Dan Tong

KRAS mutation analysis in ovarian samples using a high sensitivity biochip assay

BackgroundMutations in the KRAS gene are one of the most frequent genetic abnormalities in ovarian carcinoma. They are of renewed interest as new epidermal growth factor receptor (EGFR)-targeted therapies are being investigated for use in ovarian carcinoma. As KRAS mutations are associated with poor response and resistance to EGFR-targeting drugs, this study was conducted to obtain more information on the spectrum of KRAS mutations in ovarian carcinoma.MethodsThe presence of KRAS mutations in codon 12 and 13 was analyzed in frozen and formalin-fixed paraffin-embedded (FFPE) tissue with a low density biochip platform. 381 malignant (29 borderline malignancy, 270 primary carcinomas, and 82 recurrent carcinomas) and 22 benign tissue samples from a total of 394 patients were examined. KRAS mutational status of each sample was correlated with dignity, FIGO stage, grade, histology, and survival.ResultsKRAS mutations were found in 60 (15%) samples with 58 samples deriving from malignant tissue and 2 samples deriving from benign tissue. In 55 (92%) samples codon 12 was found to be mutated. Frozen and FFPE samples concurred with respect to KRAS mutation status.ConclusionKRAS mutation is a common event in ovarian cancer primarily in carcinomas of lower grade, lower FIGO stage, and mucinous histotype. The KRAS mutational status is no prognostic factor for patients treated with standard therapy. However, in line with experience from colorectal cancer and non-small-cell-lung cancer (NSCLC), it may be important for prediction of response to EGFR-targeted therapies.

Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients

BackgroundThe presence of circulating tumor cells (CTC) in the peripheral blood of cancer patients has been described for various solid tumors and their clinical relevance has been shown. CTC detection based on the analysis of epithelial antigens might be hampered by the genetic heterogeneity of the primary tumor and loss of epithelial antigens. Therefore, we aimed to identify new gene markers for the PCR-based detection of CTC in female cancer patients.MethodsGene expression of 38 cancer cell lines (breast, ovarian, cervical and endometrial) and of 10 peripheral blood mononuclear cell (PBMC) samples from healthy female donors was measured using microarray technology (Applied Biosystems). Differentially expressed genes were identified using the maxT test and the 50% one-sided trimmed maxT-test. Confirmatory RT-qPCR was performed for 380 gene targets using the AB TaqMan® Low Density Arrays. Then, 93 gene targets were analyzed using the same RT-qPCR platform in tumor tissues of 126 patients with primary breast, ovarian or endometrial cancer. Finally, blood samples from 26 healthy women and from 125 patients (primary breast, ovarian, cervical, or endometrial cancer, and advanced breast cancer) were analyzed following OncoQuick enrichment and RNA pre-amplification. Likewise, hMAM and EpCAM gene expression was analyzed in the blood of breast and ovarian cancer patients. For each gene, a cut-off threshold value was set at three standard deviations from the mean expression level of the healthy controls to identify potential markers for CTC detection.ResultsSix genes were over-expressed in blood samples from 81% of patients with advanced and 29% of patients with primary breast cancer. EpCAM gene expression was detected in 19% and 5% of patients, respectively, whereas hMAM gene expression was observed in the advanced group (39%) only. Multimarker analysis using the new six gene panel positively identified 44% of the cervical, 64% of the endometrial and 19% of the ovarian cancer patients.ConclusionsThe panel of six genes was found superior to EpCAM and hMAM for the detection of circulating tumor cells in the blood of breast cancer, and they may serve as potential markers for CTC derived from endometrial, cervical, and ovarian cancers.

Expression of organic anion-transporting polypeptides 1B1 and 1B3 in ovarian cancer cells: relevance for paclitaxel transport.

PURPOSEnOvarian cancer remains a deadly malignancy because most patients develop recurrent disease that is resistant to chemotherapy. Organic anion-transporting polypeptides (OATPs) mediate the uptake of clinically important drugs thereby effecting intracellular drug accumulation. In this study, we investigated whether OATPs may also contribute to paclitaxel transport in estrogen-responsive and estrogen-independent ovarian carcinoma cell lines and tumor tissue.nnnMETHODSnExpression of all 11 human OATPs in human ovarian cancer tissue samples and in the ovarian carcinoma cell lines OVCAR-3 and SK-OV-3 was investigated using real-time RT-PCR. Kinetic analysis of paclitaxel uptake was characterized in both cell lines and in OATP-transfected Xenopus laevis oocytes. Cytotoxicity of paclitaxel in OVCAR-3, SK-OV-3 and OATP1B1- and OATP1B3-transfected SK-OV-3 cells was performed using the CellTiter-Glo assay.nnnRESULTSnOATP1B1 and OATP1B3 are active paclitaxel transporters in transfected X.xa0laevis oocytes. Real-time RT-PCR analysis revealed expression of both OATPs in human ovarian cancer tissue specimens and in cancer cell lines. The higher mRNA levels for OATP1B1 and OATP1B3 found in SK-OV-3 cells correlated with higher initial uptake rates for paclitaxel. In addition, cytotoxicity studies with OATP1B1- and OATP1B3-transfected SK-OV-3 cells demonstrated lower IC(50) values compared to cells transfected with the empty vector.nnnCONCLUSIONSnOur results revealed OATP1B1 and OATP1B3 as high-affinity paclitaxel transporters expressed in ovarian cancer cell lines and tumor tissues, suggesting a role for these polypeptides in the disposition of paclitaxel during therapy.

Methylation Status of TUSC3 Is a Prognostic Factor in Ovarian Cancer

Current prognostic information in ovarian cancer is based on tumor stage, tumor grade, and postoperative tumor size. Reliable molecular prognostic markers are scarce. In this article, the authors describe epigenetic events in a frequently deleted region on chromosome 8p22 that influence the expression of tumor suppressor candidate 3 (TUSC3), a putative tumor suppressor gene in ovarian cancer.

Role of TRAP1 and estrogen receptor alpha in patients with ovarian cancer -A study of the OVCAD consortium

BackgroundThe role of the tumor necrosis factor receptor associated protein 1 (TRAP1) – supposed to be involved in protection of cells from apoptosis and oxidative stress – has just started to be investigated in ovarian cancer. TRAP1 has been shown to be estrogen up-regulated in estrogen receptor α (ERα) positive ovarian cancer cells. The clinical impact of TRAP1 is not clear so far and the significance of ERα expression as therapeutic and prognostic marker is still controversial. Therefore, we investigated the importance of TRAP1 together with ERα in regard to clinicopathological parameters, chemotherapy response, and survival.Methods and resultsExpressions of TRAP1 and ERα were evaluated by immunohistochemical staining of tissue microarrays comprised of 208 ovarian cancer samples. TRAP1 was highly expressed in 55% and ERα was expressed in 52% of all cases. High TRAP1 expression correlated significantly with ERα (pu2009<u20090.001) but high TRAP1 expression was also found in 42% of ERα negative cases. High TRAP1 expression correlated significantly with favorable chemotherapy-response (HR = 0.48; 95%CI 0.24-0.96, p=0.037) and showed a significant impact on overall survival (OS) (HR = 0.65; 95%CI 0.43-0.99, p = 0.044). ERα expression was a favorable prognostic factor for OS in univariate and multivariate analyses. Interestingly, the combined pattern (ERα positive and/or TRAP1-high) revealed the strongest independent and significant positive influence on OS (HRu2009=u20090.41; 95%CI 0.27-0.64).ConclusionImmunohistochemical evaluation of TRAP1 together with ERα provides significant prognostic information. TRAP1 alone is significantly associated with chemotherapy response and overall survival, rendering TRAP1 as interesting scientific and therapeutic target.

Validating the impact of a molecular subtype in ovarian cancer on outcomes: A study of the OVCAD Consortium

Most patients with epithelial ovarian cancer (EOC) are diagnosed at advanced stage and have a poor prognosis. However, a small proportion of these patients will survive, whereas others will die very quickly. Clinicopathological factors do not allow precise identification of these subgroups. Thus, we have validated a molecular subclassification as new prognostic factor in EOC. One hundred and ninety‐four patients with Stage II–IV EOC were characterized by whole‐genome expression profiling of tumor tissues and were classified using a published 112 gene set, derived from an International Federation of Gynecology and Obstetrics (FIGO) stage‐directed supervised classification approach. The 194 tumor samples were classified into two subclasses comprising 95 (Subclass 1) and 99 (Subclass 2) tumors. All nine FIGO II tumors were grouped in Subclass 1 (P = 0.001). Subclass 2 (54% of advanced‐stage tumors) was significantly correlated with peritoneal carcinomatosis and non‐optimal debulking. Patients with Subclass 2 tumors had a worse overall survival for both serous and non‐serous histological subtypes, as revealed by univariate analysis (hazard ratios [HR] of 3.17 and 17.11, respectively; P ≤ 0.001) and in models corrected for relevant clinicopathologic parameters (HR 2.87 and 12.42, respectively; P ≤ 0.023). Significance analysis of microarrays revealed 2082 genes that were differentially expressed in advanced‐grade serous tumors of both subclasses and the focal adhesion pathway as the most deregulated pathway. In the present validation study, we have shown that, in advanced‐stage serous ovarian cancer, two approximately equally large molecular subtypes exist, independent of classical clinocopathological parameters and presenting with highly different whole‐genome expression profiles and a markedly different overall survival. Similar results were obtained in a small cohort of patients with non‐serous tumors. (Cancer Sci 2012; 103: 1334–1341)

Genetic variations of interleukin-1 and -6 genes and risk of cervical intraepithelial neoplasia.

OBJECTIVEnTo evaluate the association between five interleukin-1 (IL-1) and -6 gene polymorphisms and risk of high grade cervical intraepithelial neoplasia (CIN 2-3).nnnMETHODSnThis case-control study investigates five common IL-1 and IL-6 gene polymorphisms in 131 women with CIN 2-3 and 209 controls by pyrosequencing and polymerase chain reaction. Associations between gene polymorphisms and risk of CIN 2-3 are analysed by univariate and multivariable models. Their combined effect on the risk of CIN is evaluated by haplotype analysis.nnnRESULTSnIn a multivariable regression model IL1A -889 (odds ratio 0.3 [95% confidence interval 0.1-0.8], p=0.01) and smoking (4.0 [1.7-9.1], p=0.001) are independently associated with the risk of high grade CIN. Haplotype analysis does not reveal any high-risk combinations for the susceptibility of CIN.nnnCONCLUSIONnThe single nucleotide polymorphism IL1A -889 is independently associated with risk of high grade CIN.

A combined blood based gene expression and plasma protein abundance signature for diagnosis of epithelial ovarian cancer - a study of the OVCAD consortium

BackgroundThe immune system is a key player in fighting cancer. Thus, we sought to identify a molecular ‘immune response signature’ indicating the presence of epithelial ovarian cancer (EOC) and to combine this with a serum protein biomarker panel to increase the specificity and sensitivity for earlier detection of EOC.MethodsComparing the expression of 32,000 genes in a leukocytes fraction from 44 EOC patients and 19 controls, three uncorrelated shrunken centroid models were selected, comprised of 7, 14, and 6 genes. A second selection step using RT-qPCR data and significance analysis of microarrays yielded 13 genes (AP2A1, B4GALT1, C1orf63, CCR2, CFP, DIS3, NEAT1, NOXA1, OSM, PAPOLG, PRIC285, ZNF419, and BC037918) which were finally used in 343 samples (90 healthy, six cystadenoma, eight low malignant potential tumor, 19 FIGO I/II, and 220 FIGO III/IV EOC patients). Using new 65 controls and 224 EOC patients (thereof 14 FIGO I/II) the abundances of six plasma proteins (MIF, prolactin, CA125, leptin, osteopondin, and IGF2) was determined and used in combination with the expression values from the 13 genes for diagnosis of EOC.ResultsCombined diagnostic models using either each five gene expression and plasma protein abundance values or 13 gene expression and six plasma protein abundance values can discriminate controls from patients with EOC with Receiver Operator Characteristics Area Under the Curve values of 0.998 and bootstrap .632+ validated classification errors of 3.1% and 2.8%, respectively. The sensitivities were 97.8% and 95.6%, respectively, at a set specificity of 99.6%.ConclusionsThe combination of gene expression and plasma protein based blood derived biomarkers in one diagnostic model increases the sensitivity and the specificity significantly. Such a diagnostic test may allow earlier diagnosis of epithelial ovarian cancer.

The prognostic value of estrogen receptor beta and proline-, glutamic acid- and leucine-rich protein 1 (PELP1) expression in ovarian cancer

BackgroundProline-, glutamic acid-, and leucine-rich protein 1 (PELP1), a coregulator of the estrogen receptors (ERs) alpha and beta, is a potential proto-oncogene in hormone dependent gynecological malignancies. To better understand the role of PELP1 in epithelial ovarian cancer (EOC), the protein expression and prognostic significance of PELP1 was evaluated together with ERalpha and ERbeta in EOC tissues.MethodsThe expression of PELP1, ERalpha, and ERbeta was characterized in tumor tissues of 63 EOC patients. The prognostic value was calculated performing log-rank tests and multivariate Cox-Regression analysis. In a second step, validation analysis in an independent set of 86 serous EOC patients was performed.ResultsNuclear PELP1 expression was present in 76.2% of the samples. Prevalence of PELP1 expression in mucinous tumors was significantly lower (37.5%) compared to serous (85.7%) and endometrioid tumors (86.7%). A significant association between PELP1 expression and nuclear ERbeta staining was found (p=0.01). Positive PELP1 expression was associated with better disease-free survival (DFS) (p=0.004) and overall survival (OS) (p=0.04). The combined expression of ERbeta+/PELP1+ revealed an independent association with better DFS (HR 0.3 [0.1-0.7], p=0.004) and OS (HR 0.3 [0.1-0.7], p=0.005). In the validation set, the combined expression of ERbeta+/PELP1+ was not associated with DFS (HR 0.7 [0.4-1.3], p=0.3) and OS (HR 0.7 [0.3-1.4], p=0.3).ConclusionPositive immunohistochemical staining for the ER coregulator PELP1, alone and in combination with ERbeta, might be of prognostic relevance in EOC.

Gene expression of PMP22 is an independent prognostic factor for disease-free and overall survival in breast cancer patients

BackgroundGene expression of peripheral myelin protein 22 (PMP22) and the epithelial membrane proteins (EMPs) was found to be differentially expressed in invasive and non-invasive breast cell lines in a previous study. We want to evaluate the prognostic impact of the expression of these genes on breast cancer.MethodsIn a retrospective multicenter study, gene expression of PMP22 and the EMPs was measured in 249 primary breast tumors by real-time PCR. Results were statistically analyzed together with clinical data.ResultsIn univariable Cox regression analyses PMP22 and the EMPs were not associated with disease-free survival or tumor-related mortality. However, multivariable Cox regression revealed that patients with higher than median PMP22 gene expression have a 3.47 times higher risk to die of cancer compared to patients with equal values on clinical covariables but lower PMP22 expression. They also have a 1.77 times higher risk to relapse than those with lower PMP22 expression. The proportion of explained variation in overall survival due to PMP22 gene expression was 6.5% and thus PMP22 contributes equally to prognosis of overall survival as nodal status and estrogen receptor status. Cross validation demonstrates that 5-years survival rates can be refined by incorporating PMP22 into the prediction model.ConclusionsPMP22 gene expression is a novel independent prognostic factor for disease-free survival and overall survival for breast cancer patients. Including it into a model with established prognostic factors will increase the accuracy of prognosis.