Shina Oranratanaphan

Human Epididymis Protein 4 (HE4) and Cancer Antigen 125 (CA125) for Prediction of Optimal Primary Surgery in Non-Mucinous Epithelial Ovarian Cancer

To determine the relationship between pre-operative HE4 and CA125 levels in non-mucinous epithelial ovarian cancer cases (EOC) and outcomes of primary surgery for prediction of optimal surgery. A retrospective study was performed on non-mucinous EOC who underwent primary surgery at King Chulalongkorn Memorial Hospital from 2016 to 2020. Demographic and clinical characters were collected. Histopathology and pre-operative tumor markers namely HE4 and CA125 were also recruited. Primary surgical outcomes were classified as optimal (OS) and suboptimal surgery (SS). One hundred and seventy patients were enrolled in the study. There were 130 and 40 cases in OS and SS, respectively. Average age and body mass index (BMI) of EOC were 54.2 years old and 23.1 Kg/m2, respectively. Both groups had comparable demographic characteristics. Two-thirds (103/170) and one-third (63/170) had early stage and clear cell histopathology, respectively. The median level of HE4 were 118.60 and 603.45 pmol/L in OS and SS, respectively. OS and SS had average CA125 at 146.95 and 814.70 U/L, respectively. The best cut-off point of HE4 and CA125 less than 170.95 pmol/L and 316.4 U/mL gave predicting OS with area under curve (AUC) at 0.78 and 0.75, respectively. HE4 and CA125 cut-off point had sensitivity, specificity, positive predict value (PPV) and negative predictive value (NPV) at percentage of 60.8/60.8, 87.5/82.5, 94.1/91.9 and 40.7/39.3, respectively. HE4 and CA125 of non-mucinous EOC among OS had significantly less than SS and could be the predicting of optimal surgery.

Role of Cyclooxygenase-2 (COX-2) Expression as a Prediction of Persistent Cervical Low Grade Squamous Intraepithelial Lesion (LSIL)

Cervical cancer rates have been decreasing due to improved screening programs targeting HPV infections. Cervical Intraepithelial Neoplasia (CIN), including CIN 1, can regress, persist, or progress, leading to patient anxiety. The expression of Cyclooxygenase-2 (COX-2) may serve as an indicator of poor cancer outcomes and could potentially predict the persistence of CIN 1. To assess the relationship between COX-2 expression and the persistence of low-grade squamous intraepithelial lesions (LSIL) or CIN 1. Additionally, to compare baseline characteristics between patients with persistent and regressive LSIL/CIN 1. This case-control study included patients diagnosed with CIN 1 at least 12 months prior to the study started and followed up between May 2019 and April 2020. Pelvic examination and liquid-based cytology collection were performed. Participants were divided into two groups: regressive and persistent, based on current examination results. Previous cervical biopsy slides were reviewed by two gynecologic pathologists to confirm the CIN 1 diagnosis. Paraffin blocks from selected samples underwent immunohistochemistry staining to evaluate COX-2 expression, which was assessed using the Allred score. Clinical risk factors, cervical cytology, HPV genotype, and Allred scores were analyzed. Of the 161 patients recruited, 132 were in the regressive group and 29 in the persistent group, yielding a regression rate of 81.99%. COX-2 expression was observed in 83.8% of the patients. In the regressive group, 110 out of 132 patients tested positive for COX-2, while 25 out of 29 patients in the persistent group were COX-2 positive. Median Allred scores were similar between the groups, with no significant correlation between COX-2 expression and persistent LSIL/CIN 1 (p = 0.663). Furthermore, there was no significant correlation between Allred scores, high-risk HPV infection, and high-risk HPV status (p = 0.66 and p = 0.80). Persistent detection of high-risk HPV was found to be a significant risk factor for persistent LSIL in univariate analysis (p = 0.001), but not in multivariate analysis. COX-2 expression and HPV status do not appear to predict persistent LSIL/CIN 1. Further research is needed to identify reliable predictors for the persistence of LSIL/CIN 1.

CyclinA1 Promoter Methylation in Self-Sampling Test

Self sampled HPV testing is a cervical cancer screening method . However, cytology in self-sampled specimen cannot be used as a triage test.  Therefore, other methods for triage should be considered. CyclinA1 (CCNA1) promoter methylation has strong association with cervical precancerous and cancerous lesion. The objective of this study was to compare the diagnostic value of CCNA1 and self-sampled specimen for detecting high-grade cervical intraepithelial lesions or worse (CIN2+). A cross sectional study was conducted. Women with abnormal cytology or positive for high risk HPV (hrHPV) indicated for colposcopic examination were enrolled.  Self-collected sampling for hrHPV DNA (SS-HPV) and CCNA1 were performed. hrHPV DNA testing was done by Cobas 4800 method. CCNA1 promoter methylation was detected by CCNA1 duplex methylation specific PCR. Histopathologic result as CIN2+ obtaining from colposcopic directed biopsy or excisional procedure  was considered as positive a gold standard. The results of hrHPV and CCNA1 were reported as positive or negative. Sensitivity specificity, positive predictive value, and negative predictive value of SS-HPV and CCNA1 were calculated by comparing the results with the gold standard. Two hundreds and eighty women were recruited. High-grade cervical lesions and cervical cancer (CIN2+) were diagnosed in 21.8% (61 cases) of the patients. The most common type of hrHPV was non 16, 18 subtype, followed by HPV16 and 18. CCNA1 was positive in 13 patients out of whom, twelve were CIN2+. Sensitivity of CCNA1 was 19.7 % and its  specificity and accuracy were 99.5% and 82.14%, respectively.  The sensitivity of SS-HPV was 70.5%, and its  specificity and accuracy were 39.2% and 43.3%, respectively. Due to high specificity and positive predictive value of CCNA1, it can be used as alarming sign of having high-grade cervical intraepithelial lesions, especially in patient who has positive hrHPV DNA test based on self-collected sampling..

Value of CCNA1 promoter methylation in triaging ASC-US cytology

Using HPV testing to triage ASC-US still has some problems of unnecessary colposcopy in many cases. A previous study reported that methylation of CCNA1, a tumor suppressor gene, can differentiate between low and high grade lesions. This study was designed to evaluate the diagnostic values and application of CCNA1 methylation in the patients with ASC-US group.Materials and methods:Cross sectional analytic study was conducted in the patients with ASC-US cytology. HPV DNA testing and CCNA1 promoter methylation testing were performed. The patients were sent for colposcopic examination and biopsy. Biopsy results were considered as gold standard. Diagnostic test of HPV test and CCNA1 methylation test were calculated for sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), likelihood ratio for test positive and negative and 95% confidence interval.Results:One hundred and seventy patients were enrolled. Mean age was 39.7 years old. HR-HPV was positive in 70% of the patients. HPV type 16, type 18 and non-16,18 were 12.4%, 4.7% and 42.4%, respectively. CIN2+ were found in 12.4% (21 cases). CCNA1 promoter methylation was positive in 5 cases. CCNA1 had high specificity 99.3%, NPV 89.2% and PPV 80% in detection of CIN2+ but sensitivity was 19%. Likelihood ratio for positive test was 28.4 and likelihood ratio for negative test was 0.8. HPV test had sensitivity of 90.5% and NPV of 95.9% but low specificity and PPV as 31.5% and 15.7%, respectively.Conclusion: CCNA1 promoter methylation testing had very high specificity, likelihood ratio for the positive test and PPV (99.3%, 28.4 and 80.0, respectively). Therefore, CCNA1 promoter methylation test may be used in the HPV DNA positive cases to classify the urgency of colposcopy and the colposcopist should pay more attention to CCNA1 positive patients because of their higher chance to identify the significant lesions.

High-Risk Human Papillomavirus Detection via Cobas® 4800 and REBA HPV-ID® Assays

Persistent infection with high-risk human papillomaviruses (HR-HPVs), particularly HPV16 and 18, has long been known to induce cervical cancer progression. However, given that a minority of HPV-infected women develop cancer, analysis of HR-HPV-infected women could help to predict who is at risk of acquiring cervical cancer. Therefore, to improve HR-HPVs detection, we used the FDA-approved cobas® 4800 HPV and REBA HPV-ID® HPV assays to detect HR-HPVs in colposcopy-derived cervical cells from 303 patients, detecting 72.28% (219) and 71.62% (217) of HR-HPVs positive cases, with HPV16 detection rates of 35.64% (108) and 30.69% (93), respectively. Of the HPV16-positive cases, cobas® 4800 and REBA HPV-ID® identified 28.81% (51) and 25.42% (45) of the CIN1 cases, and 55% (33) and 50% (30) of the 60 CIN2/3 cases, respectively. HPV-diagnostic concordance was 82.17% overall (kappa = 0.488), 87.45% for HR-HPVs (kappa = 0.689), and 88.33% for CIN2/3 (kappa = 0.51). The HR-HPVs detection rates of these assays were comparable. Our findings reveal that the FDA-approved HR-HPVs detection assay is appropriate for screening women with HR-HPVs infection, and for predicting increased risk of cervical cancer progression. REBA HPV-ID® can be used to detect low risk-HPV types in high-grade cervical lesions that are HR-HPV negative as well as in the distribution of HPV types.

Cervical Microbiome in Women Infected with HPV16 and High-Risk HPVs

Human papillomavirus type 16 (HPV16) and/or high-risk (Hr-) HPV are the main causes of cervical cancer. Another element that may contribute to the development of cervical cancer is the microbiota. To date, no study has investigated the entire cervical microbiome, which consists of bacteria, fungi, and viruses. In this study, cervical samples with different histopathology (CIN1, CIN2, and CIN3), with or without HPV16 and Hr-HPVs infection, were enrolled. From bacterial community analysis, 115 bacterial species were found and separated into 2 distinct categories based on Lactobacillus abundance: Lactobacilli-dominated (LD) and non-Lactobacilli-dominated (NLD) groups. The LD group had significantly less bacterial diversity than the NLD group. In addition, the variety of bacteria was contingent on the prevalence of HPV infection. Among distinct histological groups, an abundance of L. iners (>60% of total Lactobacillus spp.) was discovered in both groups. A few fungi, e.g., C. albicans, were identified in the fungal community. The viral community analysis revealed that the presence of HPV considerably reduced the diversity of human viruses. Taken together, when we analyzed all our results collectively, we discovered that HPV infection was a significant determinant in the diversity of bacteria and human viruses in the cervix.

Human Virome in Cervix Controlled by the Domination of Human Papillomavirus

Although other co-viral infections could also be considered influencing factors, cervical human papillomavirus (HPV) infection is the main cause of cervical cancer. Metagenomics have been employed in the NGS era to study the microbial community in each habitat. Thus, in this investigation, virome capture sequencing was used to examine the virome composition in the HPV-infected cervix. Based on the amount of HPV present in each sample, the results revealed that the cervical virome of HPV-infected individuals could be split into two categories: HPV-dominated (HD; ≥60%) and non-HPV-dominated (NHD; <60%). Cervical samples contained traces of several human viral species, including the molluscum contagiosum virus (MCV), human herpesvirus 4 (HHV4), torque teno virus (TTV), and influenza A virus. When compared to the HD group, the NHD group had a higher abundance of several viruses. Human viral diversity appears to be influenced by HPV dominance. This is the first proof that the diversity of human viruses in the cervix is impacted by HPV abundance. However, more research is required to determine whether human viral variety and the emergence of cancer are related.

Efficiency of CIN2+ Detection by Thyrotropin-Releasing Hormone (TRH) Site-Specific Methylation

Cervical cancer screening typically involves a Pap smear combined with high-risk human papillomavirus (hr-HPV) detection. Women with hr-HPV positivity but normal cytology, as well as those with precancerous abnormal cytology, such as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL), are referred for colposcopy and histology examination to identify abnormal lesions, such as cervical intraepithelial neoplasia (CIN) and cervical cancer. However, in order to enhance the accuracy of detection, bioinformatics analysis of a microarray database was performed, which identified cg01009664, a methylation marker of the thyrotropin-releasing hormone (TRH). Consequently, a real-time PCR assay was developed to distinguish CIN2+ (CIN2, CIN3, and cervical cancer) from CIN2- (CIN1 and normal cervical epithelia). The real-time PCR assay utilized specific primers targeting methylated cg01009664 sites, whereas an unmethylated reaction was used to check the DNA quality. A cut-off value for the methylated reaction of Ct < 33 was established, resulting in improved precision in identifying CIN2+. In the first cohort group, the assay demonstrated a sensitivity of 93.7% and a specificity of 98.6%. In the cytology samples identified as atypical squamous cells of undetermined significance (ASC-US) and LSIL, the sensitivity and specificity for detecting CIN2+ were 95.0% and 98.9%, respectively. However, when self-collected samples from women with confirmed histology were tested, the sensitivity for CIN2+ detection dropped to 49.15%, while maintaining a specificity of 100%. Notably, the use of clinician-collected samples increased the sensitivity of TRH methylation testing. TRH methylation analysis can effectively identify women who require referral for colposcopy examinations, aiding in the detection of CIN2+.