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Molecular Biology

Our Services

  • Our sample repository includes DNA and RNA extracts from the tumor and cell line collection.
  • Delivery of Affymetrix data and mutational data to guide tumor selection for in vitro and in vivo studies.
  • Biomarkers of interest may be investigated additionally by quantitative PCR (RT-qPCR) or protein analysis.

Your Benefit

Oncotest offers a comprehensive collection of molecular biology services, sample repositories as well as molecular characterization data of the patient-derived tumor and cell line collection. The sample repository includes purified DNA and RNA available upon request. Affymetrix whole genome expression as well as mutational data are available for guiding the tumor selection for respective studies. Individual genes of interest may be investigated by quantitative RT-PCR (qPCR). Affymetrix gene expression data in context of in vivo efficacy results are used to identify predictive gene signatures to be used for tumor type and patient stratification in clinical trials. We are open to respond to individual requests in terms of mutational analysis or new genomic profiling arrays.

Genomic, array-based analyses

Gene expression analysis
Affymetrix data based on the HG U133 Plus 2.0 array are currently available for more than 250 tumors and respective cell lines, used for example to analyze target expression in order to guide model selection, and for delineation of predictive gene signatures.

As ONCOTEST tumors grow as direct patient implants in nude mice, the malignant phenotype and tumor sensitivity to anti-cancer drugs is preserved.
Testing therapeutic compounds therefore guarantees a high degree of clinical validity. Affymetrix data combined with in vivo results allows to identify gene signatures used to predict tumor responses towards multiple standard-of-care (SOC) drugs. As sensitive and resistant patient-derived xenografts were correctly predicted, this strategy is suitable for patient stratification in clinical trials. Typically, any SOC or investigational drug is analyzed in vivo in a training set of ~ 50 patient-derived tumor xenografts.

 SNP/ CNV Analysis
We are currently profling the ONCOTEST tumor collection using the Affymetrix SNP V6.0 array bearing copy number and genotyping probes. This array based analysis allows to quantify copy number variations (CNV) of selected genes as well as the identification of single nucleotide polymorphisms (SNPs) and data are available soon.

affy

Gene signatures consist of 20 to 100 genes. They are validated by the leave-oneout crossvalidation and an independent „validation set“ of about 25 additional tumor models. Data for standard drugs like Cyclophosphamide have been published and studies with targeted agents like Avastin and Erbitux have been carried out.

Mutational Analysis

To identify mutations in specific genes, the relevant exons are amplified by PCR using genomic DNA and proofreading Taq polymerase. Both DNA strands of the amplification product are sequenced using the “dye terminator cycle sequencing” method. Comprehensive mutational analysis has already been carried out for EGFR, K- and H-ras, B-raf, Akt1 and PI3K. New relevant drug targets and pathways are currently being studied. As an example, the response to the EGFR targeting antibody Erbitux (Cetuximab) was correlated to K-ras mutational status as well as ligand expression.

Response to Erbitux treatment, KRAS status and EGFR ligand expression

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The in vivo response towards Erbitux treatment, K-ras mutational status and gene expression of EGFR ligands Epiregulin (EREG) and Amphiregulin (AREG) is plotted for 74 patient-derived tumor models. Tumors with wild-type K-ras status are distributed within the left two quadrants, with mutant K-ras within the right two quadrants. EREG and AREG expression as determined by Affymetrix arrays analysis is plotted on the y-axis, with the horizontal line representing the median ligand expression level. Tumors responding strongly to Erbitux treatment (optimal tumor/control (T/C) values 37%) are indicated in red. Comparable to the clinical situation, in most cases only tumors with wild-type K-ras and high ligand expression strongly respond to Erbitux therapy.

Quantitative PCR (qRT-PCR)

qRT-PCR We offer qPCR analysis of a target of choice using RNA from our RNA repository. As an example, the tumor stroma in xenograft tumors was evaluated by traditional histology and species-discriminating qPCR. A very good correlation was seen with murine tissue content ranking from 2% to 65% in this experiment.