Relapse Prediction By Flow Cytometric Minimal Residual Disease Assessment in Infants with Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) in infants (less than 1 year old) is a unique tumor with distinct biological features and poor outcome even when modern treatment schemes are used. Rearrangementsof MLL-gene, located in 11q23 region, are detected in approximately 75-80% of infants with ALL and lead to treatment resistance and high relapse rate. Nevertheless patients with germline MLL also demonstrate inferior outcome compared to ALL in older children. Thus additional risk factors implementation is one of the crucial points in infant ALL management. Minimal residual disease (MRD) measurement by multicolor flow cytometry (FCM) or various PCR technics is a well-standardized method of treatment response evaluation in childhood ALL, although in infants MRD data is not so widely applied. The aim of the study was to evaluate the relapse prediction feasibility in infant ALL by FCM MRD assessment during remission induction of MLL-Baby protocol.

Methods. Totally 89 infants aged from 5 days to 11 months were enrolled in the present study. In 23 cases (25.8%) MLL gene was germline (MLL-g group), 33 patients (37.1%) had MLL-AF4 fusion while in remaining 33 cases (37.1%) other types of MLL-rearrangements were found. All patients were diagnosed as B-cell precursor ALL and all were treated by well established in Russia and Belarus MLL-Baby protocol, which is specially designed for infant ALL management. MRD was measured by 6-10-color FCM in bone marrow (BM) samples obtained at day 15 and at the end of remission induction (day 36). The availability of samples at at least one of these time-points was the only criteria for study group completion. In 43 patients with known types of MLL-rearrangements MRD was also assessed by fusion gene transcript (FGT) detection in RQ-PCR after first consolidation or first high risk block (for intermediate risk and high risk groups respectively). Relapse risk was investigated by cumulative incidence of relapse (CIR) estimation. Median of follow-up was 3 years 10 months.

Results. Finally at day 15 MRD was studied in 71 cases. 17 patients (23.9%) were tested MRD-negative while remaining ones displayed various levels of MRD-positivity: 8 cases (11.3%) - from 0.01% to 0.1%; 14 (19.7%) - from 0.1% to 1%; 22 (31.0%) - from 1% to 10%; 10 (14.1%) - more than 10%. Proportion of MRD-positivity was lower in MLL-g group compared to MLL-rearranged (MLL-r) patients (58.8% and 81.5% respectively, p=0.06). Prognostic impact of day 15 MRD differed due to MLL-status. In MLL-r group significant differences between MRD(+) and MRD(-) patients were observed (n=10, CIR 0.28(0.18) and n=37, CIR 0.67(0.08) respectively, p=0.025). At the same time in MLL-g group these outcome differences were not significant (n=7, CIR 0 and n=9, CIR 0.22(0.14) correspondingly, p=0.197). Interestingly, in patients carrying MLL-AF4 fusion, known to be one of the most adverse types of MLL-rearrangements, day 15 MRD-negativity predicted low relapse incidence (n=5, CIR 0 and n=16, CIR 0.68(0.12) respectively, p=0.045). Thus day 15 MRD-negativity allows to detect low-risk MLL-r infants but it is not applicable in MLL-g group. It was previously shown that any detectable level of FGT after first consolidation or first high risk block predicts very poor outcome in MLL-r cases (G. Tsaur et al, ASH-2011). In current series RQ-PCR data in patients, FCM MRD(+) at day 15, distinguished groups of intermediate and very high relapse risk (n=17, CIR 0.51(0.13) and n=18, CIR 0.84(0.09) respectively, p=0.017). At day 36 FCM MRD was assessed in 82 infants. Among them 35 (42.7%) were tested negative while remaining 47 (57.3%) were MRD(+) at various levels. Prognostic value of day 36 FCM MRD data in MLL-r group was not significant: MRD(+) patients (n=23) had CIR of 0.71(0.08) while in MRD(-) cases (n=33) CIR was 0.49(0.12), p=0.153. Conversely, in MLL-g group low end-induction MRD (less than 0.1%) lead to excellent outcome compared to patients with higher MRD (n=14, CIR 0 and n=7, CIR 0.22(0.20) respectively, p=0.010).

Conclusions. Thus FCM MRD data could distinguish infants with low risk of ALL relapse, but in MLL-r and MLL-g groups different time-points are prognosticaly significant. In MLL-g patients tandem application of FCM at early time-point and RQ-PCR later could help to define groups with low, intermediate and high relapse risk. MRD data could be added to MLL-Baby protocol risk group stratification, which is currently based on type of MLL-rearrangement.