Dysregulation of Transcription Factor Networks Unveils Different Pathways in Human Papillomavirus 16-Positive Squamous Cell Carcinoma and Adenocarcinoma of the Uterine Cervix

Squamous cell carcinoma (SCC) and adenocarcinoma (ADC) are the most common histological types of cervical cancer (CC). The worse prognosis of ADC cases highlights the need for better molecular characterization regarding differences between these CC types. RNA-Seq analysis of seven SCC and three ADC human papillomavirus 16-positive samples and the comparison with public data from non-tumoral human papillomavirus-negative cervical tissue samples revealed pathways exclusive to each histological type, such as the epithelial maintenance in SCC and the maturity-onset diabetes of the young (MODY) pathway in ADC. The transcriptional regulatory network analysis of cervical SCC samples unveiled a set of six transcription factor (TF) genes with the potential to positively regulate long non-coding RNA genes DSG1-AS1, CALML3-AS1, IGFL2-AS1, and TINCR. Additional analysis revealed a set of MODY TFs regulated in the sequence predicted to be repressed bymiR-96-5p ormiR-28-3p in ADC. These microRNAs were previously described to target LINC02381, which was predicted to be positively regulated by two MODY TFs upregulated in cervical ADC. Therefore, we hypothesize LINC02381might act by decreasing the levels ofmiR-96-5p andmiR-28-3p, promoting the MODY activation in cervical ADC. The novel TF networks here described should be explored for the development of more efficient diagnostic tools

High-level of viral genomic diversity in cervical cancers: a Brazilian study on human papillomavirus type 16

Invasive cervical cancer (ICC) is the third most frequent cancer among women worldwide and is associated with persistent infection by carcinogenic human papillomaviruses (HPVs). The combination of large populations of viral progeny and decades of sustained infection may allow for the generation of intra-patient diversity, in spite of the assumedly low mutation rates of PVs. While the natural history of chronic HPVs infections has been comprehensively described, within-host viral diversity remains largely unexplored. In this study we have applied next generation sequencing to the analysis of intra-host genetic diversity in ten ICC and one condyloma cases associated to single HPV16 infection. We retrieved from all cases near full-length genomic sequences. All samples analyzed contained polymorphic sites, ranging from 3 to 125 polymorphic positions per genome, and the median probability of a viral genome picked at random to be identical to the consensus sequence in the lesion was only 40%. We have also identified two independent putative duplication events in two samples, spanning the L2 and the L1 gene, respectively. Finally, we have identified with good support a chimera of human and viral DNA. We propose that viral diversity generated during HPVs chronic infection may be fueled by innate and adaptive immune pressures. Further research will be needed to understand the dynamics of viral DNA variability, differentially in benign and malignant lesions, as well as in tissues with differential intensity of immune surveillance. Finally, the impact of intralesion viral diversity on the long-term oncogenic potential may deserve closer attention.Funded by Grants # 2011/24035-2 and # 2012/23290-1, São Paulo Research Foundation (FAPESP

Pharmacokinetics and tumor uptake of a derivatized form of paclitaxel associated to a cholesterol-rich nanoemulsion (LDE) in patients with gynecologic cancers

O paclitaxel é utilizado amplamente no carcinoma de ovário, nos casos refratários de carcinoma de endométrio e quimioterapia exclusiva para carcinoma avançado de colo uterino. A associação de paclitaxel a uma nanoemulsião rica em colesterol, denominada LDE, mostrou toxicidade menor e aumento da atividade antitumoral do fármaco em cobaias. No presente estudo, investigou-se os parâmetros farmacocinéticos do oleato de LDE-paclitaxel e a habilidade da LDE de concentrar o fármaco no tumor em oito pacientes com câncer do trato genital feminino. O oleate de paclitaxel associado a LDE é estável na circulação e tem uma meia-vida plasmática maior do que o paclitaxel comercial. A LDE concentra 3,6 mais paclitaxel em tecidos tumorais do que nos tecidos normais. Esta associação parece ser uma alternativa no tratamento dos tumores ginecológicosA cholesterol-rich nanoemulsion termed LDE concentrates in cancer tissues after injection into the bloodstream. The association of a derivatized paclitaxel to LDE showed lower toxicity and increased antitumoral activity as tested in mice. Here, the pharmacokinetics of LDE-paclitaxel oleate and the ability of LDE to concentrate the drug in the tumor were investigated in eight patients with gynecologic cancers. Fractional clearance rate (FCR) and pharmacokinetic parameters were calculated by compartmental analysis. Also, specimens of tumors and the normal tissues were excised during the surgery for radioactivity measurement. LDE concentrates 3.5 more paclitaxel in malignant tissues than in the normal tissues. Therefore, association to LDE is an interesting strategy for using paclitaxel to treat gynecologic cancer

Genotyping of Human Papillomavirus (HPV) in uterine cervical cancer patients of the Cancer Institute of the State of São Paulo in the period from 2008 to 2012

INTRODUÇÃO: O câncer do colo uterino é a terceira neoplasia maligna que mais afeta as mulheres brasileiras e, quando não detectada precocemente, apresenta prognóstico reservado. O câncer do colo uterino é consequência da infecção pelo Papilomavírus humano (HPV). Pouco é conhecido sobre a influência dos genótipos do HPV na apresentação clínica e o seu impacto na taxa de sobrevida no câncer do colo uterino. Os objetivos do estudo foram identificar os genótipos de HPV no tecido tumoral da população atendida no Instituto do Câncer do Estado de São Paulo e associar os genótipos de HPV aos fatores de risco conhecidos para o câncer do colo uterino. MÉTODOS: Foram incluídas mulheres com diagnóstico de câncer do colo uterino atendidas no Instituto do Câncer do Estado de São Paulo (ICESP) entre maio de 2008 e junho de 2012. A análise do material tumoral parafinado confirmou histologicamente o diagnóstico de câncer do colo uterino. O DNA tumoral foi extraído de três fragmentos de 10?m de espessura do bloco de parafina de carcinoma do colo uterino e submetido ao ensaio clínico Onclarity (sistema automatizado da BD Viper LT) para detecção e genotipagem do HPV. Idade ao diagnóstico, estadiamento clínico, tipo histológico e tempo de sobrevida foram obtidos a partir de registros do prontuário até dezembro de 2015. RESULTADOS: Foram analisadas 414 pacientes. As frequências dos genótipos estudados foram HPV16 (54%) HPV18(9%), HPV33-­58 (6%), HPV45 (5%), HPV31 (3%), HPV39-­68-­ 35 (3%), HPV59-­56-­66(3%), HPV52 (2%) e HPVnegativo (14%). A idade da população estudada variou de 17 a 87 anos, com média etária de 50,8 (DP=13,8 anos). Os tipos histológicos foram carcinoma de células escamosas (75%), adenocarcinoma (21%) e outros tipos histológicos (4%). Conforme o estadiamento clínico adotado pela FIGO (2009), 35% foi classificado como 1A1, 1A2 e 1B1, 17% como 1B2 e 2A e 48% como 2B a 4B. O genótipo do HPV apresentou distribuição diferente quanto à idade ao diagnóstico, tipo histológico e estadiamento. A mediana do tempo de sobrevida global desta coorte de pacientes com câncer do colo uterino foi de 37 meses [12-­53 meses]. A sobrevida global acumulada em 5 anos após o diagnóstico de câncer do colo uterino foi de 55%. Ocorreram 119 (38%) óbitos no período e 133 (42%) recidivas subdivididas em três grupos: local (12%), regional (30%) e à distância (58%). Curvas de sobrevida de Kaplan-­Meier e estatística de Log-­rank demonstraram que os genótipos de HPV 16 e 18 (59%) não se relacionaram a um pior prognóstico em comparação com outros genótipos de HPV (41%) (P=0,17). Idade ao diagnóstico, estadiamento clínico, tipo histológico, invasão vascular, metástase linfonodal, tamanho do tumor e os genótipos HPV16, HPV18 e HPVoutros foram analisados individualmente em um modelo de regressão de Cox. O genótipo do HPV se associou a pior taxa de sobrevida global apenas quando detectado mais de um HPV no material tumoral analisado. CONCLUSÃO: Apesar das diferentes distribuições dos os genótipos do HPV quanto à idade ao diagnóstico, tipo histológico e estadiamento, o genótipo do HPV não se mostrou como fator prognóstico independente no câncer do colo uterinoINTRODUCTION: Uterine cervical cancer is the third most common malignant neoplasm affecting Brazilian women. Prognosis is poor when diagnosis is delayed. Cervical cancer is a consequence of human papillomavirus (HPV) infection. Little is known about the influence of HPV genotypes in Brazil and its impact on cancer survival rate The purpose of the present study were to identify HPV genotypes of tumoral tissue from the affected population and to examine the association between HPV genotype and traditional cervical cancer risk factors. METHODS: Women diagnosed with cervical cancer at the Cancer Institute of the State of São Paulo (ICESP) between May 2008 and June 2012 were included in the study. Tumor specimens were reviewed to confirm the diagnosis of cervical cancer. Tumor DNA was extracted from three 10?m-­thick paraffin block fragments of each subject. HPV genotype was detected using the Onclarity system (BD Viper LT automated system). Age at diagnosis, clinical staging, histological type and survival time were obtained from the hospital electronic data records until December 2015. RESULTS: 414 patients were analyzed. The HPV genotypes studied were HPV16 (54%) HPV18 (9%), HPV33-­58 (6%), HPV45 (5%), HPV31 (3%), HPV39-­68-­35 (3%), HPV59-­56-­ 66(3%), HPV52 (2%) and HPVnegative (14%). The age of the study population ranged from 17 to 87 years, mean age= 50.8 (SD=13.8 years). Histological types were classified as squamous cell carcinoma (75%), adenocarcinoma (21%) and other histological types (4%). According to the 2009 FIGO clinical staging, 35% were classified as 1A1, 1A2 and 1B1, 1B2 and 17% as 2A and 48% as 2B the 4B. HPV genotypes showed different distributions regarding age, histologic tumor types and clinical staging. The median overall survival time was 37 months [12-­53 months]. The cumulative overall survival at 5 years after diagnosis of cervical cancer was 55%. There were 119 (38%) deaths during the study period and 133 (42%) recurrences subdivided into three groups: local (12%), regional (30%) and distant (58%). Kaplan-­Meier survival curves and Log-­rank statistics showed that HPV 16/18 (59%) did not influence prognosis compared to other HPV subtypes (41%) (P=0.17). Age at diagnosis, clinical stage, histological type, vascular invasion, lymph node metastasis, tumor size and HPV16 genotypes, HPV18 and HPVothers were individually analyzed in a Cox regression model. HPV genotype was associated with poorer overall survival rate only when multiple HPV infection was detected in the tumoral specimen. CONCLUSION: Although HPV genotype showed different distribution regarding age at diagnosis, histological type and clinical staging, HPV genotype was not an independent prognostic factor of cervical cancer in the study populatio

Dysregulation of Transcription Factor Networks Unveils Different Pathways in Human Papillomavirus 16-Positive Squamous Cell Carcinoma and Adenocarcinoma of the Uterine Cervix

Squamous cell carcinoma (SCC) and adenocarcinoma (ADC) are the most common histological types of cervical cancer (CC). The worse prognosis of ADC cases highlights the need for better molecular characterization regarding differences between these CC types. RNA-Seq analysis of seven SCC and three ADC human papillomavirus 16-positive samples and the comparison with public data from non-tumoral human papillomavirus-negative cervical tissue samples revealed pathways exclusive to each histological type, such as the epithelial maintenance in SCC and the maturity-onset diabetes of the young (MODY) pathway in ADC. The transcriptional regulatory network analysis of cervical SCC samples unveiled a set of six transcription factor (TF) genes with the potential to positively regulate long non-coding RNA genes DSG1-AS1, CALML3-AS1, IGFL2-AS1, and TINCR. Additional analysis revealed a set of MODY TFs regulated in the sequence predicted to be repressed bymiR-96-5p ormiR-28-3p in ADC. These microRNAs were previously described to target LINC02381, which was predicted to be positively regulated by two MODY TFs upregulated in cervical ADC. Therefore, we hypothesize LINC02381might act by decreasing the levels ofmiR-96-5p andmiR-28-3p, promoting the MODY activation in cervical ADC. The novel TF networks here described should be explored for the development of more efficient diagnostic tools

Dysregulation of Transcription Factor Networks Unveils Different Pathways in Human Papillomavirus 16-Positive Squamous Cell Carcinoma and Adenocarcinoma of the Uterine Cervix

Squamous cell carcinoma (SCC) and adenocarcinoma (ADC) are the most common histological types of cervical cancer (CC). The worse prognosis of ADC cases highlights the need for better molecular characterization regarding differences between these CC types. RNA-Seq analysis of seven SCC and three ADC human papillomavirus 16-positive samples and the comparison with public data from non-tumoral human papillomavirus-negative cervical tissue samples revealed pathways exclusive to each histological type, such as the epithelial maintenance in SCC and the maturity-onset diabetes of the young (MODY) pathway in ADC. The transcriptional regulatory network analysis of cervical SCC samples unveiled a set of six transcription factor (TF) genes with the potential to positively regulate long non-coding RNA genes DSG1-AS1, CALML3-AS1, IGFL2-AS1, and TINCR. Additional analysis revealed a set of MODY TFs regulated in the sequence predicted to be repressed bymiR-96-5p ormiR-28-3p in ADC. These microRNAs were previously described to target LINC02381, which was predicted to be positively regulated by two MODY TFs upregulated in cervical ADC. Therefore, we hypothesize LINC02381might act by decreasing the levels ofmiR-96-5p andmiR-28-3p, promoting the MODY activation in cervical ADC. The novel TF networks here described should be explored for the development of more efficient diagnostic tools

Multiple HPV genotype infection impact on invasive cervical cancer presentation and survival

<div><p>Background</p><p>Invasive cervical cancer (ICC) is the third most common malignant neoplasm affecting Brazilian women. Little is known about the impact of specific HPV genotypes in the prognosis of ICC. We hypothesized that HPV genotype would impact ICC clinical presentation and survival.</p><p>Methods</p><p>Women diagnosed with ICC at the Instituto do Câncer do Estado de São Paulo (ICESP) between May 2008 and June 2012 were included in the study and were followed until December 2015. HPV genotype was detected from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples using Onclarity™ system (BD Viper™ LT automated system).</p><p>Results</p><p>292 patients aged 50±14 years were analyzed. HPVDNA was detected in 84% of patients. The HPV genotypes studied were: HPV16 (64%), HPV18 (10%), HPV33-58 (7%), HPV45 (5%), HPV31 (4%) and other high-risk HPV genotypes (11%). HPV genotypes showed different distributions regarding histological type and clinical stage. Patients were followed for 35±21 months. The overall survival at 5 years after diagnosis of cervical cancer was 54%. Age, clinical staging, histological type and multiple HPV genotypes infection detected in the same tumor specimen were associated with poorer overall survival on multivariate Cox proportional hazard analysis (p<0.05). No specific HPV genotype affected survival.</p><p>Conclusion</p><p>Multiple HPV genotype infection was associated with poorer ICC survival in our study, compared with single genotype infection. HPV genotyping from FFPE tumor tissue using an automated assay such as the Onclarity BD™ assay provides a simpler alternative for routine clinical use.</p><p>Impact</p><p>This is the largest study employing an automated HPV genotyping assay using FFPE of ICC. Multiple HPV genotype infection adversely influenced survival.</p></div