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.
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.
Ankara University, Biotechnology Institute, Ankara, Turkey.
Nilgun
N
Tekin
Department of Hematology, University of Cardiff School of Medicine, UK.
Nader
N
Omidvar
Medical Research Council (MRC) Clinical Trials Unit at University College (UCL), London, UK.
Tim Peter
TP
Morris
Facultad de Medicina Clínica Alemana - Universidad del Desarrollo Santiago, Chile.
Paulette
P
Conget
Facultad de Medicina Clínica Alemana - Universidad del Desarrollo Santiago, Chile.
Flavia
F
Bruna
Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
Botond
B
Timar
Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
Eva
E
Gagyi
Departments of Medical Oncology & Pathology, Tata Memorial Hospital, Mumbai, India.
Ranjan
R
Basak
Departments of Medical Oncology & Pathology, Tata Memorial Hospital, Mumbai, India.
Omkar
O
Naik
Chulabhorn Cancer Centre and Faculty of Medicine Siriraj Hospital, Bangkok, Thailand.
Chirayu
C
Auewarakul
Chulabhorn Cancer Centre and Faculty of Medicine Siriraj Hospital, Bangkok, Thailand.
Narongrit
N
Sritana
Laboratory of Genetics and Molecular Hematology (LIM31), University of São Paulo School of Medicine, São Paulo/SP, Brazil.
Debora
D
Levy
Department of Nuclear Medicine, Quanta - Diagnóstico e Terapia, Curitiba, Brazil.
Juliano Julio
JJ
Cerci
Laboratory of Genetics and Molecular Hematology (LIM31), University of São Paulo School of Medicine, São Paulo/SP, Brazil.
Sergio Paulo
SP
Bydlowski
Laboratory of Genetics and Molecular Hematology (LIM31), University of São Paulo School of Medicine, São Paulo/SP, Brazil.
Juliana
J
Pereira
Research and Biotechnology Division, St Luke's Medical Centre, Manila, Philippines.
Mark Pierre
MP
Dimamay
Research and Biotechnology Division, St Luke's Medical Centre, Manila, Philippines.
Filipinas
F
Natividad
Department of Nuclear Medicine, Seoul National University Hospital, Seoul, South Korea.
June-Key
JK
Chung
Ankara University, Biotechnology Institute, Ankara, Turkey.
Nevin
N
Belder
Department of Pathology, Ankara University School of Medicine, Ankara, Turkey.
Isinsu
I
Kuzu
Department of Nuclear Sciences and Application, Division of Human Health, International Atomic Energy Agency, Vienna, Austria.
Diana
D
Paez
Department of Nuclear Sciences and Application, Division of Human Health, International Atomic Energy Agency, Vienna, Austria.
Maurizio
M
Dondi
Department of Haematology, Guy's & St Thomas' Hospital, King's College, London, UK.
Robert
R
Carr
Ankara University, Biotechnology Institute, Ankara, Turkey.
Hilal
H
Ozdag
Institut National de la Sante et de la Recherche Médicale (INSERM) Unité 1131, Université Paris-Diderot, Institut Universitaire d'Hématologie, Hôpital Saint-Louis, Paris, France.
Rose Ann
RA
Padua
D000368:Aged; D001208:Asia; D014408:Biomarkers, Tumor / Q000235:genetics*; D001706:Biopsy; D018572:Disease-Free Survival; D005060:Europe; D005260:Female; D005404:Fixatives / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry*; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D020022:Genetic Predisposition to Disease; D006801:Humans; D053208:Kaplan-Meier Estimate; D016403:Lymphoma, Large B-Cell, Diffuse / Q000188:drug therapy / Q000235:genetics* / Q000401:mortality / Q000473:pathology; D008297:Male; D008875:Middle Aged; D016612:Paraffin Embedding; D010641:Phenotype; D011237:Predictive Value of Tests; D016016:Proportional Hazards Models; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards; D013020:South America; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*; D059467:Transcriptome
D014408:Biomarkers, Tumor; D005404:Fixatives; D005557:Formaldehyde
6-gene predictor score; diffuse large B-cell lymphoma (DLBCL); formalin fixed paraffin embedded tissue (FFPE)