High-risk Human Papillomavirus Status Research Articles

Abstract 1854: P16 immunoreactivity in endocervical tubo-endometrial metaplasia (TEM) and correlation with high risk HPV (HRHPV) status by chromogenic in-situ hybridization (HPV-CISH) and hybrid capture 2 assay

Abstract Background Several studies have shown that endocervical TEM can intensively over express p16 and involve more than 50% of the cells. It could be speculated that TEM may be a precursor lesion of cervical glandular intraepithelial neoplasm. The aim of this study is to evaluate endocervical TEM p16 overexpression pattern and further investigate its potential driven mechanisms by correlating with HRHPV status. Method Our search included cervical biopsy, LEEP, conization with TEM diagnosis from January 2005-May 2013. Only cases with concurrent Pap smear with HRHPV testing were selected. HRHPV detected in liquid based cytology is by Hybrid Capture 2 Assay. Surgical specimens containing concurrent squamous dysplasia were excluded due to potential confounding by HRHPV positivity in dysplastic squamous epithelium. P16 stain was performed and graded as negative, focal+ (<50% cytoplasmic and nuclear stain) and diffuse+ (>50% cytoplasmic and nuclear stain). HRHPV data was collected from Pap reports. HPV-CISH assays will be performed for all cased with positive HRHPV detected in liquid based cytology. Result: Our search generated 159 cases with TEM. Only 81 cases had concurrent Pap smear with HRHPV testing. 48 cases were excluded due to squamous dysplasia. A total of 33 patients with an age range of 20-59 were available for the study. Twenty nine out of 33 had negative HRHPV in liquid based cytology; in which, p16 stain results as: 8/29 negative, 14/29 focally +. 7/29 diffusely +. Four out of 33 had positive HRHPV detected in liquid based cytology; in which, p16 stain results as: 1/4 negative, 3/4 diffusely +. HPV-CISH assays were performed in all 4 cases and they were negative. Conclusions P16 overexpression in TEM can be intense and involve more than 50% of the cells but the pattern is heterogenous and positivity never involves 100% of the cells. Neither HPV-CISH nor Hybrid Capture 2 Assay detects HRHPV in TEM. P16 overexpression in TEM is most likely HRHPV-independent mechanisms. . Note: This abstract was not presented at the meeting. Citation Format: Shobhana Talukdar, Gomez-Gelvez Juan, Thomas E Buekers, Ziying Zhang. P16 immunoreactivity in endocervical tubo-endometrial metaplasia (TEM) and correlation with high risk HPV (HRHPV) status by chromogenic in-situ hybridization (HPV-CISH) and hybrid capture 2 assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1854. doi:10.1158/1538-7445.AM2014-1854

Abstract 2490: Integrative systems analysis of HPV+ and HPV- oropharyngeal carcinomas

Abstract BACKGROUND: Over half of oropharyngeal carcinomas (OPCs) are linked to high-risk human papillomavirus (HPV) infection. As compared to their HPV-positive (HPV+) counterparts, HPV-negative (HPV-) OPCs are associated with significantly worse survival, inferior treatment response and a distinct molecular expression profile. There are currently considerable efforts underway to personalize treatment based on HPV status; however, this remains challenging as the current understanding of the different biological mechanisms in HPV+ vs. HPV- OPCs is limited. Hence, a comprehensive proteomic analysis was performed, utilizing archival OPC tissues, in order to acquire greater insights into the distinct biological pathways. METHODS: Global protein expression profiling was conducted on 26 HPV- and 27 HPV+ pre-treatment formalin-fixed paraffin-embedded (FFPE) OPCs using an automated 5-step Multidimensional Protein Identification Technology (MudPIT) procedure on a LTQ-Orbitrap XL. Protein quantification was achieved by adjusted spectral counting. Differentially expressed proteins (p<0.05) were classified into functionally enriched categories and interaction networks using GeneMania. Finally, differential expression of 4 proteins was evaluated by immunohistochemistry (IHC) on an independent cohort of 17 HPV- and 12 HPV+ pre-treatment FFPE OPC biopsies. RESULTS: Among the 2633 high confidence protein groups identified, 174 were significantly differentially expressed in HPV+ vs. HPV- OPCs. Functional enrichment analysis of these proteins demonstrated enrichment of cell cycle, DNA repair, cytokine signaling, apoptosis, DNA packaging, and metabolism proteins. These results were in concordance with a previous publication of pooled OPCs (Slebos et al, 2012). In addition, utilizing The Cancer Genome Atlas (TCGA) data, we determined that the relative expression levels of many of these proteins could be attributed to changes in copy number, methylation, mRNA, or mutation profiles. Next, the top 4 proteins were selected for verification based on concordance with these datasets and reported biological relevance. Selected proteins are involved in actin assembly, purine metabolism, differentiation, and cell migration and invasion. As a result, the differential expression of 2 proteins was successfully validated by IHC, while 2 additional proteins exhibited the same trend as in the discovery cohort. CONCLUSION: This study provides the most in-depth and integrated protein-centric view of HPV+ and HPV- OPC. As a key finding, we highlight the identification of an actin assembly oncoprotein reported to be involved in a targetable radioresistance pathway. We hypothesize that this pathway is preferentially activated in HPV- OPCs. Hence, future experiments will focus on validating the in vivo observed expression pattern of this protein in vitro, and manipulating its expression to overcome radioresistance in OPC models. Citation Format: Lusia Sepiashvili, Angela Hui, Wei Shi, Wei Xu, Daryl Waggot, Paul Boutros, Alex Ignatchenko, Vladimir Ignatchenko, Shao Hui (Sophie) Huang, John Waldron, Brian O'Sullivan, Jonathan C. Irish, Fei-Fei Liu, Thomas Kislinger. Integrative systems analysis of HPV+ and HPV- oropharyngeal carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2014-2490

Lack of P16ink4a Over Expression in Penile Squamous Cell Carcinoma is Associated with Recurrence after Lymph Node Dissection

There have been conflicting data in studies on the prognostic role of high risk human papillomavirus in penile squamous cell carcinoma. Using P16(ink4a) over expression as a surrogate marker for high risk human papillomavirus, we evaluated high risk human papillomavirus status with respect to various clinical features, including recurrence and overall survival, among others. P16(ink4a) over expression was evaluated by immunohistochemistry for 119 consecutive patients with penile squamous cell carcinoma. Several variables were recorded including age, stage, histological grade, lymph node status, lymphovascular invasion, metastasis and recurrence. Median followup was 30 months. P16(ink4a) over expression was detected in 49.5% (59 of 119) of samples. There was no significant difference between P16(ink4a) negative and P16(ink4a) positive tumors in terms of stage (p = 0.518), histological grade (p = 0.225), lymphovascular invasion (p = 0.388), overall survival (p = 0.156) or lymph node metastasis (p = 0.748). P16(ink4a) negative tumors were more likely to recur overall (p = 0.04), especially if patients had positive lymph nodes at diagnosis (p = 0.002). These data suggest that P16(ink4a)/high risk human papillomavirus status is associated with recurrence, especially in patients with positive lymph nodes at diagnosis. Thus, patients with P16(ink4a) negative penile cancer, particularly those with lymph node metastases, may warrant closer observation after surgery.

Epithelial-mesenchymal transition in human papillomavirus-positive and -negative oropharyngeal squamous cell carcinoma.

High-risk human papillomavirus (HPV) infection is associated with carcinogenesis in oropharyngeal squamous cell carcinoma (OPSCC) and patients with HPV-positive tumors have a significantly favorable prognosis. However, the underlying mechanism of this favorable clinical outcome remains unclear. Epithelial-mesenchymal transition (EMT) causes aggressiveness of cancer cells and we investigated the expression of the EMT markers and analyzed their correlation with HPV status and prognosis in order to examine the treatment response of HPV-positive OPSCCs. A total of 79 patients with OPSCC were examined in the present study. All high-risk HPV infections were determined with the multiplex PCR kit from each formalin-fixed paraffin-embedded (FFPE) sample. We performed immunohistochemical staining for E-cadherin and vimentin. Expression of the markers was graded and we statistically analyzed the correlation between tumor, node, metastasis (TNM) stages and prognosis. High-risk HPV-positive tumors were detected in 23 cases. The five‑year survival rate in HPV-positive and -negative tumors was 82.7 and 48.3%, respectively. High E-cadherin expression rate in HPV-negative samples was 76.7% and 43.4% in HPV-positive samples (p=0.007). Vimentin expression did not show a difference between HPV-positive and -negative tumors. HPV-negative patients presented significantly greater heterogeneity of E-cadherin expression compared to HPV-positive patients (p=0.0349). HPV-positive OPSCCs originally lost their epithelial cell phenotype compared with HPV-negative tumors. Therefore, the paradoxical favorable prognosis of HPV-positive OPSCC may be due to the intratumor homogeneity in EMT.

Amplification of 3q26 and 5p15 regions in cervical intraepithelial neoplasia.

To analyze different amplification patterns of 3q26 and 5p15 regions in low-grade and high-grade cervical intraepithelial neoplasia. Experimental research. Department of Obstetrics and Gynecology at a medical faculty in Slovakia. A group of 83 patients referred for colposcopic examination. Amplification of 3q26 and 5p15 regions was analyzed on the 100 most atypical cells from a cervical cytology slide by fluorescent in situ hybridization using a multicolor hybridization probe. Chi-squared and Man-Whitney U-tests were used for statistical analysis. Liquid-based cytology samples and biopsy samples obtained during colposcopic examination correlated with high-risk human papillomavirus status and with amplification patterns of selected regions analyzed by fluorescent in situ hybridization. The number of cells with 3q26 and 5p15 gain rises with the severity of the lesion p < 0.01. The sensitivity of 3q26 amplification for CIN2+ lesions was 72.1% (95% confidence interval 56.3-84.7) and specificity was 90.0% (95% confidence interval 76.3-97.1). The sensitivity of 5p15 amplification for CIN2+ lesions was 69.8% (95% confidence interval 53.9-82.8) and specificity was 85.0% (95% confidence interval 70.2-94.3). Evaluation of telomerase components can help in differential diagnosis of low-grade and high-grade cervical lesions and in individualized management of these patients.

Association between the standardized uptake value and high-risk HPV in hypopharyngeal squamous cell carcinoma

Conclusion: Median 18F-FDG PET/CT maximum standardized uptake values (SUVmax) cut-off values of 7.9 or greater were associated with high-risk human papillomavirus (HPV) negativity in patients with hypopharyngeal squamous cell carcinoma (HPSCC). Furthermore, median 18F-FDG PET/CT SUVmax cut-off values of 7.9 or greater and high-risk HPV negativity were associated with adverse outcomes. Objectives: We studied the association and the potential prognostic significance of 18F-FDG PET/CT and high-risk HPV status in HPSCC. Methods: The medical records of 45 patients who underwent 18F-FDG PET/CT for HPSCC before surgery were reviewed. High-risk HPV in situ hybridization was performed to detect HPV infection. Results: The median SUVmax was 9.91 ± 4.91 (range 1.9–22.1) and the positive rate of high-risk HPV in situ hybridization was 11% (5 of 45). The SUVmax values of negativity for the high-risk HPV subtypes (10.47 ± 4.87) and positivity (5.48 ± 2.45) were found to be significantly different (p = 0.030). The SUVmax cut-off value for differentiating negativity for the high-risk HPV subtypes from positivity was 7.9, with a sensitivity of 65% and a specificity of 80%. The 5-year disease-specific survival rate (DSSR) in our cohort was 57%. Patients with an SUVmax value higher than 7.9 (p = 0.005) and high-risk HPV negativity (p = 0.047) had decreased 5-year DSSR.

Mitotically active microglandular hyperplasia of the cervix: a case series with implications for the differential diagnosis.

Microglandular hyperplasia (MGH) is a benign proliferation of endocervical glands with relatively uniform columnar or cuboidal nuclei, and rare to absent mitoses. Endometrial adenocarcinomas with mucinous differentiation or a microglandular pattern can closely mimic MGH, often resulting in a diagnostic dilemma in small biopsy specimens. Rare unusual morphologic features-mild to moderate nuclear atypia, solid or reticular growth pattern, hobnail and signet ring cells-have been previously reported in MGH. We present 9 cases of unusual, mitotically active-between 5 and 11 mitotic figures per 10 HPF-MGH, all of which presented as endocervical polyps and had morphologic features otherwise typical of MGH. The patients' age ranged between 35 and 56 yr, 2 patients were postmenopausal. High-risk human papillomavirus status was available in 7 patients, all of which were negative. The Ki-67 proliferation index ranged between 1% and 15%, and all cases were negative for p16, carcinoembryonic antigen, and vimentin immunostains. The clinical follow-up ranged from 3 to 76.2 mo, with a median of 40.7 mo, all patients were doing well without evidence of endocervical or endometrial malignancy. In summary, this case series documents the presence of rare cases of MGH demonstrating significant mitotic activity (up to 11/10 HPF) without a negative impact on the clinical prognosis. Mitotic activity alone should be interpreted with caution in small biopsy specimens with microglandular growth pattern. Immunohistochemical stains, especially p16, carcinoembryonic antigen, and vimentin, may be helpful-in addition to the patient's clinical history and human papillomavirus status to rule out endocervical or endometrial malignancy.

Human Papillomavirus among Veterans Treated for Oropharyngeal Squamous Cell Carcinoma

Objectives: The prevalence and significance of high-risk oral human papillomavirus (HPV) among veterans treated for oropharyngeal squamous cell carcinoma (OPSCC) remains unknown. In this study, we aimed to (1) define the prevalence of HPV among veterans with OPSCC and (2) compare outcomes of veterans with OPSCC relative to high-risk HPV status and other well-known prognostic factors. Methods: Patients from a high-volume Veterans Administra-tion (VA) medical center with OPSCC were identified from 2001-2006. Demographic, surgical, and pathological data were extracted from the electronic medical record. P16 immunohistochemistry testing was performed on archived pathological samples as a surrogate marker for HPV status. Outcomes were compared between HPV-positive (+) and negative (–) patients using Kaplan-Meier estimates. Results: Seventy-six veteran patients with available tumor tissue and follow-up data were included. All of the patients were male (100%) and the vast majority were ever smokers (89%). Most patients presented with advanced stage disease (61, 80%) and were treated nonsurgically (64, 84%). The overall prevalence of p16 positivity was 52 out of 76 (68%). Overall survival at 2 and 5 years was 60% and 38%, respectively, for the entire cohort. HPV+ veteran patients had significantly better survival at 2 years (58% vs 33%) and 5 years (40% vs 17%) than HPV– veteran patients ( P = .014). Conclusions: Using p16 as a surrogate marker, HPV is prevalent among veteran patients treated for OPSCC. Veteran patients with HPV-associated OPSCC have improved survival relative to HPV-negative patients. However, their prognosis remains poor compared to a nonveteran population.

Analysis of High-Risk HPV Status in Squamous Cell Carcinomas of the Head and Neck from June 2013-March 2014

Analysis of High-Risk HPV Status in Squamous Cell Carcinomas of the Head and Neck from June 2013-March 2014

Prognostic scores after surgical treatment for cervical intraepithelial neoplasia: a proposed model and possible implications for post-operative follow-up.

To develop a prognostic model for women who underwent surgical treatment for cervical intraepithelial neoplasia. Cohort study. Patient inclusion and follow-up occurred retrospectively and prospectively. Barretos Cancer Hospital, Barretos, São Paulo, Brazil. Women (n=242) diagnosed with cervical intraepithelial neoplasia who were submitted to conization. Immediately prior to surgical treatment, a cervical cytology sample was collected from each individual included in the study by endocervical brushing and stored in a preservative solution with methanol. A human papilloma virus-DNA test was conducted using an aliquot of the endocervical brushings. The surgical specimens were subjected to immunohistochemical analysis of p16 (immunohistochemical analysis 4a) protein expression. Two-year disease-free survival rates calculated for each study variable. Identified variables in the multivariate Cox model were used for elaboration of prognostic scores. Variables associated with outcome included age (p=0.033), tobacco use (p<0.001), final histopathological diagnosis (p=0.007), surgical margins (p<0.001), high-risk human papilloma virus status (p=0.008), human papilloma virus-16 status (p<0.001) and immunoexpression of p16 in the cytoplasm (p=0.049). By the Cox model, independent risk factors for disease recurrence/persistence were: tobacco use (hazard risk=3.0; 95% confidence interval 1.6-5.6), positive surgical margins (hazard risk=3.2; 95% confidence interval 1.6-6.1), human papilloma virus-16 (hazard risk=3.3; 95% confidence interval 1.6-6.9) and age over 45years (hazard risk=2.7; 95% confidence interval 1.1-6.6). Establishment of a prognostic score can represent a valuable tool for determining the risk of cervical intraepithelial neoplasia recurrence after conization. The use of clinical (age and tobacco use), pathological (surgical margins) and molecular (human papilloma virus-16 genotyping) factors can facilitate more appropriate patient follow up according to risk stratification.

Prevalence and genotyping of high risk human papillomavirus in cervical cancer samples from Punjab, Pakistan.

Cervical cancer is the third most common cause of cancer-related death in women worldwide. Infection with high-risk human papillomavirus (HPV) is established as the cause of cervical carcinoma, therefore, high risk HPV detection may have prognostic significance for the women who are at increased risk of disease progression. The paucity of data on the incidence of cervical cancer in Pakistan makes it difficult to determine disease burden. Even less information is available regarding the prevalent HPV strains in cervical specimens collected from this region. Cervical cancer is a neglected disease in Pakistan in terms of screening, prevention, and vaccination. Identification and accurate genotyping of the virus burden in cancer specimens is important to inform intervention policies for future management of HPV associated disease and to potentially stratify patients dependent on HPV status. In this study, detection and genotyping of HPV types 16 and 18 from 77 cervical specimens were carried out. Consensus primers GP5+/GP6+, which detect 44 genital HPV types, and type specific primers (TS16 and TS18) were used in conjunction with newly designed type specific primers. Using a combination of these methods of detection, a total of 94.81% (95% CI ±4.95) of cervical lesions were positive for HPV. Single infections of HPV16 were detected in 24.68% (95% CI ±9.63) of total samples and HPV18 was found in 25.97% (95% CI ±9.79) samples. Interestingly, a high proportion of samples (40.26%, 95% CI ±10.95) was positive for both HPV16 and 18, indicating a higher incidence of co-infection than previously reported for similar ethnic regions. The HPV genotype of 3.90% of HPV positive samples remained undetected, although these samples were positive with the GP5+/GP6+ primer set indicating infection with an HPV type other than 16 or 18. These data indicate that the overall incidence of high risk HPV infection in cervical cancer and intraepithelial neoplasia specimens in Punjab, Pakistan is in line with the worldwide prevalence, but that the incidence of HPV16 and 18 co-infections in our cohort is higher than that previously reported.

Postcolposcopy Management of Women With Histologically Proven CIN 1

This study aimed to determine during 36 months of follow-up the (1) clinical outcomes and (2) influence of high-risk human papillomavirus (HPV) status on the risk of progression to cervical intraepithelial neoplasia 2+ (CIN 2+), among women with histologically proven CIN 1. This is an ad hoc analysis of women with CIN 1 within TOMBOLA, a randomized trial of the management of women with low-grade cervical cytology. Women from the colposcopy arm with CIN 1 confirmed on punch biopsies and managed conservatively by cytology every 6 months in primary care were included. Sociodemographic data and a sample for HPV testing were collected at recruitment. Data on the sample women were extracted to calculate the cumulative incidence of CIN 2+ and the performance characteristics of the baseline HPV test. Detection of CIN 2 or worse (CIN 2+) during follow-up or at exit colposcopy was analyzed. A total of 171 women were included. Their median age was 29 years. Fifty-two percent were high-risk HPV positive, 17% were HPV-16 positive, and 11% were HPV-18 positive. Overall, 21 women (12%) developed CIN 2+, with a median time to detection of 25 months. Factors associated with progression to CIN 2+ were presence of HPV-18 (relative risk = 3.04; 95% CI = 1.09-8.44) and HPV-16 and/or HPV-18 at recruitment (relative risk = 3.98; 95% CI = 1.60-9.90). The sensitivity and specificity of a combined HPV-16/HPV-18 test for the detection of CIN 2+ during 3 years were 58% and 78%, respectively. Our results suggest that women with confirmed CIN 1 have low rates of progression to high-grade CIN within 3 years. Because the median time to progression was 25 months, conservative management could recommend the next repeat cytology at 2 years.

High-risk HPV and survival in patients with oral and oropharyngeal squamous cell carcinoma – 5-year follow up of a population-based study

Conclusion: No statistically significant 5-year survival difference was seen in patients with oral and oropharyngeal squamous cell carcinoma (OOPSCC) between high-risk HPV-positive and -negative groups in this population-based study. Objectives: To see if the formerly observed higher risk for recurrence or second primary tumour (SPT) in high-risk HPV-positive patients with OOPSCC corresponds to worse survival. Methods: A total of 128 consecutive, previously untreated patients with OOPSCC, who were part of a population-based case-control study in southern Sweden during 2000–2004, were included. A mouthwash sample was collected and exfoliated cells were collected with cotton-tipped swabs from the tonsillar fossa and the tumour. Specimens were analysed for HPV DNA using nested polymerase chain reaction (PCR). Disease-specific survival (DSS) and DSS difference between HPV-negative and HPV-positive patients were calculated. The relationship between age, stage, high-risk HPV status and DSS was assessed. Oral and oropharyngeal tumours were assessed separately. Results: Mean DSS in months was 80.7/68.6 (high-risk HPV-negative/high-risk HPV-positive) for oral cavity tumours (p = 0.18) and 67.6/78.3 (high-risk HPV-negative/high-risk HPV-positive) for oropharyngeal tumours (p = 0.47). For oral cavity tumours, age, T status, N status and stage all showed significant differences in DSS. For oropharyngeal tumours, no significant difference regarding DSS was found.

Oropharyngeal squamous cell carcinoma treatment: current standards and future directions.

To discuss the changing landscape and significant developments in the diagnosis and management of oropharyngeal squamous cell carcinoma. High-risk human papilloma viruses (HPVs) have been recognized as important causative factors for oropharyngeal cancer. The diagnosis is established with type-specific and broad-spectrum in-situ hybridization probes and/or p16 immunohistochemistry assays on fresh frozen paraffin-embedded tissue blocks. HPV-associated tumors have superior response and outcomes compared with HPV-unrelated tumors. Retrospective studies have been able to stratify oropharyngeal squamous cell carcinoma based on HPV status, tumor stage, nodal stage, and smoking history into risk groups with differing risks of death or distant disease. Selected patients, nonsmokers with less advanced nodal stage, may be overtreated with current treatment paradigms, and deintensification of curative therapy is a current research focus for these patients. Smokers, patients with advanced nodal or tumor stage, and those with HPV-unrelated cancers have a less favorable prognosis and the search for novel targets is particularly important for these patients. The present review will highlight the current standards and the future direction of novel therapies in both HPV-associated and HPV-unrelated cancers.

Basal Cell Carcinomas of the Vulva

Basal cell carcinoma (BCC) of the vulva is rare and may be confused with the much more commonly encountered high-risk human papillomavirus (HPV)-related basaloid squamous cell carcinoma (SCC). The HPV status of BCCs is not well established. This study assesses the utility of p16 and BerEP4 expression patterns and high-risk HPV detection for distinction of these tumors. Thirteen cases of vulvar BCC were analyzed by immunohistochemistry for p16 and BerEP4 expression. HPV status was assessed by in situ hybridization (ISH) with a high-risk HPV wide-spectrum probe and HPV 16 and 18 type-specific probes. All tumors (13/13) demonstrated patchy p16 positivity, with <50% of tumor cells expressing p16 in all cases. None demonstrated the diffuse p16 expression characteristic of high-risk HPV-associated lesions. No high-risk HPV was detected by ISH (0/13). Eleven of 13 (85%) vulvar BCCs showed diffuse, intense expression of BerEP4. The 2 BerEP4-negative cases were notably squamatized. The lack of diffuse p16 expression and failure to detect high-risk HPV by ISH in vulvar BCCs indicate that these tumors are unrelated to high-risk HPV. Thus, these ancillary techniques, particularly p16 immunohistochemistry, are useful for distinguishing vulvar BCCs from basaloid forms of high-risk HPV-related vulvar SCC. BerEP4 expression can help in distinction of these tumors except in cases of BCC with extensive squamatization. Distinction of vulvar BCC from basaloid SCC is important because of differences in extent of surgical treatment for these entities.

High-risk human papillomavirus and lymph node size in patients with single node metastasis of oral and oropharyngeal cancer

Conclusion: High-risk human papillomavirus (HPV) infection was significantly related to lymph node size in patients with single node metastasis of oral and oropharyngeal cancer. Objective: The purpose of this study was to examine the relationship between high-risk HPV infection and lymph node size in patients with single node metastasis of oral and oropharyngeal carcinoma. Methods: This study included 48 patients with oral and oropharyngeal carcinoma. Pathologic lymph node stages comprised 36 N1 and 12 N2a. Results: High-risk HPV in situ hybridization was positive in 29% of patients (14/48). Of those patients with high-risk HPV, there was a significant difference (p = 0.008) between oral (9.5%) and oropharyngeal (44.4%) cancers. Average lymph node diameter was 20.7 ± 12.6 mm (range 5–54 mm). We found a positive correlation between high-risk HPV status and lymph node size (p = 0.018). Mean lymph node diameter in high-risk HPV-positive cases was 27.3 ± 13.1 mm and 18.0 ± 11.5 mm in high-risk HPV-negative cases. Extracapsular spread (p = 0.030) and cystic nodal metastases (p = 0.019) were also significantly related to lymph node size. High-risk HPV negative status (p = 0.043), advanced tumor stage (p = 0.009), and extracapsular spread (p = 0.038) all had significant adverse effects on 5-year disease-specific survival.

Hybrid Capture 2 Assay Based Evaluation of High-Risk HPV Status in Healthy Women of North-East India

High risk HPV (HR-HPV) testing has been recommended as an effective tool along with cytology screening in identification of cervical intraepithelial lesions (CINs) and prevention of their progress towards invasive cervical cancer. The aim of this study was to assess the HR-HPV DNA status by Hybrid Capture 2 (HC2) assay in healthy asymptomatic women of North-East India. This study examined cervical cell samples of forty three (n=43) healthy women by HC2 assay. A High Risk HPV DNA kit (Qiagen) was used which can detect 13 high risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. The mean relative light units (RLU) for samples was in the range of 141-5, 94, 619. HR-HPV DNA was confirmed in 16% (7/43) of participant women samples. Among demographic and clinical parameters, menstrual irregularity (p=0.039) and infection history (p=0.028) has shown statistically significant differences between the HR-HPV-positive and negative groups. In the HR-HPV positive group, two women were confirmed for CINs after colposcopy and histopathologic examination. We suggest that there may be an association between irregular menstruation and infection history of the urogenital tract with HR-HPV DNA prevalence in North-East Indian asymptomatic women. HC2 assay can be a valuable tool for HR-HPV screening.

Utility of P16 expression and Ki‐67 proliferation index in ASCUS and ASC‐H pap tests

Current cervical screening uses a combination of cytology and high-risk human papillomavirus (HR-HPV) analysis in cases of atypical squamous cells of undetermined significance (ASCUS) and atypical squamous cells cannot exclude high-grade intraepithelial lesion (ASC-H). These diagnoses are subject to interobserver variability and HR-HPV analysis can be limited by sampling inadequacy. This study correlates immunoexpression of P16 and Ki-67 in residual cervicovaginal material against cytology category and HR-HPV status. Eighteen pap tests were selected: 8 ASCUS, 4 ASC-H, and 6 controls (2 LSIL and 4 HSIL). Digene Hybrid Capture II test was used to detect HR-HPV. The cytospins were stained for P16/Ki-67. Pap tests, P16, Ki-67, HR-HPV result and available biopsies were correlated. P16 expression correlated with HR-HPV status in 15/17 cases. Discordant cases (1 ASCUS and 1 ASC-H) were +P16/-HR-HPV. Ki-67 correlated with HR-HPV in 8/15 cases. Discordant cases were +HR-HPV/- Ki-67 (HSIL, LSIL, and ASC-H one each), and -HR-HPV/+Ki-67 (3 ASCUS, 1 LSIL, 1 ASC-H). Two cases were + P16/+ Ki-67/- HR-HPV. None were - P16/- Ki-67/+ HR-HPV. Histologic follow-up in 13 cases varied from benign to CIN III. Two cases of +P16/ - Ki-67/- HR-HPV had benign cervical biopcies. Although a small sample size, our findings show a utility for adjunct P16/ Ki-67 in addition to HR-HPV testing in cases of squamous atypia when HR-HPVs are non-detected due to low DNA copies, or missed lesions in cervical biopsies.

Transcriptionally-Active Human Papillomavirus is Consistently Retained in the Distant Metastases of Primary Oropharyngeal Carcinomas

High-risk human papillomavirus (HPV) is both causative and prognostic in the majority of oropharyngeal squamous cell carcinomas (OPSCCs). The aim of this study was to evaluate for transcriptionally-active HPV in matched primary OPSCCs and their distant metastases given the implications of HPV status for diagnosis and treatment. Twenty matched pairs of primary OPSCC and their distant metastases were retrieved from departmental files. Two study pathologists reviewed all cases to confirm the diagnoses and to evaluate histologic features. Real-time PCR (RT-PCR) for detection of E6/E7 mRNA for all major high-risk HPV types and p16 immunohistochemistry were performed. Distant metastases were to lung (70%), bone (20%), non-regional lymph nodes (5%) and pericardium (5%). Histologically, 15 primary tumors were nonkeratinizing, 3 nonkeratinizing with maturation, one basaloid, and one keratinizing. Seventeen (85%) of the metastases had the same histologic type as the primary tumor. All 20 matched pairs were concordant for HPV status by RT-PCR and for p16 expression with 19 of 20 cases positive for high risk HPV and one negative. HPV types were concordant in all cases. These findings show that the distant metastases from HPV-related primary OPSCCs uniformly retain transcriptionally-active HPV and p16 overexpression. They also retain similar morphology. This argues that HPV status can be utilized to differentiate metastatic OPSCC from separate, new, primary squamous cell carcinomas in other organs, and that therapies specifically targeting HPV or virus-related proteins in patients with distant metastases can be utilized.

Performance of the Roche cobas 4800 high‐risk human papillomavirus test in cytologic preparations of squamous cell carcinoma of the head and neck

Determining high-risk human papillomavirus (HR-HPV) status of head and neck squamous cell carcinoma (HNSCC) defines a tumor subset with important clinical implications. Cytologic sampling often provides the sentinel or sole diagnostic specimen. The authors assessed the performance characteristics for the Roche cobas 4800 HPV real-time polymerase chain reaction (PCR)-based system (cobas) on cytologic specimens of HNSCC compared with standard methods of in situ hybridization (ISH) for HR-HPV and immunohistochemistry (IHC) for p16 on formalin-fixed, paraffin-embedded (FFPE) tissue. Samples of HNSCC were collected by fine-needle aspiration and from surgical biopsies or resections, fixed, and processed with the cobas system. Available corresponding FFPE samples were synchronously evaluated for HR-HPV using ISH and IHC. Discrepant cases underwent additional PCR studies for adjudication. Thirty-six samples from 33 patients were analyzed. Forty-two percent (n = 15) of tumors were positive for HR-HPV according to cobas. Corresponding histology with ISH (n = 30) was concordant in 91% of samples. Compared with the adjudication PCR standard, there were 3 false-positive cases according to cobas. Ninety-two percent (n = 12) of cases were the HPV16 subtype. The overall sensitivity for the cobas system was 100%, and the specificity was 86%. Concordance in HNSCC HR-HPV status between cobas and ISH/IHC was > 90%, and cobas demonstrated a sensitivity of 100% and a specificity of 86%, broadening options for HR-HPV testing of fine-needle aspiration samples. Advantages for this system include subtyping of HR-HPV and the ability to discern HR-HPV status earlier in a patient's treatment course.