Probes for INS

Background Oropharyngeal squamous cell carcinoma (OPSCC) incidence is rising worldwide, especially human papillomavirus (HPV)-associated disease. Historically, high levels of protein kinase CK2 were linked with poor outcomes in head and neck squamous cell carcinoma (HNSCC), without consideration of HPV status. This retrospective study examined tumor CK2α protein expression levels and related clinical outcomes in a cohort of Veteran OPSCC patient tumors which were determined to be predominantly HPV(+). Methods Patients at the Minneapolis VA Health Care System with newly diagnosed primary OPSCC from January 2005 to December 2015 were identified. A total of 119 OPSCC patient tumors were stained for CK2α, p16 and Ki-67 proteins and E6/E7 RNA. CK2α protein levels in tumors and correlations with HPV status and Ki-67 index were assessed. Overall survival (OS) analysis was performed stratified by CK2α protein score and separately by HPV status, followed by Cox regression controlling for smoking status. To strengthen the limited HPV(−) data, survival analysis for HPV(−) HNSCC patients in the publicly available The Cancer Genome Atlas (TCGA) PanCancer RNA-seq dataset was determined for CSNK2A1. Results The patients in the study population were all male and had a predominant history of tobacco and alcohol use. This cohort comprised 84 HPV(+) and 35 HPV(−) tumors. CK2α levels were higher in HPV(+) tumors compared to HPV(−) tumors. Higher CK2α scores positively correlated with higher Ki-67 index. OS improved with increasing CK2α score and separately OS was significantly better for those with HPV(+) as opposed to HPV(−) OPSCC. Both remained significant after controlling for smoking status. High CSNK2A1 mRNA levels from TCGA data associated with worse patient survival in HPV(−) HNSCC. Conclusions High CK2α protein levels are detected in HPV(+) OPSCC tumors and demonstrate an unexpected association with improved survival in a strongly HPV(+) OPSCC cohort. Worse survival outcomes for high CSNK2A1 mRNA levels in HPV(−) HNSCC are consistent with historical data. Given these surprising findings and the rising incidence of HPV(+) OPSCC, further study is needed to understand the biological roles of CK2 in HPV(+) and HPV(−) HNSCC and the potential utility for therapeutic targeting of CK2 in these two disease states.

EVs have emerged as an important component in tumour initiation, progression and metastasis. Although notable progresses have been made, the detection of EV cargoes remain significantly challenging for researchers to practically use; faster and more convenient methods are required to validate the EV cargoes, especially as biomarkers. Here we show, the possibility of examining embedded EVs as substrates to be used for detecting DNA amplification through ultrasensitive in situ hybridization (ISH). This methodology allows the visualization of DNA targets in a more direct manner, without time consuming optimization steps or particular expertise. Additionally, formalin-fixed paraffin-embedded (FFPE) blocks of EVs allows long-term preservation of samples, permitting future studies. We report here: (i) the successful isolation of EVs from liposarcoma tissues; (ii) the EV embedding in FFPE blocks (iii) the successful selective, specific ultrasensitive ISH examination of EVs derived from tissues, cell line, and sera; (iv) and the detection of MDM2 DNA amplification in EVs from liposarcoma tissues, cell lines and sera. Ultrasensitive ISH on EVs would enable cargo study while the application of ISH to serum EVs, could represent a possible novel methodology for diagnostic confirmation. Modification of probes may enable researchers to detect targets and specific DNA alterations directly in tumour EVs, thereby facilitating detection, diagnosis, and improved understanding of tumour biology relevant to many cancer types.

Viruses are well known drivers of several human malignancies. A causative factor for oral cavity squamous cell carcinoma (OSCC) in patients with limited exposure to traditional risk factors, including tobacco use, is yet to be identified. Our study aimed to comprehensively evaluate the role of viral drivers in OSCC patients with low cumulative exposure to traditional risk factors. Patients under 50 years of age with OSCC, defined using strict anatomic criteria were selected for WGS. The WGS data was interrogated using viral detection tools (Kraken 2 and BLASTN), together examining >700,000 viruses. The findings were further verified using tissue microarrays of OSCC samples using both immunohistochemistry and RNA in situ hybridisation (ISH). 28 patients underwent WGS and comprehensive viral profiling. One 49-year-old male patient with OSCC of the hard palate demonstrated HPV35 integration. 657 cases of OSCC were then evaluated for the presence of HPV integration through immunohistochemistry for p16 and HPV RNA ISH. HPV integration was seen in 8 (1.2%) patients, all middle-aged men with predominant floor of mouth involvement. In summary, a wide-ranging interrogation of >700,000 viruses using OSCC WGS data showed HPV integration in a minority of male OSCC patients and did not carry any prognostic significance.

The main objective of our study was to understand the impact of immune cell composition and the tumor-reactivity of tumor infiltrating lymphocytes (TIL) in HPV-positive (HPV+) and HPV-negative (HPV-) head and neck squamous cell carcinoma (HNSCC). TIL cultures were established from primary HNSCC tumors, the T cell subsets were phenotypically characterized using flow cytometry, and Interferon (IFN)-γ ELISA assay was used to determine TIL function. NanoString Immune Profiler was used to determine an immune signature by HPV-status, and multiplex immunohistochemistry (MIHC) was used to quantify immune cell distributions and their spatial relationships. Results showed that HPV+ and HPV- HNSCC had similar capacity to expand IFN-γ reactive TIL populations, and these TIL populations had similar characteristics. NanoString analysis revealed increased differential expression of genes related to B cell functions in HPV+ HNSCC, which were significant at a Benjamini-Yekutieli adjusted p-value of < 0.001. MIHC also displayed increased CD8+ T cell and CD19/CD20+ B cell densities in the tumor region of HPV+ HNSCC as opposed to HPV- HNSCC (p < 0.01). Increases in a combined metric of tumor B cell content and stromal plasma cell content was associated with increased progression-free survival in HPV- HNSCC patients treated with immune checkpoint inhibitor therapy (p = 0.03). In summary, TIL populations expanded from HPV+ and HPV- HNSCC displayed similar IFN-γ reactivity. However, we identified a strong B-cell signature present within HPV+ HNSCC, and higher B and plasma cell content associated with improved PFS in HPV- HNSCC patients treated with immune checkpoint inhibitors.

Sarcomas are rare malignancies, the number of reports is limited, and this rarity makes further research difficult even though liposarcoma is one of major sarcomas. 2D cell culture remains an important role in establishing basic tumor biology research, but its various shortcomings and limitations are still of concern, and it is now well-accepted that the behavior of 3D-cultured cells is more reflective of in vivo cellular responses compared to 2D models. This study aimed to establish 3D cell culture of liposarcomas using two different methods: scaffold-based (Matrigel extracellular matrix [ECM] scaffold method) and scaffold-free (Ultra-low attachment [ULA] plate). Lipo246, Lipo224 and Lipo863 cell lines were cultured, and distinctive differences in structures were observed in Matrigel 3D model: Lipo224 and Lipo863 formed spheroids, whereas Lipo246 grew radially without forming spheres. In ULA plate approaches, all cell lines formed spheroids, but Lipo224 and Lipo863 spheroids showed bigger size and looser aggregation than Lipo246. Formalin fixed, paraffin embedded (FFPE) blocks were obtained from all 3D models, confirming the spheroid structures. The expression of MDM2, Ki-67 positivity and MDM2 amplification were confirmed by IHC and DNAscope , respectively. Protein and DNA were extracted from all samples and MDM2 upregulation was confirmed by western blot and qPCR analysis. After treatment with MDM2 inhibitor SAR405838, DDLPS spheroids demonstrated different sensitivity patterns from 2D models. Taken together, we believed that 3D models would have a possibility to provide us a new predictability of efficacy and toxicity, and considered as one important process in in vitro pre-clinical phase prior to moving forward to clinical trials.

Introduction: Inverted papillomas (IPs) are rare, benign, sinonasal tumors with the ability to undergo malignant transformation. While rare, they are the most common type of papilloma within the sinonasal cavity and represent up to 5% of primary nasal cavity tumors. There have been many studies attempting to define a causal link between HPV and malignant transformation of IPs with mixed results. Additionally, these tumors have a high recurrence rate, and their malignant transformation potential has spurred significant investigation into their etiology, disease course, and treatment. Prior meta-analyses of HPV-mediated transformation of IPs have suggested a nearly 50% prevalence of HPV in IPSCC and strong bias toward the high-risk virus types, HPV16 and HPV18, in IP malignant transformation. In this study, we have identified a large, retrospective cohort of benign IPs, IP-SCC, and control sinonasal polyp tissues that have been tested for high-risk HPV types to determine the prevalence in both benign and malignant IPs. Methods: A total of 94 IP tumors, 22 IP-SCC, and 13 sinonasal polyps were stained with HPV16/18 RNAscope and imaged with fluorescence to determine HPV status. Formalin-fixed slides were processed via standard antigen retrieval protocols and anti-HPV RNA staining was performed. Imaging was performed via confocal and bright-field microscopy. Results: We demonstrated significant HPV-positivity in IP-SCC versus benign IP tumors (p 

HDAC inhibitors show therapeutic promise for skin malignancies; however, the roles of specific HDACs in adult epidermal homeostasis and disease are poorly understood. We find that homozygous epidermal co-deletion of Hdac1 and Hdac2 in adult mouse epidermis causes reduced basal cell proliferation, apoptosis, inappropriate differentiation, and eventual loss of Hdac1/2-null keratinocytes. Hdac1/2 deficient epidermis displays elevated acetylated p53 and increased expression of the senescence gene p16. Loss of p53 partially restores basal proliferation, whereas p16 deletion promotes long-term survival of Hdac1/2-null keratinocytes. In activated GLI2-driven pre-basal cell carcinoma, Hdac1/2 deletion dramatically reduces proliferation and increases apoptosis, and knockout of either p53 or p16 partially rescues both proliferation and basal cell viability. Topical application of the HDAC inhibitor Romidepsin to normal epidermis or GLI2ΔN-driven lesions produces similar defects to genetic Hdac1/2 deletion, and these are partially rescued by loss of p16. These data reveal essential roles for HDAC1/2 in maintaining proliferation and survival of adult epidermal and basal cell carcinoma progenitors and suggest efficacy of therapeutic HDAC1/2 inhibition will depend in part on the mutational status of p53 and p16.

The distinction of atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) from its benign counterpart, lipoma, may represent a challenge. MDM2 DNA amplification is used as the gold standard as MDM2 immunohistochemistry lacks specificity and sensitivity. Herein, we investigate the diagnostic utility of MDM2 RNA in situ hybridization (RNA-ISH) and compare the test with MDM2 immunohistochemistry and MDM2 DNA fluorescence in situ hybridization (FISH) in benign and malignant lipomatous neoplasms. We evaluated 109 neoplasms including 27 lipomas, 25 spindle cell lipomas, 32 ALTs/WDLs, and 25 dedifferentiated liposarcomas (DDL). The validation cohort included 14 lipoma-like neoplasms that lacked unequivocal features of ALT/WDL and in which MDM2 immunohistochemistry was either equivocal, negative or falsely positive. Immunohistochemistry, automated RNA-ISH and DNA-FISH for MDM2 were performed. Tumors with diffuse nuclear staining or >50 dots per cell on RNA-ISH were considered positive. All lipomas and lipoma variants were negative for RNA-ISH while all ALTs/WDLs and DDLs were positive. Eighty percent (24/30) and 92% (22/24) of ALTs/WDLs and DDLs were positive for MDM2 immunohistochemistry. Lipomas and its variants were negative for MDM2 amplification; 92% and 100% of ALTs/WDLs and DDLs showed MDM2 DNA amplification. The mean percentage of ALT/WDL tumor cells showing MDM2 RNA-ISH positivity was 73% compared with 24% on MDM2 immunohistochemistry. RNA-ISH correctly classified all 10 ALTs/WDLs and all 4 lipomas in the validation cohort. The performance of MDM2 RNA-ISH and MDM2 DNA-FISH are equivalent. MDM2 RNA-ISH can be of diagnostic value in histologically challenging lipomatous neoplasms. The automated MDM2 RNA-ISH assay should allow for more widespread use of MDM2 testing and for a more sensitive and specific diagnosis of ALT/WDL.

Human papillomavirus (HPV) is a ubiquitous human pathogen that can be cleared by host immunity. Nonetheless, a small percentage of the patients develop persistent infection with oncogenic HPV, which poses an increased risk of developing HPV-associated malignancy. While cell-mediated immunity is a known systemic factor, local factors that influence persistent HPV infection have not been fully investigated. HPV-related head/neck cancers have a strong site preference for the oropharynx, suggesting the existence of unique local factors that promote HPV-induced oncogenesis. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Because butyrate is a potent epigenetic modulator, it could be an environmental factor influencing the development of HPV-positive oropharyngeal malignancy. In this study, we showed that butyrate treatment changed the property of HPV16 E6/E7-immortalized keratinocytes. In vitro, the treatment increased the cells' migration ability, slowed the growth, and increased the genotoxic resistance. When implanted in the syngeneic mice, the treated keratinocytes survived longer and exhibited a different growth pattern. The survival advantage obtained after butyrate exposure potentially can increase the susceptibility of HPV-infected oropharyngeal keratinocytes to further malignant transformation. Our results suggest that tonsillar bacteria's fermentation products may play an important role in the long-term persistence of high risk-HPV infection, which is a critical risk factor for developing HPV-positive oropharyngeal malignancy.