Scattering-Based Light-Sheet Microscopy Imaging of Human Papillomavirus-Associated Squamous Lesions of the Anal Canal: A Proof-of-Principle Study.

Demand for anal cancer screening is expected to rise following the recent publication of the Anal Cancer-HSIL Outcomes Research trial, which showed that treatment of high-grade squamous intraepithelial lesions significantly reduces the rate of progression to anal cancer. While screening for human papillomavirus-associated squamous lesions in the cervix is well established and effective, this is less true for other sites in the lower anogenital tract. Current anal cancer screening and prevention rely on high-resolution anoscopy with biopsies. This procedure has a steep learning curve for providers and may cause patient discomfort. Scattering-based light-sheet microscopy (sLSM) is a novel imaging modality with the potential to mitigate these challenges through real-time, microscopic visualization of disease-susceptible tissue. Here, we report a proof-of-principle study that establishes feasibility of dysplasia detection using an sLSM device. We imaged 110 anal biopsy specimens collected prospectively at our institution's dysplasia clinic (including 30 nondysplastic, 40 low-grade squamous intraepithelial lesion, and 40 high-grade squamous intraepithelial lesion specimens) and found that these optical images are highly interpretable and accurately recapitulate histopathologic features traditionally used for the diagnosis of human papillomavirus-associated squamous dysplasia. A reader study to assess diagnostic accuracy suggests that sLSM images are noninferior to hematoxylin and eosin images for the detection of anal dysplasia (sLSM accuracy = 0.87; hematoxylin and eosin accuracy = 0.80; P = .066). Given these results, we believe that sLSM technology holds great potential to enhance the efficacy of anal cancer screening by allowing accurate sampling of diagnostic tissue at the time of anoscopy. While the current imaging study was performed on ex vivo biopsy specimens, we are currently developing a handheld device for in vivo imaging that will provide immediate microscopic guidance to high-resolution anoscopy providers.

Genetic variation in the E6 and E7 genes of human papillomavirus type 16 in northeastern Argentina.

The province of Misiones is considered a region with a high mortality rate due to cervical cancer (CC). To gain insight into this problem, we explored the association between genetic variation in the E6 and E7 oncogenes of HPV16 and the risk of CC. We studied 160 women with cytological diagnoses of negative for intraepithelial lesion or malignity, low-grade squamous intraepithelial lesion, and high-grade squamous intraepithelial lesion/CC and a positive test for HPV16 infection. The genetic characterization of E6 and E7 genes was undertaken through PCR amplification and direct Sanger sequencing. Phylogenetic classification was conducted using Bayesian methods. To estimate the odds ratio (OR) for an association between genetic variants in the E6 and E7 genes and the risk of CC, we used ordinal logistic regression adjusted by age. The final data set comprised 112 samples. Diagnostic single-nucleotide polymorphisms (SNPs) and phylogenetic trees confirmed the presence of Lineage A (95.5%) and D (4.5%) in the samples. For the E6 gene, we identified eleven different sequences, with the most common ones being Lineage A E6 350G (58.9%) and E6 350T (37.5%). The E6 350G was associated with progression to HSIL/CC, with an OR of 19.41 (4.95-76.10). The E7 gene was more conserved than E6, probably due to the functional constraints of this small protein. Our results confirmed the association of the E6 350G SNP with a higher risk of developing CC. These data will contribute to understanding the biological bases of CC incidence in this region.

Lineage analysis of human papillomavirus types 31 and 45 in cervical samples of Iranian women.

Knowing the regional lineages/sublineages of human papillomavirus 31 (HPV 31) and 45 would be of great importance for further evolutionary, epidemiological, and biological analysis. In this regard, to characterize more common lineages and sublineages of HPV 31 and 45, the sequence variations of E6 gene were investigated in normal, premalignant, and malignant samples collected from the cervix in Iran. In total, 54 HPV 31- and 24 HPV 45-positive samples were analyzed by hemi-nested polymerase chain reaction (PCR) and nested-PCR, respectively. All PCR products were subjected to direct sequencing analysis. The results indicated that all three lineages A, B, and C were detected in HPV 31-positive samples; among which HPV 31 lineage A was dominant as it was found in 66.7% of all samples. HPV 31 lineages B and C were identified in 5.5% and 27.8% of samples, respectively. In HPV 45-infected samples, lineage B comprised of 62.5% of all samples and the remaining 37.5%  belonged to lineage A. In conclusion, our findings showed that lineage A of HPV 31 was predominant in Iran. Lineage B of HPV 45 was also dominant among Iranian women. However, further studies with larger sample size should be addressed to estimate the pathogenicity risk of HPV 31 or HPV 45 lineages/sublineages in the development of cervical cancer among Iranian women.

Characteristics of human papillomavirus infection among women with cervical cytological abnormalities in the Zhoupu District, Shanghai City, China, 2014-2019.

BACKGROUND: Human papillomavirus (HPV) infection is currently the main cause of cervical cancer and precancerous lesions in female patients. By analyzing 6-year patient data from Shanghai Zhoupu Hospital in China, we retrospectively analyzed the epidemiological characteristics of women to determine the relationship between HPV genotype and cytological test results. METHODS: From 2014 to 2019, 23,724 cases of cervical shedding were collected from Zhoupu Hospital in Shanghai, China. By comparing the results of HPV and ThinPrep cytology test (TCT), the HPV infection rate of patients was retrospectively analyzed. HPV genotyping using commercial kits can detect 21 HPV subtypes (15 high-risk and 6 low-risk). According to the definition of the Bethesda system, seven types of cervical cytology results were involved. RESULTS: 3816 among 23,724 women, nearly 16.08%, were infected with HPV. The top three highest HPV prevalence rates were high-risk type infection, including HPV52 (3.19%), 58 (2.47%) and 16 (2.34%). The number of single-type HPV infections (3480 (91.20%)) was much larger than the number of multi-type ones (336 (8.8%)). Single-type infections were mainly in women aged 50-60 (16.63%) and women under 30 (15.37%), while multi-type infections were more common in women over 60 (2.67%). By analyzing the long-term trends, between 2014 and 2019, HPV52, 58, and 16 subtypes changed significantly, and the HPV positive rate also changed significantly during this period. Among 4502 TCT positive women, 15 (4.04%), 125 (2.64%),159 (1.54%), 4202 (17.71%) and 1 (0.004%) had atypical glandular cells (AGC), high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL), atypical squamous cells (ASC)and cervical adenocarcinoma, respectively. The HPV infection rates were 66.08%, 63.99%, 115.20%, 119.50%, and 31.72% for NILM, AGCs, HSILs LSILs and ASCs, respectively. CONCLUSIONS: HPV and TCT screening were very important steps in the secondary prevention of cervical cancer. Through the tracking and analysis of HPV and TCT results in this study, it can provide valuable information for Shanghai's HPV screening and prevention strategies, and provide references for clinical decision-making in the treatment of cervical cancer and precancerous lesions.

Expanding the Morphologic, Immunohistochemical, and HPV Genotypic Features of High-grade Squamous Intraepithelial Lesions of the Vulva With Morphology Mimicking Differentiated Vulvar Intraepithelial Neoplasia and/or Lichen Sclerosus.

Squamous cell carcinoma of the vulva can arise through 2 pathways: human papillomavirus (HPV)-dependent high-grade squamous intraepithelial lesions (previously termed usual vulvar intraepithelial neoplasia) or HPV-independent (differentiated vulvar intraepithelial neoplasia, dVIN). Distinguishing between the 2 types can be clinically and histologically difficult. A subset of high-grade squamous intraepithelial lesions with superimposed chronic inflammation mimicking dVIN has recently been reported; p53 shows characteristic mid-epithelial staining (with basal sparing) in such cases. The pathology databases of 2 academic institutions were searched for vulva specimens with corresponding p53 and p16 immunohistochemical stains, yielding 38 specimens (from 27 patients). In situ hybridization and multiplex polymerase chain reaction-MassArray for high-risk HPV were performed on at least 1 block from each patient. All cases resembled dVIN or lichen sclerosus morphologically, but with a higher degree of atypia. All but 1 case demonstrated mid-epithelial p53 staining with basal sparing by immunohistochemistry. All cases showed block positivity for p16 and at least patchy positivity by HPV in situ hybridization. Of the 23 cases with valid HPV DNA polymerase chain reaction results, 15 were positive and 8 were negative. Of the positive cases, HPV16 was identified in 10 cases, with other high-risk types in the remaining 5. To our knowledge, this is the largest cohort of high-grade squamous intraepithelial lesions mimicking dVIN reported to date. Prior studies reported positivity for HPV16 in all cases tested, however, we found HPV16 in only 67% of HPV positive cases. This case series highlights the importance of immunohistochemistry, and occasionally HPV in situ hybridization, for accurate diagnosis, and expands the spectrum of associated HPV types.

Electrocautery ablation of anal high-grade squamous intraepithelial lesions: Effectiveness and key factors associated with outcomes.

BACKGROUND: Electrocautery ablation (EA) is a common treatment modality for patients with anal high-grade squamous intraepithelial lesions (HSILs), but to the authors' knowledge its effectiveness has been understudied. The objective of the current study was to determine ablation outcomes and to identify clinicopathological factors associated with postablation disease recurrence. METHODS: A total of 330 people living with HIV with de novo intra-anal HSIL who were treated with EA from 2009 to 2016 were studied retrospectively. Using long-term, surveillance high-resolution anoscopy biopsy data, treatment failures were classified as local recurrence (HSIL noted at the treated site at the time of surveillance) or overall recurrence (HSIL noted at treated or untreated sites). The associations between these outcomes and clinical factors were analyzed using Cox proportional hazards models. RESULTS: Approximately 88% of participants were men who have sex with men. The median age of study  participants was 45.5 years (range, 35-51 years) and approximately 49% had multiple index HSILs (range, 2-6 index HSILs). At a median of 12.2 months postablation (range, 6.3-20.9 months postablation), approximately 45% of participants had developed local recurrence whereas 60% had developed overall recurrence. Current cigarette smoking, HIV viremia (HIV-1 RNA ≥100 copies/mL), and multiple index HSILs were found to be predictive of local recurrence. Overall recurrence was more common in current smokers and those with multiple index lesions. In multivariable models that included human papillomavirus (HPV) genotypes, baseline and persistent infections with HPV-16 and/or HPV-18 were found to be significantly associated with both local and overall recurrence. CONCLUSIONS: EA is an effective treatment modality for anal HSIL in people living with HIV, but rates of disease recurrence are substantial. Multiple index HSILs, HIV viremia, current cigarette smoking, and both baseline and persistent infection with HPV-16 and/or HPV-18 appear to negatively impact treatment success. Ongoing surveillance is imperative to capture recurrence early and improve long-term treatment outcomes.

HIV-positive women with anal high-grade squamous intraepithelial lesions: a study of 153 cases with long-term anogenital surveillance.

Women living with HIV (WLHIV) are at increased risk for human papillomavirus (HPV)-associated anal cancer. Given the "field effect" of HPV pathogenesis, some recommend that anal cancer screening should be limited to WLHIV with prior genital disease. This study aimed to characterize the relationship between anal and genital disease in WLHIV in order to better inform anal cancer screening guidelines. We retrospectively studied 153 WLHIV with biopsy-proven anal high-grade squamous intraepithelial lesions (AHSIL) and long-term evaluable cervical/vaginal/vulvar histopathology. Based on the absence or presence of genital HSIL, subjects were categorized as having isolated AHSIL or multicentric HSIL. Demographics, HIV parameters and cervical/anal HPV status were recorded. Chi-square test was used for bivariate analyses. Of 153 WLHIV with AHSIL, 110 (72%) had isolated AHSIL, while 43 (28%) had multicentric HSIL (28 cervical, 16 vulvar, and 8 vaginal HSIL). The median genital surveillance was 8 years (range 1-27). Cervical HPV16/18 infection was associated with multicentric disease (P = 0.001). Overall, 53% of multicentric cases presented genital HSIL preceding AHSIL with median interval 13 years (range 2-23). Paired anal and cervical high-risk HPV results were available for 60 women within 12 months of AHSIL diagnosis: 30 (50%) had anal infection alone, while 30 (50%) had anal/cervical coinfection by 16/18 (15%), non-16/18 (13%), or different types (22%). In conclusion, WLHIV frequently develop AHSILs without pre-existing genital disease or after long latency following a genital HSIL diagnosis. Our findings support anal cancer screening for WLHIV irrespective of prior genital disease.

Genetic variability and functional implication of HPV16 from cervical intraepithelial neoplasia in Shanghai women.

Human papillomavirus (HPV)16 gene mutation is usually associated with persistent HPV infection and cervical intraepithelial neoplasia (CIN). However, the functional implications of HPV16 mutations remain poorly understood.145 LCR/E6/E7 of the HPV16 isolates were amplified and sequenced, and HPV16 integration status was detected. In total, 89 SNPs (68 in the LCR, 13 in E6, 8 in E7) were discovered, 11 of which were nonsynonymous mutations (8 in E6, 3 in E7). The H85Y and E120D variants in E6 were significantly reduced in the high-grade squamous intraepithelial lesion (HSIL) group compared to the T," a potential binding site for TATA-binding protein, is the most common in LCR variants. A4 (Asian) was associated with an increased risk of HSIL compared to A1-3(P = .009). The H85/E120 in E6 and N29 in HPV16 E7 might play a critical role in carcinogenesis by disrupting p53 and Rb degradation due to affecting their interaction, respectively. In a word, the findings in this study provide preventative and therapeutic interventions of HPV16 -related cervical lesions/cancer.

The prevalence of VAIN, CIN, and related HPV genotypes in Japanese women with abnormal cytology.

Vaginal intraepithelial neoplasia (VAIN) is often found by chance. We investigated the prevalence of VAIN and related human papillomavirus (HPV) types in comparison with cervical intraepithelial neoplasia (CIN). This study enrolled 648 women who were referred to the outpatient clinic of Kanazawa Medical University Hospital for abnormal cytology from January 2009 to January 2019. HPV genotypes were determined using Genosearch-31 + 4, which can detect 35 different HPV types. Colposcopy was performed at the first visit by an experienced gynecological oncologist. Among 611 subjects with squamous cell lesions, 107 (17.5%) VAIN cases were identified, and 67 (11.0%) women had both VAIN and CIN. Ultimately, 72 VAIN1, 15 VAIN2/3, 203 CIN1, 249 CIN2/3, 32 cervical squamous cell carcinomas (SCC), and one vaginal SCC (Vag-SCC) were identified. The prevalences of VAIN1, VAIN2/3, and Vag-SCC were 35.5%, 6.0%, and 3.1% of equivalent cervical lesions, respectively. The VAIN patients were older than the CIN patients (P = .002). About half of the VAIN cases were diagnosed during the follow-up. Multiple HPV infections were found in 42.9% of the VAIN and CIN patients. HPV52, 16, 51, 53, and 56 were the most common types in VAIN, whereas HPV16, 52, 58, 51, and 31 predominated in CIN. HPV18 was rare in VAIN, HPV58 was more common in CIN than in VAIN, and HPV53 and HPV73 were more common in VAIN. In conclusion, VAIN1 was identified more frequently than we expected. Various HPV types were identified in the vagina, which is likely a reservoir for HPV.

14-type HPV mRNA test in triage of HPV DNA-positive postmenopausal women with normal cytology.

BACKGROUND: During 2013 and 2016 the region of Skåne, Sweden started to analyse human papillomavirus (HPV) and cytology in postmenopausal women 60-65 years of age. Our aim was to evaluate high-risk (HR) HPV mRNA testing for the triage of HPV DNA-positive postmenopausal women with normal cytology. METHODS: A total of 271 women, 60-65 years of age, underwent liquid-based cytology (LBC) and HPV testing by using the HR-HPV DNA MGP-PCR-Luminex assay. HR-HPV DNA-positive women with normal cytology underwent complimentary HPV mRNA testing (Aptima, Hologic Inc.). Over a period of 49 months (SD 11.0) the women received regular follow-ups at intervals of 12-18 months. Women with abnormal cytology and/or a positive HR-HPV DNA and/or mRNA result at two subsequent visits were scheduled for colposcopy and clinical examination. RESULTS: Over the surveillance period, 3.6% (10/271) of the HR-HPV DNA-positive women developed histologically confirmed high-grade squamous intraepithelial lesions (HSILs) or worse. The cumulative incidence rates (CIR) were 29.7% (CI 24.8-30.1) for HSIL or worse among HPV mRNA-positive women at enrolment (39.5% 107/271) and 0% among HPV mRNA-negative women (60.5%, 164/271), (p = 0.002). CONCLUSIONS: Postmenopausal women with normal cytology testing positive for HR-HPV mRNA are at increased risk for the development of high-grade cervical intraepithelial neoplasia (CIN), in contrast to women with a negative HR-HPV mRNA outcome. The HR-HPV mRNA APTIMA assay detecting 14 HR-HPV types may be a useful triage method among HPV DNA-positive postmenopausal women with normal cytology.

Genetic variability, phylogeny and functional implication of the long control region in human papillomavirus type 16, 18 and 58 in Chengdu, China.

BACKGROUND: Long control region (LCR) of human papillomavirus (HPV) has shown multiple functions on regulating viral transcription. The variations of LCR related to different lineages/sub-lineages have been found to affect viral persistence and cervical cancer progression differently. In this study, we focused on gene polymorphism of HPV16/18/58 LCR to assess the effect variations caused on transcription factor binding sites (TFBS) and provided more data for further study of LCR in Southwest China. METHODS: LCR of HPV16/18/58 were amplified and sequenced to do polymorphic and phylogenetic anlysis. Sequences of each type were aligned with the reference sequence by MEGA 6.0 to identify SNPs. Neighbor-joining phylogenetic trees were constructed using MEGA 6.0. Transcription factor binding sites were predicted by JASPAR database. RESULTS: The prevalence of these three HPVs ranked as HPV16 (12.8%) > HPV58 (12.6%) > HPV18 (3.5%) in Chengdu, Southwest China. 59 SNPs were identified in HPV16-LCR, 18 of them were novel mutations. 30 SNP were found in HPV18-LCR, 8 of them were novel. 55 SNPs were detected in HPV58-LCR, 18 of them were novel. Also, an insertion (CTTGTCAGTTTC) was detected in HPV58-LCR between position 7279 and 7280. As shown in the neighbor-joining phylogenetic trees, most isolates of HPV16/18/58 were clustered into lineage A. In addition, one isolate of HPV16 was classified into lineage C and 3 isolates of HPV58 were classified as lineage B. JASPAR results suggested that TFBS were potentially influenced by 7/6 mutations on LCR of HPV16/18. The insertion and 5 mutations were shown effects in LCR of HPV58. CONCLUSION: This study provides more data for understanding the relation among LCR mutations, lineages and carcinogenesis. It also helps performing further study to demonstrate biological function of LCR and find potential marker for diagnosis and therapy.

Possibly carcinogenic HPV subtypes are a cause of HSIL and negative clinical HPV tests - A European prospective single center study.

OBJECTIVE: To correlate p16ink4a positive cervical precancers of 388 consecutive patients from a single European center with the preceding clinical HPV-DNA and HPV E6/E7 mRNA screening test. METHOD: 374/388 patients had a HSIL (CIN 2/3) and 14/388 AIS (6 pure and 8 combined AIS/HSIL). Lesional tissues of HSIL/AIS with negative Cobas and/or Aptima HPV tests underwent HPV genotyping with CHIPRON HPV 3.5 LCD-array. Selected cases were subjected to a cancer hot spot analysis. RESULTS: The Aptima test missed 10/388 (2.6%) and the Cobas test seven of 388 (1.8%) precancers associated HPV-HR. Both HPV tests were negative in 20/374 precancers (5.3%; 17 HSIL/CIN3, two HSIL/CIN2, one AIS). Due to insufficient DNA four of 20 double negative cases (three HSIL, one AIS) were not genotyped. In the remaining cases, two of 20 (10%) HSIL genotyping detected HR-HPV subtypes. 10/20 (50%) HSIL were associated with possibly carcinogenic and low risk HPV (four x HPV73, three x HPV 53, one x HPV 82, one x HPV 67 and one x HPV 6), all of which are not included in both HPV tests. Two of 20 (10%) HSIL were negative with all HPV tests; one of these HSIL had a somatic PIK3CA gene mutation and the other had a single nucleotide variant in the APC gene. Three of 20 HSIL (15%) were thin HSIL (≤9 cell layers thick). CONCLUSIONS: Possibly carcinogenic HPV subtypes not included in the clinical HPV tests may account for the small gap of missed HSIL in clinical HPV screening. True HPV negative HSIL are exceedingly rare. Expanding HPV testing to include more possibly carcinogenic HPV subtypes may further reduce cervical cancer.

Significance of p53-Binding Protein 1 Nuclear Foci in Cervical Squamous Intraepithelial Lesions: Association With High-Risk Human Papillomavirus Infection and P16INK4a Expression.

As p53-binding protein 1 (53BP1) localizes to the sites of DNA double-strand breaks and rapidly forms nuclear foci (NF), and its presence may be an indicator of endogenous genomic instability (GIN). We previously showed that 53BP1 NF in cervical cells increase with neoplastic progression, indicating the significance of 53BP1 expression for the estimation of malignant potential during cervical carcinogenesis. This study aimed to further elucidate the impact of 53BP1 expression as a biomarker for cervical squamous intraepithelial lesion (SIL). A total of 81 tissue samples, including 17 of normal cervical epithelium, 22 of cervical intraepithelial neoplasia (CIN) 1, 21 of CIN2, and 21 of CIN3, from patients positive for high-risk human papillomavirus (HR-HPV) were used for double-label immunofluorescence of 53BP1 and Ki-67/p16INK4a expression and HR-HPV in situ hybridization. We analyzed associations between 53BP1 expression type with parameters such as CIN grade, HR-HPV infection status, p16INK4a expression, and CIN prognosis. Expression type of 53BP1 was significantly associated with histological grade of CIN and HR-HPV in situ hybridization signal pattern (P < .0001). There was a significant correlation between 53BP1 and p16INK4a expression levels (r = .73, P < .0001). However, there was no association between 53BP1 expression type and CIN prognosis. We propose that 53BP1 expression type is a valuable biomarker for SIL, which can help estimate the grade and GIN of cervical lesions reflecting replication stress caused by the integration of HR-HPV to the host genome.

HIV-Infected Patients With Anal Cancer Precursors: Clinicopathological Characteristics and Human Papillomavirus Subtype Distribution.

BACKGROUND: People living with HIV have high rates of anal human papillomavirus infection and anal precancer/cancer. OBJECTIVE: This study aims to: 1) determine human papillomavirus subtype distribution among people living with HIV with anal high-grade squamous intraepithelial lesions; 2) compare the clinicopathological characteristics of patients with anal high-grade squamous intraepithelial lesions by human papillomavirus 16 status; and 3) investigate high-risk human papillomavirus negative anal high-grade squamous intraepithelial lesion cases. DESIGN: In this retrospective study, 700 people living with HIV who have biopsy-proven anal high-grade squamous intraepithelial lesions were reviewed for demographics, cytological diagnoses, and human papillomavirus testing results for human papillomavirus 16, 18, and 12 other high-risk types. For human papillomavirus-negative subjects, corresponding biopsies were genotyped by using real-time polymerase chain reaction. SETTINGS: This study was conducted in a large urban HIV clinic system and major referral center for anal cancer screening. PATIENTS: Median age was 46 years (range, 20-76). Ninety-one percent of the patients were men who have sex with men. MAIN OUTCOME MEASURES: The primary outcome measure was the association between demographic variables and human papillomavirus 16 status. RESULTS: Anal cytology was unsatisfactory (5%), benign (13%), atypical squamous cells of undetermined significance (35%), low-grade squamous intraepithelial lesion (36%), and high-grade squamous intraepithelial lesions (11%). Human papillomavirus cotesting results were negative (n = 38, 5%), human papillomavirus 16 (n = 303, 43%), human papillomavirus 18 (n = 78, 11%), or exclusively non-16/18 types (n = 281, 40%). Human papillomavirus 16 positivity was associated with ≥3 high-grade lesions and ≥ low-grade squamous intraepithelial lesion cytology (p < 0.001). Age, race/ethnicity, sex, smoking, CD4+ T-cell count, and HIV viral load did not differ by status of human papillomavirus 16 (p > 0.05). For human papillomavirus-negative cases, human papillomavirus genotyping of biopsies was positive for high-risk (n = 14, 36%) or possibly carcinogenic types (n = 12, 32%), or negative (n = 12, 32%). LIMITATIONS: This was a retrospective data analysis, and it pooled the results for 12 high-risk human papillomavirus types rather than individual types. CONCLUSIONS: Nearly all people living with HIV and anal high-grade squamous intraepithelial lesions test positive for high-risk human papillomavirus on anal swabs; negative results may be due to sampling error, L1-based polymerase chain reaction assay, or human papillomavirus types not captured by standard clinical assays. Patients who have human papillomavirus 16-positive anal high-grade squamous intraepithelial lesions are indistinguishable from others based on demographic and clinical characteristics, underscoring the potential role of human papillomavirus testing for anal cancer screening. See Video Abstract at http://links.lww.com/DCR/B208. PACIENTES PORTADORES DE VIH CON PRECURSORES DE CÁNCER DE ANO: CARACTERÍSTICAS CLINICOPATOLÓGICAS Y DISTRIBUCIÓN DEL SUBTIPO VPH: Los pacientes portadores de VIH tienen altas tasas de infección por VPH y alto riesgo de desarrolar lesiones precáncerosas / cáncerosas del ano.(1) Determinar la distribución del subtipo de VPH entre las personas portadoras de VIH con lesiones intraepiteliales escamosas anales de alto grado. (2) Comparar las características clinicopatológicas de pacientes con lesiones intraepiteliales escamosas anales de alto grado del subtipo VPH 16. (3) Investigar casos de lesiones intraepiteliales escamosas anales de alto grado negativas para el VPH de alto riesgo.Estudio retrospectivo sobre 700 personas portadoras de VIH con lesiones intraepiteliales escamosas anales de alto grado confirmadas por biopsia. Los datos fueron revisados para determinar información demográfica, diagnósticos citológicos y resultados de tipización en el VPH subtipos 16 y 18, y otros 12 tipos de alto riesgo. Para los individuos negativos al VPH, se analizó el genotipo en las biopsias correspondientes mediante test de PCR en tiempo real.Extenso sistema de clinicas urbanas tratando VIH y un importante centro de referencia para la detección del cáncer analla mediana de edad poblacional fue de 46 años (rango, 20-76). 91% eran hombres que tenían sexo con hombres.Asociación entre las variables demográficas y el estado del VPH subtipo16.la citología anal fue insatisfactoria (5%), benigna (13%), células escamosas atípicas de importancia indeterminada (35%), lesión intraepitelial escamosa de bajo grado (36%) y lesiones intraepiteliales escamosas de alto grado (11%). Los resultados de la prueba conjunta del VPH fueron negativos (n = 38, 5%), el virus del VPH subtipo 16 (n = 303, 43%), el VPH subtipo 18 (n = 78, 11%) o los subtipos exclusivamente no 16/18 (n = 281, 40%). La positividad del VPH subtipo 16 se encotraba asociado con ≥3 lesiones de alto grado y ≥ células escamosas atípicas en la prueba de citología de indeterminada importancia (p < 0.001). La edad, la raza / etnia, el sexo, el tabaquismo, el recuento de células T CD4 + y la carga viral del VIH no difirieron según el estado del VPH subtipo 16 (p > 0.05). Para los casos negativos al VPH, el genotipo del virus del papiloma humano de las biopsias fue positivo para los tipos de alto riesgo (n = 14, 36%) o posiblemente carcinogénicos (n = 12, 32%), o negativo (n = 12, 32%).Análisis de datos retrospectivos, con resultados agrupados para 12 tipos de VPH de alto riesgo en lugar de tipos individuales.Casi todas las personas portadoras de VIH con lesiones intraepiteliales escamosas anales de alto grado dan positivo para el VPH de alto riesgo al muestreo de hisopos anales; Los resultados negativos pueden deberse a un error en el muestreo y al análisis de PCR basado en L1 o subtipos de VPH no obtenidos en los ensayos clínicos estándar. Los pacientes con lesiones intraepiteliales escamosas anales de alto grado positivas para el VPH subtipo 16 no son identificables de los demás, en función de las características demográficas y clínicas, lo que minimiza el rol potencial de la prueba del VPH en la detección del cáncer anal. Consulte Video Resumen en http://links.lww.com/DCR/B208. (Traducción-Dr. Xavier Delgadillo).

Alternations in mitochondrial genome in carcinogenesis of HPV positive cervix.

OBJECTIVE: It is well known that mitochondrial dysfunctions are involved in tumorigenesis. A special interest of scientists is mitochondrial ND1 gene (mtND1). Recently detected mutations in the mtND1 can disrupt the normal activity of complex I and affect oxidative phosphorylation, thus leading to increase reactive oxygen species production. This study was undertaken to determine the alternations in the mtND1 and evaluate their association with development of precancerous lesions and cervical cancer. METHODS: In the study 29 cervical cancer, 28 low grade squamous intraepithelial lesion (L-SIL) and 30 high grade squamous intraepithelial lesion (H-SIL) HPV positive fragments tissue were screened for the presence of mtND1 mutations. RESULTS: Our study showed that mutations in the mtND1 gene were detected in patients with precancerous stage, as well as cervical cancer. We have identified 12 point mutations in 116 analyzed precancerous and cancer samples HPV positive. Most detected missense mutations were previously described, except one (p. M156K) with Grantham value 95. The lower expression of mRNA ND1 was detected in cervical cancer cases and in all samples in which mtND1 mutations were identified. Our analyses revealed that level of ROS production was higher in cervical cancer tissues and all cases characterized by mtND1 mutations. CONCLUSIONS: We hypothesize that mutations in MT-ND1 observed in H-SIL and cancer could activate cervical carcinogenesis by increased ROS production.

Evaluation of the intraoperative human papillomavirus test as a marker of early cure at 12 months after electrosurgical excision procedure in women with cervical high-grade squamous intraepithelial lesion: a prospective cohort study.

OBJECTIVE: To evaluate if the intraoperative human papillomavirus (IOP-HPV) test has the same prognostic value as the HPV test performed at 6 months after treatment of high-grade squamous intraepithelial lesion (HSIL) to predict treatment failure. DESIGN: Prospective cohort study. SETTING: Barcelona, Spain. POPULATION: A cohort of 216 women diagnosed with HSIL and treated with loop electrosurgical excision procedure (LEEP). METHODS: After LEEP, an HPV test was performed using the Hybrid Capture 2 system. If this was positive, genotyping was performed with the CLART HPV2 technique. The IOP-HPV test was compared with HPV test at 6 months and with surgical margins. MAIN OUTCOME MEASURE: Treatment failure. RESULTS: Recurrence rate of HSIL was 6%. There was a strong association between a positive IOP-HPV test, a positive 6-month HPV test, positive HPV 16 genotype, positive surgical margins and HSIL recurrence. Sensitivity, specificity, and positive and negative predictive values of the IOP-HPV test were 85.7, 80.8,24.0 and 98.8% and of the HPV test at 6 months were 76.9, 75.8, 17.2 and 98.0%. CONCLUSION: Intraoperative HPV test accurately predicts treatment failure in women with cervical intraepithelial neoplasia grade 2/3. This new approach may allow early identification of patients with recurrent disease, which will not delay the treatment. Genotyping could be useful in detecting high-risk patients. TWEETABLE ABSTRACT: IOP-HPV test accurately predicts treatment failure in women with CIN 2/3.

Triage of low-grade squamous intraepithelial lesions using human papillomavirus messenger ribonucleic acid tests-A prospective population-based register study.

INTRODUCTION: The optimal clinical management of women diagnosed with low-grade squamous intraepithelial lesions (LSIL) during cervical cancer screening remains unclear. In this prospective cohort study, we compared the clinical performance of two human papillomavirus (HPV) mRNA tests for triage of women with LSIL in Denmark. MATERIAL AND METHODS: In a nationwide pathology register, we identified women aged 23-65 years with LSIL during 2008-2012. We included women tested for HPV mRNA with the PreTect HPV Proofer test for five high-risk HPV types (n = 2176) or the Aptima assay for 14 high-risk HPV types (n = 426). Subsequent histological diagnoses of cervical intraepithelial neoplasia grades 2, 3 or cancer (CIN2+) were identified in the register. We calculated the sensitivity and specificity for CIN2+ at 18 and 36 months of follow up, and the cumulative incidence of CIN2+ among women testing positive and negative, overall and by age (23-29, 30-39, 40-65 years). RESULTS: The proportion of women with a positive mRNA test at baseline was higher in women tested with Aptima (66.7%) than in women tested with Proofer (42.8%). After 18 months, Aptima had higher sensitivity for CIN2+ than Proofer (98% [95% CI 94% to 100%] vs 85% [95% CI 82% to 88%]), whereas Proofer showed higher specificity than Aptima (67% [95% CI 64% to 70%]) vs (40% [95% CI 33% to 46%]). Aptima had particularly low specificity in women aged <40 years (23-29: 19% [95% CI 5% to 36%]; 30-39: 10% [95% CI 0% to 33%]). The 36-month cumulative incidence of CIN2+ was higher in Proofer positive (54.3% [95% CI 50.9% to 57.8%]) than in Aptima positive women (37.6% [95% CI 31.2% to 44.8%]). In women with a negative mRNA test, the 36-month cumulative incidences of CIN2+ were 13.1% (95% CI 10.8% to 15.8%) and 5.9% (95% CI 1.7% to 19.0%) for Proofer and Aptima, respectively. CONCLUSIONS: In women with LSIL, Aptima had high sensitivity for CIN2+, but low specificity, especially in women aged <40 years. The Proofer test may be useful to limit immediate colposcopy referrals in younger women with LSIL, but given its low sensitivity and negative predictive value, Proofer negative women must be followed with repeat cytology.

Laryngeal cytology: A cytological, histological, P16 and human papillomavirus study.

BACKGROUND: Invasive diagnostic methods, such as punch biopsies, have a potential to produce undesirable side effects in the larynx, such as scarring and vocal dysfunction. This study is an attempt to assess the diagnostic potential of cytology to efficiently diagnose premalignant and malignant laryngeal lesions, while sparing patients the risk of complications of punch biopsies. METHODS: Laryngeal smears, using endocervical-type brushes, and punch biopsies were procured from each patient. Smears were prepared and the brush was cut and put in Surepath preservative solution for cytological analysis and human papillomavirus (HPV) DNA testing. A Real-TM Quant kit that detects 14 HPV types was used for genotyping. Immunohistochemical staining for p16 was performed on cytological and histological specimens. RESULTS: Cytological diagnosis was correct in 84.6%, 100% and 100% of cases with a histological diagnosis of squamous cell carcinomas, high-grade squamous intraepithelial lesions and benign lesions, respectively. However, cytological interpretation was correct only in 25% of low-grade squamous intraepithelial lesions. HPV DNA test was positive in only one case, which was a laryngeal polyp. Testing for p16 was negative in all the cytological and histological material. CONCLUSION: Laryngeal cytology is a useful diagnostic tool in establishing the diagnosis of high-grade squamous epithelial cell abnormalities. Recognition of low-grade lesions, however, is challenging. HPV genotyping and p16 staining do not seem to be helpful ancillary techniques in cytological material procured from the larynx.

Evaluation of the accuracy of colposcopy in detecting high-grade squamous intraepithelial lesion and cervical cancer.

PURPOSE: The primary aim of this study was to evaluate the diagnostic accuracy of colposcopy in identifying high-grade squamous intraepithelial lesion or worse (HSIL+) and the characteristic performance of colposcopic images with various severity levels of cervical lesions. METHODS: The medical records from 1828 women who underwent colposcopy at Affiliated Hospital of Tongji University from February 2016 to March 2019 were reviewed. Human papilloma virus (HPV) GenoArray test kit (HybriBio Ltd) and Thinprep cytologic test (TCT, Hologic, USA) were used to perform HPV genotyping and cytology. All colposcopic images were collected from the standard-of-care colposcope (Leisegang 3ML LED) and evaluated based on the 2011 International Federation of Cervical Pathology and Colposcopy (IFCPC) Colposcopy Standards. The linear by linear association, Pearson χ2 test, χ2 test, Kappa test, McNemar test and risk test were used to perform statistical analyses. RESULTS: The consistency between colposcopy and biopsy pathology was 59.35% with the moderate strength of kappa coefficient of 0.464. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of colposcopy and cytology for HSIL+ were 56.29%, 93.82%, 77.47%, 85.04% and 37.13%, 98.49%, 90.29%, 80.58%, respectively. The colposcopic features of HSIL+ were as follows: (1) thick or bulgy acetowhite epithelium with sharp border; (2) completely nonstained of Lugol's iodine; (3) type III/IV/V of gland openings; (4) punctation or atypical vessels. CONCLUSION: The data and findings herein provide the resource for evaluating the diagnostic value of colposcopy, and suggested that the accuracy of colposcopy is required to be further improved.

Anal squamous intraepithelial lesions: an update and proposed management algorithm.

The term anal squamous intraepithelial lesion (ASIL) is used to describe premalignant change of anal squamous cells that precede the development of squamous cell carcinoma. Pathophysiology is driven by the human papilloma virus (HPV), and progression and regression of ASIL being well described, with 12% of high-grade lesions progressing to invasive cancer within 5 years. Vaccination against HPV is effective for primary prevention. Management consists of identification and treatment of high-grade lesions to prevent progression to squamous cell carcinoma. Management of established ASIL aims to avoid the progression to invasive cancer and maintain fecal continence. A combination of surveillance, excision, ablative, or topical therapies is used to achieve this. The aim of the present study was to review the contemporary evidence about ASIL and to suggest a management algorithm.