Neoadjuvant in situ gene-mediated cytotoxic immunotherapy improves postoperative outcomes in novel syngeneic esophageal carcinoma models

Esophageal carcinoma is the most rapidly increasing tumor in the United States and has a dismal 15% 5-year survival. Immunotherapy has been proposed to improve patient outcomes; however, no immunocompetent esophageal carcinoma model exists to date to test this approach. We developed two mouse models of esophageal cancer by inoculating immunocompetent mice with syngeneic esophageal cell lines transformed by cyclin-D1 or mutant HRASG12V and loss of p53. Similar to humans, surgery and adjuvant chemotherapy (cisplatin and 5-fluorouracil) demonstrated limited efficacy. Gene-mediated cyototoxic immunotherapy (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir; AdV-tk/GCV) demonstrated high levels of in vitro transduction and efficacy. Using in vivo syngeneic esophageal carcinoma models, combining surgery, chemotherapy and AdV-tk/GCV improved survival (P=0.007) and decreased disease recurrence (P<0.001). Mechanistic studies suggested that AdV-tk/GCV mediated a direct cytotoxic effect and an increased intra-tumoral trafficking of CD8 T cells (8.15% vs 14.89%, P=0.02). These data provide the first preclinical evidence that augmenting standard of care with immunotherapy may improve outcomes in the management of esophageal carcinoma

Progress in gene therapy for neurological disorders

Diseases of the nervous system have devastating effects and are widely distributed among the population, being especially prevalent in the elderly. These diseases are often caused by inherited genetic mutations that result in abnormal nervous system development, neurodegeneration, or impaired neuronal function. Other causes of neurological diseases include genetic and epigenetic changes induced by environmental insults, injury, disease-related events or inflammatory processes. Standard medical and surgical practice has not proved effective in curing or treating these diseases, and appropriate pharmaceuticals do not exist or are insufficient to slow disease progression. Gene therapy is emerging as a powerful approach with potential to treat and even cure some of the most common diseases of the nervous system. Gene therapy for neurological diseases has been made possible through progress in understanding the underlying disease mechanisms, particularly those involving sensory neurons, and also by improvement of gene vector design, therapeutic gene selection, and methods of delivery. Progress in the field has renewed our optimism for gene therapy as a treatment modality that can be used by neurologists, ophthalmologists and neurosurgeons. In this Review, we describe the promising gene therapy strategies that have the potential to treat patients with neurological diseases and discuss prospects for future development of gene therapy

Abstract P6-07-03: Broken promise of liquid biopsy: Plasma DNA does not accurately reflect tumor DNA in metastatic breast cancer

Abstract Background: Circulating tumor DNA in plasma may present a minimally invasive approach to identify tumor-derived mutations that could be used to inform the selection of targeted therapies for individual patients, particularly in cases of metastatic disease where biopsy is often difficult. We hypothesized that plasma DNA will genetically reflect DNA derived from multiple tumors in patients with metastatic breast cancer. To test this hypothesis and assess the utility of plasma DNA obtained as a “liquid biopsy” for precision medicine, we sought to determine whether massively parallel sequencing of plasma DNA is a reliable surrogate for sequencing of DNA from tissue biopsies in patients with metastatic breast cancer. Methods: Blood samples were obtained from 7 patients with multiple advanced breast cancer lesions (recurrent breast and metastatic tumors), and tumor specimens were obtained thereafter by biopsy or surgical excision. DNA extracted from plasma, buffy coat of blood, and tumor tissues was used for probe-directed capture of all exons in 196 genes followed by massively parallel sequencing with an average coverage of 3000x for plasma DNA. Tumor and plasma DNA sequences were bioinformatically compared to buffy coat controls, and high-confidence somatic mutations were called. One patient with extensive metastatic disease was evaluated in further detail to study the contribution of different tumors to the overall plasma DNA pool. In this patient, 9 metastatic tumors were sampled in an axillary lymph node, heart, kidney (2), liver, omentum (3), and ovary by biopsy or at autopsy. Results: Mutations were found in plasma that were represented in one or more tumors in each patient. Three classes of mutations were discovered: 1) mutations overlapping between both plasma and tumors (e.g., TP53 p.R273C and SRC p.E527K); 2) mutations found in plasma but not tumors (e.g., AKT p.E17K and multiple known and novel ESR1 mutations); 3) mutations found in tumors but not plasma (e.g., PIK3CA p.H1047R, p.D350G, and p.N345K). The presence of mutations in each of these classes was validated in plasma and/or tumors using mutation-specific droplet digital PCR (ddPCR). In the patient with extensive metastatic disease, DNA sequencing revealed heterogeneity of tumor contribution to plasma DNA, with some tumors better represented than others. No correlation was found between tumor size (measured by CT scan) and mutational burden in plasma. Interestingly, a significant correlation was found between blood perfusion to the organ where the tumor resides and mutational burden in plasma, with the greatest tumor contribution coming from the heart metastasis (Pearson's r = 0.835, p=0.039). Conclusions: Plasma DNA sequencing adds a layer of depth to sequencing analysis of tumor biopsy samples, and serves to both confirm tumor-derived mutations as well as detect new mutations. However, plasma DNA profiling does not comprehensively reflect the mutational profiles of tumors in patients with metastatic breast cancer, and thus is unlikely to serve as a surrogate for tumor biopsy as a source of DNA for genetic profiling. Furthermore, plasma DNA contains many mutations not found in tumors, which will confound treatment decision-making and precision medicine.Background: Circulating tumor DNA in plasma may present a minimally invasive approach to identify tumor-derived mutations that could be used to inform the selection of targeted therapies for individual patients, particularly in cases of metastatic disease where biopsy is often difficult. We hypothesized that plasma DNA will genetically reflect DNA derived from multiple tumors in patients with metastatic breast cancer. To test this hypothesis and assess the utility of plasma DNA obtained as a “liquid biopsy” for precision medicine, we sought to determine whether massively parallel sequencing of plasma DNA is a reliable surrogate for sequencing of DNA from tissue biopsies in patients with metastatic breast cancer. Methods: Blood samples were obtained from 7 patients with multiple advanced breast cancer lesions (recurrent breast and metastatic tumors), and tumor specimens were obtained thereafter by biopsy or surgical excision. DNA extracted from plasma, buffy coat of blood, and tumor tissues was used for probe-directed capture of all exons in 196 genes followed by massively parallel sequencing with an average coverage of 3000x for plasma DNA. Tumor and plasma DNA sequences were bioinformatically compared to buffy coat controls, and high-confidence somatic mutations were called. One patient with extensive metastatic disease was evaluated in further detail to study the contribution of different tumors to the overall plasma DNA pool. In this patient, 9 metastatic tumors were sampled in an axillary lymph node, heart, kidney (2), liver, omentum (3), and ovary by biopsy or at autopsy. Results: Mutations were found in plasma that were represented in one or more tumors in each patient. Three classes of mutations were discovered: 1) mutations overlapping between both plasma and tumors (e.g., TP53 p.R273C and SRC p.E527K); 2) mutations found in plasma but not tumors (e.g., AKT p.E17K and multiple known and novel ESR1 mutations); 3) mutations found in tumors but not plasma (e.g., PIK3CA p.H1047R, p.D350G, and p.N345K). The presence of mutations in each of these classes was validated in plasma and/or tumors using mutation-specific droplet digital PCR (ddPCR). In the patient with extensive metastatic disease, DNA sequencing revealed heterogeneity of tumor contribution to plasma DNA, with some tumors better represented than others. No correlation was found between tumor size (measured by CT scan) and mutational burden in plasma. Interestingly, a significant correlation was found between blood perfusion to the organ where the tumor resides and mutational burden in plasma, with the greatest tumor contribution coming from the heart metastasis (Pearson's r = 0.835, p=0.039). Conclusions: Plasma DNA sequencing adds a layer of depth to sequencing analysis of tumor biopsy samples, and serves to both confirm tumor-derived mutations as well as detect new mutations. However, plasma DNA profiling does not comprehensively reflect the mutational profiles of tumors in patients with metastatic breast cancer, and thus is unlikely to serve as a surrogate for tumor biopsy as a source of DNA for genetic profiling. Furthermore, plasma DNA contains many mutations not found in tumors, which will confound treatment decision-making and precision medicine. Citation Format: Shee K, Chamberlin MD, Varn FS, Bean JR, Marotti JD, Wells WA, Trask HW, Hamilton JS, West RJ, Kaufman PA, Schwartz GN, Gemery JM, McNulty NJ, Tsapakos MJ, Barth RJ, Arrick BA, Gui J, Cheng C, Miller TW. Broken promise of liquid biopsy: Plasma DNA does not accurately reflect tumor DNA in metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-03.</jats:p