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.

Interobserver reproducibility of cervical histology interpretation with and without p16 immunohistochemistry.

OBJECTIVES: Histopathological diagnosis of colposcopically identified cervical lesions is a critical step for the recognition of cervical cancer precursors requiring treatment. Although there have been efforts to standardize the histologic diagnosis of cervical biopsy specimens, in terms of terminology and use of biomarkers, there is no uniform approach in the pathology community. Adjunctive p16 immunohistochemistry (IHC) can highlight precancer diagnoses, with use recommendations outlined by the Lower Anogenital Squamous Terminology project. METHODS: We assessed the diagnostic reproducibility of cervical histopathological biopsy specimens with and without p16 staining among 2 expert pathologists. RESULTS: Interpretation of p16 IHC as positive vs negative was highly reproducible (92.5% agreement, κ = 0.85); greater variation was seen in the choice of which biopsy specimens required adjunctive p16 staining (78.0% agreement, κ = 0.43). Adjunctive p16 IHC did not significantly increase diagnostic agreement under multitiered grading systems (benign vs cervical intraepithelial neoplasia [CIN] 1/low-grade squamous intraepithelial lesion vs atypical squamous metaplasia vs CIN2/high-grade squamous intraepithelial lesion [HSIL] vs CIN3/HSIL-CIN3 vs cancer) (65.5% agreement, κ = 0.56 without p16; 70.0% agreement, κ = 0.58 with p16). However, when dichotomizing diagnoses based on clinical management (less than HSIL vs HSIL+), diagnostic agreement increased with p16 IHC (90.5% agreement, κ = 0.79 without p16; 92.0% agreement, κ = 0.84 with p16). For biopsy specimens taken from women positive for human papillomavirus (HPV) type 16, agreement was similar with or without adjunctive p16 (κ = 0.80 without p16; κ = 0.78-0.80 with p16). In contrast, p16 IHC substantially improved diagnostic agreement for cervical biopsy specimens taken from women positive for other high-risk HPV strains, producing improvements in κ from 0.03 to 0.24. CONCLUSIONS: Adjunctive p16 immunostaining provides useful information in the evaluation of cervical biopsies for precancer. In our study, we have demonstrated that it is highly reproducible between 2 pathologists, although the decision of which biopsies warrant its use is less so. Furthermore, although p16 IHC showed a limited increase in diagnostic reproducibility for all biopsies included in our study, it did demonstrate a more sizable gain in biopsies negative for HPV 16 but positive for other high-risk genotypes. Further studies are needed to clarify the role of p16 IHC and how it can be optimized for the detection of cervical precancer, particularly in HPV-vaccinated populations where types other than HPV 16 are relatively more important.

Recommendations for Use of p16/Ki67 Dual Stain for Management of Individuals Testing Positive for Human Papillomavirus.

OBJECTIVES: The Enduring Consensus Cervical Cancer Screening and Management Guidelines Committee developed recommendations for dual stain (DS) testing with CINtec PLUS Cytology for use of DS to triage high-risk human papillomavirus (HPV)-positive results. METHODS: Risks of cervical intraepithelial neoplasia grade 3 or worse were calculated according to DS results among individuals testing HPV-positive using data from the Kaiser Permanente Northern California cohort and the STudying Risk to Improve DisparitiES study in Mississippi. Management recommendations were based on clinical action thresholds developed for the 2019 American Society for Colposcopy and Cervical Pathology Risk-Based Management Consensus Guidelines. Resource usage metrics were calculated to support decision-making. Risk estimates in relation to clinical action thresholds were reviewed and used as the basis for draft recommendations. After an open comment period, recommendations were finalized and ratified through a vote by the Consensus Stakeholder Group. RESULTS: For triage of positive HPV results from screening with primary HPV testing (with or without genotyping) or with cytology cotesting, colposcopy is recommended for individuals testing DS-positive. One-year follow-up with HPV-based testing is recommended for individuals testing DS-negative, except for HPV16- and HPV18-positive results, or high-grade cytology in cotesting, where immediate colposcopy referral is recommended. Risk estimates were similar between the Kaiser Permanente Northern California and STudying Risk to Improve DisparitiES populations. In general, resource usage metrics suggest that compared with cytology, DS requires fewer colposcopies and detects cervical intraepithelial neoplasia grade 3 or worse earlier. CONCLUSIONS: Dual stain testing with CINtec PLUS Cytology is acceptable for triage of HPV-positive test results. Risk estimates are portable across different populations.

Two-Year Incidence and Cumulative Risk and Predictors of Anal High-Grade Squamous Intraepithelial Lesions (Anal Precancer) Among Women With Human Immunodeficiency Virus.

BACKGROUND: Detection and treatment of anal histologic high-grade squamous intraepithelial lesions (hHSIL) prevents anal cancer. However, anal hHSIL incidence among women with human immunodeficiency virus (HIV, WHIV) remains unknown. Performance of anal high-risk human papillomavirus ([hr]HPV), anal cytology (anal-cyt), and both for hHSIL detection longitudinally over 2 years also remains undetermined. METHODS: We determined 2-year incidence and cumulative risk estimates (2-y-CR) of anal hHSIL among WHIV using prevalence and incidence (per 100 person-years [py]) observations stratified by baseline hrHPV and/or anal-cyt results. RESULTS: In total, 229 WHIV with complete baseline data were included in the analysis; 114 women without prevalent anal hHSIL were followed with 2 annual evaluations. Median age was 51, 63% were Black, and 23% were Hispanic. Anal hrHPV or abnormal anal-cyt was associated with an increased risk of incident anal hHSIL at 2 years (18.9/100py [95% confidence interval {CI} 11.4-31.3] and 13.4/100py [95% CI 8.0-22.7], respectively) compared with no detection of anal HPV or negative cytology (2.8/100py [95% CI 1.1-7.4] and 4.2 [95% CI, 1.8-10.2]) The presence of anal hrHPV with abnormal cytology was associated with 2-y-CR of anal hHSIL of 65.6% (95% CI 55.4%-75%); negative hrHPV with negative cytology was associated with 2-y-CR of anal hHSIL of 9.2% (95% CI 7.0-16.0). CONCLUSIONS: Detection of anal hrHPV or abnormal anal cytology are comparable predictors for 2-y-CR of anal hHSIL. The absence of anal hrHPV combined with negative cytology was predictive of a lower (but measurable) risk of developing anal hHSIL. These findings provide important data to inform anal cancer screening guidelines for WHIV.

Cumulative detection of anal high-grade squamous intraepithelial lesions over two-year follow-up in men who have sex with men living with HIV in France.

We assessed cumulative detection and determinants of anal high-grade squamous intraepithelial lesions (HSIL) in men who have sex with men living with HIV who underwent three visits over two years, with cytology and high-resolution anoscopy (HRA), within the ANRS-EP57-APACHES study. Cumulative HSIL detection was 33% (134/410), of which 48% were detected at baseline. HSIL detection varied considerably by center (13-51%). Strongest HSIL determinants were baseline HPV16 (adjusted odds ratio [aOR] 8.2; 95% confidence interval [95%CI] 3.6-18.9), and p16/Ki67 (aOR 4.6; 95%CI 2.3-9.1). Repeat annual cytology and HRA improved HSIL detection but did not fully compensate between-center heterogeneity.

Cervical Cancer Screening and Treatment algorithms using Human Papillomavirus testing - lessons learnt from a South African pilot randomized controlled trial.

BACKGROUND: To report quantitative and qualitative results on cervical cancer (CC) HPV-based screening and treatment algorithms, with/out triage with visual inspection after acetic acid (VIA), followed by ablative treatment (AT). METHODS: Women 30-54 years-old from Durban, South Africa were recruited, regardless of HIV status, randomized into one of two study arms and screened for HPV. VIA-triage arm: HPV-positive women were triaged using VIA, biopsied and received AT if VIA-positive and eligible; no-triage arm: eligible HPV-positive women received AT. Women ineligible for AT were referred to colposcopy. Women were asked about side effects immediately and one week after AT. Retention to screening and treatment algorithms was compared between arms. RESULTS: 350 women (275 HIV-uninfected and 75 women living with HIV, (WLWH)) were allocated to receive HPV testing with VIA-triage (n=175) or no-triage (n=175). HPV prevalence was 28% (95%CI=23-33); WLWH: 52% (95%CI=40-64) vs HIV-uninfected: 21% (95%CI=17-27) (p<0.05). Among women who underwent VIA triage with histological diagnosis, 3/17 were VIA negative with CIN2+; 14/18 were VIA positive with

Human papillomavirus testing on self-collected samples to detect high-grade cervical lesions in rural Bhutan: The REACH-Bhutan study.

BACKGROUND: "REACH-Bhutan" aimed to evaluate the feasibility and clinical performance of a community-based screening program for cervical cancer in rural Bhutan using self-collected samples for high-risk human papillomavirus (HR-HPV) testing. METHODS: In April/May 2016, 2590 women aged 30-60 years were screened across rural Bhutan by providing a self-collected sample for careHPV testing. All careHPV-positive women, plus a random sample of careHPV-negative women, were recalled for colposcopy and biopsy. Self-samples also underwent GP5+/6+ polymerase chain reaction (PCR)-based HR-HPV DNA detection and genotyping. Cross-sectional screening indices were estimated against histological high-grade squamous intraepithelial lesions or worse (hHSIL+), including imputation of hHSIL+ in women without colposcopy. RESULTS: HR-HPV positivity was 10.2% by careHPV and 14.8% by GP5+/6+ PCR. Twenty-two cases of hHSIL+ were histologically diagnosed, including one invasive cancer; an additional 7 hHSIL+ were imputed in women without colposcopy. HR-HPV testing by GP5+/6+ showed higher sensitivity for hHSIL+ (89.7%, 95% CI 72.6-97.8) than careHPV (75.9%, 95% CI 56.5-89.7). Negative predictive value was also slightly higher for GP5+/6+ (99.9%, 95% CI 99.6-100) than careHPV (99.7%, 95% CI 99.4-99.9). Specificity, however, was lower for GP5+/6+ (86.1%, 95% CI 84.6-87.4) than careHPV (90.6%, 95% CI 89.4-91.7), as was positive predictive value (6.9%, 95% CI 4.5-9.9 vs. 8.5%, 95% CI 5.4-12.6). Of 377 HR-HPV-positive women by GP5+/6+, 173 (45.9%) were careHPV-positive, including 54.7% HPV16-positive and 30.2% HPV18-positive women. CONCLUSIONS: The final REACH-Bhutan results show that screening for cervical cancer with self-collection of samples and HR-HPV testing, in addition to our previous report of achieving high participation, can also perform well to detect women with hHSIL+.

Performance of standardised colposcopy to detect cervical precancer and cancer for triage of women testing positive for human papillomavirus: results from the ESTAMPA multicentric screening study.

BACKGROUND: Colposcopy, currently included in WHO recommendations as an option to triage human papillomavirus (HPV)-positive women, remains as the reference standard to guide both biopsy for confirmation of cervical precancer and cancer and treatment approaches. We aim to evaluate the performance of colposcopy to detect cervical precancer and cancer for triage in HPV-positive women. METHODS: This cross-sectional, multicentric screening study was conducted at 12 centres (including primary and secondary care centres, hospitals, laboratories, and universities) in Latin America (Argentina, Bolivia, Colombia, Costa Rica, Honduras, Mexico, Paraguay, Peru, and Uruguay). Eligible women were aged 30-64 years, sexually active, did not have a history of cervical cancer or treatment for cervical precancer or a hysterectomy, and were not planning to move outside of the study area. Women were screened with HPV DNA testing and cytology. HPV-positive women were referred to colposcopy using a standardised protocol, including biopsy collection of observed lesions, endocervical sampling for transformation zone (TZ) type 3, and treatment as needed. Women with initial normal colposcopy or no high-grade cervical lesions on histology (less than cervical intraepithelial neoplasia [CIN] grade 2) were recalled after 18 months for another HPV test to complete disease ascertainment; HPV-positive women were referred for a second colposcopy with biopsy and treatment as needed. Diagnostic accuracy of colposcopy was assessed by considering a positive test result when the colposcopic impression at the initial colposcopy was positive minor, positive major, or suspected cancer, and was considered negative otherwise. The main study outcome was histologically confirmed CIN3+ (defined as grade 3 or worse) detected at the initial visit or 18-month visit. FINDINGS: Between Dec 12, 2012, and Dec 3, 2021, 42 502 women were recruited, and 5985 (14·1%) tested positive for HPV. 4499 participants with complete disease ascertainment and follow-up were included in the analysis, with a median age of 40·6 years (IQR 34·7-49·9). CIN3+ was detected in 669 (14·9%) of 4499 women at the initial visit or 18-month visit (3530 [78·5%] negative or CIN1, 300 [6·7%] CIN2, 616 [13·7%] CIN3, and 53 [1·2%] cancers). Sensitivity was 91·2% (95% CI 88·9-93·2) for CIN3+, whereas specificity was 50·1% (48·5-51·8) for less than CIN2 and 47·1% (45·5-48·7) for less than CIN3. Sensitivity for CIN3+ significantly decreased in older women (93·5% [95% CI 91·3-95·3] in those aged 30-49 years vs 77·6% [68·6-85·0] in those aged 50-65 years; p<0·0001), whereas specificity for less than CIN2 significantly increased (45·7% [43·8-47·6] vs 61·8% [58·7-64·8]; p<0·0001). Sensitivity for CIN3+ was also significantly lower in women with negative cytology than in those with abnormal cytology (p<0·0001). INTERPRETATION: Colposcopy is accurate for CIN3+ detection in HPV-positive women. These results reflect ESTAMPA efforts in an 18-month follow-up strategy to maximise disease detection with an internationally validated clinical management protocol and regular training, including quality improvement practices. We showed that colposcopy can be optimised with proper standardisation to be used as triage in HPV-positive women. FUNDING: WHO; Pan American Health Organization; Union for International Cancer Control; National Cancer Institute (NCI); NCI Center for Global Health; National Agency for the Promotion of Research, Technological Development, and Innovation; NCI of Argentina and Colombia; Caja Costarricense de Seguro Social; National Council for Science and Technology of Paraguay; International Agency for Research on Cancer; and all local collaborative institutions.

HPV Type Distribution in Benign, High-Grade Squamous Intraepithelial Lesions and Squamous Cell Cancers of the Anus by HIV Status.

The incidence of anal cancer is increasing, especially in high-risk groups, such as PLWH. HPV 16, a high-risk (HR) HPV genotype, is the most common genotype in anal high-grade squamous intraepithelial lesions (HSIL) and squamous cell carcinoma (SCC) in the general population. However, few studies have described the distribution of HR HPV genotypes other than HPV 16 in the anus of PLWH. HPV genotyping was performed by DNA amplification followed by dot-blot hybridization to identify the HR and low-risk (LR) genotypes in benign anal lesions (n = 34), HSIL (n = 30), and SCC (n = 51) of PLWH and HIV-negative individuals. HPV 16 was the most prominent HR HPV identified, but it was less common in HSIL and SCC from PLWH compared with HIV-negative individuals, and other non-HPV 16 HR HPV (non-16 HR HPV) types were more prevalent in samples from PLWH. A higher proportion of clinically normal tissues from PLWH were positive for one or more HPV genotypes. Multiple HPV infection was a hallmark feature for all tissues (benign, HSIL, SCC) of PLWH. These results indicate that the development of anal screening approaches based on HPV DNA testing need to include non-16 HR HPVs along with HPV 16, especially for PLWH. Along with anal cytology, these updated screening approaches may help to identify and prevent anal disease progression in PLWH.

Human Papillomavirus Detection in Scrotal Squamous Cell Carcinoma: Case Series from a Population-Based Cancer Registry.

Introduction: Scrotal squamous cell carcinomas (SCCs) are rare malignancies that are not considered to be associated with the human papillomavirus (HPV) by the International Agency for Research on Cancer. However, recent studies have detected HPV in these cancers. We sought to determine the presence of HPV types among scrotal cancer cases identified through population-based cancer registries. Methods: Primary scrotal SCCs diagnosed from 2014 to 2015 were identified, and tissue sections from formalin-fixed, paraffin-embedded tissue blocks were obtained for laboratory testing. A pathology review was performed to confirm morphology. HPV testing was performed using L1 consensus polymerase chain reaction analysis. Immunohistochemistry was used to evaluate p16INK4a (p16) expression. Results: Five cases of scrotal SCC were identified from 1 cancer registry. Age at diagnosis ranged from 34 to 75 years (median, 56 years). Four cases were non-Hispanic White, and 1 was non-Hispanic Black. The morphologic subtype of 4 cases was keratinizing (usual), and 1 case was verrucous (warty) histologic subtype. Two of the usual cases of SCC were HPV-negative and p16-negative, and 2 were positive for HPV16 and p16. The verrucous (warty) SCC subtype case was HPV6-positive and p16-negative. Conclusions: The presence of HPV16 and p16 overexpression in the examined tissue specimens lends additional support for the role of HPV in the etiology of scrotal SCC.

Treatment of Anal High-Grade Squamous Intraepithelial Lesions to Prevent Anal Cancer.

BACKGROUND: The incidence of anal cancer is substantially higher among persons living with the human immunodeficiency virus (HIV) than in the general population. Similar to cervical cancer, anal cancer is preceded by high-grade squamous intraepithelial lesions (HSILs). Treatment for cervical HSIL reduces progression to cervical cancer; however, data from prospective studies of treatment for anal HSIL to prevent anal cancer are lacking. METHODS: We conducted a phase 3 trial at 25 U.S. sites. Persons living with HIV who were 35 years of age or older and who had biopsy-proven anal HSIL were randomly assigned, in a 1:1 ratio, to receive either HSIL treatment or active monitoring without treatment. Treatment included office-based ablative procedures, ablation or excision under anesthesia, or the administration of topical fluorouracil or imiquimod. The primary outcome was progression to anal cancer in a time-to-event analysis. Participants in the treatment group were treated until HSIL was completely resolved. All the participants underwent high-resolution anoscopy at least every 6 months; biopsy was also performed for suspected ongoing HSIL in the treatment group, annually in the active-monitoring group, or any time there was concern for cancer. RESULTS: Of 4459 participants who underwent randomization, 4446 (99.7%) were included in the analysis of the time to progression to cancer. With a median follow-up of 25.8 months, 9 cases were diagnosed in the treatment group (173 per 100,000 person-years; 95% confidence interval [CI], 90 to 332) and 21 cases in the active-monitoring group (402 per 100,000 person-years; 95% CI, 262 to 616). The rate of progression to anal cancer was lower in the treatment group than in the active-monitoring group by 57% (95% CI, 6 to 80; P = 0.03 by log-rank test). CONCLUSIONS: Among participants with biopsy-proven anal HSIL, the risk of anal cancer was significantly lower with treatment for anal HSIL than with active monitoring. (Funded by the National Cancer Institute; ClinicalTrials.gov number, NCT02135419.).

Performance of Cervical Screening a Decade Following HPV Vaccination: The Costa Rica Vaccine Trial.

BACKGROUND: We investigated the impact of human papillomavirus (HPV) vaccination on the performance of cytology-based and HPV-based screening for detection of cervical precancer among women vaccinated as young adults and reaching screening age. METHODS: A total of 4632 women aged 25-36 years from the Costa Rica HPV Vaccine Trial were included (2418 HPV-vaccinated as young adults and 2214 unvaccinated). We assessed the performance of cytology- and HPV-based cervical screening modalities in vaccinated and unvaccinated women to detect high-grade cervical precancers diagnosed over 4 years and the absolute risk of cumulative cervical precancers by screening results at entry. RESULTS: We detected 95 cervical intraepithelial neoplasia grade 3 or worse (52 in unvaccinated and 43 in vaccinated women). HPV16/18/31/33/45 was predominant (69%) among unvaccinated participants, and HPV35/52/58/39/51/56/59/66/68 predominated (65%) among vaccinated participants. Sensitivity and specificity of cervical screening approaches were comparable between women vaccinated as young adults and unvaccinated women. Colposcopy referral rates were lower in the vaccinated group for HPV-based screening modalities, but the positive predictive value was comparable between the 2 groups. CONCLUSIONS: Among women approaching screening ages, vaccinated as young adults, and with a history of intensive screening, the expected reduction in the positive predictive value of HPV testing, associated with dropping prevalence of HPV-associated lesions, was not observed. This is likely due to the presence of high-grade lesions associated with nonvaccine HPV types, which may be less likely to progress to cancer.

Automated Evaluation of p16/Ki-67 Dual-Stain Cytology as a Biomarker for Detection of Anal Precancer in Men Who Have Sex With Men and Are Living With Human Immunodeficiency Virus.

BACKGROUND: Human papillomavirus-related biomarkers such as p16/Ki-67 "dual-stain" (DS) cytology have shown promising clinical performance for anal cancer screening. Here, we assessed the performance of automated evaluation of DS cytology (automated DS) to detect anal precancer in men who have sex with men (MSM) and are living with human immunodeficiency virus (HIV). METHODS: We conducted a cross-sectional analysis of 320 MSM with HIV undergoing anal cancer screening and high-resolution anoscopy (HRA) in 2009-2010. We evaluated the performance of automated DS based on a deep-learning classifier compared to manual evaluation of DS cytology (manual DS) to detect anal intraepithelial neoplasia grade 2 or 3 (AIN2+) and grade 3 (AIN3). We evaluated different DS-positive cell thresholds quantified by the automated approach and modeled performance compared with other screening strategies in a hypothetical population of MSM with HIV. RESULTS: Compared with manual DS, automated DS had significantly higher specificity (50.9% vs 42.2%; P < .001) and similar sensitivity (93.2% vs 92.1%) for detection of AIN2+. Human papillomavirus testing with automated DS triage was significantly more specific than automated DS alone (56.5% vs 50.9%; P < .001), with the same sensitivity (93.2%). In a modeled analysis assuming a 20% AIN2+ prevalence, automated DS detected more precancers than manual DS and anal cytology (186, 184, and 162, respectively) and had the lowest HRA referral rate per AIN2+ case detected (3.1, 3.5, and 3.3, respectively). CONCLUSIONS: Compared with manual DS, automated DS detects the same number of precancers, with a lower HRA referral rate.

Design of the ANal Cancer/HSIL Outcomes Research study (ANCHOR study): A randomized study to prevent anal cancer among persons living with HIV.

It is well established that persons living with HIV (PLWH) have highly elevated rates of anal HSIL and anal cancer compared with those who are not living with HIV. The 5-year risk of anal cancer following anal HSIL has been reported to be as high as 14.1% among PLWH compared with 3.2% among those who are not living with HIV. To address these concerns, the AIDS Malignancy Consortium completed a large-scale, randomized trial to compare strategies for the prevention of anal cancer among PLWH with anal HSIL. The objective of the study was to determine whether treating anal HSIL was effective in reducing the incidence of anal cancer in PLWH compared with active monitoring. This paper describes the design of the ANal Cancer/HSIL Outcomes Research Study (ANCHOR) with respect to estimating the anal cancer event rate in this high risk population.

p16 Immunoreactivity Correlates With Morphologic Diagnosis of HPV-associated Anal Intraepithelial Neoplasia: A Study of 1000 Biopsies.

p16 is the most useful diagnostic marker for human papillomavirus (HPV)-associated anogenital lesions. In the cervix, the pattern of p16 immunoreactivity generally correlates with lesion severity. p16 expression in anal intraepithelial neoplasia (AIN) is far less studied. Whether such correlation holds true has to be determined. We correlated the degree and pattern of p16 immunohistochemistry (IHC) results with morphologic diagnoses of 1000 anal squamous and transitional zone biopsy specimens. Using the Lower Anogenital Squamous Terminology criteria, p16 IHC results were classified as block staining, partial staining, or negative. Among 150 samples without morphologic evidence of AIN, p16 was negative in 85% and partial staining in 15%. AIN 1 (n=400) revealed diverse results: 28% negative, 35% partial, and 37% block staining. Among AIN 2 (n=298), 89% were block, 9% partial staining, and 2% negative. AIN 3 (n=152) revealed block (95%) or partial staining (5%). For the detection of AIN 2/3, p16 block staining yielded 91% sensitivity, 73% specificity, 80% positive predictive value, 91% negative predictive value, and a Youden Index of 0.64. Combining block staining and partial staining slightly increased sensitivity (99%) and negative predictive value (98%), but significantly decreased specificity (43%), positive predictive value (59%) and Youden Index (0.42, P<0.001). As with the cervix, p16 immunoreactivity correlates with morphologic diagnoses of AIN. Block staining offers the optimal diagnostic value for AIN 2/3. Caution is required since AIN 1 frequently exhibits block staining; the prognostic value of p16 warrants further investigation.

High Prevalence of Anal High-Grade Squamous Intraepithelial Lesions, and Prevention Through Human Papillomavirus Vaccination, in Young Men Who Have Sex With Men Living With Human Immunodeficiency Virus.

BACKGROUND: Men who have sex with men (MSM) are at high risk for human papillomavirus (HPV)-related anal cancer. Little is known about the prevalence of low-grade squamous intraepithelial lesions (LSILs) and the anal cancer precursor, high-grade squamous intraepithelial lesions (HSILs), among young MSM with HIV (MSMLWH). HPV vaccination is recommended in this group, but its safety, immunogenicity, and protection against vaccine-type HPV infection and associated LSILs/HSILs have not been studied. METHODS: Two hundred and sixty MSMLWH aged 18-26 years were screened at 17 US sites for a clinical trial of the quadrivalent (HPV6,11,16,18) HPV (qHPV) vaccine. Those without HSILs were vaccinated at 0, 2, and 6 months. Cytology, high-resolution anoscopy with biopsies of lesions, serology, and HPV testing of the mouth/penis/scrotum/anus/perianus were performed at screening/month 0 and months 7, 12, and 24. RESULTS: Among 260 MSMLWH screened, the most common reason for exclusion was detection of HSILs in 88/260 (34%). 144 MSMLWH were enrolled. 47% of enrollees were previously exposed to HPV16. No incident qHPV type-associated anal LSILs/HSILs were detected among men naive to that type, compared with 11.1, 2.2, 4.5, and 2.8 cases/100 person-years for HPV6,11,16,18-associated LSILs/HSILs, respectively, among those previously exposed to that type. qHPV was immunogenic and safe with no vaccine-associated serious adverse events. CONCLUSIONS: 18-26-year-old MSMLWH naive to qHPV vaccine types were protected against incident qHPV type-associated LSILs/HSILs. Given their high prevalence of HSILs, there is an urgent need to vaccinate young MSMLWH before exposure to vaccine HPV types, before initiating sexual activity, and to perform catch-up vaccination.

Gastric-type endocervical adenocarcinoma and cervical cytology: Experience at a general hospital and review of the literature.

INTRODUCTION: Gastric-type endocervical adenocarcinoma (GAS) is an uncommon type of endocervical adenocarcinoma that is not associated with human papillomavirus infection. This diagnosis is relatively rare and may portend a worse prognosis than usual-type endocervical adenocarcinoma. Subtle morphological features make it an under-recognised diagnostic challenge. Study of the cytological features of individual cases is valuable in order to increase awareness of this entity. METHODS: The pathology database of our institution was searched for the diagnosis of GAS and all cytological and surgical specimens for each patient were reviewed. The original cytological interpretation was compared to a retrospective central review interpretation. Clinical history and follow-up results were obtained from the electronic medical record. RESULTS: Four cases of GAS were identified. The findings on initial cervical cytology varied, with GAS found in both patients with negative cervical cytology and those with atypical glandular cells. Cytological findings included endocervical cells arranged in three-dimensional clusters and honeycomb sheets with abundant vacuolar cytoplasm, and in two patients, moderate nuclear atypia with irregular nuclear membranes, coarse chromatin, hyperchromatic nuclei, and prominent nucleoli. In one patient, GAS was incidentally discovered via thorough sampling of a cystic lesion in the superior portion of the endocervical canal. CONCLUSIONS: GAS is an aggressive human papillomavirus-independent type of endocervical adenocarcinoma with subtle morphological features and, as our study shows, varying clinical presentation. Given the aggressive nature of GAS and the difficulties in initial diagnosis, increased awareness of this entity among pathologists is crucial.

Classifying Anal Intraepithelial Neoplasia 2 Based on LAST Recommendations.

OBJECTIVES: The Lower Anogenital Squamous Terminology (LAST) recommendations classify human papillomavirus-associated squamous lesions into low- and high-grade squamous intraepithelial lesions (LSILs/HSILs). Our study aimed to assess interobserver agreement among 6 experienced pathologists in assigning 40 anal lesions previously diagnosed as anal intraepithelial neoplasia 2 (AIN 2) to either HSIL or non-HSIL categories. METHODS: Agreement based on photomicrographs of H&E alone or H&E plus p16 immunohistochemistry was calculated using κ coefficients. RESULTS: Agreement was fair based on H&E alone (κ = 0.42; 95% confidence interval [CI], 0.34-0.52). Adding p16 improved agreement to moderate (κ = 0.55; 95% CI, 0.54-0.62). On final diagnosis, 21 cases (53%) had unanimous diagnoses, and 19 (47%) were divided. When designating p16 results as positive or negative, agreement was excellent (κ = 0.92; 95% CI, 0.83-0.95). Among variables (staining location, extent, and intensity), staining of the basal/parabasal layers was a consistent feature in cases with consensus for positive results (20/20). Of the 67 H&E diagnoses with conflicting p16 results, participants modified 32 (48%), downgrading 23 HSILs and upgrading 9 non-HSILs. CONCLUSIONS: Although p16 increased interobserver agreement, disagreement remained considerable regarding intermediate lesions. p16 expression, particularly if negative, can reduce unwarranted HSIL diagnoses and unnecessary treatment.

Efficacy of the bivalent HPV vaccine against HPV 16/18-associated precancer: long-term follow-up results from the Costa Rica Vaccine Trial.

BACKGROUND: Oncogenic human papillomavirus (HPV) infections cause most cases of cervical cancer. Here, we report long-term follow-up results for the Costa Rica Vaccine Trial (publicly funded and initiated before licensure of the HPV vaccines), with the aim of assessing the efficacy of the bivalent HPV vaccine for preventing HPV 16/18-associated cervical intraepithelial neoplasia grade 2 or worse (CIN2+). METHODS: Women aged 18-25 years were enrolled in a randomised, double-blind, controlled trial in Costa Rica, between June 28, 2004, and Dec 21, 2005, designed to assess the efficacy of a bivalent vaccine for the prevention of infection with HPV 16/18 and associated precancerous lesions at the cervix. Participants were randomly assigned (1:1) to receive an HPV 16/18 AS04-adjuvanted vaccine or control hepatitis A vaccine. Vaccines were administered intramuscularly in three 0·5 mL doses at 0, 1, and 6 months and participants were followed up annually for 4 years. After the blinded phase, women in the HPV vaccine group were invited to enrol in the long-term follow-up study, which extended follow-up for 7 additional years. The control group received HPV vaccine and was replaced with a new unvaccinated control group. Women were followed up every 2 years until year 11. Investigators and patients were aware of treatment allocation for the follow-up phase. At each visit, clinicians collected cervical cells from sexually active women for cytology and HPV testing. Women with abnormal cytology were referred to colposcopy, biopsy, and treatment as needed. Women with negative results at the last screening visit (year 11) exited the long-term follow-up study. The analytical cohort for vaccine efficacy included women who were HPV 16/18 DNA-negative at vaccination. The primary outcome of this analysis was defined as histopathologically confirmed CIN2+ or cervical intraepithelial neoplasia grade 3 or worse associated with HPV 16/18 cervical infection detected at colposcopy referral. We calculated vaccine efficacy by year and cumulatively. This long-term follow-up study is registered with ClinicalTrials.gov, NCT00867464. FINDINGS: 7466 women were enrolled in the Costa Rica Vaccine Trial; 3727 received the HPV vaccine and 3739 received the control vaccine. Between March 30, 2009, and July 5, 2012, 2635 women in the HPV vaccine group and 2836 women in the new unvaccinated control group were enrolled in the long-term follow-up study. 2635 women in the HPV vaccine group and 2677 women in the control group were included in the analysis cohort for years 0-4, and 2073 women from the HPV vaccine group and 2530 women from the new unvaccinated control group were included in the analysis cohort for years 7-11. Median follow-up time for the HPV group was 11·1 years (IQR 9·1-11·7), 4·6 years (4·3-5·3) for the original control group, and 6·2 years (5·5-6·9) for the new unvaccinated control group. At year 11, vaccine efficacy against incident HPV 16/18-associated CIN2+ was 100% (95% CI 89·2-100·0); 34 (1·5%) of 2233 unvaccinated women had a CIN2+ outcome compared with none of 1913 women in the HPV group. Cumulative vaccine efficacy against HPV 16/18-associated CIN2+ over the 11-year period was 97·4% (95% CI 88·0-99·6). Similar protection was observed against HPV 16/18-associated CIN3-specifically at year 11, vaccine efficacy was 100% (95% CI 78·8-100·0) and cumulative vaccine efficacy was 94·9% (73·7-99·4). During the long-term follow-up, no serious adverse events occurred that were deemed related to the HPV vaccine. The most common grade 3 or worse serious adverse events were pregnancy, puerperium, and perinatal conditions (in 255 [10%] of 2530 women in the unvaccinated control group and 201 [10%] of 2073 women in the HPV vaccine group). Four women in the unvaccinated control group and three in the HPV vaccine group died; no deaths were deemed to be related to the HPV vaccine. INTERPRETATION: The bivalent HPV vaccine has high efficacy against HPV 16/18-associated precancer for more than a decade after initial vaccination, supporting the notion that invasive cervical cancer is preventable. FUNDING: US National Cancer Institute.

Screening strategies for the detection of anal high-grade squamous intraepithelial lesions in women living with HIV.

OBJECTIVE: HIV-infected women (WLHIV) have more than 10-fold higher risk for squamous cell cancer of the anus. Experts suggest cytology-based strategies developed for cervical cancer screening may prevent anal cancer by detecting anal cytologic or histological high-grade squamous intraepithelial lesion (hHSIL) for treatment. Currently, there is no consensus on anal-hHSIL screening strategies for WLHIV. DESIGN: Between 2014 and 2016, 276 WLHIV were recruited at 12 US AIDS Malignancy Consortium clinical trials sites to evaluate hHSIL prevalence and (test) screening strategies. METHODS: Participants completed detailed questionnaire, underwent anal assessments including high-risk human papillomavirus (hrHPV) testing using hrHPV-Hybrid Capture 2 (HC2) and hrHPV-APTIMA, anal cytology, and concurrent high-resolution anoscopy. Screening test characteristics for predicting hHSIL validated by central review of histologic diagnosis were estimated sensitivity, specificity, positive predictive value, and false-omission rate. Paired analyses compared sensitivity and specificity for hrHPV single tests to anal cytology alone. RESULTS: 83% (229/276) of enrolled WLHIV had complete anal assessment data and were included in this analysis. Mean age was 50, 62% black and 60 (26%) had hHSIL. Anal cyotology (>atypical squamous cells of undetermined significance), hrHPV-HC2, and hrHPV-APTIMA sensitivity estimates were similarly high (83, 77, and 75%, respectively, P values > 0.2). Specificity was higher for both hrHPV-APTIMA and hrHPV-HC2 compared with anal cytology (67 vs. 50%, P < 0.001) and (61 vs. 50%, P = 0.020), respectively. CONCLUSION: Anal hrHPV testing demonstrated similar sensitivity for anal cytology (>atypical squamous cells of undetermined significance) to predict anal hHSIL. Among tests with similar sensitivity, the specificity was significantly higher for hrHPV-APTIMA and hrHPV-HC2. Thus, anal hrHPV testing may be an important alternative strategy to anal cytology for anal hHSIL screening among WLHIV.