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Objectives: Based on differences in chemosensitivity and clinical outcomes, we hypothesize that clear cell ovarian carcinoma (CCCO) may display a unique tumor immune microenvironment compared to high grade serous ovarian carcinoma (HGSOC). In addition, micro-satellite instability (MSI) and ARID1DA mutations are more common in clear cell ovarian carcinoma suggesting improved outcomes with checkpoint blockade immunotherapy use. Thus, our objective is to compare the immune landscape of CCCO to HGSOC using imaging mass cytometry (IMC). Methods: Primary untreated ovarian tumors were collected under an IRB-approved protocol. Formalin fixed paraffin embedded (FFPE) tissue sections were stained with 34 metal-tagged antibodies to detect various cell specific and immune related markers using the Fluidigm protocol. IMC data was obtained by the Fluidigm Helios CyTOF instrument utilizing the Hyperion Imaging System laser ablation module. Images from a 1mm2 area of each tissue section were processed and converted to tiff files using MCD Viewer (Fluidigm). The multiplexed images were analyzed using a deep learning based data analytic pipeline beginning with cell segmentation then multiple rounds of phenotype clustering for cell type annotation. Various cell phenotypes were then measured for cell density analysis, or the frequency of a cell phenotype, within a tumor enriched area. Spearman correlation was performed between cell densities of CCCO and HGSOC. Download : Download high-res image (359KB) Download : Download full-size image Results: Samples from 51 patients were used for the study: 10 with clear cell histology and 41 with high grade serous histology. In the CCCO group, stage of disease ranged from stage I (70%) to III (10%), while all the HGSOC had stage III (92.7%) or IV (7.3%) disease (p Conclusions: Clear cell ovarian tumors harbor a distinct tumor immune microenvironment compared to high grade serous ovarian tumors. In our cohort, despite most having stage I disease, CCCO demonstrated features associated with poor prognosis including decreased intratumoral CD8+ T cells, higher density of a subtype of actively dividing B7-H4mid ki67hi cancer cells, and higher density of CD73hi cells. Since B7-H4 and CD73 both act as immune checkpoint inhibitors that can confer chemoresistance, therapeutic strategies targeting these molecules may enhance CD8+ T cell mediated antitumor immune response and chemosensitivity to ultimately improve survival rates in patients with CCCO.

To validate the overexpression of insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) in low-grade serous ovarian carcinoma (SOC), and to investigate whether the IGF-1 pathway is a potential therapeutic target for low-grade SOC.Gene expression profiling was performed on serous borderline ovarian tumors (SBOTs) and low-grade SOC, and overexpression of IGF-1 in low-grade SOC was validated by RT-PCR and immunohistochemistry. The effect of exogenous IGF-1 on cell proliferation was determined in cell lines by cell proliferation assays, cell migration assays, and Western blot. Signaling pathways downstream of IGF-1 and the effects of the AKT inhibitor MK-2206 were investigated by Western blot analysis and by generating IGF-1R short hairpin RNA stable knockdown cell lines. Low- and high-grade cell lines were treated with the dual IGF-1R- and insulin receptor-directed tyrosine kinase inhibitor OSI-906, and cellular proliferation was measured.mRNA analysis and immunostaining revealed significantly higher IGF-1 expression in low-grade SOCs than in SBOTs or high-grade SOCs. In response to exogenous treatment with IGF-1, low-grade cell lines exhibited more intense upregulation of phosphorylated AKT than did high-grade cell lines, an effect that was diminished with IGF-1R knockdown and MK-2206 treatment. Low-grade SOC cell lines were more sensitive to growth inhibition with OSI-906 than were high-grade cell lines.IGF-1 is overexpressed in low-grade SOCs compared with SBOTs and high-grade SOCs. Additionally, low-grade SOC cell lines were more responsive to IGF-1 stimulation and IGF-1R inhibition than were high-grade lines. The IGF-1 pathway is therefore a potential therapeutic target in low-grade SOC.

The purpose of this study is to examine the role of versican (VCAN) in advanced stage serous ovarian cancer by investigating its expression, its function, and its correlation with clinical outcomes.Microarray analysis was performed on RNA isolated from tumor and stromal components of advanced stage serous ovarian cancer and normal ovarian epithelial tissue to identify genes up-regulated in ovarian tumor stroma. Validation studies using immunohistochemistry and quantitative real-time PCR (Q-RT-PCR) was performed on one of the up-regulated genes, VCAN. Immunolocalization of VCAN (n=111) and CD31 (n=56) was done on serous ovarian tumors. CD31 staining was performed to examine microvessel density (MVD). Q-RT-PCR was performed on 65 samples to evaluate the differential expression of VCAN isoforms. Cell proliferation and invasion assays were performed to examine how V1-treated ovarian cancer cell lines and an endothelial cell line would differ from controls. Univariate survival analyses were done with VCAN expression. Correlation analysis was done with CD31, platinum resistance, and clinical data.Validation studies using Q-RT-PCR and immunohistochemistry showed significantly higher VCAN V1 isoform expression in ovarian cancer stroma compared with normal ovarian stroma and ovarian cancer cells. Correlation studies showed stromal VCAN expression was associated with poorer overall and progression-free survival, platinum resistance, and increased MVD. VCAN-treated ovarian cancer and endothelial cells showed increased invasion potential.VCAN overexpression is associated with increased MVD and invasion potential, which may lead to poorer overall and progression-free survival and platinum resistance.

RUNX family genes, including RUNX3, are developmental regulators that are important in human cancers. The purpose of this study was to evaluate expression and oncogenic potential of RUNX3 in ovarian carcinoma.Immunohistochemical staining was performed on 60 malignant, 14 borderline, and 5 normal ovarian specimens. Correlation between RUNX3 expression with tumor histology was performed. RUNX3 expression was evaluated by quantitative real-time polymerase chain reaction (QRT-PCR) in microdissected normal and malignant epithelial ovarian tissues. Cell proliferation and viability studies were performed on cells expressing RUNX3 by lentiviral infection and cells with silenced RUNX3 expression by siRNA.RUNX3 expression by immunohistochemistry was higher in serous ovarian carcinomas versus normal ovarian epithelium (P0.001). Immunofluorescent staining confirmed upregulation of cytoplasmic RUNX3 in ovarian cancer cell lines and tissues. QRT-PCR showed higher RUNX3 mRNA expression in microdissected borderline and malignant ovarian tumor tissues compared with the normal ovarian surface epithelial cells (HOSE) (P=0.006 and P=0.023). Forced RUNX3 expression by lentiviral gene delivery in ovarian cancer cells, SKOV3, that initially showed undetectable RUNX3 expression, resulted in increased cell viability (P=0.043). Silencing RUNX3 expression by siRNA transfection into ovarian cancer cells, OVCAR429, initially expressing high levels of endogenous RUNX3 resulted in a decrease in proliferation (P=0.021).These results suggest that RUNX3 has a role in cell proliferation and viability in ovarian cancer.

Objective Identifying markers specific for mucinous ovarian neoplasms (MON) is important for cancer diagnosis and surveillance, and will help improve our general understanding of the pathobiology of these tumors. CEACAM6 overexpression appears to be an early molecular event with prognostic significance in gastrointestinal carcinomas. Microarray analysis previously demonstrated high CEACAM6 overexpression in MON's and this study sought to validate this finding. Methods Western blot compared CEACAM6 expression in normal human ovarian surface epithelium (HOSE) and ovarian cancer cell lines. Quantitative RT-PCR (qRT-PCR) was performed on 75 laser-microdissected HOSE and ovarian cancer tissue samples. Immunohistochemistry (IHC) was performed and slides were analyzed in a semi-quantitative manner. Results CEACAM6 was expressed in 2 of 3 mucinous cancer cell lines. Expression was absent in all 2 HOSE, 7 serous cancer, and 2 clear cell cancer cell lines. 100-fold CEACAM6 overexpression (qRT-PCR) was demonstrated in 13/16 (81%) borderline, low-grade, and high-grade invasive MON's, compared to 5/50 (10%) serous and 1/5 (20%) benign mucinous samples. CEACAM6 expression was not different between borderline and invasive MON's ( p =0.55) or across tumor stage ( p =0.76). CEACAM6 staining was present in 24/28 (86%) borderline, low-grade, and high-grade invasive MON's; 13/28 (46%) exhibited moderate-strong staining. Neither CEACAM6 expression ( p =0.36) nor staining intensity ( p =0.51) was different between borderline and invasive MON's. None of the serous or benign mucinous tumors exhibited CEACAM6 staining. Conclusions CEACAM6 is overexpressed in borderline and invasive MON's.

Objectives. Laminin receptor 1 (LR1), a non-integrin-type laminin receptor, has been described in several tumors and may play a role in tumor invasion. It is well known that LR1 can modify the conformation and degradation of laminin thus enhancing tumor cell invasion. LR1 may play a role in controlling trophoblast invasion in normal and molar pregnancies. Materials and methods. Real-time polymerase chain reaction (RT-PCR) was performed on total cDNA from 17 gestational age-controlled normal placentas, 10 complete moles (CM), and 4 partial moles (PM). Immunolocalization of LR1 was performed on paraffin sections prepared from 17 age-controlled placentas, 17 PM, and 19 CM. Results. RT-PCR demonstrated a 13-fold increase in LR1 mRNA expression in molar tissues (PM and CM combined) versus normal placentas ( p =0.012). Immunohistochemical analysis revealed LR1 localized to the decidual cells. In normal placenta LR1 localized to the decidual cell membrane. However, in PM and CM, LR1 was additionally noted in the cytoplasm of decidual cells. Interestingly, with immunohistochemistry method, we found that PM demonstrated higher protein expression of LR1 than either normal placenta or CM ( p =0.001 and p =0.024, respectively). Conclusion. LR1 is expressed in both the decidual cells of normal placenta and mole (CM and PM). Decidual cells in CM and PM express LR1 significantly greater than the decidual cells in placenta. The underlying mechanism of how molar tissue may be associated with enhanced expression of LR1 in the maternal endometrium is unclear.

Objectives Cytotoxic T cells (Tc) and natural killer (NK) cells may play a role in controlling trophoblast invasion. This study was undertaken to determine the level of infiltration and antigen profile of immune cells and explore their relationship in normal placenta (NP) and molar tissues to better understand the biology of gestational trophoblastic diseases. Materials and methods Immunolocalization of CD8, Granzyme B (GrB), and FoxP3 was performed on sections prepared from 11 gestational age-matched NP, 19 partial moles (PM), and 18 complete moles (CM) to characterize effector (GrB+CD8+) and GrB− (GrB−CD8+) Tc cells, GrB+ NK cells (GrB+CD8−), and Treg (FoxP3+) cells. Results Immune cells infiltrated into the implantation site of normal placenta, PM, and CM with increasing frequency. Effector and GrB− Tc, GrB+ NK and Treg infiltration in the CM were significantly stronger than seen in the normal placenta ( p =0.002, p =0.007, p =0.002, respectively). Immune cell infiltration was not detected in the villi or trophoblast of gestational tissues. Treg infiltration in the implantation site was only observed in PM and CM. In CM and PM Tc infiltration positively correlated with Treg infiltration ( p =0.035), but Treg infiltration did not correlate with the Tc effector ratio (effector Tc cells/all Tc cells). Conclusion In CM the cellular immune response in the implantation site was significantly more vigorous than seen in normal placenta. These observations demonstrate that in the implantation site of CM, the number of effector Tc and GrB+ NK cells are increased and Treg cells may negatively regulate T lymphocyte activation.

Objective. Unlike other histological types of epithelial ovarian carcinoma, ovarian clear cell carcinoma is known to have very poor response to therapy even when discovered in its early stages. Since tumor hypoxia has been shown to be strongly associated with poor prognosis, deregulation of the representative factor of tissue hypoxia; hypoxia-inducible factor 1 alpha (HIF-1α) and related protein; Von Hippel-Lindau (VHL) may be associated with poor prognosis of ovarian clear cell carcinoma. Methods. Immunolocalization of both HIF-1α and VHL was performed on 56 cases of paraffin-embedded tissue sections of four different histological types of epithelial ovarian carcinoma and 5 cases of benign ovarian tumors as a control. Quantitative RT-PCR analysis of both HIF1A and VHL was performed on RNA isolated from 61 microdissected frozen tissues of four different histological types of epithelial ovarian carcinoma and 6 cases of normal ovarian epithelial cells. Expression levels of HIF-1α and VHL in different histological types and correlation between HIF-1α and VHL were determined by nonparametric analysis by Kruskal–Wallis and Spearman's test. Results. HIF-1α expression levels were significantly higher in ovarian clear cell carcinoma than in other histological types ( P =0.001). We found no correlation between mRNA and protein expression level in any type of carcinoma specimens. Among endometrioid, serous, and mucinous carcinoma, there were no differences in HIF-1α expression ( P =0.643). There was a negative correlation between HIF-1α and VHL in serous ( r =−0.661, P =0.027) and in endometrioid carcinoma ( r =−0.657 P =0.039), but no correlation was found between HIF-1α and VHL expression levels in ovarian clear cell carcinoma ( P =0.60). Conclusions. The results suggest that the role of hypoxia may change according to the histological type of ovarian carcinoma. High expression of HIF-1α and its independence from VHL in ovarian clear cell carcinoma may confer chemoresistance in this histological type.

To measure clusterin expression in cervical cancer tissues and cell lines and to evaluate whether clusterin confers resistance to paclitaxel in cervical cancer cells.Immunohistochemical staining for clusterin was performed on 15 normal cervical tissues and 32 primary cervical cancer tissues, and clusterin expression in cervical cancer cell lines was quantified by Western blotting. The correlation between clusterin expression level and paclitaxel IC50 in cervical cancer cell lines was evaluated. The effect of clusterin siRNA on paclitaxel resistance was evaluated by XTT assays.Cervical cancer tissues expressed significantly higher levels of clusterin than did normal cervical tissues (4.08 vs. 1.35, P0.05). Clusterin expression levels were correlated with paclitaxel resistance in cervical cancer cell lines, and transfection of clusterin siRNA into HeLaS3 cells significantly decreased their resistance to paclitaxel (P0.05).Our finding that clusterin expression was significantly higher in cervical cancer than in normal cervical tissues suggests that clusterin may confer paclitaxel resistance in cervical cancer cells.

Background. When ovarian carcinoma is diagnosed in stage I, up to 90% of patients can be cured with surgery and currently available chemotherapy. At present, less than 25% of cases are diagnosed at this stage. To increase the fraction of ovarian cancers detected at an early stage, screening strategies have been devised that utilize a rising serum CA125 level to trigger the performance of transvaginal sonography. One limitation of CA125 as an initial step in such a screening strategy is that up to 20% of ovarian cancers lack expression of the antigen. Serum tumor markers that can be detected in ovarian cancers that lack CA125 expression might improve the sensitivity for early detection. Methods. From 296 ovarian cancers, 65 (22%) were found to have weak or absent CA125 expression on immunoperoxidase staining. Tissue expression of CA125 was compared to serum CA125 levels. Using immunoperoxidase staining of tissue arrays, we have assessed expression of 10 potential serum tumor markers in the 65 epithelial ovarian cancers with little or no CA125 expression and in ovarian cystadenomas, tumors of low malignant potential, normal ovaries, and 16 other normal tissues. Results. Low or absent expression of CA125 in surgical specimens of epithelial ovarian cancer was associated with low levels of serum CA125 in pre-operative serum specimens. In ovarian cancers that lacked CA125, all specimens (100%) expressed human kallikrein 10 (HK10), human kallikrein 6 (HK6), osteopontin (OPN), and claudin 3. A smaller fraction of CA125-deficient ovarian cancers expressed DF3 (95%), vascular endothelial growth factor (VEGF) (81%), MUC1 (62%), mesothelin (MES) (34%), HE4 (32%), and CA19-9 (29%). When reactivity with normal tissues was considered, however, MES and HE4 showed the greatest specificity. Differential expression was also found for HK10, OPN, DF3, and MUC1. Conclusions. At the level of tissue expression, each of 10 potential serum markers could be detected in 29–100% of ovarian cancers that had low or absent expression of CA125. Several markers exhibited more intense expression in cancers than in normal organs. Further investigation is needed to demonstrate complementary expression of markers in serum.

Introduction Surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS) is a novel method for biomarker discovery that can provide a rapid protein expression profile from a variety of biological samples. Since SELDI-MS requires a small amount of biological material, this technique is ideal for analyzing proteins isolated from microdissected tissue samples. The current study was undertaken to investigate potential differences in protein expression between normal and molar trophoblast procured by laser capture microdissection (LCM) utilizing SELDI ProteinChip array technology. Further knowledge of protein expression in complete mole may advance our understanding of the pathogenesis of gestational trophoblastic diseases. Materials and methods Laser capture microdissected trophoblast cells from nine fresh complete moles were analyzed and compared to the trophoblast cells from 10 fresh normal placentas of comparable gestational age, using SELDI ProteinChip to identify potential differences in protein expression. Results Three metal binding polypeptides were identified with the estimated molecular weights of 11.3, 13.8, and 14.0 kDa, which appeared in significantly lower levels in complete mole as compared to normal trophoblast cells ( P P P Discussion While further characterization of these protein peaks is important and necessary, our current work clearly demonstrates that the combined technology of SELDI and LCM is effective in distinguishing protein expression between normal placenta and complete mole. Further knowledge of protein expression in complete mole may advance our understanding of molecular mechanisms and improve management in gestational trophoblastic diseases.

Objectives. We have previously described that bioactive lysophospholipids—lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC)—are present in ascitic fluids from patients with ovarian cancer. To understand the role of these lipids in ovarian cancer, we investigated the effects of these lipids on interleukin-8 (IL-8) production in ovarian cancer cells. IL-8 is a proinflammatory and proangiogenic factor, which is potentially involved in ovarian cancer development. Methods. The Clontech PCR-Select cDNA subtraction method (Clontech Laboratories, Inc., Palo Alto, CA) was used to identify genes potentially regulated by LPA in HEY and OCC1 ovarian cancer cell lines. Northern blot analysis was used to confirm and examine IL-8 mRNA regulation by lysolipids. Enzyme-linked immunosorbent assay (ELISA) was used for detecting secreted IL-8. Results. We describe here that LPA, S1P, and SPC increased mRNA levels (2- to 7-fold) and protein secretion (2- to 12-fold) of IL-8 from ovarian cancer cells (HEY, OCC1, and SKOV3) in vitro. These regulations were both dose- and time-dependent. All three lipids increased the stability IL-8 mRNA in HEY cells. In contrast to malignant ovarian cancer cells, immortalized human ovarian epithelial cells did not respond to any of these lipids to increase the secretion of IL-8, although these cells secreted similar basal levels of IL-8 (310 pg/ml/10,000 cells). Two breast cancer cell lines (MCF7 and T47D) secreted lower basal levels of IL-8 (48-80 pg/ml/10,000 cells), compared with ovarian cancer cells (200–500 pg/ml/10,000 cells). MCF7 cells responded to LPA, but not S1P and SPC, by increasing the secretion of IL-8. T47D and MCF10A, an immortalized breast cell line, did not respond to LPA, S1P, or SPC to increase IL-8 secretion. Conclusions. LPA, S1P, and SPC regulate the mRNA and protein levels of the proinflammatory and proangiogenic factor IL-8 in ovarian cancer cells. The pathological significance of these regulations in ovarian cancer remains to be further investigated.

Objective. Matrix metalloproteinase-9 (MMP-9) can degrade gelatin and type IV collagen and is known to play an important role in tumor cell invasion across the basement membrane. The tissue inhibitor of metalloproteinase-1 (TIMP-1) is able to prevent activation of pro-MMP-9 and forms a 1:1 complex with the active form of MMP-9. The aim of the present study was to investigate the expression of MMP-9 and TIMP-1 in benign, borderline, and invasive epithelial ovarian tumors. Materials and methods. A total of 90 patients with epithelial ovarian tumor were treated at the Brigham and Women's Hospital and were used as the study population. Immunohistochemistry and in situ hybridization were performed to detect protein and mRNA expression of MMP-9 and TIMP-1. Results. In the 90 epithelial ovarian tumors tested, MMP-9 expression in tumor cells was found to be significantly enhanced in serous and mucinous ovarian carcinomas compared with benign and borderline tumors. We also observed the immunostaining of MMP-9 in stromal cells of benign, borderline, and invasive epithelial ovarian tumors. Moreover, the expression levels of TIMP-1 in tumor cells were significantly higher in borderline and invasive ovarian tumors than in benign tumors. Conclusion. Using an in situ hybridization technique, we disclosed a direct correlation between the presence of mRNA and protein expression for both MMP-9 and TIMP-1. The present data suggest that high levels of MMP-9 protein in invasive epithelial ovarian carcinoma are strongly associated with tumor cell invasion. Enhanced expression of TIMP-1 protein in borderline and invasive tumors indicates that endogenous TIMP-1 protein may play a paradoxical role in ovarian tumor progression.

Objective. The ras genes are a well-studied family of proto-oncogenes whose involvement in many cancers has been delineated. However, K- ras mutations have not previously been examined in papillary serous carcinoma of the peritoneum (PSCP), a tumor which resembles serous epithelial ovarian carcinoma (SEOC) both in histology and epidemiology. In this study we examine the role of the K- ras oncogene in PSCP compared to SEOC. Methods. Using single-strand conformational polymorphism analysis and cycle sequencing protocols, we evaluated our collection of 51 cases of PSCP for K- ras mutations and compared these findings with the experience in SEOC. We then examined 5 cases of PSCP for activation of ras proteins and MAP kinase to evaluate the potential involvement of the ras pathway in PSCP tumorigenesis. Results. We found only one K- ras mutation in our 51 cases (2%) of PSCP compared to three mutations in 46 cases (6.5%) of high-grade, late-stage SEOC. This was not significantly different ( P > 0.10). In the single PSCP case with a K- ras mutation, the mutation was found in only one of five tumor sites tested. All four mutations involved a single nucleotide alteration in codon 12 (GGT to GTT, Gly to Val). To evaluate the ras pathway in PSCP, we used the known activated ras binding domain on Raf-1 to perform an assay to test for activated ras. We identified ras activation in 4 of 5 PSCP cases tested and, to confirm that the activation was functional, we tested and found similar activation of MAP kinase, a downstream mediator for K- ras expression. Conclusions. K- ras mutations occur at low rates in both PSCP and high-grade, late-stage SEOC, and therefore K- ras mutations are not involved in the development of these two diseases. Finding the mutation in only one of multiple tumor sites in the PSCP case supports growing evidence for a multifocal origin of PSCP. Our findings of ras activation in four of five cases of PSCP suggest that ras activation by mechanisms other than genetic mutation is important for PSCP tumorigenesis.

Objective. Our purpose was to identify potential differences in gene expression between normal trophoblast and choriocarcinoma cells. Methods. The Atlas human cDNA expression array hybridization technique was used to study the gene expression pattern in normal trophoblast and choriocarcinoma cell lines. Furthermore, to confirm heat shock protein-27 (Hsp-27) expression data, reverse transcriptase-PCR (RT-PCR), Western blot, and immunohistochemical analyses were used in vitro with cell lines and in vivo with paraffin sections. Results. The expression of nine genes was strongly different comparing a normal trophoblast cell line with choriocarcinoma cells on the Atlas membranes. Compared to normal trophoblast cells, six genes were upregulated and three were downregulated in choriocarcinoma cells. Furthermore, the downregulation of Hsp-27 in choriocarcinoma cells was confirmed both in vitro with cell lines and in vivo with paraffin sections using RT-PCR, Western blot, and immunohistochemical techniques. Conclusion. cDNA expression array is a useful technique for identifying differentially expressed gene patterns in normal trophoblast and choriocarcinoma cells. The strong expression of Hsp-27 in placental villous trophoblast cells may play a role in trophoblast differentiation. The downregulation of Hsp-27 in choriocarcinoma may contribute to the extreme sensitivity of trophoblastic tumors to chemotherapy.

Our purpose was to investigate the expression of matrix metalloproteinases (MMPs) in gestational trophoblastic diseases and normal first-trimester placenta.Paraffin sections of 16 partial moles, 25 complete moles, 10 gestational choriocarcinomas, and 11 normal first-trimester placentas were studied immunohistochemically for expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and tissue inhibitor of metalloproteinase-1 (TIMP-1).Nine (90.0%) of the choriocarcinoma cases showed strong intensity of staining for MMP-1. Choriocarcinoma exhibited significantly stronger staining for MMP-1 than syncytiotrophoblast in normal placenta (P0.01), partial mole (P0.01), and complete mole (P0.01). Choriocarcinoma also showed significantly stronger staining for MMP-1 than the extravillous trophoblast in placenta (P0.05). MMP-2 was expressed only in syncytio- and extravillous trophoblasts in normal placenta, partial mole, and complete mole. Choriocarcinoma and the extravillous trophoblast in partial mole and complete mole had significantly stronger staining for MMP-2 than the extravillous trophoblast in placenta (P0.05, P0.01, P0.01, respectively). Choriocarcinoma also exhibited significantly stronger staining for MMP-2 than syncytiotrophoblasts in placenta (P0.01), partial mole (P = 0.05), and complete mole (P0.01). The expression of MMP-3, MMP-9, and MMP-13 was similar in all four tissues with the predominance of syncytiotrophoblast for MMP-3 and MMP-13 and cytotrophoblast for MMP-9. While 8 (73.0%) placentas, 14 (87.5%) partial moles, and 19 (76.0%) complete moles showed strong immunoreactivity for TIMP-1 in syncytiotrophoblasts, no strong staining was found in choriocarcinomas (P0.01, P0.01, P0.01, respectively).The extravillous trophoblast of first-trimester placenta has significantly less expression of MMP-1 than choriocarcinoma and significantly less expression of MMP-2 than choriocarcinoma and extravillous trophoblast of partial and complete mole. The expression of TIMP-1 was significantly less in choriocarcinoma than the syncytiotrophoblast of normal placenta, partial mole, and complete mole. MMPs and their inhibitors may play a role in the pathogenesis of gestational trophoblastic diseases.

Objective The objective of this study is to investigate whether wild-type TP53 status in high-grade serous ovarian carcinoma is associated with poorer survival. Methods Clinical and genomic data of 316 sequenced samples from The Cancer Genome Atlas (TCGA) ovarian high-grade serous carcinoma study were downloaded from TCGA data portal. Association between wild-type TP53 and survival was analyzed with Kaplan Meier method and Cox regression. The diagnosis of high-grade serous carcinomas was evaluated by reviewing pathological reports and high-resolution hematoxylin and eosin (H&E) images from frozen sections. The authenticity of wild-type TP53 in these tumor samples was assessed by analyzing SNP array data with ASCAT algorithm, reverse phase protein array (RPPA) data and RNAseq data. Results Fifteen patients with high grade serous ovarian carcinomas were identified to have wild-type TP53 , which had significantly shorter survival and higher chemoresistance than those with mutated TP53 . The authenticity of wild-type TP53 status in these fifteen patients was supported by SNP array, RPPA, and RNAseq data. Except four cases with mixed histology, the classification as high grade serous carcinomas was supported by pathological reports and H&E images. Using RNAseq data, it was found that EDA2R gene, a direct target of wild-type TP53, was highly up-regulated in samples with wild-type TP53 in comparison to samples with either nonsense or missense TP53 mutations. Conclusion Although patients with wild-type TP53 ovarian cancer were rare in the TCGA high grade ovarian serous carcinomas cohort, these patients appeared to have a poorer survival and were more chemoresistant than those with mutated TP53 . Differentially expressed genes in these TP53 wild-type tumors may provide insight in the molecular mechanism in chemotherapy resistance.

A DNA-fingerprinting approach has been adapted to detect differentially expressed genes in human ovarian carcinoma. This method is based on the use of arbitrary primers to generate fingerprints from total RNA isolated from normal ovarian epithelial cells and ovarian carcinoma cells by polymerase chain reaction (PCR). Using this method, we cloned two cDNA fragments (DOC-1 and DOC-2) which were present in normal ovarian surface epithelial cells but consistently absent in all of the ovarian cancer cell lines from the differential display. In addition, we also identified a cDNA fragment (LF4.0) which is overexpressed in most of the tumor cell lines and tumor tissues in comparison to the normal ovarian surface epithelial cells. The differential expression of the genes in the tumor cell lines as well as in the tumor tissues was also confirmed by Northern analysis. The clone DOC-2, which is a 800-bp cDNA fragment, has one open reading frame suggesting that the gene may be translated. Assuming that this frame is the sense strand, we generated both sense and antisense riboprobe for in situ mRNA hybridization. Only the antisense DOC-2 riboprobe revealed a hybridization signal which was restricted to the human surface ovarian epithelium. The potential functional roles of these genes is now under investigation.