Citations

Abstract Purpose: Thyroid cancer cell lines are valuable models but have been neglected in pancancer genomic studies. Moreover, their misidentification has been a significant problem. We aim to provide a validated dataset for thyroid cancer researchers. Experimental Design: We performed next-generation sequencing (NGS) and analyzed the transcriptome of 60 authenticated thyroid cell lines and compared our findings with the known genomic defects in human thyroid cancers. Results: Unsupervised transcriptomic analysis showed that 94% of thyroid cell lines clustered distinctly from other lineages. Thyroid cancer cell line mutations recapitulate those found in primary tumors (e.g., BRAF, RAS, or gene fusions). Mutations in the TERT promoter (83%) and TP53 (71%) were highly prevalent. There were frequent alterations in PTEN, PIK3CA, and of members of the SWI/SNF chromatin remodeling complex, mismatch repair, cell-cycle checkpoint, and histone methyl- and acetyltransferase functional groups. Copy number alterations (CNA) were more prevalent in cell lines derived from advanced versus differentiated cancers, as reported in primary tumors, although the precise CNAs were only partially recapitulated. Transcriptomic analysis showed that all cell lines were profoundly dedifferentiated, regardless of their derivation, making them good models for advanced disease. However, they maintained the BRAFV600E versus RAS-dependent consequences on MAPK transcriptional output, which correlated with differential sensitivity to MEK inhibitors. Paired primary tumor-cell line samples showed high concordance of mutations. Complete loss of p53 function in TP53 heterozygous tumors was the most prominent event selected during in vitro immortalization. Conclusions: This cell line resource will help inform future preclinical studies exploring tumor-specific dependencies.

Abstract We have previously shown that a vesicular stomatitis virus-based cDNA library expressing xenogeneic altered self-epitopes led to T-cell mediated rejection of prostate cancer, glioma and melanoma. The use of a library of altered self-peptides which reflects the transcriptome of the tumor circumvents the necessity for a priori knowledge of which antigens may be immunogenic in a patient, and primes a polyclonal T-cell response that provides strong cumulative antitumor selective pressure. We have expanded this concept of altered-self library vaccination using tumor vaccines modified by the overexpression of APOBEC3B, a cytosine deaminase that generates C to T transition mutations. A freeze-thawed whole tumor cell vaccine prepared from B16 melanoma cells stably overexpressing human APOBEC3B treated established subcutaneous parental B16 tumors and, when combined with PD-1 checkpoint blockade, cured between 75%-100% of mice. T-cells from mice treated with the vaccine and anti-PD1 produced high levels of IFN-γ when restimulated with parental unmodified B16 melanoma cells in vitro. Whole-genome sequencing of B16APOBEC3B overexpressing cells identified 301 C to T or G to A missense mutations unique to the APOBEC3B line. Using an in silico MHC binding affinity algorithm, we identified and experimentally validated a short list of 10 APOBEC3B- induced heteroclitic peptides. We have also shown that this approach can be extended to an orthotopic brainstem model of high grade glioma. GL261 tumors stereotactically implanted into the brainstem were significantly responsive to treatment with an APOBEC3B- modified GL261 vaccine in combination with anti-PD-1 checkpoint blockade. In summary, when overexpressed in tumor cells, APOBEC3B generates a library of heteroclitic sequences that primes both CD4 and CD8 T-cells that have escaped central tolerance and that recognize both newly mutated antigens from the vaccine, and the corresponding unaltered self epitopes expressed on the tumor cells. Citation Format: Richard Vile, Laura Evgin, Timothy Kottke, Matthew Schuelke, Christopher B. Driscoll, Amanda L. Huff, Jill Thompson, Amy Molan, Reuben S. Harris, Jose S. Pulido, Phonphimon Wongthida. Cancer immunotherapy with APOBEC3B-induced heteroclitic library tumor cell vaccines and immune checkpoint blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B138.

Abstract Tumor cells are able to evade cytotoxic therapies through the accumulation of genomic mutations and rapid evolution. In the case of oncolytic virotherapy, understanding the mechanisms by which cancer cells develop resistance to infection and lysis is critical to the development of more effective viral-based platforms. Here, we identify APOBEC3 as an important factor involved in the restriction of vesicular stomatitis virus (VSV). We show that VSV infection of B16 murine melanoma cells upregulated APOBEC3 in an IFNβ-dependent manner, which was responsible for the evolution of virus-resistanT-cell populations and suggested that APOBEC3 expression promoted the acquisition of a virus-resistant phenotype. ShRNA knockdown of APOBEC3 in B16 cells diminished their capacity to develop resistance to VSV infection in vitro, and enhanced the therapeutic effect of VSV in vivo. Similarly, overexpression of human APOBEC3B promoted the acquisition of resistance to oncolytic VSV in murine melanoma and glioblastoma lines, as well as in a human melanoma cell line in vitro and in vivo. Finally, we demonstrate that progeny virus obtained after passage through APOBEC3B overexpressing cells contained more defective interfering particles, thereby indicating that APOBEC3B directly affected the fitness of VSV, an RNA virus that has not previously been identified to be restricted by APOBEC3B. This research identifies APOBEC3 enzymes as key players to target in order to improve the efficacy of oncolytic viruses as well as broader nucleic acid-based therapeutic platforms. Citation Format: Amanda L. Huff, Phonphimon Wongthida, Timothy Kottke, Jill Thompson, Christopher B. Driscoll, Matthew Schuelke, Kevin G. Shim, Reuben S. Harris, Amy Molan, Jose S. Pulido, Peter J. Selby, Kevin J. Harrington, Alan Melcher, Laura Evgin, Richard Vile. APOBEC3 confers resistance to oncolytic VSV therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A133.

Abstract Although the chimeric antigen receptor (CAR) T-cell platform has experienced clinical success in patients with hematologic malignancies, CAR T-cells specific to solid tumor targets have met with more limited efficacy. The highly inflammatory nature of oncolytic viruses and their ability to remodel the tumor microenvironment suggested to us that they would provide a complementary mechanism of action to both recruit and potentiate the functionality of CAR T-cells. VSVmIFNβ injection into B16EGFRvIII tumors increased the expression of chemokines such as CXCL10 and CCL5, which we would expect to recruit CXCR3+and CCR5+ EGFRvIII specific murine CAR T-cells. However, we did not observe an increase in overall survival or tumor control using the combination strategy compared to monotherapy with CAR T-cells. We recovered fewer viable CD8+ CAR T-cells from tumors injected with VSVmIFNβ early after adoptive transfer, and observed a similar reduction in the number of CD8+ CAR T-cells which persisted long term in the blood. We have shown that type I interferon increases the expression of the CAR from the retroviral LTR and in turn sensitizes these cells to tonic signaling mediated exhaustion and apoptosis. Correspondingly, CAR T-cells prepared from IFNAR KO T-cells were protected from the deleterious effect of type I IFN in vivo. We are currently investigating strategies to overcome the interference between these two modalities and to uncouple the regulation of the expression of the CAR from type I IFN signaling. Citation Format: Laura Evgin, Amanda L. Huff, Phonphimon Wongthida, Jill Thompson, Timothy Kottke, John Sampson, Luis Sanchez Perez, Richard Vile. Unexpected antagonism between oncolytic virus derived type I interferon and EGFRvIII CAR T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A029.

AimsInverted appendices are rare, but have the potential to cause diagnostic confusion among endoscopists and pathologists. The aim of this study was to describe the clinicopathological features of inverted appendices seen at our institution over the last 30 years.Methods and resultsTwenty‐one inverted appendices were identified and the clinical and pathological features reviewed. Patients were predominantly middle‐aged women. Most cases were detected incidentally on colonoscopy. Endoscopically, inverted appendices appeared polypoid in the proximal caecum. All resections featured associated pathological processes, including endometriosis (n = 3), inflammatory mucocoele (n = 1), low‐grade appendiceal mucinous neoplasm (n = 2), traditional serrated adenoma (n = 1) and inflammatory fibroid polyp (n = 1). Five cases were endoscopically mischaracterised as caecal polyps and removed via polypectomy; initial pathological impressions were erroneous in most cases. All polypectomies featured a dome‐like configuration covered by mucosa on the convex surface; the majority had aggregates of ganglion cells and neural plexi embedded in muscularis propria. The vast majority of cases, regardless of the procedure, showed lymphoid aggregates. Among post‐polypectomy patients with follow‐up, none experienced perforation‐associated morbidity despite the histological presence of muscularis propria.ConclusionsThe diagnosis of an inverted appendix should be considered in polypectomy specimens from the caecum or appendiceal orifice with (i) dome‐like tissue configuration covered by mucosa on the convex surface, (ii) a deep, robust smooth muscle component with ganglion cells (muscularis propria) and (iii) associated lymphoid aggregates. Prompt recognition on H&E will avoid unnecessary time and resource investment.

AbstractBackgroundIt is important to identify patients with inflammatory bowel disease [IBD] refractory to anti-tumour necrosis factor [TNF] therapy, to avoid potential adverse effects and to adopt different treatment strategies. We aimed to identify and validate clinical and genetic factors to predict anti-TNF response in patients with IBD.Materials and MethodsMayo Clinic and Washington University IBD genetic association study cohorts were used as discovery and replicate datasets, respectively. Clinical factors included sex, age at diagnosis, disease duration and phenotype, disease location, bowel resection, tobacco use, family history of IBD, extraintestinal manifestations, and response to anti-TNF therapy.ResultsOf 474 patients with IBD treated with anti-TNF therapy, 41 [8.7%] were refractory to therapy and 433 [91.3%] had response. Multivariate analysis showed history of immunomodulator use (odds ratio 10.2, p = 8.73E-4) and bowel resection (odds ratio 3.24, p = 4.38E-4) were associated with refractory response to anti-TNF agents. Among genetic loci, two [rs116724455 in TNFSF4/18, rs2228416 in PLIN2] were successfully replicated and another four [rs762787, rs9572250, rs144256942, rs523781] with suggestive evidence were found. An exploratory risk model predictability [area under the curve] increased from 0.72 [clinical predictors] to 0.89 after adding genetic predictors. Through identified clinical and genetic predictors, we constructed a preliminary anti-TNF refractory score to differentiate anti-TNF non-responders (mean [standard deviation] score, 5.49 [0.99]) from responders (2.65 [0.39]; p = 4.33E-23).ConclusionsNovel and validated genetic loci, including variants in TNFSF, were found associated with anti-TNF response in patients with IBD. Future validation of the exploratory risk model in a large prospective cohort is warranted.