Quantitative methylation analysis of human papillomavirus 16L1 gene reveals potential biomarker for cervical cancer progression.

Human papillomavirus 16 is the most prevalent type found in cervical cancer worldwide, accounting for >50% of all cases. Quantitative methylation analysis of human papillomavirus 16L1 gene within 5' (CpGs 5600, 5606, 5609, 5615) and 3' (7136 and 7145) regions to determine potential biomarker for cervical cancer progression was performed in exfoliated cervical cells collected from 101 Thai women of precancerous and cancerous lesions. Intermediate to high methylation levels (>20%) were detected in HPV16 5'L1 regions especially CpG 5600 of all cancerous (100%) and 50% of CIN3 samples, whereas normal/CIN1 samples (80%) showed methylation levels <20%. Our results indicate the potential use of HPV 16L1 gene methylation at specific site as a biomarker for prognostic cervical cancer screening, however, suitable cutoff should be further evaluated in a larger sample size.

Cost-Effectiveness Analysis of Different Management Strategies for Detection CIN2+ of Women with Atypical Squamous Cells of Undetermined Significance (ASC-US) Pap Smear in Thailand.

BACKGROUND: To identify the optimal cost effective strategy for the management of women having ASC-US who attended at King Chulalongkorn Memorial Hospital (KMCH). DESIGN: An Economical Analysis based on a retrospective study. SUBJECT: The women who were referred to the gynecological department due to screening result of ASC-US at King Chulalongkorn Memorial Hospital, a general and tertiary referral center in Bangkok Thailand, from Jan 2008 - Dec 2012. MATERIALS AND METHODS: A decision tree-based was constructed to evaluate the cost effectiveness of three follow up strategies in the management of ASC-US results: repeat cytology, triage with HPV testing and immediate colposcopy. Each ASC-US woman made the decision of each strategy after receiving all details about this algorithm, advantages and disadvantages of each strategy from a doctor. The model compared the incremental costs per case of high-grade cervical intraepithelial neoplasia (CIN2+) detected as measured by incremental cost-effectiveness ratio (ICER). RESULTS: From the provider's perspective, immediate colposcopy is the least costly strategy and also the most effective option among the three follow up strategies. Compared with HPV triage, repeat cytology triage is less costly than HPV triage, whereas the latter provides a more effective option at an incremental cost-effectiveness ratio (ICER) of 56,048 Baht per additional case of CIN 2+ detected. From the patient's perspective, the least costly and least effective is repeat cytology triage. Repeat colposcopy has an incremental cost-effectiveness (ICER) of 2,500 Baht per additional case of CIN2+ detected when compared to colposcopy. From the sensitivity analysis, immediate colposcopy triage is no longer cost effective when the cost exceeds 2,250 Baht or the cost of cytology is less than 50 Baht (1USD = 31.58 THB). CONCLUSIONS: In women with ASC-US cytology, colposcopy is more cost-effective than repeat cytology or triage with HPV testing for both provider and patient perspectives.

Comparison of Unsatisfactory Rates and Detection of Abnormal Cervical Cytology Between Conventional Papanicolaou Smear and Liquid-Based Cytology (Sure Path®).

PURPOSE: To compare unsatisfactory rates and detection of abnormal cervical cytology between conventional cytology or Papanicolaou smear (CC) and liquid-based cytology (LBC). MATERIALS AND METHODS: A total of 23,030 cases of cervical cytology performed at King Chulalongkorn Memorial Hospital during 2012-2013 were reviewed. The percentage unsatisfactory and detection rates of abnormal cytology were compared between CC and LBC methods. RESULTS: There was no difference in unsatisfactory rates between CC and LBC methods (0.1% vs. 0.1%, p = 0.84). The detection rate for squamous cell abnormalities was significantly higher with the LBC method (7.7% vs. 11.5%, p < 0.001), but those for overall abnormal glandular epithelium were similar (0.4% vs. 0.6%, p = 0.13). Low grade squamous lesion (ASC-US and LSIL) were more frequently detected by the LBC method (6.1% vs. 9.5%, p < 0.001). However, there was no difference in high gradd squamous lesions (1.1% vs. 1.1%, p = 0.95). When comparing between types of glandular abnormality, there was no significant difference the groups. CONCLUSIONS: There was no difference in unsatisfactory rates between the conventional smear and LBC. However, LBC could detect low grade squamous cell abnormalities more than CC, while there were similar rates of detection of high grade squamous cell lesions and glandular cell abnormalities.

Cytotoxic function of gamma delta (gamma/delta) T cells against pamidronate-treated cervical cancer cells.

The cytotoxic function of polyclonal expanded gamma/delta T cells against pamidronate-treated cervical cancer cells in vitro and in vivo were determined. The gamma/delta T cells were isolated and purified from PBMCs by using miniMACS and were later treated with 10 microM pamidronate. The expansion of gamma/delta T cells was 15 times more than the non-stimulated cells. Among the expanded gamma/delta T cells, 47% were Vgamma9/Vdelta2 T cells with a purity of 87%. Analyzing the cytotoxic function of gamma/delta T cells against 3 cervical cancer cells in vitro by LDH cytotoxicity test revealed that the killing efficacy increased if the cervical cancer cells (HeLa, SiHa and CaSki) were pretreated with pamidronate. The presence of CD107 on gamma/delta T cells indicated the degranulation of perforin and granzyme pathway is one of the mechanisms used by the gamma/delta T cells to kill cancer cells. The killing ability of gamma/delta T cells against cancer cells in vivo was preliminary assessed by using mouse baring HeLa cells. The results demonstrated that gamma/delta T cells induce apoptosis in tumor cells. Our study supports the usefulness of gamma/delta T cells in future development of immunotherapy for cervical cancer.

Detection of human papillomavirus in male and female urine by electrochemical DNA chip and PCR sequencing.

BACKGROUND: Cervical cancer is the second most common cancer in Thai women after breast cancer. Currently, the Papanicolaou (Pap) smear is the recommended procedure for cervical cancer screening in Thailand, but only a relatively small percentage of women follow this screening program. An alternative method to detect HPV genotypes associated with cervical cancer is self-sampling of urine, which is a more widely accepted method. Our study aimed to evaluate the prevalence of HPV in Thai women using urine and cervical swabs and prevalence of HPV in Thai men using urine samples. MATERIALS AND METHODS: Tumorigenic HPV detection was accomplished by electrochemical DNA chip and PCR/direct sequencing. In addition to HPV prevalence, we report the concordance between different methods and sample types. One-hundred and sixteen women and 100 men were recruited. Histological examination revealed normal cytology in 52 women, atypical squamous cells of undetermined significance (ASCUS) in 9, low-grade squamous intraepithelial lesions (LSIL) in 24, and high-grade squamous intraepithelial lesions (HSIL) in 31. One-hundred men were classified as heterosexuals (n=45) and homosexuals (n=55). RESULTS: The most prevalent HPV genotype in our study was HPV16. The HPV detection rate was generally lower in urine samples compared with cervical samples. Overall, there was good agreement for the detection of carcinogenic HPV from female cervical samples between the DNA chip and PCR/ sequencing, with 88.8% total agreement and a kappa value of 0.76. In male urine samples, the level of agreement was higher in heterosexuals compared with homosexuals. CONCLUSIONS: Further improvement is required to increase an overall yield of HPV DNA detection in urine samples before clinical application of a urine-based HPV screening program. The electrochemical DNA chip test is a promising technique for carcinogenic HPV detection.

Effects of Acanthus ebracteatus Vahl on tumor angiogenesis and on tumor growth in nude mice implanted with cervical cancer.

PURPOSE: The aim of this study was to examine the effects of the crude extract of Acanthus ebracteatus Vahl (AE) on tumor growth and angiogenesis by utilizing a tumor model in which nude mice were implanted with cervical cancer cells containing human papillomavirus 16 DNA (HPV-16 DNA). MATERIALS AND METHODS: The growth-inhibitory effect of AE was investigated in four different cell types: CaSki (HPV-16 positive), HeLa (HPV-18 positive), hepatocellular carcinoma cells (HepG2), and human dermal fibroblast cells (HDFs). The cell viabilities and IC(50) values of AE were determined in cells incubated with AE for different lengths of time. To conduct studies in vivo, female BALB/c nude mice (aged 6-7 weeks, weighing 20-25 g) were used. A cervical cancer-derived cell line (CaSki) with integrated HPV-16 DNA was injected subcutaneously (1 × 10(7) cells/200 µL) in the middle dorsum of each animal (HPV group). One week after injection, mice were fed orally with AE crude extract at either 300 or 3000 mg/kg body weight/day for 14 or 28 days (HPV-AE groups). Tumor microvasculature and capillary vascularity were determined using laser scanning confocal microscopy. Tumor tissue was collected from each mouse to evaluate tumor histology and vascular endothelial growth factor (VEGF) immunostaining. RESULTS: The time-response curves of AE and the dose-dependent effect of AE on growth inhibition were determined. After a 48-hour incubation period, the IC(50) of AE in CaSki was discovered to be significantly different from that of HDFs (P < 0.05). A microvascular network was observed around the tumor area in the HPV group on days 21 and 35. Tumor capillary vascularity in the HPV group was significantly increased compared with the control group (P < 0.001). High-dose treatment of AE extract (HPV-3000AE group) significantly attenuated the increase in VEGF expression and tumor angiogenesis in mice that received either the 14- or 28-day treatment period (P < 0.001). CONCLUSION: Our novel findings demonstrated that AE crude extract could inhibit cervical cancer growth, VEGF expression, and angiogenesis in a CaSki-cell transplant model in mice.

Comparison between direct sequencing and INNO-LiPA methods for HPV detection and genotyping in Thai Women.

Human papillomaviruses (HPVs) have been recognized as etiologic factors in cervical carcinoma and several other anogenital cancers in females and males. HPV are classified as low risk (LR), probable high risk and high risk (HR) on the basis of their oncogenic potential. HPV genotypes, which are crucial for diagnosis and relationship with carcinogenesis, have been determined by several genotyping methods. In this study, two genotyping methods were compared: direct sequencing and INNO-LiPA. In total, 2,494 cervical specimens were tested and 27.2 % of these were found to be HPV DNA positive with 24.5% showing normal cytology. Specimens were divided into four groups according to their pathological cytology as normal, LSIL, HSIL and cancer and 134 specimens were selected for HPV genotyping by both methods. HPV genotyping results showed 87.5% positive correlation. With 17 specimens, the results were discordant, 12 specimens showed different genotypes. Others had genotypes that could not be typed by the INNO-LiPA method. Neither did direct sequencing in 3 different regions yield unequivocal results. Both genotyping methods have advantages and disadvantages. Consequently, the method most suitable for the study objective, budget and predominance of HPV genotype in any given area should be selected.

Human papillomavirus genotypes among infected Thai women with different cytological findings by analysis of E1 genes.

Cervical cytological data may not be sufficient for cervical cancer screening and prevention. In this project, we determined HPV genotype among infected Thai women with different cytological findings by characterization of E1 genes. Five hundred and thirty-five specimens were tested by PCR amplification of the E1 genes. HPV genotypes were determined by sequencing, comparison with the GenBank database and were analyzed in relation to different cytological findings. HPV-DNA by PCR were typed and revealed 32 different genotypes. HR-HPV (HPV16, 18 or 52) was detected in all samples with cervical cancer cytology. HPV16 was most prevalent irrespective of cervical cytology. Moreover, HPV31 and 52 were most prevalent in the HSIL and LSIL groups whereas HPV66 was found mostly in the LSIL group. The LSIL group displayed the highest variation of HPV genotypes. Moreover, HPV31 and 52 predominated in the HSIL and LSIL groups especially HPV52 which was found in cancer samples. We hoped that these data of HPV genotypes can be used as preliminary data of HPV in Thailand and can serve as basic data for future research into the HPV genotype in south-east Asia.

Prevalence and genotypes of human papillomavirus among Thai women.

One of the most common cancers in women worldwide is cervical cancer, with death rates highest in less developed countries, including Thailand. This study was conducted to explore the prevalence of human papillomavirus (HPV) and its related cytological abnormalities among women attending cervical screening clinics in Thailand using the polymerase chain reaction (PCR). LBC specimens (ThinPrep, Hologic, West Sussex, UK) were subjected to PCR of the E1 region to identify the most prevalent HPV types. Information on age and cytology grade was also collected. Among a total of 1,662 women, 29 different HPV types were found and the overall HPV prevalence was 8.7%. HPV prevalence among the general population amounted to 7.8%. The following HPV types were identified: HPV16 (17.9%), HPV90 (16.6%) and HPV71 (10.3%). The rates of other types were as follows; HPV66 (6.9%), HPV52 (6.2%), HPV34 (5.5%), HPV31 (5.3%), HPV42 (4.8%) and HPV39 (3.4%). HPV infection peaked in women aged around 20-39 years and thereafter gradually declined. As expected, HPV DNA can be found in normal cytology specimens. These results which elucidate HPV distribution in Thailand could be useful for vaccine development and the national cervical cancer prevention program.

Whole-genome sequence analysis of human papillomavirus type 18 from infected Thai women.

OBJECTIVE: The aim of this study was to attain molecular knowledge of human papillomavirus type 18 (HPV18) by sequencing the whole genome of HPV18 isolated from Thai women at various clinical stages of disease progression. METHOD: Our group analyzed 9 samples of whole-genome HPV18 in infected women ranging from normal to cervical cancer by PCR, a sequencing method and bioinformatics programs. RESULTS: Phylogenetic analysis based on the whole genome showed that HPV18 samples were more closely related to the European and Asian-American type than the African type. The vaccine strain's L1 nucleotide (US patent 5820870) showed a close relationship to the African type. However, our data cannot indicate the correlation between cytological data and nucleotide or amino acid variation. CONCLUSION: Our group cannot draw any inference between the clinical stage of disease progression and amino acid alterations as there were only 1 or 2 samples available for each clinical trial. However, we hope that these new data on the HPV genome, which are representative of the entire genome of HPV in Southeast Asia, can serve as basis data for future research on the pathogenesis of cervical cancer. Additionally, the second-generation HPV18 vaccines should be tested on both HPV18-L1 and HPV18-L2 for increasing potential protection.

Mouse acquired HPV tumor using dorsal skin-fold window chamber.

Human papillomavirus (HPV) plays important role in developing several types of cancer especially cervical cancer. In order to understand the viral pathogenesis, the animal model of HPV infection is very necessary. This communication reports establishment of an animal model carrying implanted HeLa cells, a human cervical cancer cell line via dorsal skinfold window chambers. Nude mice were divided into 4 groups; each group contained different amount of HeLa cells, 2.5 x 10(5), 5 x 10(5), and 1 x 10(6) cells, and cell free medium (control), respectively. The results showed that even using the low number of HeLa cells (2.5 x l0(5)), the tumor microvasculature was developed at 2 weeks after implantation with the enlarged tumor margin which then progressed to tumor mass in the following week. The existing tumor was confirmed to be HeLa-cell type by PCR, in situ hybridization, and HPV genotyping. By using linear regression analysis, it indicated that means of tumor size from each group significantly increased in relation to number of HeLa cells used (R2 = 0.98, y = 0.1171x + 4.35). This mouse model will be useful for the further HPV studies particularly anti-cancer drugs efficacy.

Human telomerase reverse transcriptase (hTERT) expression in borderline ovarian tumors: an immunohistochemical study.

OBJECTIVE: To investigate the expression of human telomerase reverse transcriptase (hTERT) in epithelial borderline ovarian tumor (BOT) by immunohistochemistry with correlation to clinicopathologic variables. MATERIAL AND METHOD: Paraffin-embedded tissue sections of 62 borderline ovarian tumors (47 mucinous, 14 serous, and 1 clear cell) and 12 epthelial ovarian carcinomas were immunostained with antibodies to hTERT. The intensity and quantity of the immunostaining was determined and analyzed with clinicopathological characteristics. RESULTS: hTERT expression was detected in 48.4% of BOT and all cases of epithelial ovarian carcinoma. In immunoreactive BOT 50% of cases were scored as high expression. Serous BOT had the highest rate of hTERT expression. There was no significant statistical difference of hTERT immunoreactivity between histologic types of BOT. No hTERT immunoreactivity was observed in the benign parts of the same slides of each immunoreactive case. hTERT immunoreactivity was positively correlated with FIGO stage (p = 0.04), but not with other variables. The mean follow-up time of BOT cases was 81.63 months and no recurrence or death was noted. CONCLUSION: hTERT expression was found in half of BOT and all of epithelial ovarian carcinoma. High hTERT expression was associated with FIGO stage.

Comparison between adenocarcinoma in both endocervical and endometrial specimens from fractional curettage and pathologic findings in subsequent hysterectomy specimens.

OBJECTIVE: To evaluate the hysterectomy specimen findings in the patients who underwent fractional curettage (F&C) with presence of adenocarcinoma in both endocervical and endometrial specimens. MATERIAL AND METHOD: Forty-one patients who had adenocarcinoma in both endocervical and endometrial specimens from F&C and underwent subsequent hysterectomy for surgical staging without pre-operative radiotherapy or chemotherapy at King Chulalongkorn Memorial Hospital between 1999 and 2007 were evaluated Histologic slides from both F&C and hysterectomy specimens were reviewed and assessed All cases of endometrial adenocarcinoma with cervical involvement (stage 2) in hysterectomy specimens were also assessed and compared to the results in F&C specimens. RESULTS: Fifteen patients (36.6%) with both positive endocervical and endometrial specimens from F&C were diagnosed as endometrial adenocarcinoma within uterine cavity with lower uterine segment involvement. Only 34.1% of cases were endometrial carcinomas with cervical involvement. In the 35 cases with endometrial carcinoma stage 2, 60% had adenocarcinoma in both endocervical and endometrial specimens from F&C. CONCLUSION: In the patients who had adenocarcinoma in both endocervical and endometrial specimens from fractional curettage, the most common final pathological diagnosis from hysterectomy specimens was endometrial adenocarcinoma within uterine cavity with lower uterine segment involvement. Therefore, only 60% of endometrial carcinoma stage 2 revealed positive adenocarcinoma in both endocervical and endometrial specimens from fractional curettage.

Detection of PTEN immunoreactivity in endometrial hyperplasia and adenocarcinoma.

OBJECTIVE: To investigate PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression in endometrial hyperplasia and adenocarcinoma as analyzed by immunohistochemistry. MATERIAL AND METHOD: PTEN protein expression was evaluated by immunohistrochemical study of 70 paraffin-embedded curettage endometrial tissue samples (10 normal endometrium, 55 endometrial hyperplasia, and 15 endometrial adenocarcinomas) selected from surgical pathology files of the Division of Gynecologic Pathology, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, from 2001 to 2004. Intensity of epithelial staining of PTEN immunoreactivity in different histologic types was determined. RESULTS: Absence of PTEN protein expression was detected in 60% of endometrial carcinoma, 60% of atypical endometrial hyperplasia, and 24% of typical endometrial hyperplasia. In endometrial hyperplasia without atypia group, the majority of cases revealed moderate to strong PTEN expression, with 70% in simple hyperplasia and 47% in complex hyperplasia. There is a significant statistical difference of PTEN immunoreactivity among proliferative endometrium, endometrial hyperplasia and endometrial carcinoma group (p = 0.004). CONCLUSION: Complete loss of PTEN protein expression was most commonly found in endometrial carcinoma and hyperplasia with cytologic atypia.

The effects of GnRH antagonist on the endometrium of normally menstruating women.

PURPOSE: To study the effects of GnRH antagonist (ganirelix-Orgalutran) on the endometrium of regularly menstruating women. MATERIALS AND METHODS: Prospective, self-controlled study. The thirty-five volunteers were studied for two cycles: one as a control and the other, GnRH antagonist-treated cycles in which ganirelix 0.25 mg/d was given daily for 3 days, starting when the largest follicle reached 15 mm. In both cycles, serum estradiol, LH and endometrial thickness were measured when the largest follicle was > or =18 mm. Endometrial biopsy was performed on day 6 after ovulation for histological dating and morphometric study. RESULTS: No statistical differences between histological dating and the endometrial thickness in the control and GnRH antagonist-treated cycles. All morphometric parameters were also not different. Serum estradiol and LH levels were significantly lower in GnRH antagonist-treated cycles. CONCLUSION: GnRH antagonist has no effect on the endometrium of regularly menstruating women as assessed by either histological dating or morphometric analysis.

Pyomyoma in a perimenopausal woman with intrauterine device.

Pyomyoma (suppurative leiomyoma) is a rare disease, which is considered to be a serious complication of uterine leiomyoma. Since 1945, only 18 patients have been reported and ours is the 19th. Although it is frequently reported in pregnant women or postmenopausal women who have vascular disease, our case is a 42-year-old woman in the perimenopausal period who presented with fever and a tender lower abdominal mass. She used the intrauterine device as a contraceptive method but leiomyoma had never been found before. Ultrasonographic findings suggested an ovarian tumor. She was diagnosed as having infected malignant ovarian cancer with an elevated CA 125 level that was initially treated with broad spectrum antibiotics; then she underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy. Pathological findings showed acute and chronic inflammation of the endometrium with abscess formation in an intramural leiomyoma. The intrauterine device might be the origin of pyomyoma due to a direct spread of the infection from the uterine cavity. Pyomyoma may be difficult to diagnose especially in those with a nonspecific clinical presentation without any history of leiomyoma. Delayed diagnosis may result in serious complications, whereas adequate surgery and broad spectrum antibiotics may decrease serious morbidity and mortality.

Cyclin A1 promoter hypermethylation in human papillomavirus-associated cervical cancer.

BACKGROUND: The aim of this study was to evaluate epigenetic status of cyclin A1 in human papillomavirus-associated cervical cancer. Y. Tokumaru et al., Cancer Res 64, 5982-7 (Sep 1, 2004)demonstrated in head and neck squamous-cell cancer an inverse correlation between cyclin A1 promoter hypermethylation and TP53 mutation. Human papillomavirus-associated cervical cancer, however, is deprived of TP53 function by a different mechanism. Therefore, it was of interest to investigate the epigenetic alterations during multistep cervical cancer development. METHODS: In this study, we performed duplex methylation-specific PCR and reverse transcriptase PCR on several cervical cancer cell lines and microdissected cervical cancers. Furthermore, the incidence of cyclin A1 methylation was studied in 43 samples of white blood cells, 25 normal cervices, and 24, 5 and 30 human papillomavirus-associated premalignant, microinvasive and invasive cervical lesions, respectively. RESULTS: We demonstrated cyclin A1 methylation to be commonly found in cervical cancer, both in vitro and in vivo, with its physiological role being to decrease gene expression. More important, this study demonstrated that not only is cyclin A1 promoter hypermethylation strikingly common in cervical cancer, but is also specific to the invasive phenotype in comparison with other histopathological stages during multistep carcinogenesis. None of the normal cells and low-grade squamous intraepithelial lesions exhibited methylation. In contrast, 36.6%, 60% and 93.3% of high-grade squamous intraepithelial lesions, microinvasive and invasive cancers, respectively, showed methylation. CONCLUSION: This methylation study indicated that cyclin A1 is a potential tumor marker for early diagnosis of invasive cervical cancer.

Survey of human papillomavirus infection in cervical intraepithelial neoplasia in Thai women.

HPV infection is known to be associated with cervical cancer development. Precancerous lesions named cervical intraepithelial neoplasia (CIN) are divided into 3 grades, i.e., CIN-1, CIN-2, and CIN-3. Here, HPV infection determined by PCR and dot hybridization was observed in these 3 different grades of formalin-fixed paraffin embedded tissues. The HPV infection was demonstrated in 33.3 per cent of CIN-1, 36.8 per cent of CIN-2 and 75 per cent of CIN-3. Using type specific probes for HPV-6, 11, 16, 18 and 33, HPV-16 was the most prevalent type (44.44%) followed by HPV-18 (16.05%) in CIN-3. Only one HPV-18 was identified in CIN-1 while CIN-2 contained one HPV-6 and one HPV-18. Mixed infection was found in CIN-3 (12.35%). All of them had HPV-16. The cervicitis cases with normal histopathology were included as control. Only 2.7 per cent of HPV infection was shown. The relative risk of HPV infection was high in CIN-3 (OR = 107.25, 95% CI = 50.29-228.73). Our data confirm the association between high-risk HPV types and development of CIN.