The Expression of HPV E6/E7 mRNA in Situ Hybridization in HPV Typing-negative Cervical Cancer

Background: High-risk human papilloma virus (HR-HPV) persistent infection is the major tumorigenesis factor for cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). However, the incidence of HPV-negative CC is approximately 5%-30% with different HPV detection methods. HR-HPV E6/E7 mRNA in situ hybridization (RISH) can detect HPV-driven tumours. Our study aimed to explore whether HPV typing-negative CC was caused by HPV infection.Methods: The records of CC patients with HPV typing results who had undergone cervical biopsies, cervical conization or hysterectomies were collected from April 2018 to September 2021 at the Affiliated Hospital of Weifang Medical University. RISH was detected using RNAscope chromogenicin in the tissues of patients with CC. Immunohistochemistry (IHC) was performed to evaluate the expression of p16INK4a and Ki67. CC patients with positive HPV typing in the same period were collected as controls.Results: A total of 308 women with HPV typing results were enrolled, and 30 (9.74%) cases of HPV typing were negative, including 22/30 (73.3%) cases of squamous cell carcinoma (SCC) and 8/30 (26.7%) cases of adenocarcinoma. In the CC patients who were HPV typing-negative, 28/30 (93.3%) were positive for RISH and p16INK4a block+ staining, which contained 22/22 (100%) SCCs and 6/8 (75%) adenocarcinomas. RISH was positive in 278/278 (100%) HPV typing-positive CCs, which included 232/232 (100%) SCCs and 46/46 (100%) adenocarcinomas. While 273/278 (98.2%) showed p16INK4a block+ staining, the five negative cases were SCC. Positive RISH in HPV typing-negative CC was significantly lower than in the HPV typing-positive group (P=0.002, 95% CI: 0.848-1.027). However, this significant difference only existed in adenocarcinoma, and positive RISH in HPV typing-negative SCC was the same as in the HPV typing-positive group. No significant differences were seen in the expression of p16INK4a and Ki67 (P=0.291, 95% CI: 0.863-1.047 and P=0.174, 95% CI: 0.905-1.033).Conclusions: HPV typing may cause misdiagnosis in 9.74% of CC patients, and HPV E6/E7 mRNA can detect the majority of CC with HPV-related status even in HPV typing-negative patients. This approach could provide a novel option to accurately detect HR-HPVs in cervical tumours and help to eliminate the percentage of misdiagnosed HPV-related cases.

HPV Genotyping by Molecular Mapping of Tissue Samples in Vaginal Squamous Intraepithelial Neoplasia (VaIN) and Vaginal Squamous Cell Carcinoma (VaSCC)

HPV genotypes were determined in 63 vaginal squamous intraepithelial neoplasia (VaIN) and 7 vaginal squamous cell carcinomas (VaSCC). Of these, 37 cases had VaIN alone, and 26 cases had both VaIN and cervical intraepithelial neoplasia (CIN) or condyloma. HPV typing was performed in scraped cells by Genosearch-31 (GS-31) and in the archived tissues by uniplex E6/E7 PCR. In a total of 49 VaIN1, 17 VaIN2/3, and 7 VaSCC tissues, the prevalence of HPV was 91.2% in VaIN (VaIN1: 87.8%, VaIN2/3: 100%) and 85.7% in VaSCC. Comparing HPV results in scraped cell and tissue, 46.2% of high-risk (HR) types and 68.1% of any HPV types that had been identified in cell samples were not present in corresponding tissues. HPV types in VaIN and CIN lesions differed in 92.3% (24/26) of cases with multiple lesions. These results suggest that there are many preclinical HPV infections in the vagina or the cervix, and VaIN and CIN are independently developed. The manual microdissection procedure of tissue revealed one HPV type in one lesion. Seventeen HPV types, including high-risk (HR), possible high-risk (pHR), and low-risk (LR), were identified in 43 VaIN1 lesions. In higher grade lesions, six HR (HPV16, 18, 51, 52, 56, 58), one pHR (HPV66), and one LR (HPV42) HPV types were identified in 17 VaIN2/3, and six HPV types, including HPV16, 45, 58, and 68 (HR), and HPV53 and 67 (pHR), were detected in each case of VaSCC. The vagina appears to be the reservoir for any mucosal HPV type, and HR- or pHR-HPV types are causative agents for vaginal malignancies.

Recurrent respiratory papillomatosis with malignant transformation

Recurrent respiratory papillomatosis (RRP) is a rare disease with an unpredictable course. The etiology is associated with the human papillomavirus (HPV). Malignant transformation of papillomas is extremely rare. Two clinical cases of RRP malignization are presented. In the first case, 22-year-old women with a long history of RRP and the lower respiratory tract lesions, squamous cell lung cancer developed. Second case - development of squamous cell carcinoma from the tracheal papilloma in a 35-year-old patient. Aggressive local endobronchial treatment was carried out; however, the prognosis remains pessimistic due to the absence of etiopathogenetic therapy for RRP. Careful monitoring including HPV typing is mandatory, since infection with aggressive HPV strains leads to rapid proliferation of papillomas and malignant transformation.

The Prognostic Value of High-risk Human Papillomavirus Infection Status in Surgically Treated Stage IB1-IIA2 Cervical Squamous Cell Carcinoma

Background: High-risk HPV(hr-HPV) infection is important for the development of invasive cervical cancer. As a developing country with the largest population in the world, China has a great burden of cervical cancer. The cervical cancer screening strategies consisted of cytology and virology, which has been studied by HPV-typing test and HPV-DNA quantitative test since decades ago. The cervical cancer incidence rate has been declined due to the early treatment of precancerous lesions, but postoperative HPV infection is still an issue for cervical cancer patients. This study aims to investigate the association between HPV infection status and recurrence of early-stage cervical cancer through a novel PCR-based HPV test, which could do both HPV typing-test and DNA quantitative-test. Methods: Patients diagnosed with cervical cancer staged IB1- IIA2, who were treated by radical hysterectomy and lymphadenectomy at Cancer Hospital Chinese Academy of Medical Sciences(CAMS) between January 2014 and December 2016 were accrued. The clinicopathological factors, pre- and post-operative HPV infection status, and the prognosis were investigated. Cox regression was used to identify factors associated with LRFS, MFS and OS. Results: A total of 312 patients were enrolled in this study, who were treated by radical resection and accepted pre- and post-operative HPV tests, with a median follow-up time of 60 months(range 14~79 months). The 5-year LRFS rate, MFS rate, and OS rate were 97.8%, 98.4%, and 98.7%. The pre-operative HPV infection rate was 85.3%(266/312), 74 patients had a high level of HPV-DNA(>5x106 copy number/104 cells). Twenty-nine patients had a postoperative persistent high level of HPV-DNA(9.3%). Postoperative persistent high level of HPV-DNA within 12 months(p=0.013), postoperative persistence of HPV-16/18 within 24 months(p=0.004), and deep stromal invasion(>2/3)(p=0.007) were associated with a poor LRFS.Conclusion: Pre-operative HPV-16/18 infection and high level of HPV-DNA were not associated with local recurrence of cervical cancer. Most initial HPV-positive patients had HPV cleared within 24 months postoperatively. Postoperative HPV-16/18 persistence within 24 months, postoperative persistence of high HPV-DNA level within 24 months, and deep stromal invasion(>2/3) were independent risk factors for local recurrence of cervical cancer.

Prevalence of High-Risk Human Papillomavirus Types Among Women Screened for Cervical Cancer in Yazd, Iran, and Comparison of Cytology, Histology, and Colposcopy Results

Background: Human Papillomavirus (HPV) is a DNA virus with more than 100 genotypes, at least 12 of which are high-risk and associated with high-grade cervical lesions. Data on the prevalence of high-risk HPV genotypes among women are not yet available for the total regions of Iran. Objectives: The present study aimed to determine the prevalence of high-risk HPV types among women screened for cervical carcinoma in Yazd and compare the cytology, histology, and colposcopy results. Methods: In this cross-sectional study, 402 women referring to gynecology clinics of Shahid Sadoughi University of Medical Sciences, Yazd, Iran, were selected. The Pap smear and HPV typing were performed on cervical samples. The high-risk HPV types were detected by the polymerase chain reaction (PCR)-based reverse blot hybridization assay. Colposcopy was carried out on patients with high-risk HPV types, and biopsies were taken for histological examination. Results: Among 402 women screened by HPV-PCR, 32 (7.97%) women were positive for high-risk HPV types. Human papillomavirus 16 and HPV18 were the most frequent genotypes (46.9%). The cytology, histology, and colposcopy results were abnormal in 56.2%, 29.1%, and 71.9% of patients, respectively. Pap smear had 100% sensitivity and 58.3% specificity for the detection of high-grade cervical lesions, while these values for colposcopy were 75% and 87.5%, respectively. Conclusions: The frequency of high-risk HPV types was relatively low among women living in Yazd than in those from other provinces of Iran. A significant percentage of patients with HPV had normal cervical cytology and histology. Therefore, HPV typing is recommended to decrease the development of cervical cancer. Colposcopy had acceptable sensitivity and specificity for the detection of high-grade cervical lesions.

Designing low-cost, accurate cervical screening strategies that take into account COVID-19: a role for self-sampled HPV typing

Background We propose an economical cervical screening research and implementation strategy designed to take into account the typically slow natural history of cervical cancer and the severe but hopefully temporary impact of COVID-19. The commentary introduces the practical validation of some critical components of the strategy, described in three manuscripts detailing recent project results in Asia and Africa. The main phases of a cervical screening program are 1) primary screening of women in the general population, 2) triage testing of the small minority of women that screen positive to determine need for treatment, and 3) treatment of triage-positive women thought to be at highest risk of precancer or even cancer. In each phase, attention must now be paid to safety in relation to SARS-CoV-2 transmission. The new imperatives of the COVID-19 pandemic support self-sampled HPV testing as the primary cervical screening method. Most women can be reassured for several years by a negative test performed on a self-sample collected at home, without need of clinic visit and speculum examination. The advent of relatively inexpensive, rapid and accurate HPV DNA testing makes it possible to return screening results from self-sampling very soon after specimen collection, minimizing loss to follow-up. Partial HPV typing provides important risk stratification useful for triage of HPV-positive women. A second “triage” test is often useful to guide management. In lower-resource settings, visual inspection with acetic acid (VIA) is still proposed but it is inaccurate and poorly reproducible, misclassifying the risk stratification gained by primary HPV testing. A deep-learning based approach to recognizing cervical precancer, adaptable to a smartphone camera, is being validated to improve VIA performance. The advent and approval of thermal ablation permits quick, affordable and safe, immediate treatment at the triage clinic of the majority of HPV-positive, triage-positive women. Conclusions Overall, only a small percentage of women in cervical screening programs need to attend the hospital clinic for a surgical procedure, particularly when screening is targeted to the optimal age range for detection of precancer rather than older ages with decreased visual screening performance and higher risks of hard-to-treat outcomes including invasive cancer.

Hypercalcemia with invasive pulmonary papillomatosis and microinvasive squamous carcinoma

Juvenile respiratory papillomatosis is a rare pediatric disease in which benign papillomata develop in the respiratory tract, most commonly involving the larynx and tracheobronchial tree. Invasive pulmonary papillomatosis is an aggressive form in which the papillomata extend into the lung parenchyma. We report a case of a 22-year-old man with a long-standing juvenile respiratory tract papillomatosis, initially diagnosed at age 2, who subsequently developed invasive pulmonary papillomatosis and underwent partial surgical resection for his pulmonary disease. Hypercalcemia complicated the patient’s final hospitalizations. HPV typing performed on a laryngeal papilloma was positive for HPV 6/11. The lobectomy specimen revealed malignant transformation of invasive pulmonary papillomatosis characterized by the presence of microinvasive nests of squamous carcinoma. Immunohistochemical stain for parathyroid hormone on the invasive component was negative. Eventually, the patient succumbed to his disease and while the family refused post-mortem examination, Positron Emission Tomography (PET) performed during the patient’s terminal course suggested the possibility of metastasis to liver and periaortic lymph nodes. There was no evidence of bony metastasis.

Bioinformatics Pipeline for Human Papillomavirus Short Read Genomic Sequences Classification Using Support Vector Machine

We recently developed a test based on the Agilent SureSelect target enrichment system capturing genomic fragments from 191 human papillomaviruses (HPV) types for Illumina sequencing. This enriched whole genome sequencing (eWGS) assay provides an approach to identify all HPV types in a sample. Here we present a machine learning algorithm that calls HPV types based on the eWGS output. The algorithm based on the support vector machine (SVM) technique was trained on eWGS data from 122 control samples with known HPV types. The new algorithm demonstrated good performance in HPV type detection for designed samples with 25 or greater HPV plasmid copies per sample. We compared the results of HPV typing made by the new algorithm for 261 residual epidemiologic samples with the results of the typing delivered by the standard HPV Linear Array (LA). The agreement between methods (97.4%) was substantial (kappa = 0.783). However, the new algorithm identified additionally 428 instances of HPV types not detectable by the LA assay by design. Overall, we have demonstrated that the bioinformatics pipeline is an accurate tool for calling HPV types by analyzing data generated by eWGS processing of DNA fragments extracted from control and epidemiological samples.