Pb2376: assay development and validation of ig-hts to monitor mrd in plasma of patients with diffuse large b-cell lymphoma (dlbcl)

Topic: 20. Lymphoma Biology & Translational Research Background: Ongoing development of novel therapies in lymphoid malignancies has led to improvements in outcomes. However, relapses can occur after achievement of an imaging scan negative complete remission (CR). General for NHL, surveillance imaging does not improve outcomes and is associated with increased radiation exposure, high costs, and anxiety to patients. Therefore, there is an unmet need to early predict treatment refractoriness and relapse in order to improve treatment approaches before clinical or imaging evidence of disease. For other hematologic malignancies, such as leukemias, monitoring minimal residual disease (MRD) has become a very important process during disease treatment. The importance of MRD in lymphomas such as DLBCL has also attract the attention of clinicians, and several studies have found that MRD monitoring could predict early molecular relapse before clinical evidence of disease for DLBCL. However, a clinical available assay with well validated performances in reference standards and clinical samples are still warranted, to monitor MRD in lymphoma patients with high sensitivity and accuracy. Aims: Immunoglobulin gene rearrangements high throughput sequencing (Ig-HTS) in plasma cell-free DNA (cfDNA) is an alternate approach for minimal residual disease (MRD) detection. In this study, we aim to design and develop an optimized assay for MRD detection in plasma cfDNA sample, and evaluate/validate its performance of accuracy, sensitivity, specificity, linearity, and precision in both reference standard materials and clinical specimen of DLBCL patients was assessed. Methods: A series of reference standard materials are designed and developed from simulated cell free DNA samples to investigate and validate the analytical performance of NEO-MRD Test. Precision was reported as %CV for each tested MRD frequency at each DNA input. Limit of Detection (LoD) was defined as 95% detection rate for each sample. Limit of Quantitation (LoQ) was defined as the lowest number of malignant cells whose MRD value can be quantitatively determined with an accuracy of less than 300% relative bias. To define the linear range, the correlation coefficient (r) of the expected value and observed value was calculated and a linear plot was drawn with r > 0.9. And we assessed the accuracy to evaluate the therapeutic effect by detecting cfDNA-MRD at the interim and end of induction treatment in 20 DLBCL patients, and comparing the MRD result with clinical or PET-CT data. Results: The analytical validation of the NEO-MRD Test demonstrated high sensitivity for detection of cfDNA MRD. The LoD was determined to be 3 tumor copies according to 95% detection rate for each sample. The LoQ was determined to be 5 tumor copies. Linear range improved with increasing cfDNA input from 20ng-200ng. For 200ng cf DNA input, the linear range of MRD level is between 0.017%-100% (170-10000 tumor copies per 10000 diploid genome equivalent). Among 18 patients with newly diagnosed or relapsed/refractory DLBCL, at least one tumor clonotype was successfully detected in 89% (16/18) patients. The overall sensitivity, specificity, positive predictive value, and negative predictive value of the MRD assay was 77%(20/26), 81%(17/21), 83%(20/24), and 74%(17/23), respectively. Summary/Conclusion: We have developed and validated a standard assay to monitor MRD in plasma cfDNA of lymphoma patients, with potentiality for future clinical utility. Accuracy, precision, and linear range improve with increasing cfDNA mass input. Studies are ongoing to demonstrate clinical value of the NEO-MRD product in over 500 newly diagnosed and relapsed/refractory DLBCL patients.Keywords: DLBCL, ctDNA, Minimal residual disease (MRD)