Impact of biospecimens handling on biomarker research in breast cancer.

Author(s): De Cecco L, Musella V, Veneroni S, Cappelletti V, Bongarzone I, Callari M, Valeri B, Pierotti MA, Daidone MG

Publication: BMC Cancer, 2009, Vol. 9, Page 409

PubMed ID: 19930681 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to assess the effects of cold ischemia (the interim at room temperature between surgical excision and snap freezing) on RNA quality, and mRNA expression in frozen breast cancer specimens.

   Summary of Findings:

   Ischemia-dependent alterations in gene expression where observed in breast cancer specimens despite modest levels of RNA degradation. While the magnitude of change did not interfere with patient-specific microarray clustering, time-dependent in expression alterations (up- and down-regulation) were observed, with 0.76% genes modulated after 2 h and 4.1% after 24 h of ischemia compared to immediately frozen (0 h) case-matched controls. Affected genes included those functioning in inflammation, immunological disease and cell cycle. A comparison of microarray results with those reported for four other studies was conducted. Real-time quantitative RT-PCR analysis of 4 genes produced results that were significantly correlated to microarray results.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	DNA microarray
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	0 h 2 h 6 h 24 h
   Real-time qRT-PCR Specific	Targeted nucleic acid	FGFR4 FBLN2 ESR1 ERBB2

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2. Study Purpose

   The purpose of this study was to assess the effects of cold ischemia (the interim at room temperature between surgical excision and snap freezing) on protein phosphorylation status in frozen breast cancer specimens.

   Summary of Findings:

   Levels of phosphorylated ERBB2 and phosphotyrosine decreased progressively, declining to nondetectable levels after 24 h of cold ischemia as measured by Western blot. Although evidence of protein degradation, FAK levels, was also observed after 24 h of cold ischemia, orthovadate-mediated phosphatase inhibition attenuated the observed decrease in phosphorylation for specimens subjected to 2-6 h of cold ischemia but not 24 h, suggesting phosphoprotein levels may be influenced by protein degradation after 24 h of cold ischemia. Importantly, the authors note that ERBB2 gene expression was not altered in case-matched specimens.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	0 h 2 h 6 h 24 h
   Western blot Specific	Targeted peptide/protein	Phosphotyrosine ERBB2 FAK

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