Research Papers:

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

Nilgun Tekin _, Nader Omidvar, Tim Peter Morris, Paulette Conget, Flavia Bruna, Botond Timar, Eva Gagyi, Ranjan Basak, Omkar Naik, Chirayu Auewarakul, Narongrit Sritana, Debora Levy, Juliano Cerci, Sergio Paulo Bydlowski, Juliana Pereira, Mark Pierre Dimamay, Filipinas Natividad, June-Key hung, Nevin Belder, Isinsu Kuzu, Diana Paez, Maurizio Dondi, Robert Carr, Hilal Ozdag and Rose Ann Padua

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Oncotarget. 2016; 7:83319-83329. https://doi.org/10.18632/oncotarget.13066

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Nilgun Tekin1,*, Nader Omidvar2,**, Tim Peter Morris3,**, Paulette Conget4, Flavia Bruna4, Botond Timar5, Eva Gagyi5, Ranjan Basak6, Omkar Naik6, Chirayu Auewarakul7, Narongrit Sritana7, Debora Levy8, Juliano Julio Cerci9, Sergio Paulo Bydlowski8, Juliana Pereira8, Mark Pierre Dimamay10, Filipinas Natividad10, June-Key Chung11, Nevin Belder1, Isinsu Kuzu12, Diana Paez13, Maurizio Dondi13, Robert Carr14, Hilal Ozdag1,**,#, Rose Ann Padua15,**,#

1Ankara University, Biotechnology Institute, Ankara, Turkey

2Department of Hematology, University of Cardiff School of Medicine, UK

3Medical Research Council (MRC) Clinical Trials Unit at University College (UCL), London, UK

4Facultad de Medicina Clínica Alemana - Universidad del Desarrollo Santiago, Chile

5Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary

6Departments of Medical Oncology & Pathology, Tata Memorial Hospital, Mumbai, India

7Chulabhorn Cancer Centre and Faculty of Medicine Siriraj Hospital, Bangkok, Thailand

8Laboratory of Genetics and Molecular Hematology (LIM31), University of São Paulo School of Medicine, São Paulo/SP, Brazil

9Department of Nuclear Medicine, Quanta - Diagnóstico e Terapia, Curitiba, Brazil

10Research and Biotechnology Division, St Luke’s Medical Centre, Manila, Philippines

11Department of Nuclear Medicine, Seoul National University Hospital, Seoul, South Korea

12 Department of Pathology, Ankara University School of Medicine, Ankara, Turkey

13Department of Nuclear Sciences and Application, Division of Human Health, International Atomic Energy Agency, Vienna, Austria

14Department of Haematology, Guy’s & St Thomas’ Hospital, King’s College, London, UK

15Institut National de la Sante et de la Recherche Médicale (INSERM) Unité 1131, Université Paris-Diderot, Institut Universitaire d'Hématologie, Hôpital Saint-Louis, Paris, France

*Present address: Heinrich-Pette Institute, Leibniz Institute for Experimental Virology, Department of Viral Transformation, Hamburg, Germany

**These authors have contributed equally to this work

#These authors should be considered as co-senior author

Correspondence to:

Rose Ann Padua, email: [email protected]

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

Received: November 10, 2015    Accepted: September 24, 2016    Published: November 04, 2016


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.

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