Objective To explore the sensitivity and specificity of colposcopy directed biopsy (CDB) and the value of loop electrosurgical excision procedure (LEEP) for the diagnosis and treatment of microinvasive cervical cancer (MCC). Methods One hundred and thirty five patients with MCC were diagnosed with LEEP in Obstetrics and Gynecology Hospital, Fudan University from April 2008 to November 2010, and were retrospectively analyzed on CDB diagnoses and following treatment after LEEP. According to patient′s desire for preservation of fertility and cone margin status, following strategies after LEEP included follow-up, second LEEP, hysterectomy, modified radical hysterectomy and radical hysterectomy. Single and multiple factors related to residual lesions after LEEP were analysed with Pearson Chi-square test and logistic regression model, respectively. Results CDB diagnosed MCC with a sensitivity of 4.4%(6/135), specificity of 100.0%(4 680/4 680), and false negative rate of 95.6%(129/135). Among the 135 patients, 29 did not receive further treatment in our hospital and lost contact. One hundred and six patients had secondary treatment or follow-up in our hospital, 4 of among which were closely followed up;one hundred and two received further treatment, which included 6 cases with second LEEP (3 received extrafascial hysterectomy after repeat LEEP), 59 cases hysterectomy, 14 cases modified radical hysterectomy and 26 cases radical hysterectomy. For factors related to residual lesions after LEEP, single factor analysis showed that the ratio of residual lesion in patients aged 27-39, 40-49 and 50-65 years were respectively 19.0%(11/58), 15.4%(10/65) and 5/12 (χ2=4.505, P=0.105). Residual lesions occurred in 24.7%(23/93) of patients with positive LEEP margins, which was more than that 7.1%(3/42) of patients with negative LEEP margins (χ2=5.756, P=0.016). The ratio of residual lesions in patients with positive endocervical, ectocervical and deep stromal margins were respectively 29.6%(8/27), 17.1%(7/41)and 30.6%(11/36;χ2=2.275, P=0.321). Residual lesions in patients with or without lymph vascular space invasion (LVSI) were 2/7 and 18.8%(24/128), respectively (χ2=0.412, P=0.521). The ratio of residual lesions in patients with invasion depth of<1 mm was 17.1%(7/41), 1-<3 mm was 19.0%(16/84), and 3-5 mm was 3/10, with no significant difference among three groups (χ2=0.870, P=0.647). Logistic regression analysis showed positive cone margin (OR=5.069, P=0.014) and age (OR=1.080, P=0.024) were the independent risk factors of residual lesions after LEEP conization. Conclusions CDB alone is not adequate for the diagnosis of MCC. For young patients who desire to preserve fertility with a negative cone margin, close follow-up is acceptable. Cone margin status and age are two independent risk factors for residual lesions after LEEP.

Objective To explore the role of toll-like receptor (TLR)/nitric oxide (NO) pathway in cervical tumor with high risk human papillomavirus (hrHPV) infection.Methods (1)Study was based on 36 women with nonmalignantcervical tissue as control group and 36 women with squamous cell cervical cancer (SCC),all with hrHPV infection which were assessed by using 14 types hrHPV E6/E7 mRNA real-time PCR kit.The amount of NO was detected by Griess reaction,the expression of inducible nitric oxide synthase (iNOS) was detected by immunohistochemistry (IHC).The mRNA expression of TLR3,TLR4,TLR7,TLR8,TLR9,nuclear factor-κB (NF-κB) p65 and iNOS in control and SCC epithelium which was captured by laser capture microdissection (LCM) were determined.(2)The expressions of TLR4 in CaSki,HeLa and C33a were detected by cell immunofluorescence method.The mRNA and protein expression of TLR/NO pathway transduction molecules including TLR4,NF-κBp65 and iNOS in CaSki,HeLa and C33a cell lines were detected by real-time PCR and western blot.Results (1)The level of NO was much higher in SCC group than that in control group [(42.92±0.36) μmol/L vs (15.49±0.24) μmol/L;P＜0.05].iNOS was detected in 75％ (27 cases) of patients with squamous cervical carcinoma,while only 6％ (2 cases) of normal controls were confirmed with positve result (P＜0.05).TLR/NO pathway maybe activated in SCC,for the mRNA levels of TLR3,TLR4,TLR7,TLR8,NF-κBp65 and iNOS increased significantly when compared to control group (all P＜0.05),and the greatest change in the expression level of TLR in SCC was spotted on TLR4(7.41±0.39 vs 1.86±0.21).(2)The results of immunofluorescence showed that TLR4 was located at plasma membrance of hrHPV positive HeLa and CaSki cells,while the integral optical density of TLR4 in HeLa cells (3 599±427) or CaSki cells (2 080±456) were higher than that in C33a cells (730±96;P＜0.05).The mRNA and protein level of TLR4,NF-κBp65 and iNOS in HeLa and CaSki cells were higher than those of C33a cells (P＜0.05).Conclusion TLR4/NO pathway is highly expressed in cervical cancer with hrHPV infection,while the pathway may be involved in cervical tumorigenesis with hrHPV infection.

This study was designed to explore the RNA interference technique in inhibition of the expression of the mouse fibrinogen like protein 2 (mfgl2), which has been reported to be involved in the development a variety of diseases including fulminant viral hepatitis. A plasmid named p-mfgl2shRNA,complementary to the sequence of mfgl2 was constructed, while another short hairpin RNA (shRNA)which was a mutated form of the mfgl2shRNA sequences was used as a control. A plasmid named pEGFP-mfgl2 expressing the mfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-mfgl2shRNA on mfgl2 expression. By cotransfection of p-mfgl2shRNA and pEGFP-mfgl2 or pcDNA3.1-mfgl2 expression construct into CHO cells or HeLa cells, the inhibition of mfgl2 expression by mfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, RT-PCR and immunohistochemistry staining. The experiments showed the significant inhibitory effect of p-mfgl2shRNA on mfgl2 expression at 48h post-transfection in both CHO and Hela cell lines with the inhibitory efficiency as high as 80.1%. The study demonstrated that the construct of p-mfgl2shRNA successfully interfered with the mfgl2 expression in vitro.

Objective To investigate mutation rate and types of KRAS in colorectal carcinoma , and to analyze the relationship between KRAS mutation and clinicopathological parameters in patients with colorectal carcinoma ( CRC) .Methods Scorpions Amplification Refractory Mutation System ( ARMS) fluorescence quantitative PCR was performed to detect the mutations in codons 12 and 13 of KRAS and to correlate between clinicopathological characteristics and the presence of various KRAS mutations of colorectal carcinoma .Results KRAS mutations were identified in 518 patients ( 42.92%) , including G12D ( 197 cases, 16.32%) , G12V ( 125 cases, 10.36%),G12C(40 cases, 3.31%),G12S(29 cases, 2.40%),G12A(21cases, 1.74%),G12R(7 cases, 0.58%),13D(117 cases, 9.69%).Female patients had a higher KRAS mutation rate than male (46.21%, 238/515 vs.40.46%, 280/692, P<0.05 ) .KRAS mutation was significantly higher in right colon cancer (46.45%,131/282)and in rectal cancer(44.50%,255/573)than that in left colon cancer(37.50%,132/352) (P<0.05,P<0.05).Conclusions There are many types of KRAS mutations in colorectal cancer, and many mutation types exist simultaneously .The detection rate of KRAS mutation is higher in female CRC patients than in the males.The detection rate of KRAS mutation is significantly higher in right colon cancer and in rectal cancer than that in left colon cancer.

Objective:To estimate risks of cervical intraepithelial neoplasia (CIN) Ⅱ or worse (CINⅡ +) on loop electrosurgical excisional procedure (LEEP) specimens with the diagnosis of endocervical curettage (ECC) CINⅠ compared with biopsy CINⅠ, and also to investigate the hierarchical management scheme of ECC CINⅠ based on the relevant factors of CINⅡ + risk. Methods:(1) A retrospective computer-based research for subjects enrolled in the Obstetrics and Gynecology Hospital, Fudan University from Jan. 2013 to Jun. 2021 was performed. The case group comprised women with an ECC CINⅠ (ECC results of CINⅠ with colposcopy-directed biopsy results ≤CINⅠ), and the control group comprised women with a biopsy CINⅠ (colposcopy-directed biopsy results of CINⅠ with negative ECC findings) were divided after LEEP surgery and diagnosis in the next three months. The clinical data of all patients before LEEP were analyzed, and the pathological diagnosis between two groups after LEEP was compared. (2) Variables, including age, cytology, high-risk human papillomavirus (HR-HPV), ECC results, cervical transformation zone (TZ) and colposcopy impression, were included to describe the characteristics and compare the incidence of LEEP CINⅡ +. (3) Univariate analysis and Multivariate logistic regression method were used to analyze the related factors that affect the LEEP CINⅡ + in CINⅠ patients. Further, the specific risks caused by related factors and conduct a stratified study in LEEP CINⅡ + were analyzed. Results:(1) Overall, 2 581 women with ECC CINⅠ or biopsy CINⅠ diagnosis who underwent LEEP participated in the study with the mean age (43.6±9.5) years old. Chi square test found that the age and cytology of patients in ECC CINⅠ group were statistically different from those of biopsy CINⅠ group (all P<0.05). There was no significant difference in HR-HPV detection, TZ type and colposcopy impression between the two groups (all P>0.05). ECC CINⅠ comprised 957 women, with LEEP histopathology results revealing 288 (30.1%, 288/957) CINⅡ +, which was significantly higher than that of biopsy CINⅠ which was comprised 1 624 women, with LEEP histopathology results showing 333 (20.5%, 333/1 624) CINⅡ + ( χ2=30.31, P<0.001). (2) Compared by LEEP CINⅡ + with LEEP ≤CINⅠ group, there were no significant difference in the age, HR-HPV, colposcopy impression (all P>0.05); but there were significantly differences in cytology, ECC CINⅠ, type Ⅲ TZ (all P<0.001). Multivariate logistic regression analysis showed that atypical squamous epithelial cells (ASC-H; OR=2.77, 95% CI: 2.04-3.77), high-grade squamous intraepithelial lesions and worse (HSIL +; OR=2.93, 95% CI: 2.24-3.81), ECC CINⅠ ( OR=1.89, 95% CI: 1.56-2.29) and type Ⅲ of TZ ( OR=1.76, 95% CI: 1.45-2.11) were independent risk factors for LEEP CINⅡ + (all P<0.05). (3) When cytology was ≤low-grade squamous intraepithelial lesion (LSIL) and ≥ASC-H, the detection rate of CINⅡ + in ECC CINⅠ was significantly higher than that of biopsy CINⅠ (all P<0.001). In ECC CINⅠ, the rate of CINⅡ + with cytology ≤LSIL was significantly lower than that in cytology ≥ASC-H (56.0% vs 25.9%; χ2=49.38, P<0.001). In type Ⅰ/Ⅱ of TZ, the detection rate of CINⅡ + between ECC CINⅠand biopsy CINⅠ had no significantly different; while in type Ⅲ of TZ, there was significantly different (72.7% vs 46.2%; χ2=4.02, P=0.045). In ECC CINⅠ, type Ⅲof TZ was significantly higher in the rate of CINⅡ + than that of type Ⅰ/Ⅱ of TZ (72.7% vs 21.7%; χ2=16.38, P<0.001). When cytology ≥ASC-H, type Ⅲ of TZ and colposcopy impression of HSIL were combined, the rate of CINⅡ + in ECC CINⅠ was 6/6 while 1/3 in biopsy CINⅠ. Conclusions:Cytology ≥ASC-H, ECC CINⅠ and type Ⅲ TZ are the risk factors of LEEP CINⅡ +. However, cytology ≥ASC-H is more valuable in predicting LEEP CINⅡ + than ECC CINⅠ. For patients with ECC CINⅠ to perform LEEP, it is recommended that cytology ≥ASC-H is taken as the first level stratification, and type Ⅲ TZ is taken as the second level stratification. The colposcopy impression of patients is recommended for a reference parameter.

Objective:To explore the detection rate, clinical characteristics of vulvar squamous intraepithelial lesion (SIL).Methods:Women diagnosed with vulvar high-grade squamous intraepithelial lesions (HSIL) through colposcopy-guided biopsy from January 1, 2018 to August 31, 2022 in Obstetrics and Gynecology Hospital of Fudan University were included in a 1∶1 ratio with patients diagnosed with vulvar low-grade squamous intraepithelial lesions (LSIL) during the same period. Clinical characteristics including human papillomavirus (HPV) infection rate, genotype, cytology result, colposcopy impression, and lesion location were retrospectively analyzed.Results:(1) The proportion of vulvar SIL detected by colposcopy-guided biopsy increased annually from 2018 to 2022, with rates of 1.64% (740/45 057), 2.34% (1 110/47 402), 2.68% (1 108/41 335), 3.26% (1 536/47 078), 3.31% (667/20 155), with an average rate of 2.57% (5 161/201 027). (2) A total of 1 096 cases of vulvar HSIL and 1 096 cases of vulvar LSIL were included. The overall infection rate of HPV was 92.7% (1 993/2 150), with higher infection rate in vulvar HSIL patients than that in vulvar LSIL patients [96.0% (1 012/1 054) vs 89.5% (981/1 096); χ2=33.62, P<0.001]. Among vulvar HSIL patients, the common HPV genotype from high to low were HPV 16 (66.7%), HPV 52 (14.3%), and HPV 58 (10.0%). For vulvar LSIL patients, the most common HPV genotype were respectively HPV 16 (24.9%), HPV 6 (20.1%) and HPV 52 (17.1%). The overall sensitivity rate of cytology was 53.6%, with no significance difference between vulvar LSIL and HSIL groups (54.3% vs 52.9%; χ2=0.40, P=0.526). The accuracy of colposcopy impression for vulvar HSIL was lower than that for vulvar LSIL [40.2% (163/405) vs 81.7% (380/465); χ2=158.72, P<0.001]. About 57.3% (1 257/2 192) of the patients had concomitant cervical and vaginal lesions, with a higher rate in vulvar HSIL group than that in vulvar LSIL group [62.6% (686/1 096) vs 52.1% (571/1 096); χ2=24.67, P<0.001]. Unifocal lesion was the main type, with no significance difference between vulvar LSIL and HSIL groups [81.4% (381/468) vs 82.5% (386/468); χ2=0.18, P=0.671]. The most common lesion locations were the posterior commissure, followed by labia minora, vaginal vestibule, labia majora, perianal and clitoris. Conclusions:The detection rate of vulvar SIL under colposcopy is about 3%, and the infection rate of HPV is 92.7%. Vulvar SIL, especially vulvar HSIL, is likely to cause concomitant cervical and vaginal lesions. The accuracy of colposcopy in diagnosing vulvar HSIL is low. Therefore a comprehensive and careful examination of the vulva is necessary and suspicious vulvar lesions should be undergone colposcopy-guided biopsy for diagnosis.

OBJECTIVETo develop a rapid duplex Real-time reverse transcription PCR (rRT-PCR) method to detect E119V mutation on neuraminidase (NA) of influenza A(H3N2) subtype with drug resistance to oseltamivir.

METHODSTwenty-six NA genes of influenza A(H3N2) virus between 2000 and 2012 in GenBank database were selected as the target genes, and specific TaqMan-MGB probe was designed to target the E119V amino acid change in neuraminidase protein. rRT-PCR was then performed and evaluated for the sensitivity, specificity and reproducibility using virus with E119V mutation and clinical samples.

RESULTSThis study described the validation of a highly sensitive and specific duplex rRT-PCR for detection of substitutions leading to the E119V amino acid change in NA protein of influenza A(H3N2). Fluorescence signals could be detected even when diluted a A (H3N2) virus (HA = 8) into 10(-5) and linear correlation between the logarithm of the viral titer with the Ct values was observed. In addition, the assay was highly specific in that there was no cross-react with other respiratory viruses, nor did two TaqMan-MGB probes. E119V substitution in quasispecies with both sensitive and resistant viruses could be detected as well. The limit of detection was 5% for quasispecies with high concentrations and 50% for quasispecies with low concentrations. The average coefficient of variation (CV) for within-run assays was 2.32% and 0.57% for H3N2-119E and H3N2-119V primer/probe sets separately, 1.77% and 0.97% for average CV of between-run assays, which exhibited good repeatability. Sequence analysis of twenty NA genes verified glutamic acid (E) at amino acid site 119, which was in consistent with the results from our rRT-PCR method.

CONCLUSIONThe assay developed in this study is highly sensitive and specific, and easy to operate; thereby it could be used for identification of A(H3N2) virus with E119V amino acid change in NA protein.

Amino Acid Substitution ; Drug Resistance, Viral ; Influenza A Virus, H3N2 Subtype ; drug effects ; enzymology ; genetics ; Mutation ; Neuraminidase ; genetics ; Nucleic Acid Probes ; Reverse Transcriptase Polymerase Chain Reaction ; methods

As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected for EGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cell line DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies of EGFR, KRAS, PIK3CA, PTEN, and MEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in the HER2, NRAS, or BRAF genes. Three of the 51 mutant samples harbored double mutations: two PIK3CA mutations coexisted with KRAS or EGFR mutations, and another KRAS mutation coexisted with a PTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay is a sensitive and easily customized assay for multigene mutation testing in clinical practice.
Adenocarcinoma ; genetics ; Carcinoma, Non-Small-Cell Lung ; genetics ; Class I Phosphatidylinositol 3-Kinases ; Genes, erbB-1 ; Genes, erbB-2 ; Genes, ras ; Humans ; Mutation ; PTEN Phosphohydrolase ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins ; Proto-Oncogene Proteins B-raf ; Proto-Oncogene Proteins p21(ras) ; Retrospective Studies ; ras Proteins

To study the prevalence and variation of influenza A(H3N2) viruses, the antigenic and genetic characteristics of influenza A(H3N2) viruses circulating in Mainland China during April 2011 to March 2012 were analyzed. The results showed that influenza A(H3N2) viruses increased gradually since 2012 and became the dominant strain since March. The viruses were antigenically closely related to the vaccine strain A/PER/16/09 (87.2%) and the representative virus A/FJ/196/09 (76.0%) in Mainland China. The genetic characteristics analysis results showed that recently isolated viruses belonged to the Vic/208 clade, and most of the low reaction strains also fell into the same clade. Crystal structure analysis of HA protein found that, compared with the vaccine strain A/PER/16/09, the recently isolated viruses had amino acid substitutions in the antigenic site A, B and C areas, in addition to gaining potential glycosylation sites at the amino acid position of 45 of HA and 367 of NA. Although the majority of circulating influenza A (H3N2) viruses in 2011-2012 season in Mainland China were antigeniclly matched by current influenza vaccine strain and the selected representative viruses, low reaction strains have increased since 2012, therefore it is necessary to strengthen the surveillance on the variation of influenza virus and to provide solid information for the vaccine strain selection.
Amino Acid Sequence ; China ; epidemiology ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; isolation & purification ; physiology ; Influenza, Human ; epidemiology ; virology ; Models, Molecular ; Molecular Sequence Data ; Phylogeny

Pdm09 virus outbreak occurred in Mainland China in May 2009, a few months later, the prevalence of seasonal H1N1(sH1N1) influenza virus that already circulated in human for tens of years began to decline and disappeared afterwards. To identify the reason for the rapid decline of sH1N1 in mainland China, we sequenced the HA1 of sH1N1 during 2006-2011, and then analyzed the selective pressure in different phases. Our results showed before Pdm09 outbreak, the omega value was 0. 36 while after Pdm09 outbreak the omega value was 0. 28 and significant difference (t test, P<0. 05) was identified. We concluded that sH1N1 obtained stronger purifying selection after Pdm09 outbreak in China. This might one of the major reasons causing the disappearance of sH1N1 in human.
China ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Phylogeny ; Seasons ; Selection, Genetic