Paola De Lorenzo

A treatment protocol for infants younger than 1 year with acute lymphoblastic leukaemia (Interfant-99): an observational study and a multicentre randomised trial

BACKGROUND Acute lymphoblastic leukaemia in infants younger than 1 year is rare, and infants with the disease have worse outcomes than do older children. We initiated an international study to investigate the effects of a new hybrid treatment protocol with elements designed to treat both acute lymphoblastic leukaemia and acute myeloid leukaemia, and to identify any prognostic factors for outcome in infants. We also did a randomised trial to establish the value of a late intensification course. METHODS Patients aged 0-12 months were enrolled by 17 study groups in 22 countries between 1999 and 2005. Eligible patients were stratified for risk according to their peripheral blood response to a 7-day prednisone prophase, and then given a hybrid regimen based on the standard protocol for acute lymphoblastic leukaemia, with some elements designed for treatment of acute myeloid leukaemia. Before the maintenance phase, a subset of patients in complete remission were randomly assigned to receive either standard treatment or a more intensive chemotherapy course with high-dose cytarabine and methotrexate. The primary outcomes were event-free survival (EFS) for the initial cohort of patients and disease-free survival (DFS) for the patients randomly assigned to a treatment group. Data were analysed on an intention-to-treat basis. This trial was registered with ClinicalTrials.gov, number NCT 00015873, and at controlled-trials.com, number ISRCTN24251487. FINDINGS In the 482 enrolled patients who underwent hybrid treatment, 260 (58%) were in complete remission at a median follow-up of 38 (range 1-78) months, and EFS at 4 years was 47.0% (SE 2.6, 95% CI 41.9-52.1). Of 445 patients in complete remission after 5 weeks of induction treatment, 191 were randomised: 95 patients to receive a late intensification course, and 96 to a control group. At a median follow-up of 42 (range 1-73) months, 60 patients in the treatment group and 57 controls were disease-free. DFS at 4 years did not differ between the two groups (60.9% [SE 5.2] for treatment group vs 57.0% [5.5] for controls; p=0.81). During the intensification phase, of 71 patients randomly assigned to the treatment group, and for whom toxicity data were available, 35 (49%) had infections, 21 (30%) patients had mucositis, 22 (31%) patients had toxic effects on the liver, and 2 (3%) had neurotoxicity. All types of rearrangements in the (mixed lineage leukaemia) MLL gene, very high white blood cell count, age of younger than 6 months, and a poor response to the prednisone prophase were independently associated with inferior outcomes. INTERPRETATION Patients treated with our hybrid protocol, and especially those who responded poorly to prednisone, had higher EFS than most reported outcomes for treatment of infant ALL. Delayed intensification of chemotherapy did not benefit patients.

Imatinib after induction for treatment of children and adolescents with Philadelphia-chromosome-positive acute lymphoblastic leukaemia (EsPhALL): a randomised, open-label, intergroup study.

Summary Background Trials of imatinib have provided evidence of activity in adults with Philadelphia-chromosome-positive acute lymphoblastic leukaemia (ALL), but the drugs role when given with multidrug chemotherapy to children is unknown. This study assesses the safety and efficacy of oral imatinib in association with a Berlin–Frankfurt–Munster intensive chemotherapy regimen and allogeneic stem-cell transplantation for paediatric patients with Philadelphia-chromosome-positive ALL. Methods Patients aged 1–18 years recruited to national trials of front-line treatment for ALL were eligible if they had t(9;22)(q34;q11). Patients with abnormal renal or hepatic function, or an active systemic infection, were ineligible. Patients were enrolled by ten study groups between 2004 and 2009, and were classified as good risk or poor risk according to early response to induction treatment. Good-risk patients were randomly assigned by a web-based system with permuted blocks (size four) to receive post-induction imatinib with chemotherapy or chemotherapy only in a 1:1 ratio, while all poor-risk patients received post-induction imatinib with chemotherapy. Patients were stratified by study group. The chemotherapy regimen was modelled on a Berlin–Frankfurt–Munster high-risk backbone; all received four post-induction blocks of chemotherapy after which they became eligible for stem-cell transplantation. The primary endpoints were disease-free survival at 4 years in the good-risk group and event-free survival at 4 years in the poor-risk group, analysed by intention to treat and a secondary analysis of patients as treated. The trial is registered with EudraCT (2004-001647-30) and ClinicalTrials.gov, number NCT00287105. Findings Between Jan 1, 2004, and Dec 31, 2009, we screened 229 patients and enrolled 178: 108 were good risk and 70 poor risk. 46 good-risk patients were assigned to receive imatinib and 44 to receive no imatinib. Median follow-up was 3·1 years (IQR 2·0–4·6). 4-year disease-free survival was 72·9% (95% CI 56·1–84·1) in the good-risk, imatinib group versus 61·7% (45·0–74·7) in the good-risk, no imatinib group (p=0·24). The hazard ratio (HR) for failure, adjusted for minimal residual disease, was 0·63 (0·28–1·41; p=0·26). The as-treated analysis showed 4-year disease-free survival was 75·2% (61·0–84·9) for good-risk patients receiving imatinib and 55·9% (36·1–71·7) for those who did not receive imatinib (p=0·06). 4-year event-free survival for poor-risk patients was 53·5% (40·4–65·0). Serious adverse events were much the same in the good-risk groups, with infections caused by myelosuppression the most common. 16 patients in the good-risk imatinib group versus ten in the good-risk, no imatinib group (p=0·64), and 24 in the poor-risk group, had a serious adverse event. Interpretation Our results suggests that imatinib in conjunction with intensive chemotherapy is well tolerated and might be beneficial for treatment of children with Philadelphia-chromosome-positive ALL. Funding Projet Hospitalier de Recherche Clinique-Cancer (France), Fondazione Tettamanti-De Marchi and Associazione Italiana per la Ricerca sul Cancro (Italy), Novartis Germany, Cancer Research UK, Leukaemia Lymphoma Research, and Central Manchester University Hospitals Foundation Trust.

Chemotherapy versus allogeneic transplantation for very-high-risk childhood acute lymphoblastic leukaemia in first complete remission: comparison by genetic randomisation in an international prospective study

BACKGROUND The dismal prognosis of very-high-risk childhood acute lymphoblastic leukaemia could be improved by allogeneic haemopoietic cell transplantation. We compared this strategy with intensified chemotherapy protocols, with the aim to improve the outcome of children with very-high-risk acute lymphoblastic leukaemia in first complete remission. METHODS A cooperative prospective study was set up in seven countries. Very-high-risk acute lymphoblastic leukaemia in first complete remission was defined by the presence of at least one of the following criteria: (1) failure to achieve complete remission after the first four-drug induction phase; (2) t(9;22) or t(4;11) clonal abnormalities; and (3) poor response to prednisone associated with T immunophenotype, white-blood-cell count of 100x10(9)/L or greater, or both. Children were allocated treatment by genetic chance, according to the availability of a compatible related donor, and assigned chemotherapy or haemopoietic-cell transplantation. The primary outcome was disease-free survival and analysis was by intention to treat. FINDINGS Between April, 1995, and December, 2000, 357 children entered the study, of whom 280 were assigned chemotherapy and 77 related-donor haemopoietic-cell transplantation. 5-year disease-free survival was 40.6% (SE 3.1) in children allocated chemotherapy and 56.7% (5.7) in those assigned transplantation (hazard ratio 0.67 [95% CI 0.46-0.99]; p=0.02); 5-year survival was 50.1% (3.1) and 56.4% (5.9), respectively (0.73 [0.49-1.09]; p=0.12). INTERPRETATION Children with very-high-risk acute lymphoblastic leukaemia benefit from related-donor haemopoietic-cell transplantation compared with chemotherapy. The gap between the two strategies increases as the risk profile of the patient worsens.

Specific promoter methylation identifies different subgroups of MLL-rearranged infant acute lymphoblastic leukemia, influences clinical outcome, and provides therapeutic options

MLL-rearranged infant acute lymphoblastic leukemia (ALL) remains the most aggressive type of childhood leukemia, displaying a unique gene expression profile. Here we hypothesized that this characteristic gene expression signature may have been established by potentially reversible epigenetic modifications. To test this hypothesis, we used differential methylation hybridization to explore the DNA methylation patterns underlying MLL-rearranged ALL in infants. The obtained results were correlated with gene expression data to confirm gene silencing as a result of promoter hypermethylation. Distinct promoter CpG island methylation patterns separated different genetic subtypes of MLL-rearranged ALL in infants. MLL translocations t(4;11) and t(11;19) characterized extensively hypermethylated leukemias, whereas t(9;11)-positive infant ALL and infant ALL carrying wild-type MLL genes epigenetically resembled normal bone marrow. Furthermore, the degree of promoter hypermethylation among infant ALL patients carrying t(4;11) or t(11;19) appeared to influence relapse-free survival, with patients displaying accentuated methylation being at high relapse risk. Finally, we show that the demethylating agent zebularine reverses aberrant DNA methylation and effectively induces apoptosis in MLL-rearranged ALL cells. Collectively these data suggest that aberrant DNA methylation occurs in the majority of MLL-rearranged infant ALL cases and guides clinical outcome. Therefore, inhibition of aberrant DNA methylation may be an important novel therapeutic strategy for MLL-rearranged ALL in infants.

Gene expression profiling-based dissection of MLL translocated and MLL germline acute lymphoblastic leukemia in infants

Acute lymphoblastic leukemia (ALL) in infants (< 1 year) is characterized by a poor prognosis and a high incidence of MLL translocations. Several studies demonstrated the unique gene expression profile associated with MLL-rearranged ALL, but generally small cohorts were analyzed as uniform patient groups regardless of the type of MLL translocation, whereas the analysis of translocation-negative infant ALL remained unacknowledged. Here we generated and analyzed primary infant ALL expression profiles (n = 73) typified by translocations t(4;11), t(11;19), and t(9;11), or the absence of MLL translocations. Our data show that MLL germline infant ALL specifies a gene expression pattern that is different from both MLL-rearranged infant ALL and pediatric precursor B-ALL. Moreover, we demonstrate that, apart from a fundamental signature shared by all MLL-rearranged infant ALL samples, each type of MLL translocation is associated with a translocation-specific gene expression signature. Finally, we show the existence of 2 distinct subgroups among t(4;11)-positive infant ALL cases characterized by the absence or presence of HOXA expression, and that patients lacking HOXA expression are at extreme high risk of disease relapse. These gene expression profiles should provide important novel insights in the complex biology of MLL-rearranged infant ALL and boost our progress in finding novel therapeutic solutions.

IKZF1 status as a prognostic feature in BCR-ABL1–positive childhood ALL

Childhood BCR-ABL1-positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has an unfavorable outcome and shows high frequency of IKZF1 deletions. The prognostic value of IKZF1 deletions was evaluated in 2 cohorts of BCR-ABL1-positive BCP-ALL patients, before tyrosine kinase inhibitors (pre-TKI) and after introduction of imatinib (in the European Study for Philadelphia-Acute Lymphoblastic Leukemia [EsPhALL]). In 126/191 (66%) cases an IKZF1 deletion was detected. In the pre-TKI cohort, IKZF1-deleted patients had an unfavorable outcome compared with wild-type patients (4-year disease-free survival [DFS] of 30.0 ± 6.8% vs 57.5 ± 9.4%; P = .01). In the EsPhALL cohort, the IKZF1 deletions were associated with an unfavorable prognosis in patients stratified in the good-risk arm based on early clinical response (4-year DFS of 51.9 ± 8.8% for IKZF1-deleted vs 78.6 ± 13.9% for IKZF1 wild-type; P = .03), even when treated with imatinib (4-year DFS of 55.5 ± 9.5% for IKZF1-deleted vs 75.0 ± 21.7% for IKZF1 wild-type; P = .05). In conclusion, the highly unfavorable outcome for childhood BCR-ABL1-positive BCP-ALL with IKZF1 deletions, irrespective of imatinib exposure, underscores the need for alternative therapies. In contrast, good-risk patients with IKZF1 wild-type responded remarkably well to imatinib-containing regimens, providing a rationale to potentially avoid hematopoietic stem-cell transplantation in this subset of patients.

Improved outcome with hematopoietic stem cell transplantation in a poor prognostic subgroup of infants with mixed-lineage-leukemia (MLL)-rearranged acute lymphoblastic leukemia: results from the Interfant-99 Study.

To define a role for hematopoietic stem cell transplantation (HSCT) in infants with acute lymphoblastic leukemia and rearrangements of the mixed-lineage-leukemia gene (MLL(+)), we compared the outcome of MLL(+) patients from trial Interfant-99 who either received chemotherapy only or HSCT. Of 376 patients with a known MLL status in the trial, 297 (79%) were MLL(+). Among the 277 of 297 MLL(+) patients (93%) in first remission (CR), there appeared to be a significant difference in disease-free survival (adjusted by waiting time to HSCT) between the 37 (13%) who received HSCT and the 240 (87%) who received chemotherapy only (P = .03). However, the advantage was restricted to a subgroup with 2 additional unfavorable prognostic features: age less than 6 months and either poor response to steroids at day 8 or leukocytes more than or equal to 300 g/L. Ninety-seven of 297 MLL(+) patients (33%) had such high-risk criteria, with 87 achieving CR. In this group, HSCT was associated with a 64% reduction in the risk of failure resulting from relapse or death in CR (hazard ratio = 0.36, 95% confidence interval, 0.15-0.86). In the remaining patients, there was no advantage for HSCT over chemotherapy only. In summary, HSCT seems to be a valuable option for a subgroup of infant MLL(+) acute lymphoblastic leukemia carrying further poor prognostic factors. The trial was registered at www.clinicaltrials.gov as #NCT00015873 and at www.controlled-trials.com as #ISRCTN24251487.

Association of high-level MCL-1 expression with in vitro and in vivo prednisone resistance in MLL-rearranged infant acute lymphoblastic leukemia

MLL-rearranged acute lymphoblastic leukemia (ALL) represents an unfavorable type of leukemia that often is highly resistant to glucocorticoids such as prednisone and dexamethasone. Because response to prednisone largely determines clinical outcome of pediatric patients with ALL, overcoming resistance to this drug may be an important step toward improving prognosis. Here, we show how gene expression profiling identifies high-level MCL-1 expression to be associated with prednisolone resistance in MLL-rearranged infant ALL, as well as in more favorable types of childhood ALL. To validate this observation, we determined MCL-1 expression with quantitative reverse transcription-polymerase chain reaction in a cohort of MLL-rearranged infant ALL and pediatric noninfant ALL samples and confirmed that high-level MCL-1 expression is associated with prednisolone resistance in vitro. In addition, MCL-1 expression appeared to be significantly higher in MLL-rearranged infant patients who showed a poor response to prednisone in vivo compared with prednisone good responders. Finally, down-regulation of MCL-1 in prednisolone-resistant MLL-rearranged leukemia cells by RNA interference, to some extent, led to prednisolone sensitization. Collectively, our findings suggest a potential role for MCL-1 in glucocorticoid resistance in MLL-rearranged infant ALL, but at the same time strongly imply that high-level MCL-1 expression is not the sole mechanism providing resistance to these drugs.

Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants

Acute lymphoblastic leukemia in infants represents an aggressive malignancy associated with a high incidence (approx. 80%) of translocations involving the Mixed Lineage Leukemia (MLL) gene. Attempts to mimic Mixed Lineage Leukemia fusion driven leukemogenesis in mice raised the question whether these fusion proteins require secondary hits. RAS mutations are suggested as candidates. Earlier results on the incidence of RAS mutations in Mixed Lineage Leukemia-rearranged acute lymphoblastic leukemia are inconclusive. Therefore, we studied frequencies and relation with clinical parameters of RAS mutations in a large cohort of infant acute lymphoblastic leukemia patients. Using conventional sequencing analysis, we screened neuroblastoma RAS viral (v-ras) oncogene homolog gene (NRAS), v-Ki-ras Kirsten rat sarcoma viral oncogene homolog gene (KRAS), and v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) for mutations in a large cohort (n=109) of infant acute lymphoblastic leukemia patients and studied the mutations in relation to several clinical parameters, and in relation to Homeobox gene A9 expression and the presence of ALL1 fused gene 4-Mixed Lineage Leukemia (AF4-MLL). Mutations were detected in approximately 14% of all cases, with a higher frequency of approximately 24% in t(4;11)-positive patients (P=0.04). Furthermore, we identified RAS mutations as an independent predictor (P=0.019) for poor outcome in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia, with a hazard ratio of 3.194 (95% confidence interval (CI):1.211–8.429). Also, RAS-mutated infants have higher white blood cell counts at diagnosis (P=0.013), and are more resistant to glucocorticoids in vitro (P<0.05). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients.

Outcome of congenital acute lymphoblastic leukemia treated on the Interfant-99 protocol

Acute lymphoblastic leukemia (ALL) diagnosed in the first month of life (congenital ALL) is very rare. Although congenital ALL is often assumed to be fatal, no studies have been published on outcome except for case reports. The present study reports the outcome of 30 patients with congenital ALL treated with the uniform Interfant-99 protocol, a hybrid regimen combining ALL treatment with elements designed for treatment of acute myeloid leukemia. Congenital ALL was characterized by a higher white blood cell count and a strong trend for higher incidence of MLL rearrangements and CD10-negative B-lineage ALL compared with older infants. Induction failure rate was 13% and not significantly different from that in older infants (7%, P = .14), but relapse rate was significantly higher in congenital ALL patients (2-year cumulative incidence [SE] was 60.0 [9.3] vs 34.2 [2.3], P < .001). Two-year event-free survival and survival of congenital ALL patients treated with this protocol was 20% (SE 9.1%). Early death in complete remission and treatment delays resulting from toxicity were not different. The survival of 17% after last follow-up, combined with a toxicity profile comparable with that in older infants, justifies treating congenital ALL with curative intent. This trial was registered at www.clinicaltrials.gov as no. NCT 00015873, and at www.controlled-trials.com as no. ISRCTN24251487.