Arild Holth

NK- and B-Cell Infiltration Correlates With Worse Outcome in Metastatic Ovarian Carcinoma

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.

Cleaved caspase-3 and nuclear factor-κB p65 are prognostic factors in metastatic serous 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.

Gene expression signatures differentiate ovarian/peritoneal serous carcinoma from breast carcinoma in 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.

Mammalian target of rapamycin is a biomarker of poor survival in metastatic serous ovarian carcinoma

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.

The diagnostic role of claudins in serous 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.

Gene expression signatures of primary and metastatic uterine leiomyosarcoma

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.

Evaluation of Cell Surface Expression of Phosphatidylserine in Ovarian Carcinoma Effusions Using the Annexin-V/7-AAD Assay: Clinical Relevance and Comparison With Other Apoptosis Parameters

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.

Gene expression signatures differentiate uterine endometrial stromal sarcoma from leiomyosarcoma

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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 clinical role of epithelial-mesenchymal transition and stem cell markers in advanced-stage ovarian serous carcinoma effusions

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.

Flow Cytometric Immunphenotyping of Epithelial Cancer Cells in Effusions—Technical Considerations and Pitfalls

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.