Detection Of p16INK4a Research Articles

Prevalence of human papillomavirus DNA and p16INK4a positivity in vulvar cancer and vulvar intraepithelial neoplasia: a systematic review and meta-analysis.

Human papillomavirus (HPV) DNA and p16INK4a positivity have crucial roles in the pathogenesis of vulvar cancer and vulvar intraepithelial neoplasia. We aimed to examine the pooled prevalence of HPV DNA and p16INK4a positivity in vulvar cancer and vulvar intraepithelial neoplasia worldwide. In this systematic review and meta-analysis, we searched PubMed, Embase, and the Cochrane Library databases for studies published between Jan 1, 1986, and May 6, 2022, that reported the prevalence of HPV DNA, or p16INK4a positivity, or both, in histologically verified vulvar cancer or vulvar intraepithelial neoplasia. Studies on a minimum of five cases were included. Study-level data were extracted from the published studies. Random effect models were used to examine the pooled prevalence of HPV DNA and p16INK4a positivity in both vulvar cancer and vulvar intraepithelial neoplasia, which were further investigated using stratified analyses by histological subtype, geographical region, HPV DNA or p16INK4a detection method, tissue sample type, HPV genotype, publication year, and age at diagnosis. Additionally, meta-regression was applied to explore sources of heterogeneity. We retrieved 6393 search results, of which 6233 were excluded for being duplicates or after application of our inclusion and exclusion criteria. We also identified two studies from manual searches of references lists. 162 studies were eligible for inclusion in the systematic review and meta-analysis. The prevalence of HPV in vulvar cancer (91 studies; n=8200) was 39·1% (95% CI 35·3-42·9) and in vulvar intraepithelial neoplasia (60 studies; n=3140) was 76·1% (70·7-81·1). The most predominant HPV genotype in vulvar cancer was HPV16 (78·1% [95% CI 73·5-82·3]), followed by HPV33 (7·5% [4·9-10·7]). Similarly, HPV16 (80·8% [95% CI 75·9-85·2]) and HPV33 (6·3% [3·9-9·2]) were also the most two predominant HPV genotypes in vulvar intraepithelial neoplasia. The distribution of type-specific HPV genotypes in vulvar cancer among geographical regions was different, with HPV16 varying between regions, showing a high prevalence in Oceania (89·0% [95% CI 67·6-99·5]) and a low prevalence in South America (54·3% [30·2-77·4]). The prevalence of p16INK4a positivity in patients with vulvar cancer was 34·1% (95% CI 30·9-37·4; 52 studies; n=6352), and it was 65·7% (52·5-77·7; 23 studies; n=896) in patients with vulvar intraepithelial neoplasia. Furthermore, among patients with HPV-positive vulvar cancer, p16INK4a positivity prevalence was 73·3% (95% CI 64·7-81·2), compared with 13·8% (10·0-18·1) in HPV-negative vulvar cancer. The prevalence of double positivity for HPV and p16INK4a was 19·6% (95% CI 16·3-23·0) in vulvar cancer and 44·2% (26·3-62·8) in vulvar intraepithelial neoplasia. Most analyses had large heterogeneity (I2>75%). The high prevalence of HPV16 and HPV33 in vulvar cancer and vulvar intraepithelial neoplasia emphasised the importance of nine-valent HPV vaccination in preventing vulvar neoplasm. Additionally, this study highlighted the potential clinical significance of double positivity for HPV DNA and p16INK4a in vulvar neoplasm. Taishan Scholar Youth Project of Shandong Province, China.

Triaging abnormal cervical cancer screening tests using p16INK4a detection by ELISA on fresh cervical samples.

Cervical cancer screening strategies in the United States include cotesting (human papillomavirus (HPV) with cytology), primary HPV with genotyping and reflex cytology, and cytology alone. An ongoing challenge is the appropriate triage of patients to colposcopy to those at highest risk. We investigated whether incorporation of p16INK4a immunodetection by enzyme-linked immunosorbent assay (ELISA) on fresh cervical samples obtained at the time of screening could improve appropriate referral to colposcopy. A derivation group comprised of cervical swabs collected from subjects with high-grade dysplasia or cancer (positive control) and from subjects with negative screening history (negative control). Samples collected from colposcopy were used to evaluate the existing screening strategies individually and with incorporation of p16INK4a ELISA. Histology was used as the gold standard. Among 163 subjects recruited, 138 were included. In the derivation group, mean p16INK4a level was 2.86ng/mL (n=31) and 0.58ng/mL (n=20) among positive and negative controls respectively (p=0.002) with an area under the receiver operator characteristic curve of 0.79 (p<0.001). Among colposcopy subjects, sensitivity/specificity for cotesting, primary HPV, and cytology were 94%/42%, 88%/45%, and 88%/49%, respectively. Incorporation of p16INK4a resulted in similar sensitivity and improved specificity (cotesting+p16 88%/58%, primary HPV+p16 88%/57%, cytology+p16 81%/62%; p=0.23/p=0.008) with decrease in colposcopy referrals by 15% to 22% (p=0.01). These results demonstrate the feasibility of quantifying p16INK4a by ELISA in fresh cervical samples, and its potential as an adjunct to existing screening strategies in the identification of high grade-dysplasia while reducing the number of colposcopic referrals.

HPV Infection in Middle Ear Squamous Cell Carcinoma: Prevalence, Genotyping and Prognostic Impact.

Middle ear squamous cell carcinoma (MESCC) is rare. Human Papilloma Virus (HPV) infection has been found in a significant number of cases of MESCC. Despite the emerging role of HPV in oncogenesis, its role in the pathogenesis and prognosis of MESCC is not known. This study aims to identify the prognostic impact of alpha and beta HPV in MESCC and its correlation with p16 protein. We retrospectively investigated 33 patients with MESCC surgically treated between 2004 and 2016. HPV DNA was ascertained by polymerase chain reaction (PCR) and P16INK4a detection was performed. Disease-specific survival (DSS) and cumulative incidence of recurrence were calculated in relation to HPV presence and genotype. p16 sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in predicting HPV infection were calculated. HPV was detected in 66.7% of patients (36.4% alpha HPV, 63.6% beta HPV). Five-year DSS was 55.0% and was not statistically related to HPV presence (p = 0.55) or genotype (p = 0.87). Five-year cumulative incidence of recurrence was 46 %, and was not statistically related to HPV presence (p = 0.22) or genotype (p = 0.44). p16 sensitivity, specificity, PPV, and NPV in predicting HPV infection were 27.3%, 36.4%, 46.2%, and 20.0%, respectively. In our experience, beta HPV was more frequent than alpha HPV in MESCC. Neither HPV presence nor HPV genotypes relate to DSS or cumulative incidence of recurrence. p16 expression was not predictive for HPV infection in MESCC. The role of HPV infection in oncogenesis, maintenance, and prognosis of MESCC seems to be different from that in oropharynx and skin cancer.

Clinical value of p16INK4a immunocytochemistry in cervical cancer screening

Objective: To evaluate the value of p16INK4a detected by p16INK4a immunostaining as a new generation of cervical cytology for primary screening and secondary screening in population-based cervical cancer screening, and in improving cytological diagnosis. Methods: Between 2016 and 2018, 5 747 non-pregnant women aged 25-65 years with sexual history were recruited and underwent cervical cancer screening via high-risk (HR)-HPV/liquid-based cytological test (LCT) test in Shenzhen and surrounding areas. All slides were immuno-stained using p16INK4a technology, among them, 902 cases were offered p16INK4a detection during primary screening, and the remaining 4 845 cases were called-back by the virtue of abnormal HR-HPV and LCT results for p16INK4a staining. Participants with complete LCT examination, HR-HPV test, p16INK4a staining and histopathological examination results were included in this study. The performance of p16INK4a in primary and secondary screening, and in assisting cytology to detect high grade squamous intraepithelial lesion [HSIL, including cervical intraepithelial neoplasia (CIN) Ⅱ or Ⅲ] or worse [HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+] were analyzed. Results: (1) One-thousand and ninety-seven cases with complete data of p16INK4a and histology were included. Pathological diagnosis: 995 cases of normal cervix, 37 cases of low grade squamous intraepithelial lesion (LSIL), 64 cases of HSIL and one case of cervical cancer were found. Among them, 65 cases of HSIL (CIN Ⅱ)+ and 34 cases of HSIL (CIN Ⅲ)+ were detected. The positive rate of p16INK4a in HSIL (CIN Ⅱ)+ was higher than that in CINⅠ or normal pathology (89.2% vs 10.2%; P<0.01). (2) p16INK4a as primary screening for HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+ was equally sensitive to primary HR-HPV screening (89.2% vs 95.4%, 94.1% vs 94.1%; P>0.05), but more specific than HR-HPV screening (89.8% vs 82.5%, 87.7% vs 80.2%; P<0.05). p16INK4a was equally sensitive and similarly specific to cytology (≥LSIL; P>0.05). (3) The specificity of LCT adjunctive p16INK4a for detecting HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+ were higher than that of LCT alone or adjunctive HR-HPV (P<0.01), while the sensitivity were similar (P>0.05). (4) p16INK4a staining as secondary screening: p16INK4a was significantly more specific (94.1% vs 89.7%, 91.9% vs 87.4%; P<0.01) and comparably sensitive (84.6% vs 90.8%, 88.2% vs 91.2%; P>0.05) to cytology for triaging primary HR-HPV screening. HPV 16/18 to colposcopy and triage other HR-HPV with p16INK4a was equally sensitive (88.2% vs 94.1%; P=0.500) and more specific (88.3% vs 83.0%; P<0.01) than HPV 16/18 to colposcopy and triage other HR-HPV with LCT≥ atypical squamous cells of undetermined significance (ASCUS), and the referral rate decreased (14.0% vs 19.4%; P=0.005). Conclusions: For primary screening, p16INK4a is equally specific to cytology and equally sensitive to HR-HPV screening. p16INK4a alone could be an efficient triage after primary HR-HPV screening. In addition, p16INK4a immunostaining could be used as an ancillary tool to cervical cytological diagnosis, and improves its accuracy in cervical cancer screening.

Viral markers in nasopharyngeal carcinoma: A systematic review and meta-analysis on the detection of p16INK4a, human papillomavirus (HPV), and Ebstein-Barr virus (EBV).

This study aimed to conduct a meta-analysis to investigate the distribution of EBV and HPV stratified according to histological NPC type. We performed a meta-analysis to produce pooled prevalence estimates in a random-effects model. We also performed calculations for attributable fractions of viral combinations in NPC, stratified according to histological type. There was a higher prevalence of HPV DNA in WHO Type I (34.4%) versus WHO Type II/III (18.4%). The attributable fractions of WHO Type I NPC was predominantly double negative EBV(-) HPV(-) NPC (56.4%), and EBV(-) HPV(+) NPC (21.5%), in contrast to the predominant infection in WHO Type II/III which was EBV(+) HPV(-) NPC (87.5%). Co-infection of both EBV and HPV was uncommon, and double-negative infection was more common in WHO Type I NPC. A significant proportion of WHO Type I NPC was either double-negative EBV(-)HPV(-) or EBV(-)HPV(+).

Analysis of salivary detection of P16INK4A and RASSF1A promoter gene methylation and its association with oral squamous cell carcinoma in a Colombian population.

BackgroundEpigenetic factors play a fundamental role in the etiopathogenesis of oral squamous cell carcinoma (OSCC). This study evaluated if salivary detection of P16INK4A/RASSF1A gene promoter methylation might be linked to the clinical/histological features of OSCC in a Colombian population. Material and MethodsMethylation-specific polymerase chain reaction (MSP-PCR) was used to detect the methylation frequency of P16INK4A/RASSF1A genes in DNA obtained from whole saliva collected of 40 healthy controls (HC) and 43 OSCC patients. Determination of the clinical performance of MSP-PCR assay was based on standard algorithms derived from two-way contingency table analysis. The association of methylation status of targeted genes with OSCC was analyzed in a multivariate binary logistic regression model. ResultsThere were significantly higher proportions of promoter methylation of these target genes in OSCC patients when compared with HC. The analysis of single methylated genes showed high specificity, good positive and negative predictive values, but was accompanied by a low sensitivity. OSCC cases with clinical stage III/IV, poorly differentiated, and severe cellular atypia showed a significantly greater proportion of methylated than that of unmethylated targeted genes in saliva samples. Logistic regression analysis indicated an independent association of P16INK4A and RASSF1A promoter methylation with OSCC diagnosis. A significant interaction effect between ageing and P16INK4A promoter methylation was also detected. ConclusionsSalivary detection of P16INK4A and RASSF1A promoter methylation appears to be independently associated with OSCC and may be linked to the tumor activity in the present population. Consequently, the targeting of these genes in saliva samples might constitute an important tool for diagnosis and prognosis purposes. Key words:Gene methylation, oral squamous cell carcinoma, P16INK4A, RASSF1A, saliva.

Prevalence of human papillomavirus DNA and p16INK4a in penile cancer and penile intraepithelial neoplasia: a systematic review and meta-analysis

Although previous meta-analyses have examined human papillomavirus (HPV) DNA prevalence in penile cancer, none, to our knowledge, have assessed pooled HPV DNA prevalence in penile intraepithelial neoplasia or p16INK4a percent positivity in penile cancer and penile intraepithelial neoplasia. Therefore, we aimed to examine the prevalence of HPV DNA and p16INK4a positivity in penile cancer and penile intraepithelial neoplasia worldwide. In this systematic review and meta-analysis, we searched PubMed, Embase, and the Cochrane Library until July 24, 2017, for English-language articles published from Jan 1, 1986, onwards reporting the prevalence of HPV DNA and p16INK4a positivity, either alone or in combination, in at least five cases of penile cancer or penile intraepithelial neoplasia. Only studies that used PCR or hybrid capture for the detection of HPV DNA and immunohistochemical staining or methylation for the detection of p16INK4a were included. Data were extracted and subsequently crosschecked, and inconsistencies were discussed to reach consensus. Using random-effects models, we estimated the pooled prevalence and 95% CI of HPV DNA and p16INK4a positivity in penile cancer and penile intraepithelial neoplasia, stratifying by histological subtype and HPV DNA or p16INK4a detection method. Type-specific prevalence of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, and HPV45 in penile cancer was estimated. Our searches identified 1836 non-duplicate records, of which 73 relevant papers (71 studies) were found to be eligible. The pooled HPV DNA prevalence in penile cancer (52 studies; n=4199) was 50·8% (95% CI 44·8-56·7; I2=92·6%, pheterogeneity<0·0001). A high pooled HPV DNA prevalence was seen in basaloid squamous cell carcinomas (84·0%, 95% CI 71·0-93·6; I2=48·0%, pheterogeneity=0·0197) and in warty-basaloid carcinoma (75·7%, 70·1-81·0; I2=0%, pheterogeneity=0·52). The predominant oncogenic HPV type in penile cancer was HPV16 (68·3%, 95% CI 58·9-77·1), followed by HPV6 (8·1%, 4·0-13·7) and HPV18 (6·9%, 2·9-12·4). The pooled HPV DNA prevalence in penile intraepithelial neoplasia (19 studies; n=445) was 79·8% (95% CI 69·3-88·6; I2=83·2%, pheterogeneity<0·0001). The pooled p16INK4a percent positivity in penile cancer (24 studies; n=2295) was 41·6% (95% CI 36·2-47·0; I2=80·6%, pheterogeneity<0·0001), with a high pooled p16INK4a percent positivity in HPV-related squamous cell carcinoma (85·8%, 95% CI 72·1-95·4; I2=56·4%, pheterogeneity=0·0011) as compared with non-HPV-related squamous cell carcinoma (17·1%, 7·9-29·1; I2=78·3%, pheterogeneity<0·0001). Moreover, among HPV-positive cases of penile cancer, the p16INK4a percent positivity was 79·6% (95% CI 65·7-90·7; I2=89·9%, pheterogeneity<0·0001), compared with 18·5% (9·6-29·6; I2=89·3%, pheterogeneity<0·0001) in HPV-negative penile cancers. The pooled p16INK4a percent positivity in penile intraepithelial neoplasia (six studies; n=167) was 49·5% (95% CI 18·6-80·7). A large proportion of penile cancers and penile intraepithelial neoplasias are associated with infection with HPV DNA (predominantly HPV16), emphasising the possible benefits of HPV vaccination in men and boys. None.

Role of p16(INK4A) in detection of human papillomavirus DNA and stratification of cancer-specific mortality on the basis of long term follow-up data of 34 patients with invasive penile cancer

Human papillomavirus (HPV) infection plays an important role in the pathogenesis of penile squamous cell carcinoma (PSCC) in about one third of the affected patients. Initial data indicate that HPV status could facilitate optimised risk stratification and individualised targeted therapy. The polymerase chain reaction (PCR) assay is the reference method for detection of HPV DNA. It is unclear if alternatives such as in situ hybridisation (ISH) or various surrogate markers (defined as immunohistochemical detection of p16INK4a, histological subtype, tumour invasion front, koilocytosis) are sufficient to determine HPV status METHODS: In this single centre study on 34 patients with PSCC, multiplex nested PCR and ISH were conducted for HPV detection and identification of HPV DNA genotypes. Various histological criteria and p16INK4a were determined by central review. The influence of different criteria on cancer-specific mortality (CSM) was investigated with the Cox proportional hazards models (FU: 92 mo.). Furthermore, the discriminative qualities of various tumour invasion patterns (i. e., pT-classification 7th vs. 8th ed.) for CSM prediction were compared. Pursuant to PCR assay, HPV DNA was detected in 26 % of patients (n = 9). ISH and the examined histological criteria were of inadequate quality in the prediction of HPV status (p > ;0.3). Test parameters of p16INK4a were calculated as follows: sensitivity 66.7 %, specificity 84 %, positive predictive value 60 %, negative predictive value 87.5 %, overall agreement 79.4 % (Area-Under-Curve: 0.753, p = 0.026). None of the examined HPV criteria significantly influenced CSM. In comparison to the 7th pT-edition, the 8th version was superior in CSM prediction (c-indices 70.2 % vs. 72.9 %). In addition to penile corpora invasion, infiltration of the urethra had no independent predictive value. Regrouping of the corpora invasion patterns, as proposed by us, resulted in an increase in discriminative quality (c-index 77.9 %). In contrast to ISH and the examined histological criteria, p16INK4a allows a reasonable prediction of HPV status. Neither HPV DNA nor its surrogate markers independently impacted CSM. As urethral tumour invasion does not independently influence CSM, the recent pT classification can be considered useful for prognosis.

Application of a time-resolved fluoroimmunoassay for detection of p16INK4a in cervix.

To establish a time-resolved fluoroimmunoassay (TRFIA) method to detect p16INK4a. 119 cases of exfoliative cells following Thinprep cytologic test (TCT) test were selected for TRFIA and biopsy tissues of these patients according to TCT results were collected for immunohistochemical (IHC) staining. There were 40 cases with cervicitis, 16 cases of cervical intraepithelial neoplasia grade 1 (CIN1), 15 cases of CIN2, 26 cases of CIN3, and 22 cases of squamous cell carcinoma (SCC). For each group, TCT results displaying above ASC-US were 7, 6, 12, 23, and 21 cases. IHC results were positive for 2, 13, 14, 26, and 21 cases, while the TRFIA results were positive for 5, 12, 13, 25, and 21 cases, respectively. The sensitivity and specificity were not significantly different between TCT and TRFIA test due to the limited cases. Significantly more CIN1 cases were positive by TRFIA, as compared with cervicitis (75.00% vs. 12.50%, χ2=21.116, p<0.001). Most (93.65%) CIN2/3 and SCC cases were positive for p16INK4a (higher than CIN1, χ2=4.877 and p=0.027). Detecting p16INK4a protein by TRFIA is suitable to discriminate cervical dysplastic/neoplastic lesions from cervicitis. A commercial kit which created in the future might be a rapid, simple, and low-cost method to obtain objective results with an instrument automatically on a large scale.

Comparison of p16ink4a immunostaining in benign and malignant HPV-related lesion

Objective: We aim to analyze the difference between p16inka immunostaining in normal epithelium, two benign HPV-related lesions (papilloma and condyloma acuminatum) and one malignant HPV-related lesion (oropharynx carcinoma).Methods: Five normal oral mucosas, fifteen papiloma, fifteen condyloma acuminatum and fifteen HPV-positive OSCC were included in the present study. Histological sections were stained anti-p16ink4a by immunohistochemistry. For the positive stain, score was based on a scale of - to 3+, as follows: - negative stain; 1+ less than 25% of positivity and focal distribution; 2+ 26-50% positivity and focal distribution; and 3+ 50-75% of positive cells and diffuse distribution. As for the evaluation of the intensity score was based on: - negative; 1- low intensity; 2- moderate intensity; 3- intensive.Results: The results showed no significant differences between the scores (positive x intensity) of p16ink4a in normal epithelium, papilloma and condyloma acuminatum. All these 3 lesions showed significant differences when compared with the oropharynx squamous cell carcinoma.Relevance: There are differences in the expression of p16ink4a between benign HPV-lesions and malignant HPV-lesions. Also, it is not possible to identify the presence of HPV by the detection of p16ink4a using IHC in benign lesions, as has been used in OSCC.

A piezoelectric immunosensor for the rapid detection of p16INK4a expression in liquid-based cervical cytology specimens

A piezoelectric immunosensor was developed for the detection of p16INK4a, a protein that is expressed in liquid-based cervical cytology specimens and has been proved to be linked cervical cancer. Mouse polyclonal antibody against p16INK4a was bound onto the piezoelectric crystal surface in an ordered orientation using protein A. The antibody on the crystal could specifically adsorb p16INK4a and changes in the mass of the crystal would led to frequency shift. The supernatants from different clinical samples, including various cervical lesions, containing different concentration of p16INK4a were used for testing. Under the optimized conditions, the frequency shifts were linearly dependent on the antigen concentration in the range of 50–1200ngmL−1. The device showed a short analysis time (less than 30min), good reproducibility (could be reused six times without any detectable loss of activity), stability (the immunosensor was stable for more than eight weeks when stored over silica gel blue at 4°C), and specificity. Attempts have been made to use the sensor for the initial screening for cervical lesion in cervical cytology specimen, and the relationship between cytologic and histologic findings, p16INK4a expression in various cervical lesions, including carcinoma been investigated.

The p16INK4a Antibody Immobilization Method for Immonosensor Application.

The p16INK4a is a protein that expressed in Liquid-based cervical cytology specimens and has been proved link to cervical cancer. The p16INK4a could be detection by piezoelectric immunosensor and the immobilization of the p16INK4a antibody influence the sensitivity of the piezoelectric immunosensor. 5μL mouse polyclonal antibody against p16INK4a was bound onto the surface of immonosensor through two methods. (directly immobilized method; protein A method). Absorb of the p16INK4a antibody on the surface of immonosensor caused a shift in the resonant frequency of the immunosensor and The frequency changes recorded showed a better reproducibility. The activity of the immobilization antibody with the directly method and protein A method was tested with p16INK4a antigen. The resonant frequency for different antibody immobilization methods were different, and the sensitivity for p16INK4a detection also different. The protein A method was found to be much more better than the directly method for the immobilization of the p16INK4A antibody on the gold electrode of the quartz crystal for cervical lesion detection. The Protein A method created more reproducible and stable immobilization antibody layers with p16INK4A antigen.

HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis

We aimed to provide updated information about the global estimates of attributable fraction and type distribution of human papillomavirus (HPV) in head and neck squamous cell carcinomas by doing a systematic review and meta-analysis. We did a literature search on PubMed to identify studies that used PCR for detection of HPV DNA in head and neck squamous cell carcinomas with information about HPV genotype distribution. We included studies that tested 20 or more biopsies per cancer site and were published between July 15, 1990, and Feb 29, 2012. We collected information about sex, risk factors, HPV detection methods, and biomarkers of potentially HPV-induced carcinogenesis (E6/E7 mRNA and p16(INK4a)). If it was not possible to abstract the required information directly from the paper, we contacted the authors. We did a meta-analysis to produce pooled prevalence estimates including a meta-regression to explore sources of heterogeneity. 148 studies were included, contributing data for 12 163 cases of head and neck squamous cell carcinoma from 44 countries. HPV DNA was detected in 3837 cases. HPV16 accounted for 82·2% (95% CI 77·7-86·4) of all HPV DNA positive cases. By cancer site, pooled HPV DNA prevalence estimates were 45·8% (95% CI 38·9-52·9) for oropharynx, 22·1% (16·4-28·3) for larynx (including hypopharynx), and 24·2% (18·7-30·2) for oral cavity. The percent positivity of p16(INK4a) positive cases in HPV-positive oropharyngeal cancer cases was 86·7% (95% CI 79·2-92·9) and of E6/E7 mRNA positive cases was 86·9% (73·2-96·8). The estimate of HPV attributable fraction in oropharyngeal cancer defined by expression of positive cases of E6/E7 mRNA was 39·8% and of p16(INK4a) was 39·7%. Of subsites, tonsils (53·9%, 95% CI 46·4-61·3) had the highest HPV DNA prevalence. HPV DNA prevalence varied significantly by anatomical site, geographic region, but not by sex or tobacco or alcohol consumption. The contribution of HPV prevalence in head and neck squamous cell carcinoma and in particular that of HPV16 in the oropharynx shows the potential benefit of prophylactic vaccines. European Commission.

P16INK4A en el diagnóstico diferencial entre atrofia cervicouterina y neoplasia intraepitelial cervical de alto grado (NIC III): reporte de caso

Introducción: En pacientes posmenopáusicas los cambios atróficos del cérvix dificultan el diagnóstico colposcópico y cito-histológico, pues pueden simular lesiones premalignas, por lo cual, la detección inmunohistoquimica de p16INK4a sería útil como herramienta diagnóstica diferencial entre estos cambios; sin embargo, es necesario aportar evidencias desde la perspectiva clínica. Caso clínico: Paciente posmenopáusica con colposcopia insatisfactoria y hallazgos cito-histológicos sugestivos pero no conclusivos de neoplasia intraepitelial cervical de alto grado (NIC III). La detección inmunohistoquímica de p16INK4a fue negativa. El estudio histológico de la conización diagnóstica/terapéutica no reportó lesión intraepitelial o maligna. Este caso permite aportar evidencia a favor del p16INK4a como biomarcador diferencial entre cambios atróficos y premalignos, con lo cual se podrían evitar intervenciones innecesarias. Para el desarrollo del manuscrito se contó con consentimiento informado de la paciente objeto de estudio y consentimiento institucional de la Universidad de Cartagena y la subdirección científica de la E.S.E. Clínica “Maternidad Rafael Calvo”.

OP041: Impact of human papillomavirus (HPV) and p16 status on survival and response with cisplatin plus 5-FU and cetuximab in recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN): Analysis of the phase III extreme trial

Purpose Retrospective analysis to assess the role of tumor HPV/p16 status in patients (pts) with R/M SCCHN receiving cisplatin+5-FU chemotherapy (CT)+/−cetuximab. Material and methods Cervista HPV 16/18 and HPV high-risk assays and IHC detection of p16INK4A were used to determine tumor HPV/p16 status. Results Of 442 pts in the trial, p16 status was available for 381 and HPV status for 321. For survival, hazard ratios in favor of CT+cetuximab were seen in HPV−, HPV+, p16− and p16+ subgroups ( Table 1 ). Interaction tests confirmed the treatment effect of CT+cetuximab vs CT was independent of tumor HPV or p16 status. Similar findings were made for PFS (interaction test p = 0.98 for HPV and p = 0.43 for p16: no significant interaction was noted). CT+cetuximab also improved the chances of achieving a response across all subgroups ( Table 1 ). Within the CT+cetuximab and CT arms, HPV+/p16+ status was associated with a more favorable outcome than HPV−/p16− status. Conclusion Although the number of HPV+/p16+ pts was low, the results suggest that the survival benefits of CT+cetuximab vs CT are independent of tumor HPV/p16 status but that HPV+/p16+ status seems associated with a more favorable outcome than HPV−/p16− status.

Immunohistochemical Detection of p16 INK4a in Leukoplakia and Oral Squamous Cell Carcinoma

Over-expression of p16(INK4a) has been reported in tissues of oral squamous cell carcinoma (SCC) associated with Human Papillomaviruses (HPVs). Immunohistochemical (IHC) detection of p16(INK4a) is an easy technique than molecular detection of HPVs, hence we investigated the presence of this protein in the most common pre-malignant and malignant oral lesions i.e. leukoplakia and SCC respectively. We performed IHC detection of p16(INK4a) in sections of paraffin embedded formalin fixed tissues of leukoplakia with or without dysplasia (n= 21) and SCC lesions (n= 69) and correlated with various patterns of p16(INK4a) positivity with respect to histological diagnosis. In the present study, 71% cases of oral SCC cases were positive for p16(INK4a), of which the most common pattern was diffuse nuclear and cytoplasmic staining. Among the cases with leukoplakia, 57.1% were positive for overexpression of p16(INK4a), wherein diffuse and sporadic pattern was observed among 23.8 percent each. In the present study, significant number of oral SCC cases observed overexpressing p16(INK4a) . However HPV DNA detection based studies are needed to validate the utility of IHC detection of p16(INK4a) as a surrogate marker for HPV associated oral SCC.

1018O - Safety and Efficacy of Cisplatin Plus 5-Fu and Cetuximab in HPV-Positive and HPV-Negative Recurrent and/or Metastatic Squamous Cell Carcinoma of the Head and Neck (R/M SCCHN): Analysis of the Phase III Extreme Trial

ABSTRACT Background Tumor human papillomavirus (HPV) status is a strong predictor of survival and response to treatment in patients (pts) with early and locally advanced oropharyngeal cancer, but its effects in R/M SCCHN remain to be clarified. This retrospective analysis of data from the EXTREME trial assessed the role of tumor HPV status in pts with R/M SCCHN receiving cisplatin + 5-FU chemotherapy (CT) alone or in combination with cetuximab. Material and methods: Immunohistochemical detection of p16INK4A was used to determine HPV status: p16-positive and p16-negative disease is referred to as HPV-positive (HPV+) and HPV-negative (HPV-) disease, respectively. Results 196/222 (88%) pts in the CT + cetuximab and 185/220 (84%) pts in the CT arm had tissue evaluable for p16. Of these, 91% and 88%, respectively, had HPV- tumors. Among HPV- pts, CT + cetuximab (n = 178) improved overall survival (OS), progression-free survival (PFS) and overall response rate (ORR), compared with CT (n = 162) (Table). A similar pattern was observed among HPV+ pts, between CT + cetuximab (n = 18) and CT (n = 23) for OS, PFS and ORR. Interaction tests between treatment and HPV status for OS and PFS confirmed that the treatment effect of CT + cetuximab vs CT was independent of tumor HPV status (p = 0.482 and p = 0.430, respectively: no significant interaction was noted). Within the CT + cetuximab and CT arms, HPV (p16) expression is associated with a more favorable outcome than HPV- (Table). In the CT + cetuximab arm, the overall incidence of grade 3/4 adverse events was slightly higher among HPV+ than HPV- pts. Conclusions Although the number of HPV+ pts in this analysis was low, the results suggest that in R/M SCCHN pts in the EXTREME trial, the survival benefits ofCT + cetuximab vs CT are independent of tumor HPV status and HPV (p16) expression is associated with a more favorable outcome than HPV-. Table: 1018O: Tumor HPV status: effects on prognosis and efficacy of CT + cetuximab and CT in R/M SCCHN. Population Comparison OS PFS ORR CT + cetuximab vs CT HPV- (n = 340) Median (months)/rate (%) 9.7 7.3 5.7 3.1 37 17 HR/odds ratio 95% CI p 0.822 (0.647–1.043) 0.1064 0.486 (0.376–0.628) 2.753 (1.655–4.579) HPV+ (n = 41) Median (months)/rate (%) 12.6 9.6 5.6 3.6 50 22 HR/odds ratio 95% CI p 0.628 (0.295–1.338) 0.2241 0.730 (0.363–1.469) 0.3761 3.600 (0.929–13.953) 0.0614 HPV+ vs HPV- CT + cetuximab (n = 196) Median (months)/rate (%) 12.6 9.7 5.6 5.7 50 37 HR/odds ratio 95% CI p 0.592 (0.319–1.098) 0.0925 1.172 (0.685–2.005) 0.5624 1.738 (0.657–4.600) 0.2620 CT (n = 185) Median (months)/rate (%) 9.6 7.3 3.6 3.1 22 17 HR/odds ratio 95% CI p 0.827 (0.504–1.355) 0.4492 0.871 (0.529–1.434) 0.5870 1.329 (0.455–3.880) 0.6025 Data are medians and hazard ratios (HR) for PFS and OS and rates and odds ratios for ORR. p: log-rank test for OS/PFS; Cochran-Mantel-Haenszel test for ORR. Disclosure B. De Blas: The author is an employee of Merck KGaA. I. Celik: The author is an employee of Merck KGaA J.B. Vermorken: I have participated in advisory boards of Merck-Serono, Amgen, Boehringer-Ingelheim, Genentech and Sanofi-aventis and have lectured (compensated) for Merck-Serono, Amgen, Bristol-Myers Squibb and Sanofi-aventis All other authors have declared no conflicts of interest.

P16/Ki-67 dual staining in cervico-vaginal cytology: Correlation with histology, Human Papillomavirus detection and genotyping in women undergoing colposcopy

ObjectivesTo evaluate the CINtec® PLUS assay (mtm laboratories), a new immunocytochemical method for the simultaneous detection of p16INK4a and Ki-67, in liquid-based cervico-vaginal cytology, investigating the association of the dual staining with HPV infection and genotyping as well as cytological and histological abnormalities. Methods140 women with a cervico-vaginal sample obtained immediately before the colposcopy were enrolled. This cytological sample was used for HPV testing with the Linear Array HPV Genotyping Test, the dual staining with the CINtec® PLUS kit and the morphology assessment. ResultsCytology results were 38 NILM, 16 ASC-US, 32L-SIL, 54H-SIL or worse. 113 patients also had a colposcopy-guided biopsy, classified as 14 negative, 35 CIN1, 24 CIN2, 37 CIN3, 3 invasive SCC. A strong association between p16/Ki-67 and HR-HPV infection was found (COR=6.86, 95% CI: 1.84–31.14). Importantly, the association between p16/Ki-67 positivity and HPV16 and/or 18 infection was 2-fold stronger compared to that with the infection by other HR-HPV types (COR=9.92, 95% CI: 2.39–47.77 vs COR=4.20, 95% CI: 0.99–20.87). In addition, p16/Ki-67 positivity rate significantly increased with the severity of the cytological and histological abnormalities (p<0.05 in both cases). p16/Ki-67 positivity resulted strongly associated with a CIN2+ diagnosis (COR=10.86 95% CI: 4.16–29.12). ConclusionsThis preliminary study evidenced that p16/Ki-67 immunostaining might have a relevant clinical role, since the dual staining was significantly associated with HR-HPV infection, particularly with HPV 16 and 18, and the increasing grade of the cervical lesions, the positivity for this biomarker being strongly related to the presence of a CIN2+ lesion.

Dual Stain Immunohistochemical Localization of p16INK4A and ki-67

The primary goal of this study was to assess the clinical utility of a multiplexed immunohistochemical method using colocalization of p16 and Ki-67 in identifying high-grade cervical mucosal lesions. The study included formalin-fixed cervical biopsy specimens, representative of 297 diagnostic regions. They were subjected to 2 colors immunohistochemical staining for p16 and Ki-67 using an EnVision polymer-based method. The chromogens used were of DAB brown for the detection of p16 and alkaline phosphatase blue for Ki-67. Histologic regions were scored positive for either marker based on the detection of p16 or Ki-67 in >10% of the cells of interest. Positive test results with colocalization of p16/Ki-67 were found in 20 of 40 cases of cervical intraepithelial neoplasia 1 (n=40) and in all cases of cervical intraepithelial neoplasia 2/3 (n=32), squamous cell carcinoma (n=11), adenocarcinoma in situ (n=10), and invasive adenocarcinoma (n=8). Colocalization of p16/Ki-67 was also detected in few cells in 1 of 19 sections with tuboendometrial metaplasia but was not detected in normal squamous mucosa (n=78), normal endocervical mucosa (n=76), immature squamous metaplasia (n=13), or in microglandular hyperplasia (n=9). The p16 and Ki-67 are coexpressed in virtually 100% of cases of high-grade squamous and glandular lesions, but they are rarely coexpressed in normal tissues or in benign lesions of the squamous and glandular mucosa. Thus, multiplexed colocalization of p16 and Ki-67 is a practical and potentially powerful diagnostic approach to enhance the accuracy of cervical histopathologic diagnosis.

Detection of p16INK4a Promoter Methylation Status &amp;1QJ;in Non-small Cell Lung Cancer by a Fluorescence Polarization Assay

The detection of the p16INK4a promoter methylation status has a good value for the prognosis, early detection, and individualized management of patients with non-small cell lung cancer. A novel method detecting the p16INK4a promoter methylation status of primary carcinoma tissue samples by a fluorescence polarization assay was developed in this research. A pair of general primers was used to amplify a 305-basepair fragment in the promoter region of p16INK4a. Two probes specific for either methylated p16INK4a or unmethylated p16INK4a DNA labeled with either tetramethyl 6-carboxyrhodamine or 6-carboxy-fluorescein hybridized, respectively, with their target amplicons, and the hybridization increased the fluorescence polarization values. The p16INK4a promoter methylation status was determined by the analysis of the fluorescence polarization values. One hundred and twenty-nine non-small cell lung cancer samples were analyzed in parallel with a fluorescence polarization assay and a gel-based methylation-specific polymerase chain reaction (PCR) assay. There was no significant difference between the results of the p16INK4a promoter methylation status obtained with the fluorescence polarization assay and the results obtained with the gel-based methylation-specific PCR assay. The minimum detection level of the fluorescence polarization assay was 25 copies/μL. The fluorescence polarization assay allowed the semiautomated detection of the methylated p16INK4a and unmethylated p16INK4a promoters directly in the solution with 1 PCR cycle, and it was much simpler than methylation-specific PCR and methylation-specific multiplex ligation-dependent probe amplification assays.