RESUMEN
Methylation markers have shown potential for triaging high-risk HPV-positive (hrHPV+) women to identify those at increased risk of invasive cervical cancer (ICC). Our aim was to assess the performance of the S5 DNA methylation classifier for predicting incident high-grade cervical intraepithelial neoplasia (CIN) and ICC among hrHPV+ women in the ARTISTIC screening trial cohort. The S5 classifier, comprising target regions of tumour suppressor gene EPB41L3 and L1 and L2 regions of HPV16, HPV18, HPV31, and HPV33, was assayed by pyrosequencing in archived hrHPV+ liquid-based samples from 343 women with high-grade disease (139 CIN2, 186 CIN3, and 18 ICC) compared to 800 hrHPV+ controls. S5 DNA methylation correlated directly with increasing severity of disease and inversely with lead time to diagnosis. S5 could discriminate between hrHPV+ women who developed CIN3 or ICC and hrHPV+ controls (p <.0001) using samples taken on average 5 years before diagnosis. This relationship was independent of cytology at baseline. The S5 test showed much higher sensitivity than HPV16/18 genotyping for identifying prevalent CIN3 (93% vs. 61%, p = .01) but lower specificity (50% vs. 66%, p <.0001). The S5 classifier identified most women at high risk of developing precancer and missed very few prevalent advanced lesions thus appearing to be an objective test for triage of hrHPV+ women. The combination of methylation of host and HPV genes enables S5 to combine the predictive power of methylation with HPV genotyping to identify hrHPV-positive women who are at highest risk of developing CIN3 and ICC in the future.
Asunto(s)
Metilación de ADN , Infecciones por Papillomavirus , Displasia del Cuello del Útero , Neoplasias del Cuello Uterino , Humanos , Femenino , Displasia del Cuello del Útero/virología , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/diagnóstico , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/virología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/diagnóstico , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/complicaciones , Adulto , Persona de Mediana Edad , Estudios de Cohortes , Detección Precoz del Cáncer/métodos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/aislamiento & purificaciónRESUMEN
Cervical cancer screening programs, including triage tests, need redesigning as human papillomavirus (HPV)-vaccinated women are entering the programs. Methylation markers offer a potential solution to reduce false-positive rates by identifying clinically relevant cervical lesions with progressive potential. In a nested case-control study, 9242 women who received the three-dose HPV16/18-vaccine at ages 12-15 or 18 in a community-randomized trial were included. Subsequently, they were re-randomized for either frequent or infrequent cervical cancer screening trials. Over a 15-year post-vaccination follow-up until 2022, 17 high-grade squamous intraepithelial lesion (HSIL) and 15 low-grade (LSIL) cases were identified at the 25-year screening round, alongside 371 age and community-matched HPV16/18-vaccinated controls. Methylation analyses were performed on cervical samples collected at age 25, preceding histologically confirmed LSIL or HSIL diagnoses. DNA methylation of viral (HPV16/18/31/33) and host-cell genes (EPB41L3, FAM19A4, and miR124-2) was measured, along with HPV-genotyping. No HPV16/18 HSIL cases were observed. The predominant HPV-genotypes were HPV52 (29.4%), HPV59/HPV51/HPV58 (each 23.5%), and HPV33 (17.7%). Methylation levels were generally low, with no significant differences in mean methylation levels of viral or host-cell genes between the LSIL/HSIL and controls. However, a significant difference in methylation levels was found between HSIL cases and controls when considering a combination of viral genes and EPB41L3 (p value = .0001). HPV-vaccinated women with HSIL had HPV infections with uncommon HPV types that very rarely cause cancer and displayed low methylation levels. Further investigation is warranted to understand the likely regressive nature of HSIL among HPV-vaccinated women and its implications for management.
RESUMEN
The shift towards primary human papillomavirus (HPV)-based screening has necessitated the search for a secondary triage test that provides sufficient sensitivity to detect high-grade cervical intraepithelial neoplasia (CIN) and cancer, but also brings an improved specificity to avoid unnecessary clinical work and colposcopy referrals. We evaluated the performance of the previously described DNA-methylation test (S5) in detecting CIN3 and cancers from diverse geographic settings in high-, medium- and low-income countries, using the cut-off of 0.80 and exploratory cut-offs of 2.62 and 3.70. Assays were performed using exfoliated cervical specimens (n = 808) and formalin-fixed biopsies (n = 166) from women diagnosed with cytology-negative results (n = 220), CIN3 (n = 204) and cancer stages I (n = 245), II (n = 249), III (n = 28) and IV (n = 22). Methylation increased proportionally with disease severity (Cuzick test for trend, P < .0001). S5 accurately separated women with negative-histology from CIN3 or cancer (P < .0001). At the 0.80 cut-off, 543/544 cancers were correctly identified as S5 positive (99.81%). At cut-off 3.70, S5 showed a sensitivity of 95.77% with improved specificity. The S5 odds ratios of women negative for cervical disease vs CIN3+ were significantly higher than for HPV16/18 genotyping at all cut-offs (all P < .0001). At S5 cut-off 0.80, 96.15% of consistently high-risk human papillomavirus (hrHPV)-negative cancers (tested with multiple hrHPV-genotyping assay) were positive by S5. These cancers may have been missed in current primary hrHPV-screening programmes. The S5 test can accurately detect CIN3 and malignancy irrespective of geographic context and setting. The test can be used as a screening and triage tool. Adjustment of the S5 cut-off can be performed considering the relative importance given to sensitivity vs specificity.
Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 18/aislamiento & purificación , Infecciones por Papillomavirus/complicaciones , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Estudios Transversales , Detección Precoz del Cáncer , Femenino , Estudios de Seguimiento , Salud Global , Humanos , Persona de Mediana Edad , Infecciones por Papillomavirus/virología , Pronóstico , Estudios Retrospectivos , Neoplasias del Cuello Uterino/epidemiología , Neoplasias del Cuello Uterino/genética , Adulto Joven , Displasia del Cuello del Útero/epidemiología , Displasia del Cuello del Útero/genéticaRESUMEN
Methylation of host and viral genes is promising for triage of women with high-risk human papillomavirus infections (hrHPV). Using a population-based sample of hrHPV positive women with cervical biopsies within 12 months after cervical screening, the clinical value of the S5 methylation classifier (S5), HPV genotyping and cytology were compared as potential triage tests, for outcomes of cervical intraepithelial neoplasia (CIN) grade 3 or greater (CIN3+), CIN2+ and CIN2, and the area under the curve (AUC) calculated. S5 scores increased with histopathology severity (Ptrend < .001). For CIN3+, the AUC was 0.780 suggesting S5 provides good discrimination between Asunto(s)
Infecciones por Papillomavirus
, Displasia del Cuello del Útero
, Neoplasias del Cuello Uterino
, Metilación de ADN
, Detección Precoz del Cáncer
, Femenino
, Papillomavirus Humano 16/genética
, Papillomavirus Humano 18/genética
, Humanos
, Papillomaviridae/genética
, Infecciones por Papillomavirus/complicaciones
, Infecciones por Papillomavirus/diagnóstico
, Infecciones por Papillomavirus/genética
, Triaje
, Neoplasias del Cuello Uterino/diagnóstico
, Neoplasias del Cuello Uterino/genética
, Neoplasias del Cuello Uterino/patología
, Displasia del Cuello del Útero/diagnóstico
, Displasia del Cuello del Útero/genética
, Displasia del Cuello del Útero/patología
RESUMEN
High-risk human papillomavirus (hrHPV) DNA tests have excellent sensitivity for detection of cervical intraepithelial neoplasia 2 or higher (CIN2+). A drawback of hrHPV screening, however, is modest specificity. Therefore, hrHPV-positive women might need triage to reduce adverse events and costs associated with unnecessary colposcopy. We compared the performance of HPV16/18 genotyping with a predefined DNA methylation triage test (S5) based on target regions of the human gene EPB41L3, and viral late gene regions of HPV16, HPV18, HPV31 and HPV33. Assays were run using exfoliated cervical specimens from 710 women attending routine screening, of whom 38 were diagnosed with CIN2+ within a year after triage to colposcopy based on cytology and 341 were hrHPV positive. Sensitivity and specificity of the investigated triage methods were compared by McNemar's test. At the predefined cutoff, S5 showed better sensitivity than HPV16/18 genotyping (74% vs 54%, P = 0.04) in identifying CIN2+ in hrHPV-positive women, and similar specificity (65% vs 71%, P = 0.07). When the S5 cutoff was altered to allow equal sensitivity to that of genotyping, a significantly higher specificity of 91% was reached (P < 0.0001). Thus, a DNA methylation test for the triage of hrHPV-positive women on original screening specimens might be a valid approach with better performance than genotyping.
Asunto(s)
Metilación de ADN/genética , Displasia del Cuello del Útero/diagnóstico , Displasia del Cuello del Útero/genética , Femenino , Genotipo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 16/patogenicidad , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/aislamiento & purificación , Papillomavirus Humano 18/patogenicidad , Papillomavirus Humano 31/genética , Papillomavirus Humano 31/aislamiento & purificación , Papillomavirus Humano 31/patogenicidad , Humanos , Proteínas de Microfilamentos/genética , Estadificación de Neoplasias , Embarazo , Sensibilidad y Especificidad , Displasia del Cuello del Útero/virologíaRESUMEN
Methylation of human papillomavirus (HPV) and host genes may predict cervical cancer risk. We examined the methylation status of selected sites in HPV16 and human genes in DNA extracted from exfoliated cervical cell samples of 244 women harboring HPV16-positive cancer or cervical intraepithelial neoplasia (CIN) or negative for intraepithelial lesions or malignancy (NILM). We quantified the methylation of CpG sites in the HPV16 L1 gene (CpG 6367 and 6389) and in the human genes EPB41L3 (CpG 438, 427, 425) and LMX1 (CpG 260, 262, 266, 274) following bisulfite treatment and pyrosequencing. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic utility of methylation level for the different sites and for a joint predictor score. Methylation in all sites significantly increased with lesion severity (p < 0.0001). Area under the curve (AUC) was highest among the CIN2/3 vs. cancer ranging from 0.786 to 0.853 among the different sites. Site-specific methylation levels strongly discriminated CIN2/3 from NILM/CIN1 and cancer from CIN2/3 (range of odds ratios [OR]: 3.69-12.76, range of lower 95% confidence bounds: 1.03-4.01). When methylation levels were mutually adjusted for each other EPB41L3 was the only independent predictor of CIN2/3 vs. NILM/CIN1 contrasts (OR = 9.94, 95%CI: 2.46-40.27). High methylation levels of viral and host genes are common among precancerous and cancer lesions and can serve as independent risk biomarkers. Methylation of host genes LMX1 and EPB41L3 and of the viral HPV16 L1 sites has the potential to distinguish among precancerous lesions and to distinguish the latter from invasive disease.
Asunto(s)
Metilación de ADN , Papillomavirus Humano 16/genética , Displasia del Cuello del Útero/genética , Neoplasias del Cuello Uterino/genética , Adulto , Femenino , Humanos , Curva ROC , Índice de Severidad de la Enfermedad , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/virologíaRESUMEN
Testing for high-risk (hr) types of human papillomavirus (HPV) is highly sensitive as a screening test of high-grade cervical intraepithelial neoplastic (CIN2/3) disease, the precursor of cervical cancer. However, it has a relatively low specificity. Our objective was to develop a prediction rule with a higher specificity, using combinations of human and HPV DNA methylation. Exfoliated cervical specimens from colposcopy-referral cohorts in London were analyzed for DNA methylation levels by pyrosequencing in the L1 and L2 regions of HPV16, HPV18, HPV31 and human genes EPB41L3, DPYS and MAL. Samples from 1,493 hrHPV-positive women were assessed and of these 556 were found to have CIN2/3 at biopsy; 556 tested positive for HPV16 (323 CIN2/3), 201 for HPV18 (73 CIN2/3) and 202 for HPV31 (98 CIN2/3). The prediction rule included EPB41L3 and HPV and had area under curve 0.80 (95% CI 0.78-0.82). For 90% sensitivity, specificity was 36% (33-40) and positive predictive value (PPV) was 46% (43-48). By HPV type, 90% sensitivity corresponded to the following specificities and PPV, respectively: HPV16, 38% (32-45) and 67% (63-71); HPV18, 53% (45-62) and 52% (45-59); HPV31, 39% (31-49) and 58% (51-65); HPV16, 18 or 31, 44% (40-49) and 62% (59-65) and other hrHPV 17% (14-21) and 21% (18-24). We conclude that a methylation assay in hrHPV-positive women might improve PPV with minimal sensitivity loss.
Asunto(s)
Metilación de ADN , Papillomaviridae/genética , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Estudios de Cohortes , Islas de CpG , ADN de Neoplasias/genética , ADN Viral/genética , Femenino , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Humanos , Neoplasias del Cuello Uterino/clasificación , Displasia del Cuello del Útero/clasificaciónRESUMEN
OBJECTIVE: Testing for high risk human papillomavirus (HR-HPV) is increasing; however due to limitations in specificity there remains a need for better triage tests. Research efforts have focused recently on methylation of human genes which show promise as diagnostic classifiers. METHODS: Methylation of 26 genes: APC, CADM1, CCND2, CDH13, CDKN2A, CTNNB1, DAPK1, DPYS, EDNRB, EPB41L3, ESR1, GSTP1, HIN1, JAM3, LMX1, MAL, MDR1, PAX1, PTGS2, RARB, RASSF1, SLIT2, SOX1, SPARC, TERT and TWIST1 was measured by pyrosequencing in cytology specimens from a pilot set of women with normal or cervical intraepithelial neoplasia grade 3 (CIN3) histology. Six genes were selected for testing in Predictors 1, a colposcopy referral study comprising 799 women. The three genes EPB41L3, DPYS and MAL were further tested in a second colposcopy referral study, Predictors 2, comprising 884 women. RESULTS: The six genes selected from the pilot: EPB41L3, EDNRB, LMX1, DPYS, MAL and CADM1 showed significantly elevated methylation in CIN2 and CIN3 (CIN2/3) versus ≤CIN1 in Predictors 1 (p<0.01). Highest methylation was observed in cancer tissues. EPB41L3 methylation was the best single classifier of CIN2/3 in both HR-HPV positive (p<0.0001) and negative samples (p=0.02). Logistic regression modeling showed that other genes did not add significantly to EPB41L3 and in Predictors 2, its classifier value was validated with AUC 0.69 (95% CI 0.65-0.73). CONCLUSION: Several methylated genes show promise for detecting CIN2/3 of which EPB41L3 seems the best. Methylated human gene biomarkers used in combination may be clinically useful for triage of women with HR-HPV infections.
Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Infecciones por Papillomavirus/genética , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Amidohidrolasas/genética , Femenino , Humanos , Proteínas de Microfilamentos/genética , Infecciones por Papillomavirus/patología , Reproducibilidad de los ResultadosRESUMEN
DNA methylation changes in human papillomavirus type 16 (HPV16) DNA are common and might be important for identifying women at increased risk of cervical cancer. Using recently published data from Costa Rica we developed a classification score to differentiate women with cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) from those with no evident high-grade lesions. Here, we aim to investigate the performance of the score using data from the UK. Exfoliated cervical cells at baseline and 6-months follow-up were analyzed in 84 women selected from a randomized clinical trial of women undergoing surveillance for low-grade cytology. Selection of women for the methylation study was based on detectable HPV16 in the baseline sample. Purified DNA was bisulfite converted, amplified and pyrosequenced at selected CpG sites in the viral genome (URR, E6, L1 and L2), with blinding of laboratory personnel to the clinical data. The primary measure was a predefined score combining the mean methylation in L1 and any methylation in L2. At the second follow-up visit, 73/84 (87%) women were HPV16 positive and of these 25 had a histopathological diagnosis of CIN2/3. The score was significantly associated with CIN2/3 (area under curve = 0.74, p = 0.002). For a cutoff with 92% sensitivity, colposcopy could have been avoided in 40% (95% CI 27-54%) of HPV16 positive women without CIN2/3; positive predictive value was 44% (32-58%) and negative predictive value was 90% (71-97%). We conclude that quantitative DNA methylation assays could help to improve triage among HPV16 positive women.
Asunto(s)
Proteínas de la Cápside/genética , Metilación de ADN/genética , Proteínas Oncogénicas Virales/genética , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Cuello del Útero/citología , Colposcopía , ADN Viral/genética , Femenino , Papillomavirus Humano 16/genética , Humanos , Indoles/uso terapéutico , Tamizaje Masivo , Clasificación del Tumor , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virologíaRESUMEN
We explored the association of human papillomavirus type 16 (HPV16) DNA methylation with age, viral load, viral persistence and risk of incident and prevalent high grade CIN (CIN2+) in serially collected specimens from the Guanacaste, Costa Rica cohort. 273 exfoliated cervical cell specimens (diagnostic and pre-diagnostic) were selected: (1) 92 with HPV16 DNA clearance (controls), (2) 72 with HPV16 DNA persistence (without CIN2+) and (3) 109 with CIN2+. DNA was extracted, bisulfite converted and methylation was quantified using pyrosequencing assays at 66 CpGs across the HPV genome. The Kruskal-Wallis test was used to determine significant differences among groups, and receiver operating characteristic curve analyses were used to evaluate how well methylation identified women with CIN2+. In diagnostic specimens, 88% of CpG sites had significantly higher methylation levels in CIN2+ after correction for multiple tests compared with controls. The highest area under the ROC curve (AUC) was 0.82 for CpG site 6457 in L1, and a diagnostic sensitivity of 91% corresponded to a specificity of 60% for CIN2+. Prospectively, 17% of CpG sites had significantly higher methylation in pre-diagnostic CIN2+ specimens (median time of 3 years before diagnosis) versus controls. The strongest pre-diagnostic CpG site was 6367 in L1 with an AUC of 0.76. Age-stratified analyses suggested that women older than the median age of 28 years have an increased risk of precancer associated with high methylation. Higher methylation in CIN2+ cases was not explained by higher viral load. We conclude that elevated levels of HPV16 DNA methylation may be useful to predict concurrently diagnosed as well as future CIN2+.
Asunto(s)
Metilación de ADN , ADN Viral/metabolismo , Papillomavirus Humano 16/genética , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Adolescente , Adulto , Área Bajo la Curva , Islas de CpG , Femenino , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias del Cuello Uterino/etiología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/etiología , Displasia del Cuello del Útero/virologíaRESUMEN
UNLABELLED: Oxygen free radicals and their reactive derivatives participate in formation of chronic inflammation states, which facilitate development of gastrointestinal tract tumors. Oxidative stress is one of the main causes of damage to cell membranes in result of exacerbated lipid peroxidation process. End products of lipid peroxidation (aldehydes, organic peroxides) react with important biological macromolecules such as DNA and proteins, cause changes in cell membrane structure and properties leading to loss of its integrity. Intensification of the lipid peroxidation process is a factor which may also lead to a malfunction in the antioxidant barrier, which further weakens the defense of cells against oxygen free radicals and promotes the onset and development of cancer. The aim of the study was the determination of lipid peroxidation level in gastrointestinal tract tumors (stomach, liver, colon, and colorectal cancer to liver metastases). MATERIAL AND METHODS: Materials for studies were obtained from 150 patients with gastrointestinal tract tumors: 10 with stomach cancer, 30 with malignant and benign liver cancers, 60 with primary colorectal cancer, and 50 with metachronous colorectal cancer liver metastases. We also investigated 25 patients with liver cirrhosis, which was treated as a pre-cancerous condition. In total, 175 patients were examined. Tumor specimens, and normal adjacent tissues (6-7 cm from the edge of the tumor), which served as control tissue in studies, were collected from patients (with their consent) during surgery. Additionally, liver specimens were collected from patients with liver cirrhosis. Lipid peroxidation level was determined spectrophotometrically as a concentration of final lipid peroxidation products, which in reaction with tiobarbituric acid (TBA) form colour complex (thiobarbituric acid-reactive substances - TBARS). RESULTS: The study showed the highest concentration of TBARS in benign, and the lowest in malignant liver tumors. Other types of gastrointestinal tumors studied, were characterized by similar levels of lipid peroxidation. TBARS concentration in these tumors was approximately 2-fold higher than in malignant liver tumors and much lower than in benign tumors. In all cancers of the digestive tract with the exception of malignant liver tumors increased level of TBARS was found, comparing with control tissue. The concentration of TBARS in cirrhotic liver was lower than in control. The level of lipid peroxidation in liver cirrhosis and malignant liver tumors was similar. There were no significant differences in TBARS concentration in the tumors of particular sections of the intestine and normal colon. The highest concentration of TBARS was found in G1 grade of colorectal cancer. In subsequent grades of cells differentiation (G2 and G3) its concentration was lower. The highest level of lipid peroxidation, expressed as the concentration of TBARS was found in the I stage of colorectal cancer clinical advancement. The significantly lowest concentration of TBARS was shown for stage II (UICC). CONCLUSIONS: The level of lipid peroxidation in cancerous cells of gastrointestinal tract indicates increased oxidative stress. The changes of lipid peroxidation level--a marker of oxidative stress in gastrointestinal tumors appear to be closely associated with their development stages (liver cirrhosis/malignant liver cancer; colorectal cancer/colorectal cancer liver metastases) and are likely to create such conditions, in which cancerous cells may proliferate, undergo gradual dedifferentiation and malignancy, and generate metastases.
Asunto(s)
Neoplasias Gastrointestinales/metabolismo , Peroxidación de Lípido , Neoplasias Hepáticas/secundario , Adulto , Anciano , Femenino , Neoplasias Gastrointestinales/patología , Humanos , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Persona de Mediana Edad , Estrés OxidativoRESUMEN
INTRODUCTION: Clinical and experimental studies indicate oxygen free radicals and their reactive derivatives participation in formation of chronic inflammation states, which facilitate gastrointestinal tract tumors development. During malignant changes formation in epithelium of gastrointestinal tract increased oxygen radicals generation initiates lipid peroxidation and DNA and proteins oxidation processes. Final lipid peroxidation products (saturated and unsaturated aldehydes) are highly reactive and characterized by greatly longer half life time than reactive oxygen species and capability to diffuse from places of their formation to distant cell areas. In cells they react with important biological macromolecules such as DNA and proteins causing their structural and functional damages. The effects of changes in cell membranes structure are increase in their permeability, depolarization, decrease of hydrophobicity and inhibition of enzymes, membrane channels and transporters. These changes lead to the loss of cell integrity. STUDY AIM: Determination of lipid peroxidation level in blood serum patients with gastrointestinal tract tumors. MATERIALS AND METHODS: Materials for studies were obtained from 170 patients with gastrointestinal tract tumors: 10 with stomach cancer, 20 with pancreatic cancer, 30 with primary liver cancer, 60 with primary colorectal cancer and 50 with metachronic colorectal cancer liver metastases. Blood was taken from patients 1 day before and 6 days after surgery. Control blood was obtained from 53 healthy blood donors. Lipid peroxidation level was determined spectrophotometrically as a concentration of final lipid peroxidation products, which in reaction with tiobarbituric acid (TBA) form colour complex (thiobarbituric acid-reactive substances, TBARS). RESULTS: Higher lipid peroxidation level was observed in pre- and postoperative blood sera patients with gastrointestinal tumors in comparison to serum from healthy blood donors. CONCLUSIONS: Increased lipid peroxidation level in peripheral blood of patients with gastrointestinal tract tumors is evidence of intensive oxidative stress and might indicate impairment of antioxidant defense mechanisms in organism.
Asunto(s)
Neoplasias Gastrointestinales/sangre , Peroxidación de Lípido , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Periodo Posoperatorio , Periodo PreoperatorioRESUMEN
Reactive oxygen species (ROS) formation are a constant element of a cell's oxygen metabolism. They are its normal products and in physiological concentrations they play important roles in a variety of cell processes. Disturbances in the balance between ROS formation and the efficiency of antioxidant mechanisms lead to oxidative stress. Oxidative stress causes damage to important macromolecules, such as DNA, proteins, and lipids. Growing evidence indicates the participation of ROS in the cancerous transformation of cells. Oxidative stress was also found in cancer cells, but the mechanisms responsible for its induction have not been definitively explained. It is known that they include inflammation and cytokine action, oncogenic signals, intensive metabolism related to constant proliferation, mutations in mitochondrial DNA, and malfunction in the respiratory chain. A high level of ROS in cancer cells may lead to a variety of biological responses, such as cell adaptation, increased proliferation rate, formation of DNA mutations and genetic instability, and resistance to some drugs used in anticancer therapy. Therefore, oxidative stress in cancer cells promotes tumor development, but it can also be useful in the search for new therapeutic strategies of cancer treatment.
Asunto(s)
Neoplasias/fisiopatología , Estrés Oxidativo , Especies Reactivas de Oxígeno/efectos adversos , Proliferación Celular , Resistencia a Antineoplásicos , HumanosRESUMEN
BACKGROUND: Vigilant management of women with high-risk human papillomavirus (hrHPV) is necessary in cancer screening programs. To this end, we evaluated the performance of S5 (targeting DNA methylation in HPV16, HPV18, HPV31, HPV33, and human gene EPB41L3) to predict cervical intraepithelial neoplasia grade 2 or higher (CIN2+) in a sample of hrHPV-infected women referred to colposcopy in the FRIDA Study, a large screening trial in Mexico. A nested case-control sample with women referred to colposcopy either by atypical squamous cells of undetermined significance or higher (ASCUS+) in cytology and/or positive for HPV types 16 or 18 was tested by S5. Seventy-nine cases of CIN2+ were age-matched to 237 controls without a diagnosis of CIN2+ (Asunto(s)
Metilación de ADN
, Papillomavirus Humano 16/genética
, Papillomavirus Humano 18/genética
, Proteínas de Microfilamentos/genética
, Infecciones por Papillomavirus/genética
, Displasia del Cuello del Útero/diagnóstico
, Neoplasias del Cuello Uterino/diagnóstico
, Adulto
, Área Bajo la Curva
, Estudios de Casos y Controles
, Ensayos Clínicos como Asunto
, Colposcopía
, ADN Viral/genética
, Detección Precoz del Cáncer/métodos
, Epigénesis Genética
, Femenino
, Genotipo
, Humanos
, México
, Persona de Mediana Edad
, Infecciones por Papillomavirus/complicaciones
, Sensibilidad y Especificidad
, Análisis de Secuencia de ADN
, Triaje
, Neoplasias del Cuello Uterino/genética
, Neoplasias del Cuello Uterino/virología
, Displasia del Cuello del Útero/genética
, Displasia del Cuello del Útero/virología
RESUMEN
UNLABELLED: Transcription factors AP-1 and NF-kappaB control important cell processes like proliferation, apoptosis and differentiation. Disturbance of this processes is often a reason of carcinogenesis. Therefore AP-1 and NF-kappaB might have an important role in arising and development of tumors. THE AIM OF THE STUDY: An evaluation of changes of AP-1 and NF-kappaB protein level in selected human alimentary tract tumors. Material and methods. Materials were obtained from patients with alimentary tract tumors (stomach, liver; colon), diagnosed by routine histopathological examination. Normal tissues were taken more than 6-7 cm away from tumor border (histologically examined). Protein level of AP-1 and NF-kappaB was detected by standard Western blotting technique. RESULTS: In all examined alimentary tract tumors (gastric adenocarcinoma, hepatocellular carcinoma, colorectal adenocarcinoma and liver metastases) we observed changes in AP-1 and NF-kappaB protein level comparing with normal tissues. CONCLUSIONS: Changes in AP-1 and NF-kappaB expression indicate, that they are important factors not only in initiation, but also in further development of digestive tract tumors.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Gastrointestinales/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Persistent infection %by with high risk human papillomavirus (hrHPV) types causes cervical cancer but most women who test positive are at very low risk of neoplasia. Strategies are needed which can retain high sensitivity of hrHPV testing but reduce the number of false-positives. We showed previously that a combination DNA methylation triage assay for HPV types 16, 18 and 31 and human gene EPB41L3 was useful to identify high grade cervical lesions. OBJECTIVE: Assess whether measurement of DNA methylation in HPV type 33 can improve the previous classifier. METHODS: A London colposcopy referral group of 1493 women of whom 556 (37%) had histologically-confirmed CIN (cervical intraepithelial neoplasia) 2 or 3 that included 114 HPV33 positive women with methylation measured for three L2 CpGs 5557, 5560 and 5566. Discrimination performance was assessed for the new classifier S5, built by adding HPV33 to the earlier classifier. RESULTS: HPV33 methylation measurement improved prediction among HPV33 positive women. Receiver operating characteristic analyses showed an area under the curve (AUC) for HPV33 methylation of 0.68 (95% CI 0.57-0.78). The earlier risk score was significantly improved by HPV33 methytlation (AUC = 0.82 vs 0.80; P < 0.001). For 90% sensitivity the specificity for CIN2/3 was 49% (95% CI 46-52%). CONCLUSIONS: Measurement of HPV33 DNA methylation contributes independent diagnostic information to EPB41L3 and HPV16, HPV18 and HPV31, and is superior to genotyping. Other HPV and human methylation target regions might be useful to further improve S5.
Asunto(s)
Metilación de ADN/genética , ADN Viral/genética , Genotipo , Neoplasias del Cuello Uterino/genética , Detección Precoz del Cáncer , Femenino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidad , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/patogenicidad , Papillomavirus Humano 31/genética , Papillomavirus Humano 31/patogenicidad , Humanos , Papillomaviridae/genética , Papillomaviridae/patogenicidad , Embarazo , Factores de Riesgo , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
BACKGROUND: High risk human papillomavirus (HR-HPV) infection is common and only a small minority of infections become persistent and lead to cervical cancers. Women positive for HR-HPV usually require a second test to avoid unnecessary colposcopies and over treatment. Elevated DNA methylation of HR-HPV L1 and L2 genes in high grade disease has emerged as a promising molecular triage tool. OBJECTIVES: Our aim was to accurately measure methylation levels at selected CpG positions in the HPV18, HPV31 and HPV33 genomes. We focused on the L2, L1, URR and E6 regions because these were previously shown to be interesting areas for study. STUDY DESIGN: Pyrosequencing was used to measure methylation in 208 HPV18, 207 HPV31, and 126 HPV33 positive women selected from a London colposcopy referral population. RESULTS: After adjustment for multiple testing, at FDR 5%, elevated methylation was significantly associated with cervical intraepithelial neoplasia grades 2 or worse (CIN2+) in all investigated CpGs in HPV18 L2 and L1. Two of 6 L2 and 12 of 15 L1 sites in HPV31 and 6 of 8 L2 and 3 of 13 L1 sites in HPV33 showed significantly elevated methylation in CIN2+. Methylation of CpG sites in the URR and E6 region of the HPV types was low and most differences were not significant. CONCLUSION: Elevated methylation of CpG sites in the L1 and L2 regions of HPV18, HPV31 and HPV33 is associated with CIN2+ and a panel test may be useful for triage of women with HR-HPV infections.
Asunto(s)
Alphapapillomavirus/genética , Metilación de ADN/genética , Genoma Viral/genética , Lesiones Precancerosas/virología , Neoplasias del Cuello Uterino/virología , Adulto , Estudios de Cohortes , ADN Viral/análisis , ADN Viral/genética , Femenino , Humanos , Análisis de Secuencia de ADN , Estadísticas no ParamétricasRESUMEN
BACKGROUND: The need for new prognostic factors in breast cancer is ever increasing as breast cancer management evolves. Aberrant DNA methylation plays a pivotal role in cancer development and progression; DNA methylation-based biomarkers may provide independent prognostic information. We used pyrosequencing to investigate the prognostic potential of quantitative DNA methylation of a large set of candidate genes in a Korean single-institution series of operable breast cancer. METHODS: Absolute DNA methylation in 20 candidate genes from an initial set of 30 genes was measured by pyrosequencing of bisulfite converted DNA in 121 fresh frozen breast cancer cases. Survival analyses used continuous and categorized (quintile-based) gene methylation data with time to recurrence (TTR) as an endpoint. Prognostic abilities of gene-only and risk-score models were explored. RESULTS: Median follow-up was 5.1 years; 25 recurrences (21%) were observed. Nodal status, methylation of TWIST1, SLIT2 (both as continuous and categorized variables) and APC, HLA-A, NKX2-5, SERPINB5, SFN (as categorized variables) were significantly prognostic; grade showed a prognostic trend. A multivariate model containing nodal status, grade and TWIST1 was a best fit (p< 0.001) in stepwise regression; risk-score based on this model separated patients into 3 distinct risk-groups (p< 0.001). A gene-only model based on TWIST1 and SFN also classified patients into distinct risk-groups (p=0.009). CONCLUSIONS: This study shows that accurate quantitative measurement of DNA methylation by pyrosequencing identifies a small set of genes with independent prognostic potential in breast cancer. These genes complement the current clinico-pathological prognostic factors and appear to be potential biomarkers that warrant further validation.