Association of Clinical Status of Follicular Lymphoma Patients after Autologous Stem Cell Transplant and Quantitative Assessment of Lymphoma in Blood and Bone Marrow as Measured by SYBR Green I Polymerase Chain Reaction

Abstract

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. (J

Rituximab purging and maintenance combined with auto-SCT: long-term molecular remissions and prolonged hypogammaglobulinemia in relapsed follicular lymphoma

“…Nonetheless, compared with results from another study at our center using the same stem cell collection procedure but without the in vivo rituximab purge, where we detected a median of 0.1% contamination, the addition of rituximab decreased contamination to 0.002%, a 50-fold reduction (a median of 0.1% contamination reduced to 0.002%). 31,33 We observed a significant incidence of non-fatal pulmonary toxicity. Single agent rituximab has been associated with interstitial pneumonitis, 60 and the risk of pulmonary toxicity with carmustine is well described.…”

“…Real-time PCR was performed with QuantiTect SYBR Green I (Qiagen, Mississauga, ON, Canada) on a DNA Engine Opticon I (Bio-Rad, Mississauga, ON, Canada). 31 Molecular monitoring for MRD was performed on all follow-up PB and BM samples in patients in whom a molecular marker could be detected at baseline. Similar analyses were performed on the stem cell graft to detect the presence of occult contamination.…”

“…30 Quantitative PCR assays are capable of detecting as few as one malignant cell in 10 5 cells. 31 As a result, molecular remission status has been used as a surrogate marker of FL disease burden after treatment. 32 Patients who become PCR negative for BCL2/JH rearrangements in peripheral blood (PB) or BM after treatment are reported to have superior survival compared with those who remain positive.…”

Commercial LightCycler®-based quantitative real-time PCR compared to nested PCR for monitoring of Bcl-2/IgH rearrangement in patients with follicular lymphoma

“…Quantitative PCR may offer additional benefits as it may (a) allow for the identification of patients with an increase in MRD after therapy, being at an increased risk of relapse [33][34][35] and (b) stratify patients according to the initial tumour load in BM, thereby predicting response to therapy and long-term outcome [36]. Our results suggest that the relatively low sensitivity of the LigthCycler assay may be disadvantageous for the prediction of early relapse after therapy.…”

In situ localization of follicular lymphoma: evidence for subclinical systemic disease with detection of an identical BCL-2/IGH fusion gene in blood and lymph node

“…Amplification using primers for the RAG2 gene served as a control for amplifiable DNA quantity. 1 The PCR test was then repeated on the DNA extracted earlier from the sections of the intact lymph node. The BCL-2/IGH fusion gene was detected in high copy numbers in the lymph node DNA.…”