Janine Stutterheim

PURPOSE: About 60% of patients with high-risk neuroblastoma relapse. Specific mRNA detection in bone marrow (BM) by reverse transcriptase quantitative PCR (RT-qPCR) is associated with survival outcomes. Peripheral blood (PB) sampling is less invasive. Therefore, we prospectively validated an RT-qPCR panel of neuroblastoma mRNA in PB of patients with high-risk neuroblastoma, treated in NB2004-HR (GPOH) and NBL2009 (DCOG). METHODS: From 312 patients, 634 PB samples were prospectively collected (2009-2017) at diagnosis, after two cycles and end-of-induction therapy. RT-qPCR was performed using our panel of neuroblastoma mRNA markers: PHOX2B , TH , DDC , CHRNA3 , and DBH . Results were compared with paired BM samples. The association between neuroblastoma detection and event-free survival (EFS) and overall survival (OS) was estimated using Kaplan-Meier's methodology and multivariable Cox regression model. RESULTS: Clear correlation between calculated infiltration by neuroblastoma mRNA expression in PB and BM was seen at diagnosis ( r _s 0.70 [95% CI, 0.62 to 0.76]), and heterogeneity was seen after two cycles (0.37 [95% CI, 0.12 to 0.58]) and end of induction (0.61 [95% CI, 0.10 to 0.87]). mRNA expression was significantly lower in PB compared with BM. At diagnosis, PB infiltration ≥1% was a prognostic factor for survival: adjusted hazard ratio (HR) was 2.37 [95% CI, 1.56 to 3.60] for EFS and 2.60 [1.65-4.08] for OS. At the end of induction, PHOX2B positivity in PB samples (n = 11) was associated with poor outcomes: HR 3.06 [1.51-6.20] for EFS and 2.88 [1.36-6.11] for OS. In patients with ≥10% BM infiltration at diagnosis, detection of the mRNA panel in PB samples could significantly distinguish between survival groups; the adjusted HR of PB infiltration ≥1% was 2.09 [1.01-4.30] for OS. CONCLUSION: PB infiltration is associated with EFS and OS at diagnosis; it is also significantly associated with survival outcomes of patients with ≥10% BM infiltration at diagnosis. During follow-up, neuroblastoma mRNA detection in PB can be of added value, when BM analysis is not possible.

Summary: Infant acute lymphoblastic leukaemia (ALL) is a rare and aggressive type of leukaemia, especially when a KMT2A rearrangement is involved. Historical treatment strategies have included intensification of conventional ALL chemotherapy in combination with acute myeloid leukaemia treatment elements. However, outcomes have remained consistently poor compared to the advances that have been seen in childhood ALL. The emergence of novel immunotherapies has transformed treatment strategies, with blinatumomab now incorporated into clinical trials for infants with KMT2A ‐rearranged ALL, promising significant advancements. Additionally, deeper insights into the unique biology of KMT2A ‐rearranged ALL have facilitated the development of targeted therapies, such as venetoclax and menin inhibitors, which are under clinical evaluation. These efforts offer renewed hope for better outcomes. This review highlights the substantial progress in understanding and treating K MT2A ‐rearranged ALL, fuelling optimism for future therapeutic success. In addition, we highlight the challenges that might be faced by using immunotherapy.

Chromosomal translocation of the KMT2A gene represents the cytogenetic hallmark of acute lymphoblastic leukemia diagnosed in infants (<1 year of age), driving a highly aggressive malignancy. For decades the event-free survival rates for these very young patients were at best ~40%. However, recent advances adding immunotherapy in the form of the bi-specific T-cell engager blinatumomab to the treatment led to encouraging results. In the present review we describe the current progress made, as well as the challenges that still lie ahead in terms of drug-related toxicity, the implementation of less toxic agents, acquired drug resistance, central nervous system involvement, and lineage switches. In addition, we touch on the benefit of preclinical models that can assist in guiding new treatment strategies.

Measurable residual disease (MRD) is regularly tested at later timepoints after the end of first consolidation (EOC) in children with acute lymphoblastic leukemia (ALL). The question remains whether this is useful for detecting (molecular) relapse. We investigated the clinical relevance of MRD after EOC in intermediate risk patients treated on DCOG-ALL-10 (n = 271) and DCOG-ALL-9 (n = 122), with MRD n = 178) had excellent outcomes, irrespective of MRD results at later timepoints; 6-year cumulative incidence of relapse (6-y CIR) of 7.4% (95% CI, 3.9%–12.3%) for those with MRD negativity at all later timepoints compared to 3.8% (95% CI, 0.3%–16.8%) for those with one or more later timepoints being positive (p = 0.51). Patients with positive EOC MRD (n = 91) of whom the subsequent timepoints were MRD negative (n = 43), had comparable good outcomes, 6-y CIR of 7.0% (95% CI, 1.8%–17.2%). In contrast, patients being MRD positive at EOC and MRD positive at one or more subsequent timepoints (n = 48) had a higher risk of relapse, 6-y CIR 29.4% (95% CI, 17.2%–42.8%), p < 0.001. These findings were confirmed in the validation cohort of ALL-9 as well as using the updated EuroMRD guidelines. In EOC MRD-negative patients, subsequent MRD measurements can be abandoned. For EOC MRD-positive patients the subsequent MRD measurement might be informative for further risk stratification.

Aberrant expression of HOX and MEIS1 family genes, as seen in KMT2A-rearranged, NUP98-rearranged, or NPM1-mutated leukemias leads to arrested differentiation and leukemia development. HOX family genes are essential gatekeepers of physiologic hematopoiesis, and their expression is regulated by the interaction between KMT2A and menin. Menin inhibitors block this interaction, downregulate the abnormal expression of MEIS1 and other transcription factors and thereby release the differentiation block. Menin inhibitors show significant clinical efficacy against KMT2A-rearranged and NPM1-mutated acute leukemias, with promising potential to address unmet needs in various pediatric leukemia subtypes. In this collaborative initiative, pediatric and adult hematologists/oncologists, and stem cell transplant physicians have united their expertise to explore the potential of menin inhibitors in pediatric leukemia treatment internationally. Our efforts aim to provide a comprehensive clinical overview of menin inhibitors, integrating preclinical evidence and insights from ongoing global clinical trials. Additionally, we propose future international, inclusive, and efficient clinical trial designs, integrating pediatric populations in adult trials, to ensure broad access to this promising therapy for all children and adolescents with menin-dependent leukemias.

Pediatric acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) exhibit favorable survival rates. However, for AML and ALL patients carrying KMT2A gene translocations clinical outcome remains unsatisfactory. Key players in KMT2A-fusion-driven leukemogenesis include menin and DOT1L. Recently, menin inhibitors like revumenib have garnered attention for their potential therapeutic efficacy in treating KMT2A-rearranged acute leukemias. However, resistance to menin inhibition poses challenges, and identifying which patients would benefit from revumenib treatment is crucial. Here, we investigated the in vitro response to revumenib in KMT2A-rearranged ALL and AML. While ALL samples show rapid, dose-dependent induction of leukemic cell death, AML responses are much slower and promote myeloid differentiation. Furthermore, we reveal that acquired resistance to revumenib in KMT2A-rearranged ALL cells can occur either through the acquisition of MEN1 mutations or independently of mutations in MEN1. Finally, we demonstrate significant synergy between revumenib and the DOT1L inhibitor pinometostat in KMT2A-rearranged ALL, suggesting that such drug combinations represent a potent therapeutic strategy for these patients. Collectively, our findings underscore the complexity of resistance mechanisms and advocate for precise patient stratification to optimize the use of menin inhibitors in KMT2A-rearranged acute leukemia.

Minimal residual disease (MRD) is regularly measured at later timepoints after end of first consolidation (EOC) in children with acute lymphoblastic leukemia (ALL). The question remains whether this is useful for detecting (molecular) relapse. We investigated the clinical relevance of MRD after EOC in intermediate risk patients treated on DCOG-ALL-10 (n = 271) and DCOG-ALL-9 (n = 122), with MRD < 0.05% at EOC. EOC MRD negative patients (n = 178) had excellent outcomes, irrespective of MRD results at later timepoints; 6-years relapse free survival (6y-RFS) of 92.0% (95% CI:88–97) for those with MRD negativity at all later timepoints compared to 96.2% (95% CI:89–100) for those with one or more later timepoints being positive (p = 0.46). Patients with positive EOC MRD (n = 91) of whom the subsequent timepoints were MRD negative (n = 43), had comparable good outcomes, 6y-RFS of 93.0% (95% CI:85–100). Patients being MRD positive at EOC and MRD positivity at one or more subsequent timepoints (n = 48) had a higher risk of relapse, 6y-RFS 70.5% (95% CI:58–84), p = 0.002. These findings were confirmed in the validation cohort of ALL-9. In patients who are MRD negative at EOC, MRD measurements at later timepoints can be abandoned. For patients who are EOC MRD positive the subsequent MRD measurement might be informative for further risk stratification.

Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls (n = 40) and neuroblastoma patients with localized (n = 10) and metastatic disease (n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel (PHOX2B, TH, CHRNA3) and a cell cycle regulation panel (E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further.

PURPOSE: Total cell-free DNA (cfDNA) and tumor-derived cfDNA (ctDNA) can be used to study tumor-derived genetic aberrations. We analyzed the diagnostic and prognostic potential of cfDNA and ctDNA, obtained from pediatric patients with rhabdomyosarcoma. METHODS: cfDNA was isolated from diagnostic plasma samples from 57 patients enrolled in the EpSSG RMS2005 study. To study the diagnostic potential, shallow whole genome sequencing (shWGS) and cell-free reduced representation bisulphite sequencing (cfRRBS) were performed in a subset of samples and all samples were tested using droplet digital polymerase chain reaction to detect methylated RASSF1A ( RASSF1A-M). Correlation with outcome was studied by combining cfDNA RASSF1A-M detection with analysis of our rhabdomyosarcoma-specific RNA panel in paired cellular blood and bone marrow fractions and survival analysis in 56 patients. RESULTS: At diagnosis, ctDNA was detected in 16 of 30 and 24 of 26 patients using shallow whole genome sequencing and cfRRBS, respectively. Furthermore, 21 of 25 samples were correctly classified as embryonal by cfRRBS. RASSF1A-M was detected in 21 of 57 patients. The presence of RASSF1A-M was significantly correlated with poor outcome (the 5-year event-free survival [EFS] rate was 46.2% for 21 RASSF1A-M ‒positive patients, compared with 84.9% for 36 RASSF1A-M ‒negative patients [ P < .001]). RASSF1A-M positivity had the highest prognostic effect among patients with metastatic disease. Patients both negative for RASSF1A-M and the rhabdomyosarcoma-specific RNA panel (28 of 56 patients) had excellent outcome (5-year EFS 92.9%), while double-positive patients (11/56) had poor outcome (5-year EFS 13.6%, P < .001). CONCLUSION: Analyzing ctDNA at diagnosis using various techniques is feasible in pediatric rhabdomyosarcoma and has potential for clinical use. Measuring RASSF1A-M in plasma at initial diagnosis correlated significantly with outcome, particularly when combined with paired analysis of blood and bone marrow using a rhabdomyosarcoma-specific RNA panel.