Human Papillomavirus In Situ Hybridization Research Articles

P16 and HPV status in head and neck sarcomas and sarcomatoid carcinomas

Human papillomavirus (HPV)-positive oropharyngeal carcinoma is a distinct type of head and neck carcinoma with improved prognosis. p16 immunostaining is often used as a surrogate marker for HPV infection in this particular setting. The aim of this study is to estimate the prevalence of p16 staining and HPV infection in head and neck sarcomatoid carcinomas as well as head and neck sarcomas. 21 sarcomatoid carcinomas and 28 head and neck sarcomas were tested for p16 positivity using immunohistochemical staining, and for high-risk HPV infection using In situ hybridization (ISH). 24 % of sarcomatoid carcinomas and 21 % of sarcomas were positive for p16 staining. All 49 cases were negative for HPV ISH. The results confirm that p16 staining is not specific and may not be associated with HPV infection in non-oropharyngeal head and neck sites. They also indicate that non-oropharyngeal head and neck sarcomatoid carcinomas are not likely to be HPV related.

Incidence and Clinicopathologic Characteristics of Human Papillomavirus-independent Invasive Squamous Cell Carcinomas of the Cervix: A Morphologic, Immunohistochemical, and Human Papilloma-Virologic Study of 670 Cases.

We aimed to determine the frequency of human papillomavirus-independent (HPVI) cervical squamous cell carcinoma (SCC) and to describe clinicopathologic characteristics. Among 670 patients with surgically treated SCCs in an established multi-institutional cohort, 447 had available tissue. Tissue microarrays were constructed and studied by in situ hybridization (ISH) for high-risk and low-risk human papillomavirus (HPV) mRNA and immunohistochemistry for p16 and p53. Tumors were HPVI if negative by HPV ISH and they failed to show diffuse p16 positivity by immunohistochemistry, and human papillomavirus-associated (HPVA) if positive by HPV ISH. Ten HPVI SCCs and 435 HPVA SCCs were identified; 2 cases were equivocal and excluded from analysis. The overall rate of HPVI SCC was low (2%) but was higher among older patients (7% in patients above 60y of age and 17% in patients above 70y of age). Compared with HPVA, patients with HPVI SCC were significantly older (median age, 72 vs. 49, P <0.001) and diagnosed at a higher stage (40% vs. 18% with stage III/IV disease, P =0.055). p53 expression was varied; 2 cases (20%) had null expression and 8 (80%) had wild-type expression. HPVI SCCs were heterogenous, with keratinizing, nonkeratinizing, and warty morphologies observed. Several cases had a precursor lesion reminiscent of differentiated vulvar intraepithelial neoplasia, with prominent basal atypia and hypereosinophilia or a basaloid-like morphology. Two patients (20%) had distant recurrences within 12 months, and 3 (30%) died of disease during follow-up. HPVI SCCs are rare tumors that are more common among older patients with higher stage disease and have important clinical and histologic differences from HPVA SCCs.

Classification of Vulvar Squamous Cell Carcinoma and Precursor Lesions by p16 and p53 Immunohistochemistry: Considerations, Caveats, and an Algorithmic Approach

There is emerging evidence that vulvar squamous cell carcinoma (VSCC) can be prognostically subclassified into 3 groups based on human papillomavirus (HPV) and p53 status: HPV-associated (HPV+), HPV-independent/p53 wild-type (HPV−/p53wt), or HPV-independent/p53 abnormal (HPV−/p53abn). Our goal was to assess the feasibility of separating VSCC and its precursors into these 3 groups using p16 and p53 immunohistochemistry (IHC). A tissue microarray containing 225 VSCC, 43 usual vulvar intraepithelial neoplasia (uVIN/HSIL), 10 verruciform acanthotic vulvar intraepithelial neoplasia (vaVIN), and 34 differentiated VIN (dVIN), was stained for p16 and p53. Noncomplementary p16 and p53 patterns were resolved by repeating p53 IHC and HPV RNA in situ hybridization (ISH) on whole sections, and sequencing for TP53. Of 82 p16-positive VSCC, 73 (89%) had complementary p16 and p53 patterns and were classified into the HPV+ group, 4 (4.9%) had wild-type p53 staining, positive HPV ISH and were classified into the HPV+ group, whereas 5 (6.1%) had p53 abnormal IHC patterns (1 null, 4 overexpression), negativity for HPV ISH, and harbored TP53 mutations (1 splice site, 4 missense); they were classified as HPV−/p53abn. Of 143 p16-negative VSCC, 142 (99.3%) had complementary p53 and p16 patterns: 115 (80.4%) HPV−/p53abn and 27 (18.9%) HPV−/p53wt. One had a basal-sparing p53 pattern, positivity for HPV ISH and was negative for TP53 mutations—HPV+ category. The use of IHC also led to revised diagnoses—HSIL to dVIN (3/43), dVIN to vaVIN (8/34), and dVIN to HSIL (3/34). Overall, 215/225 VSCC (95.6%) could be easily classifiable into 3 groups with p16 and p53 IHC. We identified several caveats, with the major caveat being that “double-positive” p16/p53 should be classified as HPV−/p53abn. We propose an algorithm that will facilitate the application of p16 and p53 IHC to classify VSCC in pathology practice.

Prognosis of P16 and HPV Discordant Oropharyngeal Cancers: Natural Language Processing to Extract Data from Free-Text Pathology Reports

<h3>Purpose/Objective(s)</h3> P16 immunohistochemistry (IHC) is not as specific for detecting human papillomavirus (HPV) related oropharyngeal cancers (OPC) as in situ hybridization (ISH). However, given its high sensitivity and wide availability, P16 IHC is a standard of care requirement for OPC staging and is also used for clinical trial eligibility. As both P16 and HPV testing are not required for routine clinical management, there are varying reports on the prognosis of P16 and HPV discordant tumors. We aimed to assess the prognosis of P16 and HPV discordant OPC tumors, using natural language processing (NLP) techniques to extract results from free-text OPC pathology reports. <h3>Materials/Methods</h3> We reviewed our institutional OPC radiation database and included a series of patients from 7/1994-6/2020 with digitized pathology reports. Patients were excluded if they did not receive curative intent radiation, did not have both P16 IHC and HPV ISH, or if either test was equivocal. 422 patients were included with a median follow-up of 87.8 months (0.3-241.0). We performed text extraction and classification to ascertain P16 and HPV results from free-text pathology reports. All reports were manually evaluated by two independent reviewers as our gold standard. Four groups were identified: P16-negative/HPV-negative (P16-/HPV-, n=60), P16-/HPV-positive (P16-/HPV+, n=9), P16-positive/HPV- (P16+/HPV-, n=49), and P16+/HPV+ (P16+/HPV+, n=304). The Kaplan-Meier method was used to estimate cancer-specific survival (CSS) and overall survival (OS). <h3>Results</h3> 39% of reports could not be processed by our NLP techniques. The algorithm accurately coded 92.6% of P16 and 94.2% of HPV results. Positive predictive value/precision, sensitivity/recall, and F-score for P16/HPV were: 99.5%/99.0%, 91.7%/94.0%, and 95.5%/96.5%. 13.7% of tumors were discordant (P16-/HPV+ or P16+/HPV-). As expected, P16-/HPV- tumors had the worst CSS and OS, and P16+/HPV+ had excellent prognosis (Table 1). P16-/HPV+ constituted only 2.1% of the cohort but outcomes performed favorably with P16+/HPV+. P16+/HPV- tumors had favorable outcomes in the first two years, but with longer follow-up clustered between P16-/HPV- and P16+/HPV+ tumors. <h3>Conclusion</h3> P16 and HPV results were accurately abstracted from the majority of free-text OPC pathology reports. We provide the first example to our knowledge of employing NLP techniques to extract data from head and neck cancer pathology reports and correlate results with clinical outcomes. P16 and HPV discordant tumors constitute a minority of patients, but their divergent prognoses suggest that performing both tests may be of utility—especially for clinical trial eligibility or treatment de-escalation.

P53 and p16ink4a As Predictive and Prognostic Biomarkers for Nodal metastasis and Survival in A Contemporary Cohort of Penile Squamous Cell Carcinoma.

Human papilloma virus (HPV) infection is implicated in a proportion of invasive squamous cell carcinoma of the penis (PC). A subset of PC involves dysregulation of the p53 pathway. HPV in situ hybridization (ISH) and p16ink4a positivity are surrogate markers for HPV infection, and p53 immunohistochemistry (IHC) denotes abnormality in the p53 pathway. There remains an ambiguity with regard to the contribution of both the pathways in the prognosis of PC. We sought to analyze the clinicopathologic characteristics of a cohort of Indian PC patients with respect to p16 ink4a and p53 expression. A cohort of 123 PC patients was studied for p16ink4aand p53IHC and HPVISH. The results of these biomarkers were correlated with various clinicopathologic parameters. p16ink4a and HPV ISH were positive in 47% and 53% of the tumors, respectively. The proportion of warty, basaloid, or mixed warty-basaloid tumor subtypes showed significant p16ink4apositivity (P< .0001) compared to other subtypes. Twenty-eight patients were dual negative (p53- /p16ink4a-), 32 were dual positive (p53+/p16ink4a+), 38 were p53+/p16ink4a-, and 25 were p53-/p16ink4a +. In patients where p16ink4a was negative, a p53-positive phenotype had a higher propensity for lymph node metastases (OR, 5.42; 95% CI, 1.75-16.80; P=.003). Similarly, p53 positivity dictates nodal involvement in the p16ink4a-positive subset of tumors (OR, 5.00; 95% CI, 1.23-20.17; P=.024). On multivariate analyses, pathologic subtypes (warty, warty-basaloid, and basaloid) (P < .0001), p16ink4aexpression (P < .0001), and absence of nodal metastasis (P < .0001) were significant predictors of improved overall (OS) and cancer specific survival (CSS). In Kaplan-Meier analysis, the OS was significantly longer in patients with p16ink4a+tumors (P < .0001), as was the CSS (P < .0001). Patients with dual positive tumors had a significantly higher OS (P < .001) and CSS (P=.012), in the entire cohort. In the node positive patients, dual positivity was associated with significantly higher OS (P < .0001); however, the median CSS for p53+/p16ink4a+tumors were not significantly different compared to p53- /p16ink4a- tumors (P=.064), although there was a trend towards improved CSS. There is a strong concordance between p16ink4aIHC and HPV ISH results. p16ink4a status is an independent predictor of survival (OS and CSS) in our cohort of PCs. p53 is a predictor of nodal metastasis irrespective of p16 status. Dual positive tumors have a significantly better outcome in comparison to dual negative tumors.

4MO p53 and p16ink4a as predictive and prognostic biomarkers for nodal metastasis and survival in a contemporary cohort of penile squamous cell carcinoma

Human papilloma virus (HPV) infection is implicated in a proportion of invasive squamous cell carcinoma of the penis (PC). A subset of PC involves dysregulation of the p53 pathway. HPV in situ hybridization (ISH) and p16 ink4a positivity are surrogate markers for HPV infection, and p53 immunohistochemistry (IHC) denotes abnormality in the p53 pathway. Clinicopathologic characteristics of a cohort of Indian PC patients with respect to p16 and p53 expression were analyzed. 123 PC patients were studied for p16 and p53 IHC and HPV ISH and the results were correlated with various clinicopathologic parameters. p16 and HPV ISH were positive in 47% and 53% of the tumors, respectively. The proportion of warty, basaloid, or mixed warty-basaloid tumor subtypes showed significant p16 positivity (p < 0.0001) compared to other subtypes. In patients where p16 was negative, a p53-positive phenotype had a higher propensity for lymph node metastases (OR, 5.4; 95% CI, 1.7 to16.8; p=0.003). Similarly, p53 positivity dictates nodal involvement in the p16-positive subset of tumors (OR, 5; 95% CI, 1.2 to 20.1; p=0.024). On multivariate analyses, pathologic subtypes (warty, warty-basaloid, and basaloid) (p<0.0001), p16 expression (p<0.0001), and absence of nodal metastasis (p<0.0001) were significant predictors of improved overall (OS) and cancer specific survival (CSS). The OS was significantly longer in patients with p16+ tumors (p<0.0001), as was the CSS (p<0.0001). Patients with dual positive tumors had a significantly higher OS (p<0.001) and CSS (p=0.01), in the entire cohort. In the node positive patients, dual positivity was associated with significantly higher OS (p<0.0001); however, the median CSS for p53+/p16+tumors was not significantly different compared to p53- /p16- tumors (p=0.064), although there was a trend towards improved CSS. There is a strong concordance between p16 IHC and HPV ISH results. p16 status is an independent predictor of survival (OS and CSS) in our cohort of PCs. p53 is a predictor of nodal metastasis irrespective of p16 status. Dual positive tumors have a significantly better outcome in comparison to dual negative tumors.

P53 Immunohistochemical patterns in HPV-related neoplasms of the female lower genital tract can be mistaken for TP53 null or missense mutational patterns.

We have recently encountered p53 immunohistochemical (IHC) patterns in human papillomavirus (HPV)-associated carcinomas of the gynecologic tract, which were confused with absent (null) or overexpression TP53 mutational staining. We therefore evaluated p53 and p16 IHC in 25 squamous cell carcinomas (SCC) (16 vulva, 4 Bartholin's gland, and 5 cervix), 20 endocervical adenocarcinomas (EDAC), 14 high-grade squamous intraepithelial lesions (HSIL), 2 adenocarcinoma in situ (AIS), all of which exhibited morphologic features of HPV. Only cases showing diffuse/strong block-like p16 staining were included for further study. All EDACs underwent TP53 sequencing and HPV in situ hybridization (ISH) was performed in selected cases. p53 IHC staining fell into two main patterns. The most common was designated as "markedly reduced (null-like)" (absence or significantly attenuated staining in >70% of cells), which could be confused with true null mutational pattern. This was present in 14/25 (56%) SCCs, 7/14 (50%) HSILs, and 18/20 (90%) EDACs. The second notable pattern was "mid-epithelial (basal sparing)" (distinct absence of staining in basal cells juxtaposed with strong staining in parabasal cells), seen in 10/25 (40%) SCC, 7/14 (50%) HSIL, and none of the EDACs. There was scattered weak to moderate p53 staining (conventional wild type) in 1/25 (4%) SCC and 2/20 (10%) EDAC. No cases showed strong/diffuse overexpression. One EDAC had a TP53 missense mutation and exhibited "markedly reduced (null-like)" staining. HPV ISH revealed an inverse relationship with p53, cells positive for HPV mRNA were negative for p53. Knowledge of these patterns can help pathologists avoid misinterpreting p53 status in the setting of HPVA cancers.

Low prevalence of p16-positive HPV-related head-neck cancers in Thailand: tertiary referral center experience

BackgroundThere has been a sharp rise in the incidence of human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) in many countries. Patients with HPV-positive OPSCC have a more favorable prognosis compared with HPV-negative OPSCC, leading to investigation and adoption of de-escalation treatment protocols. The baseline rate of HPV prevalence in certain populations is of epidemiologic significance. We aimed to evaluate the rate of high-risk HPV in a large cohort of Thai patients, including OPSCC, oral SCC (OSCC) and laryngeal SCC (LSCC).MethodsIn total, 504 patients with HN cancer (110 OPSCC, 260 OSCC and 134 LSCC) who had been treated in Chulalongkorn University between 2010 and 2016 formed the sample set. All histological slides were reviewed to validate the diagnosis and render the histological type as keratinizing (K), non-keratinizing (NK) or non-keratinizing with maturation (NK-M). Immunohistochemistry with p16 was performed in all cases and scored semiquantatively. Positive and equivocal cases were tested by the high-risk HPV DNA in situ hybridization (ISH). Validation with quantitative polymerase-chain reaction (qPCR) was performed in p16-positive OPSCC.ResultsThe OPSCC were represented by NK (7.3%), NK-M (16.4%) and K (76.4%) types, with an HPV incidence of 100, 22.2 and 4.7%, respectively. The average HPV prevalence in OPSCC was 14.5%. The concordance with p16/ISH was 51.6%, while concordance of the NK morphology with positive HPV ISH was 100%. ISH-qPCR concordance in p16-positive OPSCC was 72.7%. Patients with HPV-positive OPSCC had significantly more tumors with a NK histologic type, tonsillar location, earlier clinical stage, less association with smoking, and, finally, better outcome and longer survival time. In non-OPSCC, p16-positive HPV-associated cancers were found in only 1.5% of OSCC (4/260) and LSCC (2/134).ConclusionA low rate of HPV-related OPSCC was found in Thai patients. The NK morphology was an excellent predictor of high-risk HPV infection in OPSCC. For OPSCC patients, HPV-positive ones had a significantly longer survival time than HPV-negative ones. There was a lack of p16-positive HPV-related OSCC and LSCC. Morphology and p16 status had a poor predictive value for detecting HPV in OSCC and LSCC.

Morphology, p16, HPV, and outcomes in squamous cell carcinoma of the penis: a multi-institutional study

Our objective was to evaluate the pathologic features and clinical outcomes in cases of invasive penile squamous cell carcinoma (SCC) and the association with p16 immunohistochemistry (IHC) and human papilloma virus (HPV) in situ hybridization (ISH). A retrospective multi-institutional database search was conducted for invasive SCC of the penis diagnosed between 2007 and 2018 that had undergone surgical resection. Pathologic features, p16 IHC, and HPV ISH were investigated with clinical outcomes. A total of 102 patients were included in the study. The average age was 63 ± 13.3 years. Based on histology, 46% of tumors displayed an HPV-related subtype, whereas p16 was positive in 52% of all cases. Tumor histology correlated well with p16 positivity (P < .001), and p16 IHC accurately predicted the presence of HPV in 25/26 (96%) cases. On multivariate analysis, perineural invasion was associated with local disease recurrence (P = .02), whereas lymphovascular invasion was associated with progression to metastatic disease (P = .002) and increased overall mortality (P = .02). Urethral involvement was also associated with increased overall mortality (P = .02). In addition, HPV-related tumors based on histologic features correlated with lower rates of metastatic disease (P = .007). HPV is a common cause of penile SCC and can be diagnosed by tumor histology and confirmed by overexpression of p16 on IHC. The presence of lymphovascular invasion, perineural invasion, and urethral involvement are poor prognostic indicators, whereas HPV-related tumors based on histology may have lower risk for metastatic disease.

Cytopathologic characteristics of HPV-related small cell carcinoma of the oropharynx.

Human papillomavirus (HPV)-related squamous cell carcinoma (SqCC) of the oropharynx is an epidemiologically and clinically distinct form of SqCC that is associated with an improved prognosis. However, HPV-related small cell carcinoma of the oropharynx is a rare and newly described variant that is associated with aggressive clinical behavior and poor outcomes. To date, fewer than 2 dozen reports of this entity exist in the literature, and there is no discussion of cytopathologic features. This article reports 6 cases and discusses the salient cytomorphologic findings, ancillary studies, and challenges when this entity is encountered. Anatomic pathology archives were searched to identify patients with a diagnosis of HPV-related small cell carcinoma of the oropharynx. Medical records were reviewed to document the following: age, sex, smoking status, other relevant clinical history, primary location, treatment, and clinical outcome. Both p16 and high-risk HPV in situ hybridization (ISH) studies were positive in at least 1 specimen from each patient. The pathologic diagnoses, cytomorphologic characteristics, immunocytochemical stains, and HPV ISH studies were reviewed and recorded for all available cases. Six patients with 11 cytopathology specimens of HPV-related small cell carcinoma of the oropharynx were identified. The mean age was 61.3 years, and all patients died with widely metastatic disease (mean, 23 months; range, 12-48 months). Mixed small cell carcinoma and SqCC components were present in half of the cases. The identification of a small cell component can be reliably performed with cytology preparations and is crucial because this (and not the HPV status) determines the prognosis.

Normal ve tümöral orofaringeal dokuda in situ hibridizasyon ve p16 ekspresyonu ile human papilloma virüsü varlığının değerlendirilmesi ve klinikopatolojik önemi

Objective: The rise in the number of cancer cases with human papilloma virus (HPV)-positive squamous carcinoma of the oropharynx makes the detection of HPV clinically important. We aimed to investigate the HPV positivity in our patients who have oropharyngeal cancer and compare the two different HPV detection methods, which are HPV in situ hybridization (ISH) and p16 immunohistochemistry (IHC), and show the staining patterns. Methods: Twenty-three specimens of oropharyngeal cancer patients and ten tonsillectomy specimens that revealed no cancerous tissue (control group) were collected from retrospective file analysis. All specimens were evaluated by both p16 IHC and HPV ISH on paraffin blocks. Results: Seven of 23 cases showed p16 expression. Of all these 7 cases that showed p16 expression, six showed high p16 expression and one showed low p16 expression. All six cases that showed high p16 expression were HPV ISH (+). One case that showed low expression of p16 was HPV ISH (-). All cases that were p16 (+) showed diffuse p16 expression and none of the cases showed focal p16 expression. Conclusion: High p16 expression (>70%) is a reliable marker of HPV positivity. Combining p16 IHC with HPV ISH will further improve its specificity. All p16 positive cases showed diffuse p16 expression, thus did not show tumor heterogeneity, suggesting that even a biopsy specimen showing diffuse p16 expression shows p16 positivity of the whole tumoral tissue.

RNA chromogenic in situ hybridization assay with clinical automated platform is a sensitive method in detecting high-risk human papillomavirus in squamous cell carcinoma

Detection of active human papillomavirus (HPV) is clinically important because its presence has been shown to correlate with favorable clinical outcomes and better response to treatment in oropharyngeal squamous cell carcinomas. Using a clinical automated platform, we compared the performance of commercially available HPV DNA and RNA in situ hybridization (ISH) probes in archival tissues of 57 squamous cell carcinomas. Importantly, a clinical automated platform gives (1) consistent and reproducible results for HPV ISH and (2) better standardization across clinical laboratories. Compared with polymerase chain reaction results, RNA ISH exhibited 93% concordance versus 81% of DNA ISH. RNA ISH was more sensitive than DNA ISH (100% versus 88%) and more specific (87% versus 74%). When only accounting for 2+-3+ positivity, sensitivity was 92% for RNA ISH versus 73% for DNA ISH, highlighting the ease of interpretation. p16 exhibited 96% sensitivity, whereas specificity was only 55%. In 3 cases, both RNA and DNA ISH were positive, whereas polymerase chain reaction results were negative, suggesting that ISH methods might be a more sensitive method. Performing on a clinical automated platform, RNA ISH is sensitive in determining high-risk HPV status in formalin-fixed, paraffin-embedded tissues and has the potential of being a standalone clinical test.

Expression of cell cycle-related proteins in oropharyngeal squamous cell carcinoma based on human papilloma virus status.

The molecular association between human papilloma virus (HPV) and cell cycle-related (CCR) proteins is not fully understood in oropharyngeal squamous cell carcinoma (OPSCC). Herein, we examined the expression levels of CCRs in OPSCCs based on HPV status. Insitu hybridization (ISH)- and polymerase chain reaction (PCR)-based assays were used to detect HPV status in 98OPSCCs, and CCRs were detected by immunostaining. Of 98tumors, 47had HPV-positive tumors by either ISH- or PCR-based assays. Concordance analysis between ISH- and PCR-based tests showed a good agreement in OPSCC. Expression of p16 was moderate, consistent with HPV positivity as determined by ISH-based analysis. High p53-, phospho (p)-Rb-, SKP2-, cyclinD1-, and p-c-myc-positive rates were correlated with HPV-negative tumors, whereas high p16- and p27-positive rates were associated with HPV-positive tumors. The positive rates for p21 and cyclinA did not differ between HPV-positive and HPV-negative tumors. Finally, Ki-67 positivity was commonly observed for both types of OPSCCs. Although expression of p16 is thought to be a marker of HPV infection, ISH- or PCR-based tests should be used for HPV detection. In addition, our results regarding CCRs may be helpful for understanding the carcinogenesis of OPSCC.

Cell Block Preparation versus Liquid-Based Thin-Layer Cervical Cytology: A Comparative Study Evaluating Human Papillomavirus Testing by Hybrid Capture-2/Cervista, in situ Hybridization and p16 Immunohistochemistry

Objective: Cell block (CB) preparations from residual liquid-based Pap samples have been shown to be of diagnostic value. In this study we evaluated human papillomavirus (HPV) in situ hybridization (ISH) and p16 immunohistochemistry (IHC) on CB preparations and compared the results with the primary diagnosis and standard HPV tests. Design: In total, 197 HE-stained CB slides prepared from CBs from residual Pap samples (152 ASCUS, 2 ASC-H, 32 LGSIL, 4 HGSIL, 1 AGUS and 6 normal) were analyzed. Hybrid Capture-2 (HC-2)/Cervista testing and HPV ISH and p16 IHC were performed on the CB samples. The test performance characteristics were compared with HPV and p16 assay performances. Results: The cellular architecture was well maintained in CBs with excellent consistency. HPV ISH testing had an excellent concordance with the HC-2/Cervista methods (85%) with high sensitivity (82.6%; 95% CI 75.9, 89.4) and specificity (89.3%). Of all the p16 tests, 38% were positive (60 out of 159 samples). The overall concordance between p16 and HC-2/Cervista (64%), or between p16 and ISH (68%), was lower than the concordance between ISH and HC-2/Cervista (85%). Conclusions: HPV ISH and p16 IHC testing is feasible, cost effective and practical. A combination of the two tests would ultimately improve diagnostic accuracy, leading to better therapeutic decisions.

Clinical Significance of p53 and p16ink4a Status in a Contemporary North American Penile Carcinoma Cohort

BackgroundBecause of the low incidence of penile carcinoma (PC), the value of p16ink4a, p53, and human papilloma virus (HPV) infection status in clinical practice remains unclear. Herein, we report our experience with potential clinical utility of these markers in men with PC treated at our institution. Patients and MethodsTissue microarrays of 57 cases of invasive penile squamous cell carcinomas were immunohistochemically stained for p16 and p53. HPV in situ hybridization (ISH) for high-risk subtypes was also performed. Association between marker status, nodal disease, overall (OS) and cancer-specific survival (CSS) were assessed. Resultsp16 and HPV ISH were positive in 23 (40%) and 24 (42%) of the cohort, respectively. The proportion of warty, basaloid, or mixed warty basaloid tumor subtypes were significantly greater in the p16-positive patients (48% vs. 3%; P < .01). p53 expression was negative in 31 (54%) cases. Only in p16-negative patients, positive p53 status was associated with pN+ disease (odds ratio, 4.4 [95% confidence interval (CI), 1.04-18.6]). In Kaplan–Meier analysis, the unadjusted estimated OS was insignificantly longer in p16-positive patients (median OS, 75 vs. 27 months; P = .27) and median CSS was not reached (P = .16). In a multivariable Cox proportional hazard model, when controlling for pathological nodal status and adjuvant chemotherapy, p16 status was a significant predictor for improved CSS (hazard ratio, 0.36 [95% CI, 0.13-0.99]). The worst CSS was seen in pN+ patients with double negative p16 and p53 expression (8 vs. 34 months; P = .01). ConclusionIn this current cohort, p53 and p16 status showed clinical utility in predicting nodal disease as well as survival.

β-Catenin Expression in Oropharyngeal Squamous Cell Carcinomas: Comparison and Correlation with p16 and Human Papillomavirus in situ Hybridization

Background: The Wnt/β-catenin signaling pathway has been noted to be upregulated in head and neck cancers, including oropharyngeal squamous cell carcinoma (OSCC). This study compared the efficacy of β-catenin immunohistochemistry (IHC), p16 IHC and automated human papillomavirus (HPV) in situ hybridization (ISH) in OSCC. Methods: Sixty-eight OSCCs (48 surgical specimens and 20 fine-needle aspirations) were evaluated. Nuclear staining only of β-catenin was assessed as 0-3+ intensity (relative to controls of benign squamous mucosa). p16 was interpreted as positive if 70% of tumor cells showed brown nuclear and cytoplasmic staining. HPV ISH was interpreted as positive if a minimum of one tumor cell showed brown punctate dot-like nuclear positivity. p16 IHC and HPV ISH were then correlated with β-catenin staining. HPV ISH was used as the gold standard. Results: Twenty-five of 48 surgical specimens (52.1%) and 11 of 20 cell blocks (55%) stained positively for β-catenin, making a total of 36 of 68 (52.9%) staining positively for β-catenin, as compared to 61.7% positive for p16 IHC and 70.6% positive by automated HPV ISH, the gold standard method for OSCC diagnosis. χ² analysis revealed no significant correlation between β-catenin and HPV ISH (p > 0.05) and demonstrated a strong correlation between p16 and HPV ISH (p < 0.05). Conclusion: β-Catenin IHC is not a sensitive or specific marker of HPV and is unlikely to be a useful adjunct to p16 IHC or HPV ISH in the setting of advanced OSCC. However, as this study focused on samples of advanced OSCC, β-catenin IHC may still find some use in the diagnosis of early-stage OSCC.

Genomic alterations in head and neck squamous cell carcinoma determined by cancer gene-targeted sequencing

To determine genomic alterations in head and neck squamous cell carcinoma (HNSCC) using formalin-fixed, paraffin-embedded (FFPE) tumors obtained through routine clinical practice, selected cancer-related genes were evaluated and compared with alterations seen in frozen tumors obtained through research studies. DNA samples obtained from 252 FFPE HNSCC were analyzed using next-generation sequencing-based (NGS) clinical assay to determine sequence and copy number variations in 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. Human papillomavirus (HPV) status was determined by presence of the HPV DNA sequence in all samples and corroborated with high-risk HPV in situ hybridization (ISH) and p16 immunohistochemical (IHC) staining in a subset of tumors. Sequencing data from 399 frozen tumors in The Cancer Genome Atlas and University of Chicago public datasets were analyzed for comparison. Among 252 FFPE HNSCC, 84 (33%) were HPV positive and 168 (67%) were HPV negative by sequencing. A subset of 40 tumors with HPV ISH and p16 IHC results showed complete concordance with NGS-derived HPV status. The most common genes with genomic alterations were PIK3CA and PTEN in HPV-positive tumors and TP53 and CDKN2A/B in HPV-negative tumors. In the pathway analysis, the PI3K pathway in HPV-positive tumors and DNA repair-p53 and cell cycle pathways in HPV-negative tumors were frequently altered. The HPV-positive oropharynx and HPV-positive nasal cavity/paranasal sinus carcinoma shared similar mutational profiles. The genomic profile of FFPE HNSCC tumors obtained through routine clinical practice is comparable with frozen tumors studied in research setting, demonstrating the feasibility of comprehensive genomic profiling in a clinical setting. However, the clinical significance of these genomic alterations requires further investigation through application of these genomic profiles as integral biomarkers in clinical trials.

Prognostic significance of human papillomavirus in recurrent or metastatic head and neck cancer: an analysis of Eastern Cooperative Oncology Group trials.

The purpose of this article was to study the association of human papillomavirus (HPV) with clinical outcomes in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN). Archival baseline tumor specimens were obtained from patients treated on two clinical trials in recurrent or metastatic SCCHN: E1395, a phase III trial of cisplatin and paclitaxel versus cisplatin and 5-fluorouracil, and E3301, a phase II trial of irinotecan and docetaxel. HPV DNA was detected by in situ hybridization (ISH) with a wide-spectrum probe. p16 status was evaluated by immunohistochemistry. Clinical outcomes of interest were objective response, progression-free survival (PFS) and overall survival (OS). We analyzed 64 patients for HPV ISH and 65 for p16. Eleven tumors (17%) were HPV+, 12 (18%) were p16+, whereas 52 (80%) were both HPV- and p16-. The objective response rate was 55% for HPV-positive versus 19% for HPV-negative (P = 0.022), and 50% for p16-positive versus 19% for p16-negative (P = 0.057). The median survival was 12.9 versus 6.7 months for HPV-positive versus HPV-negative patients (P = 0.014), and 11.9 versus 6.7 months for p16-positive versus p16-negative patients (P = 0.027). After adjusting for other covariates, hazard ratio for OS was 2.69 (P = 0.048) and 2.17 (P = 0.10), favoring HPV-positive and p16-positive patients, respectively. The other unfavorable risk factor for OS was loss of ≥5% weight in previous 6 months (P = 0.0021 and 0.023 for HPV and p16 models, respectively). HPV is a favorable prognostic factor in recurrent or metastatic SCCHN that should be considered in the design of clinical trials in this setting. NCT01487733 Clinicaltrials.gov.

Sparing Bilateral IB in Node-Positive Oropharyngeal Carcinoma Improves Xerostomia Outcomes: Definitive Management of Head-and-Neck Squamous Cell Carcinoma

Purpose/Objective(s): Human papilloma virus (HPV) is considered to play a major role in the pathogenesis of oropharyngeal squamous cell carcinoma (OPSCC). The vast majority of patients with HPV-related OPSCC exhibit p16 overexpression; in fact, some investigators have suggested that the p16 immunohistochemistry (IHC) is a better predictor of disease specific and overall survival than the presence of HPV DNA since p16+/HPVcases demonstrate clinical behavior that resembles p16+/ HPV+ cases. Among the several laboratory testing platforms available to determine the presence of HPV in formalin-fixed paraffin-embedded (FFPE) tissue, most clinical laboratories employ HPV in situ hybridization (ISH), p16 IHC, or a combination thereof. Although elucidating viral transcriptional activity by detecting HPV E6/E7 mRNA provides greater insight into molecular events than identifying HPV DNA alone, its clinical utility has not been adequately evaluated using surgical material. Materials/Methods: Retrospective clinical and histopathologic review of 75 cases of OPSCC that underwent surgical resection at Mayo Clinic Rochester in 2009 was performed. Clinicopathologic data including patient age and sex, tumor site and size, treatment (surgery, radiation therapy and/ or chemotherapy), and clinical outcome parameters were abstracted from the medical records. Surgical pathology archival material was re-examined histopathologically using HE sixty-three cases (84%) were positive for HPV E6/E7 mRNA; and fifty-one cases (68%) were positive for HPV DNA. Concordance between HPV RNA and p16, HPV DNA and p16, and HPV RNA and DNAwas 92% (kappaZ 0.74), 89% (kappaZ 0.73), and 81% (kappa Z 0.51), respectively. The sensitivity of HPV E6/E7 mRNA ISH to detect p16-positive cases was 98% (58/59), whereas that of HPV DNA ISH was 86% (51/59). Conclusions: Consideration should be given to detecting HPV E6/E7 mRNA using ISH and p16 overexpression using IHC in the setting of OPSCC to determine appropriate surveillance and treatment algorithms. Importantly, utilization of HPV E6/E7 mRNA ISH provides insight into transcriptional activity and reduces discordance between HPV ISH and p16 IHC results. Author Disclosure: J.J. Garcia: None. M.G. Keeney: None. M.E. Law: None. D.S. Wismayer: None. M. Rivera: None. J.L. Kasperbauer: None. E.J. Moore: None. X. Ma: A. Employee; Advanced Cell Diagnostics. D.J. Ma: None. K.A. Price: None.

Automated and Manual Human Papilloma Virus in situ Hybridization and p16 Immunohistochemistry: Comparison in Metastatic Oropharyngeal Carcinoma

Objectives: We compared the efficacy of automated and manual human papilloma virus (HPV) in situ hybridization (ISH) and p16 immunohistochemical staining (IHC) in fine needle aspiration (FNA) of metastatic oropharyngeal carcinoma. Study Design: A total of 41 FNA cell blocks (CB) were evaluated. HPV ISH was interpreted as positive if a minimum of one tumor cell showed punctate dot-like nuclear positivity. p16 was interpreted as positive if ≥70% of tumor cells showed brown nuclear and cytoplasmic staining. Results: Thirty of 41 CB (73%) were positive by automated HPV ISH, 25 of 41 CB (60%) with manual HPV ISH. Eighteen of 41 CB (43%) were positive for p16 IHC. Twelve of 41 CB (29%) with automated HPV ISH and 2 of 41 CB (4%) with the manual method were positive at 10× magnification. Three of 41 CB (7%) with automated HPV ISH and 14 of 41 CB (34%) with the manual method were positive at 20× magnification. Fifteen of 41 CB (36%) with automated HPV ISH and 9 of 41 CB (21%) with the manual method were positive at 40-60× magnification. Conclusion: Automated HPV ISH plays a more significant role in determining the HPV status in CB. However, the failure to use high magnification in the evaluation can give false-negative results.