Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH in ovarian cancer

Objective:Hematogenous metastasis is essential for the progression of ovarian cancer(OC),and circulating tumor cells(CTCs)are part of the metastatic cascade.However,the detection rate of CTC is low due to the use of less sensitive detection methods.Therefore,this study aimed to detect CTCs and circulating tumorigenic endothelial cells(CTECs)in patients with OC using subtraction enrichment and immunostaining and fluorescence in situ hybridization(SE-iFISH).Methods:We enrolled a total of 56 subjects,including 20 OC patients and 36 ovarian benign tumor patients.CTCs and CTECs were captured by subtraction enrichment(SE)and counted and classified according to immunofluorescence staining of tumor markers(TMs)carbohydrate antigen 125(CA125)and human epididymis protein 4(HE4)combined with fluorescence in situ hybridization(iFISH)of chromosome 8(Chr8)aneuploidy.The diagnostic value and subtype characteristics of CTCs and CTECs were investigated.Results:The detection rate of CTCs by SE-iFISH was high.Compared with CA125 and HE4,Chr8 aneuploidy was the major identification feature of CTC.CTC counts in OC were statistically higher than those in benign groups.CTC and CTEC with≥pentaploidy were detected in both groups,illustrating the poor diagnostic value of CTC or CTEC.Distributions of triploid and tetraploid CTC subtypes were significantly different,and combined detection of triploid and tetraploid CTCs showed the best diagnostic value.In contrast,the distribution of CTECs in the OC and benign groups had no statistically significant difference.Small CTCs accounted for over 1/3 of the total CTC count.We also found that small CTCs and CTECs primarily comprised triploid cells,while large CTCs and CTECs mainly comprised pentaploidy and beyond.Conclusions:The application of SE-iFISH offered a more comprehensive understanding of heterogeneous CTCs and CTECs in OC.Analysis of subclass characteristics of the CTCs and CTECs according to Chr8 aneuploidy and cell size may broaden their potential clinical utility and deepen mechanistic studies in OC.

Evaluation of COC183B2 antibody targeting ovarian cancer by near-infrared fluorescence imaging

Objective: To evaluate the imaging potential of a novel near-infrared(NIR) probe conjugated to COC183 B2 monoclonal antibodies(MAb) in ovarian cancer(OC).Methods: The expression of OC183 B2 antigen in OC was determined by immunohistochemical(IHC) staining using tissue microarrays with the H-score system and immunofluorescence(IF) staining of tumor cell lines.Imaging probes with the NIR fluorescent dye cyanine 7(Cy7) conjugated to COC183 B2 Mab were chemically engineered. OC183 B2-positive human OC cells(SKOV3-Luc) were injected subcutaneously into BALB/c nude mice. Bioluminescent imaging(BLI) was performed to detect tumor location and growth. COC183 B2-Cy7 at 1.1,3.3, 10, or 30 μg were used for in vivo fluorescence imaging, and phosphate-buffered saline(PBS), free Cy7 dye and mouse isotype immunoglobulin G(IgG)-Cy7(delivered at the same doses as COC183 B2-Cy7) were used as controls.Results: The expression of OC183 B2 with a high H-score was more prevalent in OC tissue than fallopian tube(FT) tissue. Among 417 OC patients, the expression of OC183 B2 was significantly correlated with the histological subtype, histological grade, residual tumor size, relapse state and survival status. IF staining demonstrated that COC183 B2 specifically expressed in SKOV3 cells but not HeLa cells. In vivo NIR fluorescence imaging indicated that COC183 B2-Cy7 was mainly distributed in the xenograft and liver with optimal tumor-to-background(T/B)ratios in the xenograft at 30 μg dose. The highest fluorescent signals in the tumor were observed at 96 h postinjection(hpi). Ex vivo fluorescence imaging revealed the fluorescent signals mainly from the tumor and liver. IHC analysis confirmed that xenografts were OC183 B2 positive.Conclusions: COC183 B2 is a good candidate for NIR fluorescence imaging and imaging-guided surgery in OC.

Clinical implications and prognostic value of five biomarkers in endometrial carcinoma

Objective： The aim was to identify the relationship between ER, PR, P53, Ki-67, PTEN, the association with clinicopathological parameters and the correlation with survival. Methods： We studied 190 cases of primary endornetrial carcinoma in which ER, PR, Ki-67, P53, PTEN antigens were investigated with the use of immunohistochemical methods. To evaluate the correlations among immunohistochemical staining and the age, menopause status, histological type, FIGO stage, grading, depth of invasion, lymph nodes involvement and serum tumor marker. Survival analysis was assessed within single and combined biomarkers types. Results： The percentage of Ki-67 and P53 positive endometrial tumors was signifi- cantly higher in ER negative vs ER positive tumors （both P = 0.000）. The same trend was evident in PR positive and nega- tive group. The percentage of PTEN positive tumors was significantly higher in PR positive versus PR negative tumors （P = 0.021） but was no difference in different ER status. ER and PR status were significant predictors with FIGO staging, grading and recurrence. There was no clear association between PTEN positivity and clinicopathological parameters except more relevance with endometrioid histotype （P = 0.013）. Positive Ki-67 or P53 was found to be strictly related to more aggressive features. There was statistically significant difference in different status of P53 and Ki-67 in survival time. Conclusion： ER and PR positive tumors showed a statistically significant association with better clinical outcome, PR has more significant influ- ence on prognosis. The percentage of positive Ki-67 or P53 was significantly higher in hormone-independent group versus in hormone-dependent group and combined Ki-67 and P53 may have more effect on prognosis in former group.

Platelet RNA enables accurate detection of ovarian cancer:an intercontinental,biomarker identification study

Platelets are reprogrammed by cancer via a process called education,which favors cancer development.The transcriptional profile of tumor-educated platelets(TEPs)is skewed and therefore practicable for cancer detection.This intercontinental,hospital-based,diagnostic study included 761 treatment-naive inpatients with histologically confirmed adnexal masses and 167 healthy controls from nine medical centers(China,n=3;Netherlands,n=5;Poland,n=1)between September 2016 and May 2019.The main outcomes were the performance of TEPs and their combination with CA125 in two Chinese(VC1 and VC2)and the European(VC3)validation cohorts collectively and independently.Exploratory outcome was the value of TEPs in public pan-cancer platelet transcriptome datasets.The AUCs for TEPs in the combined validation cohort,VC1,VC2,and VC3 were 0.918(95%CI 0.889-0.948),0.923(0.855-0.990),0.918(0.872-0.963),and 0.887(0.813-0.960),respectively.Combination of TEPs and CA125 demonstrated an AUC of 0.922(0.889-0.955)in the combined validation cohort;0.955(0.912-0.997)in VC1;0.939(0.901-0.977)in VC2;0.917(0.824-1.000)in VC3.For subgroup analysis,TEPs exhibited an AUC of o.858,0.859,and 0.920 to detect early-stage,borderline,non-epithelial diseases and 0.899 to discriminate ovarian cancer from endometriosis.TEPs had robustness,compatibility,and universality for preop.erative diagnosis of ovarian cancer since it withstood validations in populations of different ethnicities,heterogeneous histoiogical subtypes,and early-stage ovarian cancer.However,these observations warrant prospective validations in a larger population beforeclinicalutilities.

Characterization of candidate factors associated with the metastasis and progression of high-grade serous ovarian cancer

Background:High-grade serous ovarian cancer(HGSOC)is the biggest cause of gynecological cancer-related mortality because of its extremely metastatic nature.This study aimed to explore and evaluate the characteristics of candidate factors associated with the metastasis and progression of HGSOC.Methods:Transcriptomic data of HGSOC patients’samples collected from primary tumors and matched omental metastatic tumors were obtained from three independent studies in the National Center for Biotechnology Information(NCBI)Gene Expression Omnibus(GEO)database.Differentially expressed genes(DEGs)were selected to evaluate the effects on the prognosis and progression of ovarian cancer using data from The Cancer Genome Atlas(TCGA)database.Hub genes’immune landscapes were estimated by the Tumor Immune Estimation Resource(TIMER)database.Finally,using 25 HGSOC patients'cancer tissues and 10 normal fallopian tube tissues,immunohistochemistry(IHC)was performed to quantify the expression levels of hub genes associated with International Federation of Gynecology and Obstetrics(FIGO)stages.Results:Fourteen DEGs,ADIPOQ,ALPK2,BARX1,CD37,CNR2,COL5A3,FABP4,FAP,GPR68,ITGBL1,MOXD1,PODNL1,SFRP2,and TRAF3IP3,were upregulated in metastatic tumors in every database while CADPS,GATA4,STAR,and TSPAN8 were downregulated.ALPK2,FAP,SFRP2,GATA4,STAR,and TSPAN8 were selected as hub genes significantly associated with survival and recurrence.All hub genes were correlated with tumor microenvironment infiltration,especially cancer-associated fibroblasts and natural killer(NK)cells.Furthermore,the expression of FAP and SFRP2 was positively correlated with the International Federation of Gynecology and Obstetrics(FIGO)stage,and their increased protein expression levels in metastatic samples compared with primary tumor samples and normal tissues were confirmed by IHC(P=0.0002 and P=0.0001,respectively).Conclusions:This study describes screening for DEGs in HGSOC primary tumors and matched metastasis tumors using integrated bioinformatics analyses.We identified six hub genes that were correlated with the progression of HGSOC,particularly FAP and SFRP2,which might provide effective targets to predict prognosis and provide novel insights into individual therapeutic strategies for HGSOC.

An experimental study on autologous transplantation of fresh ovarian tissue in sheep

Objective:To investigate current autologous transplantation methods and sites of ovarian tissues in sheep.Methods:Sheep ovaries were resected.Ovarian cortices were sliced and transplanted orthotopically into the ovarian mesangial latum and heterotopically into the greater omentum and under groin skin.The grafts were removed two months after transplantation and examined to evaluate the survival of follicles(hematoxylin-eosin staining)and help determining feasible graft sites and transplantation methods.Results:Graft nodules were found in the transplanted sites.HE staining of the grafts showed that multiple primordial follicles were able to survive in the grafts on both sides of the ovarian mesangial latum,the right side of the greater omentum,and the left inguinal subcutaneous tissue.Secondary or cystic follicles were found in almost all of the grafts.Conclusion:The ovarian mesangial latum,the greater omentum and the inguinal subcutaneous tissue can be used as autologous transplantation sites,where sheep ovarian tissue can survive and the follicles grow and develop in good condition.

Age and menopausal status are important factors influencing the serum human epididymis secretory protein 4 level:a prospective cross-sectional study in healthy Chinese people

Background::Human epididymis secretory protein 4(HE4)is a new ovarian cancer biomarker.The factors influencing HE4 levels are not clear,and the reference data in China are limited.Here,we aim to evaluate the effects of menopause and age on HE4 levels and to provide a possible reference value for HE4 in healthy Chinese people.Methods::A total of 2493 healthy females aged 40 years or older were recruited from March 2013 to March 2017 with the cooperation of four medical institutions across Beijing,China.The serum levels of HE4 and cancer antigen 125(CA125)were measured by enzyme-linked immunosorbent assay.The Wilcoxon rank-sum test of variance and a stratified analysis were used to analyze the relationships among age,menopausal status,and levels of HE4 or CA125.Confidence intervals(5%-95%)were determined for reference ranges in different populations.Results::There was a statistically significant difference in median HE4 levels between the post-menopausal(n=2168)and premenopausal groups(n=325)(36.46 vs.24.04 pmol/L,Z=-14.41,P<0.001).HE4 increased significantly with age in the post-menopausal groups(H=408.18,P<0.001)but not in the pre-menopausal subjects(Z=-0.43,P=0.67).The upper 95th percentile of HE4 levels were 44.63 pmol/L for pre-menopausal women,78.17 pmol/L for post-menopausal women,and 73.3 pmol/L for all women.In the post-menopausal population,the HE4 reference ranges were 13.15 to 47.31,14.31 to 58.04,17.06 to 73.51,24.50 to 115.25,and 35.71 to 212.37 pmol/L for different age groups from forty divided by decade.The CA125 level was affected mainly by menopausal status and not age.Conclusions::Menopausal status and age were both important factors influencing the level of HE4,and age affected HE4 levels mainly in post-menopausal women.The HE4 level was higher in the post-menopausal population than in the pre-menopausal population and increased with age.