Biopsy vs. brushing: comparison of two sampling methods for the detection of HPV-DNA in squamous cell carcinoma of the oral cavity

Background: HR HPV infection was proposed as aetiological factor of oral squamous cell carcinomas (OSCC). HPV frequency in OSCC is highly variable, due to the discrepancy in oral sampling procedures, HPV testing methods and inclusion criteria regarding tumour site (strictly oral cavity vs. nearby structures). Our aim was to compare HPV DNA frequency and type-specific distribution in paired cytological and histological samples of SCC strictly located in oral cavity. The correlation between HPV detection rate by each method of sampling and demographical, behavioural and clinical-pathological variables was also examined. Patients and methods: HPV DNA was detected in brushed cells and formalin-fixed paraffin-embedded biopsies obtained from 83 consecutive unselected immunocompetent adults with OSCC. HPV DNA detection was performed in all samples by nPCR followed by direct DNA sequencing and the assay INNO-LiPA HPV Genotyping. Univariate and multivariate statistics were used, including Cohen κ index to evaluate agreement between two methods and association between HPV infection and demographical, behavioural and clinical-pathological variables for each method of sampling (p < 0.05 statistically significant). Results: HPV DNA was detected in 15.7% (13/83) of brushings and 12.1% (10/83) of biopsies (p > 0.05). High risk HPV 51, 16 and 39 were genotypes more frequently detected, especially among biopsies; no concordance between two methods was found (Cohen κ index = 0.04, p = 0.34). Conclusion: A fraction of OSCC could be linked to HR HPV infection in the Mediterranean area. Although without a statistical significance, biopsy specimen demonstrated more accurate for HR HPV detection than brushing in OSCC

The role of the innate immune response in HPV-related oral and oropharyngeal cancer

Introduction. During the last 20 years, the incidence of HPV-associated oropharyngeal cancer is increased. Principal actors of the innate immune response against HPV are represented by the TLRs (Toll like receptors). On the other hand different studies have reported that HPV can directly inhibit the functions of the TLRs pathway through interferons (IFNs). There are very few preliminary studies on the role of TLRs mediated HPV clearance in human oncology. Our study aim has been to evaluate whether TLR4 identifies HR-HPV integration state in OSCC. Methods. Protein levels of TLR4 in OSCC were assessed using Immunohistochemistry (IHC). In situ hybridization (ISH) for HPV-DNA detection in morphological context and Pyro-sequencing method have been performed in order to detect viral integration or episomic status. The relationship between TLR expression with or without HPV infection has been elucidated. Results. ISH HPV positive samples have reported lower TLR4 intensity than negative samples and it has confirmed by statistically significant difference (p = .002). There is no statistical correlation between TLR4 intensity and PCR HPV results (p > 0.05). Point-biserial correlation coefficient revealed statistically significant association between TLR4 expression and HR-HPV integration status (p = .0001) and between TLR4 expression index and HR-HPV infection (p = .001). Conclusions. We retain that TLR4 down-regulation is not associated to the histological tumoral grade but rather to HPV-16 infection and to its integration state into the host DNA

POSSIBLE ROLE OF CRY1 AND CRY2 IN ORAL CARCINOGENESIS

Aim. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. It has been shown recently that the occurrence, development, prognosis, and treatment of cancer are closely related to the abnormal expression of certain circadian-clock genes. CRY1 and CRY2 circadian-clock gene plays an important role in the regulation of many normal hysiological rhythms. This proteins act as light-independent inhibitors of CLOCK-BMAL1 components of the circadian clock. It has been revealed recently that abnormal expression of CRY1 and CRY2 correlate closely with the occurrence and development of many cancers. However, the expression and significance of this proteins in oral squamous cell carcinoma (OSCC) remains unknown. The aim of this study was to evaluate the expression levels of CRY1 and CRY2 in oral cancer. Materials and methods. CRY1 and CRY2 expression in cancerous and peritumoral tissues (when it was present) from 27 patients with OSCC was detected by immunohistochemistry techniques. Of all samples were received medical records (age, sex, grading, TNM, site of localization of the tumor). Immunohistochemistry was then performed on two sections for each of 27 sample mounted on poly-Llysine-coated glass slides to evaluate respectively the expression of CRY1 and CRY2.Results. In this study, out of the 27 cases, 11 were +/- positive in tumor area for CRY1 (most of which are well (differentiated), while out of 23 cases in which we evaluated the peritumoral tissue present in the section, 18 were positive. Also in the cases of positive tumor, almost always cytoplasmic, the CRY1 appears to be more strongly positive in dysplastic areas or even more in healthy epithelium, with a negative regulation in the areas most undifferentiated. As for the CRY2, out of the 27 cases analyzed, 17 were positive in the tumor area while about 23 cases in which we evaluated in peritumoral tissue present in the sections, 20 cases were positive. In tumor epithelium were found positivity also medium / high, present in tumors of different degree of differentiation, in some cases in other nuclear or cytoplasmic and nuclear/cytoplasmic, but when present the CRY2 is expressed, in most cases, in a manner similar or more intensely in peritumoral dysplastic epithelium. In the case of CRY2, there were no positivity in healthy epithelium (when present), but only in dysplastic epithelium. In addition, the positivity observed especially in peritumoral epithelium were present in states intermediate/surface. Conclusions. In conclusion, abnormal expression levels of CRY1 and CRY2 in OSCC tissue compared to healthy or dysplastic tissue may be related to the process of tumorigenesis. Further research focusing on these genes may, from the perspective of biological rhythms, provide novel ideas and methods for a better understanding of the occurrence and development of tumors, and for treatment of oral cancer

A Troubling Diagnosis of Verrucous Squamous Cell Carcinoma (“the Bad Kind” of Keratosis) and the Need of Clinical and Pathological Correlations: A Review of the Literature with a Case Report

Verrucous carcinoma (also known as Ackerman tumor) is an uncommon exophytic low-grade well-differentiated variant of squamous cell carcinoma. This neoplasm typically involves the oral cavity, larynx, genitalia, skin, and esophagus. It is well known for its locally aggressiveness and for its clinically slow-growing behaviour with minimal metastatic potential. Verrucous carcinoma of oral cavity is so closely aligned with the use of snuff and chewing tobacco that it has been called the “snuff dipper's cancer”. Recent studies have proved the role of HPV. The typical clinical presentation of oral verrucous carcinoma has long been known, as its remarkably innocuous appearance and biological behaviour. In this work, we report a review of the scientific literature and describe a troublesome case of oral verrucous cancer

A Novel GAN-Based Anomaly Detection and Localization Method for Aerial Video Surveillance at Low Altitude

The last two decades have seen an incessant growth in the use of Unmanned Aerial Vehicles (UAVs) equipped with HD cameras for developing aerial vision-based systems to support civilian and military tasks, including land monitoring, change detection, and object classification. To perform most of these tasks, the artificial intelligence algorithms usually need to know, a priori, what to look for, identify. or recognize. Actually, in most operational scenarios, such as war zones or post-disaster situations, areas and objects of interest are not decidable a priori since their shape and visual features may have been altered by events or even intentionally disguised (e.g., improvised explosive devices (IEDs)). For these reasons, in recent years, more and more research groups are investigating the design of original anomaly detection methods, which, in short, are focused on detecting samples that differ from the others in terms of visual appearance and occurrences with respect to a given environment. In this paper, we present a novel two-branch Generative Adversarial Network (GAN)-based method for low-altitude RGB aerial video surveillance to detect and localize anomalies. We have chosen to focus on the low-altitude sequences as we are interested in complex operational scenarios where even a small object or device can represent a reason for danger or attention. The proposed model was tested on the UAV Mosaicking and Change Detection (UMCD) dataset, a one-of-a-kind collection of challenging videos whose sequences were acquired between 6 and 15 m above sea level on three types of ground (i.e., urban, dirt, and countryside). Results demonstrated the effectiveness of the model in terms of Area Under the Receiving Operating Curve (AUROC) and Structural Similarity Index (SSIM), achieving an average of 97.2% and 95.7%, respectively, thus suggesting that the system can be deployed in real-world applications

HSP 27 aspossible prognostic factor in patients with oral squamous cell carcinoma.

Summary. HSP27 belongs to the Heat shock protein (HSP) family, which plays essential functions in cells under physiological conditions and prevents stressinduced cellular damage. The aim of this study was to investigate the biological role of HSP27 in oral tumorigenesis. Materials and methods: Seventy-nine cases of oral squamous cell carcinoma and 10 cases of normal mucosa were analysed for HSP27 expression by immunohistochemistry. Moreover, the western blot analysis was performed on two cases of normal mucosa and five cases of OSCC. Results: Normal oral mucosa showed a suprabasal expression of HSP27. Twenty-four cases of SCC (30.7%) showed a diffuse staining for HSP27, and 48 cases (60.3%) showed instead a decrease in staining, which was diffuse, homogeneous, or with alternation of positive and negative areas in a single tumor (“mosaic” pattern). Only 7 cases of OSCC (7.5%) were completely negative for HSP27. Frequency of lymph node metastases was higher in HSP27-negative tumours (3/7, 42.8%) than in HSP-reduced (16/48, 33.3%) or positive ones (5/26, 19.2%). Regard staging, stages I and II had a higher score than stages III and IV (stage I > stage II > stage III > stage IV). There was also a statistically significant correlation between HSP27 expression and grade: HSP27 expression was reduced in poorly differentiated tumours (P < 0.05). When analysed for prognostic significance, patients with negative/reduced HSP27 expression had poorer survival rates than the group with positive HSP27 expression (P < 0.05). The statistical analysis of these findings showed no significant correlation between HSP27 expression, sex, and tumour size. Conclusion: Cases with reduced expression were more aggressive and poorly differentiated. These data suggest that HSP27 expression may be useful in order to identify cases of oral squamous cell carcinoma with more aggressive and invasive phenotype providing novel diagnostic and prognostic information on individual patient survival with oral cancers

The proliferation marker Chromatin Assembly Factor-1 is of clinical value in predicting the biological behaviour of salivary gland tumours.

Salivary gland tumours (SGT) constitute a diagnostically challenging group of neoplasms with frequently unpredictable clinical outcome. The proliferation rate facilitates the identification of aggressive SGT. The Chromatin Assembly Factor-1 (CAF-1) is a major epigenetic regulator of nuclear chromatin organization during DNA replication. It plays a critical function in human tumourigenesis and has been proposed as a new proliferation and prognostic marker for some malignancies. This study focused on the role of CAF-1/p60 protein as a marker of clinical value for SGT. The expression of CAF-1/p60 was evaluated by immunohistochemistry on a retrospective series of 362 surgically excised benign and malignant SGT with different histogenesis and, when available, on fine-needle pre-surgical cytological biopsies. The resulting data were compared with traditional prognostic parameters, including the expression of the routine proliferation marker ki67/MIB1. CAF-1/p60 was detectable in all SGT, with highest degree of expression in metastasizing malignant tumours. Moreover, the cases of benign tumours which progressed to carcinoma during the follow-up, showed significantly higher CAF-1/p60 expression than non-progressing benign SGT, both on histological sections and cytological smears of the primary tumour. Cox's multiple regression analysis selected CAF-1/p60 expression as the best independent predictor of cancer development for benign SGT (p<0.0001), and the best independent predictor of metastasis onset for malignant tumours (p<0.0004). Overexpression of CAF-1/p60, on histological and/or cytological samples, characterizes malignant SGT with aggressive behaviour, irrespective of their specific histotype, and allows the early diagnosis of progression toward malignancy of morphologically benign tumours

Targeting oncogenic Src homology 2 domain-containing phosphatase 2 (SHP2) by inhibiting its protein-protein interactions

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2

Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study

Background: Human papillomavirus (HPV) is the causative agent in cervical cancer and HPV genotypes 16 and 18 cause the majority of these cancers. Natural killer (NK) cells destroy virally infected and tumour cells via killer immunoglobulin-like receptors (KIR) that recognize decreased MHC class I expression. These NK cells may contribute to clearance of HPV infected and/or dysplastic cells, however since KIR controls NK cell activity, KIR gene variation may determine outcome of infection.Methods: KIR gene frequencies were compared between 147 patients with a history of high-grade cervical intraepithelial neoplasia (CIN) and a control population of 187, to determine if any KIR genes are associated with high-grade CIN. In addition a comparison was also made between cases of high grade CIN derived from 30 patients infected with HPV 16/18 and 29 patients infected with non-16/18 HPV to determine if KIR variation contributes to the disproportional carcinogenesis derived from HPV 16/18 infection.Results: High-grade CIN was weakly associated with the absence of KIR2DL2 and KIR2DS2 (p = 0.046 and 0.049 respectively, OR 0.6; 95% CI 0.4 – 0.9) but this association was lost after correction for multi-gene statistical analysis.No difference in KIR gene frequencies was found between high-grade CIN caused by HPV 16/18 and non-16/18.Conclusion: No strong association between KIR genes, high-grade CIN and HPV genotype was found in the Western Australian population