Flow Cytometry Based MRD Monitoring Is an Accurate Risk Stratification Tool in Acute T-Lymphoblastic Leukemia

Abstract

▪Minimal residual disease (MRD) measured by PCR of clonal TCR/IGH rearrangements during early phases of chemotherapy is a strong predictor of relapse risk in acute T-lymphoblastic leukemia (T-ALL), and has thus served as the most important tool for stratification to reduced therapy (to avoid toxicity) or intensified therapy. In 8-15 % of T-ALL patients no useful PCR marker is available and flow cytometry (FCM) has been used for monitoring by some collaborative groups. However, studies of the usefulness of FCM-based MRD measurements are scarce, limiting the widespread use of the method. Furthermore, it is unknown whether the generally applied end-of-induction PCR MRD cut-off level of 10-4 should be the same for FCM-based MRD. In addition, it is not known whether earlier identification (day15) of low risk patients by FCM is possible, in which case it would allow elimination of anthracyclines during the latter part of induction therapy thus lowering risk of cardiotoxicity.Aim: To evaluate flow and PCR based MRD measurements as risk stratification tools in T-ALL.Material and methods: The study included 274 patients with T-ALL (193 children (<18y) and 81 adults (18-45y)) diagnosed between July 2008 and February 2016 and treated according to NOPHO ALL2008 in the Nordic and Baltic countries. Median follow up was 32 (range 0-103) months. MRD was measured by 6-colour FCM and by RT-QPCR at days 15, 29 (end of induction) and 79 (only intermediate risk, IR) and prior to and post high risk (HR) blocks. End-of-induction MRD (cut-off 10-3) was used to stratify for IR or HR consolidation therapy. In analyses of day29 MRD data and prognosis, patients who were stratified for HR blocks at day 15 or directly to SCT at day29 were excluded from analyses.Results: Overall 5y EFS was 72.7% (CI 67-79%) with relapse risk (RR) of 15.5% (CI 8-16%). End of induction FCM MRD separated patient risk groups well (5y EFS 84.2% (CI 78-91%) and RR 8.3 % (CI 4-13%) for MRD<10-3 versus EFS 63.4% (CI 50.1-80.3%) and RR 21.8% (CI 9-35%) for MRD>=10-3 (p=0.002 for EFS, p=0.049 for RR) although PCR-MRD gave better discrimination (5y EFS 90.2 (CI 85-96%) and RR 5.5% (CI 1-10%) for MRD<10-3 versus EFS 62.5 (CI 51-78%) and RR 23.4% (CI 12-35%) for MRD>=10-3 (p=0.0002 for EFS, p=0.005 for RR).Adjusting for WBC and age at diagnosis, patients with end-of-induction FCM-MRD >=10-3 had a higher risk of relapse (hazard rate 3.04, p=0.025) compared with MRD <10-3. For PCR the hazard rate was 4.4 (p=0.007). Day29 MRD was measured by both methods in 159 patients. There was an overall good correlation between day29 FCM- and PCR-MRD: (r=0.72, p<0.0001 (Spearman)), though PCR values were generally higher. These differences would give discrepant day29 risk classification in 43/159 patients (36 with PCR>FCM, 7 with FCM>PCR) with the 10-3 cut-off.For both FCM- and PCR-MRD a cut-off level of 10-3 was superior to 10-4 in separating risk groups at end of induction. Patients with d29 PCR MRD between 10-3 and 10-4 were stratified to IR therapy in NOPHO ALL2008 without decreasing EFS or relapse risk (5y EFS 91.4% (CI 82.6-100%), 0 relapses, n=35) compared to patients with MRD<10-4 (EFS 89.6% (CI 83-97%), relapse risk 7.9%, n=81).A day 15 FCM-MRD cut-off of 10-2 provided good prediction of the final risk groups (5y EFS 87% (CI 81-93%) and RR 8% (CI 3-13%) for day 15 FCM-MRD<10-2 vs. EFS 65.4% (CI 53-80%) and RR 16% (CI 6-26%) for FCM-MRD between 10-2 and 2.5x10-1 vs. EFS 24% (CI 8-71) and RR 45.8% (CI 19-73%) for flow MRD>=2.5x10-1 (patients with MRD>=2.5x10-1 went to HR therapy day 15)), but only 35 of the 145 patients with day15 flow MRD<10-2 had no detectable PCR MRD at day29. Only 27 out of 62 patients with day15 FCM-MRD <10-4 had no detectable PCR MRD at day29. Three out of these 62 patients experienced relapse, but none of these had a fully informative FCM marker at diagnosis.Conclusion: End-of-induction FCM-MRD provides a reliable indicator of outcome in T-ALL and can serve as second line method when no suitable PCR marker is found. Regardless of method, 10-3 seems to be superior to 10-4 as end of induction stratification cut-point, allowing patients with PCR MRD between 10-3 and 10-4 to receive intermediate risk therapy without increased relapse risk. Stratification at day 15 does not allow early and unambiguous identification of low risk patients eligible for reduced intensity induction therapy, but randomized studies are needed to clarify this. DisclosuresNo relevant conflicts of interest to declare.