Qualitative and Quantitative PCR Monitoring of MRD: Practical Implications of a Surrogate Marker for GVL Effect after RIC Allogeneic Transplantation in Relapsed Chronic Lymphocytic Leukemia.
Authors: Farina Lucia , Carniti Cristiana , Dodero Anna , Vendramin Antonio , Raganato Anna , Patriarca Francesca , Narni Franco , Montefusco Vittorio , Benedetti Fabio , Olivieri Attilio , Corradini Paolo 
Published: April 2020

Abstract
Reduced intensity allogeneic stem cell transplantation (RIC alloSCT) is a therapeutic option for poor risk relapsed chronic lymphocytic leukemia (CLL) and can lead to 50% of progression-free survivors. The aims of this study were:
to assess the “molecular quality” of clinical remission after RIC alloSCT in CLL patients; to investigate whether molecular remission (MR) can correlate with a lower relapse risk; to understand the clinical role of molecular monitoring in post-transplant immunotherapy. Twenty-nine patients with a molecular marker (heavy chain gene immunoglobulin rearrangement, IgH) were monitored for minimal residual disease (MRD).
All patients had a relapsed disease; 75% had an unmutated IgH; cytogenetic analysis was available in 13 patients at first relapse, and 5 of them (38%) showed a 17p deletion. Median age at transplant was 60 years (range, 44–69). Median number of previous chemotherapy was 3 (range, 1–6) and 29% of patients failed autologous transplant. Eleven patients (38%) were chemorefractory before transplant. The conditioning regimen included thiotepa-cyclophosphamide-fludarabine in HLA identical sibling transplants (n=21), and an in vivo T cell depletion in haploidentical and unrelated alloSCT (n=8). Molecular monitoring was performed by nested-PCR on bone marrow using CDR-2 and CDR-3-derived patient-specific primers. For real-time PCR a FR3-derived probe was used. Post-transplant samples were amplified including the pre-transplant sample as positive control, and ΔCT was calculated after normalization for GAPDH gene. Eight patients (28%) were PCR-negative: 4 of them (14%) have been always PCR-negative while 4 patients (14%) experienced a delayed clearance of MRD during the first year after transplant. All these patients are alive and in CR at the median follow-up of 24 months (range, 3–71). Seven patients (24%) showed a mixed pattern of PCR positivity and negativity: one patient died of secondary acute leukemia, another patient had a nodal relapse, the others are in CR at a median follow-up of 30 months (range, 6–60). Fourteen patients (48%) were always PCR-positive: 7 of them relapsed after a median time of 9 months (range, 3–12); 2 patients died of TRM in MR; 5 patients are alive and in CR after a median follow-up of 15 months (range, 3–24). Relapse risk was higher for PCR-positive patients compared to PCR-mixed/negative patients (p=0.014). Eighty percent of PCR-mixed/negative patients experienced GVHD compared to 43% of PCR-positive patients (p=0.06). In 5 PCR-positive patients real-time PCR was carried out. In 3 patients that did not relapse a decreasing tumor load was detected; these patients were affected by extensive chronic GVHD. In the other 2 patients after an initial reduction, the tumor load increased on day +300 and +270: both patients relapsed on days +420. In conclusion, in poor risk relapsed CLL clinical and molecular remission can be achieved in a sizeable fraction of patients after RIC alloSCT. Persistent PCR positivity correlates with a high incidence of relapse and requires novel treatments, while a mixed-PCR pattern can be observed without clinical relapse.