This study evaluated the effectiveness of using high resolution melting curve analysis (HRM) to detect immunoglobulin heavy chain gene rearrangement (FR3) in malignant lymphoma samples from Thailand. DNA samples from 26 cases of B-cell non-Hodgkin lymphoma were analyzed using PCR and HRM. The results showed that HRM can detect monoclonal bands in samples with as little as 3.125% tumor cells. HRM was found to be a low-cost, rapid and simple method for diagnosing malignant lymphoma.

1. DANIEL PALACIO GÓMEZ MEDICINE STUDENT

2. INTRODUCTION Kummalue et al ., Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis Diagnostic Pathology 2010, 5:31 DETECTION OF MONOCLONAL IMMUNOGLOBULIN HEAVY CHAIN GENE REARRANGEMENT (FR3) IN THAI MALIGNANT LYMPHOMA BY HIGH RESOLUTION MELTING CURVE ANALYSIS Immunoglobulin: Plasma protein that acts as an antibody . It can be found in the serum and other body tissues. There are five types: IgA, IgD, IgE, IgG, IgM. Monoclonal Antibody: Antibody produced by a cell source to a specific antigen. It is mainly used for diagnostic purposes. Lymphoma: Neoplasia of lymphoid tissue, in some cases benign, but in general it’s from malignant nature. FR3: Immunoglobulin heavy chain (IgH) gene region.

3. GENERAL OBJECTIVE Kummalue et al ., Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis Diagnostic Pathology 2010, 5:31 DETECTION OF MONOCLONAL IMMUNOGLOBULIN HEAVY CHAIN GENE REARRANGEMENT (FR3) IN THAI MALIGNANT LYMPHOMA BY HIGH RESOLUTION MELTING CURVE ANALYSIS Determine the effectiveness of High Resolution Melting curve analysis (HRM) as a tool in the detection of immunoglobulin heavy chain gene rearrangement (FR3) for the diagnosis of malignant lymphoma

6. MATERIALES Y MÉTODOS Kummalue et al ., Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis Diagnostic Pathology 2010, 5:31 DETECTION OF MONOCLONAL IMMUNOGLOBULIN HEAVY CHAIN GENE REARRANGEMENT (FR3) IN THAI MALIGNANT LYMPHOMA BY HIGH RESOLUTION MELTING CURVE ANALYSIS PCR: Método de amplificación enzimática de un gen o un fragmento específico de DNA o RNA . Se utilizó para obtener las secuencias de DNA conservadas que circundaban la única region hipervariable del antígeno de unión.

7. MATERIALES Y MÉTODOS DETECTION OF MONOCLONAL IMMUNOGLOBULIN HEAVY CHAIN GENE REARRANGEMENT (FR3) IN THAI MALIGNANT LYMPHOMA BY HIGH RESOLUTION MELTING CURVE ANALYSIS Kummalue et al ., Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis Diagnostic Pathology 2010, 5:31 Electroforesis con Gel de Poliacrilamida (PAGE): Método de estudio basado en el movimiento de las partículas cargadas y suspendidas en un medio líquido, en respuesta a los cambios de un campo eléctrico. Se utilizó para evaluar el porcentaje mínimo al cual se detectaba el DNA monoclonal con inmunoglobulina de cadena pesada (IgH).

8. RESULTADOS Kummalue et al ., Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis Diagnostic Pathology 2010, 5:31 DETECTION OF MONOCLONAL IMMUNOGLOBULIN HEAVY CHAIN GENE REARRANGEMENT (FR3) IN THAI MALIGNANT LYMPHOMA BY HIGH RESOLUTION MELTING CURVE ANALYSIS Figure 2 Minimal detection of polyacrylamide gel electrophoresis analysis and ethidium bromide staining of monoclonal IgH gene rearrangement . The distinct band consistent with monoclonal IgH gene rearrangement could be detected at concentration as low as 3.125%. Lane 1 = GeneRuler 50 bp DNA ladder marker, Lane 2 = positive control, Lane 3 = negative control, Lane 4-10 = serial dilutions 50%, 25%, 12.5%, 6.25%, 3.125%, 1.56%, and 0.78%, respectively.

9. RESULTADOS Kummalue et al ., Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis Diagnostic Pathology 2010, 5:31 DETECTION OF MONOCLONAL IMMUNOGLOBULIN HEAVY CHAIN GENE REARRANGEMENT (FR3) IN THAI MALIGNANT LYMPHOMA BY HIGH RESOLUTION MELTING CURVE ANALYSIS Figure 5 IgH gene rearrangement showing the distinct band at 100-170 bp on ethidium bromide stained acrylamide gel. (arrow) M = GeneRuler 50 bp DNA Ladder Marker (Fermentas), PT = Positive control, NT = Negative control, 1-5 = Patients samples.

10. DISCUSSION Kummalue et al ., Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis Diagnostic Pathology 2010, 5:31 DETECTION OF MONOCLONAL IMMUNOGLOBULIN HEAVY CHAIN GENE REARRANGEMENT (FR3) IN THAI MALIGNANT LYMPHOMA BY HIGH RESOLUTION MELTING CURVE ANALYSIS RESEARCHER ARGUMENT CHECK Kiyoi H, Naoe T. “ Immunoglobulin heavy chain gene rearrangement has been widely used for diagnosis on B cell monoclonality” Agree Uemura A, Mori S, Sugahara K, et al. “ From a previous report, using PCR-MCA (LightCycler Technology with high-speed amplification, and Idaho-Technology with rapid and high-resolution melting curve analysis- MCA) showed high sensitivity and specificity at 89.2% and 100% respectively” Agree Retamales E, Rodriguez L, Guzman L, et al. “ Another study of 10 gastric MALT lymphomas using melting curve analysis with SYBR Green I showed similar sensitivity and specificity at 95.2% and 100% respectively” Agree Lyon E, Wittwer CT. “ LightCycler technology has been used in many areas of clinical testing because of its rapid turn around time and accuracy. High Resolution Melting curve analysis, which is faster, simpler and less expensive, was developed as the latest method for product analysis in molecular diagnostics” Agree

12. DETECTION OF MONOCLONAL IMMUNOGLOBULIN HEAVY CHAIN GENE REARRANGEMENT (FR3) IN THAI MALIGNANT LYMPHOMA BY HIGH RESOLUTION MELTING CURVE ANALYSIS Kummalue et al ., Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis Diagnostic Pathology 2010, 5:31