Dataset Information

Preservation of biomolecules in breast cancer tissue by a formalin-free histology system.

ABSTRACT: BACKGROUND:The potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer - estrogen receptor and HER2 - at the RNA and protein levels. METHODS:Parallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR. RESULTS:The immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p < 0.05). Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05). The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative. Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens. CONCLUSION:The formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.

SUBMITTER: Nassiri M

PROVIDER: S-EPMC2267798 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Preservation of biomolecules in breast cancer tissue by a formalin-free histology system.

Nassiri Mehdi M Ramos Sharon S Zohourian Hajir H Vincek Vladimir V Morales Azorides R AR Nadji Mehrdad M

BMC clinical pathology 20080129

<h4>Background</h4>The potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer - estrogen receptor and HER2 - at the RNA and protein levels.<h4>Methods</h4>Parallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fi ...[more]

PMID: 18230182

Similar Datasets

Project description:BackgroundTissue handling can alter global gene expression potentially affecting the analytical performance of genomic signatures, but such effects have not been systematically evaluated.MethodsTissue samples from 11 previously untreated breast tumors were minced and aliquots were either snap frozen or placed in RNAlater immediately or after 20, 40, 60, 120 or 180 min at room temperature. RNA was profiled on Affymetrix HG-U133A arrays. We used probe-set-wise hierarchical models to evaluate the effect of preservation method on transcript expression and linear mixed effects models to assess the effect of cold ischemic delay on the expression of individual probe sets. Gene set enrichment analysis identified pathways overrepresented in the affected transcripts. We combined the levels of 41 most sensitive transcripts to develop an index of ischemic stress.ResultsConcordance in global gene expression between the baseline and 40 min delay was higher for samples preserved in RNAlater (average concordance correlation coefficient CCC = 0.92 compared to 0.88 for snap frozen). Overall, 481 transcripts (3%) were significantly affected by the preservation method, most of them involved in processes important in cancer. Prolonged cold ischemic delay of up to 3 h induced marginal global gene expression changes (average CCC = 0.90 between baseline and 3 h delay). However 41 transcripts were significantly affected by cold ischemic delay. Among the induced transcripts were stress response genes, apoptotic response genes; among the downregulated were genes involved in metabolism, protein processing and cell cycle regulation. An index combining the expression levels of these genes was proportional to the cold ischemic delay.ConclusionsProlonged cold ischemia induces significant transcriptional changes in a small subset of transcripts in the tissue. Furthermore, the expression level of about 3% of the transcripts is affected by the preservation method. These sensitive transcripts should not be included in genomic signatures for more reliable analytical performance.

Project description:We evaluated (a) the feasibility of whole genome cDNA-mediated Annealing, Selection, extension and Ligation (DASL) assay on formalin-fixed paraffin-embedded (FFPE) tissue and (b) whether similar conclusions can be drawn by examining FFPE samples as proxies for fresh frozen (FF) tissues. We used a whole genome DASL assay (addressing 18,391 genes) on a total of 72 samples from paired breast tumor and surrounding healthy tissues from both FF and FFPE samples.Gene detection was very good with comparable success between the FFPE and FF samples. Reproducibility was also high (r2 = 0.98); however, concordance between the two types of samples was low. Only one-third of the differentially expressed genes in tumor tissues (compared to corresponding normal) from FF samples could be detected in FFPE samples and conversely only one-fourth of the differentially expressed genes from FFPE samples could be detected in FF samples. GO-enrichment analysis, gene set enrichment analysis (GSEA) and GO-ANOVA analyses also suggested small overlap between the lead functional groups that were differentially expressed in tumor detectable by examining FFPE and FF samples. In other words, FFPE samples may not be ideal for picking individual target gene(s), but may be used to identify some of the lead functional group(s) of genes that are differentially expressed in tumor. The differentially expressed genes in breast cancer found in our study were biologically meaningful. The "cell cycle" & "cell division" related genes were up-regulated and genes related to "regulation of epithelial cell proliferation" were down-regulated.Gene expression experiments using the DASL assay can efficiently handle fragmentation issues in the FFPE tissues. However, formalin fixation seems to change RNA and consequently significantly alters gene expression in a number of genes which may not be uniform between tumor and normal tissues. Therefore, considerable caution needs to be taken when interpreting gene expression data from FFPE tissues, especially in relation to specific genes.

Dataset Information

Preservation of biomolecules in breast cancer tissue by a formalin-free histology system.

Publications

Preservation of biomolecules in breast cancer tissue by a formalin-free histology system.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure