Sodium valproate affects the expression of p16INK4a and p21WAFI/Cip1 cyclin­dependent kinase inhibitors in HeLa cells.

p16INK4a and p21WAF1/Cip1 are cyclin-dependent kinase inhibitors involved in cell cycle control, which can function as oncogenes or tumor suppressors, depending on the context of various extracellular and intracellular signals, and cell type. In human papillomavirus-induced cervical cancer, p16 INK4a shows oncogenic activity and functions as a diagnostic marker of cervical neoplasia, whereas p21 WAF1/Cip1 acts as a tumor suppressor and its downregulation is associated with the progression of malignant transformation. Several histone deacetylase (HDAC) inhibitors promote the positive and negative regulation of a number of genes, including p16 INK4a and p21 WAF1/Cip1; however, the effects of sodium valproate (VPA) on these genes and on the proteins they encode remain uncertain in HeLa cervical cancer cells. In the present study, these effects were investigated in HeLa cells treated with 0.5 or 2 mM VPA for 24 h, using reverse transcription-quantitative PCR, confocal microscopy and western blotting. The results revealed a decrease in the mRNA expression levels of p16 INK4a and a tendency for p16INK4a protein abundance to decrease in the presence of 2 mM VPA. By contrast, an increase in the protein expression levels of p21WAF1/Cip1 was detected in the presence of 0.5 and 2 mM VPA. Furthermore, VPA was confirmed to inhibit HDAC activity and induce global hyperacetylation of histone H3. Notably, VPA was shown to suppress p16 INK4a, a biomarker gene of cervical carcinoma, and to increase the abundance of the tumor suppressor protein p21WAF1/Cip1, thus contributing to the basic knowledge regarding the antitumorigenic potential of VPA. Exploration of epigenetic changes associated with the promoters of p16 INK4a and p21 WAF1/Cip1, such as histone H3 methylation, may provide further information and improve the understanding of these findings.

Findings and Challenges in Replacing Traditional Uterine Cervical Cancer Diagnosis with Molecular Tools in Private Gynecological Practice in Mexico.

We have been encouraging practicing gynecologists to adopt molecular diagnostics tests, PCR, and cancer biomarkers, as alternatives enabled by these platforms, to traditional Papanicolaou and colposcopy tests, respectively. An aliquot of liquid-based cytology was used for the molecular test [high-risk HPV types, (HR HPV)], another for the PAP test, and one more for p16/Ki67 dual-stain cytology. A total of 4499 laboratory samples were evaluated, and we found that 25.1% of low-grade samples and 47.9% of high-grade samples after PAP testing had a negative HR HPV-PCR result. In those cases, reported as Pap-negative, 22.1% had a positive HR HPV-PCR result. Dual staining with p16/Ki67 biomarkers in samples was positive for HR HPV, and 31.7% were also positive for these markers. Out of the PCR results that were positive for any of these HR HPV subtypes, n 68.3%, we did not find evidence for the presence of cancerous cells, highlighting the importance of performing dual staining with p16/Ki67 after PCR to avoid unnecessary colposcopies. The encountered challenges are a deep-rooted social reluctance in Mexico to abandon traditional Pap smears and the opinion of many specialists. Therefore, we still believe that colposcopy continues to be a preferred procedure over the dual-staining protocol.

Primary hepatic pleomorphic liposarcoma: Case report and literature review.

Primary hepatic liposarcoma is an extremely rare malignant tumour derived from adipocytes and is part of the group of mesenchymal tumours. We present the case of a 43-year-old Hispanic male patient with a pleomorphic hepatic liposarcoma and absence of MDM2 gene amplification. Two years and six months after surgery, the patient is asymptomatic. The present case is the first report of this entity with positive immunohistochemical testing for p16, p53, S100, vimentin and absence of MDM2 gene amplification.

Oral HPV-associated dysplasia: is koilocytic dysplasia a separate entity?

Oral epithelial dysplasia associated with high-risk HPV infection has received different names since its initial description, such as oral Bowenoid lesions, HPV-associated intraepithelial neoplasia, and oral koilocytic dysplasia. Some features, identified in more or less quantity in some of the descriptions, like apoptotic keratinocytes, karyorrhexis, and mitosoid figures, are intricately connected to viral transcriptional status and, consequently, viral load. Since the variety in terminology has introduced diagnostic confusion within medical and research communities, establishing a uniform and standardized approach to diagnosing HPV-oral epithelial dysplasia is crucial for accurate and early diagnoses and holds significant implications for patient outcomes, particularly in high-risk individuals.

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.

Influence of p16 Protein Immunostaining on Histopathological Features of Pleomorphic Adenoma and Carcinoma ex- Pleomorphic Adenoma.

The objective of this study was to evaluate the role of p16 in histologic characteristics and transition of Pleomorphic Adenoma (PA) to Carcinoma ex-PA (CxPA). So, 60 PA and 4 CxPA were histologic reviewed based on microscopic characteristics proposed by Hellquist, Triantafyllou and Dulguerov (PA) and Morais, Antony and Toluie (CxPA). Immunostaining for p16 was associated in different parenchyma and stroma of both tumors and Fisher's/chi-square tests and Mann-Whitney test were performed (SPSS v20.0, p<0.05). In PA the periductal cells were predominantly p16- and that ductal and myoepithelial cells showed a significant increase in p16+ cells (p<0.001). In CxPA, none of the cases showed p16+ in periductal cells, most parotid cases showed p16+ in ductal cells, and one case of parotid and the submandibular case showed mild immunostaining for myoepithelial cells. There was a small reduction in p16+ in CxPA compared to PA (p=0.537), but in both tumors there was less p16+ cells in solid stroma than other (p<0.001). The p16+ cases of PA had a higher capsular thickness (p=0.047). So, the loss of p16 immunostaining does not seem to be associated with the transition from PA to CxPA, but in both tumors the loss of p16+ cells are related to microscopic aggressiveness.

Human Papillomavirus-Associated Oral Epithelial Dysplasia: Report of 5 Illustrative Cases from Latin America.

BACKGROUND: Human papillomavirus-associated oral epithelial dysplasia (HPV-OED) is a distinct oral epithelial disorder characterized by viral cytopathic changes caused by transcriptionally active high-risk HPV. The aim of the present study was to report 5 additional cases from Latin America. METHODS: Clinical data from five patients with HPV-OED were obtained from the archives of three oral pathology services from Brazil and Chile. All cases were submitted to morphological, p16 expression and in situ hybridization (ISH) for HPV analyses. RESULTS: Four patients were male and one patient was female, with a mean age of 55.4 years. Four patients were HIV seropositive and two were smokers. Three cases affected the buccal mucosa and commissure, one of which had an additional plaque in the soft palate, and one case each occurred on the floor of mouth and lower labial mucosa. Most cases presented as well-demarcated white plaques with a verrucous surface. One case presented multiple lesions ranging from normal to white-colored slightly elevated plaques with a cobblestone surface. Peripheral mucosal pigmentation was observed in two cases. All five cases presented with the characteristic microscopic features of HPV-OED, including severe dysplasia with numerous karyorrhectic and apoptotic cells, full-thickness "block positivity" for p16 and high Ki-67 index (> 90%) sharply demarcated from the adjacent non-dysplastic epithelium. Wide-spectrum DNA ISH-HPV was positive in 4 cases. All patients were treated with conservative surgical excision with no signs of recurrence after a mean of 39-month follow-up. CONCLUSION: This represents the first series of HPV-OED from Latin America; most cases presented as well-demarcated papillary white plaques affecting the buccal mucosa and commissure of HIV-positive middle-aged men, two of them exhibiting peripheral pigmentation caused by reactive melanocytes. The typical microscopic findings of HPV-OED were observed in all cases, which also showed strong p16 positivity in a continuous band through the full thickness of the epithelium and high Ki67.

DNA high-risk HPV, mRNA HPV and P16 tests for diagnosis of anal cancer and precursor lesions: a systematic review and meta-analysis.

Background: Anal cancer prevention has two critical points: the incidence rate is several fold higher for some groups, such as people living with human immunodeficiency virus (HIV) and men who have sex with men (MSM), and there is not a well-defined guideline for its screening. This systematic review evaluates the accuracy of DNA HRHPV (high-risk human papillomavirus), mRNA HPV, DNA HPV16 isolated and p16 staining biomarkers in anal canal smears for identifying anal intraepithelial neoplasia (AIN) 2 or 3, summarised as anal high-grade squamous intraepithelial lesions (aHSIL), and cancer. Methods: We searched the MEDLINE, Cochrane Library and Embase electronic databases as well as Grey literature to identify eligible papers published up to 31st July 2022. This systematic review and meta-analysis included observational studies comparing biomarker tests to histopathology after HRA (High-resolution Anoscopy) as a reference standard. We (ACM, TF) analysed studies in which patients of both sexes were screened for anal cancer using DNA HRHPV, mRNA HPV, DNA HPV16 and/or p16 biomarkers. The analysis was performed in pairs, for instance AIN2 or worse (AIN2+) vs. AIN1, HPV infection and normal (AIN1-). PROSPERO CRD42015024201. Findings: We included 21 studies with 7445 patients. DNA HR HPV showed a higher sensitivity 92.4% (95% CI 84.2-96.5), specificity 41.7% (95% CI 33.9-44.9) and AUC 0.67, followed by the mRNA HPV test, with a sensitivity 77.3% (95% CI 73.2%-80.9%), specificity 61.9% (95% CI 56.6-66.9) and AUC 0.78. DNA HPV16 showed higher specificity 71.7% (95% CI 55.3-83.8), followed by p16 test, 64.1% (95% CI 51.0-75.4); Sensitivity of DNA HPV16 was 53.3% (95% CI 35.4-70.3) and AUC 0.69, while p16 had a sensitivity of 68.8% (95% CI 47.9-84.1) and AUC 0.74. Subgroup analysis of MSM with HIV, with 13 studies and 5123 patients, showed similar accuracy, with a bit higher sensitivities and lower specificities. Considering the measure of the total between-study variability, mRNA HPV tests showed the smallest area of the 95% prediction ellipse, 6.0%, influenced by the low logit sensitivity, 0.011. All other groups of tests exceed 50% prediction ellipse area, which represent a high heterogeneity. Interpretation: Our findings suggested that DNA HR HPV can be a useful tool for screening for aHSIL and anal cancer if followed by biomarker with a higher specificity. As an isolated test, mRNA HPV had better performance. Funding: There was no funding source for this study.

p16INK4a and pRb expression in laryngeal squamous cell carcinoma with and without infection by EBV or different genotypes of HPV: a retrospective study.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) represents one of the principal tumors of the head and neck. Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are considered risk factors for the development and the clinical prognosis of LSCC. High levels of p16INK4a are suggested as a surrogate marker of HPV or EBV infection in some head and neck tumors but in LSCC is still controversial. Furthermore, pRb expression may be considered an additional biomarker but it has not been clearly defined. This work aimed to compare the expression of pRb and p16INK4a as possible biomarkers in tumor tissues with and without infection by EBV or different genotypes of HPV from patients with LSCC. METHODS: Tumor samples from 103 patients with LSCC were previously investigated for the presence and genotypes of HPV using the INNO-LiPA line probe assay and for the infection of EBV by qPCR. p16 INK4a and pRb expression was assessed by immunohistochemistry. RESULTS: Of the 103 tumor samples, expression of p16INK4a was positive in 55 (53.4%) and of this, 32 (56.1%) were positive for HPV whereas 11 (39.3%) were EBV positive but both without a significantly difference (p > 0.05). pRb expression was positive in 78 (75.7%) and a higher frequency of this expression was observed in HPV negative samples (87.0%) (p = 0.021) and in high-risk HPV negative samples (85.2%) (p = 0.010). No difference was observed when comparing pRb expression and EBV infection status (p > 0.05). CONCLUSION: Our results support the suggestion that p16INK4a is not a reliable surrogate marker for identifying HPV or EBV infection in LSCC. On the other hand, most of our samples had pRb expression, which was more frequent in tumors without HPV, suggesting that pRb could indicate HPV negativity. However, more studies with a larger number of cases are required, including controls without LSCC and evaluating other molecular markers to determine the real role of p16INK4a and pRb in LSCC.

Immunostaining of ßA-Activin and Follistatin Is Decreased in HPV(+) Cervical Pre-Neoplastic and Neoplastic Lesions.

The activin-follistatin system regulates several cellular processes, including differentiation and tumorigenesis. We hypothesized that the immunostaining of ßA-activin and follistatin varies in neoplastic cervical lesions. Cervical paraffin-embedded tissues from 162 patients sorted in control (n = 15), cervical intraepithelial neoplasia (CIN) grade 1 (n = 38), CIN2 (n = 37), CIN3 (n = 39), and squamous cell carcinoma (SCC; n = 33) groups were examined for ßA-activin and follistatin immunostaining. Human papillomavirus (HPV) detection and genotyping were performed by PCR and immunohistochemistry. Sixteen samples were inconclusive for HPV detection. In total, 93% of the specimens exhibited HPV positivity, which increased with patient age. The most detected high-risk (HR)-HPV type was HPV16 (41.2%) followed by HPV18 (16%). The immunostaining of cytoplasmatic ßA-activin and follistatin was higher than nuclear immunostaining in all cervical epithelium layers of the CIN1, CIN2, CIN3, and SCC groups. A significant decrease (p < 0.05) in the cytoplasmic and nuclear immunostaining of ßA-activin was detected in all cervical epithelial layers from the control to the CIN1, CIN2, CIN3, and SCC groups. Only nuclear follistatin immunostaining exhibited a significant reduction (p < 0.05) in specific epithelial layers of cervical tissues from CIN1, CIN2, CIN3, and SCC compared to the control. Decreased immunostaining of cervical ßA-activin and follistatin at specific stages of CIN progression suggests that the activin-follistatin system participates in the loss of the differentiation control of pre-neoplastic and neoplastic cervical specimens predominantly positive for HPV.

Tumor risk markers in recurrent respiratory papillomatosis

Abstract Objectives: This study aims to investigate the pattern of immunoexpression of proteins p16, p53 and Ki-67 in RRP, as well as to evaluate its influence on the number of surgeries that patients have undergone to date and to analyze the benefit of immunohistochemistry in this disease. Methods: Clinical-demographic data and tumor samples were obtained from 33 patients with RRP. The expression of proteins p16, p53 and Ki-67 was analyzed by immunohistochemical method. Results: Most patients had already undergone more than one surgery. The p16 marker was negative in 24.2% of the cases, with little positivity in 27.3% of the cases, moderate in 36.4% and intense in 12.1%. The p53 marker was positive in all cases, with little immunoexpression in 39.4% of cases, moderate in 36.4% and intense in 24.2%. The Ki-67 marker showed nuclear positivity in all lesions, although in varying degrees, with a mean proliferative index ±SD (standard deviation) of 51.7±26. Conclusions: The papillomatous lesions had varying degrees of immunoexpression of proteins p16, p53 and Ki-67, but no specific immunohistochemical pattern was observed. It was found, with statistical difference, that the number of surgeries was higher in cases with greater intensity of p53 expression, without correlation with the other markers. The benefit of immunohistochemistry in recurrent respiratory papillomatosis may lie in the prognostic assessment. However, further studies are needed to evaluate the use of this technique for this purpose. Level of evidence:: 4.

Penile intraepithelial neoplasia: Distribution of subtypes, HPV genotypes and p16INK4a in 84 international cases.

There are few pathologic or molecular studies of penile precancerous lesions, and the majority refers to lesions associated with invasive carcinomas. Penile Intraepithelial Neoplasia (PeIN) is classified in two morphologically and distinctive molecular groups, non-HPV and HPV-related with special subtypes. The primary purpose of this international series was to classify PeIN morphologically, detect HPV genotypes and determine their distribution according to PeIN subtypes. A secondary aim was to evaluate the p16INK4a immunostaining as a possible HPV surrogate for high-risk HPV infection in penile precancerous lesions. Samples consisted of 84 PeIN cases, part of a retrospective cross-sectional analysis of 1095 penile carcinomas designed to estimate the HPV DNA prevalence in penile cancers using PCR and p16INK4a immunostaining. Penile Intraepithelial Neoplasia (PeIN) was classified in HPV-related (basaloid, warty-basaloid, warty, hybrid, and mixed subtypes) and non-HPV-related (differentiated), the former being the most frequent. PeIN subtypes were differentiated (non-HPV-related) and basaloid, warty-basaloid, warty, hybrid and mixed (HPV-related). Basaloid PeIN was the most commonly diagnosed subtype, and HPV16 was the most frequent HPV genotype detected. Warty-basaloid and warty PeIN showed a more heterogeneous genotypic composition. Most HPV genotypes were high-risk but low-risk HPV genotypes were also present in a few cases (4%). A single HPV genotype was detected in 82% of HPV positive cases. In contrast, multiple genotypes were detected in the remaining 18% of cases. The findings in this study support the paradigm that penile in situ neoplasia, like its invasive counterparts, is HPV dependent or independent and has distinctive morphological subtypes readily identified in routine practice. Considering that HPV16 is clearly the predominant type, and that the three available vaccines have HPV16, all of them will be suitable for vaccination programs; the price of the vaccines will be probably the main determinant to choose the vaccine.

Tumor risk markers in recurrent respiratory papillomatosis.

OBJECTIVES: This study aims to investigate the pattern of immunoexpression of proteins p16, p53 and Ki-67 in RRP, as well as to evaluate its influence on the number of surgeries that patients have undergone to date and to analyze the benefit of immunohistochemistry in this disease. METHODS: Clinical-demographic data and tumor samples were obtained from 33 patients with RRP. The expression of proteins p16, p53 and Ki-67 was analyzed by immunohistochemical method. RESULTS: Most patients had already undergone more than one surgery. The p16 marker was negative in 24.2% of the cases, with little positivity in 27.3% of the cases, moderate in 36.4% and intense in 12.1%. The p53 marker was positive in all cases, with little immunoexpression in 39.4% of cases, moderate in 36.4% and intense in 24.2%. The Ki-67 marker showed nuclear positivity in all lesions, although in varying degrees, with a mean proliferative index ±â€¯SD (standard deviation) of 51.7 ±â€¯26. CONCLUSIONS: The papillomatous lesions had varying degrees of immunoexpression of proteins p16, p53 and Ki-67, but no specific immunohistochemical pattern was observed. It was found, with statistical difference, that the number of surgeries was higher in cases with greater intensity of p53 expression, without correlation with the other markers. The benefit of immunohistochemistry in recurrent respiratory papillomatosis may lie in the prognostic assessment. However, further studies are needed to evaluate the use of this technique for this purpose. LEVEL OF EVIDENCE: 4.

HPV Prevalence and Predictive Biomarkers for Oropharyngeal Squamous Cell Carcinoma in Mexican Patients.

BACKGROUND: Worldwide prevalence of Oropharyngeal Squamous Cell Carcinoma (OPSCC) has increased, affecting mostly young males. OPSCC associated with Human Papillomavirus (HPV) infection exhibits particular characteristics in terms of response to treatment, hence HPV has been proposed as a prognostic factor. The impact of HPV positivity and associated biomarkers on OPSCC in the Mexican population has not been addressed. Therefore, the analysis of OPSCC prognostic markers in the Mexican population is necessary. METHODS: Retrolective study in Mexican OPSCC patients, where HPV prevalence, p16 and EGFR levels were assessed using INNO-LiPA and immunohistochemistry. RESULTS: We found an HPV prevalence of 57.6% in OPSCC cases treated at a reference center in Mexico. HPV and p16 positivity, as well as EGFR, associate with better outcomes in OPSCC patients, and they also promote reduced death risk. Notably, HPV presence and p16 positivity showed a significant association with disease-free survival (DFS), with a HR of 0.15 (p = 0.006) and a HR of 0.17 (p = 0.012), respectively, indicating a possible role as predictive biomarkers in Mexican OPSCC patients. CONCLUSIONS: Our results reflect the clinical utility of p16 analysis to improve overall survival (OS) and to predict recurrence in oropharyngeal cancer. These results position p16 and HPV as predictive biomarkers for OPSCC.

HPV infection and 5mC/5hmC epigenetic markers in penile squamous cell carcinoma: new insights into prognostics.

BACKGROUND: Penile cancer is one of the most aggressive male tumors. Although it is preventable, the main etiologic causes are lifestyle behaviors and viral infection, such as human papillomavirus (HPV). Long-term epigenetic changes due to environmental factors change cell fate and promote carcinogenesis, being an important marker of prognosis. We evaluated epidemiological aspects of penile squamous cell carcinoma (SCC) and the prevalence of HPV infection using high-risk HPV (hrHPV) and p16INK4A expression of 224 participants. Global DNA methylation was evaluated through 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). RESULTS: The incidence of HPV was 53.2% for hrHPV and 22.32% for p16INK4a. hrHPV was not related to systemic or lymph node metastasis and locoregional recurrence, nor influenced the survival rate. P16INK4a seems to be a protective factor for death, which does not affect metastasis or tumor recurrence. Lymph node and systemic metastases and locoregional recurrence increase the risk of death. An increased 5mC mark was observed in penile SCC regardless of HPV infection. However, there is a reduction of the 5hmC mark for p16INK4a + (P = 0.024). Increased 5mC/5hmC ratio (> 1) was observed in 94.2% of penile SCC, irrespective of HPV infection. Despite the increase in 5mC, it seems not to affect the survival rate (HR = 1.06; 95% CI 0.33-3.38). CONCLUSIONS: P16INK4a seems to be a good prognosis marker for penile SCC and the increase in 5mC, an epigenetic mark of genomic stability, may support tumor progression leading to poor prognosis.

A comprehensive analysis of penile cancer in the region with the highest worldwide incidence reveals new insights into the disease.

BACKGROUND: Although penile cancer (PC) is uncommon in developed countries, it is widespread in developing countries. The state of Maranhão (Northeast, Brazil) has the highest global incidence recorded for PC, and, despite its socioeconomic vulnerability, it has been attributed to human papillomavirus (HPV) infection. This study aimed to determine the histopathological features, the prevalence of HPV infection, and the immunohistochemical profile of PC in Maranhão. METHODS: A retrospective cohort of 200 PC cases were evaluated. HPV detection was performed using nested-PCR followed by direct sequencing for genotyping. Immunohistochemistry (IHC) was performed using monoclonal antibodies anti-p16INK4a, p53, and ki-67. RESULTS: Our data revealed a delay of 17 months in diagnosis, a high rate of penile amputation (96.5%), and HPV infection (80.5%) in patients from Maranhão (Molecular detection). We demonstrated the high rate of HPV in PC also by histopathological and IHC analysis. Most patients presented koilocytosis (75.5%), which was associated with those reporting more than 10 different sexual partners during their lifetime (p = 0.001). IHC revealed frequent p16INK4a overexpression (26.0%) associated with basaloid (p < 0.001) and high-grade tumors (p = 0.008). Interestingly, p16 appears not to be a better prognostic factor in our disease-free survival analysis, as previously reported. We also demonstrated high ki-67 and p53 expression in a subset of cases, which was related to worse prognostic factors such as high-grade tumors, angiolymphatic and perineural invasion, and lymph node metastasis. We found a significant impact of high ki-67 (p = 0.002, log-rank) and p53 (p = 0.032, log-rank) expression on decreasing patients' survival, as well as grade, pT, stage, pattern, and depth of invasion (p < 0.05, log-rank). CONCLUSIONS: Our data reaffirmed the high incidence of HPV infection in PC cases from Maranhão and offer new insights into potential factors that may contribute to the high PC incidence in the region. We highlighted the possible association of HPV with worse clinical prognosis factors, differently from what was observed in other regions. Furthermore, our IHC analysis reinforces p16, ki-67, and p53 expression as important diagnosis and/or prognosis biomarkers, potentially used in the clinical setting in emerging countries such as Brazil.

Transcriptional regulation of CDKN2A/p16 by sirtuin 7 in senescence.

Cell senescence is a state of limited cell proliferation during a stress response or as part of a programmed process. When a senescent cell stops dividing, maintaining metabolic activity contributes to cellular homeostasis maintenance. In this process, the cell cycle is arrested at the G0/G1 phase. p16INK4A protein is a key regulator of this process via its cyclin­dependent kinase inhibitor (CDKI) function. CDKI 2A (CDKN2A)/p16 gene expression is regulated by DNA methylation and histone acetylation. Sirtuins (SIRTs) are nicotinamide dinucleotide (NAD+)­dependent deacetylases that have properties which prevent diseases and reverse certain aspects of aging (such as immune, metabolic and cardiovascular diseases). By performing quantitative PCR, Western blot, ChIP, and siRNAs assays, in this study it was demonstrated that CDKN2A/p16 gene transcriptional activation and repression were accompanied by selective deposition and elimination of histone acetylation during the senescence of MRC5 cells. Specifically, significant H3K9Ac and H3K18Ac enrichment in cells with a senescent phenotype concomitant with CDKN2A/p16 gene overexpression was demonstrated compared with the non­senescent phenotype. Furthermore, the presence of H3K18Ac in deacetyl­transferase SIRT7 knockdown MRC5 cells allowed CDKN2A/p16 promoter activation. These results suggested that SIRT7 served as a critical component of an epigenetic mechanism involved in senescence mediated by the CDKN2A/p16 gene.

Human Papillomavirus, Cytomegalovirus Infection and P16 Staining in Breast Tumors from Peruvian Women.

OBJECTIVE: To evaluate the frequency distribution of viral infections in Peruvian Breast Cancer (BC) lesions and its association with clinicopathological features. Additionally, a prospective evaluation of p16 and Tumor-infiltrating lymphocytes (TIL) levels were performed for developing a comprehensive analysis. METHODS: Detection of high risk- human papillomavirus (HR- HPV) through qPCR was performed in 447 BC and 79 non-cancer frozen samples. Paired paraffin samples from 238 BC were stained with Human cytomegalovirus (HCMV) and p16 immunohistochemistry. TIL was calculated in 397 BC cases. RESULTS: HCMV was positive in 72.5%. HR- HPV was detected in 2.9% of BC and 1.3% of non-malignant samples. P16+ was found in 28.15% and median TIL percentage was 30. HR- HPV infection was associated with non-ductal histology (p=0.003) and p16+ (p=0.017). Positive P16+ was associated with higher T stage (p=0.022), grade (p=0.009), TIL level (p=0.002), and triple-negative phenotype (p=0.021). CONCLUSION: HCMV is frequent, but HR- HPV infection is unusual in Peruvian BC. P16+ is associated with HR- PVH infection, high TIL and aggressive features.

Evaluation of genome-wide association signals for nonsyndromic cleft lip with or without cleft palate in a multiethnic Brazilian population.

OBJECTIVE: To evaluate previous nonsyndromic cleft lip with or without cleft palate (NSCL±P) associated signals in 4p16.2, 8p11.23, 12q13.13, 12q13.2 and 17q21.32 in a multiethnic Brazilian cohort. DESIGN: The single nucleotide polymorphisms (SNPs) rs34246903 in 4p16.2, rs13317 in 8p11.23 (FGFR1, fibroblast growth factor receptor 1), rs3741442 in 12q13.13, rs705704 in 12q13.2 and rs4968247 in 17q21.32 were genotyped with TaqMan allelic discrimination assays in a case-control sample including 801 NSCL±P patients [233 nonsyndromic cleft lip (NSCLO) and 568 nonsyndromic cleft lip and palate (NSCLP)] and 881 healthy controls. Multiple logistic regression analyses, considering sex and genomic ancestry as covariates, were conducted, and the p value was adjusted with Bonferroni multiple correction testing (p ≤ 0.01). RESULTS: Although several associations were identified, those that resisted the multiple correction testing involved the alleles and genotypes of rs34246903 and rs13317. The NSCLO group had a lower frequency of the minor C allele of rs34246903 compared to controls, giving an odds ratio (OR) of 0.74 [95% confidence interval (CI): 0.59-0.93, p = 0.01]. The rs34246903 CC genotype (homozygous) and the recessive model revealed significant protective associations with NSCLO, yielding ORs of 0.50 (95% CI: 0.29-0.85, p = 0.005) and 0.55 (95% CI: 0.33-0.93, p = 0.01) respectively. The presence of C variant allele of rs13317 (OR: 0.81, 95% CI: 0.69-0.96, p = 0.01) as well the TC genotype (OR: 0.77, 95% CI: 0.62-0.94, p = 0.01) and the dominant model (OR: 0.77, 95% CI: 0.63-0.94, p = 0.009) showed significant associations with reduced risk of NSCL±P. CONCLUSION: Our study is the first to support the association of rs34246903 (4p16.2) with NSCLO and rs13317 within FGFR1 with NSCL±P in the highly admixed Brazilian population. Further studies are needed to determine the functionality of those SNPs or to identify the causal markers in linkage disequilibrium with those susceptibility markers.

Hierarchical evaluation of histology and p16-labeling can improve the risk assessment on cervical intraepithelial neoplasia progression.

OBJECTIVE: High-grade cervical lesions (HSIL) are associated with the presence of high-risk HPV types, tissue expression of p16, and increased chance of malignant progression, requiring surgical intervention. To improve risk evaluation, we assessed the discriminatory power of the histological findings associated with p16 immunohistochemistry (IHC) staining to classify the low-grade cervical lesion (LSIL) and HSIL. METHODS: We collected cervical biopsies from colposcopy-visible lesions and non-affected tissue (adjacent to the lesions) of 62 Brazilian women and labeled them with anti-p16 antibodies. In addition to the observational pattern and labeling to define the latent classes (affected vs. non-affected), a computational tool was used for semi-quantitative analysis of p16 expression. The intensity of staining of the nucleus or cytoplasm was captured using the Gimp 2.10 software. ROC curves were used to determine cutoff values for p16 expression in patients classified as LSIL and HSIL by latent class statistics for each labeling stratum. RESULTS: p16 nuclear labeling showed the best sensitivity and specificity to discriminate LSIL with low p16 expression (62%) and HSIL with high p16 expression (37%). Many patients whose lesions had intermediate levels of p16 nuclear staining were subsequently stratified according to the expression of p16 in the cytoplasm, indicating that five of 21 LSIL were at risk of progression, and 13 of 41 HSIL at risk of regression. CONCLUSIONS: We suggest a hierarchical analysis, with histology at the first level, followed by a labeling analysis in the nucleus and then in the cytoplasm to increase the accuracy of the HPV cervical lesion stratification.