Next-Generation Diagnostics

Development of novel biomarkers to diagnose and predict prostate cancer

Flow Cell for high-throughput sequencing
© Fraunhofer IZI

Flow Cell for high-throughput sequencing.

Semi-automated extraction of nucleic acids (RNA and DNA)
© Fraunhofer IZI

Semi-automated extraction of nucleic acids (RNA and DNA).

The changing demographic landscape is causing a steady rise in the number of oncological, chronic-inflammatory and degenerative diseases. Despite a similarly growing number of therapeutic options, treating these diseases often proves unsatisfactory. Personalized therapy can bring about fundamental progress here. For this to work, the molecular basis of a disease first needs to be precisely determined and the case-specific disease progression and response to therapy has to be predicted. Ever since the human genome was sequenced in full in 2001, the decoding of disease-relevant genes has opened up new options for developing tailor-made approaches to therapy. Alongside evidence of changes in DNA patterns (e.g. mutations), the investigation of RNA gene expression patterns by means of transcriptome-wide sequencing is increasingly shifting into focus.

As part of the RIBOLUTION project, funded by the Fraunhofer Future Foundation, new biomarkers were identified for prostate cancer based on transcriptome-wide (RNA) sequencing together with microarray analyses. Biomarkers were identified here that can diagnose the disease and also predict the aggressiveness of the cancer.

In order to validate and subsequently use these biomarkers for diagnostic purposes, a manageable number of biomarkers is to be identified using a simple test. To do this, the RNA Biomarker Unit has come up with a workflow for detecting diagnostic biomarkers in the urine, using quantitative real-time PCR (qPCR). For optimization purposes, suitable reference and target regions as well as primers and probes were tested in depth and the reaction conditions were adapted, among other things. For the assessment, the selected biomarkers were investigated in close cooperation with the Bioinformatics Unit using a specially developed algorithm.

For the more complicated issue of predicting the cancer, the Next-Generation Diagnostics Unit developed a workflow based on RNA sequencing from FFPE biopsy material. The aim here was to identify a broad spectrum of potential biomarkers in clinically available samples. In the interests of reducing time and costs, sequencing was optimized in terms of sensitivity and robustness. Based on this established method, the transcriptome-wide sequencing of a large patient cohort (n>150) is currently being carried out to validate the identified biomarkers.

The workflows developed in this project are to be transferred to other indications in the future.

RIBOLUTION

The "RIBOLUTION – integrated platform for the identification and validation of innovative RNA-based biomarkers for personalized medicine" project is a research association supported by the “Fraunhofer-Zukunftsstiftung” (Fraunhofer Future Foundation). The project is coordinated by Prof. Dr. Friedemann Horn at Fraunhofer IZI.

New RNA biomarkers for complex disorders (e.g. oncological, chronic-inflammatory and degenerative disorders) are being identified in the RIBOLUTION project by genome-wide screening processes. RIBOLUTION also includes so-called non-coding RNAs whose significance as potential biological markers have only been investigated in the recent past. Additionally, the process of the biological marker screening by RIBOLUTION is optimized and perfected with the help of technical innovations.

Subsequent to the screening phase, potential biomarkers are evaluated with regard to their diagnostic and prognostic significance. Biomarkers with the ability to indicate a disorder or its course or forecast the response to therapies are of special interest.

Endocarditis

In cooperation with the Heart Center Leipzig and the Institute for Microbiology of the University of Leipzig, the topic of improved pathogen diagnostics on infectious endocarditis is being investigated.

By means of genome-wide sequencing, pathogens are identified both directly on the infected cardiac valves as well as in the blood of the patient and compared with the findings of the microbiological analysis laboratory.

In addition to the already available microbiological analyses, the objective is to devise an optimized treatment strategy for every individual patient.

Mammary carcinoma

In a joint project with the University Gynecological Hospital Essen, genes are examined that are relevant for the formation and metastatic spread of mammary carcinoma, with respect to their expression pattern and the occurrence of mutations.

The unit developed a special DNA capture approach that permits disease-relevant DNA sections from primary tumors and metastases of patient to be selectively enriched and subsequently sequenced. Simultaneously at the Essen Hospital, circulating tumor cells (CTCs) are isolated from patient blood and the expression of mammary carcinoma-relevant genes examined by means of PCR.

The data from these studies can show how primary tumors and metastases differ in their mutation and expression patterns. Moreover, the project investigates whether CTCs can be employed for the diagnosis of mammary carcinoma and deliver indications for a target-specific treatment. As a result, complicated surgical removal of tumor tissues could become redundant.

Functional analyses for the role of non-coded RNAs in multiple myeloma, glioblastoma and Alzheimer's disease

In the past years, genome-wide next-generation sequencing has identified a multitude of non-protein coded RNAs (ncRNAs). Many of these ncRNAs apparently influence the formation and course of diseases. Nevertheless, a large part of these transcripts are hardly functionally characterized.

The main focus of this project therefore is comprehensive functional analyses of these ncRNAs in various immunological and neurologically relevant cell systems. With the selective knockdown of an ncRNA, subsequent examinations allow the determination of their influence on vitality, apoptosis rate, proliferation, adhesion and cell migration. Furthermore, the expression and localization of ncRNAs within tissues, but also on the single-cell level, can be visualized and quantified. In vivo interaction studies have deepened the identification of ncRNA-binding partners on the RNA, DNA and protein level. The objective is to clarify the molecular and cellular therapeutic mechanism of ncRNAs to better understand their role in diverse disease models and to establish possible therapeutic approaches.

Completed project: Diagnostics of circulating tumor cells

This SAB- funded R&D association project was carried out by ApoCell Europe GmbH together with various units at Fraunhofer IZI. The objective was to develop innovative technologies for collecting and characterizing circulating tumor cells, to support the development of significantly improved tumor diagnostics and innovative therapies.