Somchai Niruthisard

Human papillomavirus DNA in plasma of patients with cervical cancer

BackgroundHuman papillomavirus (HPV) is a crucial etiological factor for cervical cancer (CC) development. From a diagnostic view-point, the consistent presence of HPV in CC allows the viral DNA to be used as a genetic marker. The aims of this study were to evaluate the presence, physical status and clinical significant of HPV DNA in circulation of CC patients.ResultsWhereas 6 out of 50 (12%) HPV positive CC patients revealed plasma HPV DNA, it was detected in none of 20 normal controls or 13 HPV negative CC cases. The plasma DNA exhibited an HPV type identical to the HPV in the primary tumors and the DNA from both sources was integrated into host genome. Interestingly, several findings suggested an association between plasma HPV DNA and metastasis. First, three of the HPV DNA positive cases were CC patients with clinical stage IVB or recurrence with distance metastases (P = 0.001, RR = 15.67). Second, the amount of plasma HPV DNA from metastatic patients to be three times more than three other patients without metastases. Finally, the later cases had tendency to develop recurrence distant metastases within one year after complete treatment when compared with other HPV associated CC patients with the same stage but without the present of plasma HPV DNA.ConclusionsThe plasma HPV DNA originated from the CC, was associated with metastasis and could be used as a marker representing the circulating free CC DNA.

Cyclin A1 promoter hypermethylation in human papillomavirus-associated cervical cancer

BackgroundThe aim of this study was to evaluate epigenetic status of cyclin A1 in human papillomavirus-associated cervical cancer. Y. Tokumaru et al., Cancer Res64, 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.MethodsIn 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.ResultsWe 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.ConclusionThis methylation study indicated that cyclin A1 is a potential tumor marker for early diagnosis of invasive cervical cancer.

Telomerase activity and human papillomavirus in malignant, premalignant and benign cervical lesions.

The purpose of this study was to define a correlation between telomerase activity and human papillomavirus (HPV) in normal control tissue and in benign, premalignant and malignant cervical lesions. Telomerase activity was detectable in 33 out of 34 cases of squamous-cell carcinoma, five out of six cases of microinvasive carcinoma, 8 out of 20 cases and two out of six cases of high- and low-grade squamous intraepithelial lesions (SILs) respectively. The higher frequency of positive telomerase in invasive carcinoma compared with SILs was observed in both HPV-associated and non-associated groups. Whereas 92.6% of HPV-positive and 100% of HPV-negative invasive lesions expressed telomerase, only 50% of HPV-positive and 25% of HPV-negative SILs did. Interestingly, telomerase activity was also detectable in 13 out of 28 cases of benign lesions regardless of the presence of HPV. In conclusion, there may be two roles of telomerase in the cervix. The first one would present in benign lesions; the second is associated with cancer development and activated during the late stage of multistep carcinogenesis in both HPV-positive and -negative groups.

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.

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.

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 IC50 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 × 107 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 IC50 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.

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

PurposeTo study the effects of GnRH antagonist (ganirelix-Orgalutran®) on the endometrium of regularly menstruating women.Materials and methodsProspective, 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 0.25xa0mg/d was given daily for 3xa0days, starting when the largest follicle reached 15xa0mm. In both cycles, serum estradiol, LH and endometrial thickness were measured when the largest follicle was ≥18xa0mm. Endometrial biopsy was performed on day 6 after ovulation for histological dating and morphometric study.ResultsNo 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.ConclusionGnRH antagonist has no effect on the endometrium of regularly menstruating women as assessed by either histological dating or morphometric analysis.

Detection of Human Papillomavirus in Male and Female Urine by Electrochemical DNA Chip and PCR Sequencing

BACKGROUNDnCervical 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.nnnMATERIALS AND METHODSnTumorigenic 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).nnnRESULTSnThe 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.nnnCONCLUSIONSnFurther 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.

Comparison of detection sensitivity for human papillomavirus between self-collected vaginal swabs and physician-collected cervical swabs by electrochemical DNA chip.

BACKGROUNDnHuman papillomavirus (HPV) DNA testing is an effective method to screen for precancerous changes in the cervix. Samples from self-collection rather than Pap smear can potentially be used to test for HPV as they are more acceptable and preferred for use in certain settings. The objective of this study was to compare HPV DNA testing from self-collected vaginal swabs and physician-collected cervical swabs.nnnMATERIALS AND METHODSnA total of 101 self-collected vaginal and physician-collected cervical swabs of known cytology from Thai women were tested by electrochemical DNA chip assay. The specimens were divided into 4 groups: 29 with normal cytology, 14 with atypical squamous cells of undetermined significance (ASCUS), 48 with low- grade squamous intraepithelial lesion (LSIL), and 10 with high-grade squamous intraepithelial lesion (HSIL).nnnRESULTSnPositive detection rates of HPV from self-collected swabs were similar to those from physician-collected swabs. Among specimens with abnormal cytology, HPV was found in 50% of self-collected swabs and 47.2% of physician-collected swabs. In specimens with normal cytology, 17.2% of self-collected swabs and 24.1% of physician-collected swabs were positive for HPV. Concordance was relatively high between results from self- collected and physician-collected samples. The most common HPV genotype detected was HPV 51.nnnCONCLUSIONSnHPV DNA testing using self-collected swabs is a feasible alternative to encourage and increase screening for cervical cancer in a population who might otherwise avoid this important preventive examination due to embarrassment, discomfort, and anxiety.

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

PURPOSEnTo compare unsatisfactory rates and detection of abnormal cervical cytology between conventional cytology or Papanicolaou smear (CC) and liquid-based cytology (LBC).nnnMATERIALS AND METHODSnA 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.nnnRESULTSnThere 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.nnnCONCLUSIONSnThere 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.