sybr-green-i and Lymphoma--Follicular

Molecular remission in the autograft and bone marrow after transplant are predictive of durable clinical remission in relapsed follicular lymphoma. Thus, a simple reliable method to quantify minimal residual disease (MRD) would improve prognostication in these patients. Fluorescent hybridization probes have been used in real-time quantitative polymerase chain reaction (RQ-PCR) to monitor MRD with a reproducible sensitivity of 0.01%; however, these techniques are expensive and require additional experiments to examine clonality. We describe a SYBR Green I detection method that is more universal, checks clonal identity, yields the same sensitivity for monitoring MRD, and is more economically attractive. Using this method to follow 14 follicular lymphoma patients treated with autologous stem cell transplantation, molecular markers were successfully defined for 12 patients. Median contamination of stem-cell grafts was 0.1% (range, 0 to 13%). Six patients with measurable graft contamination became PCR-negative in blood and bone marrow within 12 months after autologous stem cell transplantation. Three patients free of disease progression (median follow-up of 75 months) are in molecular remission. Increasing fractions of RQ-PCR-positive blood and bone marrow cells reliably predicted morphological and clinical relapse. In one case, both clinical relapse and spontaneous regression were reflected by changes in MRD levels. Thus, our RQ-PCR method reproducibly distinguishes different levels of MRD.

Topics: Adolescent; Adult; Benzothiazoles; Biomarkers, Tumor; Bone Marrow; Diamines; Fluorescent Dyes; Humans; Lymphoma, Follicular; Middle Aged; Neoplasm, Residual; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Sensitivity and Specificity; Stem Cell Transplantation; Transplantation, Autologous

Follicular lymphoma is characterized by the t(14;18)(q32;q21) translocation, which juxtaposes Ig heavy chain gene (IgH) sequences with the BclII gene. Several publications have highlighted the importance of molecular follow-up in follicular lymphoma, demonstrating that the detection of cells bearing the BclII-IgH rearrangement by real-time quantitative polymerase chain reaction (RQ-PCR) can anticipate a clinical relapse. In this context, we developed a BclII-IgH RQ-PCR. We began with SYBR Green I detection technology but observed that this system does not allow an accurate measurement of the tumor load when working with genomic DNA. While we were designing the assay using Taqman technology, Moppett et al (Moppett J, van der Velden VHJ, Wijkhuijs AJM, Hancock J, van Dongen JJM, Goulden N: Inhibition affecting RQ-PCR-based assessment of minimal residual disease in acute lymphoblastic leukemia: reversal by addition of bovine serum albumin. Leukemia 2003, 17:268-270) reported PCR inhibition problems in around 15% of blood and bone marrow samples, affecting the DNA quantification and thus the assessment of minimal residual disease. They demonstrated that this PCR inhibition could be partially resolved by adding nonacetylated bovine serum albumin. In our studies, we observed the same phenomenon in a single follicular lymphoma case and extended our study to other available cases. As a result, we suggest a new RQ-PCR procedure that is based on Taqman probe technology and that takes into account the PCR inhibition problems, making this assay more reliable in a routine molecular laboratory.

Topics: Aged; Benzothiazoles; Diamines; Gene Rearrangement; Humans; Immunoglobulin Heavy Chains; Lymphoma, B-Cell; Lymphoma, Follicular; Male; Neoplasm, Residual; Organic Chemicals; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Quinolines; Sensitivity and Specificity; Tumor Burden