC alone. Patients in the L-BLP25 arm received subcutaneous vaccinations of L-BLP25 930 μg weekly for 8 weeks, followed by maintenance vaccinations at 6-week intervals. Median survival time was 4.2 months longer in patients receiving L-BLP25 plus BSC (n = 88) than in those receiving BSC alone (n = 83; 17.2 months vs. 13.0 months, respectively; hazard ratio [HR] 0.745, 95% confidence interval [CI] 0.533–1.042). The 3-year survival rate was 31% in patients receiving L-BLP25 plus BSC and 17% in those receiving BSC (P = 0.035). In the stratified subset of patients with stage IIIB loco-regional (LR) disease, median survival time was 17.3 months longer in patients receiving L-BLP25 plus BSC (n = 35) than in those receiving BSC (n = 30; 30.6 months vs. 13.3 months, respectively; HR 0.548, 95% CI 0.301–0.999). In this subgroup, 3-year survival was 49% in patients receiving L-BLP25 plus BSC and 27% in those receiving BSC (P = 0.070). Confirming the initial results, further follow-up continues to show that survival time for patients with stage IIIB/IV NSCLC was longer with L-BLP25 plus BSC compared with BSC alone, with the greatest difference seen in patients with stage IIIB LR disease.

In about 80% of Burkitt's lymphoma cases, the tumour cell harbours a reciprocal chromosomal translocation which invariably transposes the coding exons 2 and 3 of c-myc from chromosome 8 to the immunoglobulin heavy chain locus on chromosome 14. Those t(8;14) translocations which disrupt chromosome 8 within or close to the c-myc gene are well documented. In this study we have focussed on t(8;14) translocations with the chromosomal breakpoint far upstream of c-myc. We analyzed the breakpoint position in 44 BL cell lines with t(8;14) translocations of different geographical origin and identified 9 cell lines with the breakpoint more than 14 kb upstream of c-myc. In these cell lines the positions of the translocation junctions on the derivative chromosomes 8q- and 14q+ were mapped by pulsed field gel electrophoresis and multicolour fluorescence in situ hybridization. The breakpoints occur at distances between 55 and more than 340 kb upstream of c-myc with no preferential site on chromosome 8. On chromosome 14, however, the translocation breakpoints are clustered in a narrow region 5′ of the intron enhancer of the immunoglobulin heavy chain gene. In 7 of 9 cases, the enhancer is fused to the c-myc bearing sequences of chromosome 8. In two cases, the translocation has occurred in switch μ and downstream of Cμ, respectively. The impact of these results with respect to the hypothesis, that cisregulatory sequences from the immunoglobulin heavy chain locus can deregulate c-myc expression in a manner sufficient for tumour formation, is discussed.

Treating rare, primary cardiac soft-tissue sarcomas (C-STS) with heart transplantation (HTx) is controversial. Conventional tumor resection only partially alleviates the disease, and patients die of local recurrence of the tumor or of distant metastases. Heart transplantation offers the opportunity to eradicate the primary malignancy completely. In our experience of 4 patients with C-STS indicates, HTx followed by post-operative chemotherapy does not affect the long-term outcome. However, pre-operative chemotherapy can regress tumors in chemosensitive C-STS and potentially eradicate early micrometastases. Consecutive HTx for responders may then offer a chance of long-term survival.

TPS7608 Background: Tecemotide is an antigen-specific cancer immunotherapy targeting the mucinous glycoprotein MUC1, which is overexpressed and aberrantly glycosylated in a number of cancers includ...

7500 Background: L-BLP25 is a MUC1 antigen specific cancer immunotherapy. We report results from the phase III START study of L-BLP25 in patients (pts) not progressing after primary chemoradiotherapy (CRT) for stage III NSCLC. Methods: From Jan 2007 to Nov 2011, 1513 pts with unresectable stage III NSCLC that did not progress after CRT (platinum based chemo and ≥50 Gy) were randomized (2:1; double-blind) to L-BLP25 (806 µg lipopeptide) or placebo (PBO) SC weekly x 8 then Q6 weeks until disease progression or withdrawal. Cyclophosphamide 300 mg/m2 x 1 or saline was given 3 days prior to first L-BLP25/PBO dose. Primary endpoint was overall survival (OS). Results: The primary analysis population (n=1239) was defined prospectively to try to account for a clinical hold by excluding pts randomized 6 months (m) before the hold. Arms were balanced for baseline characteristics. Median age was 61 y; 38.2% had stage IIIA and 61.3% IIIB; 65% had concurrent and 35% sequential CRT. Median OS was 25.6 m with L-BLP25 vs. 22.3 m with PBO (adjusted HR 0.88, 95% CI 0.75-1.03, p=0.123). Secondary endpoints time-to-progression and time-to-symptom-progression support consistency of results: HR 0.87 (95% CI 0.75-1.00, p=0.053) and 0.85 (95% CI 0.73-0.98, p=0.023). In predefined subgroup analyses, pts with concurrent CRT (n=806) had median OS of 30.8 m (L-BLP25) vs. 20.6 m (PBO; HR 0.78, 95% CI 0.64-0.95, p=0.016), while median OS with sequential CRT was 19.4 m (L-BLP25) vs. 24.6 m (PBO; HR 1.12, 95% CI 0.87-1.44, p=0.38; interaction p=0.032, Cox PH model). Sensitivity analyses revealed that there was no OS benefit in pts randomized 6 m before the hold (HR 1.09, CI 0.75-1.56, p=0.663). LEBLP25 was well tolerated with no safety concerns identified and no emergent evidence of immune related adverse events. Conclusions: L-BLP25 maintenance therapy in stage III NSCLC was well tolerated, but did not significantly prolong OS. Sensitivity analyses showed a smaller treatment effect due to the clinical hold, suggesting that longer uninterrupted treatment with L-BLP25 is required. Clinically meaningful prolongation of OS was observed in the predefined subgroup of pts with primary concurrent CRT. Clinical trial information: NCT00409188.

To develop a transformation system with a conditional Epstein—Barr virus nuclear antigen 2 (EBNA2) gene, we fused the hormone binding domain of the oestrogen receptor to the N or C terminus of EBNA2. In promoter transactivation as well as primary B cell transformation assays these chimeric EBNA2 proteins are able to substitute for wild-type EBNA2 in the presence of oestrogen. Here we provide evidence that this transformation is the result of double infection of a cell with two virions, the P3HR1 virus genome and a mini-EBV plasmid carrying the chimeric EBNA2 gene. Unexpectedly, expression of the same EBNA2-oestrogen receptor fusion protein in established human B cell lymphoma lines resulted in growth retardation or growth arrest upon the addition of oestrogen. By titrating the oestrogen concentration in these stably transfected cells, the growth retarding and the transactivating function of the chimeric proteins could not be dissociated. We propose that growth inhibition of established B cell lymphoma lines is a novel function of EBNA2 which has not been detected in the absence of an inducible system. It remains open whether the growth retarding property of the EBNA2-oestrogen receptor fusion protein in B cell lymphoma lines is due to unphysiologically high expression of the chimeric protein or to interference with a cellular programme driving proliferation in these cell lines.

The cytokine stem cell factor (SCF) synergizes with interleukin-7 (IL- 7) to enhance the proliferation of pre-B cells. To examine the role of SCF and its receptor, c-kit, in the pathogenesis of pediatric Burkitt's lymphomas (BL), we investigated the expression of SCF and c-kit in BL cells and the mitogenic activity of SCF on BL cells. A panel of 13 BL cell lines and 7 fresh biopsy tumors was investigated. BL cells were stimulated either by Epstein-Barr virus (EBV) infection or by different reagents and cytokines, and expression of SCF and c-kit was studied on the mRNA level by Northern blot analysis and reverse-transcriptase polymerase chain reaction (RT-PCR), followed by Southern blotting. c- kit expression was also studied by fluorescence-activated cell sorting and by crosslinking of digoxigenin-labeled recombinant human SCF to the cell surface. Proliferation of BL cell lines was measured by 3H- thymidine incorporation. Low-level expression of c-kit mRNA was detected in 2 of 13 unstimulated BL cell lines and in 1 fresh BL tumor. One cell line showed upregulation of c-kit mRNA with A23187 and downregulation with phorbol myristate acetate. Neither c-kit nor SCF could be detected in any other cell line under any condition of stimulation as analyzed by Northern blot analysis, RT-PCR followed by Southern blot analysis, crosslinking, and immunofluorescence. No response to SCF was seen in 3H-thymidine incorporation assays. We conclude that most BL cells express neither SCF nor c-kit and that the low-level expression of c-kit in some BL cells most likely has no biologic significance.

Background Chromosomal rearrangements in the receptor tyrosine kinase (RTK) ROS1 define a new molecular subset of NSCLC. ALK inhibitors can inhibit ROS1 kinase activity in cell lines. We examined the efficacy and safety of crizotinib, a small molecule inhibitor of two other RTKs, MET and ALK, in patients with advanced, ROS1-rearranged NSCLC. Methods Patients with advanced NSCLC harboring ROS1 rearrangement, as determined using a break-apart FISH assay, were recruited into an expansion cohort of the original phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg BID. The objective response rate (ORR) was determined based on RECIST v1.0. The disease control rate (DCR; stable disease [SD] + partial response [PR] + complete response [CR]) was evaluated at weeks 8 and 16. Results At the time of data cut-off on April 19, 2012, 15 patients with ROS1 NSCLC had received crizotinib and 14 were evaluable for response. The median age was 54 years (range 31–72). Fourteen patients were never-smokers and all had adenocarcinoma histology. All patients were confirmed negative for ALK rearrangement. The median number of prior treatments was 1 (range 0–6). The first ROS1 patient received crizotinib on October 19, 2010. To date, the ORR is 57% (8/14; 95% CI 28.9–82.3), with 7 PR and 1 CR. There were 4 SD, one of which was unconfirmed as PR at the time of data cut-off. The DCR at 8 weeks was 79% (11/14). Median duration of treatment was 25.7 weeks (range 0.1 + to 65.3 + ), and 12 patients continue on study. The pharmacokinetics, antitumor activity and safety profile of crizotinib in this group of patients were similar to those observed in patients with ALK-positive NSCLC. Conclusions Crizotinib is associated with clinically significant antitumor activity in patients with ROS1 NSCLC. Similar to ALK, ROS1 rearrangement defines another unique molecular subset of NSCLC patients for whom crizotinib therapy may be highly effective. Importantly, this study represents the first clinical investigation of ROS1 as a driver mutation and therapeutic target in cancer. Disclosure S.I. Ou: Advisory role: Pfizer, Genentech. Compensated Honoraria: Pfizer, Genentech, Lilly. Myself Research funding: Pfizer. Myself (institution). D.R. Camidge: Advisory role: Pfizer. Myself. Honoraria: Pfizer. Myself. L. Tye: Employment: Pfizer. Clinician. Myself. Stock Ownership; Pfizer. Myself. K. Wilner: Emplyement: Pfizer. Senior Director. Myself. Compensated. Stock ownership: Pfizer. Myself. M. Varella-Garcia: Honoraria: Abbott Molecular. Myself. funding for the trial provided by NIH - yes. A.T. Shaw: Advisory role: Pfizer, Ariad, Chugai, Novartis, Daiichi-sankyo. Myself. Compensated Research funding: Astra Zeneca, Novartis. Myself. All other authors have declared no conflicts of interest.

Introduction L-BLP25 (Stimuvax®) is an antigen-specific cancer immunotherapeutic agent targeting the mucin 1 tumour-associated antigen. Phase IIb data suggest prolonged overall survival with L-BLP25 when administered after completion of chemoradiotherapy for locally advanced non-small-cell lung cancer (NSCLC; non-significant). This study was initiated in April 2005 to obtain safety data when modifications of the adjuvant component of the immunotherapeutic were performed. Here we report an updated analysis of long-term data based on the cut-off date 18 May 2011. Methods The primary objective of this open-label phase II study was to evaluate the safety of the new formulation of L-BLP25 in patients with unresectable stage IIIA/B NSCLC, with a secondary objective of assessment of survival time. Following completion of initial chemoradiotherapy, patients with stage III NSCLC received treatment with L-BLP25 starting with weekly injections over 8 weeks, followed by injections given every 6 weeks from week 13 onwards until disease progression. A single low dose of intravenous cyclophosphamide 300 mg/m2 (maximum 600 mg) was given 3 days before first vaccination. Results Twenty-two patients were enrolled. After a median follow-up of 70.3 months (median treatment duration 9.9 months [range, 1–73]), median survival time was 51.9 months (95%CI, 17.5, not estimable [NE]) and median progression-free survival was 31.4 months (95%CI, 5.7, NE). Five-year survival was 50% (95%CI, 29–71%); previously reported 1- and 2-year survival rates, 82% (95%CI, 66–98%) and 64% (95%CI, 44–84%), respectively. Four patients survived more than 72 months. Safety findings were consistent with previous reports; adverse events (AEs) consisted mainly of fatigue (59%), injection-site reactions (55%) and mild-to-moderate influenza-like illness. Two serious treatment-emergent AEs were assessed as being treatment-related: cholecystitis and pneumonia. Conclusions Long-term follow-up of this small patient population provides encouraging survival data for L-BLP25 maintenance therapy in stage III NSCLC. Long-term safety data were consistent with previous reports and did not reveal any new safety issues. Disclosure C. Butts: Charles Butts has provided consultancy and served on Speakers' Bureaux for Merck Serono. A. Maksymiuk: Andrew Maksymiuk has some stock shares in Merck Canada ( M. Falk: Martin Falk is a Merck Serono employee. A.H. Loos: Dr Loos is a Merck employee. All other authors have declared no conflicts of interest.

TPS4701 Background: In high-risk prostate cancer, radiation therapy (RT) + androgen deprivation therapy (ADT) improve survival. Nonetheless, 10-year disease specific mortality is about 25%. L-BLP25 is a cancer vaccine containing the BLP25 lipopeptide that targets MUC1 tumor antigen. It may enhance immune targeting of cells that express MUC1 (e.g. prostate cancer). In murine models, RT synergizes with vaccine-induced anti-cancer immunity (augments T-cell mediated cancer cytolysis, up-regulates cellular Fas and co-stimulatory/adhesion molecules). ADT augments T-cell trafficking to prostate. Immune response to combining the three (L-BLP25 + RT + ADT) is not known. The current trial intends to study this immune response to L-BLP25 + RT + ADT and compare it to RT+ADT alone. Using ELISPOT, endo-rectal MRI and serial prostate biopsies, this trial was designed to correlate systemic immune response with changes in tumor imaging and/or tumor microenvironment after treatment with L-BLP25. This trial may provide insight into immune response biomarkers that are most appropriate in this setting. Methods: A randomized (1:1), open-label, phase II trial of 42 pts is planned. Eligibility: Adult males with newly diagnosed high-risk prostate cancer (T3 or Gleason ≥ 8 or seminal vesicle involvement or N1 or PSA>20) and HLA-A2/A3 positivity (to allow for ELISPOT analysis). The vaccine arm will receive RT + 2-year ADT + L-BLP25. Standard arm will receive RT + 2-year ADT. L-BLP25 vaccine schedule: biweekly X 5 starting with neo-adjuvant ADT, then 6 weekly X 4 starting with RT. A single 300mg/m2 cyclophosphamide infusion (decreases suppressor T-cells) will be given 3 days before L-BLP25 to enhance immune response in the vaccine arm. The impact of L-BLP25 + RT+ADT on MUC-1-specific systemic immune response will be determined using interval peripheral blood ELISPOT assays. Endo-rectal coil MRI will be done before and after treatment to study prostate signal changes for correlative and predictive analysis. MRI-UltraSound guided lesion-targeted serial prostate biopsies will be obtained to assess immune response in tumor microenvironment. Two pts have been enrolled.

e13058 Background: Safety data are not yet available for the potential long-term treatment of NSCLC with vaccines. In a phase IIB open-label, randomized trial, treatment with the therapeutic cancer...

3055 Background: The BLP25 liposome vaccine (L-BLP25, Stimuvax) is an investigational innovative therapeutic cancer vaccine incorporating immunoadjuvant monophosphoryl lipid A and synthetic MUC1 lipopeptide in a liposomal delivery system. In an open-label phase IIb study in 171 pts with stage IIIB/IV NSCLC randomized to best supportive care (BSC) alone (n=83) or BSC + L-BLP25 (1000 μg lipopeptide) (n=88), L-BLP25 pts received weekly vaccinations for 8 weeks (wks) and could continue maintenance vaccinations every 6 wks from wk 13. Results were encouraging (Butts et al, JCO 2005) and a remarkable number of pts received prolonged treatment (tmt). As a result a global phase III study (START) has been initiated. Methods: Safety results for 16 pts from the phase IIb study treated for ≥2 years with L-BLP25 are reported. Results: In these 16 pts (median age 57.5 years, ECOG PS: 0 in 5/16 [31%] and 1 in 11/16 [69%]), there were more females (56% vs 44% [9/16 vs 76/171]) and locoregional stage IIIB disease at entry (81% vs 38% [13/16 vs 65/171]) vs the phase IIb study population. Pts received L-BLP25 for 2.0–7.7 years and 10 pts were treated for >5 years. Compliance with tmt was good: almost all (96%) maintenance vaccinations were given every 6 wks according to the tmt schedule. L-BLP25 was well tolerated: the most common tmt-emergent adverse events (TEAEs) were cough (n=12, 75%), fatigue (n=12, 75%), and dyspnea (n=11, 69%). The pattern of TEAEs did not change significantly over time and there was no rise in the incidence of TEAEs with increasing time on tmt. Among the most common TEAEs in years 1, 2, and >2 were nausea (44, 19, and 38%), fatigue (56, 18, and 38%), chest pain (38, 31, and 31%), and cough (38, 25, and 31%). Twelve pts (75%) had tmt-related TEAEs (grade 1/2 in 11/12 pts [92%] and grade 3 in 1/12 [8%]): the most common were injection-site reactions. The occurrence of tmt-related TEAEs decreased with increasing tmt duration. There was no evidence of any TEAEs related to autoimmunity. Analysis of laboratory data did not indicate any long-term renal, liver, or other toxicity. Conclusions: Long-term use of L-BLP25 was without any identifiable safety issues. In particular, there was no evidence of autoimmune reactions with prolonged use. [Table: see text]

3075 Background: L-BLP25 is an innovative cancer vaccine that incorporates a synthetic MUC1 lipopeptide in a liposomal delivery system. L-BLP25 is expected to elicit an immune response to cancer cells that express MUC1. Previous clinical studies have demonstrated that L-BLP25 has the potential to extend survival of pts with stage IIIB locoregional NSCLC (Butts C et al., JCO 2005; 23:6674–6681). The present ph I-II study was designed to assess the safety of the current formulation of L-BLP25 using a monophosphoryl lipid A in pts with unresectable stage IIIA and stage IIIB NSCLC. Methods: Pts with stable disease or an objective response to upfront radical therapy with chemoradiation for unresectable stage III NSCLC, plus ECOG 0–1 were eligible. All pts were vaccinated according to a previously described schedule (1). Maintenance immunizations were administered every 6-wks until disease progression. Primary and secondary endpoints were safety and survival respectively. Results: Twenty-two pts were recruited at 7 sites in Canada. 16 pts were evaluated for this interim safety analysis (8 stage IIIA, 8 stage IIIB, median age; 57, ECOG 0 56%, concurrent chemotherapy; 93.8%). Thirteen pts had a partial response and 3 had stable disease following chemoradiation. Thirteen pts experienced an adverse event (AE) during the first 4 vaccinations of which 7 pts had a L-BLP25 related adverse event. Grade 1/2 AEs related to L-BLP25 ≥10% included fatigue, dyspnea, insomnia, anorexia, headache, diarrhea, paresthesia, abdominal pain, influenza-like illness, urinary tract infection and peripheral neuropathy. No pts discontinued L-BLP25 due to an AE and no grade 3/4 AEs related to L-BLP25 were reported. Six pts (37.5%) had an injection site reaction. As of September 2006, 10 pts were still on study treatment. Conclusions: This formulation of L-BLP25 was well tolerated and the side effect profile was similar to that seen in previous studies (1). A controlled global multi-center phase III trial is underway to further evaluate L-BLP25 in this patient population. No significant financial relationships to disclose.

Three Hodgkin's disease-derived cell lines were analysed for the organization of immunoglobulin and T cell receptor genes, for the expression of the interleukin 2 (IL-2) receptor gene, and for the cellular oncogene c-myc. All three cell lines have characteristic genotypic markers of lymphoid cells and can be classified as immature lymphoid cells with respect to the incomplete expression of their antigen receptor genes. This immature genotype is in contrast to the expression of activation antigens Ki-1 (CD 30), IL-2 receptor (CD 25), and HLA-DR. The results obtained are in agreement with studies obtained from primary Hodgkin's lymphomas, which are activated by as yet unknown mechanisms.

Three Hodgkin-derived cell lines (L428, L540, and CO) were studied for rearrangements and expression of immunoglobulin and T-cell receptor genes, and their genotype was compared to the phenotype. As far as the genotype is concerned, all 3 cell lines have characteristics of lymphoid cells; L428 of B, and L540 and CO of T-cell origin. L428 cells have one Ig heavy chain allele rearranged to C gamma and transcribed into RNA, while the second is deleted. Furthermore, L428 cells show an unusual immunoglobulin kappa light chain gene rearrangement involving deletion of the kappa constant gene in one allele, while the remaining kappa and lambda loci are in germline configuration. L540 and CO have, in contrast to L428 cells, the immunoglobulin genes in germline and T-cell receptor genes rearranged. The T-cell receptor beta and gamma genes are rearranged in both L540 and CO, whereas a rearrangement in the alpha locus was detected in L540 cells only. RNA of the size of functional beta chain transcripts was found in CO cells and of the size of functional alpha chain transcripts in L540 cells. All 3 cell lines are classified as immature lymphoid cells with respect to the limited expression of B- and T-cell antigens, respectively, and to the incomplete expression of their antigen receptor. The immaturity of lymphoid differentiation contrasts with the expression of activation antigens, i.e. Ki-1, Ki-24, HLA-DR, and IL-2 receptor. The immaturity of the cells excludes the possibility that the cells were activated along the physiological pathway, i.e. by interaction of the cell with antigen. The results obtained on the cell lines are in accordance with in vivo studies and suggest that Hodgkin and Sternberg-Reed cells are immature lymphoid cells which are activated by a still unknown mechanism.