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OBJECTIVE: To detect the HPV genotype and integration sites in patients with high-risk HPV infection at different stages of photodynamic therapy using nanopore technology and to evaluate the treatment effect. METHODS: Four patients with HPV infection were selected and subjected to photodynamic therapy, and cervical exfoliated cell was sampled at before treatment, after three courses of treatment and six courses of treatment, their viral abundance and insertion sites were analyzed by nanopore technology, and pathological examinations were performed before and after treatment. In this study, we developed a novel assay that combined viral sequence enrichment and Nanopore sequencing for identification of HPV genotype and integration sites at once. The assay has obvious advantages over qPCR or NGS-based methods, as it has better sensitivity after viral sequences enrichment and can generate long-reads (kb to Mb) for better detection rate of structure variations, moreover, fast turn-around time for real-time viral sequencing and analysis. RESULTS: The pathological grade was reduced in all four patients after photodynamic therapy. Virus has been cleared in two cases after treatment, the virus amount reduced after treatment but not completely cleared in one case, and two type viruses were cleared and one type virus persisted after treatment in the last patient with multiple infection. Viral abundance and the number of integration sites were positively correlated. Gene enrichment analysis showed complete viral clearance in 1 patient and 3 patients required follow-up. CONCLUSION: Nanopore sequencing can effectively monitor the abundance of HPV viruses and integration sites to show the presence status of viruses, and combined with the results of gene enrichment analysis, the treatment effect can be dynamically assessed.

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BACKGROUND: High-risk HPV is clearly associated with cervical cancer. Integration of HPV DNA into the host genome is considered a key event in driving cervical carcinogenesis. However, the mechanism on how HR-HPV integration influences the host genome structure has remained enigmatic. METHODS: In our study, 25 DNA samples including 11 from fresh-frozen cervical carcinomas and 14 from fresh-frozen high-grade squamous intraepithelial lesion (HSILs) were detected using the method of HPV capture combined with next generation sequencing. RESULTS: We calculated the frequency in each viral gene or region and found that breakpoints were prone to occur in L1 and L2 instead of E2 in the cervical cancer (P = 0.0004 and P = 5.15 × 10-40) and HSIL group (P = 2.1 × 10-32 and P = 7.06 × 10-13). The results revealed that HPV16 showed a strong tendency toward intronic region (P = 5.02 × 10-64) but a subtle tendency toward intergenic region (P = 0.04). The most frequent integration site was in the MACROD2 gene (introns 2, 4, 5, 6, 8 and 9), which in MACROD2 functional domain. CONCLUSION: Our results revealed that MACROD2 is HPV hot spot integration site in cervical lesions, and its deficiency alter DNA repair and sensitivity to DNA damage thought impaired PARP1 activity resulting in chromosome instability.

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OBJECTIVES: The objective of this study was to explore a better adjuvant treatment for patients with high-grade (HG) neuroendocrine cervical carcinomas (NECC) who had undergone surgery as a primary treatment. DESIGN: A retrospective cohort study, which involved women diagnosed as HG-NECC, was conducted in the Obstetrics and Gynecology Hospital of Fudan University. All patients had undergone radical surgery and pelvic lymphadenectomy with a laparotomy or a minimally invasive surgery. An analysis was made of the prognosis of HG-NECC. METHODS: Overall survival (OS) and progression-free survival (PFS) curves were drawn using the Kaplan-Meier method to be compared via log-rank tests. A Cox proportional hazards model was used to estimate the independent prognostic factors. RESULTS: A number of 110 patients diagnosed as HG-NECC at the pathological stage IA2 to IIIC2 according to the International Federation of Gynecology and Obstetrics (FIGO) 2018 staging system were initially treated with a primary surgery between 2008 and 2020. The eligible patients had the median age of 42.5 years (range: 22-76), with the median follow-up period of 39.6 months (range: 1.0-156.6). The 5-year OS of the patients at pathological stage I, II, and III accounted for 84.9%, 85.7%, and 60.9%, respectively. The Kaplan-Meier survival curves revealed no significant differences in OS and PFS between postoperative chemoradiotherapy and chemotherapy alone (OS: p = 0.77; PFS: p = 0.41). Etoposide plus platinum therapy did not improve OS when compared with platinum plus paclitaxel therapy after surgery (p = 0.71). The univariable analysis showed that chemotherapy with cycles ≥4 presented a better prognosis than with cycles <4 (OS: p = 0.01; HR = 6.71; PFS: p = 0.02; HR = 5.18). The multivariate analysis indicated that the cycles of chemotherapy (p = 0.02; HR 0.29) were a prognostic factor for PFS. LIMITATIONS: A retrospective design and the absence of partial follow-up data are limitations of the study. CONCLUSIONS: In initially surgically treated HG-NECC, postoperative chemotherapy alone showed no inferiority when compared with chemoradiotherapy for HG-NECC, and 4+ cycles of chemotherapy tended to produce a better prognosis than 4-ones.

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The aim of the present study was to evaluate the residual lesions after conization and the clinical outcome of patients with multifocal micro-invasive squamous cell cervical carcinomas (MMSCCs) treated with different surgical strategies. A retrospective study was carried out in 98 patients with MMSCCs diagnosed by conization and treated between January 2010 and December 2016 in 2 institutions. The patients underwent further different surgeries as therapeutic conization, extrafascial hysterectomy (ES), modified radical hysterectomy (MRH), radical hysterectomy (RH) with or without pelvic lymph node dissection (PLND) and regular follow-up. The clinicopathological characteristics of all of the patients were recorded. The risk factors of residual lesions and that of the recurrence were also analyzed in the present study. The logistic regression analysis revealed that cone margins (P=0.001) were correlated with residual disease after conization whereas parameters including age, gravidity, parity, menopause, stage, LVSI and the number of lesions were not predictors of residual lesions. The cone margin status also indicated the incidence of residual disease as follows: The risk of residual disease was lower with a negative margin when compared with the margin with micro-invasive carcinoma [MIC; odds ratio (OR)=0.064, P=0.012] and was lower in margin with a high-grade intraepithelial lesion than the margin with MIC (OR=0.297, P=0.287). The Cox regression analysis revealed that there were no significant correlations between the following surgery scales and postoperative recurrences, nor were any significant correlations found between the recurrences and the gravidity and parity, postmenopausal state, stage, residual disease after conization, margin status, LVSI and number of lesions (P>0.05). Positive cone margin was the only predictive factor for residual disease in patients with MMSCCs. There were no significant correlations between the surgical scales and postoperative recurrences. This result may be due to the excellent prognosis of MICs despite multiple lesions, regardless the treatment.

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BACKGROUND: Viral infections and the burden of high-grade intraepithelial neoplasias (HIN) and invasive carcinomas (IC) associated to infections by human papillomavirus (HPV) types may be prevented by type-specific anti-HPV vaccines. This study determined the prevalence of HPV types in non-cervical HIN and IC diagnosed from 1999 to 2011 at a general hospital in San Luis Potosí, Mexico. METHODS: Review of the 67 formaldehyde-fixed paraffin-embedded non-cervical specimens initially diagnosed as HIN (n = 28) or IC (n = 39) confirmed the presence of tumor tissue in 63 of them and changed the diagnosis of 24 from HIN to low-grade intraepithelial neoplasias, that were excluded from the study. HPV DNA was detected with the SPF10-DNA enzyme immunoassay in the 39 cases included, and viral types in the HPV-positive tumors were identified with the INNO-LiPA linear probe array. RESULTS: Among the cases included, four HIN were located in the vagina (n = 3) and vulva (n = 1), and 35 IC in the oral cavity (n = 19), penis (n = 8), vagina (n = 7) and vulva (n = 1). There were 13 HPV-positive cases from the vagina (n = 7), vulva (n = 1), penis (n = 1) and oral cavity (n = 1). The viral types identified were the high-risk types HPV16 in the vagina (n = 3) and vulva (n = 3), HPV45 in the vagina (n = 2), HPV59 in the vagina (n = 1) and penis (n = 1), HPV33 in the vagina (n = 1),and HPV35 in the tongue(n = 1); and the low-risk types HPV54 in the vagina (n = 1), and HPV11 in the vulva (n = 1). CONCLUSIONS: Five high-risk viral types (HPV16, 45, 59, 33 and 35) and two low-risk types (HPV11 and 54) infect one third of the non-cervical HIN and IC included. Most infections are by a single HPV high-risk type, the most prevalent one being HPV16. Vagina is the most frequent location of the HPV-positive tumors. Vaccination against HPV16 and HPV18 could have prevented around half of the HPV-positive tumors.

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Objective To study the pathogenetic role of HPV infection and vascular endothelial growth factor-C in the development of cervical carcinomas. Methods HPV16 DNA was tested in cervical intraepithelial neoplasia(CIN) 11I, cervical carcinomas and norma1 cervical tissue by PCR.and the expression of vascular endothelial growth factor-C protein was examined by immunohistochemistry. Results The expression of VEGF-C became stronger and stronger with the progression of cervical diseases from chronical inflammation to CIN and to invasive carcinoma of cervix uteri, but no expression in normal cervical tissue. There was significant correlation between HR-HPV and VEGF-C expression in CIN and cervical carcinomas(odds ratio are 19.12 and 20.49; 95 % confidence interval are 2.31-157.8 and 3.28-226.09). Conclusions The expression of VEGF-C, maybe a sensitive maker, is closely related to HR-HPV infection.

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Objective To investigate the relationship between expression of survivin and CD44v6 and HPV16/18 infection in uterine cervical carcinogenesis.Methods Using Streptavidin-Peroxidase(S-P) immunohistochemical technique,the authors examined the expression of survivin and CD44v6 in samples.The infection of HPV type 16,18 DNA was determined by PCR.Results There were significant differences for survivin and CD44v6 between carcinomas,CIN and normal cervices(P

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OBJECTIVE: Angiogenesis, the formation of blood vessels by sprouting from pre-existing ones, is essential for the growth of solid tumors beyond 2-3mm in diameter and for tumor metastasis. Vascular endothelial growth factor (VEGF), is known as vascular permeability factor(VPF) and mediates vascularization and tumor-induced angiogenesis. This study examined the potential of growth, invasion, and metastasis of uterine cervical carcinomas associated with neovascularization. METHODS: From January 1996 to December 1999, at the Department of Obstetrics and Gynecology, Kyung-Hee University Hospital, 37 uterine cervical carcinomas and 7 normal cervical tissues were obtained and the samples were immediately frozen and stored at -70 degrees C. Immunohistochemical staining for VEGF was carried out to study VEGF localization, and the levels of VEGF subtype mRNAs were determined by quantitative RT-PCR in specimens. The relation between VEGF subtypes expression of cervical cancers was analysed. RESULTS: The positive staining for VEGF is seen dominantly in the cytoplasm of the cancer cells, and faintly in interstitial cells. The intensity of staining was stronger in squamous carcinomas than in adenocrcinomas, but there was no significant difference (p>0.05). Quantitative RT-PCR analysis demonstrated significantly increased VEGF121/VEGF165 mRNA expression levels (>0.56 / >0.72) in 21 (56.8%) and 15 (40.5%) of 37 cervical carcinomas comparing to control groups (mean: 0.28 / 0.36). There was no obvious relationship between VEGF121/VEGF165 mRNA expression levels and the clinical parameters examined including age, pathology, differentiation, tumor size, lymphovascular space invasion, LN involvement and invasion depth except clinical stage (p<0.05). CONCLUSIONS: The overexpression of VEGF mRNA may be an important contributing factor in cervical carcinomas. There is no significant differenece of VEGF mRNAs levels according to clinical parameters, so it seems that the expression of VEGF is involved in the promotion of angiogenesis on cervical cancer and plays an important role in early invasion.

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OBJECTIVE: Telomerase is a ribonucleoprotein enzyme which stabilizes chromosomal structure, thereby inducing cellular immortality. We investigated telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression in relation to high-risk human papillomavirus (HPV) DNA presence in cervical carcinomas. METHODS: From December 1995 to December 1999, at the department of obstetrics and Gynecology of Kyung-Hee University Hospital, 32 cervical carcinomas and 5 corresponding nontumor cervical tissues were obtained and the samples were immediately frozen and stored at -70 degree C. Telomerase activity was measured by using telomerase PCR ELISA, a modified version of the TRAP. Analysis of the expression of hTERT mRNA was performed by quantitative RT-PCR and the analysis of the HPV E6 gene was performed by DNA-PCR. RESULT: All of the carcinomas examined exhibited strongly positive for telomerase activity (OD>0.24), whereas telomerase activity was week or not found in the 5 corresponding nontumor cervical tissues. Quantitative RT-PCR analysis demonstrated significantly increased hTERT mRNA expression levels (>0.024) in most carcinomas comparing to control groups. There was no obvious relationship between telomerase activity levels and the clinical parameters examined including age, clinical stage, pathology, differentiation, tumour size, LN involvement and invasion depth except lymphovascular space invasion (p=0.03). In the correlation between the levels of hTERT mRNA expression and telomerase activity, correlation index which was 0.916, shows high correlation (p=0.01). According to the analysis of HPV E6 gene, 29 of 32 (90.6%) carcinomas showed HPV E6 positivity. CONCLUSION: There is a strong association between telomerase activity and hTERT mRNA expression, and up-regulation of hTERT probably plays a role in the progression of cervical carcinomas. Telomerase is at least partially activated by viral oncogenes of high-risk types. There is no obvious relationship between telomerase activity levels and the clinical parameters except LSVI (p=0.03). These findings provides that telomerase may play an important role in the early stage of carcinogenesis.

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Purpose:To investigate the relation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) to tumor progression of cervical carcinoma. Methods:The expression and localization of COX-2 and iNOS protein in the 25 patients with cervical carcinomas were determined by immunohistochemical and the gene expression of COX-2 and iNOS were examined by reverse-transcription polymerase chain reaction ( RT-PCR). Results:Immunohistochemical staining for COX-2 and iNOS expression was strongly positive in 15 of 25 (60 %) and 20 of 25 (80 %) cases,respectively. Increased COX-2 and iNOS mRNA levels were confirmed by RT-PCR. There was negative correlation between COX-2 expression and tumor cell differentiation(r=-0.420, P

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Objective:To investigate the expression of minichromosome maintenance proteins 4(MCM4)and cell division cycle 6(CDC6)in uterine cervical carcinomas and its relationship with human papilloma virus(HPV)16/18 infection.Methods:The expression of MCM4 and CDC6 was examined in 50 squamous cell carcinoma specimens,20 cervical intraepithelial neoplasia(CIN)Ⅱ-Ⅲ specimens,20 CINⅠ specimens,and 20 normal cervical tissues by immunohistochemical method.The infections of HPV type 16,18 DNA were determined by PCR.Results:(1)The expression of MCM4 and CDC6 in uterine cervical carcinoma tissues was significantly higher than that in the CIN specimens and normal cervical tissues(Both P0.05).(2)The positive rates of(HPV)16/18 were significant different between cervical carcinomas,CIN and normal tissues(P0.05).(3)MCM4 expression were positively correlated with the expression of CDC6 in uterine cervical carcimonas(r=0.390,P