Andrew Nguyen

An Msh2 conditional knockout mouse for studying intestinal cancer and testing anticancer agents.

BACKGROUND & AIMS Mutations in the DNA mismatch repair (MMR) gene MSH2 cause Lynch syndromes I and II and sporadic colorectal cancers. Msh2(null) mice predominantly develop lymphoma and do not accurately recapitulate the colorectal cancer phenotype. METHODS We generated and examined mice with a conditional Msh2 disruption (Msh2(LoxP)), permitting tissue-specific gene inactivation. ECMsh2(LoxP/LoxP) mice carried an EIIa-Cre transgene, and VCMsh2(LoxP/LoxP) mice carried a Villin-Cre transgene. We combined the VCMsh2(LoxP) allele with either Msh2(Delta7null) (VCMsh2(LoxP/null)) or Msh2(G674D) mutations (VCMsh2(LoxP/G674D)) to create allelic phase mutants. These mice were given cisplatin or 5-fluorouracil/leucovorin and oxaliplatin (FOLFOX), and their tumors were measured by magnetic resonance imaging. RESULTS Embryonic fibroblasts from ECMsh2(LoxP/LoxP) mice do not express MSH2 and are MMR deficient. Reverse transcription, polymerase chain reaction, and immunohistochemistry from VCMsh2(LoxP/LoxP) mice demonstrated specific loss of Msh2 messenger RNA and protein from epithelial cells of the intestinal tract. Microsatellite instability was observed in all VCMsh2 strains and limited to the intestinal mucosa. Resulting adenomas and adenocarcinomas had somatic truncation mutations to the adenomatous polyposis coli (Apc) gene. VCMsh2(LoxP/LoxP) mice did not develop lymphoma. Comparison of allelic phase tumors revealed significant differences in multiplicity and size. When treated with cisplatin or FOLFOX, tumor size was reduced in VCMsh2(LoxP/G674D) but not VCMsh2(LoxP/null) tumors. The apoptotic response to FOLFOX was partially sustained in the intestinal mucosa of VCMsh2(LoxP/G674D) animals. CONCLUSIONS Msh2(LoxP/LoxP) mice in combination with appropriate Cre recombinase transgenes have excellent potential for preclinical modeling of Lynch syndrome, MMR-deficient tumors of other tissue types, and use in drug development.

Inactivation of Conditional Rb by Villin-Cre Leads to Aggressive Tumors outside the Gastrointestinal Tract

We have crossed mice carrying the conditional Rb(tm2Brn) allele with a constitutive Villin-Cre transgenic mouse. The Villin promoter in these animals is highly expressed in adult intestine and kidney proximal tubules and is expressed in the gut and nephros anlagen during embryogenesis. We report here that these mice develop tumors between 12 and 17 months old outside the gastrointestinal (GI) tract. A high penetrance of pituitary tumors and medullar carcinoma of the thyroid is observed with a lower incidence of hyperplasia of pulmonary neuroendocrine cells and aggressive liver, bile duct, stomach, oral cavity tumors, and lipomas. Rb rearrangement due to ectopic Villin promoter activity in neural crest or neural crest stem cells during embryogenesis is most likely responsible for the medullar carcinoma of the thyroid phenotype. The aggressive nature of the medullar carcinoma of the thyroid and its ability to metastasize to unusual sites make the model suitable for the study of tumor progression and mechanism of metastasis. Observed sites of metastasis include the stomach, small intestine, liver, lung, kidney, pancreas, spleen, bone marrow, salivary gland, fat, lymph nodes, and dorsal root ganglion. Because the Villin promoter is highly active throughout the GI and in the nephros anlagen during development, we find that Rb inactivation is not sufficient to initiate tumorigenesis in the GI or kidneys in mice.

Abstract 640: Subsets of HLA alleles are capable of binding neoantigens derived from mutations within cancer driving genes such as KRAS and EGFR

Background: Immunoncology has shown great promise as a low toxicity tool to combat several cancers. Use of checkpoint inhibitors against PD1 or CTLA4 unlocks the immune system’s ability to recognize tumor antigens and, more specifically, neoantigens caused by random mutations within cancers. The vast majority of neoantigens consist of private mutations unique to a patient’s tumor genome, but several cancers harbor recurrent mutations. Mutations in the KRAS gene, such as p.G12V, occur in roughly 25% of colorectal cancers. Mutations in EGFR occur in 10% and 35% of patients with non-small cell lung cancer in the US and East Asia, respectively. Even more prevalent are mutations within the TP53 tumor suppressor gene, with roughly 23000 unique protein variants reported to date. If these mutations in cancer driving genes are so prevalent in cancers, why are neoantigens against these targets not more readily available? Results: We collected recurrent mutations across a variety of cancer driving genes such as KRAS, EGFR, TP53 and MYC and performed binding analysis using netMHC 3.4 to see which HLA alleles are capable of binding specific cancer mutations such as KRAS p.G12V. Using this method, we report all possible HLA alleles capable of binding these recurrent mutations within cancer genes. We further performed 3-dimensional modeling to determine whether complexes created by the HLA alleles and cancer neoepitopes are stable. Conclusions: Several HLA alleles are capable of binding recurrent cancer mutations. These include both MHC Class 1 and Class 2 alleles. The variation in alleles capable of binding commonly mutated genes such as EGFR may explain the difference in prevalence of these mutations between geographic populations. Determining whether a certain HLA allele confers resistance to common cancer gene mutations may lead to identification of immune cells within these populations that can recognize neoantigens from commonly mutated cancer genes. Citation Format: Andrew Nguyen, J Zachary Sanborn, Charles J. Vaske, Shahrooz Rabizadeh, Kayvan Niazi, Patrick Soon-Shiong, Steve Benz. Subsets of HLA alleles are capable of binding neoantigens derived from mutations within cancer driving genes such as KRAS and EGFR [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 640. doi:10.1158/1538-7445.AM2017-640

Abstract 4512: High-throughput identification of neoepitopes for the development of patient-specific cancer immunotherapies

Introduction: Immunotherapies such as checkpoint inhibitors, CAR T cells, NK cells, and therapeutic vaccines are revolutionizing cancer medicine. Although these agents have resulted in remarkable responses in some patients, many fail to respond, suggesting that a patient-specific approach is needed for immunotherapies to realize their full potential. Here we report the analysis of whole genome sequencing (WGS) and RNA sequencing (RNAseq) data from The Cancer Genome Atlas (TCGA) to identify neoepitopes that may serve as viable immunotherapeutic targets and that could be used to develop next-generation, patient-specific cancer immunotherapies. Methods: HLA-A compatible neoepitopes were identified by creating all permutations of 9-mer amino acid strings derived from identified single nucleotide variants (SNVs) occurring in expressed genes. All neoepitopes were filtered against a database of 9-mers created using every known human protein along with common variations obtained through dbSNP to reduce off-target effects. HLA typing was determined from WGS data using the HLA forest algorithm. NetMHC 3.4 was used to obtain the predicted binding affinities of neoepitopes to HLA-A alleles Results: We analyzed 750 patients across 23 cancer types using WGS data and RNAseq data, when available. Mutational and neoepitope loads varied across cancer types, with skin cutaneous melanoma and thyroid carcinoma having the highest and lowest mutational and neoepitope loads, respectively. Neoepitope counts identified by WGS correlated with neoepitope expression identified by RNAseq across a wide variety of cancers (Pearson9s r = 0.99 for all cancers combined). The distribution of HLA-A and HLA-DRB1 alleles determined from TCGA were generally comparable to that in the US population; however, the frequency of HLA-DRB1*15:01 (associated with several diseases) was ∼2-fold higher than the US population. Among patients who expressed HLA-A*02:01 (n = 143), the numbers of predicted neoepitopes identified by WGS, neoepitopes expressed per RNAseq, and neoepitopes having binding affinity to HLA-A*02:01 were 23272, 9619, and 138, respectively. Almost all neoepitopes were unique to each patient, with a maximum of only 2-shared neoepitopes among any cancer type. Among 26 triple negative breast cancer (TNBC) samples, the numbers of predicted neoepitopes, expressed neoepitopes, and neoepitopes with affinity to HLA-A*02:01 were 17925, 8184, and 228, respectively. There were no shared neoepitopes among TNBC samples. Across all cancers, ∼6% of neoepitopes occurred in cancer driver genes. Conclusions: Neoepitopes are rarely shared among cancers, and almost all are unique to each patient. For patients with limited treatment options and poor outcomes, such as TNBC, high-throughput identification of neoepitopes could serve as the basis for the development of next-generation, patient-specific immunotherapies. Citation Format: Andrew Nguyen, J Zachary Sanborn, Charles J. Vaske, Shahrooz Rabizadeh, Kayvan Niazi, Patrick Soon-Shiong, Steven C. Benz. High-throughput identification of neoepitopes for the development of patient-specific cancer immunotherapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4512.