Development of a Real-Time Reverse Transcription Polymerase Chain Reaction Assay for c-myc Expression That Allows the Identification of a Subset of c-myc+ Diffuse Large B-Cell Lymphoma

• Published in: Laboratory investigation 2003-02, Vol.83 (2), p.143-152
• Main Authors: Sáez, Ana-Isabel, Artiga, María-Jesús, Romero, Cristina, Rodríguez, Sandra, Cigudosa, Juan-Cruz, Pérez-Rosado, Alberto, Fernández, Isabel, Sánchez-Beato, Margarita, Sánchez, Esther, Mollejo, Manuela, Piris, Miguel Á
• Format: Article
• Language: English
• Subjects: B-Lymphocytes - pathology; Biological and medical sciences; Biomarkers, Tumor - analysis; Burkitt Lymphoma - genetics; Burkitt Lymphoma - metabolism; Burkitt Lymphoma - pathology; DNA, Complementary - analysis; DNA, Neoplasm - analysis; Genes, myc - genetics; Hematologic and hematopoietic diseases; Humans; In Situ Hybridization, Fluorescence; Laboratory Medicine; Leukemias. Malignant lymphomas. Malignant reticulosis. Myelofibrosis; Lymphoma, Large B-Cell, Diffuse - genetics; Lymphoma, Large B-Cell, Diffuse - metabolism; Lymphoma, Large B-Cell, Diffuse - mortality; Lymphoma, Large B-Cell, Diffuse - pathology; Medical sciences; Medicine; Medicine & Public Health; Neoplasm Proteins - analysis; Pathology; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - metabolism; Pseudolymphoma - genetics; Pseudolymphoma - metabolism; Pseudolymphoma - pathology; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Neoplasm - analysis; RNA, Ribosomal - analysis; Survival Rate
• ISSN: 0023-6837; 1530-0307
• DOI: 10.1097/01.LAB.0000057000.41585.FD

Absence of a reliable method for determining the level of c-myc expression has impeded the analysis of its biological and clinical relevance in tumors. We have standardized the conditions for a real-time reverse transcription polymerase chain reaction analysis for c-myc expression, including the selection of an endogenous reference (18S rRNA), the adequate number of measurements for each sample (2 cDNA in triplicate), and suitable controls for determining inter- and intrarun variability (standard curve and calibrator). Subsequently, in a series of 56 non-Hodgkin's lymphomas, we analyzed the expression of c-myc mRNA, using real-time reverse transcription polymerase chain reaction, and of other functionally related proteins (bcl-6, p27, cyclin D3, and p53). As expected, all eight Burkitt's lymphoma cases analyzed had high levels of c-myc mRNA expression compared with that observed in reactive lymphoid tissue. There was a wider range of expression in diffuse large B-cell lymphoma, with 30% (15 of 48) of cases overexpressing c-myc. This overexpression was largely independent of c-myc translocations (4 of 5), as demonstrated by fluorescence in situ hybridization. In this large B-cell lymphoma series, a high level of c-myc expression predicted lower survival probability, irrespectively of the International Prognostic Index risk group classification. A slightly increased frequency of p53 inactivation was observed in the cases with c-myc overexpression, which suggests a growth advantage in lymphomas with concurrent deregulation of c-myc and p53. In addition, a moderate increase in bcl-6 protein expression was observed in the c-myc–positive cases, suggesting the existence of a complex interrelationship between these two genes. These findings suggest that c-myc may play a relevant role in the pathogenesis of a subset of large B-cell lymphoma and suggest the existence of additional regulatory mechanisms of c-myc expression to c-myc rearrangements.