Analysis of MRD by use of molecular-orientated methods

Currently, for solid malignancies there is no routinely established method to detect minimal residual disease, the first indicator of therapy failure and precursor of recurrence, which inevitably leaves specific traces through out the body. There is strong need for molecular-oriented research to detect minimal disease in order to disburden patients from inefficient and toxic therapies.

Introduction
In 2003 financial support (400.000,- €) was granted to the LBI of Gynecology and Gynecological Oncology for the research project “Molecular Diagnostics in Gynecologic Oncology: Identification and Expression Characterization of Disseminated Tumor Cells for Early Detection of Malignant Disease and Relapse” by the Ministery of Science in the scope of the Austrian GenAU program. This project centres on the detection of disseminated tumor cells in blood and other bodily fluids and the molecular characterization of these in patients with ovarian, breast and endometrial cancer. By comparative gene expression analysis gene shall be identified, which are solely expressed in tumor cells, but not in white blood cells. Most of putative marker genes described in the literature so far are not useful because of lack of sensitivity or specificity. Until now, 19 candidate genes were identified which are expressed in most of established cancer cell lines and primary tumors. In a second approach 382 additional candidate genes were selected from whole genome expression array experiments. Validation by highly sensitive PCR-based methods yielded a list of 92 candidate genes which were found to be expressed in primary tumors but not in white blood cells. Currently these genes are evaluated as markers for disseminated tumor cells in 150 preoperatively drawn blood samples of patients with ovarian, breast and endometrial cancer. Final results which will consist of a selection of genes suitable for routine diagnosis will be available during the second half of 2006.
This research activities are currently boosted by the financial support (4,26 Mio € in total) of the European Union. Within the OVCAD project (Ovarian Cancer Diagnosis), which is coordinated by Robert Zeillinger, 15 international partners will define the ideal molecular diagnosis of minimal residual disease in ovarian cancer.
The results of the GenAu project and the continuous information generated in the OVCAD project will be the basis for two collaborative projects in the scope of the LBI cluster, which are described below:

 

Aim
The aim of this project is to define clinically useful molecular-orientated early detection of minimal residual disease (MRD) in breast and ovarian cancer that can identify patients not responding to the standard (state-of–the-art) therapy already at the time of surgery or very early during disease progression. This will eventually lead to alternative therapy modalities, which can really bring benefits to this group of patients.
“Molecular Signatures” that signal the presence of MRD will be investigated at the genomic, transcriptomic and epigenomic level and in a broad spectrum of biological materials from breast and ovarian cancer patients.
Specific signatures that predict patients’ outcome or indicate minimal disease, eventually suitable for early primary diagnosis, will be extracted.

Pre-existing knowledge
This project is based on the findings of a currently ongoing project of the LBI for Gynecology and Gynecologic Oncology, which is supported within the scope of the GEN-AU program of the Austrian Ministry of Science. The GEN-AU project focuses on the selection of molecular markers for the detection of disseminated tumor cells in blood and other bodily fluids. Such markers are expressed and tumor cells have not in white blood cells. The methods for enrichment of tumor cells from blood and real-time RT-PCR based analytics are well established.
Further, in this project the pre-existing knowledge of the LBI of Stem cell Transplantation and of the LBI for Surgical Oncology on breast and colorectal cancer patients will be applied for the detection of disseminated tumor cells in bone marrow (Section 3.2.4.). Veronika Buxhofer is working as a research fellow at Prof. Radich’s lab at the Fred Hutchinson Cancer Research Centre in Seattle until the end of 2006. She will acquire additional know-how in MRD analysis. After returning to Vienna she will be able to use the equipment and infrastructure of the cluster.

Methods
15-25 ml whole blood and up to 300 ml of ascitic fluid or pleural effusion will be collected from breast and ovarian cancer patients. Disseminated tumor cells will be enriched by density gradient centrifugation (“OncoQuick”) (LIT OQ). After lysis of enriched cell fractions total RNA will be extracted, transcribed to cDNA, amplified and analysed by TaqMan Real-time RT-PCR in form of single-tube reactions or TLDA (TaqMan low density array). The latter allows the analysis of up to 384 genes in parallel, on principal.
Bone marrow aspirates will be collected from breast and colorectal cancer patients only. The specimens will be processed according to standard procedures and stained with a pan-cytokeratin antibody (A45-B/B3).

Workplan
Ovarian cancer
Access to blood and ascites specimens of about 40 ovarian cancer patients will be possible per year. After initial therapy the patients are examined for their disease status every three months. Blood samples will be enriched for possibly existing tumor cells and isolated RNA stored at -80°C. In the case of relapse of a patient the respective blood sample and all earlier collected samples will be analysed for disseminated tumor cells by Real-time RT-PCR. In total we expect to analyse samples from about 30-40 patients, since patients having their initial treatment before the start of this project can also be included.
Final results will consist of markers/marker combinations which allowed the detection of MRD before clinical manifestation of relapse, and the respective frequencies.

Breast cancer
About 100 patients with advanced breast cancer will be analysed. At the time of diagnosis of progressive disease blood and pleural effusions will be collected, as well as after (and during, if possible) chemotherapy. Samples will be processed as described above.
Final results will consist of markers /marker combinations which allowed the detection of MRD after chemotherapy, and the respective frequencies. These results will be compared with established parameters to assess therapy response.

In addition, bone marrow and blood samples of about 75 patients with primary breast cancer shall be investigated. Results from immunocytochemical analysis of bone marrow aspirates and data on disseminated tumor cells in blood will be compared.
Final results will consist of frequencies of tumor-cell positive bone marrow and blood samples and will describe the concordance of the two diagnostic approaches.

Colorectal cancer
Bone marrow and blood samples of about 75 patients with primary colorectal cancer will be investigated. Results from immunocytochemical analysis of bone marrow aspirates and data on disseminated tumor cells in blood will be compared.
Final results will consist of frequencies of tumor-cell positive bone marrow and blood samples and will describe the concordance of the two diagnostic approaches.