High-grade Serous Carcinoma Effusions Research Articles

Occludin is overexpressed in tubo-ovarian high-grade serous carcinoma compared to mesothelioma and is a marker of tumor progression and chemoresistance.

The objective of this study was to analyze the expression and prognostic role of the tight junction protein occludin in high-grade serous carcinoma (HGSC). Occludin protein expression by immunohistochemistry was analyzed in 602 HGSC (417 effusions, 185 surgical specimens). Expression in mesothelioma (n = 87; 45 effusions, 42 surgical specimens) was studied for comparative purposes. Occludin protein expression was found in 587/602 (98%) HGSC vs. 40/87 (46%) mesotheliomas and was predominantly limited to < 5% of cells in the latter (p < 0.001). Occludin was additionally overexpressed in HGSC effusions compared to surgical specimens (p < 0.001) and was overexpressed in post-chemotherapyeffusionscompared tochemo-naive effusions tapped at diagnosis (p = 0.015). Occludin expression in HGSC surgical specimens was associated with poor chemoresponse (p < 0.001) and primary resistance (p = 0.001). Expression in effusions and surgical specimens was unrelated to survival (p > 0.05). In conclusion, occludin expression is higher in HGSC compared to mesothelioma, and this protein is overexpressed in HGSC effusions, possibly reflecting changes in adhesion related to anchorage-independent growth in this microenvironment. Overexpression in post-chemotherapy compared to chemo-naïve effusions suggest a role in disease progression. Occludin expression in surgical specimens may be related to chemoresistance.

Clinical significance of L1CAM expression in metastatic tubo-ovarian high-grade serous carcinoma

ObjectiveTo analyze the expression and prognostic role of L1CAM in tubo-ovarian high-grade serous carcinoma (HGSC). MethodsL1CAM protein expression by immunohistochemistry was analyzed in 644 HGSC (413 effusions, 231 surgical specimens). Expression was analyzed for association with clinicopathologic parameters and survival. ResultsL1CAM protein expression was found in 401/413 (97%) effusions and 209/231 (90%) surgical specimens, with significantly higher staining extent in effusions (p < 0.001). L1CAM protein expression in effusions was unrelated to clinicopathologic parameters (p > 0.05). In surgical specimens, higher L1CAM expression was significantly related to primary (intrinsic) chemoresistance (p = 0.017). High (>25%) L1CAM expression in HGSC effusions (p = 0.02), older patient age (p = 0.013), FIGO stage IV disease (p < 0.001) and larger residual disease volume (p = 0.001) were significantly associated with shorter overall survival (OS) in univariate analysis. In Cox multivariate analysis, only FIGO stage (p = 0.001) and residual disease volume (p = 0.003) were independent prognosticators of OS. L1CAM expression in effusions was unrelated to progression-free survival (PFS). There was no association between L1CAM expression in surgical specimens and survival. ConclusionL1CAM is overexpressed in HGSC effusions compared to surgical specimens. Its overexpression in effusions is significantly associated with shorter OS, but not independently of established prognostic factors such as FIGO stage and residual disease volume.

Claudin-10 is a new candidate prognostic marker in metastatic high-grade serous carcinoma

The objective of this study was to analyze the expression and prognostic role of the tight junction protein claudin-10 in high-grade serous carcinoma (HGSC). Claudin-10 protein expression by immunohistochemistry was analyzed in 588 HGSC (414 effusions, 174 surgical specimens). Expression in mesotheliomas (n = 97; 47 effusions, 50 surgical specimens) was studied for comparative purposes. CLDN10 mRNA expression by quantitative RT-PCR (qRT-PCR) was analyzed in 40 HGSC effusions. Claudin-10 protein expression was found in 360/588 (61%) HGSC vs. 19/97 (20%) mesotheliomas (p < 0.001), and was higher in HGSC surgical specimens compared to effusions (p < 0.001). qRT-PCR confirmed the presence of CLDN10 mRNA in HGSC effusions. High (> 25%) claudin-10 expression in HGSC effusions was significantly associated with shorter overall survival (OS; p = 0.036) and progression-free survival (PFS; p = 0.045) in univariate analysis, and was an independent prognosticator of OS in multivariate analysis (p = 0.045). In conclusion, claudin-10 protein expression is higher in HGSC compared to mesothelioma, although the diagnostic power of this marker appear to be lesser than other claudin family members. Claudin-10 expression in HGSC effusions is marker of more aggressive disease.

The phosphatase PTPN1/PTP1B is a candidate marker of better chemotherapy response in metastatic high-grade serous carcinoma.

To analyse the expression and clinical role of the phosphatase PTPN1 (PTP1B) in serous effusions. PTPN1 mRNA expression by quantitative RT-PCR was analysed in 83 high-grade serous carcinoma (HGSC) and 15 malignant mesothelioma (MM) effusions. PTP1B and phospho-PTP1B (pPTP1B) protein expression by immunohistochemistry was analysed in 62 HGSC and 44 MM effusions. PTPN1 mRNA (P=.048), PTP1B protein (P=.047) and pPTP1B protein (P<.001) were overexpressed in HGSC compared to MM effusions. PTPN1 mRNA was additionally overexpressed in post-chemotherapy HGSC effusions compared to chemo-naïve effusions (P=.005). However, pPTP1B protein expression was higher in effusions from patients with FIGO stage III compared to stage IV (P=.006), and higher expressions of both PTPN1 mRNA (P=.041) and PTP1B protein (P=.035) in HGSC effusions were associated with better (complete) chemotherapy response at diagnosis. PTPN1 RNA and protein expression was unrelated to survival in HGSC, whereas a trend for shorter overall survival (P=.06) was found for MM patients whose tumours expressed pPTP1B protein. PTPN1 is overexpressed in HGSC compared to MM effusions, and may be a marker of better chemotherapy response in the former. Whether PTPN1 activation is informative of adverse outcome in MM merits further investigation.

Expression of palladin is associated with disease progression in metastatic high-grade serous carcinoma.

To analyse the expression and clinical role of the actin-associated molecule palladin in serous effusions. PALLD mRNA expression was analysed by quantitative reverse transcription polymerase chain reaction in 83 high-grade serous carcinoma (HGSC) effusions. Fifteen malignant mesothelioma (MM) effusions and 18 surgical HGSC specimens from the ovary were studied for comparative purposes. Palladin protein expression by immunohistochemistry was analysed in another series consisting of 261 HGSC effusions. PALLD mRNA was significantly overexpressed in HGSC compared to MM effusions (P<.001). Palladin expression by immunohistochemistry was found in HGSC cells in 106/261 (41%) effusions, most commonly focally (<5% of cells). PALLD expression was additionally higher in ovarian HGSC specimens compared to HGSC effusions (P<.001). However, immunohistochemistry showed only stromal expression of this protein in surgical specimens. PALLD mRNA expression in HGSC effusions was unrelated to clinicopathological parameters, chemotherapy response or survival. Palladin protein expression was higher in post-chemotherapy, mainly disease recurrence, specimens compared to chemo-naïve effusions tapped at diagnosis (P=.018), although it was unrelated to other clinicopathological parameters or survival. PALLD mRNA is overexpressed in HGSC compared to MM effusions, and its protein product is overexpressed in post-chemotherapy compared to pre-chemotherapy HGSC effusions, suggesting upregulation along tumour progression. The presence of this molecule in HGSC effusions, at the mRNA or the protein level, is unrelated to disease outcome.

The Biological and Clinical Role of the Long Non-Coding RNA LOC642852 in Ovarian Carcinoma.

The objective of the present study was to analyze the biological and clinical role of the long non-coding RNA LOC642852 in ovarian carcinoma (OC). LOC642852 expression was analyzed in seven OC cell lines (OVCAR-3, OVCAR-8, OVCA 433, OVCA 429, OC 238, DOV13, ES-2) and 139 high-grade serous carcinoma (HGSC) specimens (85 effusions, 54 surgical specimens). Following LOC642852 knockout (KO) using the CRISPR/Cas9 system, OVCAR-8 HGSC cells were analyzed for spheroid formation, migration, invasion, proliferation, matrix metalloproteinase (MMP) activity, and expression of cell signaling proteins. OVCAR-8 cells with LOC642852 KO were significantly less motile and less invasive compared to controls, with no differences in spheroid formation, proliferation, or matrix metalloproteinase (MMP) activity. Total Akt and Erk levels were comparable in controls and KO cells, but their phosphorylation was significantly increased in the latter. In clinical specimens, LOC642852 was overexpressed in ovarian tumors and omental/peritoneal metastases compared to effusion specimens (p = 0.013). A non-significant trend for shorter overall (p = 0.109) and progression-free (p = 0.056) survival was observed in patients with HGSC effusions with high LOC642852 levels. Bioinformatics analysis showed potential roles for LOC642852 as part of the TLE3/miR-221-3p ceRNA network and in relation to the FGFR3 protein. In conclusion, LOC642852 inactivation via CRISPR/Cas9 affects cell signaling, motility, and invasion in HGSC cells. LOC642852 is differentially expressed in HGSC cells at different anatomical sites. Its potential role in regulating the TLE3/miR-221-3p ceRNA network and FGFR3 merits further research.

Death domain-associated protein (DAXX) expression is associated with poor survival in metastatic high-grade serous carcinoma

The objective of this study was to analyze the expression and clinical role of mitosis regulators α-thalassemia/mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX) in metastatic high-grade serous carcinoma (HGSC). ATRX and DAXX protein expression by immunohistochemistry was analyzed in 400 HGSC effusions. DAXX expression was additionally studied in 15 cancer cell lines, including 4 ovarian carcinoma lines, and in 81 of the 400 HGSC effusions using Western blotting. ATRX and DAXX were expressed in HGSC cells in 386/400 (96%) and 348/400 (87%) effusions, respectively. Western blotting showed DAXX expression in all 15 cell lines and in 70/81 (86%) HGSC effusions. DAXX expression by immunohistochemistry was higher in pleural compared to peritoneal effusions (p = 0.006) and in post-chemotherapy compared to pre-chemotherapy effusions (p = 0.004), and its expression was significantly associated with poor overall survival in univariate of the entire cohort (p = 0.014), as well as analysis limited to chemo-naïve effusions tapped at diagnosis (p = 0.038). The former association retained its prognostic role in Cox multivariate survival analysis (p = 0.011). ATRX expression was unrelated to clinicopathologic parameters or survival. In conclusion, DAXX is associated with disease progression and could be a prognostic marker in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

Expression of the cancer stem cell marker SSEA1 is associated with poor survival in metastatic high-grade serous carcinoma

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 (< 5% of cells). SSEA1 was overexpressed in post-chemotherapy disease recurrence specimens compared with chemo-naïve HGSC effusions tapped at diagnosis (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.

Expression of Wnt pathway molecules is associated with disease outcome in metastatic high-grade serous carcinoma

The objective of this study was to analyze the expression and clinical role of Wnt pathway molecules in metastatic high-grade serous carcinoma (HGSC). mRNA expression by qPCR of 20 molecules related to Wnt signaling (WNT1, WNT2, WNT3, WNT4, WNT5A, WNT6, WNT7, WNT11, FZD1, FZD4, FZD5, FZD6, FZD7, FZD8, FZD10, LRP5, LRP6, DKK, CCND, RUNX2) was analyzed in 87 HGSC effusions. Thirty-nine surgical specimens (19 ovarian, 20 from otherintra-abdominal sites) were analyzed for comparative purposes. Protein expression of YAP and LRP and their phosphorylated forms by western blotting were analyzed in 52 tumors. Significant differences in mRNA expression as a function of the anatomic site were observed for WNT3 (p = 0.005), WNT5A (p = 0.008), WNT7 (p < 0.001), FRZ5 (p = 0.04), and FRZ6 (p < 0.001). YAP and LRP and their phosphorylated forms were detected in HGSC specimens. FZD10 was overexpressed in effusions from patients who had complete response to chemotherapy compared with those with less favorable response (p = 0.037). WNT4 (p = 0.005), WNT7 (p = 0.047), RUNX2 (p = 0.038), LRP5 (p = 0.022), LRP6 (p = 0.011), FZD6 (p = 0.036), FZD7 (p = 0.004), and FZD10 (p = 0.015) levels were inversely related to primary chemoresistance. High FZD5 levels in pre-chemotherapy effusions tapped at diagnosis and high WNT2 levels in post-chemotherapy disease recurrence effusions were related to shorter overall survival (p = 0.018 and p = 0.011, respectively), whereas high RUNX2 (p = 0.031) and FZD1 (p = 0.029) in post-chemotherapy effusions were associated with longer overall survival. In multivariate analysis of post-chemotherapy cases, WNT2 (p = 0.002), RUNX2 (p = 0.017), FZD1 (p = 0.036), and FZD4 (p = 0.013) were independent prognosticators. In conclusion, expression of Wnt pathway molecules is anatomic site dependent. In HGSC effusions, it is informative of chemoresponse and survival.

SOX2 and SOX9 are markers of clinically aggressive disease in metastatic high-grade serous carcinoma

ObjectiveThe aim of this study was to analyze the expression, biological role and clinical relevance of cancer stem cell markers in high-grade serous carcinoma (HGSC). MethodsmRNA expression by qRT-PCR of NANOG, OCT4, SOX2, SOX4, SOX9, LIN28A and LIN28B was analyzed in 134 HGSC specimens (84 effusions, 50 surgical specimens). Nanog, OCT3/4, SOX2 and SOX9 protein expression by immunohistochemistry was analyzed in 52 HGSC effusions. Nanog protein expression in exosomes from 80 HGSC effusions was studied by Western Blotting. OVCAR3 cells underwent CRISPR/Cas9 Nanog knockout (KO), and the effect of Nanog KO on migration, invasion, proliferation and proteolytic activity was analyzed in OVCAR3 and OVCAR8 cells. ResultsOCT4 mRNA was overexpressed in effusions compared to solid specimens (p = 0.046), whereas SOX9 was overexpressed in the ovarian tumors compared to effusions and solid metastases (p = 0.003). Higher SOX2 and SOX9 expression was associated with primary (intrinsic) chemoresistance (p = 0.009 and p = 0.02, respectively). Higher SOX9 levels were associated with shorter overall survival in univariate (p = 0.04) and multivariate (p = 0.049) analysis. OCT3/4, SOX2 and SOX9 proteins were found in HGSC cells, whereas Nanog was detected only in exosomes. Higher SOX2 protein expression was associated with shorter overall survival in univariate analysis (p = 0.049). OVCAR cells exposed to OVCAR3 NANOG KO exosomes had reduced migration, invasion and MMP9 activity. ConclusionsSOX2 and SOX9 mRNA levels in HGSC effusions may be markers of clinically aggressive disease. Nanog is secreted in HGSC exosomes in effusions and modulates tumor-promoting cellular processes in vitro.

Expression and clinical role of the dipeptidyl peptidases DPP8 and DPP9 in ovarian carcinoma

Dipeptidyl peptidase 9 (DPP9) was recently identified as fusion gene in ovarian high-grade serous carcinoma (HGSC). The aim of this study was to analyze the expression and clinical relevance of DPP8 and DPP9 in ovarian carcinoma, with focus on HGSC. mRNA expression by qRT-PCR of DPP8 and DPP9 was analyzed in 232 carcinomas, including 114 effusions and 118surgical specimens (89 ovarian, 29 solid metastases). DPP8 and DPP9 protein expression was analyzed in 92 effusions. DPP8 and DPP9 mRNA was overexpressed in effusions compared to solid lesions in analysis of all histotypes (p < 0.001 both), as well as in analysis limited to HGSC (p < 0.001 for DPP9, p = 0.002 for DPP8). DPP9 mRNA was additionally overexpressed in HGSC compared to other histotypes (p = 0.021). DPP8 and DPP9 protein was expressed in carcinoma cells in 31/92 (37%) and 81/92 (88%) effusions, respectively. DPP8 protein expression in HGSC effusions was significantly related to better (complete) chemoresponse at diagnosis (p = 0.005). DPP8 and DPP9 mRNA and protein expression was unrelated to survival in analysis of the entire effusion cohort. However, higher DPP9 mRNA levels were significantly related to longer overall survival in pre-chemotherapy effusions (p = 0.049). In conclusion, DPP8 and DPP9 mRNA is frequently expressed in ovarian carcinoma, whereas DPP9 is more frequently expressed at the protein level. DPP8 and DPP9 may be related to lessaggressive disease in advanced-stage HGSC.

Expression, activation and clinical relevance of CHK1 and CHK2 in metastatic high-grade serous carcinoma

ObjectiveTo analyze the expression and clinical role of CHK1 and CHK2 in metastatic high-grade serous carcinoma (HGSC). MethodsHGSC effusions (n = 335; 280 peritoneal, 55 pleural) were analyzed for protein expression of total CHK1 and its phosphorylated forms p-ser317 and p-ser296, as well as total CHK2 and its phosphorylated form p-thr68 using immunohistochemistry. Expression was analyzed for association with clinicopathologic parameters, including chemotherapy response, and survival. ResultsCarcinoma cells stained positive, predominantly at the nuclei, in the majority of cases (range 83–100% for the five antibodies), while expression in reactive mesothelial cells and tumor-associated macrophages was more variable. Total CHK1 (p = 0.037), p-CHK1ser317 (p = 0.001), p-CHK1ser296 (p = 0.002) and p-CHK2thr68 (p < 0.001) expression was significantly higher in post-chemotherapy disease recurrence compared to pre-chemotherapy effusions obtained at diagnosis. CHK1, p-CHK1ser296, p-CHK2thr68 and p-CHK1ser317 nuclear expression was positively related to expression of the checkpoint regulator WEE1, previously studied in this cohort (p = 0.003, p = 0.013, p = 0.001 and p = 0.01, respectively). Higher total CHK1 (p = 0.007), p-CHK1ser317 (p = 0.004), CHK2 (p = 0.01) and p-CHK2thr68 (p = 0.048) expression was significantly related to shorter overall survival in univariate analysis, and CHK1ser317 was an independent prognostic marker in multivariate analysis (p = 0.025). Higher p-CHK1ser317 (p = 0.03) and CHK2 (p = 0.034) expression was additionally associated with poor progression-free survival. ConclusionsCHK1 and CHK2 and their activated forms are frequently expressed in HGSC effusions, with higher expression following exposure to chemotherapy, and their expression is related to survival.

Expression of 14-3-3 sigma and eta proteins is unrelated to survival in metastatic high-grade serous carcinoma.

The objective of this study was to analyze the expression and clinical role of 14-3-3 family proteins in high-grade serous carcinoma (HGSC). Protein expression of 14-3-3 sigma (14-3-3σ) and 14-3-3 eta (14-3-3η) by immunohistochemistry was studied in 298 HGSC specimens (249 peritoneal, 49 pleural) and was analyzed for association with clinicopathologic parameters, chemoresponse and survival. The 14-3-3σ protein was diffusely (>75% of cells) expressed in 100% of carcinomas in analysis of a pilot series and was therefore not further analyzed. The 14-3-3η protein was expressed to a variable extent in 260/298 (87%) effusions. Higher 14-3-3η protein expression was significantly related to higher CA 125 levels at diagnosis (p=0.004), but was unrelated to other clinicopathologic parameters, chemoresponse or survival. Analysis of the association between 14-3-3η and previously studied proteins regulating mitosis showed positive association with class III β-tubulin expression (p=0.025). The present study documents frequent expression of 14-3-3σ and 14-3-3η in HGSC effusions, but does not support a role for these proteins as prognostic markers or predictors of chemotherapy response in metastatic HGSC.

TGFβ splicing and canonical pathway activation in high-grade serous carcinoma

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<0.001 to p=0.05), with anatomic site-dependent expression of splice variants. Conversely, Smad2, p-Smad2, and p-Smad3 were overexpressed in solid specimens (ovarian and peritoneal) compared to those in effusions (p<0.001 for all). In univariate survival analysis, higher TGFβRI variant 1 and TGFβRIII mRNA levels were associated with a trend for shorter overall survival in patients with post-chemotherapy effusions (p=0.066 and p=0.087, respectively), and the latter was an independent prognostic marker in Cox multivariate analysis (p=0.041). Smad3 protein expression was associated with a trend for shorter overall survival in univariate survival analysis (p=0.052). TGFβ receptor splice variant expression is anatomic site-dependent in HGSC. Elevated levels of TGFβ signaling pathway mRNAs are seen in metastatic HGSC, but are not accompanied by increased Smad expression and activation in HGSC effusions, evidence of failure to activate canonical TGFβ signaling. Assessment of the prognostic role of this pathway in HGSC effusions merits further research.

MiR-29a is a candidate biomarker of better survival in metastatic high-grade serous carcinoma

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.