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Background and Objectives : Cervical cancer is one of the most common types of frequently found cancers in Thailand. One of the causative agents is the infection of the high-risk human papillomavirus (HPV) type 16 and 18. Traditional medicines are rich sources of bioactive compounds which are a valuable source for the development of novel cancer therapies. In this study, the therapeutic effects of 3 traditional medicines, Kerra TM , KS TM , and Minoza TM , were studied on HeLa and CaSki cells. Materials and Methods : The effects of Kerra TM , KS TM , and Minoza TM on cancer cells were evaluated through cytotoxicity and cell death assays. The infection assay using HPV-16 pseudovirus was also carried out. Results : All traditional medicines efficiently suppressed cell growths of HeLa and CaSki, with Kerra TM being the most potent anticancer agent followed by KS TM and Minoza TM . Kerra TM at 158 µg/mL and 261 µg/mL significantly increases the percentage inhibition of the HPV-16 pseudovirus infection in a pre-attachment step in a dose-dependent manner, while KS TM at 261 µg/mL efficiently inhibited viral infection in both pre-attachment and adsorption steps. However, Kerra TM , KS TM , and Minoza TM at subtoxic concentrations could not reduce the viral E6 mRNA expressions of HPV-16 and HPV-18. Cell death assay by acridine orange/ethidium bromide showed that Kerra TM increased population of dead cells in dose-dependent manner in both CaSki and HeLa. The percentage of secondary necrosis in Kerra TM -treated CaSki was higher than that of HeLa cells, while the percentage of late apoptotic cells in HeLa was higher than that of CaSki, indicating that HeLa was more susceptible to Kerra TM than CaSki. For KS TM and Minoza TM , these extracts at 250 µg/mL promoted autophagy over cell death. At 500 µg/mL, the percentage of dead cells in Kerra TM was higher than that of KS TM and Minoza TM . Conclusions : Kerra TM is a potent traditional medicine for promoting cancer cell death. Kerra TM is possibly useful in the prevention and treatment of cervical cancer. Further investigation will be carried out to gain a better understanding of the biochemical mechanism and the pharmacological activity underlying this effect.

Deregulated expression of viral E6 and E7 genes often caused by viral genome integration of high-risk human papillomaviruses (HR-HPVs) into host DNA and additional host genetic alterations are thought to be required for the development of cervical cancer. However, approximately 15% of invasive cervical cancer specimens contain only episomal HPV genomes. In this study, we investigated the tumorigenic potential of human cervical keratinocytes harboring only the episomal form of HPV16 (HCK1T/16epi). We found that the HPV16 episomal form is sufficient for promoting cell proliferation and colony formation of parental HCK1T cells. Ectopic expression of host oncogenes, MYC and PIK3CAE545K, enhanced clonogenic growth of both early- and late-passage HCK1T/16epi cells, but conferred tumor-initiating ability only to late-passage HCK1T/16epi cells. Interestingly, the expression levels of E6 and E7 were rather lower in late-passage than in early-passage cells. Moreover, additional introduction of a constitutively active MEK1 (MEK1DD) and/or KRASG12V into HCK1T/16epi cells resulted in generation of highly potent tumor-initiating cells. Thus an in vitro model for progression of cervical neoplasia with episomal HPV16 was established. In the model, constitutively active mutation of PIK3CA, PIK3CAE545K, and overexpression of MYC, in the cells with episomal HPV16 genome were not sufficient, but an additional event such as activation of the RAS-MEK pathway was required for progression to tumorigenicity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wongjampa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: Cervical cancer screening can effectively reduce new cervical cancer cases, including in Thailand. The abnormal results are subsequently referred for colposcopy. To avoid unnecessary colposcopy, an efficient triage is still needed for validation. This study aimed to investigate the overall positivity of cytology-based screening, HPV detection, and p16/Ki-67 dual staining and evaluate different triage strategies for predictive diagnosis of abnormal cervical lesions in northeastern Thailand., Methods: Cervical cells were collected from 191 women who came for cervical screening in the gynecological outpatient department during March 2019-February 2020. Pap smear samples were classified into 6 groups including 17 atypical glandular cells (AGC), 21 atypical squamous cells of undetermined significance (ASC-US), 7 atypical squamous cells - cannot exclude HSIL (ASC-H), 26 low-grade squamous intraepithelial lesions (LSILs), 19 high-grade SILs (HSILs) and 101 no squamous intraepithelial lesion (noSIL). Polymerase chain reaction (PCR) was performed for HPV DNA detection. HPV genotyping was determined by reverse line blot hybridization. P16/Ki-67 dual staining was performed by using CINtec PLUS Cytology kit. Biopsies from abnormal screening were collected for surgical pathology classification., Results: High-risk HPV (HR-HPV) infection was 2.97%, 29.41%, 38.10%, 57.14%, 46.15% and 84.21% in noSIL, AGC, ASC-US, ASC-H, LSIL and HSIL cytology respectively. P16/ Ki-67 in noSIL, AGC, ASC-US, ASC-H, LSIL and HSIL was 0.99%, 5.88%, 9.52%, 42.86%, 26.92% and 63.16%, respectively (P-value < 0.001). Among p16/Ki-67 positive cases, 96.15% (25/26) were infected with HPV and 84.62% (22/26) were HR-HPV. The overall positivity of each and co-testing between cytology or HPV DNA testing or p16/Ki-67 dual staining was evaluated. In each cervical lesion, primary HPV DNA testing showed the highest sensitivity, but low specificity. The combined all HPV/HR-HPV with p16/Ki-67 detection increased the specificity of abnormal cervical lesions., Conclusion: P16/Ki-67 dual stain cytology in HPV-positive women performs well for diagnosis of abnormal cervical lesions and should be considered for management of HPV-positive women to avoid unnecessary colposcopy referrals.

Regardless of the prophylactic vaccine accessibility, persistent infections of high-risk human papillomaviruses (hr-HPVs), recognized as an etiology of cervical cancers, continues to represent a major health problem for the world population. An overexpression of viral early protein 6 (E6) is linked to carcinogenesis. E6 induces anti-apoptosis by degrading tumor suppressor proteins p53 (p53) via E6-E6-associated protein (E6AP)-mediated polyubiquitination. Thus, the restoration of apoptosis by interfering with the E6 function has been proposed as a selective medicinal strategy. This study aimed to determine the activities of andrographolide (Androg) on the disturbance of E6-mediated p53 degradation in cervical cancer cell lines using a proteomic approach. These results demonstrated that Androg could restore the intracellular p53 level, leading to apoptosis-induced cell death in HPV16-positive cervical cancer cell lines, SiHa and CaSki. Mechanistically, the anti-tumor activity of Androg essentially relied on the reduction in host cell proteins, which are associated with ubiquitin-mediated proteolysis pathways, particularly HERC4 and SMURF2. They are gradually suppressed in Androg-treated HPV16-positive cervical cancer cells. Collectively, the restoration of p53 in HPV16-positive cervical cancer cells might be achieved by disruption of E3 ubiquitin ligase activity by Androg, which could be an alternative treatment for HPV-associated epithelial lesions.

Objective: We investigated human papillomavirus (HPV) infection and detected anal squamous intraepithelial lesions by modified liquid-based cytology (LBC) and p16/Ki67 dual-staining., Methods: Anal swabs (n=393) were collected from patients with HIV infection. Anal cells were kept in 95% ethyl alcohol for modified LBC. DNA was extracted from cells for HPV detection and genotyping using real-time PCR and reverse line blot hybridization., Results: Nine samples (2.3%) were unsatisfactory specimens, 74.8% (294/393) were negative for intraepithelial malignancies (NILM) and 22.9% (90/393) exhibited squamous intraepithelial lesions (SIL). In the latter category, 13.7% of samples (54/393) contained atypical squamous cells of undetermined significance (ASCUS), 6.9% (27/393) were classified as low-grade SIL (LSIL) and 2.3% (9/393) as high-grade SIL (HSIL). A total of 331 from 393 swab samples were suitable for detection of HPV infection. Among these, 34.1% (113/331) were positive. HPV 58 (15.9%) was the most common genotype, followed by HPV 18 (14.2%) and HPV 16 (11.5%). The severity of abnormal cells was significantly associated with HPV infection. Dual staining with p16/Ki-67 was performed on 130 samples: in 30.8% (40/130) of samples positive staining was significantly associated with severity of abnormal cells. Agreement between cytology, p16/Ki67 dual-staining and high-risk HPV detection was 100% in HSIL samples. Interestingly, eight apparently NIML cases might have contained abnormal cells, since they were positive by both p16/Ki67 dual-staining and high-risk HPV detection., Conclusion: Anal specimens screened using modified LBC with 95% ethyl alcohol solution as the fixative are suitable for screening anal precancerous lesions by cytology, HPV testing and p16/Ki-67 dual staining.

Objective: High-risk human papillomavirus (HR HPV) was associated with the development of cervical cancer. Asymptomatic Chlamydia trachomatis (C. trachomatis) infection is the most common bacterial, sexually-transmitted infection. This study aimed to investigate the association of C. trachomatis in positive HR HPV and the cytological results from liquid-based cytology (LBC)., Methods: 150 residual LBC specimens were collected; all of which had undergone cytology and HPV testing by Cobas. The samples were established as C. trachomatis using real-time PCR (RT-PCR) with Cryptic F/Cryptic R primers., Results: Of 150 positive HPV findings, the most common (72.7%, 109/150) were the 12 other HR HPVs (viz., 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The cervical cytology of those positive HR HPVs were mostly negative (70.0%, 105/150).  The C. trachomatis infections in positive HR HPV were 16% (24/150) HPV. The analysis of the abnormal cytology revealed that 41.6% had C. trachomatis co-infection (C. trachomatis and HPV infection) viz., LSIL (20.8%), HSIL (12.5%), and ASC-US (8.3%). A comparison with positive HPV without C. trachomatis co-infection revealed that the highest prevalence was for LSIL, ASC-US, and HSIL (11.1%, 10.3%, and 6.4%, respectively). There was no difference between the abnormalities and negative cervical cytology with negative and positive C. trachomatis co-infection in HR HPV positive (p = 0.174)., Conclusion: C. trachomatis infection was not significantly associated HR-HPV and abnormal cytology. This study confirms the increasing rate of C. trachomatis infection in asymptomatic women so routine screening for these infections has been suggested to (a) prevent complications such as the chronic pelvic pain associated with prolong infection and (b) reduce sexual transmission of the infection.

MicroRNAs (miRNAs) are small non-coding RNAs that function to down-regulate gene expression involving in various cellular processes related to carcinogenesis. Recently, miR-22 was identified as a tumor-suppressing miRNA in many human cancers. However, the regulatory mechanism and the specific function of this miRNA in cervical cancer remain unclear. In the present study, we carried out gene transfection, western blot and quantitative RT-PCR to explore the regulatory mechanism and the functional role of miR-22 in cervical cancer. We verified that miR-22 was down-regulated in cervical cancer tissues and cervical cancer cell lines relative to matched non-tumor tissues and normal human cervical keratinocyte line (HCK1T). By contrast, histone deacetylase 6 (HDAC6) was inversely correlated with miR-22 in both cervical tissues and cancer cell lines. Mechanically, HDAC6 was down-regulated by miR-22 at the post-transcriptional level, via a specific target site within the 3'UTR, identified by a luciferase reporter assay. Moreover, we also showed that the correlation between miR-22 and HDAC6 expression was regulated by an E6/p53 pathway in HCK1Ts expressing HPV16 E6. For functional study, an ectopic expression of miR-22 could inhibit cell proliferation and migration, and could induce apoptosis of cervical cancer cell lines. These findings demonstrated that miR-22 was down-regulated in cervical cancer and inversely collated with its downstream target HDAC6. MiR-22 acts as tumor suppressor by inhibiting proliferation and migration, and by inducing apoptosis of cervical cancer cell lines by targeting the 3'UTR of HDAC6. This newly identified E6/p53/miR-22/HDAC6 regulatory network might be a candidate therapeutic target for cervical cancer., Competing Interests: The authors have declared that no competing interests exist.

Human papillomavirus (HPV) E2 and L1 proteins are expressed in cervical cells during the lytic stage of infection. Overexpression of p16 INK4A is a biomarker of HPV-associated cervical neoplasia. This study investigated antibodies to HPV16 E2, HPV16 L1, and p16 INK4A in sera from women with no squamous intraepithelial lesion (No-SIL) of the cervix, low-grade SIL, high-grade SIL, and cervical squamous cell carcinoma (SCC). HPV DNA was detected by polymerase chain reaction. Anti-E2, -L1, and -p16 INK4A antibodies in sera were determined by western blot. Among 116 samples, 69 (60%) were HPV DNA-positive. Percentages seropositive for anti-E2, -L1, and -p16 INK4A antibodies were 39.6, 22.4, and 23.3%, respectively. Anti-E2 antibody was significantly correlated with HPV DNA-positive cases. Eighty-seven women (75%) were regarded as infected with HPV, having at least one positive result from HPV DNA, L1, or E2 antibody. Antibody to p16 INK4A was associated with HPV infection (odds = 5.444, 95% CI 1.203-24.629, P = 0.028) and precancerous cervical lesions (odds = 5.132, 95% CI 1.604-16.415, P = 0.006). Interestingly, the concurrent detection of anti-E2 and -p16 INK4A antibodies was significantly associated with HPV infection (odds = 1.382, 95% CI 1.228-1.555, P = 0.044). These antibodies might be good candidate biomarkers for monitoring HPV-associated cervical lesion development to cancer.

Background: Persistent infection with EBV has been linked to the development of malignancies including HPV-associated cervical carcinoma. However, the role of EBV in HPV-associated cervical cancer is still poorly understood., Objective: To determine the possible contributing role of EBV in HPV-associated cervical carcinogenesis according to HPV genotypes, HPV genome status and EBV localization., Study Design: Cervical tissues, including 82 with no squamous intraepithelial lesions (noSILs), 85 low-grade SILs (LSILs), 85 high grade SILs (HSILs) and 40 squamous cell carcinoma samples (SCC) were investigated using PCR and dot blot hybridization for EBV detection and PCR and reverse line blot hybridization for HPV genotyping. The amplification of papillomavirus oncogene transcripts assay and in situ hybridization were used to determine HPV physical status and EBV EBER localization, respectively., Results: EBV was detected increasingly from noSIL (13.4%), LSIL (29.4%) to HSIL (49.4%) samples. The prevalence of HPV-EBV co-infection was significantly higher in any grade of lesion than in noSIL samples (p<0.05) including noSIL (1.2%; 95% confidence intervals [CI]=0.0-3.6%, relative risk [RR]=1), LSIL (18.8%, 95% CI=10.5-27.1%, RR=15.4), HSIL (41.2%, 95% CI=30.7-51.6%, RR=33.8) and SCC (30.0%, 95% CI=15.8-44.2%, RR=24.6). Interestingly, HPV-EBV co-infection was more common in cases with episomal forms of high-risk (HR) HPV whereas HPV alone was more common in cases with integrated HR-HPV. In addition, EBER staining demonstrated that EBV was mainly present in infiltrating lymphocytes., Conclusion: Infiltrating EBV-infected lymphocytes may play a role in cancer progression of cervical lesion containing episomal HR-HPV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Cervical squamous cell carcinoma (CSCC) is a major cause of female mortality worldwide. This study has examined epidermal growth factor receptor (EGFR) pathway markers that represent actionable pharmacological targets., Methods: HPV16 positive CSCCs (n = 105 patients) from Madhya Pradesh, India were screened for KRAS and PIK3CA mutations by PNA-clamp real-time PCR. Immunohistochemistry (IHC) was performed for EGFR, PIK3CA, PTEN, phospho-AKT, phospho-mTOR and phospho-44/42 MAPK (ERK1/2)., Results: KRAS mutations were detected in 0/91 (0%) and PIK3CA mutations in 19/95 (20.0%) informative specimens: exon 9, E542 (n = 3) and E545 (n = 15); exon 20, H1047R (n = 1). PIK3CA mutation detection was associated with older mean patient age [48.2 vs. 56.6 years (P = 0.007)] and with post-menopausal age: 5/45 (11.1%) patients <50 years vs. 14/50 (28.0%) patients ≥50 years (P = 0.045; OR = 3.11). EGFR expression was present in 60/101 (59.4%) CSCCs and was associated with PIK3CA mutation detection (P < 0.05) but not age (P > 0.05). EGFR and phospho-AKT staining showed associations with tumor grade and/or lymph node status (P < 0.05). Significant associations were not found for the other study markers (P > 0.05)., Conclusion: These data show that PIK3CA mutation acquisition is related to patient age and EGFR expression. The absence of KRAS mutations supports the potential of anti-EGFR therapies for CSCC treatment. The relatively high PIK3CA mutation rates indicate that PI3K may be a therapeutic target for a significant subset of CSCC patients. Qualitatively distinct IHC staining profiles for the marker panel were noted patient to patient; however, across patients, consistent linear relationships between up- and downstream pathway markers were not observed. Evaluation of the expression status of potential cancer pathway targets may be of value in addition to molecular profiling for choosing among therapeutic options.

Andrographolide (Androg) has been reported to contain antiviral and antitumor activities, but the effects of Androg on human papillomavirus (HPV) infection and cervical cancer have not been elucidated. This study investigated the effects of Androg and its derivatives, namely, 14-deoxy-11,12-didehydroandrographolide (14-DDA) and 3,19-isopropylidene andrographolide (IPAD), on HPV16 pseudovirus (HPV16PsV) infectivity, HPV16 E6 oncogene expression and cervical cancer cell apoptosis. The result demonstrated that all compounds inhibited HPV16PsV infection and that 14-DDA showed the highest potency. Only Androg suppressed long control region (LCR) transcription activity of HPV16 in transiently transfected C33A cells and significantly inhibited E6 oncogene expression in SiHa cells in a dose-dependent manner. A twofold subcytotoxic concentration of IPAD exhibited an inhibitory effect on E6 oncogene expression at 48-h posttreatment. Interestingly, p53 protein was restored in a downstream process and was detected earlier by IPAD treatment than by Androg treatment. This result corresponded to the level of cell apoptosis and cell cycle arrest at the G2/M phase. E6 oncogene expression was also suppressed in CaSki cells treated with Androg and IPAD leading to cell apoptosis. These findings imply that Androg and its derivatives have different activities and may be effective agents for HPV prevention and cervical cancer treatment.

Background: Variants of human papillomavirus (HPV) show more oncogenicity than do prototypes. The HPV16 Asian variant (HPV16As) plays a major role in cervical cancer of Asian populations. Some amino acid changes in the E6 protein of HPV16 variants affect E6 functions such as p53 interaction and host immune surveillance. This study aimed to investigate activities of HPV16As E6 protein on modulation of expression of miRNA-21 as well as interferon regulatory factors (IRFs) 1, 3, 7 and c-fos., Materials and Methods: Vectors expressing E6 protein of HPV16As (E6D25E) or HPV16 prototype (E6Pro) were constructed and transfected into C33A cells. HCK1T cells expressing E6D25E or E6Pro were established by transducing retrovirus-containing E6D25E or 16E6Pro. The E6AP-binding activity of E6 and proliferation of the transfected C33A cells were determined. MiR-21 and mRNA of interesting genes were detected in the transfected C33A cells and/or the HCK1T cells, with or without treatment by culture medium from HeLa cells (HeLa-CM)., Results: E6D25E showed binding activity with E6AP similar to that of E6Pro. Interestingly, E6D25E showed a higher activity of miR-21 induction than did E6Pro in C33A cells expressing E6 protein. This result was similar to the HCK1T cells expressing E6 protein, with HeLa-CM treatment. The miR-21 up-regulation significantly corresponded to its target expression. Different levels of expression of IRFs were also observed in the HCK1T cells expressing E6 protein. Interestingly, when treated with HeLa-CM, IRFs 1, 3 and 7 as well as c-fos were significantly suppressed in the HCK1T cells expressing E6D25E, whereas those in the HCK1T cells expressing E6Pro were induced. A similar situation was seen for IFN-α and IFN-β., Conclusions: E6D25E of the HPV16As variant differed from the E6 prototype in its activities on epigenetic modulation and immune surveillance and this might be a key factor for the important role of this variant in cervical cancer progression.

To understand the potential role in cervical cancer development of the three most common high-risk human papillomavirus (HR-HPVs) in Thai women, HPV genotypes and viral genome statuses in different cervical lesions were investigated. Cervical tissues consisting of no cervical intraepithelial neoplasia (84 cases), grade I cervical intraepithelial neoplasia (176 cases), grade II-III cervical intraepithelial neoplasia (91 cases), and squamous cell carcinoma (66 cases) were subjected for HPV genotyping by polymerase chain reaction (PCR) and reverse line blot hybridization assay and for HPV genome status determination by amplification of papillomavirus oncogene transcripts (APOT) assay. HPV prevalence was 28.6% in no cervical intraepithelial neoplasia, 40.3% in grade I cervical intraepithelial neoplasia, 70.3% in grade II-III cervical intraepithelial neoplasia and 86.4% in squamous cell carcinoma cases. The three most common HR-HPV types were HPV 16, 58, and 18 which were distributed in all cervical lesions. HPV physical statuses could be investigated in 4 no cervical intraepithelial neoplasias, 2 grade I cervical intraepithelial neoplasias, 28 grade II-III cervical intraepithelial neoplasias and 31 squamous cell carcinomas. The integrated-derived transcripts were found 3.6% in grade II-III cervical intraepithelial neoplasia and 48.4% in squamous cell carcinoma, whereas no viral genome integration was found in the group of no cervical intraepithelial neoplasia or grade I cervical intraepithelial neoplasia samples. The frequencies of HR-HPV integration in squamous cell carcinoma were found 40%, 100%, 20% of HPV 16, 18, and 58. This study indicates the oncogenic potential ability of the three most common HR-HPVs associated with cervical cancer progression., (© 2014 Wiley Periodicals, Inc.)

High risk human papillomavirus (HR-HPV) E2 proteins play roles in transcriptional regulation and are commonly functionally disrupted when the HPV genome integrates into host chromosomes. Some 15-40% of cancer cases, however, contain an intact E2 gene or episomal HPV. In these cases, polymorphism of the E2 gene might be involved. This study aimed to determine polymorphisms of the E2 gene in episomal HPV16 detected in high grade squamous intraepithelial lesions and squamous cell carcinomas and altered functions compared to the E2 prototype. The E2 gene was amplified and sequenced. Two expression vectors containing E2 gene polymorphisms were constructed and transfected in SiHa and C33A cells, then E6 gene as well as Il- 10 and TNF-α expression was determined by quantitative RT-PCR. Expression vectors and reporter vectors containing the HPV16 long control region (LCR) were co-transfected and transcriptional activity was determined. The results showed that a total of 32 nucleotides and 23 amino acids were changed in all 20 cases of study, found in the transactivation (TA) domain, hinge (H) region and DNA binding (DB) domain with 14, 5 and 13 nucleotide positions. They mostly caused amino acid change. The expressing vectors containing different E2 gene polymorphisms showed E6 mRNA suppression, TNF-α mRNA suppression and IL-10 induction but no statistically significant differences when compared to the E2 prototype. Moreover, promoter activity in HPV16 LCR was not affected by E2 protein with different gene polymorphisms, in contrast to nucleotide variations in LCR that showed an effect on transcription activity. These results demonstrated that E2 gene polymorphisms of episomal HPV16 did not affect transcriptional regulation and suggested that nucleotide variation as well as epigenetic modification of the LCR might play a role in inducing malignant transformation of cells containing episomal HPV16.

Enhanced expression of the HPV 16 E6-E7 oncogenes may trigger neoplastic transformation of the squamous epithelial cells at the uterine cervix. The HPV E2 protein is a key transcriptional regulator of the E6-E7 genes. It binds to four E2 binding sites (E2BSs 1-4) in the viral upstream regulatory region (URR). Modification of E2 functions, for example, by methylation of E2BSs is hypothesized to trigger enhanced expression of the viral E6-E7 oncogenes. In the majority of HPV-transformed premalignant lesions and about half of cervical carcinomas HPV genomes persist in an extra-chromosomal, episomal state, whereas they are integrated into host cells chromosomes in the remaining lesions. Here we compared the methylation profile of E2BSs 1-4 of the HPV 16 URR in a series of 18 HPV16-positive premalignant lesions and 33 invasive cervical cancers. CpGs within the E2BSs 1, 3, and 4 were higher methylated in all lesions with only episomal HPV16 genomes compared with lesions displaying single integrated copies. Samples with multiple HPV16 integrated copies displayed high methylation levels for all CpGs suggesting that the majority of multiple copies were silenced by extensive methylation. These data support the hypothesis that differential methylation of the E2BSs 1, 3 and 4 is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state. Once the virus becomes integrated into host cell chromosomes these methylation patterns may be substantially altered due to complex epigenetic changes of integrated HPV genomes., (Copyright © 2012 UICC.)

HPV 58 is detected commonly in cervical cancer in East Asian countries. To evaluate the HPV 58 physical state, the amplification of papillomavirus oncogene transcripts (APOT) and hybridisation assays were established. Episome- and integrate-derived transcripts were confirmed by direct sequencing. Twenty-nine HPV 58 positive samples from various cervical lesions were used. The results showed that the episome-derived transcripts were recognised as two major specific amplified products (1040 and 714 bp). Two splice donor sites were mapped to the 5' splice site of the E1 gene on SD898 and SD899 and spliced to the 3' acceptor site of the E4 gene on SA3353, SA3356 and SA3365. The episome-derived transcripts were found 100% in normal cervical epithelia and low-grade lesions (9/9 cases) while the integrate-derived transcripts were detected in 13.3% of high-grade lesions (2/15 cases) and in 20% of carcinomas (1/5 cases). HPV 58 integration sites were found on chromosomes 4q21, 12q24 and 18q12. Using the established APOT assay, the results revealed not only novel information on the HPV 58 transcription patterns of episomal transcripts, but also integration site. The APOT assay is a reliable and useful tool for the detection of the HPV 58 physical state and its oncogene expression., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The variation of human papillomavirus (HPV) genes or HPV variants demonstrates different risks of cervical cancer. Mutation in the long control region (LCR) at YY1-motifs is one of the mechanisms for enhancing viral oncogene expression during the course of cancer cell progression. In Thai women, cervical cancers are almost always associated with HPV16 variant sub-lineage Asian (HPV16As); however, the mechanism involved remains elusive. The aim of this study was to understand further the oncogenic potential of HPV16As., Methods: A total of 82 HPV16-positive specimens from Thai women were selected from formalin-fixed paraffin-embedded cervical tissues, and the full length E6 gene of each specimen was amplified and sequenced. LCRs of the HPV16As-positive cases were amplified and sequenced to analyze their polymorphisms. Transcriptional activities of the HPV16As LCRs were then compared with sub-lineage European (EUR), sub-lineage Asian-American 1 (AA1) and HPV16 prototype by insertion of the LCRs into the pGL3-Basic vector., Results: The HPV16 DNA sequences were classified as HPV16 prototype (18.3%), Asian (As, 61%), Asian American-1 (AA1, 8.5%), European (EUR, 7.3%), Asian African-2 (AFR2, 3.7%) and Java-135C (J135C, 1.2%). The prevalence of HPV16As was 30% in low-grade squamous intraepithelial lesion (LSIL), while that in high-grade squamous intraepithelial lesion (HSIL) and squamous cell cervical carcinoma (SCC) were 63.9% and 66.7%, respectively, which demonstrates a significant association of HPV16As with the disease severity. LCR polymorphisms from 43 HPV16As positive cases were analyzed by PCR-sequencing. Thirty-eight nucleotide variation positions spanned nucleotide positions 7157-82. Ten new mutations found in the HPV16As LCRs were located predominantly at the enhancer and proximal to the 3'-end of the early promoter. The LCRs of the common HPV16As, EUR and AA1 showed 5, 13 and 23-fold higher activity than the HPV16 prototype LCR, while those of the new nucleotide variations of As showed 19 (As-sv1) and 30 (As-sv14) -fold higher activity than the HPV16 prototype., Conclusions: HPV16As DNA sequence variation, especially at the proximal to early promoter in the LCR, enhances transcriptional activity. This could be one of the possible mechanisms for HPV16As-associated cervical cancer development.

Human papillomavirus (HPV) is a major cause of cervical cancer. More than 100 HPV genotypes have been identified; however the distribution varies geographically and according to ethnicity. The purpose of this study was to investigate the prevalence and distribution of HPV subtypes among Northeast Thai women. Subjects included 198 cases of SCCA and 198 age-matched, healthy controls. HPV-DNA was amplified by PCR using the consensus primers GP5+/6+ system followed by reverse line blot hybridization genotyping. The prevalence of high-risk HPV infection was 21 (10.1%) and 152 (76.8%) in the controls and in the cases, respectively. High-risk HPV significantly increased the risk for cervical cancer with an OR of 42.4 (95%CI: 22.4-81.4, p<0.001) and an adjusted OR of 40.7-fold (95%CI: 21.5-76.8, p <0.001). HPV-16 was the most prevalent HPV type in the SCCA (56.2%) followed by HPV-58 (17.8%) and HPV-18 (13.6%); whereas HPV-58 (46.4%) was a prominent genotype in the controls followed by HPV-16 (39.3%) and unidentified HPV types (25.0%). These findings indicate that HPV infection remains a critical risk factor for SCCA; particularly, HPV-16, HPV-58 and HPV-18. In order to eradicate cervical cancer, sustained health education, promoted use of prophylactics and a HPV-58 vaccine should be introduced in this region.

Cervical cancer is a serious public health problem in Thailand. We investigated possible risk factors for cervical cancer including HPV infection, p53 polymorphism, smoking and reproductive history among women in Northeast Thailand using a case control study with 177 cases and age-matched controls. Among the HPV carriers, a significantly increased risk for cervical cancer with an OR of 36.97 (p<0.001) and an adjusted OR of 38.07 (p<0.001) were observed. Early age at first sexual exposure, and multiple sexual partners increased the risk of cervical cancer with ORs ranging between 1.73-2.78 (p<0.05). The interval between menarche and first sexual intercourse<6 years resulted in a significant increase in the risk for cervical cancer with ORs ranging between 3.32-4.09 and the respective adjusted OR range for the 4-5 and 2-3 year-old groups were 4.09 and 2.92. A higher risk was observed among subjects whose partner had smoking habits, whether currently or formerly; with respective ORs of 3.36 (p<0.001) and 2.17 (p<0.05); and respective adjusted ORs of 2.90 (p<0.05) and 3.55 (p<0.05). Other smoking characteristics of the partners including smoking duration≥20 years, number of cigarettes smokes≥20 pack-years and exposure time of the subject to passive smoking≥5 hrs per day were found to be statistically significant risks for cervical cancer with adjusted ORs of 3.75, 4.04 and 11.8, respectively. Our data suggest that the risk of cervical cancer in Thai women is substantially associated with smoking characteristics of the partner(s), the interval between menarche and first sexual intercourse as well as some other aspects of sexual behavior.

Background: The pattern of infection in cervical lesions with respect to HPV subtype has not been systematically studied in Thai women. The aim here was to determine HPV prevalence, genotype, and infection pattern in cervical lesions and to estimate the potential efficacy of an HPV prophylactic vaccine., Design: Formalin-fixed paraffin-embedded cervical tissue blocks of 410 Thai patients from 8 institutes in 4 regions of Thailand (northern, southern, north-eastern, and central) were studied. The samples included 169 low grade squamous intraepithelial lesions (LSILs), 121 high grade squamous intraepithelial lesions (HSILs), and 120 squamous cell carcinomas (SCCs). HPV-DNA was amplified by PCR using consensus primers GP5+ and GP6+. The HPV genotype was then determined by reverse linear blot assay that included 37 HPV-specific 5'-amino-linked oligonucleotide probes. Patterns of infection were classified as single infection (one HPV type), double infection (two HPV types), and multiple infection (three or more HPV types)., Results: The mean age of the subjects was 42 years. The prevalence of HPV infection was 88.8%. The highest HPV prevalence was found in the southern region (97.1%) and the lowest in the central region (78.6%). HPV-DNA was detected in 84.6% of LSILs, 90.1% of HSILs, and 93.3% of SCCs. A total of 20 HPV genotypes were identified. The five most common high risk HPV were HPV16 (83.2%), HPV18 (59.3%), HPV58 (9.3%), HPV52 (4.1%), and HPV45 (3.8%). In double and multiple infection patterns, the most common genotypes were HPV16/18 (27.8%) and HPV11/16/18 (54.9%). HPV6 was found only in LSIL and never in combination with other subtypes. HPV11 was most common in LSIL., Conclusion: There is no difference of HPV type distribution in women from 4 regions of Thailand with prominent HPV16 and HPV18 in all cases. The bivalent and quadrivalent vaccines have the potential to prevent 48.6 % and 74.5% of cervical cancers in Thai women. The potential of cancer prevention would rise to 87.6% if other frequent HR-HPV types (HPV58, 52, and 45) were also targeted by an HPV vaccine.

Aim: This study investigated the utility of visual inspection with acetic acid (VIA) as a method for cervical cancer screening in Thailand and examined the relationship of VIA to high-risk human papillomavirus (HR-HPV) status., Methods: Cervical cells were collected from 160 patients receiving a Pap smear. VIA was performed on the cervix of the patients by application of 5% acetic acid. HPV screening of DNA extracted from cytology samples was performed by PCR using the GP5+/6+ primer system followed by reverse line blot hybridization genotyping., Results: The majority (96.9%) of the patients were diagnosed with normal or inflammatory cytologic changes. 32.8% of normal cytology and 42.0% of inflammation cases showed positive acetowhite staining. 3.1%, 38.1% and 42.5% of subjects were positive for an abnormal Pap test, VIA test, and HPV DNA, respectively. VIA demonstrated 50% sensitivity and 66.7% specificity for abnormal histology with PPV and NPV values of 50% and 66.7%, respectively, whereas HPV DNA test showed 100% sensitivity. HPV16 was the most common (54.4%) and HR-HPV was detected in 36.3% of all cases. 48.5% of HR-HPV positive and 36.8% of HR-HPV negative cervices stained with acetowhite following the VIA test., Conclusion: The VIA test is a simple method for cervical cancer screening; however, a significant proportion of patients with normal or inflammatory cytology were positive by this test. Further, HR-HPV in women without acetowhite staining was demonstrated. Therefore, some form of HR-HPV detection test may be required for combination with cervical cell screening even in low-resource nations., (© 2010 The Authors. Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology.)

Objective: To compare the clearance rate of HPV infection among women aged older than 30 years with biopsy-confirmed cervical low-grade squamous intraepithelial lesions (LSIL) 1 year after cryotherapy with the spontaneous clearance rate (observation)., Method: HPV DNA typing by polymerase chain reaction and reverse line blot hybridization were used to identify 14 high-risk types and 23 low-risk types. HPV DNA sequencing was also used for other types., Result: Between December 2007 and March 2009, 100 women were recruited to the study and 60 cases had positive results on HPV testing. Twenty-nine patients were randomly allocated to the cryotherapy group and 31 to the observation group. At 1 year, 89.7% (26/29; 95% CI, 78.6-100%) of the cryotherapy group and 90.3% (28/31; 95% CI, 79.9-100%) of the observation group had negative results on HPV testing (0.6% difference; 95% CI, -15.8 to 14.6%, P=0.94)., Conclusion: Cryotherapy failed to increase the clearance of prevalent HPV infections among women with LSIL, although in both arms the clearance rates were above 80%. However, in coupling with visual inspection with acetic acid as a single visit approach, its effect on prevention of HSIL and cervical cancer is still promising. Therefore, cryotherapy should not be withdrawn from such programs., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Objectives: To investigate the prevalence of HPV, HPV16, and HPV16 variants in scraped cervical cells cytologically diagnosed as normal cervical cell and in formalin-fixed, paraffin-embedded tissues of cervical intraepithelial neoplasia II-III and squamous cervical carcinoma in Northeast Thailand., Methods: All samples were subjected to PCR using consensus GP5+/GP6+ primers. HPV16 was genotyped by Southern blot hybridization and reverse line blot hybridization. The HPV16 E6 gene was amplified and sequenced., Results: HPV infections were found in 33.8% of normal cervical cells, 97.3% of cervical intraepithelial neoplasia II-III, and 100% of squamous cervical carcinomas. The prevalence of HPV16 increased significantly with histological grade (normal cervical cell, 16.7%; cervical intraepithelial neoplasia II-III, 38.9%; squamous cervical carcinoma, 75%). The most common variant found was the Asian (As) (58.7%) followed by the European (E) lineage (41.3%). The HPV16 As lineages showed a risk association in 73.9% of squamous cervical cancer and 57.1% of cervical intraepithelial neoplasia II-III, while no increased risk was observed in the E lineages., Conclusion: Our study demonstrates that HPV16, in particular the As variant, was the major causative agent associated with cervical cancer in Northeast Thailand, and our study suggests that some mutations of the E6 gene in this variant, which leads to amino acid changes, may be more carcinogenic.

Objective: To evaluate the value of the combination of p16 and HPV detection in the screening for cervical cancer., Methods: 186 patients with previous abnormal cervical lesion were studied. After colposcopic examination, two conventional Pap slides were prepared: the first was Papanicolaou-stained and examined by cytologist; the second was immunocytochemically stained for p16. Cervical cells were collected by brush using for HPV detection by Hybrid Capture II. Biopsy of any colposcopically abnormal lesions was performed., Results: The 186 cervical samples were classified cytologically as normal (148), ASCUS (13), low-grade (11), high-grade (12) dysplasia and squamous cell carcinoma (2). P16 and HPV were found in all high-grade dysplasia and SCC, and in 64% and 27% of low-grade dysplasia, 62% and 0% of ASCUS and 7.4% and 3.4% of normal, respectively. 18 of p16-positive cases (11%) were HPV-negative, 14 of them in the ASCUS and normal group. Compared to histological results, all of the p16-positive cases of squamous metaplasia, CIN II/III and SCC were HR-HPV-positive. Therefore, the cases that were positive for both with normal cytology (5 cases) or low-grade dysplasia (3 cases) may comprise a high-risk group for neoplastic change., Conclusion: The combination of p16 and HPV detection may be useful in cervical cancer screening to identify high-risk patients requiring early and proper management.

The p16INK4a protein is immunocytochemically detected in liquid-based (LB) specimens as a diagnostic marker of cervical dysplasia and neoplasia. Its up-regulation is promoted by high-risk human papillomavirus (HR-HPV) infection. We aimed to detect p16INK4a on conventional Papanicolaou (Pap) test (CPT) slides and to determine the relationship between its overexpression and HR-HPV infection. CPT and LB Pap test (LBPT) slides (165 samples of each) were examined by immunocytochemical staining for p16INK4a. After polymerase chain reaction (PCR), HPV-DNA was genotyped by dot blot hybridization. The CPT slides displayed more numerous dispersed squamous cells and LBPT slides had a clearer background. Positive p16INK4a on CPT occurred in 0% (0/30), 52.5% (21/40), 54.3% (19/35), 100% (30/30), and 100% (30/30) in normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSILs), high-grade SILs (HSILs), and squamous cell carcinomas (SCCs) cases, respectively. LBPT slides showed comparable results but were less sensitive. HPV-DNA was detected in 86.7, 70, 45, 57.14, and 10% in SCCs, HSILs, ASCUS, LSILs, and normal cervical cells, respectively. Because HR-HPV was identified in all HPV+ samples of high-grade dysplasia (HSILs and SCCs) and all positive p16INK4a samples infected with HR-HPV, the association of p16INK4a overexpression with HR-HPV infection was confirmed. This study suggests that immunocytochemical staining of p16INK4a on CPT slides is convenient and cost-effective for cervical cancer screening by the detection of dysplastic cells infected with HR-HPV., (2004 Wiley-Liss, Inc.)

Objective: To develop an immunocytochemical technique for p16INK4a protein detection in scraped cervical cells for cancer screening., Study Design: We took duplicate cervical scrapes from each participant, the first for a Pap smear and the second for p16INK4a protein detection. From a 50-microL cell suspension prepared from the scrape rinsing, a 10-microL aliquot was dropped in a 5-mm-diameter circle on a glass slide, air dried and fixed in 0.1% formal saline (1 hour) and in 95% ethanol (10 minutes). Using the immunocytochemical technique, slides from 30 samples of each Pap diagnosis class were stained sequentially with mouse monoclonal anti-p16INK4a (primary antibody), biotinylated goat antimouse IgG (secondary antibody), horse-radish peroxidase-labelled streptavidin and 3,3'-diaminobenzidine and mixed hydrogen peroxide, then counterstained with hematoxylin. A positive sample had to contain > or = 3 immunoreactive cells. Results were confirmed by western blot analysis of lysates from the remaining 40 microL of each cervical cell suspension., Results: Samples were grouped as control (normal cervical cells), mild dysplasia (ASCUS, LSIL) and high abnormality (HSIL, SCC). Using the immunocytochemical technique, > 95% of the positive (SiHa cells) but 0% of the negative controls (human embryonic lung fibroblast cells) showed immunoreactive cells. All slides displayed a clear background without mucus, and positive cells were stained in both the cytoplasm and nucleus. p16INK4a Protein was detected in 17 of 30 (56.67%) ASCUS and 10 of 30 (33.33%) LSIL and increased with the degree of abnormality to 93.33% (28 of 30) and 96.67% (29 of 30) in the HSIL and SCC group, respectively. Normal cervical cells and degenerated malignant cells were nonimmunoreactive. Western blot analysis confirmed similar positive samples in the low-abnormality group, while the whole high-abnormality group was immunoreactive. A sampling error might have caused the 2 HSIL and 1 SCC sample to be negative using our immunocytochemical technique., Conclusion: p16INK4a Protein detection in scraped cervical cells using the immunocytochemical technique correlated with western blot analysis and was nontraumatic and precise. It offers a significant diagnostic adjunct to the Pap test for cervical cancer screening.