One of the most promising approaches for understanding the molecular pathogenesis of cancer is to relate genetic changes, such as mutation or altered gene expression, to metastasis, treatment outcome, and survival, using high-throughput molecular biologic and proteomic methods. In cancers where the time between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations must be obtained with formalin-fixed paraffin-embedded (FFPE) tissues. However, large-scale multiplex techniques, such as proteomic analysis, serial analysis of gene expression, and gene chip methods using FFPE tissue have previously proven unsuccessful. The long-term goal of our research program is to improve public health by using high-throughput proteomic and molecular biologic screening methods to identify the molecular and genetic signatures of cancer. The objective of this proposal is to employ tissue surrogates to identify the formaldehyde-induced chemical modifications to proteins that occur during normal histologic tissue processing and to develop methods to reverse these modifications. Tissue surrogates are highly concentrated solutions of proteins that form tissue-like plugs following formaldehyde-induced intermolecular crosslinking. These plugs can be histologically processed like normal tissue. Our central hypothesis is that formaldehyde adducts and cross-links formed during histologic tissue processing can be sequentially reversed by a carefully designed series of heating, dialysis, rehydration, and protein renaturation steps, carried out under appropriate solvation conditions. We have formulated this hypothesis on the basis of our strong preliminary data, which have shown that the reversal of formaldehyde-induced chemical changes to proteins is relatively facile in aqueous solutions but requires a different approach for tissue that has been dehydrated in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for high-throughput proteomic screening of FFPE tissues. This will improve practical interventions for the diagnosis, treatment, and prevention of cancer and will facilitate the development of therapeutic agents. Our studies are innovative because we have developed a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins that occur during histologic processing. At the completion of this project, it is our expectation to have established a comprehensive understanding of the protein modifications that occur during tissue histology, together with methods for optimally reversing these modifications. This knowledge should result in the ability to carry out proteomic analysis using FFPE tissue.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
5R33CA107844-02
Application #
6867395
Study Section
Special Emphasis Panel (ZCA1-SRRB-C (J1))
Program Officer
Liddell Huppi, Rebecca
Project Start
2004-04-01
Project End
2007-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
2
Fiscal Year
2005
Total Cost
$181,539
Indirect Cost
Name
American Registry of Pathology, Inc.
Department
Type
DUNS #
114400633
City
Washington
State
DC
Country
United States
Zip Code
20306
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O'Leary, Tj; Fowler, Cb; Evers, Dl et al. (2009) Protein fixation and antigen retrieval: chemical studies. Biotech Histochem :1-5
Fowler, Carol B; O'Leary, Timothy J; Mason, Jeffrey T (2008) Modeling formalin fixation and histological processing with ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde cross-links. Lab Invest 88:785-91
Fowler, Carol B; Cunningham, Robert E; Waybright, Timothy J et al. (2008) Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates. Lab Invest 88:185-95
Evers, David L; Fowler, Carol B; Cunningham, Robert E et al. (2007) A novel HPLC method reveals that precipitation of 2'-deoxyadenosine 5'-monophosphate with lithium perchlorate/acetone leads to base depurination. Anal Biochem 370:255-7
Fowler, Carol B; Cunningham, Robert E; O'Leary, Timothy J et al. (2007) 'Tissue surrogates'as a model for archival formalin-fixed paraffin-embedded tissues. Lab Invest 87:836-46
Rait, Vladimir K; Zhang, Qingrong; Fabris, Daniele et al. (2006) Conversions of formaldehyde-modified 2'-deoxyadenosine 5'-monophosphate in conditions modeling formalin-fixed tissue dehydration. J Histochem Cytochem 54:301-10