Stathmin is an Independent Prognostic Marker of Poor Outcome in Uterine Leiomyosarcoma.

The objective of this study was to analyze the expression and prognostic role of cancer-associated proteins in uterine leiomyosarcoma (uLMS). p53, DAXX, ATRX, HMGA2, IMP3, Stathmin, and phospho-Stathmin (p-Stathmin) protein expression by immunohistochemistry was analyzed in tissue microarrays from 244 uLMS. Expression was assessed for association with clinicopathologic parameters in 173 patients with available data. Tissue microarrays were informative in 230 cases. p53 was aberrant in 44% of tumors. DAXX, ATRX, HMGA2, IMP3, and Stathmin were expressed in 90%, 55%, 40%, 33%, and 97% uLMS, respectively. Cytoplasmic and nuclear p-Stathmin staining was seen in 77% and 68% of tumors, respectively. Stathmin expression was significantly related to higher mitotic count (P < 0.001), a higher degree of atypia (P = 0.006), and vascular invasion (P = 0.016), whereas p-Stathmin expression was significantly related to advanced stage (P < 0.001), higher mitotic count (P < 0.001), and vascular invasion (P = 0.001). In univariate survival analysis for 165 patients with informative tissue microarrays, aberrant p53 (P = 0.026) and higher IMP3 (P = 0.024), Stathmin (P < 0.001), cytoplasmic p-Stathmin (P < 0.001), and nuclear p-Stathmin (P < 0.001) expression was associated with poor disease-specific survival. Clinicopathologic parameters significantly related to poor disease-specific survival were older age (P = 0.006), extrauterine disease at diagnosis (International Federation of Gynecology and Obstetrics (FIGO) stage ≥2; P < 0.001), high mitotic count (P = 0.02), and grade 2 to 3 atypia (P = 0.017). In multivariate analysis, age (P = 0.002), FIGO stage (P < 0.001), and Stathmin expression (P < 0.001) were independent prognosticators. Stathmin was the only prognosticator in a multivariate analysis limited to patients with FIGO stage I disease (P = 0.013). In conclusion, Stathmin expression is strongly associated with poor survival in uLMS and may be a new prognostic marker in this malignancy.

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

OBJECTIVE: To analyze the expression and prognostic role of L1CAM in tubo-ovarian high-grade serous carcinoma (HGSC). METHODS: L1CAM protein expression by immunohistochemistry was analyzed in 644 HGSC (413 effusions, 231 surgical specimens). Expression was analyzed for association with clinicopathologic parameters and survival. RESULTS: L1CAM 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. CONCLUSION: L1CAM 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.

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

OBJECTIVE: To analyse the expression and clinical role of the phosphatase PTPN1 (PTP1B) in serous effusions. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

OBJECTIVE: To analyse the expression and clinical role of the actin-associated molecule palladin in serous effusions. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

OBJECTIVE: The 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). METHODS: mRNA 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. RESULTS: OCT4 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. CONCLUSIONS: SOX2 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, activation and clinical relevance of CHK1 and CHK2 in metastatic high-grade serous carcinoma.

OBJECTIVE: To analyze the expression and clinical role of CHK1 and CHK2 in metastatic high-grade serous carcinoma (HGSC). METHODS: HGSC 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. RESULTS: Carcinoma 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. CONCLUSIONS: CHK1 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.

L1CAM as a prognostic marker in stage I endometrial cancer: a validation study.

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.

CIAPIN1 and ABCA13 are markers of poor survival in metastatic ovarian serous carcinoma.

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.

Expression and clinical role of chemoresponse-associated genes in ovarian serous carcinoma.

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<0.001) and 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<0.001) survival, the latter finding retained in multivariate analysis (p=0.035). 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.

Wee1 is a novel independent prognostic marker of poor survival in post-chemotherapy ovarian carcinoma effusions.

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.

Different Frequency and Clinical Role for MTAP Loss in Pleural and Peritoneal Mesothelioma.

The objective of this study was to analyze the expression and prognostic role of methylthioadenosine phosphorylase (MTAP) in mesothelioma. MTAP protein expression by immunohistochemistry was analyzed in 113 mesotheliomas (60 pleural and 53 peritoneal), consisting of 36 effusions and 77 surgical specimens. MTAP expression was fully lost in 38 tumors and partially lost in 8 tumors. Loss of expression was significantly more common in effusions compared with biopsies/surgical resection specimens (20/36 vs. 26/77; P =0.017), and in pleural compared with peritoneal mesotheliomas (35/60 vs. 11/53; P <0.001). MTAP performed less robustly than BAP1 in comparative analysis of 57 tumors previously analyzed for expression of the latter protein (46 vs. 25 cases with loss of expression). In survival analysis for 69 patients with partial clinical data, male gender was significantly associated with shorter overall survival (OS; P =0.042), whereas loss of MTAP was associated with a trend for shorter OS ( P =0.058), with no prognostic role for patient age ( P =0.379) or anatomic site ( P =0.381). The association between loss of MTAP and poor OS became significant when survival analysis was limited to patients with pleural mesothelioma ( P =0.018). In conclusion, loss of MTAP expression is more frequent in pleural compared with peritoneal mesothelioma and has limited diagnostic relevance at the latter anatomic site. More frequent loss in effusion specimens suggests a role for this marker in effusion cytology. MTAP loss in pleural mesothelioma is associated with poor survival.

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-chemotherapy effusions compared to chemo-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.

Molecular characteristics of tubo-ovarian carcinosarcoma at different anatomic locations.

Carcinosarcoma (CS) is an uncommon and clinically aggressive malignancy. The objective of the present study was to characterize the molecular features of CS at various anatomic locations, including serous effusions. Specimens (n = 32) consisted of 25 biopsies/surgical resection specimens and 7 serous effusions (6 peritoneal, 1 pleural) from 25 patients. Fresh-frozen cell pellets and surgical specimens underwent targeted next-generation sequencing covering 50 unique genes. A total of 31 mutations were found in 25 of the 32 tumors studied, of which 1 had 3 mutations, 4 had 2 different mutations, and 20 had a single mutation. The most common mutations were in TP53 (n = 25 in 24 tumors; 1 tumor with 2 different mutations), with less common mutations found in RB1 (n = 2), MET (n = 1), KRAS (n = 1), PTEN (n = 1), and KIT (n = 1). Patient-matched specimens harbored the same TP53 mutation. Tumors with no detected mutations were more common in serous effusion specimens (3/7; 43%) compared with surgical specimens (4/25; 16%). In conclusion, the molecular landscape of CS is dominated by TP53 mutations, reinforcing the observation that the majority of these tumors develop from high-grade serous carcinoma. Whether CS cells in serous effusions differ from their counterparts in solid lesions remains uncertain.

Endometrial carcinomas with ambiguous histology often harbor TP53 mutations.

The objective of the present study was to characterize the molecular features of endometrial carcinomas with ambiguous histology. Eighteen carcinomas that could not be conclusively typed based on morphology and immunohistochemistry underwent analysis of mismatch repair (MMR) status, microsatellite status, and whole-exome sequencing. None of the tumors had pathogenic POLE mutation. Twelve tumors (67%) were microsatellite stable, and 6 (33%) had microsatellite instability. Fourteen tumors (78%) harbored TP53 mutations, and 2 (11%) had mutations in MMR genes. Eleven carcinomas (61%) were classified as copy number high and 7 (39%) as MSI-hypermutated, the latter including 3 tumors with TP53 mutation who concomitantly had MSI or mutation in a MMR gene. Other mutations that were found in > 1 tumor affected MUC16 (7 tumors), PIK3CA (6 tumors), PPP2R1A (6 tumors), ARID1A (5 tumors), PTEN (5 tumors), FAT1 (4 tumors), FAT4 (3 tumors), BRCA2 (2 tumors), ERBB2 (2 tumors), FBXW7 (2 tumors), MET (2 tumors), MTOR (2 tumors), JAK1 (2 tumors), and CSMD3 (2 tumors). At the last follow-up (median = 68.6 months), 8 patients had no evidence of disease, 1 patient was alive with disease, 8 patients were dead of disease, and 1 patient died of other cause. In conclusion, based on this series, the molecular landscape of endometrial carcinomas with ambiguous histology is dominated by TP53 mutations and the absence of POLE mutations, with heterogeneous molecular profile with respect to other genes. A high proportion of these tumors is clinically aggressive.

Migfilin, α-parvin and ß-parvin are differentially expressed in ovarian serous carcinoma effusions, primary tumors and solid metastases.

OBJECTIVE: The objective of this study is to analyze the expression and clinical role of integrin-linked kinase (ILK), α-parvin, ß-parvin and migfilin in advanced-stage serous ovarian carcinoma. METHODS: 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. RESULTS: ILK, α-parvin, ß-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 ß-parvin expression in primary carcinomas (p=0.02 and p=0.001, respectively) and solid metastases (p=0.001 and p<0.001, 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. ß-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. CONCLUSIONS: 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. ß-Parvin may be a novel marker of chemoresponse in metastatic ovarian carcinoma.

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.

HOXB8 expression in ovarian serous carcinoma effusions is associated with shorter survival.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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 (p<0.001). 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. CONCLUSIONS: 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.

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.

Lymphovascular invasion and p16 expression are independent prognostic factors in stage I vulvar squamous cell carcinoma.

The objective of this study was to identify clinicopathologic parameters associated with disease outcome in FIGO stage I vulvar squamous cell carcinoma (vSqCC). The cohort consisted of 126 patients diagnosed with vSqCC in the period 2006-2016 who underwent primary vulvar surgery and evaluation of groin lymph node status. Tumors were reviewed by an experienced gynecologic pathologist. p16 and p53 protein expression by immunohistochemistry and HPV status were analyzed in 116 tumors. Clinicopathologic parameters, protein expression and HPV status were analyzed for association with progression-free and overall survival (PFS, OS). p16 expression and aberrant p53 were found in 49 (42%) and 61 (53%) tumors, respectively. Sixty-six tumors were HPV-associated (57%). Relapse was diagnosed in 35/126 (28%) of patients, and 23 (18%) died of disease. Tumor diameter > 4 cm (p = 0.013), lymphovascular space invasion (LVSI; p < 0.001), the presence of lichen sclerosus (p = 0.019), p16 expression (p = 0.007), p53 expression (p = 0.012), HPV status (p = 0.021), lymph node metastasis (p < 0.001) and post-operative radiotherapy (p < 0.001) were significantly related to OS in univariate analysis. Tumor diameter > 4 cm (p = 0.038), LVSI (p = 0.003), the presence of lichen sclerosus (p = 0.004), p16 expression (p = 0.004), HPV status (p = 0.039), lymph node metastasis (p < 0.001) and post-operative treatment (p < 0.001), were significantly related to PFS in univariate analysis. Age, BMI and surgical resection involvement were not significantly associated with OS or PFS. In multivariate Cox analysis, LVSI and p16 expression were independent prognosticators of OS (p < 0.001 and p = 0.02, respectively) and PFS (p = 0.018, p = 0.037). In conclusion, LVSI and p16 expression are independent prognostic factors in stage I vSqCC.

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.