Correction to: Leukemia (2014) 28, xx–xx; doi:10.1038/leu.2014.87; published online 21 March 2014 Since the online publication of this article, the authors have identified an omission concerning the author contributions, namely that the following was not included: FP and GF contributed equally to this study.

Distinct from other forms of acute lymphoblastic leukemia (ALL), infant ALL with mixed lineage leukemia ( MLL ) gene rearrangement, the most common leukemia occurring within the first year of life, might arise without the need for cooperating genetic lesions. Through Ig/TCR rearrangement analysis of MLL-AF4+ infant ALL at diagnosis and xenograft leukemias from mice transplanted with the same diagnostic samples, we established that MLL-AF4+ infant ALL is composed of a branching subclonal architecture already at diagnosis, frequently driven by an Ig/TCR-rearranged founder clone. Some MLL-AF4+ clones appear to be largely quiescent at diagnosis but can reactivate and dominate when serially transplanted into immunodeficient mice, whereas other dominant clones at diagnosis can become more quiescent, suggesting a dynamic competition between actively proliferating and quiescent subclones. Investigation of paired diagnostic and relapse samples suggested that relapses often occur from subclones already present but more quiescent at diagnosis. Copy-number alterations identified at relapse might contribute to the activation and expansion of previously quiescent subclones. Finally, each of the identified subclones is able to contribute to the diverse phenotypic pool of MLL-AF4+ leukemia-propagating cells. Unraveling of the subclonal architecture and dynamics in MLL+ infant ALL may provide possible explanations for the therapy resistance and frequent relapses observed in this group of poor prognosis ALL.

The pathogenesis of infant acute lymphoblastic leukemia (ALL) is still not well defined. Short latency to leukemia and very high concordance rate for ALL in Mixed-Lineage Leukemia (MLL)-positive infant twins suggest that the MLL rearrangement itself could be sufficient for overt leukemia. Attempts to generate a suitable mouse model for MLL-AF4-positive ALL did not thoroughly resolve the issue of whether cooperating mutations are required to reduce latency and to generate overt leukemia in vivo. In this study, we applied single-nucleotide polymorphism array technology to perform genomic profiling of 28 infant ALL cases carrying t(4;11) to detect MLL-cooperating aberrations hidden to conventional techniques and to gain new insights into infant ALL pathogenesis. In contrast to pediatric, adolescent and adult ALL cases, the MLL rearrangement in infant ALL is associated with an exceptionally low frequency of copy-number abnormalities, thus confirming the unique nature of this disease. By contrast, additional genetic aberrations are acquired at disease relapse. Small-segmental uniparental disomy traits were frequently detected, mostly constitutional, and widely distributed throughout the genome. It can be argued that the MLL rearrangement as a first hit, rather than inducing the acquisition of additional genetic lesions, has a major role to drive and hasten the onset of leukemia.

Acute lymphoblastic leukemia (ALL) in infants younger than 1 year is a rare but relatively homogeneous disease (∼80% MLL gene rearranged, ∼70% CD10-negative) when compared with childhood and adult ALL. Several studies in children and adults with ALL have shown that minimal residual disease (MRD) status is a strong and independent prognostic factor. We therefore evaluated the prognostic significance of MRD in infant ALL. Ninety-nine infant patients treated according to the Interfant-99 protocol were included in this study. MRD was analyzed by real-time quantitative PCR analysis of rearranged immunoglobulin genes, T-cell receptor genes and MLL genes at various time points (TP) during therapy. Higher MRD levels at the end of induction (TP2) and consolidation (TP3) were significantly associated with lower disease-free survival. Combined MRD information at TP2 and TP3 allowed recognition of three patients groups that significantly differed in outcome. All MRD-high-risk patients (MRD levels ⩾10 −4 at TP3; 26% of patients) relapsed. MRD-low-risk patients (MRD level <10 −4 at both TP2 and TP3) constituted 44% of patients and showed a relapse-rate of only 13%, whereas remaining patients (MRD-medium-risk patients; 30% of patients) had a relapse rate of 31%. Comparison between the current Interfant-06 stratification at diagnosis and the here presented MRD-based stratification showed that both stratifications recognized different subgroups of patients. These data indicate that MRD diagnostics has added value for recognition of risk groups in infant ALL and that MRD diagnostics can be used for treatment intervention in infant ALL as well.

Mice models and prenatal studies indicate that in childhood ALL the individual genetic lesions alone are insufficient to generate a full leukemic phenotype, and cooperating oncogenic lesions are required. Recently, multiple genome-wide studies on childhood ALL (1–18 years) identified deletions at several loci, mainly affecting genes that play a critical role in regulating B cell development and differentiation. By contrast, the prenatal and postnatal steps in the pathogenesis of Infant ALL (less than 1 year at diagnosis) are not defined. Infant ALL is a very aggressive disease, with t(4;11)/MLL-AF4 fusion representing the major subgroup. Although the very short latency period suggests that leukemogenic events occur prenatally, mice models indicates that MLL-AF4 alone is not sufficient to induce leukemia, and additional mutations may occur. Also unclear is whether the molecular pathways needed for lymphoid cell differentiation are altered in cases with an MLL rearrangement and, if so, whether these alterations differ between the leukemia of infants and older children. Aim of this study was to detect MLL-cooperating aberrations, undetectable by conventional techniques, by using genome-wide single nucleotide polymorphism (SNP) genome wide analysis (100K SNP human mapping, Affymetrix). More specifically, we searched for Loss of Heterozygosity (LOH) associated or not to copy number alteration. The identification of these lesions could help identifying leukemia pathogenesis, as well as providing the basis for targeted therapy. We have analyzed 28 cases of Infant ALL with t(4;11) at diagnosis and their corresponding samples at remission, when available (n=18). SNP data were analyzed by using dChip software, and confirmed by CNAG 2.0. A more dense SNP array analysis (250K) has been applied in selected cases to confirm LOH and precisely dissect the affected chromosomal regions. Compared to older childhood ALL patients, a far limited number of deletions/amplifications has been found; only 2/28 patients showed deletions, namely 1p36.33-p36.31 in 1 patient and 3p11.1-p12.2 plus 7q22.1-q22.2 in another patient, while 26/28 Infant ALL did not present any visible structural variation. Different from older children, several segmental copy-number neutral (CNN) LOH have been detected by dChip. The extension and prevalence of the affected regions was variable; among them 6p21.32 (4/28 cases), 7q31.33-q32.1 (3/28), 8q21.12-q21.3 (2/28), 8q24.11 (2/28) and 14q21.2 (2/28). Overall, these results confirm that Infant ALL with t(4;11)/MLL-AF4 fusion represents a biologically unique disease, different from other type of leukemia occurring in older children. While in older children a multistep mechanism (with the involvement of several genes) is required for the full leukemic phenotype, MLL rearrangements per se might play a major role on the leukemogenesis. By this approach it could not be excluded that different mechanisms could cooperate with MLL in transforming cells, including point mutations. The functional role of CNN-LOH still needs to be understood: they could either reflect the duplication of oncogenic mutations, or be related to epigenetic mechanisms.

The aim of this study was to identify immunobiological subgroups in 133 infant acute lymphoblastic leukemia (ALL) cases as assessed by their immunophenotype, immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement pattern, and the presence of mixed lineage leukemia (MLL) rearrangements. About 70% of cases showed the pro-B-ALL immunophenotype, whereas the remaining cases were common ALL and pre-B-ALL. MLL translocations were found in 79% of infants, involving MLL-AF4 (41%), MLL-ENL (18%), MLL-AF9 (11%) or another MLL partner gene (10%). Detailed analysis of Ig/TCR rearrangement patterns revealed IGH, IGK and IGL rearrangements in 91, 21 and 13% of infants, respectively. Cross-lineage TCRD, TCRG and TCRB rearrangements were found in 46, 17 and 10% of cases, respectively. As compared to childhood precursor-B-ALL, Ig/TCR rearrangements in infant ALL were less frequent and more oligoclonal. MLL-AF4 and MLL-ENL-positive infants demonstrated immature rearrangements, whereas in MLL-AF9-positive leukemias more mature rearrangements predominated. The immature Ig/TCR pattern in infant ALL correlated with young age at diagnosis, CD10 negativity and predominantly with the presence and the type of MLL translocation. The high frequency of immature and oligoclonal Ig/TCR rearrangements is probably caused by early (prenatal) oncogenic transformation in immature B-lineage progenitor cells with germline Ig/TCR genes combined with a short latency period.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a detoxification enzyme that protects cells against oxidative stress and toxic quinones. A polymorphism (C609T) in the gene produces in the heterozygous individuals (C/T) a reduction and in those homozygous for the variant allele (T/T) the abolishment of NQO1 protein activity. To assess whether NQO1 inactivating polymorphism (CT/TT) was a possible risk factor for infant acute lymphoblastic leukemia (iALL), we investigated the distribution of NQO1 genotype in 50 iALL patients, 32 with MLL gene rearrangements (MLL+) and 18 without (MLL−). As controls, 106 cases of pediatric ALL (pALL), and 147 healthy subjects were also studied. Compared to normal controls, the frequency of the low/null activity NQO1 genotypes was significantly higher in the iALL MLL− (72 vs 38%, P =0.006; odds ratio (OR) 4.22, 95% confidence interval (CI) 1.43–12.49), while no differences were observed in iALL MLL+ (44 vs 38%, P =0.553; OR 1.26, 95% CI 0.58–2.74). Similar results were observed when pALL were used as control. Our results indicate that only the iALL patients without MLL rearrangements had a significantly higher frequency of NQO1 genotypes associated with low/null activity enzyme, suggesting a possible role for NQO1 gene as an MLL -independent risk factor, in the leukemogenic process of this subtype of iALL.

Immunoglobulin (Ig) and T-cell Receptor (TcR) gene rearrangements are used as patient-specific PCR targets for MRD detection in ALL. However, oligoclonality was reported in childhood ALL, and in those cases, the uncertainty of which clone is going to emerge at relapse may give rise to false negative MRD results. In particular, oligoclonality is a peculiar characteristic of Infant ALL (less than 1 year of age at diagnosis). So far, the prognostic relevance of MRD monitoring in Infant ALL has not been defined, and the assessment of oligoclonality and stability of PCR markers may have a profound impact in MRD monitoring. We successfully studied by PCR the frequency and stability of the currently used rearrangements of the Ig Heavy (IgH), Ig Kappa deleting element (IgK-Kde), TcR delta (TcRD), and TcR gamma (TcRG) gene rearrangements in 42/43 Infant ALL patients prospectively enrolled in Italy in the Interfant-99 International ALL protocol. Pro-B, common and pre-B ALL were 29 (67%), 8 (19%), and 6 (14%), respectively. Sixty-three percent of cases were prednisone good responders (PGR), 70% were MLL-rearranged, 44% were aged less than 6 months, and 21% of cases had more than 300.000 wbc at diagnosis. Overall, rearrangements of the IgH, IgK, TcRD, and TcRG genes were found in 91, 21, 40, and 21% of Infant ALL, respectively. The pattern of Ig/TcR rearrangements in immunophenotypic subgroups of Infant ALLs has been compared to the one of older children prospectively enrolled in Italy into the AIEOP-BFM ALL2000 protocol (n=649). While the overall frequency of IgH and TcRD are similar in the two age groups, IgK and TcRG are less rearranged in Infant cases, mainly due to the low percentage in the pro-B group (10% versus 41% for IgK VK-Kde, and 17% vs 36% for TcRG). In particular, none of the 29 pro-B cases showed rearrangement of the IgK intron. Sixteen Infant relapsed so far (37%), and 12 of them were successfully analyzed for Ig/TcR rearrangement at diagnosis and relapse. Ten were early (less than 18 months from diagnosis), and 2 were late relapses. None of the patients showed an identical Ig/TcR pattern at diagnosis and relapse; 7 (58%) had at least one, and 1 had at least 2 PCR targets preserved, while 4 cases (33%) presented completely different markers at relapse. This distribution was independent on age at diagnosis, site and time of relapse. Interestingly, we observed an increase of complete-IgH (from 10 alleles in 7 patients to 23 alleles in 11) and TcRG (from 4 alleles in 2 patients to14 alleles in 8) rearrangements at relapse. The stability of Ig/TcR markers was as it follows: IgH, 6/26; IgK, 3/5; TcRD, 1/9 and TcRG, 0/4. In conclusion, the oligoclonality feature of Infant ALL may potentially hamper the MRD predictivity in Infant ALL. An appropriate identification and selection of Ig/TcR MRD-PCR targets in Infant ALL is a crucial premise for obtaining clinically relevant MRD data and for preventing false-negative MRD results. Ig Kappa may be a preferential marker for MRD studies in Infant ALL, although its use is limited by the low frequency of the rearrangement. The leukemia-specific MLL genomic breakpoint may potentially overcome these limitations and improve the MRD analysis.

Point mutations affecting RAS GTPase activity promote upregulation of RAS signaling, and are among the most common genetic alterations in human cancers. Oncogenic mutations in NRAS and KRAS2 genes are found with variable prevalence in hematological malignancies, including ALL. Upregulation of RAS signaling can also results from molecular lesions in genes coding transducers controlling RAS function. We recently provided evidence that PTPN11 is mutated in approximately one-third of pediatric ALL cases. Significantly, while PTPN11 mutations appeared to be preferentially associated with the common immunophenotype, mutations in NRAS or KRAS2 were uniformly distributed among the B-cell precursor ALL subtypes. Very few are the data on RAS pathway status in pediatric ALL patients aged <1 year (Infant). Infant ALL is a rare subgroup of leukemia peculiar for several characteristics, mainly related to a possible origin from a not-fully committed stem cell. In order to explore whether alterations of RAS signaling may identify specific subgroups within this heterogeneous patient population, we analyzed 87 Infant cases prospectively enrolled in the Interfant-99 International ALL protocol. This is the largest series of Infant ALL analyzed for alteration of the RAS pathway. PTPN11 (exon 3 and 13), NRAS (exon 1 and 2), and KRAS2 (exon 1 and 2) mutation analysis was performed by DHPLC analysis and direct sequencing. None of the 87 Infant cases carried a PTPN11 somatic mutation. By contrast, a high prevalence of mutations affecting NRAS and KRAS2 was observed (approximately 30%). Thirteen cases (14.9%) were positive for a mutation in exon 1 (codons 12 and 13, 10 cases, 11.5%) or exon 2 (codon 61, 3 cases, 3.4%) of the NRAS gene. Seventeen cases (19.5%) carried a variety of exon 1 KRAS2 mutations: codons 12 and 13, n=13 (14.9% of total); insAGC(30–32), n=1; G(40)C, n=1; C(53)A, n=1; G(57)C, n=1. Interestingly, in 9/27 cases (33.3% of RAS mutated cases, 10.3% of total) DHPLC and sequencing consistently showed the presence of the RAS mutation only in a fraction (10 to 40%) of the blast cells population. In one additional case a KRAS2 mutation was observed in a subset of cells along with a full NRAS mutation. In two more cases mutations affecting NRAS and KRAS2 coexisted on the same patient; in both cases the mutations were representative of a subgroup of the total cell population. In conclusion: differently from older children, PTPN11 gene mutations are a rare event in infant ALL. This could be related to the low incidence of the “common” immunophenotype in Infants. By contrast, the overall incidence of RAS gene mutations is high, accounting for about 30% of cases. Interestingly, in 10% of cases a mutation (or even more than one) was found in a fraction of the blast cell population. This could reflect the fact that RAS mutations were secondary to other genetic events, and in any case related to the oligoclonal characteristic of this age-specific ALL subgroup. The prognostic effect of these genetic events must be evaluated in a longer follow up time of prospective series of patient, as in the Interfant Consortium.

The study of minimal residual disease (MRD) as a 'surrogate' marker of molecular response to treatment has drawn great interest because of the potential of tailoring treatment and the possibility of gaining insight into the nature of a cure. Polymerase chain reaction-based (PCR-based) detection of MRD by immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements can be applied in more than 90-95% of cases of childhood acute lymphoblastic leukaemia (ALL). Accordingly, several retrospective studies of MRD in childhood ALL have used one of the different PCR approaches for the detection of antigen-receptor gene rearrangements. The promising results on the predictivity of MRD evaluation at the end of induction treatment has raised the need of a new definition of remission. Until now, most PCR-based MRD studies have used semiquantitative methods for the detection of Ig and TCR gene rearrangements. The introduction of real-time quantitative PCR (RQ-PCR) has resulted in the improvement of sensitivity and specificity and has given better quality control of the MRD data. There is an urgent need to incorporate MRD data in clinical studies, properly designed to address treatment questions. In this context several ongoing co-operative study groups have adopted an MRD-based risk group classification to explore whether a better tailored treatment would result in further improvement in cure rates for children with ALL.