Dag Andre Nymoen

Expression of the folate receptor genes FOLR1 and FOLR3 differentiates ovarian carcinoma from breast carcinoma and malignant mesothelioma in serous effusions

The objective of this study was to analyze the diagnostic and clinical role of the folate receptor-alpha (FOLR1) and folate receptor-gamma (FOLR3) genes in effusion cytology. Expression of the FOLR1 protein product, FR-alpha, was additionally studied. Ninety-one effusions (71 ovarian carcinomas, 10 breast carcinomas, 10 malignant mesotheliomas) were assayed for FOLR1 and FOLR3 gene expression using quantitative polymerase chain reaction. FR-alpha expression was analyzed using flow cytometry. Ovarian carcinoma expression levels were analyzed for association with clinicopathologic parameters and survival. Quantitative polymerase chain reaction analysis showed significantly higher FOLR1and FOLR3 mRNA levels in ovarian carcinomas compared with both breast carcinomas and mesotheliomas (P < .001). FOLR1 and FOLR3 mRNA levels were directly interrelated in ovarian carcinoma (P < .001). FR-alpha protein levels were similarly higher in ovarian carcinoma compared with the 2 other cancer types (P < .001). FOLR1and FOLR3 mRNA and FR-alpha protein expression in ovarian carcinoma effusions showed no association with clinical parameters or survival. Our data suggest that folate receptor levels effectively differentiate ovarian carcinoma from other cancers affecting the serosal cavities and that folate receptor genes are coexpressed in this tumor. The high expression of folate receptors in ovarian carcinoma supports their validity as molecular therapeutic targets in this disease.

Tenascin-X is a novel diagnostic marker of malignant mesothelioma

Tenascin XB (TNXB) was previously identified as a gene that is more highly expressed in malignant mesothelioma compared with ovarian/peritoneal serous carcinoma based on gene expression array analysis. The objective of this study was to validate this finding at the mRNA and protein levels. Effusions (n=91; 71 ovarian carcinomas, 10 breast carcinomas, and 10 malignant mesotheliomas) were assayed for TNXB mRNA expression using quantitative polymerase chain reaction. Tenascin-X protein expression was studied in 183 effusions (137 carcinomas of different origin, 37 mesotheliomas, and 9 reactive effusions) and 178 solid lesions (122 ovarian/peritoneal carcinomas and 56 mesotheliomas) using immunohistochemistry. Quantitative polymerase chain reaction analysis showed significantly higher TNXB mRNA level in mesotheliomas compared with ovarian and breast carcinomas (P<0.001). By immunohistochemistry, tenascin-X protein expression was significantly higher in malignant mesothelioma compared with metastatic carcinoma in effusions (34 of 37 vs. 31 of 137 positive cases; sensitivity=92% and specificity=77%; P<0.001). Reactive mesothelial cells had focal or no tenascin-X expression. Tenascin-X protein was detected in 41 of 56 mesothelioma biopsy specimens and was uniformly absent from all 122 ovarian carcinomas (sensitivity=73% and specificity=100%; P<0.001). Our data suggest that tenascin-X may be a new diagnostic marker of malignant mesothelioma in the differential diagnosis of cancers involving the serosal cavities, particularly in the differential diagnosis between this tumor and ovarian/peritoneal serous carcinoma.

Population-based prevalence of CDKN2A and CDK4 mutations in patients with multiple primary melanomas

The presence of multiple primary cutaneous melanomas (MPM) has been advocated as guidance to identifying melanoma families. Frequencies of CDKN2A mutations in materials of sporadic MPM cases from pigmented lesion clinics vary between 8 and 15%. Patients with MPM have therefore been regarded as good candidates for CDKN2A mutational screening. We describe a population‐based study where all persons in Norway diagnosed with MPM between 1953 and 2004 (n = 738 alive per April 2004) were invited to participate. Three‐hundred‐and‐ninety patients (52.8%) responded confidentially. Mutations in CDKN2A were found in 6.9% of the respondents. Eighty‐one MPM patients (20.8%) reported that they belonged to melanoma families, and 17 (21.0%) of these harboured a CDKN2A mutation, compared to 3.2% of the nonfamilial cases. The probability of finding a CDKN2A mutation increased when the patients had three or more melanomas, or a young age of onset of first melanoma. We identified five novel CDKN2A variants (Ala57Gly, Pro81Arg, Ala118Val, Leu130Val, and Arg131Pro) and four that previously have been reported in melanoma families (Glu27X, Met53Ile, Arg87Trp, and Ala127Pro). A large deletion (g.13623_23772del10150) encompassing exon 1α and the 5′ part of exon 2 was detected in six patients with a family history of melanoma. Three patients, belonging to the same family, had the CDK4 Arg24His mutation. The frequency of CDKN2A mutations was lower than previously reported in other studies, an observation which probably is due to the population‐based design of our study.

Recurrent respiratory papillomatosis: HPV genotypes and risk of high-grade laryngeal neoplasia.

Patients with recurrent respiratory papillomatosis (RRP) in Norway treated between 1987 and 2009 were recruited to this cohort study. They were followed from disease onset and data recorded until January 2012. Here, we describe the distribution of human papillomavirus (HPV) genotypes, the prevalence of multiple HPV infections, and the risk of high-grade laryngeal neoplasia and respiratory tract invasive carcinoma in a large cohort of patients with RRP. We also examined whether HPV genotype, gender, age or clinical course are risk factors for this development. Clinical records and histological specimens were reviewed. Using formalin-fixed paraffin-embedded biopsies, HPV genotyping were performed by quantitative polymerase chain reaction assays identifying 15 HPV types. HPV-negative specimens were analyzed by metagenomic sequencing. Paraffin blocks were available in 224/238 patients. The DNA quality was approved in 221/224 cases. HPV DNA was detected in 207/221 patients and all were HPV 6 or HPV 11 positive, comprising HPV 6 in 133/207, HPV 11 in 40/207 cases and HPV 6/11 in 15/207 cases. Co-infection with one or two high-risk HPV types together with HPV 6 or HPV 11 was present in 19/207 patients. Metagenomic sequencing of 14 HPV-negative specimens revealed HPV 8 in one case. In total, 39/221 patients developed high-grade laryngeal neoplasia. 8/221 patients developed carcinoma of the respiratory tract (six patients with laryngeal carcinoma and two patients with lung carcinoma). High-grade laryngeal neoplasias were found more frequently in HPV-negative versus HPV-positive patients, (RR = 2.35, 95% CI 1.1, 4.99), as well as respiratory tract carcinomas (RR = 48, 95% CI 10.72, 214.91). In summary, the majority of RRP were associated with HPV 6 and/or 11. HPV-negative RRP biopsies occurred more frequently in adult-onset patients, and were associated with an increased risk of laryngeal neoplasia and carcinoma in the respiratory tract.

Expression of the Ets transcription factor EHF in serous ovarian carcinoma effusions is a marker of poor survival

The EHF (Ets homologous factor) gene was previously shown to be overexpressed in ovarian/primary peritoneal serous carcinoma compared to malignant mesothelioma using gene expression arrays. The objective of this study was to validate this finding at the mRNA level in a larger series. We analyzed the diagnostic role of EHF in 98 ovarian serous carcinoma effusions, 23 malignant mesothelioma specimens (20 effusions, 3 surgical specimens), and 28 primary ovarian serous carcinomas using quantitative real-time polymerase chain reaction. Expression levels of EHF in ovarian carcinoma were additionally investigated for association with clinicopathologic parameters and survival. Quantitative real-time polymerase chain reaction analysis showed significantly higher expression of EHF mRNA in ovarian carcinoma effusions and in primary ovarian carcinoma compared to malignant mesothelioma effusions (P < .001 for both). EHF mRNA expression was additionally higher in primary ovarian carcinomas compared to effusions of this cancer (P < .001). In univariate analysis for all patients with effusions, higher EHF mRNA levels were associated with a trend for shorter progression-free survival (P = .066), which became significant in analysis of 45 patients with primary diagnosis pre-chemotherapy effusions (P = .01). In Cox multivariate analysis, EHF mRNA expression was an independent predictor of poor progression-free survival for all patients and patients with primary diagnosis pre-chemotherapy effusions (P = .033 and P = .009, respectively). EHF mRNA levels differentiate ovarian carcinoma from malignant mesothelioma and may thus be of diagnostic value in this setting. EHF may be a novel prognostic marker in ovarian carcinoma.

SCARA3 mRNA is overexpressed in ovarian carcinoma compared with breast carcinoma effusions

Scavenger receptor class A, member 3 (SCARA3) was previously found to be overexpressed in ovarian/primary peritoneal carcinoma (OC/PPC) compared with breast carcinoma effusions by global gene expression analysis. The present study aimed to validate this finding applying quantitative PCR and analyzing the association between SCARA3 expression and clinicopathologic parameters in a large OC cohort. SCARA3 messenger RNA (mRNA) expression was analyzed in 127 effusions (103 ovarian/peritoneal/fallopian tube carcinomas, 9 breast carcinomas, 15 malignant mesotheliomas [MM]), and 30 solid primary OCs. The association between OC SCARA3 levels and clinicopathologic parameters was investigated. SCARA3 mRNA was expressed in all effusions, irrespective of tumor type. However, transcript levels were significantly higher in OC compared with breast carcinoma (P < .001) and MM (P = .011) effusions. Primary OCs and effusions had comparable expression levels. Higher SCARA3 expression was found in disease recurrence postchemotherapy compared with primary diagnosis prechemotherapy OC effusions (P = .001), and this difference was significant for treatment with both platinum agents (P = .006) and paclitaxel (P = .002). SCARA3 levels in effusions and primary carcinomas were unrelated to patient age, tumor grade, FIGO stage, residual tumor volume after surgery, response to chemotherapy, or survival (P > .05 for all). In conclusion, SCARA3 mRNA by quantitative PCR is highly expressed in OC and may aid in differentiating this tumor from other cancers, particularly breast carcinoma, in effusions. The consistently high SCARA3 levels in both primary carcinomas and metastatic cells in effusions, and its up-regulation along disease progression from diagnosis to recurrence, suggest a role in ovarian cancer biology.

Aurora B expression in metastatic effusions from advanced-stage ovarian serous carcinoma is predictive of intrinsic chemotherapy resistance

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.

MC1R, ASIP, TYR, and TYRP1 gene variants in a population-based series of multiple primary melanomas

Allelic variants of the low‐penetrance melanoma gene MC1R increase the risk of both melanoma and non‐melanoma skin cancer. Common variants of the genes ASIP, TYR, and TYRP1, which regulate the melanogenic pathway, have also been shown to associate with melanoma. In this population‐based study, we investigated SNPs of MC1R, ASIP, TYR, and TYRP1 as risk factors for development of multiple primary melanomas (MPM) in 388 Norwegian cases. The MPM patients had a significantly higher likelihood of carrying any MC1R variant than the control group of 420 blood donors [86.8 vs. 78.3%, OR = 1.73, and confidence intervals (CI) 1.18–2.52]. When MC1R variants were analyzed individually, Asp84Glu and Arg151Cys were significantly more frequent among the MPM cases than among the controls (OR = 5.77, CI 1.97–16.90, and OR = 1.80, CI 1.36–2.37, respectively). In addition, there was an allele dose‐dependent increase in MPM risk for carriers of red hair color (RHC) MC1R variants. The AH haplotype of ASIP was also a significant risk factor for MPM development (OR = 1.72 and CI 1.12–2.49), whereas no association was observed for previously reported risk variants of the TYR and TYRP1 genes. In summary, by using a population‐based material of high‐risk melanoma cases, we demonstrate a significant effect of both MC1R RHC variants and an ASIP haplotype, but could not replicate an association with postulated risk SNPs of TYR and TYRP1.

MGST1 expression in serous ovarian carcinoma differs at various anatomic sites, but is unrelated to chemoresistance or survival.

OBJECTIVE To investigate the expression of MGST1 in primary tumors, solid metastases and metastatic effusions in advanced-stage serous ovarian carcinoma (OC) and analyze the association with clinicopathologic parameters, including chemotherapy resistance and survival. METHODS MGST1 mRNA expression was investigated in 178 tumors (88 effusions, 38 primary carcinomas, 52 solid metastases) from 144 patients using real-time quantitative PCR (qRT-PCR). Forty-two of the 88 effusions were additionally analyzed for MGST1 protein expression by Western blotting. RESULTS mRNA expression of MGST1 was higher in primary carcinomas and solid metastases compared to effusions (p=0.008 and p=0.012, respectively). In patient-matched samples, mRNA expression of MGST1 was higher in solid metastases compared to effusions (p=0.023), and a trend for higher MGST1 levels in solid metastases compared to primary tumors was observed (p=0.06). Biopsies from primary carcinomas obtained from patients with >200 ml ascites at diagnosis had higher mRNA expression of MGST1 compared to samples from patients with <200 ml ascites (p=0.037). MGST1 mRNA expression was not associated with age, histological grade, tumor stage, residual disease volume, response to chemotherapy, chemotherapy resistance or survival. Western blot analysis of patient-matched effusions showed high concordance between MGST1 protein and mRNA levels measured by qRT-PCR (p<0.001). CONCLUSIONS The present study documents frequent MGST1 mRNA and protein expression in OC. The data suggest increased activity of oxidative response pathways, reflected by higher mRNA expression, in solid OC tumors compared to metastatic effusions. Additionally, a tumor microenvironment consisting of ascites may induce antioxidant activity.

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

BackgroundThe 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.MethodsAdvanced-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.ResultsABCA4 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).ConclusionsOur 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.