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Duke and Response Genetics Collaborate to Validate Expression Signatures from Fixed Tissue

A team of Duke scientists, led by Dr. David Harpole, are collaborating with service provider, Response Genetics, to profile whole-genome expression from FFPE tumor samples on Affymetrix microarrays. The collaborators are now validating molecular signatures predictive of cancer susceptibility, onset, progression and outcome; they plan to initiate multiple patient treatment trials using the models.

In a recent interview, Dr. Harpole relayed some of his experiences profiling RNA from clinical FFPE samples. The interview centered on the topics of 1) tissue availability, 2) protocol standardization and sample variability and 3) improvements in nucleic acid isolation and microarray technology.

Availability of Tissue
One of the biggest advantages of using FFPE samples is that there are estimates of up to 1 million samples banked worldwide, and many of those samples have documented clinical outcomes.

Harpole: Currently, there are challenges encountered by many fresh tissue-based microarray models. They typically require substantial amounts of fresh or frozen tissue and although many large institutions now maintain such banks, few of these have the necessary clinical follow-up data required

David Harpole Bio

for meaningful analysis. To date, there still remains a lack of standardization regarding the handling and processing of fresh or frozen tissue from institution to institution. However, it is widely accepted that there is a vast supply of formalin-fixed, paraffin-embedded tissue for which the clinical outcome is already known. In addition, the collection and storage of this tissue is standard routine practice in pathology laboratories across the country.

Standardized Methods
Minimizing sample variability through standardized tissue preservation methods is of key interest to the group.

Harpole: Although most pathology follows the Armed Forces Institute of Pathology (AFIP) standards for fixation, much variation still exists with

respect to the time the tissue is allowed to remain
in the fixative. We are evaluating these differences
to obtain the optimal fixation period for the preservation of genetic information.

Technology Improvements
Harpole’s group is committed to further advancing the clinical applications of FFPE tissue molecular profiling by developing optimized protocols for fixed-tissue microarray analysis.

Harpole: Since FFPE tissue is routinely
collected using standardized methods in pathology laboratories across the country, making the combination of FFPE tumor RNA with gene array technology seemed like a logical and critical next step to the application of molecular signatures as a broadly applicable clinical decision-making tool.

We have been doing molecular diagnostics on
FFPE tissue for 15 years, including gene expression with real-time rt-PCR and immunohistochemistry.
We were some of the early investigators involved
with the translation of fresh frozen array technology into a paraffin-based platform. Some of our work was presented at ASCO 2006. This process is evolving with much improved RNA yields today compared with last year.
FOR MORE INFORMATION

Contact:
David Harpole, M.D.
Principal Investigator
Duke University Medical Center
Box 3617
Durham, NC 27710

Mary-Beth M. Joshi
Duke Lung Cancer Prognostics Laboratory
Laboratory Manager
Duke University Medical Center
Box 3617
Durham, NC 27710

Companies:
Response Genetics Inc.

Organizations:
Duke University Medical Center
Armed Forces Institute for Pathology

Further Reading:
Joshi, et al. Differential Canonical Pathways Derived from Microarrays Using RNA from Paraffin-Embedded Non-Small Cell Lung Cancer Tissue. Poster Presentation, American Society of Clinical Oncology, General Meeting (2006).

Dressman, et al. Genomic signatures in non-  small-cell lung cancer: targeting the targeted
 therapies. Curr Oncol Rep 8:252-7 (2006).

Bild, et al. Oncogenic pathway signatures in   human cancers as a guide to targeted therapies. Nature 439:353-7 (2006).





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