Synergistic combinations of immunotherapeutic agents can improve the performance of anti-cancer therapies but may lead to immune-mediated adverse effects. These side-effects can be overcome by using a tumor-specific delivery system. Here, we report a method of targeted immunotherapy using an attenuated Salmonella typhimurium (SAM-FC) engineered to release dual payloads: cytolysin A (ClyA), a cytolytic anti-cancer agent, and Vibrio vulnificus flagellin B (FlaB), a potent inducer of anti-tumor innate immunity. Localized secretion of ClyA from SAM-FC induces immunogenic cancer cell death and promotes release of tumor-specific antigens and damage-associated molecular patterns, which establish long-term antitumor memory. Localized secretion of FlaB promotes phenotypic and functional remodeling of intratumoral macrophages that markedly inhibits tumor metastasis in mice bearing tumors of mouse and human origin. Both primary and metastatic tumors from bacteria-treated female mice are characterized by massive infiltration of anti-tumorigenic innate immune cells and activated tumor-specific effector/memory T cells; however, the percentage of immunosuppressive cells is low. Here, we show that SAM-FC induces functional reprogramming of the tumor immune microenvironment by activating both the innate and adaptive arms of the immune system and can be used for targeted delivery of multiple immunotherapeutic payloads for the establishment of potent and long-lasting antitumor immunity.

Purpose: In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (P_tetA) was 100-fold higher in expression strength than tetR promoter (P_tetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter. Procedures: In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by P_tetA and P_tetR, and Doxy releases TetR from tetO to de-repress P_tetA and P_tetR. Results: In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (P_tetA:P_tetR = 4~6:1) compared with that of pJL87 (P_tetA:P_tetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division. Conclusions: Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

E13120 Background: To determine the epidermal growth factor receptor ( EGFR) mutation status in non-small cell lung cancer (NSCLC) patients in Vietnamese population at Bach Mai Hospital, and relation of different variables to the frequency of EGFR mutations. Methods: The EGFR mutation status of 1451 tissue samples in NSCLC patients and correlation among different variables of age, gender, smoking habit and histology groups were evaluated. Results: A total of EGFR mutation analysis was performed, with an overall mutation rate of 40.7%. The most prevalent categories were in-frame deletions in exon 19 (57.2% of the overall mutations); followed by L858R and L861Q in exon 21 (35.7%); exon 18 (4,6%); double mutation: 2.9%; The T790M mutation in exon 20 represented approximately 2.5%; The pooled prevalence of EGFR mutation was higher in female (60.6%), non-smokers (61.1%), and patients with adenocarcinoma (42.2%). Conclusions: The prevalence of EGFR mutations of NSCLC in Vietnam is higher than that in the West, but comparably equal to that in some Asian countries. It is associated with female sex, non-smoker status, and adenocarcinoma, sample tissue type and age, but not depends on disease stage and tumor sampling.

E13121 Background: Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumor tissue. Here, we demonstrate that mutations can be detected in plasma samples of non-small cell lung cancer patients at Bach Mai Hospital and relation of different variables to the frequency of EGFR mutations. Methods: 193 patients with non-small cell lung cancer were identified with EGFR mutation by cobas EGFR Mutation Test v2 kit (Roche Diagnostics, USA), evaluating the EGFR mutation status with different variables. Results: The EGFR mutation rate was 43%, common in patients with adenocarcinoma, female, non-smoking, late stage disease. The rate of EGFR T790M mutation was 36.1%. Among 78 patients who was previous treated TKIs, EGFR mutation rate was 73.1% and the rate of EGFR T790M mutation was 50.8% at the time of progression. The concordance of EGFR mutation from plasma sample with tissue sample was 81%. Conclusions: EGFR mutation in plasma sample results was not different from those in tissue samples and similar with result of studies in Vietnam and worldwide.