Quantitative nuclease protection assay in paraffin-embedded tissue replicates prognostic microarray gene expression in diffuse large-B-cell lymphoma

Robin A. Roberts, Constantine M. Sabalos, Michael L. LeBlanc, Ralph R. Martel, Yvette M. Frutiger, Joseph M. Unger, Ihab W. Botros, Matthew P. Rounseville, Bruce E. Seligmann, Thomas P Miller, Thomas M. Grogan, Lisa M Rimsza

Research output: Contribution to journalArticle

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Abstract

Gene expression profiling (GEP) has identified genes whose expression levels predict patient survival in diffuse large-B-cell lymphoma (DLBCL). Such discovery techniques generally require frozen samples unavailable for most patients. We developed a quantitative nuclease protection assay to measure expression levels of prognostic DLBCL genes using formalin-fixed, paraffin-embedded (FFPE) tissue. FFPE tissue was sectioned, permeabilized, denatured in the presence of specific probes, and hybridized to mRNA in situ. Nuclease subsequently destroyed non-hybridized probe. Alkaline hydrolysis freed mRNA-bound probes from tissue, which were transferred to ArrayPlates for probe capture and chemiluminescent quantification. We validated assay performance using frozen, fresh, and FFPE DLBCL samples, then used 39 archived DLBCL, previously microarray analyzed, to correlate GEP and ArrayPlate results. We compared old (>18 years) with new (<2 months) paraffin blocks made from previously frozen tissue from the original biopsy. ArrayPlate gene expression results were confirmed with immunohistochemistry for BCL2, BCL6, and HLA-DR, showing agreement between mRNA species and the proteins they encode. Assay performance was linear to ∼1 mg sample/well. RNase and DNase treatments demonstrated assay specificity for RNA detection, both fixed and soluble RNA detection. Comparisons were excellent for lysate vs snap-frozen vs FFPE (R 2>0.98 for all comparisons). Coefficients of variation for quadruplicates on FFPE were generally <20%. Correlation between new and old paraffin blocks from the same biopsy was good (R2=0.71). Comparison of ArrayPlate to Affymetrix and cDNA microarrays showed reasonable correlations. Insufficient power from small sample size prevented successfully correlating results with patient survival, although hazard ratios trended the expected directions. We developed an assay to quantify expression levels of survival prediction genes in DLBCL using FFPE, fresh, or frozen tissue. While this technique cannot replace GEP for discovery, it indicates that expression differences identified by GEP can be replicated on a platform applicable to archived FFPE samples.

Original languageEnglish (US)
Pages (from-to)979-997
Number of pages19
JournalLaboratory Investigation
Volume87
Issue number10
DOIs
StatePublished - Oct 2007

Fingerprint

Nuclease Protection Assays
Lymphoma, Large B-Cell, Diffuse
Paraffin
Formaldehyde
Gene Expression
Gene Expression Profiling
Survival
Messenger RNA
Oligonucleotide Array Sequence Analysis
Sample Size
Genes
Hydrolysis
Biopsy

Keywords

  • DLBCL
  • Gene expression profiling
  • Lymphoma
  • Nuclease protection assay
  • Paraffin-embedded tissue
  • Prognosis

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Quantitative nuclease protection assay in paraffin-embedded tissue replicates prognostic microarray gene expression in diffuse large-B-cell lymphoma. / Roberts, Robin A.; Sabalos, Constantine M.; LeBlanc, Michael L.; Martel, Ralph R.; Frutiger, Yvette M.; Unger, Joseph M.; Botros, Ihab W.; Rounseville, Matthew P.; Seligmann, Bruce E.; Miller, Thomas P; Grogan, Thomas M.; Rimsza, Lisa M.

In: Laboratory Investigation, Vol. 87, No. 10, 10.2007, p. 979-997.

Research output: Contribution to journalArticle

Roberts, RA, Sabalos, CM, LeBlanc, ML, Martel, RR, Frutiger, YM, Unger, JM, Botros, IW, Rounseville, MP, Seligmann, BE, Miller, TP, Grogan, TM & Rimsza, LM 2007, 'Quantitative nuclease protection assay in paraffin-embedded tissue replicates prognostic microarray gene expression in diffuse large-B-cell lymphoma', Laboratory Investigation, vol. 87, no. 10, pp. 979-997. https://doi.org/10.1038/labinvest.3700665
Roberts, Robin A. ; Sabalos, Constantine M. ; LeBlanc, Michael L. ; Martel, Ralph R. ; Frutiger, Yvette M. ; Unger, Joseph M. ; Botros, Ihab W. ; Rounseville, Matthew P. ; Seligmann, Bruce E. ; Miller, Thomas P ; Grogan, Thomas M. ; Rimsza, Lisa M. / Quantitative nuclease protection assay in paraffin-embedded tissue replicates prognostic microarray gene expression in diffuse large-B-cell lymphoma. In: Laboratory Investigation. 2007 ; Vol. 87, No. 10. pp. 979-997.
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AU - Frutiger, Yvette M.

AU - Unger, Joseph M.

AU - Botros, Ihab W.

AU - Rounseville, Matthew P.

AU - Seligmann, Bruce E.

AU - Miller, Thomas P

AU - Grogan, Thomas M.

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N2 - Gene expression profiling (GEP) has identified genes whose expression levels predict patient survival in diffuse large-B-cell lymphoma (DLBCL). Such discovery techniques generally require frozen samples unavailable for most patients. We developed a quantitative nuclease protection assay to measure expression levels of prognostic DLBCL genes using formalin-fixed, paraffin-embedded (FFPE) tissue. FFPE tissue was sectioned, permeabilized, denatured in the presence of specific probes, and hybridized to mRNA in situ. Nuclease subsequently destroyed non-hybridized probe. Alkaline hydrolysis freed mRNA-bound probes from tissue, which were transferred to ArrayPlates for probe capture and chemiluminescent quantification. We validated assay performance using frozen, fresh, and FFPE DLBCL samples, then used 39 archived DLBCL, previously microarray analyzed, to correlate GEP and ArrayPlate results. We compared old (>18 years) with new (<2 months) paraffin blocks made from previously frozen tissue from the original biopsy. ArrayPlate gene expression results were confirmed with immunohistochemistry for BCL2, BCL6, and HLA-DR, showing agreement between mRNA species and the proteins they encode. Assay performance was linear to ∼1 mg sample/well. RNase and DNase treatments demonstrated assay specificity for RNA detection, both fixed and soluble RNA detection. Comparisons were excellent for lysate vs snap-frozen vs FFPE (R 2>0.98 for all comparisons). Coefficients of variation for quadruplicates on FFPE were generally <20%. Correlation between new and old paraffin blocks from the same biopsy was good (R2=0.71). Comparison of ArrayPlate to Affymetrix and cDNA microarrays showed reasonable correlations. Insufficient power from small sample size prevented successfully correlating results with patient survival, although hazard ratios trended the expected directions. We developed an assay to quantify expression levels of survival prediction genes in DLBCL using FFPE, fresh, or frozen tissue. While this technique cannot replace GEP for discovery, it indicates that expression differences identified by GEP can be replicated on a platform applicable to archived FFPE samples.

AB - Gene expression profiling (GEP) has identified genes whose expression levels predict patient survival in diffuse large-B-cell lymphoma (DLBCL). Such discovery techniques generally require frozen samples unavailable for most patients. We developed a quantitative nuclease protection assay to measure expression levels of prognostic DLBCL genes using formalin-fixed, paraffin-embedded (FFPE) tissue. FFPE tissue was sectioned, permeabilized, denatured in the presence of specific probes, and hybridized to mRNA in situ. Nuclease subsequently destroyed non-hybridized probe. Alkaline hydrolysis freed mRNA-bound probes from tissue, which were transferred to ArrayPlates for probe capture and chemiluminescent quantification. We validated assay performance using frozen, fresh, and FFPE DLBCL samples, then used 39 archived DLBCL, previously microarray analyzed, to correlate GEP and ArrayPlate results. We compared old (>18 years) with new (<2 months) paraffin blocks made from previously frozen tissue from the original biopsy. ArrayPlate gene expression results were confirmed with immunohistochemistry for BCL2, BCL6, and HLA-DR, showing agreement between mRNA species and the proteins they encode. Assay performance was linear to ∼1 mg sample/well. RNase and DNase treatments demonstrated assay specificity for RNA detection, both fixed and soluble RNA detection. Comparisons were excellent for lysate vs snap-frozen vs FFPE (R 2>0.98 for all comparisons). Coefficients of variation for quadruplicates on FFPE were generally <20%. Correlation between new and old paraffin blocks from the same biopsy was good (R2=0.71). Comparison of ArrayPlate to Affymetrix and cDNA microarrays showed reasonable correlations. Insufficient power from small sample size prevented successfully correlating results with patient survival, although hazard ratios trended the expected directions. We developed an assay to quantify expression levels of survival prediction genes in DLBCL using FFPE, fresh, or frozen tissue. While this technique cannot replace GEP for discovery, it indicates that expression differences identified by GEP can be replicated on a platform applicable to archived FFPE samples.

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