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The objective of the present study was to perform a quantitative analysis of cancer stem cell (CSC) marker expression in ovarian carcinoma effusions. The clinical role of SSEA1 in metastatic high-grade serous carcinoma (HGSC) was additionally analyzed. CD133, Nanog, SOX2, Oct3/4, SSEA1, and SSEA4 protein expressions were quantitatively analyzed using flow cytometry (FCM) in 24 effusions. SSEA1 expression by immunohistochemistry was analyzed in 384 HGSC effusions. Highly variable expression of CSC markers by FCM was observed, ranging from 0 to 78% of Ber-EP4-positive cells in the case of CD133, with the largest number of negative specimens seen for SSEA4. SSEA1 expression by immunohistochemistry was found in HGSC cells in 336/384 (89%) effusions, most commonly focally (p = 0.029). In univariate survival analysis, higher SSEA1 expression was significantly associated with poor overall survival (p = 0.047) and progression-free survival (p = 0.018), though it failed to retain its prognostic role in Cox multivariate survival analysis in which it was analyzed with clinical parameters (p = 0.059 and p = 0.111 for overall and progression-free survival, respectively). In conclusion, CSC markers are variably expressed in ovarian carcinoma effusions. SSEA1 expression is associated with disease progression and poor survival in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

The objective of this study was to validate the diagnostic and clinical role of four protein products of genes previously found to be differentially expressed in uterine low-grade endometrial stromal sarcoma (LG-ESS) compared to uterine leiomyosarcoma (LMS). Protein expression by immunohistochemistry of transgelin (TGLN), neuron navigator-2 (NAV2), fatty acid binding protein-3 (FABP3), and cyclin D2 (CCND2) was analyzed in 305 uterine sarcomas (231 LMS, 74 LG-ESS). Expression was analyzed for association with clinicopathologic parameters and survival. TGLN (p

The present study analyzed the expression and clinical role of the transforming growth factor-β (TGFβ) pathway in high-grade serous carcinoma (HGSC), with focus on malignant effusions. TGFβ1–3 and TGFβRI–III mRNA expression by qRT-PCR was analyzed in 70 HGSC effusions and 55 solid specimens (28 ovarian, 27 abdominal metastases). Protein expression of Smad2 and Smad3 and their phosphorylated forms by Western blotting was analyzed in 73 specimens (42 effusions, 13 ovarian carcinomas, 18 solid metastases). Expression was analyzed for association with anatomic site and clinical parameters, including survival. TGFβRI and TGFβRII mRNA was overexpressed in effusions and solid metastases, particularly the former, compared to that in the ovarian tumors (p

The objective of this study was to analyze the expression and clinical role of the RNA-binding molecule HuR in metastatic high-grade ovarian serous carcinoma (HGSC). HUR mRNA expression by reverse-transcription polymerase chain reaction was analyzed in 66 effusions from patients diagnosed with HGSC. Protein expression was analyzed in 262 HGSC effusions using immunohistochemistry. HUR mRNA was detected in all 66 effusions. HUR mRNA levels were unrelated to clinicopathological parameters. However, higher HUR mRNA levels were significantly related to poor overall survival in the entire cohort (P=.023), as well as in analysis limited to patients with prechemotherapy primary diagnosis specimens (P=.001) in univariate analysis. Cox multivariate analysis showed an independent prognostic role for HUR mRNA in the entire cohort (P=.033) and in patients with prechemotherapy primary diagnosis specimens (P=.002). HuR protein was detected in the nucleus and cytoplasm of tumor cells in 258 (98%) of 262 and 153 (58%) of 262 effusions, respectively. Higher HuR protein expression was associated with higher serum Cancer Antigen (CA) 125 levels at diagnosis (P=.01), but its presence at both cellular compartments was otherwise unrelated to clinicopathological parameters or survival. In conclusion, HuR is widely expressed in metastatic HGSC at both the mRNA and protein level. Higher HUR mRNA levels are associated with poor survival in metastatic HGSC, whereas protein expression has no prognostic value.

Objective To validate our earlier observation that 11 chemoresistance-associated mRNAs are molecular markers of poor overall survival in ovarian serous carcinoma. Methods Ovarian serous carcinomas (n=112) and solid metastases (n=63; total=175) were analyzed for mRNA expression of APC , BAG3 , EGFR , S100A10 , ITGAE , MAPK3 , TAP1 , BNIP3 , MMP9 , FASLG and GPX3 using quantitative real-time PCR. mRNA expression was studied for association with clinicopathologic parameters and survival. Tumor heterogeneity was assessed in 20 cases with >1 specimen per patient. APC, BAG3, S100A10 and ERK1 protein expression by immunohistochemistry was analyzed in 58 specimens (38 primary carcinomas, 20 metastases). Results BAG3 (p=0.013), TAP1 (p=0.014), BNIP3 (p MMP9 (p=0.036) were overexpressed in primary tumors, whereas S100A10 (p=0.027) and FASLG (p=0.006) were overexpressed in metastases. Analysis of patient-matched primary carcinomas and metastases showed overexpression of APC (p=0.022), MAPK3 (p=0.002) and BNIP3 (p=0.004) in the former. In primary carcinomas, higher APC (p=0.003) and MAPK3 (p=0.005) levels were related to less favorable chemoresponse. Higher S100A10 (p=0.029) and MAPK3 (p=0.041) levels were related to primary chemoresistance. Higher BAG3 (p=0.026) and APC (p=0.046) levels in primary carcinomas were significantly related to poor overall survival in univariate, though not in multivariate survival analysis. S100A10 protein expression was related to poor chemoresponse (p=0.002) and shorter overall (p=0.005) and progression-free (p Conclusions Our data provide evidence of heterogeneity in ovarian serous carcinoma and identify APC, MAPK3, BAG3 and S100A10 as potential biomarkers of poor chemotherapy response and/or poor outcome in this cancer.

We recently identified gene signatures that allow classification of ovarian carcinoma into 5 distinct clinically relevant groups. In the present study, we investigated the clinical role of 10 protein products of the discriminating genes, with focus on epithelial-mesenchymal transition and stem cell markers. Expression of E-cadherin, N-cadherin, P-cadherin, Zeb1, HMGA2, Rab25, CD24, NCAM (CD56), Sox11, and vimentin was assessed in 100 advanced-stage (International Federation of Gynecology and Obstetrics stages III-IV) serous ovarian carcinoma effusions using immunohistochemistry. Results were analyzed for association with clinicopathological parameters, including chemotherapy response, and survival. All 10 proteins were frequently expressed in carcinoma cells. HMGA2 expression was related to older age (P = .015). HMGA2 and NCAM expression was related to stage III disease (P = .011 and P = .023, respectively), and NCAM was overexpressed in peritoneal compared with pleural effusions (P = .001). Vimentin and Zeb1 expression was significantly related to poor chemotherapy response at diagnosis (P = .005 and P = .017, respectively). The associations between NCAM and peritoneal localization and of vimentin and poor chemoresponse were retained after Bonferroni correction. NCAM expression was associated with a trend for shorter overall survival in univariate survival analysis (P = .187), but emerged as an independent prognosticator in Cox multivariate analysis (P = .042). This study identifies vimentin and Zeb1 as markers of poor chemoresponse in metastatic serous ovarian carcinoma effusions and suggests NCAM as potential prognostic marker in metastatic disease. The generally limited prognostic role of the studied markers emphasizes the difficulty in applying data obtained in studies of primary ovarian carcinomas to analyses of ovarian carcinoma effusions, reflecting the unique biology of the latter.

The aim of this study was to analyze the diagnostic role of PTEN and ARID1A in effusion cytology. Effusions (n = 279), consisting of 226 carcinomas (70 ovarian, 64 breast, 36 lung, and 15 uterine corpus carcinomas; 41 carcinomas of other origin) and 53 malignant mesotheliomas, were analyzed for PTEN and ARID1A expression using immunohistochemistry. PTEN was preserved in 166 (59%) tumors, partially lost in 38 (14%), and absent in 75 (27%), with lower expression in malignant mesotheliomas compared to carcinomas, though not significantly (p = 0.084). ARID1A was preserved in 243 (88%) tumors, partially lost in 18 (6%), and absent in 18 (6%). The majority of tumors with absent ARID1A were ovarian carcinomas, predominantly of clear cell or low-grade serous type. Reactive mesothelial cells in carcinoma specimens were uniformly positive for both proteins. ARID1A mutation analysis showed no mutations in eight analyzed specimens negative by immunohistochemistry. Loss of PTEN and ARID1A expression is highly specific for malignancy in effusion pathology. Loss of PTEN is not informative of organ of origin, whereas absence of ARID1A should raise suspicion of an ovarian primary.

The involvement of VICKZ proteins has been implicated in a large number of cancers. The aim of the present study was to investigate the biological and clinical role of VICKZ proteins in ovarian carcinoma (OC). VICKZ1-3 protein expression was analyzed in 82 serous OC specimens (51 effusions, 14 primary carcinomas, 17 solid metastases) by immunoblotting. Protein localization was studied using immunohistochemistry in 101 tumors (40 effusions, 25 primary carcinomas, 36 solid metastases). The effect of VICKZ silencing using short hairpin RNA on collagenolytic activity and invasion was assessed in ES-2 OC cells. VICKZ2 was the most frequently expressed family member in serous carcinomas. VICKZ levels measured by pan-VICKZ antibody were significantly higher in primary carcinomas and solid metastases compared to effusions (P < .001). In contrast, VICKZ1 and VICKZ2 were overexpressed in effusions compared to primary carcinomas and solid metastases (P = .016 and P = .024, respectively), and higher VICKZ2 expression in effusions was associated with shorter overall survival in univariate analysis (P = .01). All 3 proteins were localized to OC cells by immunohistochemistry, with tumor-specific expression observed for VICKZ1 and VICKZ2. VICKZ silencing in ES-2 cells led to reduced matrix metalloproteinase 9 activity and reduced invasion. In conclusion, VICKZ2 is the most frequently expressed VICKZ family member in serous OCs. VICKZ1 and VICKZ2 are overexpressed in effusions compared to primary carcinomas and solid metastases, suggesting a biological role at this anatomical site, and appear to have a role in proteolysis and invasion. VICKZ2 may be a prognostic marker in ovarian serous carcinoma effusions.

The aim of the present study was to investigate the expression and clinical role of the aurora A and aurora B kinases in primary and metastatic serous ovarian carcinoma. AURKA and AURKB messenger RNA expression was investigated in 178 tumors (88 effusions, 38 primary carcinomas, and 52 solid metastases) from 144 patients with advanced-stage disease using quantitative real-time polymerase chain reaction. Aurora A and aurora B protein expression by immunohistochemistry was additionally analyzed in 147 tumors. Messenger RNA and protein expression at different anatomical sites were studied for association with clinicopathologic parameters, including chemotherapy resistance and survival. AURKA and AURKB messenger RNA and their protein product were demonstrated in all primary carcinomas, solid metastases, and effusions. The expression of AURKA messenger RNA and aurora A protein was higher in effusions compared with solid specimens (P = .003 and P = .006, respectively). AURKB messenger RNA expression was higher in primary carcinomas, and solid metastases obtained prechemotherapy compared with postchemotherapy (P.001 and P = .012, respectively), with no such difference in effusions (P.05). Low aurora B protein expression was associated with primary chemotherapy resistance (P = .006) and poor treatment response (P = .013) in prechemotherapy effusions. No significant association was found between messenger RNA levels or protein expression and progression-free or overall survival. The present study documents for the first time frequent aurora A and aurora B expression in metastatic ovarian carcinoma, suggesting a role in cancer progression, with higher aurora A expression in effusions compared with primary carcinomas and solid metastases. Low AURKB messenger RNA expression in prechemotherapy effusions might be predictive of intrinsic chemotherapy resistance.

Background L1 cell adhesion molecule (L1CAM) overexpression has been reported to be strongly associated with poor prognosis in early stage endometrial cancer (EC). We aimed at the validation of L1CAM as a marker of poor prognosis in an independent study population. Methods Patients with endometrioid EC FIGO stage I, were treated at Oslo University Hospital between 2005 and 2012. L1CAM expression was detected by immunohistochemistry with >10 % L1CAM staining defined as positive. Risks of relapse and death were estimated as hazard ratios (HRs) with 95 % confidence intervals (95 % CI). Results Of 450 patients, 388 (86 %) were evaluable for L1CAM expression and 35 (9 %) were L1CAM positive. After follow-up for a median time of 4.8 years (0.1–8.8), 33 (8 %) patients had recurred. 6/35 (17 %) L1CAM positive patients relapsed compared to 27/353 (8 %) L1CAM-negative patients. There were 7 (20 %) deaths in the L1CAM positive group, and 34 (10 %) in the negative group. In multivariate analysis, controlled for age and FIGO stage, L1CAM positivity was not significantly associated with the risk of relapse (HR 2.08, 95 % CI: 0.85–5.10, p = 0.11) or death of all-cause (HR 1.81, 95 % CI: 0.79–4.11, p = 0.16). In patients who were not treated with chemotherapy, L1CAM was significantly associated with risk of relapse (HR 2.9; 95 % CI: 1.08–7.56; p = 0.04). Conclusion Our report confirms that L1CAM is associated with a more aggressive tumortype and more distant relapses. The overall recurrence rate in this population was low as were the absolute differences between L1CAM positive and negative patients. In this independent study sample, L1CAM failed to be a clinically relevant marker of poor prognosis in stage I endometrioid endometrial carcinoma.

Summary The objective of this study was to analyze the clinical role of 9 microRNAs (miRs) previously found to be overexpressed in ovarian carcinoma effusions compared with primary ovarian carcinomas. High-grade serous carcinoma effusions (n=148) were analyzed for expression of miR-29a, miR-31, miR-99b, miR-182, miR-210, miR-221, miR-222, miR-224, and miR-342 using quantitative polymerase chain reaction. Expression levels were analyzed for association with clinicopathological parameters and survival. miR-29a and miR-31 levels were further assessed for association with protein expression of their targets Stathmin and DNA methyltransferase-3A (DNMT3A) by immunohistochemistry and Western blotting, respectively. miRNA levels were unrelated to clinicopathological parameters. However, higher miR-29a levels were significantly related to longer overall survival in univariate ( P =.007) and Cox multivariate survival analysis ( P =.045). miR-29a levels were inversely related to those of its target DNMT3A ( P =.048), and higher DNMT3A expression was significantly related to poor overall survival in univariate ( P =.03) and Cox multivariate ( P =.016) survival analysis. In contrast, miR-31 levels were directly related to cytoplasmic phospho-Stathmin expression ( P =.029) and unrelated to Stathmin and nuclear phospho-Stathmin, and both Stathmin and phospho-Stathmin expressions were unrelated to survival. miR-29a and its target DNMT3A are novel candidate biomarkers of longer and shorter survival, respectively, in metastatic high-grade serous carcinoma.

The objective of this study was to analyze the expression and clinical role of the high mobility group AT hook (HMGA) protein in advanced-stage serous ovarian carcinoma. HMGA2 protein expression was investigated in 199 effusions and in 50 patient-matched primary tumors and solid metastases using immunohistochemistry. Results were analyzed for association with clinicopathologic parameters, including chemotherapy response, and survival. HMGA2 was expressed in tumor cells in 94.5 %, 96 %, and 90 % of specimens, respectively. There was no difference in HMGA2 expression between patient-matched samples from different anatomic sites (p > 0.3). HMGA2 expression in chemo-naive samples was significantly higher in older patients (p = 0.006, p = 0.01, and p = 0.005 for effusions, primary tumors, and solid metastases, respectively). No association was found with residual disease volume. Furthermore, HMGA2 expression was not associated with FIGO stage (p > 0.2), except in chemo-naive effusions (n = 106, p = 0.016). There was no difference in HMGA2 expression between chemo-naive samples and samples obtained post-chemotherapy in effusions (p = 0.2) or primary tumors (p = 0.1). However, solid metastases obtained after chemotherapy exposure had higher HMGA2 expression compared with chemo-naive samples (p = 0.032). HMGA2 expression was unrelated to chemotherapy response or survival. However, it was directly related to protein expression of the previously studied cancer stem cell marker Nestin (p = 0.01) and the gap junction protein claudin-7 (p = 0.02) and inversely related to the mRNA level of the E-cadherin repressor SIP1 (p = 0.02). This study provides evidence that HMGA2 is universally expressed in advanced-stage ovarian serous carcinoma irrespective of anatomic site, suggesting that HMGA2 may have a clinical role as therapeutic target.

H. P. Dong, A. K. Ree Rosnes, A. J. Bock, A. Holth, V. A. Florenes, C. G. Trope’, B. Risberg and B. Davidson Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions Objective: The objective of this study was to establish a flow cytometry assay for measuring c-FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. Methods: Two c-FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c-FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal-to-noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c-FLIP expression was analysed for association with clinicopathological parameters and survival. Results: Rabbit polyclonal c-FLIP by Abcam and the IntraStain kit by Dako performed best. c-FLIP expression was detected in tumour cells in all 69 effusions (expression range 21–100%, median = 80%). No association was found between c-FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c-FLIP levels and expression of the previously studied apoptosis marker cleaved caspase-3 (P = 0.029). Conclusions: An assay for measuring c-FLIP in cytology specimens is presented. c-FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.

The AKT signaling pathway is crucial for cancer cell survival. The objective of this study was to analyze the expression and clinical role of this pathway in serous ovarian carcinoma. Phospho-AKT and phospho-mammalian target of rapamycin protein expression was studied in 269 ovarian carcinomas (159 effusions, 38 primary carcinomas, 72 solid metastases) using immunohistochemistry. The association between AKT, mammalian target of rapamycin, and DJ-1 in effusions was quantitatively analyzed using flow cytometry. AKT phosphorylation status in effusions was further studied using Western blotting. Phospho-AKT and phospho-mammalian target of rapamycin were detected in the majority of tumors at all anatomical sites. Phospho-AKT expression in effusions was higher in grade 3 versus grades 1 and 2 tumors (P = .013). Flow cytometry analysis showed association between AKT, mammalian target of rapamycin, and DJ-1 expression (P < .001). Higher phospho-AKT Thr308/pan-AKT ratio by Western blotting was associated with more advanced International Federation of Gynecology and Obstetrics stage (P = .018) and a trend for poor response to chemotherapy at first disease recurrence (P = .051). Higher phospho-mammalian target of rapamycin protein expression in effusions by immunohistochemistry was associated with poor progression-free survival for patients with postchemotherapy effusions (P = .005). Phospho-mammalian target of rapamycin was an independent predictor of poor progression-free survival for patients with postchemotherapy effusions (P = .03). The association between activated AKT and mammalian target of rapamycin expression and clinicopathologic parameters of aggressive disease, including shorter patient survival, provides further evidence regarding the central role of this signaling pathway in ovarian carcinoma.

Phosphatidylserine cell surface exposure during apoptosis can be detected by its binding to the protein annexin-V. We investigated annexin-V expression in 76 ovarian carcinoma effusions using flow cytometry. Results were analyzed for association with clinicopathologic parameters and survival. Annexin-V expression was additionally compared with the previously studied apoptotic markers cleaved caspase-3, cleaved caspase-8, and deoxyuridine triphosphate (dUTP) incorporation into DNA fragments. Annexin-V was expressed in all specimens and was more frequently detected compared with cleaved caspases and dUTP incorporation (P < .001). Annexin-V expression was higher in grade 3 vs grades 1 and 2 tumors (P = .014). A higher percentage of annexin-V–expressing cells in postchemotherapy specimens was associated with poor overall (P = .005) and progression-free (P = .013) survival. We present the first evidence of annexin-V expression in ovarian carcinoma effusions. The higher annexin-V expression compared with other apoptosis parameters and its association with high-grade disease and poor survival in postchemotherapy patients suggest a role in cell survival rather than apoptosis in effusions.

We analyzed the diagnostic role of claudins in effusion cytology in 325 effusions, including 218 ovarian, 49 breast, 15 cervical or endometrial, 10 gastrointestinal, and 8 lung adenocarcinomas and 25 malignant mesotheliomas (MMs). Specimens were analyzed for claudin-1 and claudin-3 expression using immunohistochemical analysis. Ovarian and breast adenocarcinoma were further analyzed for claudin-7 expression. Claudin-1 expression was most frequent in ovarian and cervical or endometrial adenocarcinoma compared with other adenocarcinomas and MMs ( P < .001). Claudin-3 expression was comparable in adenocarcinomas of different origin but was absent in MMs ( P < .001). Reactive mesothelial cells rarely expressed claudins. Claudin-7 expression was higher in ovarian than in breast adenocarcinoma ( P < .001). Our data suggest that expression of claudin-3 or claudin-7 is specific for adenocarcinoma and rules out the diagnosis of cells as mesothelial and that absence of claudin-1 expression excludes ovarian carcinoma as the possible origin of metastatic adenocarcinoma. Claudins may, therefore, be of diagnostic value in effusion cytology.

Background The objective of this study was to investigate the expression and clinical role of 14 genes previously shown to be associated with chemotherapy response and/or progression-free survival in a smaller series of ovarian serous carcinoma effusions. Methods Advanced-stage serous ovarian carcinoma effusions (n = 150) were analyzed for mRNA expression of AKR1C1, ABCA4, ABCA13, ABCB10, BIRC6, CASP9, CIAPIN1, FAS, MGMT, MUTYH, POLH, SRC, TBRKB and XPA using quantitative real-time PCR. mRNA expression was studied for association with clinicopathologic parameters, including chemotherapy response and survival. Results ABCA4 mRNA expression was significantly related to better (complete) chemotherapy response at diagnosis in the entire cohort (p = 0.018), whereas higher POLH mRNA levels were significantly related to better chemoresponse at diagnosis in analysis to 58 patients with pre-chemotherapy effusions treated with standard chemotherapy (carboplatin + paclitaxel; p = 0.023). In univariate survival analysis for patients with pre-chemotherapy effusions (n = 77), CIAPIN1 mRNA expression was significantly related to shorter overall (p = 0.007) and progression-free (p = 0.038) survival, whereas ABCA13 mRNA expression was significantly related to shorter OS (p = 0.024). Higher CIAPIN1 mRNA expression was an independent marker of poor overall survival in Cox multivariate analysis (p = 0.044). Conclusions Our data identify ABCA4 and POLH as markers of better chemotherapy response in metastatic serous carcinoma. CIAPIN1 and ABCA13 may be novel markers of poor outcome in pre-chemotherapy serous carcinoma effusions.

We studied the clinical role of leukocyte infiltration and chemokine receptor expression in ovarian carcinoma effusions. Expression of leukocyte markers (CD3, CD4, CD8, CD4/CD8 ratio, CD16, CD19, and CD14) and chemokine receptors (CXCR1, CXCR4, CCR2, CCR5, and CCR7) was studied in 73 effusions by using flow cytometry. CXCR4, CCR5, and CCR7 were expressed abundantly on leukocytes, but all receptors were expressed rarely on cancer cells. The presence of natural killer cells (P = .042) and International Federation of Gynecology and Obstetrics (FIGO) stage IV disease (P = .024) predicted worse overall survival (OS). A higher percentage of CD19+ cells (P = .015) and stage IV disease (P = .008) predicted poor survival for patients with postchemotherapy effusions. Only FIGO stage retained significance as a predictor of OS (P = .035) in multivariate analysis. Chemokine receptors are expressed widely on leukocytes but rarely on carcinoma cells in ovarian carcinoma effusions, arguing against an autocrine chemokine pathway in this malignancy. Immune response parameters in ovarian cancer effusions are weak predictors of outcome.

Objective Wee1-like kinase (Wee1) is a tyrosine kinase which negatively regulates entry into mitosis at the G2 to M-phase transition and has a role in inhibition of unscheduled DNA replication in S-phase. The present study investigated the clinical role of Wee1 in advanced-stage (FIGO III–IV) ovarian serous carcinoma. Methods Wee1 protein expression was analyzed in 287 effusions using immunohistochemistry. Expression was analyzed for association with clinicopathologic parameters, including survival. Forty-five effusions were additionally studied using Western blotting. Wee1 was further silenced in SKOV3 and OVCAR8 cells by siRNA knockdown and proliferation was assessed. Results Nuclear expression of Wee1 in tumor cells was observed in 265/287 (92%) and 45/45 (100%) effusions by immunohistochemistry and Western blotting, respectively. Wee1 expression by immunohistochemistry was significantly higher in post-chemotherapy disease recurrence compared to pre-chemotherapy effusions obtained at diagnosis (p=0.002). Wee1 silencing in SKOV3 and OVCAR8 cells reduced proliferation. In univariate survival analysis of the entire cohort, a trend was observed between high (>25% of cells) Wee1 expression and poor overall survival (p=0.083). Survival analysis for 109 patients with post-chemotherapy effusions showed significant association between Wee1 expression and poor overall survival (p=0.004), a finding which retained its independent prognostic role in Cox multivariate analysis (p=0.003). Conclusions Wee1 is frequently expressed in ovarian serous carcinoma effusions, and its expression is significantly higher following exposure to chemotherapy. The present study is the first to report that Wee1 is an independent prognostic marker in serous ovarian carcinoma.

Leiomyosarcoma (LMS) is the most common uterine sarcoma. Although the disease is relatively rare, it is responsible for considerable mortality due to frequent metastasis and chemoresistance. The molecular events related to LMS metastasis are unknown to date. The present study compared the global gene expression patterns of primary uterine LMSs and LMS metastases. Gene expression profiles of 13 primary and 15 metastatic uterine LMSs were analyzed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. To identify differently expressed genes between primary and metastatic tumors, we performed one-way analysis of variance with Benjamini-Hochberg correction. This led to identification of 203 unique probes that were significantly differentially expressed in the 2 tumor groups by greater than 1.58-fold with P < .01, of which 94 and 109 were overexpressed in primary and metastatic LMSs, respectively. Genes overexpressed in primary uterine LMSs included OSTN, NLGN4X, NLGN1, SLITRK4, MASP1, XRN2, ASS1, RORB, HRASLS, and TSPAN7. Genes overexpressed in LMS metastases included TNNT1, FOLR3, TDO2, CRYM, GJA1, TSPAN10, THBS1, SGK1, SHMT1, EGR2, and AGT. Quantitative real-time PCR confirmed significant anatomical site-related differences in FOLR3, OSTN, and NLGN4X levels; and immunohistochemistry showed significant differences in TDO2 expression. Gene expression profiling differentiates primary uterine LMSs from LMS metastases. The molecular signatures unique to primary and metastatic LMSs may aid in understanding tumor progression in this cancer and in providing a molecular basis for prognostic studies and therapeutic target discovery.

HOX proteins are key transcription factors in embryogenesis. HOXB5 and HOXB8 were previously shown to be overexpressed in ovarian/primary peritoneal serous carcinoma compared to breast carcinoma using gene expression arrays. The present study investigated the clinical role of HOXB5 and HOXB8 in advanced-stage (FIGO III-IV) ovarian serous carcinoma.HOXB5 and HOXB8 protein expression was analyzed in 286 effusions and 76 patient-matched solid lesions (27 primary carcinomas, 49 metastases) using immunohistochemistry. Expression was analyzed for association with clinicopathologic parameters, including survival.Cytoplasmic HOXB5 protein was detected in 268/286 (94%) effusions. HOXB8 was expressed at both the cytoplasm (252/286; 88%) and nucleus (131/286; 46%) of carcinoma cells. Cytoplasmic HOXB5, cytoplasmic HOXB8 and nuclear HOXB8 were found in 56/76 (74%), 76/76 (100%) and 30/76 (39%) solid lesions, respectively, with significantly higher HOXB5 expression in effusions (p=0.002) and higher cytoplasmic HOXB8 in solid lesions (p0.001). HOXB5 expression was higher in post-chemotherapy disease recurrence effusions compared to pre-chemotherapy effusions tapped at diagnosis (p=0.04). In univariate survival analysis of the effusion cohort, higher expression of cytoplasmic HOXB8 was associated with significantly shorter progression-free survival (p=0.033), whereas higher nuclear HOXB8 expression was associated with significantly shorter overall survival in analysis limited to patients with post-chemotherapy effusions (p=0.036). Neither finding was independent prognostic factor in Cox multivariate analysis.HOXB5 and HOXB8 are frequently expressed in ovarian serous carcinoma, with anatomic site-related differences for cytoplasmic staining. HOXB5 may be affected by chemotherapy in effusions. HOXB8 expression is associated with shorter survival in metastatic serous carcinoma.

Endometrial stromal sarcoma (ESS) and leiomyosarcoma (LMS) are the two most common uterine sarcomas, but both are rare tumors. The aim of the present study was to compare the global gene expression patterns of ESS and LMS.Gene expression profiles of 7 ESS and 13 LMS were analyzed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry.Unsupervised hierarchical clustering using all 54,675 genes in the array separated ESS from LMS samples. We identified 549 unique probes that were significantly differentially expressed in the two malignancies by greater than 2-fold with 1% FDR cutoff using one-way ANOVA with Benjamini-Hochberg correction, of which 336 and 213 were overexpressed in ESS and LMS, respectively. Genes overexpressed in ESS included SLC7A10, EFNB3, CCND2, ECEL1, ITM2A, NPW, PLAG1 and GCGR. Genes overexpressed in LMS included CDKN2A, FABP3, TAGLN, JPH2, GEM, NAV2 and RAB23. The top 100 genes overexpressed in LMS included those coding for myosin light chain and caldesmon, but not the genes coding for desmin or actin. CD10 was not overexpressed in ESS. Results for selected genes were validated by quantitative real-time PCR and immunohistochemistry.We present the first study in which gene expression profiling was shown to distinguish between ESS and LMS. The molecular signatures unique to each of these malignancies may aid in expanding the diagnostic battery for their differentiation, and may provide a molecular basis for prognostic studies and therapeutic target discovery.

The ANPEP, AZGP1, and SPDEF genes were previously found to be overexpressed in breast compared to ovarian carcinoma effusions. The present study validated this finding in a larger cohort consisting of both primary and metastatic tumors. ANPEP, AZGP1, and SPDEF mRNA expression was investigated in 83 breast carcinomas (57 primary carcinomas and 26 effusions) and 40 ovarian carcinomas (20 primary carcinomas and 20 effusions) using qPCR. ANPEP protein expression was immunohistochemically analyzed in 53 breast carcinoma effusions and patient-matched primary carcinomas (n = 25) and lymph node metastases (n = 16). mRNA and protein levels were studied for association with tumor type and anatomic site, and for clinical role in breast carcinoma. AZGP1 and SPDEF mRNA was overexpressed in breast compared to ovarian carcinoma (both p < 0.001). AZGP1 mRNA was overexpressed in primary breast carcinoma compared to effusions (p < 0.001), with opposite findings for ANPEP (p = 0.044). AZGP1 mRNA expression correlated with positive ER status (p = 0.032) and grade 1 histology (p = 0.011), whereas SPDEF mRNA levels were associated with positive ER (p = 0.002) and PR (p = 0.013) status and tamoxifen treatment (p = 0.004). ANPEP protein expression was higher in breast carcinoma effusions compared to primary tumors and lymph node metastases (both p = 0.001). ANPEP, AZGP1, and SPDEF levels were unrelated to disease-free or overall survival. This is the first study documenting ANPEP, AZGP1, and SPDEF expression in breast carcinoma effusions. AZGP1 and SPDEF may be novel molecular markers for the differentiation of breast from ovarian carcinoma. ANPEP may be involved in breast carcinoma progression in view of its overexpression in effusions compared to solid specimens.

The objective of this study is to analyze the expression and clinical role of integrin-linked kinase (ILK), α-parvin, β-parvin and migfilin in advanced-stage serous ovarian carcinoma.Expression of these 4 proteins was investigated in 205 effusions and in 94 patient-matched solid lesions (33 primary tumors and 61 solid metastases) using immunohistochemistry. Protein expression was analyzed for association with clinicopathologic parameters and survival.ILK, α-parvin, β-parvin and migfilin were expressed in tumor cells in 53%, 2%, 28% and 53% of effusions and 57%, 20%, 83% and 25% of solid lesions, respectively. Statistical analysis showed significantly higher α-parvin and β-parvin expression in primary carcinomas (p=0.02 and p=0.001, respectively) and solid metastases (p=0.001 and p0.001, respectively), compared to effusions, with opposite findings for migfilin (p=0.006 and p=0.008 for primary carcinomas and solid metastases, respectively). ILK expression was comparable at all anatomic sites. β-Parvin expression in effusions was associated with better response to chemotherapy at diagnosis (p=0.014), with no other significant association with clinicopathologic parameters or survival. Expression in primary tumors and solid metastases was similarly unrelated to clinicopathologic parameters or survival.This study provides further evidence to our previous observations that the adhesion profile of ovarian serous carcinoma cells in effusions differs from their counterparts in primary carcinomas and solid metastases. β-Parvin may be a novel marker of chemoresponse in metastatic ovarian carcinoma.

Osteopontin, a soluble protein present in all body fluids, is involved in signaling pathways related to adhesion and extracellular matrix interactions, affecting multiple cellular functions, including inflammation, angiogenesis, and tumor metastasis. We studied osteopontin expression by immunohistochemistry and its clinical relevance in 170 effusions (140 peritoneal, 30 pleural) from women with advanced-stage ovarian carcinoma. Carcinoma cells expressed osteopontin in 126 (74%) of 170 effusions. Osteopontin expression was more frequent in effusions from patients with high-grade tumors (P = .036) but was significantly associated with better debulking at primary surgery (P = .019) and complete response to chemotherapy at diagnosis (P = .021). Osteopontin expression was positively associated to that of the previously studied nuclear factor κB inhibitor IκB (P = .019) and negatively related to expression of the inhibitor of apoptosis family member XIAP (P = .008) and the angiogenic marker endoglin (CD105; P = .018). In univariate survival analysis, the presence of osteopontin in carcinoma cells in primary diagnosis prechemotherapy effusions was associated with longer progression-free survival (P = .037), a finding that did not retain its significance in multivariate Cox analysis. This study demonstrates that osteopontin is frequently expressed in ovarian carcinoma effusions. However, its presence in tumor cells at this anatomical site is unexpectedly associated with less aggressive clinical course, suggesting different and yet undefined biological roles for this protein in serous effusions.

Ovarian/primary peritoneal carcinoma and breast carcinoma are the gynaecological cancers that most frequently involve the serosal cavities. With the objective of improving on the limited diagnostic panel currently available for the differential diagnosis of these two malignancies, as well as to define tumour-specific biological targets, we compared their global gene expression patterns. Gene expression profiles of 10 serous ovarian/peritoneal and eight ductal breast carcinoma effusions were analysed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. Unsupervised hierarchical clustering using all 54,675 genes in the array separated ovarian from breast carcinoma samples. We identified 288 unique probes that were significantly differentially expressed in the two cancers by greater than 3.5-fold, of which 81 and 207 were overexpressed in breast and ovarian/peritoneal carcinoma, respectively. SAM analysis identified 1078 differentially expressed probes with false discovery rate less than 0.05. Genes overexpressed in breast carcinoma included TFF1, TFF3, FOXA1, CA12, GATA3, SDC1, PITX1, TH, EHFD1, EFEMP1, TOB1 and KLF2. Genes overexpressed in ovarian/peritoneal carcinoma included SPON1, RBP1, MFGE8, TM4SF12, MMP7, KLK5/6/7, FOLR1/3, PAX8, APOL2 and NRCAM. The differential expression of 14 genes was validated by quantitative real-time PCR, and differences in 5 gene products were confirmed by immunohistochemistry. Expression profiling distinguishes ovarian/peritoneal carcinoma from breast carcinoma and identifies genes that are differentially expressed in these two tumour types. The molecular signatures unique to these cancers may facilitate their differential diagnosis and may provide a molecular basis for therapeutic target discovery.

We recently reported on higher expression of the insulin-like growth factor pathway genes IGF-II and IGFBP3 in serous ovarian/peritoneal carcinoma compared to malignant peritoneal mesothelioma. The present study analyzed the diagnostic and clinical role of these proteins in serous effusions. Effusions (n = 327), including 294 carcinomas (205 ovarian, 48 breast, 17 cervical/endometrial, 12 lung, 12 gastrointestinal/genitourinary) and 33 malignant mesotheliomas, were immunostained for insulin-like growth factor-II and insulin-like growth factor binding protein-3. Surgical ovarian carcinoma (n = 124) and peritoneal mesothelioma (n = 18) specimens were additionally studied. Insulin-like growth factor binding protein-3 levels were measured in 148 effusion supernatants (114 ovarian carcinomas, 18 breast carcinomas, 16 mesotheliomas) using enzyme-linked immunosorbent assay. Insulin-like growth factor binding protein-3 promoter methylation was analyzed in 11 ovarian carcinoma effusions. Insulin-like growth factor binding protein-3 (P = .002) and insulin-like growth factor-II (P < .001) expression by immunohistochemistry was significantly higher in carcinomas compared to mesotheliomas, with diagnostic sensitivity of 77% and 70% and specificity of 55% and 70%, respectively. In surgical specimens, insulin-like growth factor binding protein-3 expression was higher in ovarian carcinomas compared to peritoneal mesotheliomas (P = .007), whereas insulin-like growth factor-II expression was comparable (P = .505). Insulin-like growth factor binding protein-3 levels by enzyme-linked immunosorbent assay were comparable in the 3 analyzed cancer types. Insulin-like growth factor binding protein-3 promoter methylation was found in 6 of 11 effusions. High insulin-like growth factor binding protein-3 expression in prechemotherapy and high insulin-like growth factor-II expression in postchemotherapy ovarian carcinoma effusions correlated with poor overall survival (P = .031 and P = .024, respectively). Insulin-like growth factor-II expression in postchemotherapy effusions was an independent prognostic factor in Cox multivariate analysis (P = .04). In conclusion, insulin-like growth factor-II and insulin-like growth factor binding protein-3 are more frequently expressed in metastatic carcinomas compared to mesothelioma in effusions but are less specific than currently used markers. Insulin-like growth factor-II and insulin-like growth factor binding protein-3 may be novel prognostic markers in metastatic ovarian carcinoma.

Tumor progression and treatment failure in ovarian carcinoma are frequently associated with metastasis to effusions. The present study analyzed the expression and clinical role of nuclear factor-kappaB p65, nuclear factor-kappaB inhibitor alpha, and parameters of apoptosis in serous carcinoma. Cleaved caspase-3 and caspase-8 levels and deoxyuridine triphosphate incorporation were measured in 65 effusions using flow cytometry. Effusions (n = 209) and corresponding primary carcinomas and solid metastases (n = 114) were immunohistochemically analyzed for nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expression. Effusions (n = 75) were further analyzed for nuclear factor-kappaB phospho-p65 (Ser536) levels using immunoblotting. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. Caspase cleavage and deoxyuridine triphosphate incorporation were limited to less than 10% of cells in most effusions. Nuclear factor-kappaB p65 expression was frequently detected at all anatomic sites, with less frequent cytoplasmic nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expressions. Immunoblotting showed nuclear factor-kappaB p65 phosphorylation in 72 (96%) of 75 effusions. Higher than median cleaved caspase-3 levels correlated with improved overall and progression-free survival in univariate analysis of all patients (P = .024 and P = .046, respectively) and of those with postchemotherapy effusions (P = .042 and P = .036, respectively). Cleaved caspase-3 expression was an independent predictor of longer progression-free survival for patients with postchemotherapy effusions (P = .029). Nuclear factor-kappaB p65 expression correlated with poor progression-free survival for all patients (P = .048) and for those with postchemotherapy effusions (P = .025). Ovarian carcinoma cells in effusions undergo little apoptosis, but high levels of cleaved caspase-3 are associated with improved survival. Nuclear factor-kappaB p65 is frequently expressed in advanced-stage serous ovarian carcinoma, and its nuclear localization is associated with poor progression-free survival.

Flow cytometry (FCM) immunophenotyping is frequently used as an ancillary technique for the diagnosis of hematological malignancies or for measurement of DNA content. In recent years, we applied FCM to the diagnosis of metastatic adenocarcinoma and malignant mesothelioma in effusions. We established a panel of antibodies that allows for rapid and effective differentiation between epithelial cells, mesothelial cells, and leukocytes. FCM was subsequently used for quantitative analysis of integrin subunits. Recently, we studied different parameters of the immune response, including HLA molecules and chemokine receptors, using this method. Our data suggest that FCM is an effective method for the characterization of cancer cells in clinical effusion specimens in both the diagnostic and research setting, and that this method is comparable to immunohistochemistry in terms of sensitivity and specificity, with the additional advantage of providing quantitative data. This review discusses previous work in this area and the future potential of this method in the characterization of tumor cells in serous effusions.

Claudins are tight junction proteins that are highly expressed in ovarian carcinoma (OC). The objective of this study was to analyze the anatomic site-related expression and clinical role of claudins in OC. Effusions (n = 218), corresponding primary tumors (n = 81), and solid metastases (n = 164) (total = 463 tumors) were immunostained for claudin-1, claudin-3, claudin-4, and claudin-7. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. All 4 claudins were expressed in >85% of tumors at all anatomic sites. However, staining extent of all except claudin-4 was significantly higher in effusions compared with both primary carcinomas and solid metastases (P < .001). In univariate survival analysis of the entire cohort, higher claudin-3 (P = .038) and claudin-7 (P = .035) expression in effusions correlated with shorter overall survival (OS), with similar results for claudin-7 in analysis of progression-free survival (P = .026). In separate analysis for patients with prechemotherapy effusions, higher claudin-7 expression correlated with shorter OS (P = .045). For patients with postchemotherapy effusions, higher claudin-1 (P = .018) and claudin-3 (P = .009) expression correlated with shorter OS. In multivariate survival analysis of the entire cohort, claudin-7 expression was an independent predictor of poor progression-free survival (P = .017). Claudin-3 independently predicted poor OS for patients with postchemotherapy effusions (P = .012). With the exception of claudin-4, claudins are upregulated in OC effusions compared with solid tumors, in agreement with our previous data for cadherins and integrins in this cancer type, suggesting a prosurvival role for these surface molecules. Claudin-3 and claudin-7 expression in effusions independently predicts poor survival in OC.

Background: Data regarding the role of flow cytometry (FCM) in the characterization of malignant effusions are limited to date. In the present study, we optimized the conditions for FCM immunphenotyping of effusions using a four-color analysis and investigated aspects related to the advantages and limitations of this method in this setting. Methods: FCM analysis optimization for the study of epithelial cells was undertaken using five carcinoma cell lines, and subsequently applied to malignant pleural and peritoneal effusions using antibodies against epithelial and mesothelial markers (Ber-EP4 and EMA), CD138, and integrin subunits. FCM of frozen versus fresh specimens and the performance of FCM compared to immunhistochemistry were evaluated. Results: FCM optimization was achieved and applied to clinical specimens, with resulting detection of epithelial markers and adhesion molecules on cancer cells. Frozen clinical specimens and cell lines showed reduced CD138 expression compared to fresh specimens, with conservation of the remaining epitopes. FCM generally showed comparable performance to immunhistochemistry. Conclusions: FCM is an effective method for characterization of cancer cells in clinical effusion specimens in both the diagnostic and research setting, and is comparable to immunhistochemistry in terms of sensitivity and specificity, with the additional advantage of providing quantitative data. The majority of epitopes are conserved in frozen cells, but a minority may be lost, suggesting that the thorough testing of each antibody in both conditions is mandatory. q 2007 Clinical Cytometry Society Key terms: flow cytometry; effusions; carcinorma; mesothelioma; immunhistochemistry

Leiomyosarcoma (LMS) is the most common uterine sarcoma, but is a rare cancer. The genetic events related to LMS metastasis are unknown to date. The aim of the present study was to compare the global gene expression patterns of primary uterine LMS and LMS metastases in order to identify potential biological targets. Gene expression profiles of 13 primary uterine LMS and 15 LMS metastases were analyzed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. Unsupervised hierarchical clustering using all 54,675 genes in the array separated primary from metastatic LMS samples. We identified 203 unique probes that were significantly differentially expressed in the two tumor groups by greater than 2-fold with 1% FDR cutoff using one-way ANOVA with Benjamini-Hochberg correction, of which 94 and 109 were overexpressed in primary and metastatic LMS, respectively. Genes overexpressed in primary uterine LMS included OSTN, NLGN4X, NLGN1, SLITRK4, MASP1, XRN2, ASS1, RORB, HRASLS and TSPAN7. Genes overexpressed in LMS metastases included FOLR3, TDO2, CRYM, GJA1, TSPAN10, THBS1, SGK1, SHMT1, EGR2 and AGT. Results for selected genes were validated by quantitative real-time PCR and immunohistochemistry. We present the first study in which gene expression profiling was shown to distinguish between primary uterine LMS and LMS metastases. The molecular signatures unique to primary and metastatic LMS may aid in understanding tumor progression in this cancer and in providing a molecular basis for prognostic studies and therapeutic target discovery. Citation Format: Ben Davidson, Vera Maria Abeler, Mette Førsund, Arild Holth, Ie-Ming Shih, Gunnar B. Kristensen, Li Chen, Yanqin Yang, Tian-Li Wang. Gene expression signatures differentiate primary and metastatic uterine leiomyosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 401. doi:10.1158/1538-7445.AM2013-401