High-risk Human Papillomavirus RNA Research Articles

Assessing the feasibility of a multimodal liquid biopsy for the diagnosis of HPV-associated oropharyngeal squamous cell carcinoma.

In this feasibility study, we explored the combined use of circulating tumor human papillomavirus (HPV) DNA (ctHPVDNA) and HPV serology as diagnostic tests for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Among patients with research-banked serum or plasma at diagnosis, IgG antibodies to oncoproteins from HPV types 16, 18, 31, 33, 35, 45, 52, and 58 were detected with multiplex serology. Positivity for HPV 16 was defined based on detection of combinations of anti-E6, E1, E2, and E7 and for other high-risk types on detection of anti-E6 and anti-E7. Circulating tumor HPV DNA was detected by custom digital droplet polymerase chain reaction (ddPCR) assays for HPV types 16, 18, 33, 35, and 45. p16 immunohistochemistry and high-risk HPV RNA in situ hybridization (ISH) using a cocktail of 18 high-risk HPV types were performed on tissue. Of 75 patients, 67 (89.3%) were HPV-associated (p16 and HPV RNA ISH positive) and 8 (10.7%) were HPV-independent. All 8 HPV-independent patients were seronegative and negative for ctHPVDNA (100% specificity). Serology was positive in 53 (79.1%) of 67 HPV-associated patients, while ddPCR was positive for ctHPVDNA in 59 (88.6%) of 67 HPV-associated patients. Requiring both tests to be positive resulted in a sensitivity of 50 (74.6%) of 67 while combining assays (either positive) improved sensitivity to 62 (92.6%) of 67. Compared to HPV RNA ISH, HPV serology and ctHPVDNA are sensitive and highly specific biomarkers for HPV-associated OPSCC at the time of presentation.

Utilization of p53 and p16 Immunohistochemistry in the Classification of Human Papillomavirus–Associated, p53 Wild-Type, and p53 Abnormal Oral Epithelial Dysplasia

p53 immunohistochemistry (IHC) has recently been shown to be a clinically useful marker for predicting risk of progression to invasive squamous cell carcinoma in oral epithelial dysplasia (OED). The literature supports the use of p53 IHC as a marker to identify TP53 mutation in in situ and invasive vulvar lesions and as a surrogate marker for high-risk human papillomavirus (HPV) infection, but there is little documentation for similar use in OED. The purpose of this study was to determine whether p53 IHC is a reliable surrogate marker for detecting both TP53 mutation and high-risk HPV infection in OED. We studied 57 cases of OED (11 mild, 18 moderate, and 28 severe), and all were stained for p16 and p53 IHC. High-risk HPV RNA in situ hybridization (ISH) was performed in selected cases (all p16-positive cases and all OED showing abundant apoptotic cells and karyorrhectic cells; N = 27). Targeted next-generation sequencing (NGS) was performed in 33 p16-negative cases and all high-risk HPV RNA ISH–negative cases (N = 36). We identified 21 cases with p53 basal sparing patterns (mid-epithelial and markedly reduced [null-like]), 14 cases with p53 wild-type patterns (scattered basal and patchy basal/parabasal), and 22 cases with p53 abnormal patterns (18 overexpression, 3 null, and 1 novel cytoplasmic pattern). Among cases with p53 basal sparing patterns, 20 were positive for p16 (20/21, 95%), and all were positive for high-risk HPV RNA ISH (21/21, 100%). The 36 sequenced cases had IHC patterns concordant with TP53 mutation status in 92% (33/36) of lesions. This study demonstrates that p53 IHC expression patterns are sensitive and specific for detection of both high-risk HPV infection and TP53 mutation. Coupled with selective p16 IHC testing, this IHC panel can accurately subclassify OED into HPV-associated, p53 wild-type (conventional), and p53 abnormal OED.

Clinicopathologic and Molecular Characterization of Anorectal Neuroendocrine Carcinomas Reveals Human Papillomavirus, p53, and c-Myc as Alternative Mechanisms of Carcinogenesis

Poorly differentiated neuroendocrine carcinomas (NECs) are rare malignant neoplasms with aggressive behavior. The diagnosis remains challenging due to ever-changing terminologies and morphologic overlaps with other disease entities. Herein, we seek to better define anorectal NECs by high-risk human papillomavirus (HPV) status and molecular profiling. Fourteen cases, including 3 men and 11 women with a median age of 63 years, were included. High-risk HPV RNA in situ hybridization was diffusely positive (+) in 7 cases, focal rarely positive (+/−) in 2 cases, and completely negative (−) in 5 cases. By morphology, all HPV(−) NECs were large-cell type, 3 mixed with a tubular adenoma/dysplasia or invasive adenocarcinoma. HPV-related (+ or +/−) NECs were mostly small-cell type, 3 mixed with squamous dysplasia and/or squamous cell carcinoma. Immunohistochemically, all NECs were positive for at least 2 neuroendocrine markers. The HPV(−) NECs were also positive for CDX2, whereas all HPV-related NECs were negative or only focally positive for CDX2, p40, and p63. Overexpression of p53 was found in 3 HPV(−) and 2 HPV(+/−) NECs but not in any HPV(+) NECs. Molecular analysis revealed MYC gene amplification in 4 cases: 2 HPV(−), 1 HPV(+/−), and 1 HPV(+). This was confirmed by fluorescence in situ hybridization in all but 1 HPV(−) NEC, which showed polysomy 8 but no true MYC amplification. Interestingly, only 2 of the 4 MYC amplification–bearing cases, both p53 normal/wild-type, expressed c-Myc protein by immunohistochemistry. The other 2 cases, both p53 overexpressed, did not show c-Myc expression despite true MYC amplification. Our study demonstrates that anorectal NECs arise in HPV-dependent or -independent pathways, with heterogeneous expression of other lineage markers and different molecular signatures. Expressions of p53 and c-Myc proteins appear to be mutually exclusive regardless of HPV status, likely mediating alternative mechanisms of NEC carcinogenesis.

Utility of high-risk HPV RNA chromogenic in situ hybridization in cytology smears and liquid-based preparations from metastatic head and neck squamous cell carcinoma.

High-risk human papillomavirus (HR-HPV) status is critical for the diagnosis, prognosis, and treatment of patients with oropharyngeal squamous cell carcinoma (OPSCC). Patients often present with enlarged cervical nodes, and fine-needle aspiration cytology (FNAC) is frequently the initial diagnostic procedure. Although p16 is the most widely used surrogate marker, problems with interpretation can limit its utility in FNAC. HR-HPV RNA in situ hybridization (ISH) has emerged as a specific way to assess HPV status on cell block preparations of cervical nodes. The authors evaluated the utility of HR-HPV ISH in conventional smears and liquid-based cytology (LBC) preparations of metastatic head and neck squamous cell carcinoma (SCC). Thirty-one aspirates of proven, HPV-related SCC (confirmed by p16 and/or HR-HPV ISH in corresponding surgical specimens) were selected. Ten aspirates of HPV-negative SCC were also retrieved. HR-HPV ISH was performed on 27 smears and 14 LBC preparations. All results were scored as positive, equivocal, or negative. Eighty-four percent of metastatic, HPV-related SCCs were positive for HR-HPV RNA ISH, with high number of signals (n=19) and low number of signals (n=7), whereas five HPV-related SCCs were equivocal. All metastatic, HPV-negative SCCs were negative for HR-HPV ISH. HR-HPV ISH can be reliably performed on smears or LBC preparations, particularly when cell blocks are unavailable or paucicellular. Results were easy to interpret when high numbers of signals were present but were challenging in aspirates with low or rare number of signals. The current study suggests that HR-HPV ISH could be used as the initial testing modality for determining HPV status in FNAC specimens of metastatic SCC.

AHCC® Supplementation to Support Immune Function to Clear Persistent Human Papillomavirus Infections.

ObjectiveTo determine the efficacy, safety, and durability of the use of AHCC supplementation for 6 months to support the host immune system to clear high-risk human papillomavirus (HPV) infections. The AHCC supplement is a proprietary, standardized extract of cultured lentinula edodes mycelia (AHCC®, Amino Up, Ltd., Sapporo, Japan) that has been shown to have unique immune modulatory benefits.Study DesignThis was a randomized, double-blind, placebo-controlled study (CTN: NCT02405533) in 50 women over 30 years of age with confirmed persistent high-risk HPV infections for greater than 2 years. Patients were randomized to placebo once daily for 12 months (N = 25) or AHCC 3-g supplementation by mouth once daily on empty stomach for 6 months followed by 6 months of placebo (N = 25). Every 3 months, patients were evaluated with HPV DNA and HPV RNA testing as well as a blood sample collected to evaluate a panel of immune markers including interferon-alpha, interferon-beta (IFN-β), interferon-gamma (IFN-γ), IgG1, T lymphocytes, and natural killer (NK) cell levels. At the completion of the 12-month study period, patients on the placebo arm were given the option to continue on the study to receive AHCC supplementation unblinded for 6 months with the same follow-up appointments and testing as the intervention arm.ResultsFifty women with high-risk HPV were enrolled, and 41 completed the study. Fourteen (63.6%) of the 22 patients in the AHCC supplementation arm were HPV RNA/HPV DNA negative after 6 months, with 64.3% (9/14) achieving a durable response defined as being HPV RNA/HPV DNA negative 6 months off supplementation. On the placebo arm, two (10.5%) of 19 patients were HPV negative at 12 months. In the twelve placebo arm patients who elected to continue on the unblinded study, 50% (n = 6) were HPV RNA/HPV DNA negative after 6 months of AHCC supplementation. At the time of completion of the study, there were a total of 34 patients (22 blinded and 12 unblinded) who had received AHCC supplementation with an overall response rate of 58.8% that cleared HPV persistent infections. At the time of enrollment, the mean IFN-β level was 60.5 ± 37.6 pg/ml in women with confirmed persistent HPV infections. Suppression of IFN-β to less than 20 pg/ml correlated with an increase in T lymphocytes and IFN-γ and durable clearance of HPV infections in women who received AHCC supplementation.ConclusionResults from this phase II study demonstrated that AHCC 3 g once daily was effective to support the host immune system to eliminate persistent HPV infections and was well tolerated with no significant adverse side effects reported. The duration of AHCC supplementation required beyond the first negative result needs more evaluation to optimize success for durable outcomes. The suppression of the IFN-β level to less than 20 pg/ml correlated with clearance of HPV infections and merits further evaluation as a clinical tool for monitoring patients with HPV infections.Clinical Trial Registrationclinicaltrials.gov/ct2/, identifier NCT02405533

CLINICOPATHOLOGIC FEATURES OF HUMAN PAPILLOMAVIRUS–ASSOCIATED ORAL EPITHELIAL DYSPLASIA

<h3>Background</h3> There has been growing acceptance by the oral pathology community that high-risk human papillomavirus (HPV) is associated with a subset of oral epithelial dysplasias (OEDs), but there is lack of consensus on the diagnostic criteria for HPV OED. This study describes the clinicopathologic features of this disease with the intention of adding to the current pool of knowledge to clarify diagnostic criteria and elucidate its clinical significance. <h3>Method</h3> Automated p16 and viral capsid immunohistochemistry, as well as high-risk HPV DNA and RNA in situ hybridization were undertaken in 71 archival samples from 40 patients diagnosed with HPV OED and 11 samples demonstrating similar histomorphological features. Ploidy analysis was undertaken on a subset of samples. <h3>Results</h3> The male:female ratio was 3.4:1. Five patients presented with adjacent associated invasive carcinoma, and in 4 patients there was subsequent malignant transformation. Histologic features of viral infection did not consistently colocalize with p16, and DNA or RNA in situ hybridization and were also present at varying degrees in the cases deemed HPV-negative. Capsid protein was present in 11 cases. Seven cases were DNA negative but assessed RNA positive. Of the cases subject to ploidy analysis, 9 were diploid and 6 were aneuploid. Two cases were reclassified as papillomas. <h3>Conclusions</h3> Standardized diagnostic criteria are required to differentiate HPV OED from histologic mimics. Further research is necessary to determine the histologic and ancillary factors that may be useful in predicting malignant transformation.

RB1, p16, and Human Papillomavirus in Oropharyngeal Squamous Cell Carcinoma.

While P16 immunohistochemistry (IHC) is a well-established surrogate marker of Human Papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC), Retinoblastoma 1 (RB1) loss may lead to p16 overexpression in the absence of HPV. We determined the proportion of p16-positive/HPV-negative OSCC with RB1 loss and other alterations in RB1/p16 pathway, and tested RB1 IHC as a prognostic biomarker for OSCC, along with the 8th edition of AJCC staging manual. P16 and RB1 IHC and HPV DNA in situ hybridization (ISH) were performed on 257 OSCC. High risk HPV RNA ISH, RB1 fluorescence in situ hybridization (FISH), and next generation sequencing (NGS) were done on p16-positive/HPV DNA ISH-negative OSCC. Disease free survival (DFS) was used as an endpoint. In the entire cohort and in p16-positive (n = 184) and p16-negative (n = 73) subgroups, AJCC 8th edition staging correlated with DFS (p < 0.01). RB1 IHC showed RB1 loss in p16-positive OSCC only (79/184, 43%). RB1 loss by IHC is associated with a better DFS, without providing additional prognostic information for patients with p16-positive OSCC. HPV RNA ISH was positive in 12 of 14 HPV DNA ISH-negative cases. RB1 IHC showed loss in 10 of 15 HPV DNA ISH-negative cases and in 1 of 2 HPV RNA ISH-negative cases. Overall, only one case of p16-positive/HPV RNA ISH-negative OSCC showed RB1 loss by IHC (1/184, 0.5%). Of the 10 p16-positive and HPV DNA ISH-negative cases with RB1 loss by IHC, 2 had RB1 hemizygous deletion and 3 showed Chromosome 13 monosomy by FISH. No RB1 mutations were detected by NGS. Other molecular alterations in p16-positive/HPV DNA ISH-negative cases included TP53 and TERT mutations and DDX3X loss. HPV-independent RB1 inactivation rarely results in false positive p16 IHC. RB1 inactivation by high risk HPV E7 oncoprotein may co-exist with RB1 deletion. RB1 loss is a favorable prognosticator and occurs exclusively in p16-positive OSCC. The 8th edition of the AJCC staging manual satisfactorily predicts DFS of OSCC patients.

Discrepancy of p16 immunohistochemical expression and HPV RNA in penile cancer. A multiplex in situ hybridization/immunohistochemistry approach study

BackgroundThe high-risk human papillomavirus (HPV) infection represents one of the main etiologic pathways of penile carcinogenesis in approximately 30–50 % of cases. Several techniques for the detection of HPV are currently available including Polymerase chain reaction-based techniques, DNA and RNA in situ hybridization (ISH), p16 immunohistochemistry (IHC). The multiplex HPV RNA ISH/p16 IHC is a novel technique for the simultaneous detection of HPV E6/E7 transcripts and p16INK4a overexpression on the same slide in a single assay. The main aim of this study was to evaluate the discrepancy of p16 IHC expression relatively to HPV RNA ISH in penile cancer tissue.MethodsWe collected a series of 60 PCs. HPV has been analysed through the RNA ISH, p16 IHC and the multiplex HPV RNA ISH/p16 IHC.ResultsThe multiplex HPV RNA ISH /p16 IHC results in the series were in complete agreement with the previous results obtained through the classic p16 IHC and HPV RNA scope carried out on two different slides. The multiplex HPV RNA ISH /p16 IHC showed that HPV positivity in our series is more frequently in usual squamous cell carcinoma than in special histotypes (19 out of 60 − 15 %- versus 6 out of 60 − 10 %-), in high-grade than in moderate/low grade carcinomas (6 out of 60 − 10 %- versus 4 out of 60 − 6.7 %-). In addition, our data revealed that in 5 out of 20 cases with p16 high intensity expression is not associated with HPV RNA ISH positivity.ConclusionsOur findings emphasize that the use of p16 as a surrogate of HPV positivity was unsuccessful in approximatively 8 % of cases analysed in our series. Indeed, p16 IHC showed a sensitivity of 100 % and a specificity of 71 %, with a positive predictive value (PPV) of 54 % and a negative predictive value of 100 %; when considering high intensity, p16 IHC showed a sensitivity of 100 %, a specificity of 89 %, with a PPV of 75 % and NPV of 100 %.Since HPV positivity could represent a relevant prognostic and predictive value, the correct characterization offered by this approach appears to be of paramount importance.

RNA in situ hybridization for human papillomavirus testing in oropharyngeal squamous cell carcinoma on a routine clinical diagnostic platform.

The current diagnostic standard for detection of high-risk human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma is via a two-stage algorithm, namely p16 immunohistochemistry followed by HPV DNA in situ hybridization in p16 positive cases. This study evaluated the feasibility of automated RNA in situ hybridization on a clinical platform as a single-step alternative to the two-stage algorithm within a routine diagnostic histopathology setting. Thirty-eight cases positive for both p16 and DNA in situ hybridization, 42 p16 negative cases and 20 cases positive for p16 but negative for DNA in situ hybridization were randomly selected. High-risk HPV RNA in situ hybridization was undertaken on all cases on an automated clinical platform. Manufacturer-recommended and on-slide additional p16/HPV positive and negative controls were used. Test quality assurance and diagnostic RNA in situ hybridization were independently assessed by two observers. A consensus diagnosis was reached in the presence of a third observer on discordant cases. All RNA in situ hybridization results were then correlated against p16 and DNA ISH status. Inter-slide RNA in situ hybridization staining variation was observed in control sections. RNA in situ hybridization demonstrated a high inter-observer agreement rate (κ=.897, P<.001). Following consensus review, there was full concordance between RNA in situ hybridization and the current standard. Human papillomavirus testing by standalone automated RNA in situ hybridization on a clinical diagnostic platform currently available in routine diagnostic histopathology laboratories is a feasible alternative to the two-step algorithm of p16 and DNA in situ hybridization. Control tissue staining procedures need to be adapted to achieve the most accurate results.

Role of human papillomavirus infection in the etiology of vulvar cancer in Italian women

BackgroundVulvar squamous cell carcinoma (VSCC) is a rare malignancy of the female genital tract. We aimed to determine the mucosal high-risk human papillomavirus (HPV)-attributable fraction of VSCCs from Italian women using multiple markers of viral infections.MethodsVSCCs and 8 metastatic lymph node samples from 107 Italian women were analyzed by a highly type-specific multiplex genotyping assay for the presence of DNA from 119 different HPVs. Tissues were further analyzed for HPV RNA and for upregulation of the cellular protein p16INK4a.ResultsThe rate of mucosal HPV-related tumors defined by viral DNA and RNA positivity was low (7.8%). HPV16 was the most prevalent, followed by 53, 56, and 58. Only five (4.9%) p16INK4a-positive tumors were also positive for both viral DNA and RNA. One (14.3%) metastatic lymph node sample was positive for all three markers. DNA of cutaneous HPVs was detected in only two VSCCs, i.e. genus beta types 5 and 110.ConclusionA small proportion of Italian VSCCs is putatively HPV-related, i.e. positive for both viral DNA and RNA of the same type, thus reinforcing the importance of HPV vaccination. Moreover, this study suggests that a direct role of HPV from genus beta and gamma in vulvar carcinogenesis is unlikely.

Transcriptionally Active HPV and Targetable EGFR Mutations in Sinonasal Inverted Papilloma: An Association Between Low-risk HPV, Condylomatous Morphology, and Cancer Risk?

Sinonasal inverted papillomas (IPs) commonly recur, and transform to malignancy in 5% to 10% of patients. It has long been debated whether IPs are caused by high-risk or low-risk (lr) human papillomavirus (HPV) and whether the HPV is transcriptionally active. EGFR mutations have also been recently implicated in the pathogenesis of IP with an unclear relationship to HPV status. IP cases over a 10-year period were tested for p16 by immunohistochemistry and for transcriptionally active hrHPV and lrHPV by reverse-transcriptase real-time polymerase chain reaction and RNA in situ hybridization, respectively. EGFR tyrosine kinase domain Sanger sequencing was performed on all lrHPV RNA positive and 15 randomly selected lrHPV RNA negative IPs. Seven sinonasal nonkeratinizing squamous cell carcinomas (SCCs) without associated IP were included as controls. Of the 44 IPs, 5 (11.4%) were associated with SCC, all keratinizing type. All IPs and associated SCCs were negative for p16 and hrHPV. lrHPV RNA was detected in 5/42 (12%) cases, including 3/5 (60%) with associated SCC (P=0.009). All 5 lrHPV RNA positive IPs involved the nasal cavity, had a distinct, condylomatous morphology, and were EGFR wild-type. In contrast, 11/15 (73.3%) lrHPV RNA negative IPs that were sequenced had EGFR exon 19 or 20 mutations. All control nonkeratinizing SCCs were lrHPV RNA negative, but 5/7 (71.4%) were p16 and high-risk HPV RNA positive. This study shows that a subset of IPs involving the nasal cavity have transcriptionally active lrHPV, condylomatous morphology, and possibly increased risk of malignancy. Furthermore, lrHPV positivity is mutually exclusive with EGFR mutations, which suggests alternate mechanisms of pathogenesis.

HPV-driven oropharyngeal squamous cell cancer in Croatia - Demography and survival.

ObjectivesHead and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Main HNSCC risk factors are tobacco, alcohol, and high-risk human papillomavirus (HPV). HPV+ oropharyngeal squamous cell cancer (OPSCC) usually have different etiology, increasing incidence and often show an improved survival when compared to HPV-negative cases. The objective of the current study was to retrospectively examine the influence of HPV on the survival of OPSCC patients in a non-Western population setting.Materials and methodsWe determined the presence of HPV DNA and/or RNA in 99 formalin-fixed paraffin embedded (FFPE) tissue samples of OPSCC patients treated between 2002 and 2015. Patients were compared based on laboratory, demographic, clinical, life style characteristics and survival.ResultsHPV RNA was found in 29.3% cases. However, groups based on HPV data (either RNA, DNA or retrospectively collected p16 staining) did not show significant differences. Overall, 5-year survival was 30% with minimal influence of the HPV positivity.ConclusionsThe HPV influence on survival of OPSCC patients is not identical between populations probably due to other factors overshadowing the HPV effect. This should be taken into account when treatment or policy decisions are made in each particular setting.

Invasive cervical tumors with high and low HPV titer represent molecular subgroups with different disease etiology.

Invasive cervical cancer (ICC) with very low titer of high-risk human papillomavirus (HPV) has worse clinical outcome than cases with high titer, indicating a difference in molecular etiology. Fresh-frozen ICC tumors (n = 49) were classified into high- and low-HPV-titer cases using real-time PCR-based HPV genotyping. The mutation spectra were studied using the AmpliSeq Comprehensive Cancer Panel and the expression profiles using total RNA sequencing, and the results were validated using the AmpliSeq Transcriptome assay. HPV DNA genotyping and RNA sequencing showed that 16.6% of ICC tumors contained very low levels of HPV DNA and HPV transcripts. Tumors with low HPV levels had more mutations with a high allele frequency and fewer mutations with low allele frequency relative to tumors with high HPV titer. A number of genes showed significant expression differences between HPV titer groups, including genes with somatic mutations. Gene ontology and pathway analyses implicated the enrichment of genes involved in DNA replication, cell cycle control and extracellular matrix in tumors with low HPV titer. The results indicate that in low titer tumors, HPVs act as trigger of cancer development whereas somatic mutations are clonally selected and become drivers of the tumor development process. In contrast, in tumors with high HPV titer the expression of HPV oncoproteins plays a major role in tumor development and the many low frequency somatic mutations represent passengers. This putative subdivision of invasive cervical tumors may explain the higher radiosensitivity of ICC tumors with high HPV titer and thereby have consequences for clinical management.

Transcriptionally Active High-Risk Human Papillomavirus is Not a Common Etiologic Agent in the Malignant Transformation of Inverted Schneiderian Papillomas.

The role of human papillomavirus (HPV) as an etiologic and transformational agent in inverted Schneiderian papilloma (ISP) is unclear. Indeed, reported detection rates of HPV in ISPs range from 0 to 100%. The true incidence has been confounded by a tendency to conflate high- and low-risk HPV types and by the inability to discern biologically relevant from irrelevant HPV infections. The recent development of RNA in situ hybridization for high-risk HPV E6/E7 mRNA now allows the direct visualization of transcriptionally active high-risk HPV in ISP, providing an opportunity to more definitively assess its role in the development and progression of ISPs. We performed p16 immunohistochemistry and high-risk HPV RNA in situ hybridization on 30 benign ISPs, 7 ISPs with dysplasia, 16 ISPs with carcinomatous transformation, and 7 non-keratinizing squamous cell carcinomas (SCCs) with inverted growth that were unassociated with ISP. Transcriptionally active HPV was not detected in any of the 52 ISPs including those that had undergone carcinomatous transformation, but it was detected in two of seven (29%) non-keratinizing SCCs that showed inverted growth. There was a strong correlation between high-risk HPV RNA in situ hybridization and p16 immunohistochemistry (97%; p < 0.01). These results indicate that transcriptionally active high-risk HPV does not play a common role in either the development of ISP or in its transformation into carcinoma.

Application of p16 Immunohistochemistry and RNA In Situ Hybridization in the Classification of Adenoid Basal Tumors of the Cervix.

Our understanding of adenoid basal tumors of the cervix has evolved over time. Most of the proliferations referred to as adenoid basal carcinoma have a clinically benign course--leading some to suggest the term "adenoid basal epithelioma." However, rarely, these may be associated with invasive carcinomas. These tumors have been etiologically linked with high-risk human papillomavirus (HR-HPV) infection. Here, we investigate the use of p16 immunohistochemistry and HR-HPV RNA in situ hybridization (ISH) in the classification of adenoid basal tumors of the cervix. Seventeen cases of adenoid basal tumors of the cervix were included. The patients' age ranged from 19 to 79 yr (average, 59 yr). p16 immunostain was performed on all cases and RNA ISH was performed in 4 cases with available formalin-fixed paraffin-embedded tissue. There were 11 low-grade tumors, 5 frankly invasive carcinomas, and 1 with histologic features that were intermediate between the former 2 categories. p16 immunostain was negative or showed patchy cytoplasmic staining in the low-grade tumors and was strongly and diffusely positive in the invasive carcinomas. HR-HPV RNA ISH was negative in the 3 low-grade tumors and was positive in 1 case of invasive carcinoma including the adenoid basal component. Distinct p16 immunostaining and HR-HPV RNA ISH patterns exist between low-grade adenoid basal tumors and invasive adenoid basal carcinomas. Our study indicates that p16 immunostaining and HR-HPV RNA ISH can be employed as useful ancillary tools in differentiating between noninvasive and invasive adenoid basal tumors along with careful histopathologic evaluation.

Correlation of p16 immunohistochemistry in FNA biopsies with corresponding tissue specimens in HPV-related squamous cell carcinomas of the oropharynx.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is a unique form of carcinoma that is important to identify for prognosis and treatment. Immunohistochemistry (IHC) for p16 (also known as cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1) is used as a surrogate marker for transcriptionally active, high-risk HPV. The primary objective of this study was to correlate p16 IHC of cell blocks from fine-needle aspirations (FNAs) with surgical pathology specimens of HPV-related oropharyngeal SCC. In total, 48 patients who had a diagnosis of oropharyngeal or nonoropharyngeal SCC and also had an FNA that demonstrated metastatic SCC with available cell block material were identified. IHC for p16 was evaluated on both FNA cell blocks and surgical pathology specimens. In situ hybridization for high-risk HPV messenger RNA was performed on 31 of the FNA cell blocks. Although partial p16 staining was observed in the majority of cell blocks, there was concordance in 47 of 48 FNAs (98%) with surgical pathology specimens when strong positive p16 staining of at least 15% of tumor cells in FNA cell block material was present. In addition, high-risk HPV RNA in situ hybridization demonstrated a high correlation with p16 staining in surgical pathology specimens (96%) and FNAs (93%). There was excellent correlation between p16 IHC of FNA cell blocks and surgical pathology specimens using a cutoff of at least 15% positive staining in cell blocks. The recommended threshold (70% positive staining) for surgical pathology specimens may yield a high rate of false-negative results if applied to FNA cell blocks.