Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score

Oncotarget. 2016 Dec 13;7(50):83319-83329. doi: 10.18632/oncotarget.13066.

Abstract

As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.

Keywords: 6-gene predictor score; diffuse large B-cell lymphoma (DLBCL); formalin fixed paraffin embedded tissue (FFPE).

Publication types

  • Multicenter Study
  • Validation Study

MeSH terms

  • Aged
  • Asia
  • Biomarkers, Tumor / genetics*
  • Biopsy
  • Disease-Free Survival
  • Europe
  • Female
  • Fixatives / chemistry*
  • Formaldehyde / chemistry*
  • Gene Expression Profiling / methods*
  • Gene Expression Profiling / standards
  • Genetic Predisposition to Disease
  • Humans
  • Kaplan-Meier Estimate
  • Lymphoma, Large B-Cell, Diffuse / drug therapy
  • Lymphoma, Large B-Cell, Diffuse / genetics*
  • Lymphoma, Large B-Cell, Diffuse / mortality
  • Lymphoma, Large B-Cell, Diffuse / pathology
  • Male
  • Middle Aged
  • Paraffin Embedding*
  • Phenotype
  • Predictive Value of Tests
  • Proportional Hazards Models
  • Real-Time Polymerase Chain Reaction* / standards
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction* / standards
  • South America
  • Time Factors
  • Tissue Fixation / methods*
  • Transcriptome*

Substances

  • Biomarkers, Tumor
  • Fixatives
  • Formaldehyde