Subclonality and prenatal origin of RAS mutations in KMT2A (MLL)‐rearranged infant acute lymphoblastic leukaemia

KMT2A (previously termed MLL) rearrangements (KMT2Ar) constitute the most frequent aberration in infant lymphoblastic leukaemia (iALL). Mutations in the RAS genes have previously been recognized as an important somatic abnormality in KMT2A-r-iALL (Taketani et al, 2004; Liang et al, 2006; Driessen et al, 2013; Prelle et al, 2013). Certainly, KMT2A-r in iALL are present at birth (Greaves et al, 2003). On the other hand, RAS mutations (RASmut) are suggested to be secondary and subclonal hits in the leukaemogenic pathway. In this context, our study aimed to (i) quantitatively evaluate the association between RASmut at diagnosis and the main clinical features of KMT2A-r-iALL, and (ii) analyse the mutations in matched samples [neonatal blood spots, also known as Guthrie cards (Gc), remission and/or relapse]. KRAS/NRASmut were screened by direct sequencing, and mutated cases were also subjected to pyrosequencing in order to quantify the percentage of blasts harbouring the mutation. The detailed methods performed in this study are described in the Supporting Information (Data S1). Ninety-two KMT2A-r-iALL cases were included. RASmut were found in 21 7% of cases [KRAS (14 1%) and NRAS (7 6%)]. In childhood ALL, RASmut have been diagnosed in 6–21% of patients (Liang et al, 2006; Wiemels et al, 2010). Recent studies in large cohorts of KMT2A-r cases have reported frequencies of 14–15% RASmut, with an even higher frequency in t(4;11) infants (c. 25%) (Driessen et al, 2013; Prelle et al, 2013). These previous data are therefore concordant with our study. The clinical-laboratory features of KMT2A-r-iALL according to RAS status are shown in Table SI. Sixty percent of RASmut patients were ≤6 months old, 60% harboured AFF1 as the KMT2A-translocation partner gene (TPG), and 80% presented both the KMT2A-AFF1 and AFF1-KMT2A transcripts at mRNA level. Although these differences were not statistically significant, these analyses are in agreement with previous data, such as a higher frequency of RASmut among younger and t(4;11) infants (Driessen et al, 2013; Prelle et al, 2013). Furthermore, KRAS (P = 0 033) but not NRAS (P = 0 415) mutations were associated with high white blood cell (WBC) counts. The quantitative association between RASmut at diagnosis and clinical features was analysed by pyrosequencing the mutated clones. The median percentage of mutated clones did not differ according to age group (31% vs. 27%; Fig S1A; P = 0 43) or KMT2A-TPG (28% vs. 33%; Fig S1C; P = 0 89). Significantly higher percentages of mutated clones were found in cases with WBC counts >300 x 10/l (Fig S1B; P = 0 01). The correlation of RAS-mutated clones with higher WBC count may indicate that RASmut play a role in aggressiveness. Accordingly, the presence of RASmut was an independent prognostic factor in a previous cohort (Driessen et al, 2013). Despite recent attempts, neither the role of a second-hit nor the subclonality of RASmut in KMT2A-riALL has been demonstrated (Driessen et al, 2013; Prelle et al, 2013). Nevertheless, according to pyrosequencing analyses, we checked for a putative subclonality of RASmut and found that, in 19/ 20 cases, the percentage of mutated alleles at diagnosis was lower than expected for a clonal alteration. The RASmut were monoallelic (50% in a single cell). Taking the percentage of blast cells into account, if the alteration is clonal, the percentage of mutated alleles is expected to be half of the blast count for each patient (Table SII). Then, subclonality was observed in this present study. Regarding the analysis of matched samples, 17 samples of patients who achieved remission were analysed, but no mutations were found. Among the relapse samples (n = 12), five patients lost RASmut at relapse and seven patients were wildtype at both diagnosis and relapse. Concerning the two Gc samples, in both cases the RAS-mutated allele ‒ present at diagnosis ‒ was also detected at birth (Table SII). One of these infants (Patient 13) was diagnosed with ALL 17 d after birth, with high WBC, KMT2A-MLLT10 gene fusion (Fig 1A) and KRASmut (Fig 1B) detected at birth. The intrapatient’s quantitative comparison between samples showed that the Gc sample had a higher percentage of KRASmutated clones than the diagnostic sample (42% vs. 33%, Fig 1C, D). Finding the KRASmut in this infant’s Gc sample was an expected result as the baby was presumably born with the disease and presented with 97% blasts in the peripheral blood. The other infant (Patient 15) had an NRASmut (Fig 2A) at 7-months old, with 96% peripheral blood blasts and KMT2A-AFF1. The percentage of NRAS mutated clones was 50% in both the Gc and the diagnostic sample (Fig 2C, D). This high percentage of mutated cells within the Gc sample may explain the backtracking success. The presence of RASmut at birth clearly argues against a secondary role in a two-hit model for this subgroup of leukaemias. Although former studies performed in hyperdiploid childhood leukaemia strongly indicate that RASmut are secondary hits (Case et al, 2008; Davidsson et al, 2010), this might not be a correspondence