Expression Of High-risk Human Papillomavirus Research Articles

Prognostic Significance of p16 and Its Relationship with Human Papillomavirus Status in Patients with Penile Squamous Cell Carcinoma: Results of 5 Years Follow-Up.

Penile Squamous Cell Carcinoma (PSCC) is associated with high-risk human papillomavirus (HR-HPV). The immunohistochemical (IHC) test for p16INK4a (p16) is highly correlated with HR-HPV expression in other SCCs. To investigate whether the expression of p16 IHC or HR-HPV is associated with survival in PSCC, we conducted a single institution analysis of 143 patients with a diagnosis of PSCC and, available tissue were tested for p16 IHC staining patterns, histological subtype, tumor grade, and lymphovascular invasion (LVI) by an experienced pathologist. HR-HPV status using the Cobas PCR Assay or the RNAScope high-risk HPV in situ hybridization kit were also assessed. Patient characteristics were summarized using descriptive statistics of clinico-pathologic variables. Kaplan-Meier was used to estimate median overall survival (OS), cancer specific survival (CSS) and correlated with HPV, p16, and other study variables. Patients with p16+ tumors had a significantly longer median CSS in comparison to the p16- group (p = 0.004), with respective 5-year CSS probability of 88% (95% CI; 0.84, 1) versus 58% (95% CI; 0.55, 0.76; p = 0.004). HPV status did not predict survival outcomes. Multivariable analysis with respect to OS and CSS, showed that p16+ status was associated with a lower risk of death (HR = 0.36, 95%CI; 0.20-0.67, p = 0.001), and improved CSS (HR = 0.20, 95% CI; 0.07-0.54, p = 0.002) after adjusting for covariates. In conclusion, tumor p16 status via IHC was an easy to perform independent prognostic factor for OS and CSS that correlates with HR-HPV expression.

The Expression of HPV E6/E7 mRNA In Situ Hybridization in HPV Typing-negative Cervical Cancer.

High-risk human papillomavirus (HPV) persistent infection is the major tumorigenesis factor for cervical cancer (CC). However, the incidence of HPV-negative CC is 5% to 30% with different HPV detection methods. High-risk HPV E6/E7 mRNA in situ hybridization (RISH) can detect HPV-driven tumors. Our study aimed to explore whether HPV typing-negative CC was caused by HPV infection. The tissues of CC patients with HPV typing results, collected from cervical biopsies, conization, or hysterectomies, were submitted to RISH using RNAscope chromogenicin. Immunohistochemistry was performed to evaluate the expression of p16INK4a and Ki-67. A total of 308 women with HPV typing results were enrolled, and 30 (9.74%) cases of HPV typing were negative. In HPV typing-negative CCs, 28/30 (93.3%) were positive for RISH, which contained 22/22 (100%) squamous cell carcinomas and 6/8 (75%) adenocarcinomas. RISH was positive in 278/278 (100%) HPV typing-positive CCs, which included 232/232 (100%) squamous cell carcinomas and 46/46 (100%) adenocarcinomas. Positive RISH in HPV typing-negative CC was significantly lower than in the HPV typing-positive group ( P =0.002, 95% confidence interval: 0.848-1.027). However, this significant difference only existed in adenocarcinoma. No significant differences were seen in the expression of p16INK4a and Ki-67 (all P >0.05). HPV typing may cause misdiagnosis in 9.74% of CC patients, and HPV E6/E7 mRNA can detect HPV in CC with HPV typing-negative patients. This approach could provide a novel option to accurately detect high-risk HPVs in cervical tumors and help to eliminate the percentage of misdiagnosed HPV-related cases.

Development of DNA Vaccine Targeting E6 and E7 Proteins of Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with Recombinant Vaccinia Boost and PD-1 Antibody.

Immunotherapy for cervical cancer should target high-risk human papillomavirus types 16 and 18, which cause 50% and 20% of cervical cancers, respectively. Here, we describe the construction and characterization of the pBI-11 DNA vaccine via the addition of codon-optimized human papillomavirus 18 (HPV18) E7 and HPV16 and 18 E6 genes to the HPV16 E7-targeted DNA vaccine pNGVL4a-SigE7(detox)HSP70 (DNA vaccine pBI-1). Codon optimization of the HPV16/18 E6/E7 genes in pBI-11 improved fusion protein expression compared to that in DNA vaccine pBI-10.1 that utilized the native viral sequences fused 3' to a signal sequence and 5' to the HSP70 gene of Mycobacterium tuberculosis Intramuscular vaccination of mice with pBI-11 DNA better induced HPV antigen-specific CD8+ T cell immune responses than pBI-10.1 DNA. Furthermore, intramuscular vaccination with pBI-11 DNA generated stronger therapeutic responses for C57BL/6 mice bearing HPV16 E6/E7-expressing TC-1 tumors. The HPV16/18 antigen-specific T cell-mediated immune responses generated by pBI-11 DNA vaccination were further enhanced by boosting with tissue-antigen HPV vaccine (TA-HPV). Combination of the pBI-11 DNA and TA-HPV boost vaccination with PD-1 antibody blockade significantly improved the control of TC-1 tumors and extended the survival of the mice. Finally, repeat vaccination with clinical-grade pBI-11 with or without clinical-grade TA-HPV was well tolerated in vaccinated mice. These preclinical studies suggest that the pBI-11 DNA vaccine may be used with TA-HPV in a heterologous prime-boost strategy to enhance HPV 16/18 E6/E7-specific CD8+ T cell responses, either alone or in combination with immune checkpoint blockade, to control HPV16/18-associated tumors. Our data serve as an important foundation for future clinical translation.IMPORTANCE Persistent expression of high-risk human papillomavirus (HPV) E6 and E7 is an obligate driver for several human malignancies, including cervical cancer, wherein HPV16 and HPV18 are the most common types. PD-1 antibody immunotherapy helps a subset of cervical cancer patients, and its efficacy might be improved by combination with active vaccination against E6 and/or E7. For patients with HPV16+ cervical intraepithelial neoplasia grade 2/3 (CIN2/3), the precursor of cervical cancer, intramuscular vaccination with a DNA vaccine targeting HPV16 E7 and then a recombinant vaccinia virus expressing HPV16/18 E6-E7 fusion proteins (TA-HPV) was safe, and half of the patients cleared their lesions in a small study (NCT00788164). Here, we sought to improve upon this therapeutic approach by developing a new DNA vaccine that targets E6 and E7 of HPV16 and HPV18 for administration prior to a TA-HPV booster vaccination and for application against cervical cancer in combination with a PD-1-blocking antibody.

A subset of sinonasal undifferentiated carcinoma is associated with transcriptionally active high-risk human papillomavirus by in situ hybridization: a clinical and pathologic analysis

Sinonasal undifferentiated carcinoma (SNUC) is an aggressive malignancy with a poor prognosis, and pathologically, it is a diagnosis of exclusion. Rendering this diagnosis can be challenging in practice because of the large number of diverse entities in the differential diagnosis. We encountered an index case of a sinonasal carcinoma otherwise diagnosable as SNUC which, on further investigation, demonstrated strong and diffuse P16 expression, as well as diffuse expression of high-risk human papillomavirus (hrHPV) RNA by in situ hybridization (ISH). We therefore hypothesized that a subset of cases previously diagnosed as SNUC may in fact harbor transcriptionally active hrHPV. We further investigated a cohort of 25 SNUC cases in our pathology archivesand performed ISH for hrHPV RNA on cases that demonstrated >70% nuclear and cytoplasmic P16 expression, criteria which, in other anatomic sites, correlates strongly with the presence of hrHPV. Twelve of 25 SNUC cases were P16 positive, and of these, 5 were positive for hrHPV by ISH. Thus, 20% of all SNUC cases in this cohort harbored transcriptionally active hrHPV. Herein, we report a clinical and pathologic analysis of these cases, including differential diagnostic considerations and comparison of their clinical behavior with SNUC cases that are negative for hrHPV by ISH.

MP11-16 P16INK4A EXPRESSION AND SURVIVAL OUTCOMES IN PATIENTS WITH PENILE SQUAMOUS CELL CARCINOMA: THE M.D. ANDERSON CANCER CENTER EXPERIENCE

You have accessJournal of UrologyPenile & Testicular Cancer: Penile & Testicular Cancer I (MP11)1 Apr 2020MP11-16 P16INK4A EXPRESSION AND SURVIVAL OUTCOMES IN PATIENTS WITH PENILE SQUAMOUS CELL CARCINOMA: THE M.D. ANDERSON CANCER CENTER EXPERIENCE Jad Chahoud, Rachel Pham*, Ming Guo, Curtis Pickering, Wei Qiao, Abha Khanna, Frederico Netto, Curtis Pettaway, and Priya Yao Jad ChahoudJad Chahoud More articles by this author , Rachel Pham*Rachel Pham* More articles by this author , Ming GuoMing Guo More articles by this author , Curtis PickeringCurtis Pickering More articles by this author , Wei QiaoWei Qiao More articles by this author , Abha KhannaAbha Khanna More articles by this author , Frederico NettoFrederico Netto More articles by this author , Curtis PettawayCurtis Pettaway More articles by this author , and Priya YaoPriya Yao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000831.016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Penile Squamous Cell Carcinoma (PSCC) is associated with high risk human papillomavirus (HR-HPV) in about 50% of cases. The immunohistochemical test for p16INK4a (p16) is highly correlated with HR-HPV expression and used as prognostic marker for squamous cell carcinomas in various sites. The prognostic role of this marker in PSCC remains unclear. We studied whether the expression of HPV or p16INK4a was associated with survival in a large PSCC cohort. METHODS: We conducted a single institution analysis of PSCC patients who received treatment between 1991-2017. Patients with a confirmed diagnosis of PSCC and available tissue were tested for HR-HPV status using the Cobas PCR assay. Histological subtype, tumor grade, LVI and p16 staining patterns were confirmed by an experienced pathologist. Patient characteristics were summarized using descriptive statistics of clinico-pathologic variables. Kaplan-Meier was used to estimate median overall survival (OS) and cancer specific survival (CSS). Log rank test, univariate and multivariate Cox models were applied to identify the prognostic factors for survival. RESULTS: We identified 147 patients with PSCC, with available tissue for testing. The median follow-up was 5.2 years (95% CI; 4.48, 6.68y). Patients with p16(+) tumors showed a significantly longer median OS and CSS in comparison to the p16(–) group (p = 0.038 and p = 0.012), with respective 5 year OS probability of 73% (95% CI; 0.74, 0.98) in comparison to 56% (95% CI; 0.46, 0.67) and 5 year CSS probability of 89% (95% CI; 0.7, 1) in comparison to 64% (95% CI; 0.54 , 0.75). In contrast, HPV status by PCR did not predict survival outcomes, with 5 year CSS probability for HPV(+) of 75% (95% CI; 0.61 , 0.91) compared to 65% (95% CI; 0.55, 0.78) for HPV(–) patients. Multivariable analysis to evaluate the association with CSS, showed that p16(+) along with lymph node status was associated with a lower risk of death (HR = 0.28, 95%CI; 0.09-0.8, p = 0.002), and trend toward improved OS (HR = 0.49, 95%CI; 0.19-1.24, p = 0.13) after adjusting for the covariates. CONCLUSIONS: Tumor p16 status was an independent prognostic factor for CSS in our PSCC cohort providing unique information above that of lymph node status alone. Source of Funding: Conquer Cancer Foundation of the American Society of Clinical Oncology © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e141-e142 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jad Chahoud More articles by this author Rachel Pham* More articles by this author Ming Guo More articles by this author Curtis Pickering More articles by this author Wei Qiao More articles by this author Abha Khanna More articles by this author Frederico Netto More articles by this author Curtis Pettaway More articles by this author Priya Yao More articles by this author Expand All Advertisement PDF downloadLoading ...

P16INK4a expression and survival outcomes in patients with penile squamous cell carcinoma: The M.D. Anderson Cancer Center Experience.

5 Background: Penile Squamous Cell Carcinoma (PSCC) is associated with high risk human papillomavirus (HR-HPV) in about 50% of cases. The immunohistochemical test for p16INK4a (p16) is highly correlated with HR-HPV expression and used as prognostic marker for squamous cell carcinomas in various sites. The prognostic role of this marker in PSCC remains unclear. We studied whether the expression of HPV or p16INK4a is associated with survival in a large PSCC cohort. Methods: We conducted a single institution analysis of PSCC patients who received treatment between 1991-2017.Patients with a confirmed diagnosis of PSCC and available tissue were tested for HR-HPV status using the Cobas PCR assay. Histological subtype, tumor grade, LVI and p16 staining patterns were confirmed by an experienced pathologist. Patient characteristics were summarized using descriptive statistics of clinico-pathologic variables. Kaplan-Meier was used to estimate median overall survival (OS) and cancer specific survival (CSS). Log rank test, univariate and multivariate Cox models were applied to identify the prognostic factors for survival. Results: We identified 147 patients with PSCC, with available tissue for testing. The median follow-up was 5.2 years (95% CI; 4.48, 6.68y). Patients with p16(+) tumors showed a significantly longer median OS and CSS in comparison to the p16(–) group (p=0.038 and p=0.012), with respective 5 year OS probability of 73% (95% CI; 0.74, 0.98) in comparison to 56% (95% CI; 0.46, 0.67) and 5 year CSS probability of 89% (95% CI; 0.7, 1) in comparison to 64% (95% CI; 0.54, 0.75). In contrast, HPV status by PCR did not predict survival outcomes, with 5 year CSS probability for HPV(+) of 75% (95% CI; 0.61, 0.91) compared to 65% (95% CI; 0.55, 0.78) for HPV(–) patients. Multivariable analysis to evaluate the association with CSS, showed that p16(+) along with lymph node status was associated with lower risk of death (HR=0.28, 95%CI; 0.09-0.8, p=0.002), and OS (HR=0.49, 95%CI; 0.19-1.24, p=0.13) after adjusting for the covariates. Conclusions: Tumor p16 status was an independent prognostic factor for CSS in our PSCC cohort providing unique information above that of lymph node status alone.

Correlation of p53 Expression with Histopathological and Immunohistochemical Features of Human Papillomavirus in Oral Leukoplakia.

Background:Oral cancer is strongly associated with the habit of tobacco chewing, alcohol, and betel quid consumption in India. However, sometimes, majority of the population develop oral cancer without exposure to these risk factors and are sometimes cautious about their fitness, suggesting that additional causes such as genetic predisposition, diet, and viral agents may be associated which need to explored.Aims and Objectives:The aim of this study was to establish a possible correlation between clinical types of leukoplakia with their histopathological features of high-risk human papillomavirus (HPV) infection and the presence or absence of HPV and expression of p53through immunohistochemistry (IHC).Materials and Methods:Sample comprised of 40 cases of leukoplakia and 10 cases as control group. Three sections were prepared from each biopsy and subjected to IHC and hematoxylin and eosin (H and E) stain, respectively. IHC-stained slides were used to evaluate the expression of p53 and HPV and then correlated with the features of HPV infection using H and E staining. Chi-square test with a statistical analysis software package (SPSS software Version 20.0) was used.Observation and Results:The P value for p53 against HPV (IHC) was 0.012, which indicates a significant difference between positivity proportion of P53 and HPV (IHC), whereas the P value for koilocyte and HPV (IHC) is 0.311, which is nonsignificant and indicates no significance of difference between proportion of positivity between koilocyte and HPV (IHC).Conclusion:The expression of p53 was proportionally significant to the expression of positivity of HPV, but there was no significant association between koilocyte and p53 expression.

A Retrospective 20-Year Analysis of Proliferative Verrucous Leukoplakia and Its Progression to Malignancy and Association with High-risk Human Papillomavirus.

Proliferative verrucous leukoplakia (PVL) is defined as an aggressive, relentless and recalcitrant form of leukoplakia that has a high propensity for malignant transformation. The aim of this study was to evaluate the malignant potential of PVL and determine its possible association with high-risk human papillomavirus (HPV). Twenty cases with a clinical and biopsy proven diagnosis of PVL were collected from the University of Florida Oral Medicine clinic database. Immunohistochemistry was performed to evaluate the expression of p16INK4A and p53 genes in the PVL lesions. The lesions were also tested for high-risk HPV by DNA in-situ hybridization. The average age of the patients at the time of first biopsy was 62.7years. Most patients had multiple sites of involvement, gingiva being the most common location. The lesions progressed to malignancy in approximately 50% of patients. The expression of p16INK4A gene was considered negative, with at least a 50-65% immunoreactivity observed in only three cases that progressed to malignancy. No expression of high-risk HPV was detected, whereas p53 staining was positive in less than 25% of the cells demonstrating gene expression. No definite association between PVL and high-risk HPV infection could be established. Due to the high transformation potential of PVL, early recognition with aggressive treatment, including multiple biopsies, and continued close clinical follow-up, remain the mainstay of favorable management of this condition.

Expression and clinical significance of high risk human papillomavirus and invasive gene in cervical carcinoma.

To study the expression of E6 and E7 mRNA in high-risk human papillomavirus (HPV) HPV-18 and the relationship between the expression of invasive gene and cervical carcinoma. A total of 119 patients with cervical cancer, cervical erosion and cervical HPV infection who were diagnosed in our hospital were selected and randomly divided into two groups: cervical cancer group (n=58) and non-cancerous group (n=61). Another 60 patients with uterine leiomyoma were selected as normal control group. Detection of HPV18 E6, E7 mRNA expression and invasion, migration, proliferation inhibition genes, epithelial mesenchymal transition genes and proliferation related protein content. The relative expression of E6 and E7 HPV-18 in cervical cancer group was significant higher than that in non-cancerous group and control group (mRNA) (P<0.05). The content of TRAF6 and c-FLIP in invasive cervical cancer group was significantly higher than that in non-cancerous group and control group (P<0.05). The mRNA content of CD44v6 and MMP-9 in cervical cancer group was significantly higher than that in non-cancerous group and control group (P<0.05). The content of DEC-1, IKK16, MBP-1 in cervical cancer group was significant lower than that in non-cancerous group and control group (P<0.05). The mRNA content of beta -catenin and Vimentin in cervical cancer group was significantly lower than that in non cancerous group and control group (P<0.05). The proliferation related protein E2F1 of cervical cancer group was significantly lower than that of non-cancerous group and control group, Bmi-1 content was significantly higher than non-cancerous group and control group (P<0.05). The expression of the detection of cervical cancer in high-risk human papilloma virus HPV-18 E6 and E7 mRNA, and the invasion, migration, proliferation inhibition gene, epithelial mesenchymal transition and proliferation related gene protein content, HPV expression rate of mRNA increased with the development of cervical cancer, the expression is also enhanced. The expression has a certain correlation between the level and development of cervical cancer. Through the above indicators, the development of cervical cancer monitoring and treatment to provide important clinical guidance.

HPV-16 E2 Physical Status and Molecular Evolution in Vivo in Cervical Carcinomas

A key event in the development of cervical carcinoma is the deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly due to HPV integration into host DNA. Here we explored whether HPV-16 E2 gene integrity is a biomarker of progressive disease with oncogenes expression. HPV-16 genome disruption was assessed by amplification of the entire E2 gene, while mRNA expression patterns of the E1, E2, E6, and E7 genes were evaluated by reverse transcription PCR (RT-PCR). As expected, E2 disruption was significantly higher among patients with cervical cancers than subjects with benign lesions (p=0.02). The status of the E2 gene correlated with tumorogenesis, and seemed also to correlate with the stage of the carcinomas, since integrated HPV-16 DNA was frequently detected in patients with advanced cancer stages (75% of stage III vs 60% stages I and II). In bivariate analysis, the lesions&rsquo; grade was most significantly associated with HPV-16 DNA disruption (p&lt;0.05). In cervical carcinoma the deletion pattern involved more frequently the E2 gene rather than the E1 gene (62.5% vs 45.8%). The prevalence of the E6/E7 HPV-16 transcripts in cervical carcinoma specimens and in benign cervical lesions were detected with frequencies of, respectively, 91.6% and 45.4%. The mRNA levels of the HPV-16 E6/E7 genes were expressed at approximately the same levels in each physical state. We consistently observed that E6/E7 were absent or weakly detectable in the presence of E2. However, in the absence of E2 the levels of E6/E7 markedly increased (p&lt;0.05). This study underscores the significance of investigating alternative mechanisms of E2 expression and oncogenes E6/E7 transcripts in vivo as biomarkers for disease severity in cervical carcinomas.

In vitro Progression of Human Papillomavirus 16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis

An important event in the development of cervical squamous cell carcinoma (SCC) is deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly related to viral integration into host DNA. Mechanisms of development of the approximately 15% of SCCs that contain extrachromosomal (episomal) HR-HPV are poorly understood due to limited longitudinal data. We therefore used the W12 model to study mechanisms of cervical carcinogenesis associated with episomal HPV16. In vitro progression of W12 normally occurs through selection of cells containing integrated HPV16. However, in one long-term culture, keratinocytes developed a selective growth advantage and invasive phenotype while retaining HPV16 episomes at increased copy number in the absence of transcriptionally active integrants. Longitudinal investigations revealed similarities between the episome- and integrant-associated routes of neoplastic progression. Most notable were dynamic changes in viral early gene expression in episome-retaining cells, consistent with continually changing selective pressures. An early increase in viral transcription preceded elevated episome copy number and was followed by a reduction to near baseline after the development of invasiveness. Episomal transcriptional deregulation did not require selection of a specific sequence variant of the HPV16 upstream regulatory region, although increased levels of acetylated histone H4 around the late promoter implicated a role for altered chromatin structure. Interestingly, invasive episome-retaining cells showed high levels of HPV16 E2/E6 proteins (despite decreased transcript levels) and reduced expression of IFN-stimulated genes, adaptations that support viral persistence and cell survival. Our findings suggest a unified working model for events important in cervical neoplastic progression regardless of HR-HPV physical state.

Transforming Growth Factor β1 Promotes Chromosomal Instability in Human Papillomavirus 16 E6E7–Infected Cervical Epithelial Cells

Uterine cervical cancer, the second most frequently occurring cancer in women worldwide, is tightly associated with the expression of high-risk human papillomavirus [mainly human papillomavirus (HPV)-16 and HPV18] oncogenes E6 and E7 and characteristically exhibits chromosomal instability. However, the mechanisms underlying chromosomal instability in cervical cancer are still not fully understood. In this study, we observed that two of three human cervical epithelial cell lines expressing HPV16 E6E7 became immortalized without extensive chromosomal instability and crisis. The introduction of transforming growth factor (TGF)-beta1, a multiple functional cytokine/growth factor, in the culture medium induced crisis, which was associated with massive chromosomal end-to-end fusions and other structural aberrations. The distributions of structural aberrations on individual chromosomes were significantly correlated with the profiles of telomere signal-free ends. The immortalized cells that emerged from the TGF-beta1-induced crisis showed multiple clonal structural aberrations that were not observed in cells without TGF-beta1 treatment. Overexpression of the catalytic subunit of telomerase (hTERT) abolished the effects of TGF-beta1 on chromosomal instability. Interestingly, another HPV16 E6E7-expressing cervical cell line that experienced crisis and telomere dysfunction under ordinary culture condition had a higher level of autocrine TGF-beta1 production than the other two crisis-free immortalized cell lines. Blocking the TGF-beta1 pathway by an inhibitor of TGF-beta1 receptor type I prevented the crisis and telomere-mediated chromosomal instability. In addition, more dramatic telomere shortening was observed in cervical intraepithelial neoplasias having higher expression of TGF-beta1 in vivo. These results together suggest an important role of TGF-beta1 in the early process of cervical carcinogenesis.

Estimation of Prognoses for Cervical Intraepithelial Neoplasia 2 by p16INK4aImmunoexpression and High-Risk HPV In Situ Hybridization Signal Types

The present study used immunohistochemical staining and in situ hybridization (ISH) to examine whether progression of cervical intraepithelial neoplasia, grade 2 (CIN 2) can be predicted by p16INK4a immunoexpression and high-risk human papilloma virus (HPV) ISH signal types. We studied 52 cases histologically diagnosed with CIN 2: dysplasia regressed in 28 cases; 13 cases progressed to CIN 3; and CIN 2 persisted in 11 cases. Expression of p16INK4a and high-risk HPV signal both related to grade of CIN. Stronger p16INK4a immunoexpression and a higher frequency of expression of a punctate nuclear signal were observed in CIN 2 lesions before progression compared with those before regression. CIN 2 cases in which moderate to strong immunoexpression of p16INK4a and a punctate signal were observed simultaneously progressed to CIN 3 in 10 (91%) of 11 cases. CIN 2 cases with moderate to strong immunoexpression of p16INK4a and a high-risk HPV punctate signal should be treated because of the great risk of progression.

Mammary epithelial cell transformation: insights from cell culture and mouse models

Normal human mammary epithelial cells (HMECs) have a finite life span and do not undergo spontaneous immortalization in culture. Critical to oncogenic transformation is the ability of cells to overcome the senescence checkpoints that define their replicative life span and to multiply indefinitely – a phenomenon referred to as immortalization. HMECs can be immortalized by exposing them to chemicals or radiation, or by causing them to overexpress certain cellular genes or viral oncogenes. However, the most efficient and reproducible model of HMEC immortalization remains expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Cell culture models have defined the role of tumor suppressor proteins (pRb and p53), inhibitors of cyclin-dependent kinases (p16INK4a, p21, p27 and p57), p14ARF, telomerase, and small G proteins Rap, Rho and Ras in immortalization and transformation of HMECs. These cell culture models have also provided evidence that multiple epithelial cell subtypes with distinct patterns of susceptibility to oncogenesis exist in the normal mammary tissue. Coupled with information from distinct molecular portraits of primary breast cancers, these findings suggest that various subtypes of mammary cells may be precursors of different subtypes of breast cancers. Full oncogenic transformation of HMECs in culture requires the expression of multiple gene products, such as SV40 large T and small t, hTERT (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). However, when implanted into nude mice these transformed cells typically produce poorly differentiated carcinomas and not adenocarcinomas. On the other hand, transgenic mouse models using ErbB2/neu, Ras, Myc, SV40 T or polyomavirus T develop adenocarcinomas, raising the possibility that the parental normal cell subtype may determine the pathological type of breast tumors. Availability of three-dimensional and mammosphere models has led to the identification of putative stem cells, but more studies are needed to define their biologic role and potential as precursor cells for distinct breast cancers. The combined use of transformation strategies in cell culture and mouse models together with molecular definition of human breast cancer subtypes should help to elucidate the nature of breast cancer diversity and to develop individualized therapies.

Centrosome abnormalities and genomic instability by episomal expression of human papillomavirus type 16 in raft cultures of human keratinocytes.

Primary human keratinocytes with ectopic expression of high-risk human papillomavirus (HPV) E6 and E7 oncoproteins display abnormal centrosome numbers, multipolar mitoses, and aneusomy. However, it has not been explored whether these abnormalities can occur in cells containing HPV episomes where E6 and E7 expression is under viral transcriptional control. Here, we demonstrate that centrosome abnormalities and genomic instability occur in organotypic raft cultures of human keratinocytes with episomal HPV-16 even at low copy numbers. We conclude that HPV-16 DNA, when maintained as an episome, can disturb centrosome homeostasis and subvert genomic integrity of the host cell during early stages of the viral infection.