A pilot study on salivary HPV DNA detection to monitor active disease from patients with recurrent respiratory papillomatosis.

PURPOSE: Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV)-related disease affecting the upper airway and saliva could be an important non-invasive sampling source for viral screening and clinical monitoring. We investigated whether HPV DNA could be detected in saliva (cellular pellets and supernatant) from RRP patients and influence on clinical manifestation of the disease. MATERIALS AND METHODS: In this pilot study, saliva samples from 14 RRP patients were obtained in preoperative condition (n = 7) and in disease-free interval (DFI; n = 7). Healthy donors (n = 14) were also included. HPV DNA was investigated by polymerase chain reaction (PCR)-based assays. RESULTS: From cellular pellets, HPV-positive saliva was only detected from preoperative collections (5/7; 71.4 %) and showed a mean cycle threshold (Ct) value of 24.33 (±1.25), whereas all patients in DFI were HPV-negative (Ct ≥ 32.16), revealing significant difference between these two clinical moments (p = 0.021). Patients in DFI and healthy donors showed similar Ct values. From saliva supernatant, detectable HPV cell-free DNA (cfDNA) occurred in 42.9 % (3/7) and 57.1 % (4/7) of preoperative collections using the commercial cfDNA kits from Norgen and Qiagen, respectively. Salivary cfDNA size distribution obtained by TapeStation analysis showed a predominant size range of 150 to 400 bp in both patients and healthy controls, corresponding to mononucleosomal and dinucleosomal fragments. CONCLUSIONS: In conclusion, HPV DNA screening in saliva (both cellular pellets and cfDNA) may have clinical utility to monitor active disease of RRP patients.

Clinical applications of liquid biopsy in HPV-negative and HPV-positive head and neck squamous cell carcinoma: advances and challenges.

Head and neck squamous cell carcinomas (HNSCCs) represent the most common epithelial tumors that arise from mucosa of the oral cavity, pharynx, and larynx. The development of HNSCCs is usually associated with tobacco use, alcohol consumption, and human papillomavirus (HPV) infection. Most HNSCCs are diagnosed in advanced states, leading to a worse clinical outcome. Screening tests based on potential biomarkers associated with HNSCCs could improve this scenario. Liquid biopsy has emerged as a promising area of cancer investigation, offering a minimally invasive approach to track circulating biomarkers in body fluids that could potentially contribute to the diagnosis, predict prognosis, and monitor response to treatment. This review will discuss translational studies describing the clinical applications of liquid biopsy in HPV-negative and HPV-positive HNSCCs focused on circulating nucleic acids [cell-free DNA (cfDNA) and cell-free RNA (cfRNA)], circulating tumor cells (CTCs), and extracellular vesicles (EVs), which can be found in plasma, serum, and saliva.

Prevalence of Epstein-Barr virus infection in recurrent respiratory papillomatosis and the influence on disease severity.

Recurrent respiratory papillomatosis (RRP) is characterized by benign papillomatous lesions in the upper airway associated with human papillomavirus infection. It has been proposed that viral coinfections may contribute to an aggressive clinical course of the disease. For this purpose, we investigated the prevalence of Epstein-Barr virus (EBV) infection among 40 RRP patients by polymerase chain reaction assay. EBV DNA was detected in 11 cases and disease severity was observed in 54.5% of EBV-positive patients. No significant association was found between the RRP severity categories and EBV status (P > 0.05). Regardless EBV status, disease severity showed significant association with RRP diagnosis since childhood (P = 0.009). These findings indicate an absence of direct influence of EBV infection on aggressive course of RRP. However, the development of RRP since childhood increase the susceptibility to disease severity.

Genetic and methylation status of CDKN2A (p14ARF/p16INK4A) and TP53 genes in recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a rare and chronic disease affecting the upper airway with papillomatous lesions caused by the human papillomavirus (HPV) infection, especially HPV-6 and/or HPV-11 types. Little is known about the genetic and epigenetic drivers in RRP pathophysiology. For this purpose, we analyzed 27 papillomatous lesions from patients with RRP to evaluate somatic mutations and methylation status in CDKN2A (p14ARF/p16INK4A) and TP53, which are key tumor suppressor genes for the cell cycle control. Sanger sequencing analysis revealed one somatic mutation in TP53 (c.733_734insA) and four mutations in CDKN2A (c.-30G > T, c.29_30insA, c.69delT, and c.300C > A). These mutations were observed in 10 patients, 6 of which carried double mutation. Furthermore, 50% (5/10) of these patients carrying somatic mutations had RRP severity, representing 62.5% (5/8) of the severity cases in this study, albeit no significant association was found between somatic mutations and disease severity. Methylation-specific polymerase chain reaction assays revealed p14ARF promoter hypermethylation in 100% of cases, followed by TP53 (96.3%) and p16INK4A (55.6%), suggesting the influence of HPV in the DNA methylation machinery. In conclusion, somatic mutations were not common events identified in patients with RRP. However, epigenetic modulation by high methylation rates, particularly for the p14ARF/TP53 pathway, seems to be in the course of RRP development.

Clinical and treatment course of lung carcinoma from adult-onset recurrent respiratory papillomatosis with lung involvement: A case report.

Neoplasms induced by human papillomavirus (HPV) infection are generally associated to high-risk HPV types. Recurrent respiratory papillomatosis (RRP) is a rare and chronic HPV-related disease characterized by papillomatous lesions in the respiratory tract, usually affecting larynx. RRP rarely comprises malignant transformation since the low-risk HPV-6 and/or HPV-11 are the most commonly found in the disease. We described a case of one adult-onset RRP (29-year-old, female) with HPV-6 infection affecting vocal folds and extra-laryngeal sites. Computed tomographic scan of the chest revealed papillomatosis with pulmonary spread showing multiple nodules and cavities. Lung involvement later progressed to squamous cell carcinoma of the lung. Patient's pregnancy during investigations may have accelerated lung carcinoma development due to immunological changes. Immunohistochemistry revealed PD-L1 high expression in tumor biopsy and, after pregnancy, the oncology treatment included a combination of pembrolizumab (PD-L1 inhibitor) to chemotherapy and also radiotherapy, showing considerable results. Patient died due to lung cancer complications 15 months after cancer diagnosis. RRP management associated with lung involvement and poor prognostic outcome, such as lung carcinoma, is still a big challenge. In this report, we described the clinical and treatment course of RRP progression to highlight the need for attention to future patients.

Hypermethylation in Gene Promoters Are Induced by Chronic Exposure to Benzene, Toluene, Ethylbenzene and Xylenes.

BACKGROUND AND OBJECTIVE: Gas station attendants are occupationally exposed to benzene, toluene, ethylbenzene and xylene (BTEX) compounds and thus more susceptible to the biological effects of this mixture present in gasoline, especially due to the carcinogenicity of benzene. Furthermore, the harmful effects of BTEX exposure may be potentiated by genetic and epigenetic inactivation of critical genes. The objective was to evaluate such gene-BTEX interactions accessing the promoter methylation status of p14ARF, p16INK4A and GSTP1 in peripheral blood leukocyte samples. MATERIALS AND METHODS: The 59 exposed and 68 unexposed participants from Rio de Janeiro, Brazil, were included. The promoter methylation status was accessed by methylation-specific PCR (MSP) and GSTP1 Ile105Val polymorphism was investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) technique. RESULTS: Both p14ARF and p16INK4A were significantly hypermethylated in exposed subjects compared to unexposed (p = 0.004 and p<0.001, respectively). Additionally, p16INK4A hypermethylation in the exposed group was correlated with chromosomal abnormalities (CAs) (p = 0.018), thus highlighting the influence of the gene-environment interactions on genome instability. Noteworthy, p16INK4A methylation was significantly associated with miscarriage among female attendants (p = 0.047), in which those who reported miscarriage exhibited hypermethylation in at least 2 of the 3 genes analyzed. The GSTP1 heterozygote genotype, which could affect the metabolism of benzene detoxification, was found in both groups but was more frequent in those occupationally exposed. No significant association was observed between GSTP1 genotypes and methylation status. CONCLUSION: Together, these findings indicate that gas station attendants with the aforementioned epigenetic and genetic profiles may be at greater risk of occupational BTEX exposure-induced genome instability, which could require concerted efforts to establish more preventive actions and constant biomonitoring in gas station attendants.

Impact of reduced levels of APE1 transcripts on the survival of patients with urothelial carcinoma of the bladder.

Molecular evidence indicates that alterations in genes involved in the maintenance of genome stability may be related to susceptibility to bladder carcinoma. Our goal was to evaluate the prognostic role of base excision repair (BER) genes in a cohort of patients diagnosed with primary urothelial carcinoma of the bladder (UCB). The levels of all APE1, XRCC1 and POLB transcripts were detected by quantitative real-time PCR (qPCR) technique in tumor samples from 52 patients undergoing transurethral resection (TUR) for primary UCB at the Department of Urology, Brazilian National Cancer Institute, Rio de Janeiro. Increased levels of APE1, XRCC1 and POLB transcripts were significantly associated with high-grade tumors when compared to these levels in low-grade tumors (p<0.01) and could be attributed to different mechanisms of transcriptional regulation as a response to tumorigenesis and oxidative stress. By analyzing the collected data in the present study, regardless of pathological grade or stage, univariate analysis revealed that the reduced levels of APE1 transcripts were significantly associated with cancer-specific mortality (p=0.032). Furthermore, the variant genotype (TG/GG) of the APE1 T1349G polymorphism was observed in 75% of a subset of patients who concomitantly experienced reduced levels of the APE1 transcript and death and/or recurrence events. Taken together, our data reinforce the idea that human DNA repair mechanisms must be finely regulated in order to avoid instability leading to tumorigenesis and poor clinical outcomes in UCB patients.