A Simplified Real-Time PCR Method for the Quantification of Minimal Residual Disease in Follicular Lymphoma.
Authors: Pennell Nancy , Woods Anthony , Buckstein Rena , Imrie Kevin , Spaner David , Reis Marciano , Berinstein Neil 
Published: April 2020

Abstract
High-dose therapy and autologous stem-cell transplantation (HDT/ASCT) is associated with long remissions in relapsed follicular lymphoma (FL). Molecular remission in the graft and post ASCT is predictive of durability of remission. Thus, a simple, reliable method of quantitation of minimal residual disease (MRD) would improve prognostication in these patients. Real-time polymerase chain reaction (PCR) can quantitatively detect FL markers in blood and BM with a reproducible sensitivity of 0.01%. Fluorescent probe systems are used to perform real-time PCR; however, these techniques are expensive and do not allow melt curve analysis of the PCR product. We describe here a less expensive SYBR green detection method which also enables us to check clonal identity, and yields sensitive, reliable quantitative results for monitoring MRD. We compared our real-time PCR results with previously determined conventional nested PCR results in a series of samples collected from patients enrolled in one of our clinical trials.
Methods: Patients with relapsed FL (n=14) were enrolled in a prospective study of HDT/ASCT followed by maintenance Interferon-a 3x106 U/m2 SC 3 x weekly for 2 yrs. DNA extracts were collected at enrolment from samples of lymph node, BM and blood to determine detection of BCL2/JH translocation. Stem-cell graft DNA and serial post-ASCT blood and BM DNA samples were collected. BCL2/JH was qualitatively determined by nested PCR using standard primers and analyzed by gel electrophoresis. For patients in whom BCL2/JH was not detectable, clone-specific IgH region primers were used where possible. Quantitative PCR was performed using QuantiTect SYBR Green kits. Melt curve analysis was performed to confirm amplification specificity and clonal identity of each sample. All samples were analyzed in triplicate for accuracy.
Results: At a median follow-up of 75 months (9–91), 10/14 patients are alive. Of these, 3 are free from progressive disease. Median progression-free survival is 42 months; median overall survival has not been reached. We were successful in defining molecular markers for 12/14 patients. In 11/12, we were able to PCR amplify the BCL2/JH translocation. In one patient, allele-specific clonal IgH was analyzed with patient specific oligonucleotides. Median BCL2/JH contamination of stem-cell grafts was 0.1% (range 0.01% – 13%). 6 patients who had measurable graft contamination became molecularly negative in blood and BM within 12 months after ASCT. Increasing fractions of BCL2/JH positive blood and BM cells reliably predicted morphologic and clinical relapse. Correspondingly, the 3 patients free of disease progression have sustained very low levels of molecular evidence of FL. In one illustrative case, a patient had a long-term low-level persistence of tumour cells in BM and blood with no clinical symptoms for 70 months post-ASCT. At 76 months there was a 10-fold increase in the quantity of PCR product in both BM and blood. Radiological evidence of relapse was observed 3 months later. The patient underwent spontaneous disease regression, which was paralleled by a 10-fold decrease in PCR product quantity at 82 months.
Conclusions: Our simplified method of detection using quantitative PCR yields reliable measurements of graft contamination and MRD post ASCT, which may be useful in predicting patient relapse.