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)-related oropharyngeal small cell carcinoma (OPSmCC) is a rare malignancy with aggressive behavior, whereas HPV-related oropharyngeal squamous cell carcinoma (OPSqCC) displays a favorable prognosis. Notably, these two malignancies occasionally arise in an identical tumor. In this case study, we explored the molecular characteristics that distinguishes these two carcinomas employing a rare case of HPV-related oropharyngeal carcinoma (OPC) with the combined histology of SmCC and SqCC. Immunohistochemical analysis and HPV-RNA in situ hybridization (ISH) suggested that both SmCC and SqCC were HPV-related malignancies. Targeted exome sequencing revealed that SmCC and SqCC had no significant difference in mutations of known driver genes. In contrast, RNA sequencing followed by bioinformatic analyses suggested that aberrant transcriptional programs may be responsible for the neuroendocrine differentiation of HPV-related OPC. Compared to SqCC, genes upregulated in SmCC were functionally enriched in inflammatory and immune responses (e.g., arachidonic acid metabolism). We then developed a SmCC-like gene module (top 10 upregulated genes) and found that OPC patients with high module activity showed poor prognosis in The Cancer Genome Atlas (TCGA) and GSE65858 cohort. Gene set enrichment analysis of the SmCC-like gene module suggested its link to MYC proto-oncogene in the TCGA dataset. Taken together, these findings suggest that the SmCC-like gene module may contribute to acquisition of aggressive phenotypes and tumor heterogeneity of HPV-related OPC. The present case study is the first report of genetic and transcriptomic aberrations in HPV-related OPSmCC combined with SqCC.Cold Spring Harbor Laboratory Press.

The growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly, at the periphery of the growth plate immediately adjacent to the perichondrium are 'borderline' chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single cell RNA-seq analysis revealed subpopulations of Col2a1-creER-marked neonatal chondrocytes and their cell-type specific markers. A tamoxifen pulse to Pthrp-creER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow and became Col(2.3kb)-GFP(+) osteoblasts and Cxcl12-GFP(high) reticular stromal 'CAR' cells. Interestingly, these borderline chondrocyte-derived marrow cells were short-lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage-tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. This article is protected by copyright. All rights reserved.

Abstract BACKGROUND: Nasopharyngeal carcinoma (NPC) is related to Epstein-Barr virus (EBV) in endemic areas; however, the role of viruses in nonendemic countries is unclear. Our nationwide study investigated the prevalence and prognostic significance of EBV and human papillomaviruses (HPVs) in Finnish NPC tumors. METHODS: We analyzed samples from 150 patients diagnosed between 1990 and 2009. Viral status was determined using EBV and HPV RNA in situ hybridizations, and p16 immunohistochemistry. Patient and treatment characteristics were obtained from patient records. RESULTS: In our white patient cohort, 93 of 150 (62%) patients were EBV-positive and 21/150 (14%) patients were HPV-positive with no coinfections. Thirty-six (24%) tumors were negative for both viruses. The 5-year disease-specific survival for patients with EBV-positive, HPV-positive, and EBV/HPV-negative tumors was 69%, 63%, and 39%, respectively. In multivariable-adjusted analysis, overall survival was better among patients with EBV-positive (P = .005) and HPV-positive (P = .03) tumors compared to patients with EBV/HPV-negative tumors. CONCLUSIONS: In our low-incidence population, EBV and HPV are important prognostic factors for NPC.

Skeletal stem cells regulate bone growth and homeostasis by generating diverse cell types, including chondrocytes, osteoblasts and marrow stromal cells. The emerging concept postulates that there exists a distinct type of skeletal stem cell that is closely associated with the growth plate1–4, which is a type of cartilaginous tissue that has critical roles in bone elongation5. The resting zone maintains the growth plate by expressing parathyroid hormone-related protein (PTHrP), which interacts with Indian hedgehog (Ihh) that is released from the hypertrophic zone6–10, and provides a source of other chondrocytes11. However, the identity of skeletal stem cells and how they are maintained in the growth plate are unknown. Here we show, in a mouse model, that skeletal stem cells are formed among PTHrP-positive chondrocytes within the resting zone of the postnatal growth plate. PTHrP-positive chondrocytes expressed a panel of markers for skeletal stem and progenitor cells, and uniquely possessed the properties of skeletal stem cells in cultured conditions. Cell-lineage analysis revealed that PTHrP-positive chondrocytes in the resting zone continued to form columnar chondrocytes in the long term; these chondrocytes underwent hypertrophy, and became osteoblasts and marrow stromal cells beneath the growth plate. Transit-amplifying chondrocytes in the proliferating zone—which was concertedly maintained by a forward signal from undifferentiated cells (PTHrP) and a reverse signal from hypertrophic cells (Ihh)—provided instructive cues to maintain the cell fates of PTHrP-positive chondrocytes in the resting zone. Our findings unravel a type of somatic stem cell that is initially unipotent and acquires multipotency at the post-mitotic stage, underscoring the malleable nature of the skeletal cell lineage. This system provides a model in which functionally dedicated stem cells and their niches are specified postnatally, and maintained throughout tissue growth by a tight feedback regulation system.

Genetic mutations in the Xylt1 gene are associated with Desbuquois dysplasia type II syndrome characterized by sever prenatal and postnatal short stature. However, the specific role of XylT-I in the growth plate is not completely understood. Here, we show that XylT-I is expressed and critical for the synthesis of proteoglycans in resting and proliferative but not in hypertrophic chondrocytes in the growth plate. We found that loss of XylT-I induces hypertrophic phenotype-like of chondrocytes associated with reduced interterritorial matrix. Mechanistically, deletion of XylT-I impairs the synthesis of long glycosaminoglycan chains leading to the formation of proteoglycans with shorter glycosaminoglycan chains. Histological and Second Harmonic Generation microscopy analysis revealed that deletion of XylT-I accelerated chondrocyte maturation and prevents chondrocytes columnar organization and arrangement in parallel of collagen fibers in the growth plate, suggesting that XylT-I controls chondrocyte maturation and matrix organization. Intriguingly, loss of XylT-I induced at embryonic stage E18.5 the migration of progenitor cells from the perichondrium next to the groove of Ranvier into the central part of epiphysis of E18.5 embryos. These cells characterized by higher expression of glycosaminoglycans exhibit circular organization then undergo hypertrophy and death creating a circular structure at the secondary ossification center location. Our study revealed an uncovered role of XylT-I in the synthesis of proteoglycans and provides evidence that the structure of glycosaminoglycan chains of proteoglycans controls chondrocyte maturation and matrix organization.

The current practice of determining histologic grade with a single molecular biomarker can facilitate differential diagnosis but cannot predict the risk of lesion progression. Cancer is caused by complex mechanisms, and no single biomarker can both make accurate diagnoses and predict progression risk. Modelling using multiple biomarkers can be used to derive scores for risk prediction. Mathematical models (MMs) may be capable of making predictions from biomarker data. Therefore, this study aimed to develop MM-based scores for predicting the risk of precancerous cervical lesion progression and identifying precancerous lesions in patients in northern Thailand by evaluating the expression of multiple biomarkers. The MMs (Models 1-5) were developed in the test sample set based on patient age range (five categories) and biomarker levels (cortactin, p16INK4A, and Ki-67 by immunohistochemistry [IHC], and HPV E6/E7 ribonucleic acid (RNA) by in situ hybridization [ISH]). The risk scores for the prediction of cervical lesion progression ("risk biomolecules") ranged from 2.56-2.60 in the normal and low-grade squamous intraepithelial lesion (LSIL) cases and from 3.54-3.62 in cases where precancerous lesions were predicted to progress. In Model 4, 23/86 (26.7%) normal and LSIL cases had biomolecule levels that suggested a risk of progression, while 5/86 (5.8%) cases were identified as precancerous lesions. Additionally, histologic grading with a single molecular biomarker did not identify 23 cases with risk, preventing close patient monitoring. These results suggest that biomarker level-based risk scores are useful for predicting the risk of cervical lesion progression and identifying precancerous lesion development. This multiple biomarker-based strategy may ultimately have utility for predicting cancer progression in other contexts.

HPV-associated oral cavity squamous cell carcinoma (SCC) is not well-characterized in the literature, and also has a clinical significance that is poorly understood.We gathered a cohort of oral cavity (OC) SCC with nonkeratinizing morphology, either in the invasive or in situ carcinoma (or both), tested for p16 by immunohistochemistry and high risk HPV E6/E7 mRNA by RTPCR (reference standard for transcriptionally-active high risk HPV) and gathered detailed morphologic and clinicopathologic data.Thirteen patients from two institutions were proven to be HPV-associated by combined p16 and high risk HPV mRNA positivity. All 13 patients (100%) were males, all were heavy smokers (average 57 pack/year), and most were active drinkers (9/11 or 81.8%). All 13 (100%) involved the tongue and/or floor of mouth. All had nonkeratinizing features, but maturing squamous differentiation varied widely (0-90%; mean 37.3%). Nonkeratinizing areas had high N:C ratios and larger nests, frequently with pushing borders, and minimal (or no) stromal desmoplasia. The carcinoma in situ, when present, was Bowenoid/nonkeratinizing with cells with high N:C ratios, full thickness loss of maturation, and abundant apoptosis and mitosis. HPV was type 16 in 11 patients (84.6%) and type 33 in two (15.4%). Nine patients had treatment data available. These underwent primary surgical resection with tumors ranging from 1.6 to 5.2 cm. Most had bone invasion (6/9-66.7% were T4a tumors), and most (6/9-66.7%) had extensive SCC in situ with all 6 of these patients having final margins positive for in situ carcinoma.HPV-associated OCSCC is an uncommon entity that shows certain distinct clinical and pathologic features. Recognition of these features may help pathologic diagnosis and could potentially help guide clinical management.