A triple-negative breast cancer surrogate subtype classification that correlates with gene expression subtypes.

BACKGROUND: This study developed a triple-negative breast cancer (TNBC) surrogate subtype classification that represents TNBC subtypes based on the Vanderbilt subtype classification. METHODS: Patients who underwent primary curative surgery for TNBC were included. Representative FFPE blocks were used for gene expression analysis and tissue microarray construction for immunohistochemical (IHC) staining. The Vanderbilt subtypes were re-classified into four groups: basal-like (BL), mesenchymal-like (M), immunomodulatory (IM) and luminal androgen receptor (LAR) subtype. Classification and regression tree (CART) modeling was applied to develop a surrogate subtype classification. RESULTS: A total of 145 patients were included. The study cohort was allocated to the Vanderbilt 4 subtypes as LAR (n = 22, 15.2%), IM (n = 32, 22.1%), M (n = 38, 26.2%), BL (n = 25, 17.2%) and unclassified (n = 28, 19.3%). After excluding nine (6.2%) patients due to poor IHC staining quality, CART modeling was performed. TNBC surrogate subtypes were defined as follows: LAR subtype, androgen receptor Allred score 8; IM subtype, LAR-negative with a tumor-infiltrating lymphocyte (TIL) score > 70%; M subtype, LAR-negative with a TIL score < 20%; BL subtype, LAR-negative with a TIL score 20-70% and diffuse, strong p16 staining. The study cohort was classified by the surrogate subtypes as LAR (n = 26, 17.9%), IM (n = 21, 14.5%), M (n = 44, 30.3%), BL1 (n = 27, 18.6%) and unclassified (n = 18, 12.4%). Surrogate subtypes predicted TNBC Vanderbilt 4 subtypes with an accuracy of 0.708. CONCLUSION: We have developed a TNBC surrogate subtype classification that correlates with the Vanderbilt subtype. It is a practical and accessible diagnostic test that can be easily applied in clinical practice.

[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.

[The value of p16(INK4a) cytology for early diagnosis of cervical cancer].

Objective: To investigate the use of p16(INK4a) immuno-stained cytology as the primary screening for cervical cancer prevention. Methods: From March to August 2018, 902 women from Shenzhen and surrounding area were recruited for cervical cancer screening with ThinPrep Cytologic Test (TCT), cobas4800 HPV test, and p16(INK4a) co-test. Colpo/biopsies were performed using the point of interest biopsy protocol of directed and random cervical biopsies plus endocervical curettage for all women, any of whose tests was positive. Two senior cytopathologists interpreted TCT and p16(INK4a) test. The performance of p16(INK4a) for early detection of CIN2+ and inter-observer reproducibility of the interpretation of p16(INK4a) were evaluated. Results: The positive rates of HPV test, p16(INK4a) co-test and TCT diagnosed as LSIL/AGC or higher grade were 8.1% (73/902), 6.8% (61/902) and 4.7% (42/902), respectively. Colposcopy referring rate was 79.6% (109/137), among which 10 cases were diagnosed as CIN2+ (5 cases of CIN2 and 5 cases of CIN3). The sensitivity and specificity for CIN2+ of p16(INK4a) test, TCT (LSIL/AGC or higher grade) and HPV test were 90.0%, 80.0%, 100.0% and 90.9%, 91.9%, 82.5%, respectively. Compared to TCT and HPV test, there was no significant difference in sensitivity and specificity between p16(INK4a) and TCT/HPV test (P>0.05). The Kappa value of the 2 cytopathologists in interpreting p16(INK4a) and TCT was 0.944 and 0.425, respectively (P<0.05). Conclusions: p16(INK4a) for cervical cancer screening is equally sensitive to HPV test and specific to TCT while subjective difference of cytopathologists' interpretation of p16(INK4a) is small. Therefore, p16(INK4a) can be used as a new cervical cancer screen method for its better diagnostic performance.

[Comparison of the clinicopathological features between adenoid basal cell carcinoma and adenoid cystic carcinoma of the cervix].

Objective: To compare the clinical and histopathological characteristics of cervical adenoid basal cell carcinoma and adenoid cystic carcinoma for improving the diagnosis accuracy and differential diagnosis of these tumors. Methods: A retrospective study was conducted on 9 cases of cervical adenoid basal cell carcinoma and 3 cases of adenoid cystic carcinoma which were diagnosed and consulted at the First Medical Center of PLA General Hospital from March 2009 to April 2019. Detailed clinical data were reviewed. All pathological sections and immunohistochemical results were reviewed and the clinicopathological characteristics were analyzed. Follow-up information by telephone was collected and relevant literature was consulted. Results: Both tumors were more commonly found in postmenopausal women (the age of onset ranged 43-74 years). Adenoid basal cell carcinoma was often clinical asymptomatic. Most of them presented as abnormal smears of the cervix during physical examination, and there was no definite mass in colposcopy.Adenoid cystic carcinoma was mostly presented with abnormal vaginal bleeding. A mass was seen in colposcopy.Histologically, the two tumors were characterized by nest-like growth of the tumors, consisting of basal-like tumor cells, and often surrounded by palisade structures. The two lesions might coexist, or be mixed with squamous cell carcinoma or high-grade squamous intraepithelial lesions. The difference was that adenoid basal cell carcinoma was mostly located at the junction of cervical squamous epithelium and columnar epithelium and beneath the overlying epithelium, the tumor cells were arranged in nests, with squamous differentiation in the center of the nests, or in double-layer adenoid arrangement. The cell morphology was bland with occasional mitoses, and the stromal reaction was not obvious. And adenoid cystic carcinoma cells in the nest arranged like a sieve, the homogenous red-stained and blue-stained secretions were observed in the sieve holes, with obvious cell atypia, frequent mitoses, and obvious stromal reaction.In one case of adenoid cystic carcinoma, sarcomatoid area presented around the nests.Both of them were positive in clinical HPV test. Among the 9 cases of adenoid basal cell carcinoma, 3 were tested for HPV and 5 were tested for p16, and all showed positive expression. Among the 3 cases of adenoid cystic carcinoma, 2 were tested for HPV and 3 were tested for p16, both of which showed positive expression. Telephone follow-up was conducted by June 2019(follow-up time ranged 2-37 months). No recurrence or metastasis occurred in 7 of the 9 cases of adenoid basal cell carcinoma, while 1 case had a ground-glass nodule in lung and another had recurrence of vaginal stump 32 months after the surgery.One case of adenoid cystic carcinoma developed lung metastasis 8 months after surgery and died 2 years after surgery; another case was followed up for 6 months, which showed no recurrence or metastasis; the third case was lost to follow-up. Conclusions: Both adenoid cystic carcinoma and adenoid basal cell carcinoma of the cervix are the tumors originating from cervical reserve cells and are associated with high-risk HPV infection. Due to the differences in clinical treatment and prognosis, careful histological evaluation and immunohistochemical analysis should be carried out to make accurate pathological diagnosis.

Human papillomavirus (HPV)-independent vulvar squamous cell carcinoma has a worse prognosis than HPV-associated disease: a retrospective cohort study.

AIMS: Vulvar squamous cell carcinoma (VSCC) can be subdivided by human papillomavirus (HPV) status into two clinicopathological entities. Studies on the prognostic significance of HPV in VSCC are discordant. METHODS AND RESULTS: We performed a retrospective analysis of overall survival (OS), disease-specific survival (DSS) and progression-free survival (PFS) in 217 patients with VSCC. Cases were extracted from an era of more aggressive en-bloc radical dissections (1985-95) and more localized radical surgery through separate vulvar and groin excisions (1996-2005). p16 immunohistochemistry was used as a surrogate for HPV status. HPV status could be determined in 197 tumours, 118 HPV-independent and 79 HPV-associated tumours. Patients with HPV-associated tumours were younger (mean 58.8 versus 71.6 years for HPV-independent tumours, P < 0.0001) and more likely to have prior abnormal cervical cytology (41.1 versus 5.6% for HPV-independent tumours, P < 0.0001). In univariable analysis, patients with HPV-associated tumours had superior PFS [hazard ratio (HR): 0.37, 95% confidence interval (CI): 0.18-0.70], DSS (HR: 0.19, 95% CI: 0.08-0.41) and OS (HR: 0.35, 95% CI: 0.21-0.59). This was driven by worse outcomes (PFS, DSS and OS) for patients with HPV-independent tumours compared with HPV-associated tumours who underwent surgery after 1995. After adjusting for age and stage in multivariable analysis, patients with HPV-associated tumours showed superior PFS (HR: 0.25, 95% CI: 0.07-0.77) and DSS (HR: 0.21, 95% CI: 0.04-0.78). CONCLUSIONS: VSCC can be stratified into two prognostically different diseases based on p16 immunostaining. HPV status was associated only with prognosis in the cohort that underwent surgery after 1995, suggesting that more conservative surgery may have led to worse outcomes for patients with HPV-independent tumours.

[Significance of p16/Ki-67 double immunocytochemical staining in cervical cytology ASCUS, LSIL, and ASC-H].

Objective: To investigate the application value of p16/cell proliferation associated nuclear antigen (Ki-67) double-staining and human papillomavirus mRNA in the cytological screening. Methods: Two hundred and fifty-one cases who suffered from atypical squamous cell of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cell-cannot exclude high-grade squamous intraepithelial lesion (ASC-H) in ThinPrep cytologic test (TCT) were collected in Peking University First Hospital between October 2015 and March 2016. And p16/Ki-67 double-staining and hybrid capture Ⅱ (HC-Ⅱ) detection were performed on the cervical cells. The result was compared with the pathological result of colposcope guided biopsy. All statistical analysis was completed by Stata 12.0 statistical software analysis. The results of diagnostic tests were described by using the sensitivity, specificity, positive predictive value,negative predictive value, and the area under the receiver operating characteristic (ROC) curve. Results: (1) One hundred and eight cases of liquid based cytology diagnosis of ASCUS patients, the positive rate of p16/Ki-67 was 13.9% (15/108), 102 cases of liquid based cytology diagnosis of LSIL patients, the positive rate of p16/Ki-67 was 21.6% (22/102), 41 cases of liquid based cytology diagnosis of ASC-H patients, the positive rate of p16/Ki-67 was 39.0% (16/41), compared amongthree groups, the difference was statistically significant (χ(2)=78.516, P<0.05); cervical exfoliated cells p16/Ki-67 expression rate was 13.0%(28/215) in cervical low-grade lesions [cervical intraepithelial neoplasia (CIN) Ⅰ], which was 69.4%(25/36) in high level lesions (CIN Ⅱ-Ⅲ), the difference was statistically significant (χ(2)=7.932, P<0.05). (2) The specificity of p16/Ki-67 detection and diagnosis were higher than those of HC-Ⅱ in ASCUS, LSIL, and ASC-H (89.8% vs 71.4%, 83.3% vs 15.6%, 88.9% vs 40.7%; all P<0.05), meanwhile, the positive predictive value of p16/Ki-67 detection and diagnosis exceed those of HC-Ⅱ in ASCUS, LSIL, and ASC-H (33.3% vs 26.3%, 31.8% vs 12.6%, 81.3% vs 38.5%; all P<0.05). Moreover, the ROC curve of p16/Ki-67 were bigger than those of HC-Ⅱ in ASCUS, LSIL, and ASC-H (0.799 vs 0.696, 0.708 vs 0.531, 0.909 vs 0.561; all P<0.05). Conclusion: For patients with cytological diagnosis of ASCUS, LSIL, and ASC-H, p16/Ki-67 double staining method could be used as an effective method to assist in the diagnosis of high-grade cervical lesions, and the screening efficiency is superior to that of high-rist HPV.

[Application of p16/Ki-67 immunocytochemistry in triage of patients with atypical squamous cells of undetermined significance].

Objective: To investigate the clinical value of p16/Ki-67 immunocytochemistry in patients with atypical squamous cells of undetermined significance(ASC-US). Methods: One hundred and seventy-one cases of thin-prep cytology test (TCT) diagnosed as ASC-US underwent p16/Ki-67 immunocytochemistry. All patients had colposcopy and biopsy from March 2015 to January 2016. Ninety of the 171 cases underwent high-risk HPV test at the same time. Results: p16/Ki-67 immunocytochemistry was positive in 43.9% (75/171) of the 171 cytology samples; the sensitivity and specificity of p16/Ki-67 immunocytochemistry were 77.6%(52/67) and 77.9%(81/104) in detecting CIN2+ , and the positive and negative predictive value were 69.3%(52/75) and 84.4%(81/96), respectively. The sensitivity and specificity in diagnosing CIN2+ were 100.0%(34/34) and 10.7%(6/56) for HPV test, and the positive and negative predictive value were 40.5%(34/84) and 6/6. p16/Ki-67 immunocytochemistry showed lower sensitivity but obviously higher specificity than high-risk HPV detection. Conclusion: p16/Ki-67 immunocytochemistry is a good triage test for identifying CIN2+ in ASC-US specimens.

P16 Expression in Human Breast Carcinoma and its Relationship to Clinicopathological Parameters.

Background: p16 is a cyclin-dependent kinase inhibitor and a cardinal regulator of the cell cycle. The relationship between p16 overexpression and poor prognosis of breast cancer has been reported in some studies. This study aimed to evaluate p16 expression in breast cancer in comparison to normal breast tissue and determine the association between p16 expression and clinicopathological parameters in breast cancer. Materials and Methods: Paraffin blocks of 110 samples were studied. These included 40 invasive breast carcinoma (tumor group) and normal tissue adjacent to the tumor (tumor control), as well as 30 normal mammoplasty specimens (normal control). Samples were from the pathology archive of Alzahra Hospital, Isfahan, Iran, from 2016 to 2020. p16 expression was studied and compared in these three groups using the immunohistochemistry technique. Moreover, the relationship between p16 expression and age, tumor size, carcinoma subtype, tumor grade, and lymph node involvement was investigated in the tumor group. SPSS version 16 was used to analyze data. Results: p16 expression showed a significant difference between the tumor group and the two control groups with a significantly higher expression in the tumor group. There was a significant direct relationship between the intensity of p16 expression and the number of involved lymph nodes (P < 0.001). No significant relationship was detected between p16 expression and other clinicopathological factors. Conclusion: p16 seems to have a rather significant expression in breast cancer in comparison to normal breast parenchyma. However, among clinicopathological parameters, we found only a direct relationship between lymph node involvement and intensity of p16 expression.

Prevalence of HPV Infection and p16INK4a Overexpression in Surgically Treated Laryngeal Squamous Cell Carcinoma.

Background: The mucosal high-risk (HR) human papillomavirus (HPV) is associated with oropharyngeal carcinogenesis. Aims of this study were to evaluate the prevalence of HR-HPV infection in laryngeal squamous cell carcinoma (LSCC) from different subsites, and the clinico-biological meaning of p16 overexpression. Methods: Ninety-seven LSCCs submitted to primary surgery (n = 75) or to post-irradiation salvage laryngectomy (n = 22) were evaluated for HR-HPV DNA and RNA using Luminex-based assays. p16 immunohistochemistry was performed. Results: HR-HPV DNA from HPV16 was detected in seven cases (8.75%), without significant differences between supraglottic and glottic lesions. HPV RNA was never detected. p16 overexpression correlated with HR-HPV DNA, but the kappa agreement score was poor. HPV DNA showed no impact on prognosis. p16 overexpression was associated with a better survival (OS, RFS) in primarily operated cases, while an inverse association with OS was observed in the salvage surgery group. Conclusions: HR-HPV infection appears to have a marginal role in LSCC independent of the anatomical subsite. p16 expression is deregulated in LSCC independent of HPV but displays a prognostic role in patients submitted to primary surgery. The negative predictive role of p16 overexpression in patients undergoing salvage surgery deserves more investigations for validation and elucidation of its clinical relevance.

Parathyroid hormone ameliorates temporomandibular joint osteoarthritic-like changes related to age.

OBJECTIVES: Ageing could be a contributing factor to the progression of temporomandibular joint osteoarthritis (TMJ OA), whereas its pathogenesis and potential therapeutic strategy have not been comprehensively investigated. MATERIALS AND METHODS: We generated ageing mouse models (45-week and 60-week; 12-week mice as control) and intermittently injected 45-week mice with parathyroid hormone (PTH(1-34)) or vehicle for 4 weeks. Cartilage and subchondral bone of TMJ were analysed by microCT, histological and immunostaining. Western blot, qRT-PCR, ChIP, ELISA and immunohistochemical analysis were utilized to examination the mechanism of PTH(1-34)'s function. RESULTS: We showed apparent OA-like phenotypes in ageing mice. PTH treatment could ameliorate the degenerative changes and improve bone microarchitecture in the subchondral bone by activating bone remodelling. Moreover, PTH inhibited phosphorylation level of Smad3, which can combine with p16ink4a gene promoter region, resulting in reduced senescent cells accumulation and increased cellular proliferation of marrow mesenchymal stem cells (MSCs). ELISA also showed relieved levels of specific senescent-associated secretory phenotype (SASP) in ageing mice after PTH treatment. CONCLUSIONS: In summary, PTH may reduce the accumulation of senescent cells in subchondral bone by inhibiting p16ink4a and improve bone marrow microenvironment to active bone remodelling process, indicating PTH administration could be a potential preventative and therapeutic treatment for age-related TMJ OA.

Prevalence and Detection of Sexually Transmitted Cases of Laryngeal Carcinoma.

INTRODUCTION: Human papillomavirus (HPV) is among the reported etiologies of laryngeal squamous cell carcinoma (LSCC). Understanding the impact of HPV on LSCC may help reduce its incidence. This study investigates the association between HPV and LSCC as well as the roles for different immunohistochemical stains in HPV detection. METHODS: A total of fifty-two formalin-fixed, paraffin-embedded tissue samples of LSCC, diagnosed between 2005 and 2015, were obtained from the archives of the Pathology Department. The samples were stained and processed to evaluate the relationship of HPV to LSCC. RESULTS: Patients had a mean age of 65.02 ± 14.341 years. By polymerase chain reaction (PCR), high-risk strains of HPV were detected in 15.4% of tissue samples. HPV-16 was found in 75% of the positive samples for an overall prevalence of 13.5%. P16 immunostaining was positive in 15.4% of cases while cyclin B and cyclin E were positive in 65.4% and 76.9%, respectively. Over half of the cases were histologically graded as moderately-differentiated, 28.8% as well-differentiated, and 15.4% as poorly-differentiated. DISCUSSION: The prevalence of HPV-positive LSCC was lower than previously stated in the literature. HPV-16 was the most commonly detected subtype, in concordance with the findings of multiple other studies. HPV-positive LSCC trended with higher histologic grade. P16 and cyclin E immunohistochemical stains were of limited use in identifying HPV in LSCC. In contrast, cyclin B had a high sensitivity which could be used to rule out HPV in LSCC.

p16 promotes proliferation in cervical carcinoma cells through CDK6-HuR-IL1A axis.

The Cyclin-Dependent Kinase Inhibitor p16 (p16) acts as a tumor suppressor in most cells, but for HPV transformed cervical cancer, in which oncoprotein E7 expressed by human papillomavirus (HPV) mediates the degradation of retinoblastoma protein (Rb), p16 exhibits oncogenic activity. Our study was conducted to study the mechanism underling p16 mediated promoting effect of cell proliferation in cervical cancer cell lines. CCK8 assay and EdU incorporation were conducted to evaluate cell proliferation. Loss-of-function assay was used to silence p16 in Ca Ski and SiHa cells. Next, western blot, qPCR, RNA silencing, luciferase activity assay, run-on assay, mRNA stability assay, RNA immunoprecipitation, co-immunoprecipitation Immunofluorescence were performed to examine the interaction between CDK6, HuR, and IL1A mRNA in p16 mediated proliferation promoting effect. Our results showed that: (1) Silencing p16 inhibited the proliferation of cervical cancer cells by decreasing the half-life of IL1A mRNA in CDK6 dependent manner; (2) The stabilization of IL1A mRNA was regulated by HuR which could be inactivated by p16/CDK6 mediated phosphorylation at Ser202; (3) IL1A mediated the oncogenic activity of p16 in cervical carcinoma cell lines. In conclusion, p16 promotes proliferation in cervical carcinoma cells through CDK6-HuR-IL1A axis.

Cervical Intraepithelial Neoplasia grade 2 biopsy: Do p16INK4a and Ki-67 biomarkers contribute to the decision to treat? A cross-sectional study

ABSTRACT BACKGROUND: Managing cervical intraepithelial neoplasia grade 2 (CIN2) is challenging, considering the CIN2 regression rate, perinatal risks associated with excisional procedures, and insufficient well-established risk factors to predict progression. OBJECTIVES: To determine the ability of p16INK4a and Ki-67 staining in biopsies diagnosed with CIN2 to identify patients with higher-grade lesions (CIN3 or carcinoma). DESIGN AND SETTING: Cross-sectional study conducted at a referral center for treating uterine cervical lesions. METHODS: In 79 women, we analyzed the correlation of p16INK4a and Ki-67 expression in CIN2 biopsies with the presence of a higher-grade lesions, as determined via histopathology in surgical specimens from treated women or via two colposcopies and two cytological tests during follow-up for untreated women with at least a 6-month interval. The expression of these two biomarkers was verified by at least two independent pathologists and quantified using digital algorithms. RESULTS: Thirteen (16.8%) women with CIN2 biopsy exhibited higher-grade lesions on the surgical excision specimen or during follow-up. p16INK4a expression positively and negatively predicted the presence of higher-grade lesions in 17.19% and 86.67% patients, respectively. Ki-67 expression positively and negatively predicted the presence of higher-grade lesions in 40% and 88.24% patients, respectively. CONCLUSIONS: Negative p16INK4a and Ki67 immunohistochemical staining can assure absence of a higher-grade lesion in more than 85% of patients with CIN2 biopsies and can be used to prevent overtreatment of these patients. Positive IHC staining for p16INK4a and Ki-67 did not predict CIN3 in patients with CIN2 biopsies.

DHA-enriched fish oil upregulates cyclin-dependent kinase inhibitor 2A (P16INK) expression and downregulates telomerase activity without modulating effects of PPARγ Pro12Ala polymorphism in type 2 diabetic patients: A randomized, double-blind, placebo-controlled clinical trial.

OBJECTIVE: The present study investigated the effects of docosahexaenoic acid (DHA)-enriched fish oil supplement on telomerase activity, mRNA expression of P16INK, IL-6, and TNF-α considering Pro12Ala polymorphism in the PPARγ gene. METHODS/DESIGN: In this double-blind randomized controlled trial, 72 PPARγ Pro12Ala polymorphism genotyped type 2 diabetic patients aged 30-70 years were randomly assigned to receive 2.4 gr of DHA-enriched fish oil or a placebo for 8 weeks. Genotyping of the Pro12Ala polymorphism in the PPARγ gene was assessed using polymerase chain reaction-restriction length polymorphism (PCR-RFLP), telomerase activity in the peripheral blood mononuclear cell (PBMC) was measured using PCR-ELISA based on the telomeric repeat amplification protocol (TRAP), and changes in the mRNA expression of P16, IL-6, and TNF-α were measured using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In the DHA group, telomerase activity was decreased (p = 0.001) during the intervention. In addition, between-group comparisons showed significant differences in the changes in telomerase activity (p = 0.003) and P16 mRNA expression (p = 0.028) and non-significant differences in TNF-α and IL-6 mRNA expression. The gene*DHA interaction could not affect changes in P16, IL-6, or TNF-α mRNA expression or in telomerase activity in PBMC. DISCUSSION: Short-time DHA-enriched fish oil supplementation caused increased levels of P16 expression and a decline in telomerase activity compared with the control group without modulating the effects of Pro12Ala polymorphism on the PPARγ gene. Because of the positive correlation between P16 activity and cellular senescence, the possibility of senescence stimulation by DHA is proposed.

Unilateral radiotherapy treatment for p16/human papillomavirus-positive squamous cell carcinoma of unknown primary in the head and neck.

OBJECTIVES/HYPOTHESIS: The outcomes of unilateral radiotherapy treatment for patients with p16/HPV-positive squamous cell carcinomas of unknown primary (SCCUP) affecting cervical lymph nodes are under-reported. Compared to radiating large volumes of the pharyngeal axis (the more common approach), this is potentially a much less toxic treatment for a good prognosis group. STUDY DESIGN: Retrospective cohort study. METHODS: We identified patients with SCCUP who were treated radically at our center and did not have parotid or isolated level IV or V nodal involvement. Failure-free and overall survivals were calculated using Kaplan-Meier methods. RESULTS: From 2004 to 2012, there were 49 radically treated patients with SCCUP. Fourteen patients had bilateral neck treatment (they had bilateral nodal disease or suspected lesions in the base of tongue, though not proven with biopsy), two had surgery alone, whereas 33 had unilateral radiotherapy (after neck dissection, excisional biopsy, or definitively with concurrent chemotherapy). Of the 33 patients, 21 tested positive to p16/HPV and had median follow-up of 57 months. In this group, no isolated contralateral neck failures or putative primaries emerged. There was 1/21 (4.3%) ipsilateral neck failure, 1/21 (4.3%) concurrent contralateral neck and distant failure, and 1/21 (4.3%) patient with distant failure. The 5-year freedom from failure was 78% (95% confidence interval [CI]: 56%-100%) and overall survival was 90% (95% CI: 79%-100%). CONCLUSIONS: With no emergence of putative primaries and no isolated contralateral neck failures, this single-institution experience in p16/HPV-positive SCCUP patients suggests that unilateral radiotherapy may be an underutilized management strategy. LEVELS OF EVIDENCE: 4 Laryngoscope, 128:2076-2083, 2018.

Dual p16 and Ki-67 Expression in Liquid-Based Cervical Cytological Samples Compared to Pap Cytology Findings, Biopsies, and HPV Testing in Cervical Cancer Screening: A Diagnostic Accuracy Study.

OBJECTIVE: Our objective was to verify the sensitivity and specificity of dual immunocytochemistry staining for p16 and Ki-67 in liquid-based samples (the "dual" assay) for cervical lesion screening, compared to biopsy findings and human papillomavirus (HPV) DNA molecular detection. STUDY DESIGN: Sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values for the "dual immunocytochemistry assay" were calculated and compared to histopathological results and to high-risk HPV DNA detection in adult women or teenagers submitted to cervical cancer screening. RESULTS: A total of 151 women were included. The majority (96.2%) of those with negative dual assay results had lower biopsy grades (p < 0.001). Women with cytology results suggestive of cervical cancer had positive dual immunocytochemistry assay results more frequently (p < 0.001), and these positive results were also significantly associated with biopsy findings (p < 0.001) and with high-risk genotype HPV infection (p = 0.007). Specificity and PPV for the dual assay were 0.972 (0.855-0.999) and 0.800 (0.284-0.995), respectively, and 1.000 (0.590-1.000) and 1.000 (0.631-1.000) for HPV detection. CONCLUSIONS: The dual immunocytochemistry assay had high specificity and PPV. It reveals a persistent HPV infection, avoiding the need for new tissue collections for biopsies or hybrid capture.

Human Papillomavirus Genotyping and p16(INK4a) Expression in Cervical Lesions: A Combined Test to Avoid Cervical Cancer Progression.

Cervical cancer is a major public health problem in Morocco. The cervical cancer has a long precancerous period that provides an opportunity for the screening and treatment. Improving screening tests is a priority goal for the early diagnosis of cervical cancer. This study was conducted to evaluate the combination of p16(INK4a) protein expression, human papillomavirus (HPV) typing, and histopathology for the identification of cervical lesions with high risk to progress to cervical cancer among Moroccan women. A total of 96 cervical biopsies were included in this study. Signal amplification in situ hybridization with biotinylated probes was used to detect HPV. Immunohistochemistry was used to evaluate the expression of p16(INK4a) protein. HPV DNA was detected in 74.0% of the biopsies (71/96). Of the seventy-one positive HPV cases, we detected 67.6% (48/71) of high risk (HR)-HPV (HPV 16 and 18), 24% of low risk-HPV (HPV 6 and 11), 1.4% intermediate risk-HPV (HPV 31, 33, and 35), and 7% coinfections (HPV 6/11 and 16/18). Overexpression of p16(INK4a) protein was observed in 72.9% (70/96) of the biopsies. In addition, p16(INK4a) protein detection was closely correlated with recovery of HR HPV. Our result showed that p16(INK4a) expression level is correlated with HR-HPV status.

Diagnostic value of combined detection of p16 and human papillomavirus typing in cervical intraepithelial neoplasia / 肿瘤研究与临床

Objective:To explore the application value of combined detection of p16 and human papillomavirus (HPV) typing in the diagnosis of cervical intraepithelial neoplasia (CIN).Methods:A total of 8 346 patients aged between 25 years old and 65 years old at Baoji Central Hospital of Shaanxi Province from February 2019 to February 2020 were selected. There were 2 882 patients with cervical lesions diagnosed by colposcopy biopsy. Patients were divided into the different groups based on the age range, and then the condition of HPV infection in all age groups was analyzed. Taking biopsy as the gold standard and according to the pathological results, the detection rate of p16 and HPV typing and the diagnostic value of the single and combined detection in CIN were also analyzed.Results:The age group with the highest positive rate of p16 and HPV was 31-40 years old [47.42% (1 014/2 427) and 36.84% (894/2 427), respectively], followed by 41-50 years old group [30.15% (907/2 942) and 28.11% (827/2 942)], and there were statistically significant differences in positive rate of p16 and HPV in all age groups (all P < 0.05). Among 2 882 patient with cervical lesions diagnosed by pathological examination, there were 2 572 cases (89.24%) of p16 positive, and 2 169 cases (75.26%) of HPV positive. With the disease progression of cervical lesions, the positive rate of p16 and HPV was gradually increased, and the positive rate of p16 of inflammation, CINⅠ, CINⅡ, CIN Ⅲ, cervical squamous cell carcinoma (SCC) was 11.68% (23/197), 94. 85% (1 105/1 165), 93.57% (771/824), 96.76% (538/556), 96.43% (135/140), respectively; the positive rate of HPV was 17.77% (35/197), 77.60% (904/1 165), 80.22% (661/824), 80.40% (447/556), 87.14% (122/140), respectively, and HPV infection was mostly HPV16/18 infection type with the disease progression of cervical lesions. The sensitivity, specificity, positive predictive value and negative predictive value in detecting CIN of HPV was 75.26%, 81.13%, 67.78% and 86.14%, respectively; the sensitivity, specificity, positive predictive value and negative predictive value in detecting CIN of p16 was 89.24%, 84.74%, 75.51% and 93.72%, respectively; the diagnostic efficacy of p16 was higher than that of HPV in detecting CIN, and the difference was statistically significant ( P < 0.05). The sensitivity, specificity, positive predictive value and negative predictive value of HPV combined with p16 in detecting CIN was 94.10%, 91.33%, 85.12%, 96.71%, which were higher compared with those of single detection (all P < 0.05). Conclusions:HPV infection mainly occurs in women aged 31-40 years old followed by 41-50 years old, and the infected population of CIN tends to be younger. p16 is superior to HPV in detecting the diagnostic efficacy of CIN; combined detection of p16 and HPV can increase the sensitivity and specificity, reduce the rate of misdiagnosis, and can play a key clinical value in early diagnosis and treatment of CIN.

RNA in-situ hybridization is a practical and effective method for determining HPV status of oropharyngeal squamous cell carcinoma including discordant cases that are p16 positive by immunohistochemistry but HPV negative by DNA in-situ hybridization.

OBJECTIVES: Evaluation of human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (OPSCC) has become increasingly important for prognostication and clinical trial enrollment. This assessment is confounded in OPSCCs that are p16 positive by immunohistochemistry (IHC) but HPV negative by DNA in situ hybridization (DISH). This study evaluates whether E6/E7 mRNA in situ hybridization (RISH) can detect transcriptionally active HPV in these problematic cases. MATERIALS AND METHODS: Eighty-two head and neck squamous cell carcinoma cases that had previously undergone p16 IHC and HPV DISH were evaluated with two RISH platforms and a second-generation DISH probe. The study included 21 p16+/DISH+ concordant cases, 19 p16-/DISH- concordant cases, and 42 p16+/DISH- discordant cases. RESULTS: RISH identified E6/E7 mRNA in 37 (88%) p16+/DISH- cases, 21 (100%) p16+/DISH+ cases, and 0 (0%) p16-/DISH- cases. RISH signals were clearly visible at low to medium magnification in 97% of positive cases, facilitating almost-perfect inter-observer reproducibility. The performance of the manual and automated RISH platforms were equivalent (kappa=0.915). Only 29% of carcinomas that demonstrated E6/E7 mRNA transcriptional activity were positive using the 2nd generation DISH probe. CONCLUSIONS: HPV RISH is a highly sensitive and specific platform that can clarify the HPV status of those perplexing OPSCCs that are p16 positive by IHC but HPV negative by DISH. Moreover, it is easy to interpret, readily adaptable to the clinical laboratory, and provides direct evidence of HPV transcriptional activity. E6/E7 RISH should be considered as a first-line platform for determination of HPV status in OPSCCs.

Prognostic value of p16(INK4a) as a marker of clinical evolution in patients with cervical intraepithelial neoplasia grade 3 (CIN 3) treated by cervical conization.

The aim of this study was to assess the prognostic value of p16(INK4a) as a marker of post-conization relapse in patients treated for cervical intraepithelial neoplasia grade 3 (CIN 3). A retrospective study of 76 women with CIN 3 diagnoses, treated at the Hospital of Santa Casa de Misericórdia of São Paulo (Brazil) between January 2003 and September 2004, was performed. The study samples were obtained from cervical conization procedures, where paraffin blocks containing areas with the greatest amount of neoplastic tissue were selected. Immunohistochemical techniques were used on individual paraffin blocks for each case to detect p16(INK4a) protein expression. The p16(INK4a) cell counts were performed in 10 different high-amplification fields (400x) by light microscopy and total cell count expressed as number of cells per mm(2) . Patients involved in this study were followed up at the colposcopy outpatient unit for at least 48 months after cervical conization. The correlation of p16(INK4a) values with post-conization evolution in the patients (disease relapse or disease free) was determined. A significantly higher count of cells expressing p16(INK4a) was found in those patients with disease relapse during follow-up (p < 0.001). The variables age, number of gestations, and births correlated positively with number of cells expressing p16(INK4a) cells (p < 0.001; p = 0.001; 0.009, respectively). No correlation was found for the variables menopause, hormonal contraception, or smoking (p = 0.369, 0.425 and 0.853, respectively). p16(INK4a) can be considered a biomarker of cervical intraepithelial neoplasia grade 3 cases presenting high risk of relapse or evolution to invasive carcinoma.