TCRαß/CD19 cell-depleted HLA-haploidentical transplantation to treat pediatric acute leukemia: updated final analysis.

ABSTRACT: TCRαß/CD19 cell depletion is a promising graft manipulation technique frequently used in the context of human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation (HSCT). We previously reported the results of a phase I-II clinical trial (NCT01810120) to assess the safety and the efficacy of this type of exvivo T-cell depletion in 80 children with acute leukemia, showing promising survival outcomes. We now report an updated analysis on a cohort of 213 children with a longer follow-up (median, 47.6 months for surviving patients). With a 5-year cumulative incidence of nonrelapse mortality of 5.2% (95% confidence interval [CI], 2.8%-8.8%) and a cumulative incidence of relapse of 22.7% (95% CI, 16.9%-29.2%), projected 10-year overall and disease-free survival (DFS) were 75.4% (95% CI, 68.6%-80.9%) and 71.6% (95% CI, 64.4%-77.6%), respectively. Cumulative incidence of both grade II-IV acute and chronic graft-versus-host disease were low (14.7% and 8.1%, respectively). In a multivariable analysis for DFS including type of disease, use of total body irradiation in the conditioning regimen (hazard ratio [HR], 0.5; 95% CI, 0.26-0.98; P = .04), disease status at HSCT (complete remission [CR] ≥3 vs CR 1/2; HR, 2.23; 95% CI, 1.20-4.16; P = .01), and high levels of pre-HSCT minimal residual disease (HR, 2.09; 95% CI, 1.01-4.33; P = .04) were independently associated with outcome. In summary, besides confirming the good outcome results already reported (which are almost superimposable on those of transplant from HLA-matched donors), this clinical update allows the identification of patients at higher risk of treatment failure for whom personalized approaches, aimed at reducing the risk of relapse, are warranted.

CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

FCM marker importance for MRD assessment in T-cell acute lymphoblastic leukemia: An AIEOP-BFM-ALL-FLOW study group report.

T-lineage acute lymphoblastic leukemia (T-ALL) accounts for about 15% of pediatric and about 25% of adult ALL cases. Minimal/measurable residual disease (MRD) assessed by flow cytometry (FCM) is an important prognostic indicator for risk stratification. In order to assess the MRD a limited number of antibodies directed against the most discriminative antigens must be selected. We propose a pipeline for evaluating the influence of different markers for cell population classification in FCM data. We use linear support vector machine, fitted to each sample individually to avoid issues with patient and laboratory variations. The best separating hyperplane direction as well as the influence of omitting specific markers is considered. Ninety-one bone marrow samples of 43 pediatric T-ALL patients from five reference laboratories were analyzed by FCM regarding marker importance for blast cell identification using combinations of eight different markers. For all laboratories, CD48 and CD99 were among the top three markers with strongest contribution to the optimal hyperplane, measured by median separating hyperplane coefficient size for all samples per center and time point (diagnosis, Day 15, Day 33). Based on the available limited set tested (CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99), our findings prove that CD48 and CD99 are useful markers for MRD monitoring in T-ALL. The proposed pipeline can be applied for evaluation of other marker combinations in the future.

Moving forward in target antigen discovery for immunotherapy in acute myeloid leukemia.

Not available.

The genetic basis and cell of origin of mixed phenotype acute leukaemia.

Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.

CD72 is a pan-tumor antigen associated to pediatric acute leukemia.

In the development of novel immunotherapeutic approaches, the step of target identification is a challenging process, because it aims at identifying robust tumor-associated antigens (TAAs) specific for the pathological population and causing no off-target effects. Here we propose CD72 as a novel and robust TAA for pediatric acute leukemias. We provided an outline of CD72 expression assessed by flow cytometry on a variety of cancer cell lines and primary samples, including normal bone marrow (BM) samples and hematopoietic stem and progenitor cells. We analyzed CD 72 expression on a cohort of 495 pathological pediatric BM aspirates, including: 215 B-cell precursor acute lymphoblastic leukemias (BCP-ALL), 156 acute myeloid leukemias (AMLs), 88 T-lineage ALLs or lymphoblastic lymphomas with BM infiltration, 13 B-lineage lymphoblastic lymphomas with BM infiltration, 9 myelodysplastic syndromes with increased blasts (5%-9% blasts on BM: MDS-IB1) and 14 non-hematopoietic solid tumors infiltrating BM. Results showed that CD72 is highly expressed in almost all BCP-ALL and the majority of AML at diagnosis, including BCP-ALL cases characterized by CD19 loss. These findings support a potential role for advanced diagnostics and novel immunotherapy approaches, providing a pan-ALL and AML target.

Targeting the plasticity of mesenchymal stromal cells to reroute the course of acute myeloid leukemia.

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but >30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

Molecular characterization and clinical outcome of B-cell precursor acute lymphoblastic leukemia with IG-MYC rearrangement.

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin- MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

Blinatumomab overcomes poor prognostic impact of measurable residual disease in pediatric high-risk first relapse B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Blinatumomab, a CD3/CD19 BiTE® (bispecific T cell engager) molecule, was superior to high-risk third course consolidation chemotherapy (HC3) in prolonging event-free survival (EFS) in children with high-risk first relapse B-cell precursor acute lymphoblastic leukemia (B-ALL). Here, we report results from a post hoc measurable residual disease (MRD) analysis of this phase 3 study (NCT02393859). PROCEDURE: Children >28 days and <18 years with high-risk first-relapse B-ALL in cytomorphological complete remission (M1 marrow, <5% blasts) or with M2 marrow (≥5% and <25% blasts) after induction and two cycles of high-risk consolidation chemotherapy (baseline) were enrolled in this trial. Patients received one cycle of blinatumomab (15 µg/m2 /day, 4 weeks, continuous intravenous infusion) or HC3. The primary endpoint was EFS. In this post hoc analysis, patients with MRD <10-4 by PCR were grouped as having positive but not quantifiable (pbnq) or undetectable disease. RESULTS: A higher proportion of patients with MRD <10-4 had undetectable versus pbnq disease after blinatumomab (day 29) than after HC3 (p = 0.0367). Of the 22 patients with MRD ≥10-4 at baseline who achieved MRD remission after blinatumomab, 20 (91%) achieved MRD <10-4 remission by day 15. Patients treated with blinatumomab had improved EFS and overall survival compared with those treated with HC3 independent of end-of-induction or baseline (end-of-second consolidation) MRD levels. CONCLUSIONS: Blinatumomab was more efficacious than HC3 regardless of MRD status before treatment. These data support the role of blinatumomab in inducing deep MRD remission, negating the poor prognostic value of MRD.

Relative expansion of CD19-negative very-early normal B-cell precursors in children with acute lymphoblastic leukaemia after CD19 targeting by blinatumomab and CAR-T cell therapy: implications for flow cytometric detection of minimal residual disease.

CD19-directed treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) frequently leads to the downmodulation of targeted antigens. As multicolour flow cytometry (MFC) application for minimal/measurable residual disease (MRD) assessment in BCP-ALL is based on B-cell compartment study, CD19 loss could hamper MFC-MRD monitoring after blinatumomab or chimeric antigen receptor T-cell (CAR-T) therapy. The use of other antigens (CD22, CD10, CD79a, etc.) as B-lineage gating markers allows the identification of CD19-negative leukaemia, but it could also lead to misidentification of normal very-early CD19-negative BCPs as tumour blasts. In the current study, we summarized the results of the investigation of CD19-negative normal BCPs in 106 children with BCP-ALL who underwent CD19 targeting (blinatumomab, n = 64; CAR-T, n = 25; or both, n = 17). It was found that normal CD19-negative BCPs could be found in bone marrow after CD19-directed treatment more frequently than in healthy donors and children with BCP-ALL during chemotherapy or after stem cell transplantation. Analysis of the antigen expression profile revealed that normal CD19-negative BCPs could be mixed up with residual leukaemic blasts, even in bioinformatic analyses of MFC data. The results of our study should help to investigate MFC-MRD more accurately in patients who have undergone CD19-targeted therapy, even in cases with normal CD19-negative BCP expansion.

CD56, HLA-DR, and CD45 recognize a subtype of childhood AML harboring CBFA2T3-GLIS2 fusion transcript.

The presence of CBFA2T3-GLIS2 fusion gene has been identified in childhood Acute Myeloid Leukemia (AML). In view of the genomic studies indicating a distinct gene expression profile, we evaluated the role of immunophenotyping in characterizing a rare subtype of AML-CBFA2T3-GLIS2 rearranged. Immunophenotypic data were obtained by studying a cohort of 20 pediatric CBFA2T3-GLIS2-AML and 77 AML patients not carrying the fusion transcript. Enrolled cases were included in the Associazione Italiana di Ematologia Oncologia Pediatrica (AIEOP) AML trials and immunophenotypes were compared using different statistical approaches. By multiple computational procedures, we identified two main core antigens responsible for the identification of the CBFA2T3-GLIS2-AML. CD56 showed the highest performance in single marker evaluation (AUC = 0.89) and granted the most accurate prediction when used in combination with HLA-DR (AUC = 0.97) displaying a 93% sensitivity and 99% specificity. We also observed a weak-to-negative CD45 expression, being exceptional in AML. We here provide evidence that the combination of HLA-DR negativity and intense bright CD56 expression detects a rare and aggressive pediatric AML genetic lesion improving the diagnosis performance.

Low-Blood Lymphocyte Number and Lymphocyte Decline as Key Factors in COPD Outcomes: A Longitudinal Cohort Study.

BACKGROUND: Smokers with and without chronic obstructive pulmonary disease (COPD) are at risk of severe outcomes like exacerbations, cancer, respiratory failure, and decreased survival. The mechanisms for these outcomes are unclear; however, there is evidence that blood lymphocytes (BL) number might play a role. OBJECTIVE: The objective of this study is to investigate the relationship between BL and their possible decline over time with long-term outcomes in smokers with and without COPD. METHODS: In 511 smokers, 302 with COPD (COPD) and 209 without COPD (noCOPD), followed long term, we investigated whether BL number and BL decline over time might be associated with long-term outcomes. Smokers were divided according to BL number in high-BL (≥1,800 cells/µL) and low-BL (<1,800 cells/µL). Clinical features, cancer incidence, and mortality were recorded during follow-up. BL count in multiple samples and BL decline over time were calculated and related to outcomes. RESULTS: BL count was lower in COPD (1,880 cells/µL) than noCOPD (2,300 cells/µL; p < 0.001). 43% of COPD and 23% of noCOPD had low-BL count (p < 0.001). BL decline over time was higher in COPD than noCOPD (p = 0.040). 22.5% of the whole cohort developed cancer which incidence was higher in low-BL subjects and in BL decliners than high-BL (31 vs. 18%; p = 0.001) and no decliners (32 vs. 19%; p = 0.002). 26% in the cohort died during follow-up. Furthermore, low-BL count, BL decline, and age were independent risk factors for mortality by Cox regression analysis. CONCLUSION: BL count and BL decline are related to worse outcomes in smokers with and without COPD, which suggests that BL count and decline might play a mechanistic role in outcomes deterioration. Insights into mechanisms inducing the fall in BL count could improve the understanding of COPD pathogenesis and point toward new therapeutic measures.

Unrelated donor vs HLA-haploidentical α/ß T-cell- and B-cell-depleted HSCT in children with acute leukemia.

Traditionally, hematopoietic stem cell transplantation (HSCT) from both HLA-matched related and unrelated donors (UD) has been used for treating children with acute leukemia (AL) in need of an allograft. Recently, HLA-haploidentical HSCT after αß T-cell/B-cell depletion (αßhaplo-HSCT) was shown to be effective in single-center studies. Here, we report the first multicenter retrospective analysis of 127 matched UD (MUD), 118 mismatched UD (MMUD), and 98 αßhaplo-HSCT recipients, transplanted between 2010 and 2015, in 13 Italian centers. All these AL children were transplanted in morphological remission after a myeloablative conditioning regimen. Graft failure occurred in 2% each of UD-HSCT and αßhaplo-HSCT groups. In MUD vs MMUD-HSCT recipients, the cumulative incidence of grade II to IV and grade III to IV acute graft-versus-host disease (GVHD) was 35% vs 44% and 6% vs 18%, respectively, compared with 16% and 0% in αßhaplo-HSCT recipients (P < .001). Children treated with αßhaplo-HSCT also had a significantly lower incidence of overall and extensive chronic GVHD (P < .01). Eight (6%) MUD, 32 (28%) MMUD, and 9 (9%) αßhaplo-HSCT patients died of transplant-related complications. With a median follow-up of 3.3 years, the 5-year probability of leukemia-free survival in the 3 groups was 67%, 55%, and 62%, respectively. In the 3 groups, chronic GVHD-free/relapse-free (GRFS) probability of survival was 61%, 34%, and 58%, respectively (P < .001). When compared with patients given MMUD-HSCT, αßhaplo-HSCT recipients had a lower cumulative incidence of nonrelapse mortality and a better GRFS (P < .001). These data indicate that αßhaplo-HSCT is a suitable therapeutic option for children with AL in need of transplantation, especially when an allele-matched UD is not available.

International cooperative study identifies treatment strategy in childhood ambiguous lineage leukemia.

Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Münster Study of Leukemias of Ambiguous Lineage [iBFM-AMBI2012]) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19+ leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19+ ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19- and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial.

Relapses and treatment-related events contributed equally to poor prognosis in children with ABL-class fusion positive B-cell acute lymphoblastic leukemia treated according to AIEOP-BFM protocols.

ABL-class fusions other than BCR-ABL1 characterize around 2-3% of precursor B-cell acute lymphoblastic leukemia. Case series indicated that patients suffering from these subtypes have a dismal outcome and may benefit from the introduction of tyrosine kinase inhibitors. We analyzed clinical characteristics and outcome of 46 ABL-class fusion positive cases other than BCR-ABL1 treated according to AIEOP-BFM (Associazione Italiana di Ematologia-Oncologia Pediatrica-Berlin-Frankfurt-Münster) ALL 2000 and 2009 protocols; 13 of them received a tyrosine kinase inhibitor (TKI) during different phases of treatment. ABL-class fusion positive cases had a poor early treatment response: minimal residual disease levels of ≥5×10-4 were observed in 71.4% of patients after induction treatment and in 51.2% after consolidation phase. For the entire cohort of 46 cases, the 5-year probability of event-free survival was 49.1+8.9% and that of overall survival 69.6+7.8%; the cumulative incidence of relapse was 25.6+8.2% and treatment-related mortality (TRM) 20.8+6.8%. One out of 13 cases with TKI added to chemotherapy relapsed while eight of 33 cases without TKI treatment suffered from relapse, including six in 17 patients who had not received hematopoietic stem cell transplantation. Stem cell transplantation seems to be effective in preventing relapses (only three relapses in 25 patients), but was associated with a very high TRM (6 patients). These data indicate a major need for an early identification of ABL-class fusion positive acute lymphoblastic leukemia cases and to establish a properly designed, controlled study aimed at investigating the use of TKI, the appropriate chemotherapy backbone and the role of hematopoietic stem cell transplantation. (Registered at: clinicaltrials.gov identifier: NTC00430118, NCT00613457, NCT01117441).

Next-generation sequencing of PTEN mutations for monitoring minimal residual disease in T-cell acute lymphoblastic leukemia.

Minimal residual disease (MRD) analysis has become a powerful indicator to refine therapy in acute lymphoblastic leukemia (ALL). Here, we present an MRD detection based on the next-generation sequencing of PTEN exon 7 mutations (NGS-PTEN) in 30 pediatric T-cell ALL patients. By comparing the NGS-PTEN results with current quantitative PCR of antigen receptor gene rearrangements (qPCR-Ig/TR), an overall concordance of 80% was found between the two methods. However, the NGS-PTEN qualified a lower number of high-risk patients than qPCR-Ig/TR. These findings suggest that NGS-PTEN is a promising tool that could potentially be used to support current MRD methodologies for T-ALL patients.

Flow-cytometric minimal residual disease monitoring in blood predicts relapse risk in pediatric B-cell precursor acute lymphoblastic leukemia in trial AIEOP-BFM-ALL 2000.

BACKGROUND: Flow-cytometric monitoring of minimal residual disease (MRD) in bone marrow (BM) during induction of pediatric patients with acute lymphoblastic leukemia (ALL) is widely used for outcome prognostication and treatment stratification. Utilizing peripheral blood (PB) instead of BM might be favorable, but data on its usefulness are scarce. PROCEDURE: We investigated 1303 PB samples (days 0, 8, 15, 33, and 52) and 285 BMs (day 15) from 288 pediatric ALL patients treated in trial AIEOP-BFM ALL 2000. MRD was assessed by four-color flow cytometry and evaluated as relative, absolute, and kinetic result. RESULTS: In B-ALL only, PB measures from early time points correlated with relapse incidence (CIR). Best separation occurred at threshold <1 blast/µL at day 8 (5-year CIR 0.02 ± 0.02 vs 0.12 ± 0.03; P = 0.044). Patients with highest relapse risk were not distinguishable, but PB-MRD at days 33 and 52 correlated with prednisone response and postinduction BM-MRD by PCR (P < 0.001). Kinetic assessment did not convey any advantage. In multivariate analysis including day 15 BM-MRD, PB-MRD measures lost statistical power. CONCLUSIONS: In summary, PB-MRD in pediatric B-ALL correlates with outcome and risk parameters, but its prognostic significance is not strong enough to substitute for BM assessment in AIEOP-BFM trials. It might, however, be valuable in treatment environments not using multifaceted risk stratification with other MRD measures.

The presence of mutated and deleted PTEN is associated with an increased risk of relapse in childhood T cell acute lymphoblastic leukaemia treated with AIEOP-BFM ALL protocols.

Notwithstanding the improvement in treatment results for paediatric T cell acute lymphoblastic leukaemia (T-ALL) it remains important to understand if genetic aberrations influence therapy response. PTEN tumour suppressor gene inactivation is a frequent event in T-ALL but its effect on patient therapy response is debatable. We analysed the effect of the presence of mutated PTEN on outcome in 257 children with T-ALL treated with Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP)-Berlin-Frankfürt-Münster (BFM) protocols. PTEN mutations were present in 31 (12·1%) patients and were significantly associated with increased risk of relapse. PTEN mutations also indicate a poor prognosis in T-ALL patients in the absence of NOTCH1 mutations or in the group of patients with co-presence of PTEN mutation and deletions. These results indicate that PTEN genomic aberrations and the biologically consequential PTEN inactivation contribute to adverse therapy response in T-ALL patients; PTEN status as a biomarker may contribute to the development of new molecularly-defined stratification algorithms.

Pre- and post-transplant minimal residual disease predicts relapse occurrence in children with acute lymphoblastic leukaemia.

Relapse remains the leading cause of treatment failure in children with acute lymphoblastic leukaemia (ALL) undergoing allogeneic haematopoietic stem cell transplantation (HSCT). We retrospectively investigated the prognostic role of minimal residual disease (MRD) before and after HSCT in 119 children transplanted in complete remission (CR). MRD was measured by polymerase chain reaction in bone marrow samples collected pre-HSCT and during the first and third trimesters after HSCT (post-HSCT1 and post-HSCT3). The overall event-free survival (EFS) was 50%. The cumulative incidence of relapse and non-relapse mortality was 41% and 9%. Any degree of detectable pre-HSCT MRD was associated with poor outcome: EFS was 39% and 18% in patients with MRD positivity <1 × 10-3 and ≥1 × 10-3 , respectively, versus 73% in MRD-negative patients (P < 0·001). This effect was maintained in different disease remissions, but low-level MRD had a very strong negative impact only in patients transplanted in second or further CR. Also, MRD after HSCT enabled patients to be stratified, with increasing MRD between post-HSCT1 and post-HSCT3 clearly defining cohorts with a different outcome. MRD is an important prognostic factor both before and after transplantation. Given that MRD persistence after HSCT is associated with dismal outcome, these patients could benefit from early discontinuation of immunosuppression, or pre-emptive immuno-therapy.