Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication.

Aberrant CpG-island methylation affects ovarian cancer progression. The promotor methylation changes at tumor suppressive genes in ovarian cancer stromal progenitor cells (OCSPCs) and epithelial ovarian cancer (EOC) tissues and their clinical implication remains unexplored. We systemically analyzed the promoter methylation status of 40 tumor suppressor genes (TSGs) associated with cancer in paired epithelial-like and mesenchymal-like OCSPCs and ovarian cancer cells by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The effect of DNA methylation on gene expression was confirmed using qRT-PCR. The differential frequencies of TSGs' promoter methylation among matched epithelial-like or mesenchymal-like OCSPCs from tissues and ascites and ovarian cancer tissues were further validated in cancer tissues and correlated with clinicopathological features and survival outcomes of patients. According to the promoter methylation frequencies of the 40 TSGs, promoters of RASSF1A were the only significantly hypomethylated in epithelial-like OCSPCs from tissues than those from ascites and bulk tumor cells (0% vs 38% vs 45%, P=0.039 by Fisher's exact test). The most frequencies at promotor hypermethylation of TSGs in mesenchymal-like OCSPCs from ascites which processed aggressiveness were CDKN2B (73%) followed by CCND2 (45%) and RASSF1A (45%). Forty-three percent (47/110) of RASSF1A and 45% of CCND2 were validated as a frequently hypermethylated gene in an independent set of 110 EOC tissues in contrast to none (0/60) and 12% (10/60) of benign ovarian cysts (both P<0.001). Functional experiments revealed overexpression of CCND2 or CDKN2B in MSc-OCSPCs decreases EMT, invasion, and spheroid formation in EOC, and abolishes DNMT1 and COL6A3 expression. However, for the expected 5-year overall survival (OS) for patients with methylated RASSF1A, CCND2, and CDKN2B, only RASSF1A was significantly worse than those without methylated RASSF1A (56% vs 80%, p=0.022). Taken together, overexpression of CCND2 and CDKN2B decreased the aggressiveness of mesenchymal-like OCSPCs from ascites which may represent a potential therapeutic target for EOC. Promotor hypomethylation at RASSF1A in OCSPCs from EOC tissues and changes to hypermethylation of EOC and OCSPCs from ascites could predict poor survival outcomes for EOC patients compared to without those changes of CCND2 and CDKN2B.

Isolation and characterization of stromal progenitor cells from ascites of patients with epithelial ovarian adenocarcinoma.

BACKGROUND: At least one-third of epithelial ovarian cancers are associated with the development of ascites containing heterogeneous cell populations, including tumor cells, inflammatory cells, and stromal elements. The components of ascites and their effects on the tumor cell microenvironment remain poorly understood. This study aimed to isolate and characterize stromal progenitor cells from the ascites of patients with epithelial ovarian adenocarcinoma (EOA). METHODS: Seventeen ascitic fluid samples and 7 fresh tissue samples were collected from 16 patients with EOA. The ascites samples were then cultured in vitro in varying conditions. Flow cytometry and immunocytochemistry were used to isolate and characterize 2 cell populations with different morphologies (epithelial type and mesenchymal type) deriving from the ascites samples. The in vitro cell culture model was established using conditional culture medium. RESULTS: The doubling times of the epithelial type and mesenchymal type cells were 36 h and 48 h, respectively, indicating faster growth of the epithelial type cells compared to the mesenchymal type cells. Cultured in vitro, these ascitic cells displayed the potential for self-renewal and long-term proliferation, and expressed the typical cancer stem/progenitor cell markers CD44(high), CD24(low), and AC133(+). These cells also demonstrated high BMP-2, BMP4, TGF-ß, Rex-1, and AC133 early gene expression, and expressed EGFR, integrin α2ß1, CD146, and Flt-4, which are highly associated with tumorigenesis and metastasis. The epithelial type cells demonstrated higher cytokeratin 18 and E-cadherin expression than the mesenchymal type cells. The mesenchymal type cells, in contrast, demonstrated higher AC133, CD73, CD105, CD117, EGFR, integrin α2ß1, and CD146 surface marker expression than the epithelial type cells. CONCLUSION: The established culture system provides an in vitro model for the selection of drugs that target cancer-associated stromal progenitor cells, and for the development of ovarian cancer treatments.

Value of pre-operative serum CA125 level for prediction of prognosis in patients with endometrial cancer.

BACKGROUND: High pre-operative CA125 levels in women with endometrial cancer may be related to lymph node metastases and poor prognosis. AIM: To evaluate whether pre-operative cancer antigen 125 (CA125) levels are associated with lymph node metastases and prognosis in endometrial cancer. METHODS: One hundred and twenty women with endometrial cancer were retrospectively reviewed for pre-operative CA125 levels. The results were then correlated with the clinicopathological outcome. RESULTS: An elevated CA125 (>40 U/mL) was significantly correlated with higher stage, higher grade, increased depth of myometrial invasion, lymph node metastases and the presence of lympho-vascular space involvement in endometrial cancer. Five-year overall survival (OS) and recurrence-free survival (RFS) rates were significantly higher in women with endometrial cancer with CA125 ≤ 40 U/mL than those with CA125 > 40 U/mL (P < 0.001). When women were further stratified according to CA125 levels and lymph node status, OS and RFS were highest for those with CA125 ≤ 40 U/mL and without lymph node metastases, and lowest for those with lymph node metastases and CA125 > 40 U/mL (P < 0.001). CONCLUSION: The testing of pre-operative CA125 levels may a useful prognostic tool in endometrial cancer management.

Integration of human papillomavirus correlates with high levels of viral oncogene transcripts in cervical carcinogenesis.

A prospective, cross-sectional study was conducted to investigate the correlation between the integration of high-risk human papillomavirus and disease severity of cervical lesions. 720 liquid-based cytology specimens including 422 normal cytology, 78 low-grade squamous intraepithelial lesions, 172 high-grade squamous intraepithelial lesions, and 48 women with cervical cancers were examined using HPV blot and type-specific E6 PCR. Positive HPV DNA types 16, 18, 52 and 58 were examined for viral DNA using real-time PCR. Expression of E6 transcripts was 89.5% (pure integration), 71.7% (mixed type), and 47.1% (pure episomal) (p<0.0001). Geometric mean levels ranged from 110.6 (episomal form) to 508.4 (mixed form), and 5966.2 (integration form) by real-time PCR (p<0.0001). Geometric mean levels of E6 transcript in HPV 16, 18, 52, and 58 correlated with the severity of cervical lesions and the physical integration state of the viral genome (p<0.0001). We conclude that this is the first paper to point out that integration of high-risk HPVs not only 16 and 18 but also 52 and 58 is correlated with high levels of oncogene transcripts from normal cervix, CIN to cervical cancer.

Post-coital vaginal douching is risky for non-regression of low-grade squamous intraepithelial lesion of the cervix.

BACKGROUND: Vaginal douching is a common practice worldwide. Its effect on the natural history of the early lesion of human papillomavirus (HPV) infection, low-grade squamous intraepithelial lesion (LSIL), is unknown. METHODS: In a prospective nation-wide cohort (n=1332), epidemiological variables including habit of vaginal douching after intercourse and outcomes of LSIL were studied. Colposcopy-confirmed LSIL women (n=295) were followed every 3 months. Parameters of HPV infection, sexual behavior, personal hygiene and environmental exposures were compared with the follow-up outcomes. RESULTS: There was a 15% chance of HSIL co-existing with the LSIL cytology result. Eight percent of colposcopy-confirmed LSIL were found with HSIL in 1 year. With a follow-up of up to 36 months, 83% LSIL regressed, 11% progressed and 6% persisted. The mean time (95% CIs) to regression and progression were 5.2 (4.7-5.8) and 8.0 (5.8-10.3) months, respectively. Risk factors of the non-regression of LSIL included HPV prevalence on enrollment, habit of vaginal douching after intercourse with a hygiene product and non-regular Pap screening, with odd ratio of 4.4 (1.9-10.3), 3.14 (1.04-9.49) and 2.12 (1.24-3.62), respectively. HPV prevalence and vaginal douching also conferred a slower regression of LSIL (8.0 vs. 4.1 months, P<.001 and 8.0 vs. 5.6 months, P=0.02, respectively). CONCLUSION: The study disclosed a transient but warning nature of cytological LSIL. Practicing of vaginal douching after intercourse, especially with hygiene products, is associated with non-regression of LSIL.

Promoter methylation of sFRP5 in patients with ovarian clear cell adenocarcinoma.

BACKGROUND: Specific tumour suppressor genes with promoter methylation in ovarian clear cell adenocarcinoma (OCCA) can be one important epigenetic mark distinguishing OCCA from ovarian serous adenocarcinoma (OSA), benign endometriotic cysts and normal ovarian epitheliums. MATERIALS AND METHODS: Five OCCA cell lines, 63 cancer tissues (48 OCCA and 15 OSA), 10 benign endometriotic cysts and five normal ovarian epitheliums were analysed by methylation-specific PCR using pooled DNAs to determine the methylation status of the promoter of the target genes, including genes for secreted frizzled-related proteins (sFRP1 to 5), adenomatous polyposis coli (APC), retinoblastoma protein 1 (Rb1), breast cancer 1 gene (BRCA1), p14(ARF), p15(INK4b), p16(INK4a) and survivin. Methylation frequencies of identified targets were further analysed with individual DNA samples. RESULTS: The sFRP5 promoter was significantly methylated in all OCCA cell lines, with 64.6% in OCCA tissues compared with 13.3% in OSA, and 0% in benign endometriotic cysts and normal ovarian epitheliums (P < 0.0001). With a median follow-up of 44 months, the expected 5-year overall survival (OS) for patients with methylated sFRP5 promoter were significantly worse than for those with unmethylated sFRP5 (52% vs. 88%, P = 0.03). After adjusting for age, stage, and residual disease after primary surgery, patients with unmethylated sFRP5 promoter had an independent good prognostic factor in OS (P = 0.017). CONCLUSION: The high percentage of promoter methylation in the sFRP5 gene in OCCA indicates its importance in the development of OCCA and is a potential useful marker for prognoses and target for treatment of OCCA.

Type-specific human papillomavirus oncogene messenger RNA levels correlate with the severity of cervical neoplasia.

This study aimed to evaluate whether quantitation of high-risk human papillomavirus (HR-HPV) E6 messenger RNA (mRNA) can be a potential biomarker for detecting the severity of cervical lesions. HPV genotyping was performed using a modified MY11/GP6+ PCR for HPV DNA amplification, followed by HPV genotype-specific hybridization with on a gene chip. E6 type-specific PCR was used to validate multiple infections. Quantitative real-time reverse transcriptase (QRT-PCR) and real-time PCR used to measure mRNA levels and DNA viral loads of 6 HPV oncogenic types (HPV 16, 18, 31, 33, 52 and 58) in 720 liquid-based cytology samples. The HPV DNA and RNA measurements were correlated with cervical lesions diagnosed by histopathologic examination. mRNA transcripts in the 6 types HPV DNA-positive cases was lower in normal women and

PTEN promoter methylation and LOH of 10q22-23 locus in PTEN expression of ovarian clear cell adenocarcinomas.

OBJECTIVES: Loss of phosphatase and tensin homolog (PTEN) expression is common in ovarian clear cell adenocarcinomas (OCCA), but PTEN mutations are not frequently observed in OCCA. The mechanism of PTEN gene silencing in OCCA is still not clear. MATERIALS AND METHODS: Immunohistochemical analysis of PTEN expression was performed in 40 OCCA paraffin-embedded tissues. PTEN promoter methylation in 24 OCCA tissues and 5 OCCA cell lines was examined by methylation-specific PCR. Eighteen OCCA patients and 13 serous adenocarcinomas were analyzed for loss of heterozygosity (LOH) at 10q23 with five polymorphic markers. RESULTS: Of the 40 OCCAs, 37.5% (15/40) had reduced PTEN immunoreactivity, LOH was found in 33% (6/18) of OCCAs, and 31% (4/13) of serous adenocarcinomas. In the 33% of OCCAs with LOH, only 33% (2/6) lost PTEN expression. PTEN promoter was unmethylated in 5 OCCA cell lines and 24 OCCA tissues detected by MSP-PCR. No significant correlation between PTEN expression and advanced stage disease or overall survival was found. CONCLUSION: Our results indicate that reduced PTEN expression was detected in more than one third of OCCA cases. Neither PTEN promoter methylation nor LOH at 10q23 locus is significantly related to PTEN inactivation and is not an adverse prognostic factor in OCCA.

Human papillomavirus typing with a polymerase chain reaction-based genotyping array compared with type-specific PCR.

BACKGROUND: Type-specific persistence of human papillomavirus (HPV) infection can cause invasive cervical cancer. OBJECTIVES: To evaluate the efficacy of HPV detection and typing with a general polymerase chain reaction (PCR)-based genotyping array and to compare it with a type-specific PCR assay. STUDY DESIGN: Four hundred and thirty-three cervical samples were tested with a modified MY11/GP6+ PCR-based reverse-blot assay (EasyChip HPV Blot; King Car, Taiwan [hereafter HPV Blot]) and with 20 genotypes of L1-type-specific PCR (HPV-6, -11, -16, -18, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, -59, -62, -66, -68, -70, and -71 [CP8061]). RESULTS: The concordance of the two tests in determining HPV positivity was 96.8% (419/433), with a Cohen's kappa=0.93 (95% CI: 0.90-0.97) and McNemar's test of P=1.0, which indicates excellent agreement. The overall concordance of the two tests in the identification of type-specific HPV was 91.0% (394/433). Sensitivity (90-100%), specificity (99.2-100%), and accuracy (98.6-100%) rates of HPV Blot against the gold standard were satisfactory for HPV-16, -18, -58, -33, -52, -39, -45, -31, -51, -70 while HPV-71 (63.6%) had suboptimal sensitivity. Though the kappa values between the two tests for many individual genotypes could not be reliably calculated because of low positivity, the kappa values for HPV-16, -52, and -58 were excellent (0.93, 0.96, and 0.95, respectively). CONCLUSION: The modified MY11/GP6+ PCR-based HPV Blot assay is accurate and sensitive for detection and genotyping of HPV in cervical swab samples.

Integrated human papillomavirus types 52 and 58 are infrequently found in cervical cancer, and high viral loads predict risk of cervical cancer.

OBJECTIVE: The aim of this prospective study was to analyze whether integration or high viral loads of human papillomavirus (HPV) is essential for malignant transformation of HPV types 52 and 58 as well as types 16 and 18. METHODS: Cervical swabs from 178 consecutive patients, including 81 with invasive cervical cancers and 97 with cervical intraepithelial neoplasias (CIN) II-III, were collected and examined to determine the physical status and viral load of HPV types 16, 18, 52 and 58 DNA using genechip and real-time PCR (polymerase chain reaction) analysis. RESULTS: In cervical cancer patients, the integrated form of HPV 52 and 58 DNA was found in 25.0% and 12.5% of swabs, respectively; while HPV16 and 18 DNA was found in 82.6% and 100% of swabs, respectively (P < 0.01, for pair-wise comparison of types 16, 18 versus types 52, 58). The viral loads reflected by the amount of E6 for HPV 16, 18, or 52 were significantly increased in invasive cervical cancer compared to CINII-III (P = 0.022 for type 16, P = 0.003 for type18, and P = 0.001 for type 52, respectively). Area under the receiver operating characteristic (ROC) curve for cervical cancer versus CIN II-III was 73.8%, 92.9%, and 88.5% for HPV 16, 18, and 52, respectively, indicating that real-time PCR had good diagnostic value in differentiating cervical cancer from CIN II-III. CONCLUSIONS: Infrequent integration of HPV 52 and 58 DNA in cervical cancer suggests that it is not prerequisite for progression to cervical cancer. High viral loads (E6) of HPV 16, 18, and 52 DNA may be predictive of the transition of CIN II-III to cervical cancer. Our results indicate that both viral DNA physical status and viral loads of HPV are important factors in the carcinogenesis of different HPV types.

Prevalence of cervical human papillomavirus in Taiwanese women.

PURPOSE: To define the prevalence rate of cervical human papilloma virus (HPV) using DNA oligonucleotide microarray and its correlation with risk factors in Taiwanese women in metropolitan Taipei. METHODS: Thirteen hundred and twenty healthy women, aged 21 - 65 yr without history of cervical intraepithelial neoplasia (CIN) or carcinoma were included in this prospective study. Pap smear and HPV typing using oligonucleotide microarray were performed for each woman. They were given a standardized questionnaire to obtain information about the risk factors of cervical cancer in Taiwan. RESULTS: The overall HPV positivity was 19.85% and multiple infections were found in 35.84% of the infected group, 7.92% of the whole study population. The younger the subject, the higher was the infection rate and multiple infection rates. The most common HPV types were 16, 18, 58, 52, 51 and 56, which is different from the western world. The sensitivity of the HPV DNA chip in detecting CIN and cervical carcinoma is 97.06%, and 100% in detecting CIN 2 or more lesions. Risk factors for HPV infection include earlier coitarche (P < 0.01), multiple sexual partners (P < 0.05), history of sexually transmitted disease (P < 0.05), two or more vaginal deliveries (P < 0.05) and infrequent use of condoms (P < 0.05). The association between oral contraception or cigarette smoking and HPV infection could not be determined because few women smoke or used oral contraception. There was no relationship between induced abortion and HPV infection. CONCLUSIONS: About one-fifth of adult women in metropolitan Taipei were cervical HPV positive. The popular HPV types and the risk factors of HPV infection in metropolitan Taipei are not the same as those in the western world. The sensitivity of the HPV DNA chip in detecting cervical neoplasia is very high.

Detection and quantitation of human papillomavirus type 16, 18 and 52 DNA in the peripheral blood of cervical cancer patients.

OBJECTIVE: To prospectively evaluate the feasibility of detecting human papillomavirus (HPV) type 16, 18 and 52 DNA in the peripheral blood of patients with cervical cancer using real-time polymerase chain reaction (PCR) and to determine its prognostic importance. METHODS: Blood and cervical swab specimens from 135 consecutive patients with 60 invasive cervical cancers, 10 microinvasions, 20 cervical intraepithelial neoplasias (CIN) III, 10 CIN II, 10 CIN I and 25 controls were collected and examined for HPV type 16, 18 and 52 DNA using real-time PCR to investigate the prevalence and viral load of HPV DNA at the time of diagnosis and during follow-up in patients with positive blood samples. RESULTS: Of the 60 patients with invasive cervical cancer, 27% had positive test results for HPV DNA in blood samples in contrast to 0% of patients with microinvasions, CIN III, CIN II, CIN I and normal controls. The DNA detection rates of viral subtypes in blood samples of cervical cancer patients were 5% for HPV-16, 16.7% for HPV-18, 8.3% for HPV-52, 1.7% for both HPV-16 and HPV-18 and 1.7% for both HPV-18 and HPV-52, while the detection rates in cervical swab specimens were 36.2% for HPV-16, 15.5% for HPV-18 and 17.2% for HPV-52. During follow-up, 8 of 10 cervical cancer patients with viral DNA detected in blood within 3 months after treatment had recurrence, and a high percentage (87.5%, 7/8) of this recurrence involved distant metastases. CONCLUSIONS: In this study, real-time PCR detected HPV-16, -18 or -52 DNA in the peripheral blood of more than one-fourth of invasive cervical cancer patients. The association between risk of cancer recurrence and the amount of viral DNA detected in blood among cervical cancer patients after treatment is intriguing and deserves further investigation.

Multivariate analysis of the prognostic factors and outcomes in early cervical cancer patients undergoing radical hysterectomy.

OBJECTIVE: This study was performed to identify pathologic and clinical risk factors that best predicted 5-year recurrence-free survival (RFS) among patients with early-stage cervical carcinoma, treated by radical hysterectomy and pelvic lymphadenectomy. METHODS: The records of 197 patients with early-stage invasive cervical carcinoma who underwent radical hysterectomy and pelvic lymphadenectomy from 1990 to 1999 were retrospectively reviewed. Clinical and pathologic variables including age, tumor size (TS), clinical stage, depth of invasion (DI), lymphovascular space involvement (LVSI), cell type, tumor grade, lymph node metastases (LNM), parametrial invasion, surgical margin involvement, and pattern of adjuvant therapy were analyzed using univariate and multivariate methods to define those variables that best predicted RFS. RESULTS: Outer 1/3 invasion, LVSI, and LNM were identified as independent poor prognostic factors, which were used to define three prognostic groups: patients (n = 104) with good prognoses (LVSI (-) and LNM (-)), patients (n = 46) with intermediate prognoses (either LVSI (+) without outer 1/3 invasion or LNM (+) without LVSI), and patients (n = 47) with poor prognoses (LVSI (+) patients with outer 1/3 invasion). The estimated 3-year RFS for patients with LVSI and deeply invasive tumors regardless of nodal status and/or nodal metastases receiving adjuvant CT + RT was significantly greater than that for patients who received only adjuvant radiotherapy (80% vs. 49%, P = 0.048 in the group of patients with LVSI and deeply invasive tumors with positive nodes and without positive nodes; 87% vs. 36%, P = 0.013 in the group of patients with LVSI and deeply invasive tumors with positive nodes only). CONCLUSIONS: The multivariate analysis and prognostic grouping system maximally separated patients with early-stage invasive cervical carcinoma into groups with good, intermediate, or poor prognoses, with 3-year RFSs of 90%, 82%, 67%; and 5-year RFSs of 89%, 69%, 43%, respectively. CT + RT played a role in improving RFS among patients with LVSI and deeply invasive tumors and poor prognoses.

Evaluation of complete surgical staging with pelvic and para-aortic lymphadenectomy and paclitaxel plus carboplatin chemotherapy for improvement of survival in stage I ovarian clear cell carcinoma.

OBJECTIVE: The aim was to determine the benefits of lymphadenectomy and paclitaxel plus carboplatin chemotherapy for stage I ovarian clear cell carcinoma (defined as intra-abdominal disease confined to the ovaries). METHODS: Twenty patients with stage I pure clear cell carcinoma of the ovary diagnosed between 1991 and 2001 were divided into two groups: Group A (12 patients, 1997-2001) underwent complete surgical staging including bilateral salpingo-oophorectomy, hysterectomy, omentectomy, and pelvic and para-aortic lymphadenectomy, followed by paclitaxel and carboplatin chemotherapy. Group B (8 patients, 1991-1996) underwent bilateral salpingo-oophorectomy, hysterectomy, and omentectomy without lymphadenectomy, followed by cisplatin-based chemotherapy. The survival of the two groups was compared. The clinical characteristics of the two groups were evaluated for age distribution, grade, substage, preoperative CA-125, presence or absence of endometriosis, and maximal tumor diameter. RESULTS: The estimated 4-year survival rate was 76.9%. The clinical characteristics of the two groups were similar, except for lymphadenectomy and regimen of chemotherapy. With a median follow-up of 36 months (range: 11-130 months), one of 12 patients in Group A had recurrence in comparison with 6 of 8 patients in Group B (P = 0.004). The estimated 3-year recurrence-free survival and 4-year overall survival for Group A was significantly greater than that for Group B (91.7 vs 33.3%, P = 0.014; 100 vs 50%, P = 0.014). Median time to recurrence was 8 months. CONCLUSIONS: Complete surgical staging, including pelvic and para-aortic lymphadenectomy and paclitaxel plus carboplatin chemotherapy, appeared to be capable of improving survival of patients with stage I ovarian clear cell carcinoma.