Kristina Levan

Four potential biomarkers as prognostic factors in stage III serous ovarian adenocarcinomas.

The mortality rate for patients with ovarian carcinomas is high and the available prognostic factors are insufficient. The use of biomarkers may contribute to better prediction and survival for these patients. We aimed to study the gene and protein expressions for 7 potential biomarkers, to determine if it is possible to use them as prognostic factors. Genes selected from our previous microarray analysis (2006), CLU, ITGB3, TACC1, MUC5B, CAPG, PRAME and TROAP, were analyzed in 19 of the tumors with quantitative real‐time polymerase chain reaction (QPCR). We found that CLU and ITGB3 were more expressed in tumors from survivors and PRAME and CAPG were more expressed in tumors from deceased patients. None of the other 3 genes were significantly differently expressed. The protein expressions of CLU, ITGB3, PRAME and CAPG were analyzed in 43 of the tumors with western blot for semiquantitative analysis. We established that the mRNA and protein expressions correlated and that all 4 proteins were significantly differently expressed. Further, immunohistochemistry (IHC) was used to localize the expression of the proteins in the tumor samples. According to our results, the 4 biomarkers CLU, ITGB3, PRAME and CAPG may be used as prognostic factors for patients with stage III serous ovarian adenocarcinomas.

Analysis of cytogenetic alterations in stage III serous ovarian adenocarcinoma reveals a heterogeneous group regarding survival, surgical outcome, and substage

Ovarian cancer is the leading cause of death among patients with gynecological cancers, but the biology of these tumors is still among the least understood of all major human malignancies. In this study, comparative genomic hybridization was used to determine chromosomal alterations in 98 stage III serous papillary adenocarcinomas. The tumors were grouped according to survival and the main prognostic factors stage and surgical outcome. There were chromosomal imbalances that were significantly more common in tumors from patients who died than in tumors from patients who survived: gains of 1q24–qter and losses of 4p, 4q31.1–qter, 5q12–q22, 8p, 16q, and X. Furthermore, we observed that gains of 8q23–8q24.2 and losses of 4p, 4q13–4q26, 4q31.1–qter, 5q12–q22, 8p, and 16q were significantly more common in tumors from patients with macroscopic residual tumor after primary surgery, compared to tumors from those who had undergone radical surgery. Gains of 3q13.3–qter, 6p, 7q21–q31, and 11q13–q23 and losses of 4q31.1–qter and 16q were more common in stage IIIc tumors than in stage IIIa+b tumors. On the basis of our results, we suggest that there are biological differences among the groups mentioned above and that absence of chromosomal aberrations in specific regions predicts a good clinical outcome for individual patients.

Chromosomal alterations in 98 endometrioid adenocarcinomas analyzed with comparative genomic hybridization

The aim of the present study was to investigate chromosomal alterations in a large set of homogeneous tumors, 98 endometrioid adenocarcinomas. We also wanted to evaluate differences in chromosomal alterations in the different groups of tumors in relation to stage, survival and invasive or metastatic properties of the tumors. Comparative genomic hybridization (CGH) was used to detect chromosomal alterations in tissue samples from 98 endometrioid adenocarcinomas. All chromosomes were involved in DNA copy number variations at least once in the tumor material, but certain changes were recurrent and rather specific. Among the specific changes, it was possible to identify 39 chromosomal regions displaying frequent DNA copy number alterations. The most frequent alteration was detected at 1q25→q42, in which gains were found in 30 cases (30%). Gains at 19pter→p13.1 were detected in 26 tumors (26%) and at 19q13.1→q13.3 in 19 tumors (19%). Increased copy numbers were also detected at 8q (8q21→q22 and 8q22→qter), at a relatively high rate, in 17 cases (17%). Furthermore, gains at 10q21→q23 and 10p were found in 14 (14%) and 13 cases (13%), respectively. The most common losses were found in the three regions 4q22→qter, 16q21→qter and 18q21→qter, all of which were detected in eight of the 98 tumors (8%). We also detected differences between the tumors from deceased patients and from survivors. Gain at 1q25→q42 was more commonly detected in the tumors from patients who died of cancer. We noted that the regions most affected differed in the different surgical stages (I–IV). The results of the CGH analysis identify specific chromosomal regions affected by copy number changes, appropriate objects for further genetic studies.

Diagnostic performance of the biomarkers HE4 and CA125 in type I and type II epithelial ovarian cancer

OBJECTIVE To evaluate the diagnostic performance of HE4 and CA125 in patients presenting with suspicious malignant ovarian cysts. We especially wanted to investigate the levels of HE4 and CA125 with regard to the gene and histology-unifying model of type I and type II epithelial ovarian cancer (EOC). METHODS Plasma from 373 women presenting with a suspicious malignant ovarian cyst was collected prior to surgery. Histology, grade, and stage were determined according to FIGO-classification. HE4 and CA125 were analyzed using ELISA, and the markers were evaluated for significance separately and in combination. Receiver operating curves, the area under the curve, sensitivity and specificity were estimated. RESULTS The combination of HE4 and CA125 resulted in the best diagnostic power in comparing benign tumors to EOC (ROC AUC 0.93, sensitivity 94.4% at 75% specificity) for type II. Diagnostic power in type I (ROC AUC 0.79, sensitivity 61.9% at 75% specificity) was less impressive. In particular, mucinous benign vs. malignant tumors could not significantly be separated by the dual marker combination. Impressively high ROC AUC 0.99 was found for the late stage type II EOC with 100% sensitivity at 75% specificity. CONCLUSIONS HE4 and CA125 have a good ability to diagnose the more aggressive type II tumors but a poor diagnostic ability when patients are presenting with slow-growing type I in the early stage. Our results support the hypothesis that EOC should be looked upon as several different diseases, and that we lack biomarkers for sub-groups of EOC.

Potential predictive markers of chemotherapy resistance in stage III ovarian serous carcinomas

BackgroundChemotherapy resistance remains a major obstacle in the treatment of women with ovarian cancer. Establishing predictive markers of chemoresponse would help to individualize therapy and improve survival of ovarian cancer patients. Chemotherapy resistance in ovarian cancer has been studied thoroughly and several non-overlapping single genes, gene profiles and copy number alterations have been suggested as potential markers. The objective of this study was to explore genetic alterations behind chemotherapy resistance in ovarian cancer with the ultimate aim to find potential predictive markers.MethodsTo create the best opportunities for identifying genetic alterations of importance for resistance, we selected a homogenous tumor material concerning histology, stage and chemotherapy. Using high-resolution whole genome array comparative genomic hybridization (CGH), we analyzed the tumor genomes of 40 fresh-frozen stage III ovarian serous carcinomas, all uniformly treated with combination therapy paclitaxel/carboplatin. Fishers exact test was used to identify significant differences. Subsequently, we examined four genes in the significant regions (EVI1, MDS1, SH3GL2, SH3KBP1) plus the ABCB1 gene with quantitative real-time polymerase chain reaction (QPCR) to evaluate the impact of DNA alterations on the transcriptional level.ResultsWe identified gain in 3q26.2, and losses in 6q11.2-12, 9p22.3, 9p22.2-22.1, 9p22.1-21.3, Xp22.2-22.12, Xp22.11-11.3, and Xp11.23-11.1 to be significantly associated with chemotherapy resistance. In the gene expression analysis, EVI1 expression differed between samples with gain versus without gain, exhibiting higher expression in the gain group.ConclusionIn conclusion, we detected specific genetic alterations associated with resistance, of which some might be potential predictive markers of chemotherapy resistance in advanced ovarian serous carcinomas. Thus, further studies are required to validate these findings in an independent ovarian tumor series.

Recurrent gene amplifications in human type I endometrial adenocarcinoma detected by fluorescence in situ hybridization

Determining what genes are actively involved in tumor development is important, because they may provide targets for directed therapy. Human tumors are greatly heterogeneous with respect to etiology and genetic background, which complicates the identification of common genetic aberrations. In contrast, genetic and environmental variation can be in part controlled in experimental animals, which facilitates identification of the important changes. In inbred BDII rats, which are genetically predisposed to endometrial adenocarcinomas (EAC), certain chromosome regions exhibit recurrent amplification in the tumors. Previous CGH analysis had shown that a subset of human EAC tumors exhibited increased copy numbers in the homologous chromosomal regions, located in human 2p21 approximately p25 and 7q21 approximately q31. Using fluorescence in situ hybridization analysis on imprints from 13 human EAC tumors, we determined the average copy numbers of each of 15 probes derived from cancer-related genes situated in these chromosome regions. Among the genes analyzed, those most often targeted by amplification were SDC1 and CYP1B1 in 2p21 approximately p25 and CDK6 and MET in 7q21 approximately q31, but all of the 15 genes tested were found to be amplified in at least two tumors.

Potential tumor biomarkers identified in ovarian cyst fluid by quantitative proteomic analysis, iTRAQ

BackgroundEpithelial-derived ovarian adenocarcinoma (EOC) is the most deadly gynecologic tumor, and the principle cause of the poor survival rate is diagnosis at a late stage. Screening and diagnostic biomarkers with acceptable specificity and sensitivity are lacking. Ovarian cyst fluid should harbor early ovarian cancer biomarkers because of its closeness to the tumor. We investigated ovarian cyst fluid as a source for discovering biomarkers for use in the diagnosis of EOC.ResultsUsing quantitative mass spectrometry, iTRAQ MS, we identified 837 proteins in cyst fluid from benign, EOC stage I, and EOC stage III. Only patients of serous histology were included in the study. Comparing the benign (n = 5) with the malignant (n = 10) group, 87 of the proteins were significantly (p < 0.05) differentially expressed. Two proteins, serum amyloid A-4 (SAA4) and astacin-like metalloendopeptidase (ASTL), were selected for verification of the iTRAQ method and external validation with immunoblot in a larger cohort with mixed histology, in plasma (n = 68), and cyst fluid (n = 68). The protein selections were based on either high significance and high fold change or abundant appearance and several peptide recognitions in the sample sets (p = 0.04, FC = 1.95) and (p < 0.001, FC = 8.48) for SAA4 and ASTL respectively. Both were found to be significantly expressed (p < 0.05), but the methods did not correlate concerning ASTL.ConclusionsFluid from ovarian cysts connected directly to the primary tumor harbor many possible new tumor-specific biomarkers. We have identified 87 differentially expressed proteins and validated two candidates to verify the iTRAQ method. However several of the proteins are of interest for validation in a larger setting.

Identification of a gene expression signature for survival prediction in type I endometrial carcinoma.

Endometrial cancer is the most common malignancy of the female reproductive tract. In many cases the prognosis is favorable, but 22% of affected women die from the disease. We aimed to study potential differences in gene expression between endometrioid adenocarcinomas from survivors (5-year survival) and nonsurvivors. Forty-five patients were included in the investigation, of which 21 were survivors and 24 were nonsurvivors. The tumors were analyzed with genome-wide expression array analysis, represented by 13,526 genes. Distinct differences in gene expression were found between the groups. A t-test established that 218 genes were significantly differentially expressed (p < 0.001) between the two survival groups, and in a cross-validation test 40 of the 45 (89%) tumors were classified correctly. The 218 differentially expressed genes were subjected to hierachical clustering analysis, which yielded two clusters both exhibiting over 80% homogeneity with respect to survival. When the additional constraint of fold change (FC > 2) was added the hierachical clustering yielded similar results. Stage I tumors are expected to have a favorable prognosis. However, in our tumor material there were six nonsurvivors with stage I tumors. Five out of six stage I nonsurvivors clustered in the nonsurvival fraction. Our findings suggest that a subgroup of early stage endometroid adenocarcinomas can be correctly classified as potentially aggressive by using molecular biology in combination with conventional markers, thereby providing a tool for a more accurate classification and risk evaluation of the individual patient.

External validation suggests Integrin beta 3 as prognostic biomarker in serous ovarian adenocarcinomas

BackgroundThe majority of women with ovarian cancer are diagnosed in late stages, and the mortality rate is high. The use of biomarkers as prognostic factors may improve the treatment and clinical outcome of these patients. We performed an external validation of the potential biomarkers CLU, ITGB3, CAPG, and PRAME to determine if the expression levels are relevant to use as prognostic factors.MethodsWe analysed the gene expression of CLU, ITGB3, CAPG, and PRAME in 30 advanced staged serous adenocarcinomas with quantitative real-time polymerase chain reaction (QPCR) and the protein levels were analysed in 98 serous adenocarcinomas with western blot for semiquantitative analysis. Statistical differences in mRNA and protein expressions between tumours from survivors and tumours from deceased patients were evaluated using the Mann-Whitney U test.ResultsThe gene and protein ITGB3 (Integrin beta 3) were significantly more expressed in tumours from survivors compared to tumours from deceased patients, which is in concordance with our previous results. However, no significant differences were detected for the other three genes or proteins CLU, CAPG, and PRAME.ConclusionThe loss of ITGB3 expression in tumours from deceased patients and high expression in tumours from survivors could be used as a biomarker for patients with advanced serous tumours.

Mesangial cells from patients with IgA nephropathy have increased susceptibility to galactose-deficient IgA1

BackgroundIgA nephropathy (IgAN) is the most common glomerulonephritis in the world, affecting close to a million people. Circulating galactose-deficient IgA (gd-IgA), present in patients with IgAN, form immune complex deposits in the glomerular mesangium causing local proliferation and matrix expansion. Intriguing though, individuals having gd-IgA deposits in the kidneys do not necessarily have signs of glomerular disease. Recurrence of IgAN only occurs in less than half of transplanted patients with IgAN, indicating that gd-IgA is not the only factor driving the disease. We hypothesize that, in addition to IgA complexes, patients with IgAN possess a subtype of mesangial cells highly susceptible to gd-IgA induced cell proliferation.MethodsTo test the hypothesis, we designed a technique to culture primary mesangial cells from renal biopsies obtained from IgAN patients and controls. The cell response to gd-IgA treatment was then measured both on gene and protein level and the proliferation rate of the cells in response to PDGF was investigated.ResultsWhen treated with gd-IgA, mesangial cells from patients with IgAN express and release more PDGF compared to controls. In addition, the mesangial cells from patients with IgAN were more responsive to treatment with PDGF resulting in an increased proliferation rate of the cells compared to control. Mesangial cells cultured from patients with IgAN expressed and released more IL-6 than controls and had a higher expression of matrix genes. Both mesangial cells derived from patients with IgAN and controls increased their expressed TGFβ1 and CCL5 when treated with gd-IgA.ConclusionWe conclude that mesangial cells derived from IgAN patients have a mesangioproliferative phenotype with increased reactivity to IgA and that these cellular intrinsic properties may be important for the development of IgA nephropathy.