Probes for INS

Human papilloma virus (HPV) infections are associated with almost all cervical cancers and to a lower extend also with anogenital or oropharyngeal cancers. HPV proteins expressed in HPV-associated tumors are attractive antigens for cancer vaccination strategies as self-tolerance, which is associated with most endogenous tumor-associated antigens, does not need to be overcome. In this study, we generated a live attenuated cancer vaccine based on the chimeric vesicular stomatitis virus VSV-GP, which has previously proven to be a potent vaccine vector and oncolytic virus. Genes at an earlier position in the genome more to the 3' end are expressed stronger compared to genes located further downstream. By inserting an HPV16-derived antigen cassette consisting of E2, E6 and E7 into VSV-GP either at first (HPVp1) or fifth (HPVp5) position in VSV-GP's genome we aimed to analyze the effect of vaccine antigen position and consequently expression level on viral fitness, immunogenicity, and anti-tumoral efficacy in a syngeneic mouse tumor model. HPVp1 expressed higher amounts of HPV antigens compared to HPVp5 in vitro but had a slightly delayed replication kinetic which overall translated into increased HPV-specific T cell responses upon vaccination of mice. Immunization with both vectors protected mice in prophylactic and in therapeutic TC-1 tumor models with HPVp1 being more effective in the prophylactic setting. Taken together, VSV-GP is a promising candidate as therapeutic HPV vaccine and first position of the vaccine antigen in a VSV-derived vector seems to be superior to fifth position.

Human papillomavirus (HPV) is a well-established mucosotropic carcinogen, but its impact on urothelial neoplasm is unclear. We aimed to clarify the clinical and pathological features of HPV-related urothelial carcinoma (UC).Tissue samples of 228 cases of UC were obtained from the bladder, upper and lower urinary tract, and metastatic sites to construct a tissue microarray. The samples were analyzed for the presence of HPV by a highly sensitive and specific mRNA in situ hybridization (RISH) technique (RNAscope) with a probe that can detect 18 varieties of high-risk HPV. We also conducted immunohistochemistry (IHC) for a major HPV capsid antibody and DNA-PCR.The HPV detection rates varied among the methods; probably due to low HPV copy numbers in UC tissues and the insufficient specificity and sensitivity of the IHC and PCR assays. The RISH method had the highest accuracy and identified HPV infection in 12 (5.2%) of the cases. The histopathological analysis of the HPV-positive UC showed six cases of usual type UC, five cases of UC with squamous differentiation (UC_SqD), and one case of micropapillary UC. The HPV detection rate was six-fold higher in the cases of UC_SqD than in the other variants of UC (odds ratio [OR] =8.9, p = 0.002). In addition, HPV infection showed a significant association with tumor grade (OR =9.8, p = 0.03) and stage (OR =4.7, p = 0.03) of UC. Moreover, the metastatic rate was higher in HPV-positive than in negative UC (OR =3.4).These data indicate that although the incidence of HPV infection in UC is low, it is significantly associated with squamous differentiation and poor prognosis. Furthermore, our observations show that RNAscope is an ideal method for HPV detection in UC compared with the other standard approaches such as IHC and PCR assays.

SOX10 immunoexpression is increasingly recognized in salivary gland tumors, including but not limited to those with myoepithelial, serous acinar, and intercalated duct differentiation. However, SOX10 expression has not been extensively evaluated in other epithelial tumors that can mimic salivary origin. Basaloid squamous cell carcinoma (SCC) is a unique variant of SCC that shows morphologic overlap with several salivary tumors, including adenoid cystic carcinoma, basal cell adenocarcinoma, and myoepithelial carcinoma. We performed SOX10 immunohistochemistry on 22 basaloid SCCs and 280 non-basaloid SCCs. If tissue was available, we also performed immunohistochemistry for S100 and p16, and in-situ hybridization for high-risk HPV RNA. SOX10 was positive in 13/22 basaloid SCCs (59%), including 5/6 (83%) that were HPV-positive and 6/12 (50%) that were HPV-negative. Only 2/12 basaloid SCC (17%) demonstrated focal S100 expression. All non-basaloid SCCs were SOX10 negative. Frequent positivity for SOX10 in basaloid SCC presents a significant diagnostic pitfall for distinguishing these tumors from various basaloid salivary carcinomas. The preponderance of SOX10 expression in the basaloid variant of HPV-positive SCC also presents a diagnostic challenge in separating it from HPV-related multiphenotypic sinonasal carcinoma. SOX10 may be more broadly considered a marker of basal differentiation and should not be assumed to be specific for salivary origin in epithelial head and neck tumors.

Dynamic changes in cell properties lead to intratumor heterogeneity; however, the mechanisms of nongenetic cellular plasticity remain elusive. When the fate of each cell from colorectal cancer organoids was tracked through a clonogenic growth assay, the cells showed a wide range of growth ability even within the clonal organoids, consisting of distinct subpopulations; the cells generating large spheroids and the cells generating small spheroids. The cells from the small spheroids generated only small spheroids (S-pattern), while the cells from the large spheroids generated both small and large spheroids (D-pattern), both of which were tumorigenic. Transition from the S-pattern to the D-pattern occurred by various extrinsic triggers, in which Notch signaling and Musashi-1 played a key role. The S-pattern spheroids were resistant to chemotherapy and transited to the D-pattern upon drug treatment through Notch signaling. As the transition is linked to the drug resistance, it can be a therapeutic target.