37Salmonella strains have recently been developed as antitumor agents capable of both preferentially amplifying within tumors and expressing prodrug-converting enzymes such as the herpes simplex thymidine kinase 1 . These bacteria were attenuated by auxotrophic mutations that limited their pathogenesis in normal tissues but retained high-level replication within the tumors following systemic administration. The auxotrophic requirements of these Salmonella are apparently met within the tumor environment where they then replicate, reaching up to more than 1000 times the concentration found in normal tissues.A significant limitation for safe use of systemically administered bacteria in humans is the ability of the bacteria to induce tumor necrosis factor α (TNFα)-mediated septic shock 2,3 . However, modifications in bacterial components responsible for eliciting host immune responses such as TNFα induction could interfere with tumor targeting or antitumor activity.Several mutations in lipid biosynthesis are known in Escherichia coli and Salmonella sp. that lower TNFα induction and render the bacteria nontoxic. Some mutations, such as kdo -result in the production of lipid IV A , which substantially lowers TNFα induction and acts as an antagonist to the TNFα response from wild-type lipid A 4,5 . However, these and most other lipid mutations are temperature-sensitive and conditionally lethal to the bacteria 6 , limiting the potential for tumor-based amplification seen in auxotrophic Salmonella 1 .In E. coli, the msbB (mlt) gene 7,8 is involved in the terminal myristoylation of lipid A 9,10 . Genetic disruption of this gene in E. coli results in a stable nonconditional mutation that lowers TNFα induction up to 10-fold by whole bacteria or up to 10,000-fold by purified lipopolysaccharide (LPS) 9 . A similar toxicity profile is observed when the msbB gene is disrupted in Salmonella 11 . We generated a deletion in the coding sequence of msbB within a hyperinvasive strain of Salmonella we previously used for tumor-targeting as well as the parental wild type, and examined the effect on virulence and TNFα production both in vitro and in vivo. Results indicate that msbB -mutant Salmonella retain the properties of tumor accumulation and tumor suppression in the absence of eliciting high levels of TNFα. Results Isolation and genetic disruption of the Salmonella msbB gene.DNA sequence analysis of Salmonella msbB clones obtained by DNA/DNA hybridization indicated the presence of an msbB homolog with flanking gene organization (orfU, msbB, pykA, and zwf) identical to E. coli 8 . The DNA homology of the Salmonella msbB and the E. coli msbB was determined to be 75%, and the amino acid homology 98%, confirming that the cloned Salmonella gene is an msbB homolog.Putative knockouts obtained by transformation of the linearized deletion construct were confirmed by several criteria using Southern blot analysis (Fig. 1): Two bands corresponding to the tetracycline gene were observed in the knockout construct and in the knockout clones and w...

VNP20009 is a genetically modified strain of Salmonella typhimurium possessing an excellent safety profile, including genetically stable attenuated virulence (a deletion in the purI gene), reduction of septic shock potential (a deletion in the msbB gene), and antibiotic susceptibility. VNP20009 is genetically stable after multiple generations in vitro and in vivo. In mice, VNP20009 is rapidly cleared from the blood from a peak level of 1x104 cfu/mL to undetectable levels in 24 h. In tumor-bearing mice, VNP20009 accumulates preferentially in tumors over livers at a ratio of 1000&rcolon;1. In nonhuman primates, VNP20009 was also rapidly cleared from the blood, from a peak level of 1.0x106 cfu/mL to undetectable levels in 24 h. VNP20009 was detected in the liver, spleen, and bone marrow of monkeys; the amount decreased over time, and VNP20009 was cleared from all organs by day 41; no VNP20009 could be detected in the urine or feces of the monkeys. VNP20009 is genetically stable after many generations of growth (>140) both in vitro and in vivo.

SummaryInterleukin-10 (IL-10) is a recently described pteiotropic cytokine secreted mainly by type 2 helper T cells. Previous studies have shown that IL-t0 suppresses cytokine expression by natural killer (NK) and type 1 T cells, thus down-regulating cell-mediated immunity and stimulating humoral responses. We here report that injected IL-t0 protein is an efficient inhibitor of tumor metastasis in experimental (B16-F10) and spontaneous (M27 and Lox human melanoma) metastasis models in vivo at doses that do not have toxic effects on normal or cancer cells. Histological characterization after IL-10 treatment confirmed the absence of CD8 + and CD4 + T cells and macrophages at the sites of tumor growth, but abundant NK cells were localized at these sites. This unexpected finding was confirmed by showing that IL-10 inhibits most B16-F10 and Lox metastases in mice deficient in T or B cells (SCID and nu/nu mice), but not in those deficient in NK cells (beige mice or NK cell--depleted mice). However, IL-t0 downregulation of pro-inflammatory cytokine production and/or recruitment of additional eff~ctor cells may also be involved in the anti-tumor effect at higher local concentrations of IL-t0, since transfected B16 tumor cells expressing high amounts of lL-10 were rejected by normal, nu/nu, or SCID mice at the primary tumor stage, and there was still a 33% inhibition of tumor metastasis in beige mice.

Purpose Genetic intratumoral heterogeneity has a profound influence on the selection of clinical treatment strategies and on addressing resistance to targeted therapy. The purpose of this study was to explore the potential effect of intratumoral heterogeneity on both genetic and pathologic characteristics of ALK-rearranged lung adenocarcinoma (LADC). Methods We tested ALK fusions and EGFR mutations in 629 patients with LADC by using laser-capture microdissection to capture spatially separated tumor cell subpopulations in various adenocarcinoma subtypes and to test for ALK fusions and EGFR mutations in ALK-rearranged, EGFR-mutated, and ALK/EGFR coaltered LADCs to compare the oncogenic driver status between different tumor cell subpopulations in the same primary tumor. Results Among the 629 patients, 30 (4.8%) had ALK fusions, 364 (57.9%) had EGFR mutations, and two had ALK fusions that coexisted with EGFR mutations. Intratumoral heterogeneity of ALK fusions were identified in nine patients by reverse-transcriptase polymerase chain reaction. In the two patients with an ALK/EGFR coaltered status, genetic intratumoral heterogeneity was observed both between different growth patterns and within the same growth pattern. The relative abundance of ALK and EGFR alterations was different in the same captured area. ALK fusions were positively associated with a micropapillary pattern (P = .002) and were negatively associated with a lepidic pattern (P = .008) in an expanded statistical analysis of 900 individual adenocarcinoma components, although they appeared to be more common in acinar-predominant LADCs in the analysis of 629 patients. Conclusion Intratumoral genetic heterogeneity was demonstrated to coexist with histologic heterogeneity in both single-driver and ALK/EGFR coaltered LADCs. Altered oncogenic drivers in spatially separated subclones of the same tumor may be different.

About 40 years ago, the observation was made that conditions within experimental animal tumors could favor the preferential replication of bacteria.

VNP2000 9, a genetically modified strain of Salmonella typhii?Wrium with deletions in the msbB and purl loci, exhibited antitumor activities when given systemically to tumor-bearing mice. VNP2000 9 inhibited the growth of subcutaneously implanted B16F10 murine melanoma, and the human tumor xenografts Lox, DLD-1, A549, WiDr, HTB177, and MDA MB-231. A single intravenous injection of VNP20009 , at doses ranging from 1 x Hf to 3 x 106 cfulrnouse, produced tumor growth inhibitions of 57-95%. Tumor volume doubling time, another indicator for tumor growth inhibition, also significantly increased in mice treated with VNP2000 9. Using mice with immune system deficiencies, we also demon• strated that the antitumor effects of VNP20009 did not depend on the presence ofT and B cells. In addition, VNP20009 , given intravenously, inhibited the growth of lung metastases in mice. Only live bacteria showed the antitumor effect.

ROS1 fusions occurred in ∼2.0% of Chinese patients with NSCLC and had no specific clinicopathological feature. ROS1 fusion-negative patients may have a better survival than ROS1 fusion-positive patients.

The study was designed to evaluate whether TAPET-CD, an attenuated strain of Salmonella typhimurium ex pressing Escherichia coli cytosine deaminase (CD), was capable of converting nontoxic 5-fluorocytosine (5-FC) to the active antitumor agent 5-fluorouracil (5-FU). The antitumor effect of TAPET-CD plus 5-FC against subcutaneously implanted colon tumors was also evaluated. TAPET-CD was given to tumor-bearing mice by a single bolus intravenous administration followed with 5-FC by intraperitoneal administration. TAPET-CD accumulated in tumors at levels 1000-fold higher than that in normal tissues and high levels of 5-FU were de growth by 88%-96%, compared to TAPET-CD alone, which inhibited tumor growth by 38%-79%. These data suggest that tumor-targeting Salmonella could be used to deliver prodrug-converting enzyme selectively to tumors and produced anti-tumor effects when the corresponding prodrug was also given. These studies demonstrate the potential use of attenuated Salmonella as a tumor-selective protein delivery vector.