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Human papillomavirus (HPV) infection is strongly associated with cervical cancer (CC). Genomic alterations caused by viral infection and subsequent dysregulation of cellular metabolism under hypoxic conditions could influence the response to treatment. We studied a possible influence of IGF-1Rb, hTERT, HIF1a, GLUT1 protein expression, HPV species presence and relevant clinical parameters on the response to treatment. In 21 patients, HPV infection and protein expression were detected using GP5+/GP6+PCR-RLB and immunohistochemistry, respectively. The worse response was associated with radiotherapy alone compared with chemoradiotherapy (CTX-RT), anemia and HIF1a expression. HPV16 type was the most frequent (57.1%) followed by HPV-58 (14.2%) and HPV-56 (9.5%). The HPV alpha 9 species was the most frequent (76.1%) followed by alpha 6 and alpha 7. IGF-1Rb (85.7%), HIF1a (61.9%), GLUT1 (52.3%), and hTERT expression [cytoplasm and nucleus (90.4%)] were detected. The MCA factorial map showed different relationships, standing out, expression of hTERT and alpha 9 species HPV, expression of hTERT and IGF-1Rb expression [Fisher's exact test (P = 0.04)]. A slight trend of association was observed between, GLUT1 and HIF1 a expression, hTERT and GLUT1 expression. A noteworthy finding was the subcellular localization of hTERT in the nucleus and cytoplasm of CC cells and its possible interaction with IGF-1R in presence of HPV alpha 9 species. Our findings suggest that the expression of HIF1a, hTERT, IGF-1Rb and GLUT1 proteins that interact with some HPV species may contribute to cervical cancer development, and the modu lation of treatment response.
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Infecções por Papillomavirus , Telomerase , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/genética , Transportador de Glucose Tipo 1 , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Papillomaviridae/fisiologia , Telomerase/genética , Telomerase/metabolismoRESUMO
BACKGROUND/AIM: Although some mutations of KRAS proto-oncogene, GTPase (KRAS) have been associated with the prognosis and therapeutic management of colorectal cancer (CRC), the epigenetic mechanisms (DNA methylation and microRNA expression) that regulate wild-type KRAS expression in patients with CRC are poorly known. The aim of this study was to establish whether there is a relationship between the expression of the wild-type KRAS gene, the methylation status of its distal promoter, and miR-143 and miR-18a-3p levels in samples of sporadic CRC. PATIENTS AND METHODS: A total of 51 cases of sporadic CRC with wild-type KRAS were analyzed. The expression levels of KRAS mRNA, miR-18a-3p, miR-143, and KRAS protein, as well as methylation in the distal promoter of the KRAS gene were evaluated. RESULTS: In the analyzed cases, KRAS mRNA expression was detected in 51.1%; wild-type KRAS protein was found in the membrane in 31.4% and in the cytoplasm in 98% of cases. An inverse relationship of marginal significance was observed between miR-18a-3p and KRAS protein expression in the cytoplasm (odds ratio=0.14, 95% confidence interval=0.012-1.092; p=0.08). The methylation status of the distal promoter of KRAS at four CpG islands was analyzed in 30 cases (58.8%): partial methylation of the four CpG islands evaluated was observed in two cases (6.7%). In these cases, KRAS protein expression was not evidenced at the membrane level; miR-18a-3p expression was not detected either but high expression of miR-143 was observed. CONCLUSION: No association was found between the expression levels of KRAS mRNA, miR-18a-3p, miR-143 and methylation status. Methylation status was detected with low frequency, thus being the first report of methylation in wild-type KRAS.
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Resumen Las alteraciones en la metilación de dinucleótidos CpG en regiones promotoras es uno de los mecanismos epigenéticos implicados en cáncer que tiene uso potencial como biomarcador. Su evaluación, a partir de tejidos fijados en formalina y embebidos en parafina (FFPE), representa un gran desafío dadas la degradación parcial, el entrecruzamiento y las bajas cantidades del DNA obtenido. En esta nota técnica, describimos un protocolo para el estudio del estado de metilación del promotor distal del proto-oncogén K-RAS, a partir de varias muestras obtenidas de dos tejidos FFPE de cáncer colorrectal con antigüedad de 11 años. Se empleó un protocolo de conversión con bisulfito alternativo al usual; se usó una DNA polimerasa modificada y una PCR anidada y se optimizó la secuenciación directa del DNA convertido con bisulfito. Este protocolo podría ser aplicado para determinar estados de metilación en otros genes y tipos de cáncer en tejidos FFPE.
Abstract Alterations in the methylation of CpG dinucleotides in promoter regions is one of the epigenetic mechanisms involved in cancer that has potential use as a biomarker. Its evaluation from formalin-fixed and paraffin-embedded (FFPE) tissues represents a great challenge given the partial degradation, crosslinking, and low amounts of the obtained DNA. In this technical note we describe a protocol for the study of the methylation status of the distal promoter of the K-RAS proto-oncogene from several samples obtained from two 11-years old FFPE tissues of colorectal cancer. An alternative bisulfite conversion protocol to the usual one was used; a modified DNA polymerase and a nested PCR were used and the direct sequencing of the converted DNA with bisulfite was optimized. This protocol could be applied to determine methylation states in other genes and types of cancer.
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Humanos , Parafina , Neoplasias Colorretais , Metilação de DNA , Biomarcadores , Reação em Cadeia da Polimerase , GenesRESUMO
BACKGROUND: Few studies have analyzed the association between human telomerase reverse transcriptase (hTERT) protein expression (nuclear and cytoplasmic localization), hTERT methylation status, and human papillomavirus (HPV) genotype infection in cervical cancer. PATIENTS AND METHODS: One hundred seventy-three patients with cervical cancer were analyzed. hTERT protein expression was detected by immunohistochemistry. hTERT DNA methylation analysis was performed using a PCR-RLB-hTERT assay, targeting two regions of the hTERT promoter. Type specific HPV infection was detected by using GP5+/GP6+PCR-RLB. RESULTS: hTERT protein expression was found in both cytoplasm and nucleus (78.0% of the samples showed a cytoplasmic localization and 79.8% had a nuclear localization). A statistically significant association was found between alpha 9 and 7 HPV species with a non-methylation pattern of the hTERT promoter and between these species and high expression of hTERT protein with nuclear localization. CONCLUSION: hTERT protein is found in both the nucleus and cytoplasm of patients with cervical cancer and confirm the relationship between the non-methylated status of hTERT promoter and some HPV species as well as the relationship between these species and hTERT protein expression.
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Metilação de DNA , Infecções por Papillomavirus/genética , Telomerase/metabolismo , Neoplasias do Colo do Útero/genética , Adulto , Idoso , Núcleo Celular/patologia , Colo do Útero/citologia , Colo do Útero/patologia , Colo do Útero/virologia , Quimiorradioterapia/métodos , Estudos Transversais , Citoplasma/patologia , DNA Viral/isolamento & purificação , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/terapia , Infecções por Papillomavirus/virologia , Regiões Promotoras Genéticas/genética , Telomerase/análise , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/terapia , Neoplasias do Colo do Útero/virologia , Adulto JovemRESUMO
This article is a preliminary investigational study that is aimed at giving hints about the interesting biomarkers involved in the transition process from low-grade cervix lesion to invasive cervical cancer. Our study focuses on the risk factors and tumour molecular changes in one patient. First in 1986, she was diagnosed a preinvasive cervix lesion. Then, 16 years later, she was diagnosed an invasive cervical cancer. The 2002 diagnosis was a squamous cell carcinoma of the cervix, stage IIIB (FIGO), whereas in 1986, she had been diagnosed a high-grade squamous intraepithelial cervical lesion. Retrospectively, the analysis of samples of preneoplastic lesions and invasive cervical cancer confirmed the histopathological diagnoses and detected the presence of HPV type and HPV-16 variants, as well as the overexpression of proteins such as hTERT, IGF1Rα, IGF1Rß, CAIX, and GLUT1. Finally, the Arg72Pro polymorphism was detected in TP53. The role of high-risk HPV and HPV-16 variants and of hTERT, IGF1Rα, IGF1Rß, CAIX, and GLUT1 variations seemed confirmed in the development and progression of cervical cancer. As a result, analyzing the molecular changes in one and same tumour that progresses from a low-grade cervix lesion to invasive cervical cancer could provide valuable information in order to improve detection, diagnosis, and treatment in the future.
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Certain variants of human papillomavirus (HPV)type 58 are associated with an increased risk of high grade squamous intraepithelial lesions and cervical cancer. However, little is known about the persistence of HPV58 E6/E7 variants in women with incident HPV58 infections. The aim of the present study was to evaluate the presence and persistence of HPV58 E6/E7 variants in 71 women with incident HPV58 infection throughout their follow-up. These women belonged to a cohort examined in a longitudinal study of 1,610 Colombian women, who were HPV-negative and had normal baseline cytology. E6/E7 DNA regions of HPV58-positive samples were amplified and sequenced using automated direct sequencing. A total of 639 samples were analyzed from the 71 women, and 117 samples (18.3%) were HPV58-positive. HPV58 E6/E7 variants were detected in 85.5% of the samples. The T307/A694/G744/A761 variant was identified in 88% of the samples, the T307/G744 variant was identified in 9% of samples and the T187/T307/A367/G744/G793/T798/A801/T840/C852 was identified in 3% of the samples. Overall, 50% of the HPV58 infections were present after 1 year of follow-up and all infections were cleared after 7 years. Women who had first sexual intercourse at >15 years of age had a lower clearance rate than those who had sexual intercourse for the first time at ≤15 years of age [hazard ratio (HR)=0.29; 95% confidence interval (CI)=0.09-0.92]. Likewise, parous women had a higher clearance rate than nulliparous women (HR=3.43, 95% CI=1.23-9.60). There was no difference in clearance rates between HPV58 E6/E7 variants. In conclusion, HPV58 variants were not associated with persistence of the infection in this group of women.
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BACKGROUND: There exists limited information on the role of hTERT methylation, and its association with type-specific HPV infections in cervical cancer. MATERIALS AND METHODS: Eighty-seven frozen samples were analyzed for type-specific HPV infection using a GP5+/GP6+ PCR-RLB assay (RLB). hTERT DNA methylation analysis was performed using a newly developed PCR-RLB-hTERT. RESULTS: Ninety-three percent of samples were HPV-positive and fifteen different types were detected. hTERT methylation analysis of region 1 revealed no methylation in 78.8% of the samples and partial methylation in 21.2%. In region two, 68.2% showed no methylation and 31.8% showed a pattern of partial methylation. An association between the alpha 9 and alpha 7 species with a pattern of no methylation of hTERT in the region 1 was established (p=0.02 and p=0.03, respectively). CONCLUSION: Differences in patterns of methylation of the hTERT core promoter [region 1 (nt -208 to -1) and region 2 (nt +1 to +104) relative to first ATG] are related to the HPV species present.
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Metilação de DNA/genética , Infecções por Papillomavirus/genética , Telomerase/genética , Neoplasias do Colo do Útero/genética , Feminino , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/patogenicidade , Humanos , Invasividade Neoplásica/genética , Infecções por Papillomavirus/classificação , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
High hypoxic, glycolytic and acidosis metabolisms characterize cervical cancer tumors and have been described to be involved in chemoradioresistance mechanisms. Based on these observations, the present study assessed four selected novel biomarkers on the prognosis of locally advanced cervical carcinoma. A total of 66 patients with stage IIB/IIIB cervical cancer were retrospectively included. The protein expression levels of glucose transporter 1 (GLUT1), carbonic anhydrase 9 (CAIX) and hexokinase 1 (HKII) were investigated by immunohistochemistry on tumor biopsies, hemoglobin was measured and the disease outcome was monitored. A total of 53 patients (80.3%) presented a complete response. For these patients, the protein expression levels of GLUT1, CAIX and HKII were overexpressed. A significant difference was observed (P=0.0127) for hemoglobin levels (≤11 g/dl) in responsive compared with non-responsive patients. The expression of GLUT1 is associated with a lower rate of both overall and disease-free survival, with a trend of decreased risk of 1.1x and 1.5x, respectively. Co-expression of GLUT1 and HKII is associated with a decreased trend risk of 1.6x for overall survival. Patients with hemoglobin levels ≤11 g/dl had a 4.3-fold risk (P=0.02) in decreasing both to the rate of overall and disease-free survival. The presence of anemic hypoxia (hemoglobin ≤11 g/dl) and the expression of GLUT1 and/or HKII influence treatment response and are associated with a lower overall and disease-free survival. The present results demonstrated that these biomarkers may be used as predictive markers and suggested that these metabolic pathways can be used as potential novel therapeutic targets.
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Estandarizar la técnica de citología en base líquida mediante el uso del medio fijador (BDSUREPAth) y de citocentrífuga e identificar las posibles ventajas sobre citología convencional en muestra compartida de tomas cervicouterinas. Métodos: se incluyeron 92 muestras de mujeres que asistieron a las campañas del programa de proyección social de la Fundación Universitaria de Ciencias de la Salud, Bogotá DC, Colombia. Resultados: de las 92 muestras, 89 fueron negativas para citología convencional y 75 para base líquida, observándose un mayor número de casos con anormalidades en células escamosas en citología en base líquida. Conclusiones: las células escamosas mantienen su tonalidad citoplasmática siendo basófilas o acidófilas, conservando lo translúcido de los citoplasmas; las células glandulares preservan su patrón en panal de abejas o empalizada. Mediante citología en base liquida se obtienen fondos más limpios, las atipias de las células epiteliales fueron más fáciles de identificar al igual que los microorganismos patógenos...
Objective: to standardize the use of BD SurePath and centrifuged liquid-based cytology and identify possible advantages over the use of conventional cytology by comparing results on shared cervical samples analysis. Methods: we included 92 samples collected from women who attended the social projection campaign conducted by Fundación Universitaria de Ciencias de la Salud, Bogotá DC, Colombia. Results: out of the 92 samples, 89 were negative by conventional cytology and 75 by liquid-based cytology, evidencing a greater number of cases where squamous cell anomalies were detected using liquid-based cytology. Conclusions: squamous cells cytoplasma maintains its tone, being basophilic and acidophilic, conserving translucence; glandular cells preserve their honeycomb or palisade pattern. Liquid-based cytology gives a more clean background and improves identification of epithelial cell atypia as well as pathogenic microorganisms...
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Humanos , Feminino , Biologia Celular , Papiloma , Micobactérias não Tuberculosas , Técnicas CitológicasRESUMO
AIM: The aim of this study was to evaluate the predictive utility of Insulin-like growth factor-1 receptor (IGF1R), IGF1, IGF2, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and of hemoglobin levels for tumor response to exclusive radiotherapy, in patients with locally advanced Human papillomavirus (HPV) 16-positive cervical cancer. PATIENTS AND METHODS: From 102 patients treated at our institutes, 38 patients with histologically-proven HPV16-positive cervical cancer were included in this prospective case-controlled study. All patients underwent exclusive radiotherapy-only. Complete response was defined as an absence of residual disease at clinical examination and radiological imaging, three months after the completion of treatment. Gene expression levels, assessed before radiotherapy, were compared between responders and non-responders. Controls consisted of normal cervical tissue samples from 30 patients with non-oncological indications. RESULTS: Twenty patients (52.6%) showed a complete response. Gene expressions of IGF1R (34%), IGF2 (24%), and GAPDH (median=3.26 versus 2.12) were increased in cancer patients, in comparison with the control group. Higher levels of expression of GAPDH were observed in patients co-expressing IGF2 and IGF1R, who had a hemoglobin level ≤ 11 g/dl (p=0.05). Clinical characteristics in the responder and in the non-responder groups were similar. In bi-variate and multi-variate analyses, IGF1R expression was the only factor predictive of response to radiotherapy (p=0.018). Accordingly, patients with IGF1R expression had a 28.6-fold greater risk of treatment failure. CONCLUSION: In our study, IGF1R was a strong predictive marker of lack of response to radiotherapy. Larger prospective trials are needed to validate IGF1R as a biomarker of radiation response for patients with HPV16-positive cervical cancer.
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Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/radioterapia , Expressão Gênica , Papillomavirus Humano 16/isolamento & purificação , Infecções por Papillomavirus/radioterapia , Receptor IGF Tipo 1/genética , Neoplasias do Colo do Útero/radioterapia , Adulto , Idoso , Feminino , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/biossíntese , Hemoglobinas/análise , Humanos , Fator de Crescimento Insulin-Like II/biossíntese , Pessoa de Meia-Idade , Prognóstico , Transcriptoma , Resultado do TratamentoRESUMO
UNLABELLED: Human Papillomavirus type 16 (HPV 16) DNA is regularly found in around 50% of all cervical carcinomas. Variants of this type have been found associated with different risks for cervical cancer development. Presence of HPV 16 variants in Colombia has not been previously reported. The aims of this study were to assess the feasibility of non-radioactive PCR-SSCP (polymerase chain reaction single-strand conformation polymorphism) analysis for determination of variability of ORF of E6, variability in the enhancer sequence of the LCR, and for establishment of the distribution of HPV 16 variants in invasive squamous cell carcinoma of the uterine cervix in Colombian women. Biopsies from 59 patients at the Instituto Nacional de Cancerología (INC) in Bogotá (Colombia) were collected. HPV detection was performed using universal primers. HPV 16 variants were detected by non-radioactive single-stranded conformational polymorphism (SSCP) analysis and direct sequencing. HPV 16 was detected in 57.6% of the tumors. The European branch was identified in 88.2% of the samples with the E-G350 class being the most prevalent variant (41.1%). The Asian-American branch was identified in 8.8% of the samples. Within this group it was possible to distinguish between c and a classes. It was not possible to determine the branch in 2.9% of the cases. A nucleotide transition (G to A) at position 7521 was the most prevalent variation (80%) found in the enhancer sequence of the LCR region. CONCLUSION: A non-radioactive PCR-SSCP analysis allowed us to distinguish between European and Asian-American branches of HPV 16, and to distinguish among classes in squamous cell carcinomas of the uterine cervix in Colombia. This method is an excellent alternative that can be used as a screening tool for identification of HPV 16 variants.
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Asiático , Carcinoma de Células Escamosas/virologia , Papillomavirus Humano 16/classificação , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase/métodos , Polimorfismo Conformacional de Fita Simples , Neoplasias do Colo do Útero/virologia , População Branca , Adulto , Idoso , Asiático/estatística & dados numéricos , Carcinoma de Células Escamosas/epidemiologia , Carcinoma de Células Escamosas/etnologia , Colômbia/epidemiologia , Elementos Facilitadores Genéticos , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Humanos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/etnologia , Mutação Puntual , Proteínas Repressoras/genética , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/etnologia , População Branca/estatística & dados numéricosRESUMO
INTRODUCTION: high risk HPV infections and variants presence has been associated to increase the risk of cervical cancer. However, there are few studies that analyze the presence of them in patients with cervical cancer before and alter radiotherapy treatment. OBJECTIVES: to analyse the human papilloma virus presence and E7/HPV16 variants in 60 women with cervical cancer before and after radiotherapy. MATERIALS AND METHODS: HPV detection and typing were based on a GP5+/GP6+ PCR Enzyme immune assay. E7/HPV16 variants were detected by PCR -Single strand conformation polymorphism (SSCP) and confirmed by direct sequence. RESULTS: before radiotherapy, 50/60 patients (83.3 percent) were HPV positive and HPV16 (53.3 percent) was the most prevalent type. After 3 months of radiotherapy, 55 patients attended to consult; of them, 19 (34.6 percent) were HPV positive, this decrease in the HPV detection was significant (p<0.0005). The E7/HPV16 analysis showed that 20 samples (62.5 percent) amplified before radiotherapy, 18 of them (90 percent) had identical SSCP pattern to the prototype and 2 showed a different SSCP pattern. The sequence of these two samples showed silent mutations at nt. 732 (T-to-C), 789 (T-to-C) and 795 (T-to-G). After radiotherapy, the was not detection of new mutations, 6 patients showed persistent HPV16 infection with the same SSCP pattern to the prototype, and samples that initially showed a different SSCP pattern were negative to E7/ HPV16 after radiotherapy. CONCLUSION: few E7/HPV16 variants were detected before radiotherapy and it seems that the treatment did not cause mutations in this gene