Measurable residual disease analysis in paediatric acute lymphoblastic leukaemia patients with ABL-class fusions.

BACKGROUND: ABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers. METHODS: Precise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines. RESULTS: ABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P < 0.0001) and Ig/TCR and IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P < 0.0001). In contrast, in BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P < 0.0001) and IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P < 0.0001). CONCLUSIONS: MRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL.

Sensitive Measurement of Minimal Residual Disease in Blood by HAT-PCR.

PCR is widely used to measure minimal residual disease (MRD) in lymphoid neoplasms, but its sensitivity is limited. High Adenine/Thymine PCR and High Annealing Temperature PCR (HAT-PCR) is a modified PCR designed to minimize nonspecificity and hence increase sensitivity. It was evaluated in the laboratory and the clinic, using samples from 58 patients. Of these patients, 57 were adolescents or young adults who were participating in the Australasian Leukemia and Lymphoma Group ALL06 trial in which MRD was measured in blood principally by HAT-PCR and in marrow by conventional PCR. HAT-PCR produced significantly less nonspecificity than conventional PCR, and its limit of detection was <10-6 in 90% of patients. In 196 samples, an excellent correlation was found between blood and marrow MRD. Variable partitioning of leukemic cells between blood and marrow was observed. Measurement of MRD in blood by HAT-PCR was noninferior to measurement of MRD in marrow by conventional PCR, in terms of both detecting disease and predicting clinical outcome. At a median follow-up of 3 years and for MRD levels in blood at the end of consolidation treatment, an MRD level of >10-4 cells/L significantly predicted relapse and mortality, whereas undetectable MRD significantly predicted relapse-free survival and overall survival. HAT-PCR is a simple, quick, cheap and sensitive method for measurement of MRD, and its adoption for MRD in blood may be clinically useful.

Correlation between a 10-color flow cytometric measurable residual disease (MRD) analysis and molecular MRD in adult B-acute lymphoblastic leukemia.

BACKGROUND: Measurable residual disease (MRD) monitoring in acute lymphoblastic leukemia (ALL) is an important predictive factor for patient outcome and treatment intensification. Molecular monitoring, particularly with quantitative polymerase chain reaction (qPCR) to measure immunoglobin heavy or kappa chain (Ig) or T-cell receptor (TCR) gene rearrangements, offers high sensitivity but accessibility is limited by expertise, cost, and turnaround time. Flow cytometric assays are cheaper and more widely available, and sensitivity is improved with multi-parameter flow cytometry at eight or more colors. METHODS: We developed a 10-color single tube flow cytometry assay. Samples were subject to bulk ammonium chloride lysis to maximize cell yields with a target of 1 × 106 events. Once normal maturation patterns were established, patient samples were analyzed in parallel to standard molecular monitoring. RESULTS: Flow cytometry was performed on 114 samples. An informative immunophenotype was identifiable in all 22 patients who had a diagnostic sample. MRD analysis was performed on 87 samples. The median lower limits of detection and quantification were 0.004% (range 0.0005%-0.028%) and 0.01% (range 0.001%-0.07%) respectively. Sixty-five samples had concurrent molecular MRD testing, with good correlation (r = 0.83, p < 0.001). Results were concordant in 52 samples, and discordant in 13 samples, including one case where impending relapse was detected by flow cytometry but not Ig/TCR qPCR. CONCLUSIONS: Our 10-color flow cytometric MRD assay provided adequate sensitivity and good correlation with molecular assays. This technique offers rapid and affordable testing in B-ALL patients, including cases where a suitable molecular assay cannot be developed or has reduced sensitivity.

Outcomes for Australian children with relapsed/refractory acute lymphoblastic leukaemia treated with blinatumomab.

We report on the Australian experience of blinatumomab for treatment of 24 children with relapsed/refractory precursor B-cell acute lymphoblastic leukaemia (B-ALL) and high-risk genetics, resulting in a minimal residual disease (MRD) response rate of 58%, 2-year progression-free survival (PFS) of 39% and 2-year overall survival of 63%. In total, 83% (n = 20/24) proceeded to haematopoietic stem cell transplant, directly after blinatumomab (n = 12) or following additional salvage therapy (n = 8). Four patients successfully received CD19-directed chimeric antigen receptor T-cell therapy despite prior blinatumomab exposure. Inferior 2-year PFS was associated with MRD positivity (20%, n = 15) and in KMT2A-rearranged infants (15%, n = 9). Our findings highlight that not all children with relapsed/refractory B-ALL respond to blinatumomab and factors such as blast genotype may affect prognosis.

CKLF and IL1B transcript levels at diagnosis are predictive of relapse in children with pre-B-cell acute lymphoblastic leukaemia.

Disease relapse is the greatest cause of treatment failure in paediatric B-cell acute lymphoblastic leukaemia (B-ALL). Current risk stratifications fail to capture all patients at risk of relapse. Herein, we used a machine-learning approach to identify B-ALL blast-secreted factors that are associated with poor survival outcomes. Using this approach, we identified a two-gene expression signature (CKLF and IL1B) that allowed identification of high-risk patients at diagnosis. This two-gene expression signature enhances the predictive value of current at diagnosis or end-of-induction risk stratification suggesting the model can be applied continuously to help guide implementation of risk-adapted therapies.

Enrichment of atypical hyperdiploidy and IKZF1 deletions detected by SNP-microarray in high-risk Australian AIEOP-BFM B-cell acute lymphoblastic leukaemia cohort.

Acute lymphoblastic leukaemia (ALL) is the most common childhood malignancy with the majority of patients being classified as B-cell lineage (B-ALL). The sub-classification of B-ALL is based on genomic architecture. Recent studies have demonstrated the capability of SNP-microarrays to detect genomic changes in B-ALL which cannot be observed by conventional cytogenetic methods. In current clinical trials, B-ALL patients at high risk of relapse are mainly identified by adverse cancer genomics and/or poor response to early therapy. To test the hypothesis that inclusion of SNP-microarrays in frontline diagnostics could more efficiently and accurately identify adverse genomic factors than conventional techniques, we evaluated the Australian high-risk B-ALL cohort enrolled on AIEOP-BFM ALL 2009 study (n = 33). SNP-microarray analysis identified additional aberrations in 97% of patients (32/33) compared to conventional techniques. This changed the genomic risk category of 24% (8/33) of patients. Additionally, 27% (9/33) of patients exhibited a 'hyperdiploid' genome, which is generally associated with a good genomic risk and favourable outcomes. An enrichment of IKZF1 deletions was observed with one third of the cohort affected. Our findings suggest the current classification system could be improved and highlights the need to use more sensitive techniques such as SNP-microarray for cytogenomic risk stratification in B-ALL.

A risk score including microdeletions improves relapse prediction for standard and medium risk precursor B-cell acute lymphoblastic leukaemia in children.

To prevent relapse, high risk paediatric acute lymphoblastic leukaemia (ALL) is treated very intensively. However, most patients who eventually relapse have standard or medium risk ALL with low minimal residual disease (MRD) levels. We analysed recurrent microdeletions and other clinical prognostic factors in a cohort of 475 uniformly treated non-high risk precursor B-cell ALL patients with the aim of better predicting relapse and refining risk stratification. Lower relapse-free survival at 7 years (RFS) was associated with IKZF1 intragenic deletions (P < 0·0001); P2RY8-CRLF2 gene fusion (P < 0·0004); Day 33 MRD>5 × 10-5 (P < 0·0001) and High National Cancer Institute (NCI) risk (P < 0·0001). We created a predictive model based on a risk score (RS) for deletions, MRD and NCI risk, extending from an RS of 0 (RS0) for patients with no unfavourable factors to RS2 +  for patients with 2 or 3 high risk factors. RS0, RS1, and RS2 +  groups had RFS of 93%, 78% and 49%, respectively, and overall survival (OS) of 99%, 91% and 71%. The RS provided greater discrimination than MRD-based risk stratification into standard (89% RFS, 96% OS) and medium risk groups (79% RFS, 91% OS). We conclude that this RS may enable better early therapeutic stratification and thus improve cure rates for childhood ALL.

Integration of genetic and clinical risk factors improves prognostication in relapsed childhood B-cell precursor acute lymphoblastic leukemia.

Somatic genetic abnormalities are initiators and drivers of disease and have proven clinical utility at initial diagnosis. However, the genetic landscape and its clinical utility at relapse are less well understood and have not been studied comprehensively. We analyzed cytogenetic data from 427 children with relapsed B-cell precursor ALL treated on the international trial, ALLR3. Also we screened 238 patients with a marrow relapse for selected copy number alterations (CNAs) and mutations. Cytogenetic risk groups were predictive of outcome postrelapse and survival rates at 5 years for patients with good, intermediate-, and high-risk cytogenetics were 68%, 47%, and 26%, respectively (P < .001). TP53 alterations and NR3C1/BTG1 deletions were associated with a higher risk of progression: hazard ratio 2.36 (95% confidence interval, 1.51-3.70, P < .001) and 2.15 (1.32-3.48, P = .002). NRAS mutations were associated with an increased risk of progression among standard-risk patients with high hyperdiploidy: 3.17 (1.15-8.71, P = .026). Patients classified clinically as standard and high risk had distinct genetic profiles. The outcome of clinical standard-risk patients with high-risk cytogenetics was equivalent to clinical high-risk patients. Screening patients at relapse for key genetic abnormalities will enable the integration of genetic and clinical risk factors to improve patient stratification and outcome. This study is registered at www.clinicaltrials.org as #ISCRTN45724312.

Persistent MRD before and after allogeneic BMT predicts relapse in children with acute lymphoblastic leukaemia.

Minimal residual disease (MRD) during early chemotherapy is a powerful predictor of relapse in acute lymphoblastic leukaemia (ALL) and is used in children to determine eligibility for allogeneic haematopoietic stem cell transplantation (HSCT) in first (CR1) or later complete remission (CR2/CR3). Variables affecting HSCT outcome were analysed in 81 children from the ANZCHOG ALL8 trial. The major cause of treatment failure was relapse, with a cumulative incidence of relapse at 5 years (CIR) of 32% and treatment-related mortality of 8%. Leukaemia-free survival (LFS) and overall survival (OS) were similar for HSCT in CR1 (LFS 62%, OS 83%, n = 41) or CR2/CR3 (LFS 60%, OS 72%, n = 40). Patients achieving bone marrow MRD negativity pre-HSCT had better outcomes (LFS 83%, OS 92%) than those with persistent MRD pre-HSCT (LFS 41%, OS 64%, P < 0·0001) or post-HSCT (LFS 35%, OS 55%, P < 0·0001). Patients with B-other ALL had more relapses (CIR 50%, LFS 41%) than T-ALL and the main precursor-B subtypes including BCR-ABL1, KMT2A (MLL), ETV6-RUNX1 (TEL-AML1) and hyperdiploidy >50. A Cox multivariate regression model for LFS retained both B-other ALL subtype (hazard ratio 4·1, P = 0·0062) and MRD persistence post-HSCT (hazard ratio 3·9, P = 0·0070) as independent adverse prognostic variables. Persistent MRD could be used to direct post-HSCT therapy.

Improving the identification of high risk precursor B acute lymphoblastic leukemia patients with earlier quantification of minimal residual disease.

The stratification of patients with acute lymphoblastic leukemia (ALL) into treatment risk groups based on quantification of minimal residual disease (MRD) after induction therapy is now well accepted but the relapse rate of about 20% in intermediate risk patients remains a challenge. The purpose of this study was to further improve stratification by MRD measurement at an earlier stage. MRD was measured in stored day 15 bone marrow samples for pediatric patients enrolled on ANZCHOG ALL8 using Real-time Quantitative PCR to detect immunoglobulin and T-cell receptor gene rearrangements with the same assays used at day 33 and day 79 in the original MRD stratification. MRD levels in bone marrow at day 15 and 33 were highly predictive of outcome in 223 precursor B-ALL patients (log rank Mantel-Cox tests both P<0.001) and identified patients with poor, intermediate and very good outcomes. The combined use of MRD at day 15 (≥ 1 × 10(-2)) and day 33 (≥ 5 × 1(-5)) identified a subgroup of medium risk precursor B-ALL patients as poor MRD responders with 5 year relapse-free survival of 55% compared to 84% for other medium risk patients (log rank Mantel-Cox test, P = 0.0005). Risk stratification of precursor B-ALL but not T-ALL could be improved by using MRD measurement at day 15 and day 33 instead of day 33 and day 79 in similar BFM-based protocols for children with this disease.

Primary leukocyte screens for innate immune agonists.

The innate immune system of mammals is a key defense mechanism against invading foreign pathogens. Innate immune stimulants may have applications as vaccine adjuvants as well as in the treatment of cancer and some viral diseases, and clinical studies have been performed using agonists of Toll-like receptors (TLRs) 7, 8, and 9. The high-throughput screens for such agonists have typically relied on the overexpression of a single TLR gene in an immortalized cell line and are inherently artificial systems that are restricted to the identification of agonists for a single receptor. The authors describe 2 assays for the identification of immunostimulants that employ primary human leukocytes cocultured with hepatitis C virus (HCV) replicon-expressing cells. In these assays, stimulation of innate immune pathways in leukocytes induces interferon (IFN) expression, which acts to inhibit HCV replication, providing a high-throughput and low-cost readout for leukocyte activation. These assays are highly sensitive and provide a physiologically relevant system for the identification of a broad range of immunostimulant agents.