Mod Pathol 2011;24:12951305. Am J Surg Pathol. Cost is important, but it has to be carefully considered as the impact of equivocal or incorrect diagnoses is so significant. However, it does not substitutes a physician, hospital or medical care facility. When p16 immunohistochemistry was correlated with HPV RNA in situ hybridization results (Table 2), sensitivity, specificity and positive predictive values were similar amongst all clones at all three cutoffs, although specificity was somewhat lower for the E6H4 clone at the 50 and 75% cutoffs. Studies that considered a cytoplasmic only staining as a positive result (or exclusion was not clear), immunostaining in anal cytological samples or describe simultaneously p16 and Ki-67 results were excluded. Patients were considered to have died of their disease if their cancer had recurred and they died with known disease in their bodies. Am J Obstet Gynecol. Martins and colleagues showed that up to 50 % of PC cases positive to HPV molecular assay were negative for p16 IHC expression; in addition, 21 out of 22 cases positive for p16 IHC were confirmed for the presence of HPV . For high-grade lesions, four categories were described and analyzed according to the studies description: AIN2, AIN3, HSIL (as defined by LAST [3] in a two-tiered nomenclature), and all the combined HSIL (AIN2, AIN3 and HSIL). Am J Surg Pathol. Although there may be concern for lack of representativeness of the overall tumor by these small samples, they were taken at random from each donor tumor block, the results on this microarray have shown very high prognostic value for p16 and HPV mRNA status in this and other studies,15 and, most compelling of all, is that small biopsies have been clearly proven as totally functional for p16 testing via the large number of literature studies on patients treated with primary chemoradiation after initial diagnosis on such specimens.1, 6, 20, 23, 24 In addition, Ma et al.25 specifically showed that small biopsy specimens reliably indicate p16 status in comparison with surgical resection specimens. However, each antibody clone was individually optimized to provide the best staining before use in this study. 2021 Jun 29;13(13):3260. doi: 10.3390/cancers13133260. HPV Genotyping by Molecular Mapping of Tissue Samples in Vaginal Squamous Intraepithelial Neoplasia (VaIN) and Vaginal Squamous Cell Carcinoma (VaSCC). Castle PE, Adcock R, Cuzick J, Wentzensen N, Torrez-Martinez NE, Torres SM, Stoler MH, Ronnett BM, Joste NE, Darragh TM, Gravitt PE, Schiffman M, Hunt WC, Kinney WK, Wheeler CM; New Mexico HPV Pap Registry Steering Committee; p16 IHC Study Panel. There is no recommendation for p16 immunostaining use in IN3 [3] and, such use may potentially lead to the exclusion of p16-negative samples from treatment and, consequently, leading to under-treatment. All retrospective or prospective studies that evaluated p16 immunostaining in anal and/or perianal squamous intraepithelial lesions/anal intraepithelial neoplasia (AIN) histological samples and clearly described a positive result when there was a diffuse block staining with nuclear or nuclear plus cytoplasmic staining were included. Immunohistochemical staining of p16 was performed on recuts of all original biopsies. You are using a browser version with limited support for CSS. The accuracy of ambiguous p16 immunoreactivity in predicting oncogenic HPV and HSIL outcome is significantly lower than that of the block-positive pattern but greater than negative staining. 21,38 Based on HPV risk group distribution, these cases were indeed lower risk and are less likely to progress to cancer. The results show that E6H4 provided equivalent or better performance than the other two antibodies, with equivalent or better hazard ratios for overall and disease-specific survival (Table 5 and Figures 2 and 3). p16INK4a immunohistochemistry (IHC) is widely used to facilitate the diagnosis of human papillomavirus (HPV)-associated cervical precancerous lesions. immature squamous metaplasia, atrophy), When there is professional disagreement in histologic interpretation (when the differential diagnosis includes CIN I or III). KaplanMeier survival plots were used to estimate differences in survival between these groups. Huang SH, Patel S, O'Sullivan B et al, Longer survival in patients with human papillomavirus-related head and neck cancer after positive postradiation planned neck dissection. In the new two-tiered nomenclature proposed by LAST, the LSIL category merged LSIL/IN1 and condyloma into a single low-grade category. Approximately 28% of the CP-diagnosed CIN2 tested p16 IHC negative in this study; other studies have reported the percentage of p16 IHC-negative CIN2 ranging from approximately 20% 16 to less than 10%. Morphologic CIN 2 lesions with ambiguous p16 immunoreactivity. Role of p16 testing in cervical cancer screening among HIV-infected women. This means that there is probably the equivalent amount of tumor present to many routine clinical practice oropharyngeal biopsies and to neck needle core biopsy specimens. PMC In total, 36 were potentially eligible and subsequently scrutinized in full text. Their use or mentioning on this website is only for informational purposes. Clipboard, Search History, and several other advanced features are temporarily unavailable. Background: Human papillomavirus (HPV), p16 expression, and TP53 mutations are known prognostic factors in head and neck squamous cell carcinoma, but their role in squamous cell carcinoma of the. Overdiagnosis of HSIL on cervical biopsy: errors in p16 immunohistochemistry implementation. Bhatia A, Burtness B . Fifteen studies consisting of 790 samples were included. PubMed Both nuclear and cytoplasmic staining was required for a cell to be considered 'positive' and staining distribution was read in quartiles as 124% (1), 2549% (2), 5175% (3) and 76100% (4). The current information and recommendations for HPV-associated squamous intraepithelial lesions of the lower anogenital tract come almost exclusively from cervical studies and this stands true also for p16 immunostaining. Careers. (df) Discordant p16 immunohistochemical results (d=E6H4, e=JC8, f=G175-405). Gustinucci D, Passamonti B, Cesarini E, Butera D, Palmieri EA, Bulletti S, Carlani A, Staiano M, D'Amico MR, D'Angelo V, Di Dato E, Martinelli N, Malaspina M, Spita N, Tintori B, Fulciniti F. Acta Cytol. https://doi.org/10.1038/s41379-018-0026-6, DOI: https://doi.org/10.1038/s41379-018-0026-6. Springer Nature: Switzerland, 2016, p 113121. PubMedGoogle Scholar. 2022 Apr 27;6(2):92-99. doi: 10.23922/jarc.2021-077. The aim of this systematic review and meta-analysis was to evaluate p16-positive rates according to anal squamous intraepithelial lesions/anal intraepithelial neoplasia (AIN) grade. 2015;144:11321. Would you like email updates of new search results? Although most p16 results are distinctly positive or negative, certain ones are ambiguous: they meet some but not all requirements for the "block-positive" pattern. Schache AG, Liloglou T, Risk JM et al, Validation of a novel diagnostic standard in HPV-positive oropharyngeal squamous cell carcinoma. The merit of using the immunostaining technique in IHC is that it observes the desired target in a cell or tissue sample while balancing the tissue architecture and spatial context. Positive predictive values for high-risk HPV status by RNA in situ hybridization for the E6H4, JC8 and G175-405 clones were 98%, 100% and 99% at the 75% cutoff, but negative predictive values were much more variable at 86%, 69% and 56%, respectively. Comparison of human papillomavirus in situ hybridization and p16 immunohistochemistry in the detection of human papillomavirus-associated head and neck cancer based on a prospective clinical . 2016;51:6474. Slider with three articles shown per slide. Although the numbers of positive results and negative results increased and decreased with lesser percentages of cells required for positivity for each antibody, the differences were least for the E6H4 antibody, slightly higher for the JC8 antibody, and highest for G175-405, demonstrating that the E6H4 clone has the least amount of partial reactivity. Clipboard, Search History, and several other advanced features are temporarily unavailable. PubMed K07 CA180782/CA/NCI NIH HHS/United States, International Agency for Research on Cancer - Screening Group. 2015;3:6117. This term earlier described immunohistochemical staining of tissue sections by Albert Coons in 1941. To ensure that the more accurate results were obtained, only studies that considered positivity according to the LAST definition [3] were included in our meta-analysis. The lack of improvement of results using the H-score is also not surprising as it is functionally like a low percentage cutoff. Disclaimer. Am J Surg Pathol. Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. KaplanMeier curves for overall survival for the three antibody clones and at the three different cutoffs. Cases in which there was <10% surface area consisting of tumor were excluded. Maniar KP, Nayar R. HPV-related squamous neoplasia of the lower anogenital tract: an update and review of recent guidelines. Zhang S, Saito M, Okayama K, Okodo M, Kurose N, Sakamoto J, Sasagawa T. Cancers (Basel). When compared with our meta-analysis, results in normal samples (2% vs. 2%), AIN2/CIN2 (76% vs. 68%), and AIN3/CIN3 (90% vs. 82%) were in the similar range. However, for AIN1/LSIL (excluding condylomas) and CIN1, the results were very different with positive rates of 12% in the anus and 38% in the cervix. There are sometimes pre-cancerous diseases that prepare an extra p16 as well. Approximately 75% of cases on the array had the larger (2mm) punches. f Meta-analysis of the HSIL samples (two-tiered nomenclature) stained positive for p16. Follow-up for study subjects varied from 0.06 to 12.52 years with a median follow-up of 3.35 years. Efficiency of immunohistochemical p16 expression and HPV typing in cervical squamous intraepithelial lesion grading and review of the p16 literature. Bala R, Pinsky BA, Beck AH, et al. 8600 Rockville Pike One study included only inflammatory bowel disease patients [21] and another included only hemorrhoidectomy specimens [6]. You are using a browser version with limited support for CSS. The significance of these differences was assessed with the log-rank test. The lesions were categorized based upon the above-mentioned four parameters. Careers. Virchows Arch. It binds to cyclin-dependent kinases 4 and 6 and maintains the retinoblastoma gene product in its hypophosphorylated state [1], which in turn binds to E2F transcription factor and prevents cell cycle progression. The P16 immunostain is an essential factor in the prognostic indication in both OPSCC and non-OPSCC. For HSIL results, when studies considered a two-tiered nomenclature, the positive rate was 84% (95% CI: 6696%). N Engl J Med 2010;363:2435. Rates of positive and negative results did not vary greatly between the three different cutoffs of 75%, 50% and 'any staining'. (A, C, and E: H&E, original magnification 200; B, D, and F: corresponding p16 IHC, original magnification 200). Yang EJ, Quick MC, Hanamornroongruang S, et al. In the meantime, to ensure continued support, we are displaying the site without styles Previous authors proposed HPV diagnostic algorithms for OPSCC using the p16-IHC as the first stepwise to define tumors HPV-unrelated based on exclusively p16-IHC negative. Risk stratification by p16 immunostaining of CIN1 biopsies: a retrospective study of patients from the quadrivalent HPV vaccine trials. 2008;1:713. The findings indicate that p16 immunohistochemistry is a reliable surrogate marker of CDKN2A homozygous deletion in gliomas, with recommended p16 cutoff scores of 5% for confirming and > 20% for excluding biallelic CDKN2A loss. It included 61 studies and evaluated cytological and histological specimens from the uterine cervix. Reduced intensity staining, defined as <3+, was seen more frequently with the G175-405 (33%) and JC8 (28%) clones as compared with E6H4 (15%) (Figures 1a and c). The LAST consensus [3] reported that the expected positive rates to be 8090% in IN2 and approximately 99% in IN3 cases in lower anogenital tract squamous lesions. p16ink4a expression decreases during imiquimod treatment of anal intraepithelial neoplasia in human immunodeficiency virus-infected men and correlates with the decline of lesional high-risk human papillomavirus DNA load. Cell Transplant. volume31,pages 10261035 (2018)Cite this article. Six did not describe the population from which ASIL samples were obtained [7, 16,17,18,19, 22]. Studies published only in an abstract form, case reports, evaluation of p16 expression only in anal squamous cell carcinoma (without an evaluation in ASIL) were also not included in this analysis. 2020 Jul-Sep;61(3):697-706. doi: 10.47162/RJME.61.3.08. There is a possibility that condylomas were not recognized or distinguished from AIN1 in some of the studies included in the AIN1/LSIL category. p16 immunohistochemistry has become the recommended standalone prognostic test for patients with oropharyngeal squamous cell carcinoma as it is more cost-effective and less technically cumbersome than HPV-specific testing (ie, in situ hybridization and reverse-transcriptase PCR), is widely available and has high interobserver agreement in its assessment,10 and has been consistently shown to have independent prognostic significance.8 Under the new 8th edition American Joint Commission on Cancer (AJCC) staging guidelines, p16 immunohistochemistry as a standalone test is now required in order to stage oropharyngeal squamous cell carcinoma and cervical metastatic squamous cell carcinoma of unknown primary patients,11 as p16-positive ones (as a surrogate marker for high-risk HPV status) have their own separate staging systems. Cheah PL, Koh CC, Nazarina AR, Teoh KH, Looi LM. Pirog EC, Quint KD, Yantiss RK. LEEP specimens showed that 76.2% (32 cases) women had HSIL, including 22 cases of CIN2 and 10 cases of CIN3, 14.3% (6 cases) had low-grade squamous intraepithelial lesion (CIN1), and 9.5% (4 cases) had benign cervix. Forty-two women (91.3%) had LEEP procedures done within 6 mo of the initial biopsies. In 2012, the lower anogenital squamous terminology (LAST) was published and recommended a two-tiered nomenclature for noninvasive HPV-associated lesions of the lower anogenital tract, including the terms low-grade squamous intraepithelial lesions (LSILs) and high-grade squamous intraepithelial lesions (HSILs) to replace the previous three-tiered system (intraepithelial neoplasia: IN1, IN2, IN3). Patients were treated without regard to their p16 status.17, Immunohistochemistry was performed for p16 on formalin-fixed, paraffin-embedded tissue sections. The pooled proportions and 95% confidence intervals (CIs) for p16-positive immunostaining per histological grade were computed, using the proportions described in the original studies, and the random effects model, weighted for the sample size of each study, was considered the most appropriate for the analysis. 2017 Oct 12;12(10):e0185597. MeSH BMC Womens Health. Progression of cervical low-grade squamous intraepithelial lesions: in search of prognostic biomarkers. Predictive Role of the p16 Immunostaining Pattern in Atypical Cervical Biopsies with Less Common High Risk HPV Genotypes. This website is intended for pathologists and laboratory personnel but not for patients. ISSN 0893-3952 (print), p16 immunohistochemistry in oropharyngeal squamous cell carcinoma: a comparison of antibody clones using patient outcomes and high-risk human papillomavirus RNA status, https://doi.org/10.1038/modpathol.2017.31, Evaluation of p16INK4a expression as a single marker to select patients with HPV-driven oropharyngeal cancers for treatment de-escalation, Recommendations for determining HPV status in patients with oropharyngeal cancers under TNM8 guidelines: a two-tier approach, HPV RNA CISH score identifies two prognostic groups in a p16 positive oropharyngeal squamous cell carcinoma population, Integrated analysis of cervical squamous cell carcinoma cohorts from three continents reveals conserved subtypes of prognostic significance, Intratumor heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma, Oncogenic driver mutations predict outcome in a cohort of head and neck squamous cell carcinoma (HNSCC) patients within a clinical trial, Comparison of three PD-L1 immunohistochemical assays in head and neck squamous cell carcinoma (HNSCC), Cellular states are coupled to genomic and viral heterogeneity in HPV-related oropharyngeal carcinoma, High Expression of EpCAM and Sox2 is a Positive Prognosticator of Clinical Outcome for Head and Neck Carcinoma, Computer-assisted tumor grading, validation of PD-L1 scoring, and quantification of CD8-positive immune cell density in urothelial carcinoma, a visual guide for pathologists using QuPath, Human Papillomavirus DNA Detection by Droplet Digital PCR in Formalin-Fixed Paraffin-Embedded Tumor Tissue from Oropharyngeal Squamous Cell Carcinoma Patients, Human papillomavirus as a driver of head and neck cancers. 2016 Dec;29(12):1501-1510. doi: 10.1038/modpathol.2016.141. Copyright 2017 Elsevier Inc. All rights reserved. Papillomavirus Res. J Low Genit Tract Dis. Agreement at the 75% cutoff was still best, although, for the E6H4 clone. Intensity of staining was defined as: weak=1, moderate=2 and strong=3. Before This expression is still lower than what is generally described in the cervix and, in the previous cervical meta-analysis (38%). The E6H4 clone comes 'ready-to-use' with no dilution step being required, making it the simplest to perform. p16 immunohistochemistry as a standalone test for risk stratification in oropharyngeal squamous cell carcinoma. It is used for high-risk-HPV associated dysplasias and oropharyngeal carcinomas. Huang EC, Tomic MM, Hanamornroongruang S, Meserve EE, Herfs M, Crum CP. The electronic search retrieved 207 studies of which 171 were excluded: 101 after review of the title, 5 after review of the abstract, and 65 were duplicated. 25 The results seemed to be due to the high sensitivity of JC8 immunohistochemistry on individual cells beyond the limit of detection by molecular techniques. 2014;134:171524. Dual p16 and Ki-67 Expression in Liquid-Based Cervical Cytological Samples Compared to Pap Cytology Findings, Biopsies, and HPV Testing in Cervical Cancer Screening: A Diagnostic Accuracy Study. Albuquerque et al. Pre-cancerous diseases are not only accountable for extra production of the p16 immunostain, but cancers are also responsible for their increment. Human papillomavirus or HPV is a risk factor for malignant and benign mucosal head and neck lesions. Positive staining was identified as brown, punctate dots present in the nucleus and/or cytoplasm. B, Marked koilocytic atypia. p16 IHC is a technically simple and widely available test, and this study establishes the use of p16 IHC as an alternative test to HPV PCR. p16 immunohistochemistry has become the recommended standalone prognostic test for patients with oropharyngeal squamous cell carcinoma as it is more cost-effective and less technically cumbersome than HPV-specific testing (ie, in situ hybridization and reverse-transcriptase PCR), is widely available and has high interobserver agreement in its . Currently, there are no formal recommendations for LSIL management based on p16 results [3]. P16 or INK4A is also known as MTS1 or P16, which is proteinaceous and consists solely of four ankyrin repeats. A detailed description of p16 immunoreactivity for each lesion is based; on four parameters; intensity, extent, continuity, and location. official website and that any information you provide is encrypted Only articles written in English and with p16 immunohistochemistry assessment were included. J Clin Oncol 2010;28:41428. The calculation in condylomas was not performed due to the low number of isolated anal condylomas samples and studies, in which p16 was performed/reported. The Bond Polymer Refine detection system was used for visualization. [26] showed that 34% of AIN2 are p16 negative, while Maniar et al. c Meta-analysis of the LSIL samples (AIN1/LSIL/condyloma) stained positive for p16. For all HSIL results (all classified as AIN2, AIN3, or HSIL), p16 was positive in 82% (95% CI: 7291 %), Table2 and Figs. When reducing to the any staining cutoff, a small amount more patients were positive vs negative with each antibody. PubMed Central A significant minority of tumors are p16 positive and HPV ISH negative, the significance of which is unclear. When correlating with HPV RNA in situ hybridization status, H-score results were essentially identical to those of both the 50 and 75% cutoffs and slightly superior to the any staining cutoff (Table 3). Cases were considered suitable/sufficient for interpretation if at least 10% of the cross-sectional area across the two cores consisted of tumor cells. Google Scholar. We also reviewed reference lists of retrieved articles to identify other relevant studies. Google Scholar. Immunohistochemistry for mismatch repair proteins has shown utility in the identification of Lynch syndrome, but majority of tumours with loss MLH1 expression are due to sporadic hypermethylation of the MLH1 promoter. Hum Pathol. Google Scholar. The .gov means its official. P16 Immunostain Staining For low-grade lesions, three categories were described: condyloma acuminate only (as defined by the original study), AIN1/LSIL (anal condylomas excluded or there were no anal condylomas described), and all LSIL (LSIL, condyloma, AIN1, AIN1 and condyloma described together and condyloma with AIN1/LSIL). O'Sullivan B, Lydiatt WM, Haughey BH et al, HPV-mediated (p16+) oropharyngeal cancer. Prigenzi KCK, Heinke T, Salim RC, Focchi GRA. These improved using the 50% cutoff, becoming similar for all three antibodies. Many of the conclusions for HPV-related lower anogenital tract neoplasia management, including p16 immunostaining are considered applicable across all anogenital sites, mostly based on generalizations from the cervix [3]. Clinical trials are underway that will better define specific clinical management for these patients, distinct from the current standard oropharyngeal squamous cell carcinoma recommendations and approaches.3, 4, 18 For all of these reasons, HPV/p16 testing needs to be standardized across practices. Predictive value of immunohistochemistry in pre-malignant lesions of the gastrointestinal tract. Conversely, p16-positive potentially HPV-driven OPSCC are addressed to second molecular assays, namely ISH or PCR, to confirm the presence of HR-HPV [13, 36,37,38]. p16 Immunohistochemistry is useful in confirming high-grade squamous intraepithelial lesions (HSIL) in women with negative HPV testing It is believed that almost all squamous cell carcinomas of the cervix are associated with HR-HPV infection. 2008;16:21520. The role of P16 immunostain is to prevent the change of a normal cell into a cancer cell. Am J Surg Pathol. It is certainly possible that differences in staining intensity and nonspecific staining between the antibodies may be partially because of staining conditions. . PubMed Central sharing sensitive information, make sure youre on a federal Hematol Oncol Clin North Am 2015;29:104560. Albuquerque et al. HHS Vulnerability Disclosure, Help In this study, we compared three different antibody clones in a large, well-characterized oropharyngeal squamous cell carcinoma patient cohort. Thank you for visiting nature.com. Agreement improved for all antibody clones when using the 50% cutoff so that all three were essentially comparable. Heterogeneity between studies evaluating normal anal samples was I2=0% (95% CI: 013%), in AIN1/LSIL (excluding condyloma) I2=71% (95% CI: 3986%), in all LSIL (including condyloma) I2=59% (95% CI: 2378%), AIN2 I2=77% (95% CI: 5588%), AIN3 I2=15% (95% CI: 079%), HSIL in a two-tiered nomenclature I2=85% (95% CI: 7192%), and for all combined results from HSIL I2=84% (95% CI: 7690%) (Table2). Given the clinical significance of HPV in oral squamous carcinoma, p16 IHC should be performed in all cases In situ hybridization for high-risk HPV E6/E7 mRNA had been performed and interpreted as previously described16 by hand using the RNAscope HPV kit (Advanced Cell Diagnostics, Inc, Hayward, CA, USA) according to the manufacturers instructions. Am J Surg Pathol 2011;35:13431350. Acta Pathol Microbiol Immunol Scand. C, Eroded or thin epithelium displaying abnormal nuclear features. Similarly do Carmo Alves Martins et al. Quint KD, de Koning MN, Quint WG, et al. The authors declare that they have no conflict of interest. PubMed Central The block-positive patterns fulfilled all the requirements described as a diffuse and strong immunoreactivity that extends from the basal layers more than one-third of the epithelium. Steffen Wagner, Elena-Sophie Prigge, Jens Peter Klussmann, Stephanie G. Craig, Lesley A. Anderson, Jacqueline A. James, Jrmy Augustin, Marion Mandavit, Ccile Badoual, Ankur Chakravarthy, Ian Reddin, Tim R. Fenton, Jacob H. Rasmussen, Giedrius Lelkaitis, Jeppe Friborg, Javier Fernndez-Mateos, Jssica Prez-Garca, Juan Jess Cruz-Hernndez, Emma J. de Ruiter, Frans J. Mulder, Stefan M. Willems, Sidharth V. Puram, Michael Mints, Itay Tirosh, Philipp Baumeister, Alessandra Hollmann, Olivier Gires, Modern Pathology 2012;16:20542. Internet Explorer). Correlation with high-risk HPV RNA in situ hybridization results showed significant false-negative rates (low negative predictive values or NPV) for both the JC8 (NPV=69%) and G175-405 (NPV=56%) clones at the 75% cutoff. Our study suggests that when a woman is negative for HPV and also negative for p16, diagnosis of HSIL should be very cautious in void of unnecessary LEEP procedures. 2a-g. a Meta-analysis of the normal samples stained positive for p16. Figures 2 and 3 show the cumulative overall and disease-specific mortality associated with the three antibodies at all three cutoffs (HPV RNA in situ hybridization cumulative overall and disease-specific survival curves are provided as Supplementary Figure 1). Gao G, Chernock RD, Gay HA et al, A novel RT-PCR method for quantification of human papillomavirus transcripts in archived tissues and its application in oropharyngeal cancer prognosis. Value of p16 (INK4a) as a marker of progression/ regression in cervical intraepithelial neoplasia grade 1. This disease is caused; by human papillomavirus that makes p16. Walts AE, Lechago J, Hu B, et al. Liao GD, Sellors JW, Sun HK, et al. Several p16 antibodies are available, and their performance has not been directly compared. J Am Acad Dermatol. p16ink4 and cytokeratin 7 immunostaining in predicting HSIL outcome for low-grade squamous intraepithelial lesions: a case series, literature review and commentary. Control probes for the bacterial gene DapB (negative control) and for the housekeeping gene ubiquitin C (positive control evidence of adequate RNA) were also included on each case. ASIL and AIN, LSIL and AIN1, HSIL and AIN2/3 were used synonymously; the classification used in each study was adopted. . Negative predictive values were significantly lower for the JC8 and G175-405 clones at the 75% cutoff, but increased substantially at the 50% and any staining cutoffs.