B-cell acute lymphoblastic leukemia (B-ALL) with rearrangements involving DUX4 genes (DUX4r) occurs in 4–7% of B-ALL. Due to high variability of the breakpoint loci, DUX4r is difficult to detect during routine diagnostic. This study aimed to characterize DUX4r ALL. Gene expression profiling resulted in a cohort of 132 DUX4-positive cases. Patients were stratified in standard (6%), medium (48%) and high risk (60%) treatment groups according to the ALL-BFM study protocols. In total, 89% of patients achieved remission, 4% relapsed and 5% were lost during follow-up. After induction, 91% were MRD positive (MRD1) and 8% negative, while 47% of patients were MRD-positive after consolidation (MRD2) and 46% negative. Initially relapses were stratified as medium (40%) and high (60%) risk with positive MRD1 (100%) and MRD2 (80%). Using targeted DNA sequencing of available samples, we confirmed DUX4r and identified fusion partners in 72% of cases, with breakpoints in DUX4L2 (28%), DUX4L3 (14%), IGHJ6 (15%) and IGHD7 (12%) genes being the most common. Despite high numbers of MRD2 positive cases, DUX4r relapses are rare and patients might benefit from treatment de-escalation.

Relapse of acute lymphoblastic leukemia (ALL) in children/adolescence occurs in half of male patients with an extramedullary involvement in the testis. The molecular and cellular mechanisms, why leukemic cells preferentially migrate to and survive in the testis have not been systematically assessed. We aimed at characterizing the testicular and the bone marrow niche of ALL cells. We analyzed testicular and bone marrow samples of male pediatric patients using RNASeq and imaging mass cytometry. Our data reveal the physiological immune suppressive microenvironment in the testis compared to the bone marrow. In contrast, we observed a inflammatory environment because of massive leukemic cell proliferation within the tight organ, but an additional immune suppression by the leukemia. There was an increase in apoptosis suppression and DNA repair compared to the bone marrow. We identified different pathways upregulated, supporting the survival of leukemia cells in the testis. In conclusion, the leukemia infiltrated testis is a unique physiologically and pathophysiologically microenvironment, which explains the preferential survival and chemotherapy resistance of in this compartment.

Background: Current standard karyotyping methods are labor-intensive and have severe limitations in terms of resolution and duration to results. For hematologic malignancies, but not restricted to, this represents a relevant obstacle in translating molecular findings into time critical treatment decisions. Aims: To develop a sequencing based approach for rapid and scalable cytogenetics that can be applied to a wide spectrum of hematologic malignancies to detect clinically relevant molecular markers within 48hrs Methods: For copy number alteration detection, whole genome sequencing was performed on a GridION sequencer (Oxford Nanopore Technologies, ONT, using the SQK-LSK109 kit) in a cohort of 53 hematologic neoplasms, including acute myeloid leukemia (AML, n=18), chronic lymphocytic leukemia (CLL, n=8), multiple myeloma (MM, n=17) and pediatric acute lymphocytic leukemia (ALL, n=10) samples. For the identification of balanced alterations, transcriptome sequencing data were generated using ONT (SQK-DCS109 kit), and we applied an in-house developed analysis pipeline based on minimap2 alignment followed by Blast with an ensuing filtering algorithm based on orientation, inter-read distance and length filtering. For targeted sequencing of structural aberrations, we developed a CRISPR-Cas9 based tiling approach using sgRNA pools (Integrated DNA Technologies, IDT). This approach allows to cover AML relevant targets such as t(8;21), the KMT2A (MLL) breakpoint area, and the FLT3-ITD region as well as lymphoma relevant regions such as the immunoglobulin heavy chain locus (library preparation was performed using SQK-CS9109 kit). Results: For copy number profiling a median whole-genome coverage of 2.6 was reached (range 0.27-7 fold). ONT sequencing and conventional karyotyping approaches showed a high concordance with Pearson correlation coefficients >0.95 for copy number alteration comparisons between conventional cytogenetics and ONT sequencing results for all investigated disease entities. Regarding the detection of structural aberrations transcriptome sequencing and fusion gene analysis using the above described analysis and filtering pipeline we could e.g. reliably detect the t(9;22) translocation from the K562 cell line and the t(8;21) translocation from Kasumi-1. Additionally, using this method we were able to correctly identify the primary AML sample harboring a t(8;21) translocation that was among the n=12 AML patient samples. In addition to an RNA-based work-flow, we established a CRISPR-Cas9 based DNA enrichment approach for the detection of recurrent structural aberrations from specified genomic DNA loci. The enrichment of genomic regions of interest using sgRNA libraries led e.g. to a median coverage of 44 reads (range 5-136-fold) of the t(8;21) region-of-interest in primary AML samples, which allow the detection of the exact coordinates of the breakpoints. Summary/Conclusion: Long read based sequencing based copy number alteration and structural variation detection based on combination of different long read sequencing approaches like low-coverage whole genome sequencing, transcriptome sequencing and CRISPR-Cas9 based sequencing of genomic loci of interest represents a highly promising tool for high resolution and high speed cytogenetics that has the potential to overcome many limitations of conventional cytogenetics.

Background: The testis is the second most frequent (30%) non-hematological extramedullary site of relapse in pediatric acute lymphoblastic leukemia (ALL) treated according to European protocols. Testicular relapses usually occur late (> 6 months after completion of frontline treatment), and boys with a ETV6::RUNX1 fusion gene positive ALL have a significantly higher risk of a testicular relapse. In adult ALL, only about 1% of patients have a testicular relapse. Event-free survival is between 40%-80% depending on timing of relapse, involvement of the contralateral testis and of the bone marrow. The surgical removal of the clinically-involved testis or high-dose irradiation are the only current treatment options for testicular relapses to achieve a long-term event-free survival, but it impacts long-term quality of life. The molecular mechanisms regulating leukemic cell migration, growth, and survival in the testis have not been sufficiently assessed. Aims: We aimed at investigating our hypothesis that the testis is a frequent source of extramedullary relapse in pediatric ALL because of the physiologically strong CXCL12 gradient. This allows circulating leukemia cells to migrate into the testis during pre-puberty and to survive for potentially longer periods because of the specific immune suppressive microenvironment at this unusual anatomical site. Methods: To dissect the pre-pubertal cellular requirements and molecular pathways contributing to testicular leukemic cell dissemination and survival, we combined analysis of primary human leukemias with a patient derived xenograft (PDX) mouse model. We analyzed chemokine receptor expression profiles of pediatric B-ALL samples from patients with different relapse sites, studied the crosstalk of leukemia cell-stroma in co-cultures, and established a pediatric B-ALL PDX mouse model with testicular involvement. Results: The CXCL12-CXCR4 was identified as the driving force for B-ALL cell migration and survival in the testicular leukemic niche. Analysis of primary pediatric patient samples revealed that CXCR4 was the only chemokine receptor being robustly expressed on B-ALL cells both at the time of diagnosis and relapse. One prerequisite for leukemic cell infiltration in the testis mice was high surface expression of CXCR4 on PDX-ALL cells, and CXCL12 secretion from testicular stroma. In affected patient testes, leukemic cells localized within the interstitial space in close proximity to testicular macrophages. Another requirement for migration and survival of leukemia cells in the testis was pre-pubertal age of the recipient mice. Leukemic cell interactions with specific testis macrophage subpopulations, isolated from affected testes, altered their phenotype towards a pro-tumorigenic M2-like phenotype. The blockage of CXCR4-mediated functions by anti-CXCR4 antibody treatment reduced testicular infiltration of PDX-ALL cells. Summary/Conclusion: Collectively, a pre-pubertal condition together with high CXCR4 expression are factors affecting the leukemia permissive testicular microenvironment. CXCR4 could be proposed as a promising target for therapeutic prevention of testicular relapses in childhood B-ALL.

Background: Children with high-risk first-relapse B-ALL have a poor prognosis. We previously reported that, as compared with chemotherapy, treatment with blinatumomab resulted in prolonged event-free survival (EFS) and overall survival (OS) and higher rates of minimal residual disease (MRD) remission in a phase 3 trial (JAMA 2021;325:843-54; ASH 2021:Abs 1231). Aims: Here we provide follow-up (FU) data as of Sep 2021, with a focus on survival and MRD response. Methods: In this phase 3 trial (Amgen NCT02393859), children >28 days and <18 years old with high-risk first-relapse B-ALL were randomized 1:1 after induction and 2 cycles of consolidation to receive either a third intensive multidrug chemotherapy consolidation or blinatumomab (15 μg/m2/day, 4 weeks, continuous IV infusion). Study enrollment required M1 (<5% blasts) or M2 (≥5% and <25% blasts) bone marrow (BM) at randomization. Patients with complete remission (CR, ie, M1 BM) after treatment could undergo allogeneic hematopoietic stem cell transplant (alloHSCT). EFS, the primary endpoint, was calculated from randomization to whichever occurred first of relapse or M2 BM after CR, failure to achieve a CR at the end of treatment, second malignancy, or death (any cause). Survival outcomes and response rates were analyzed by baseline MRD, ie, MRD at enrollment prior to chemotherapy or blinatumomab. Parents or a legally acceptable representative provided written informed consent. Results: Enrollment was stopped based on the interim analysis for benefit of blinatumomab (7/17/19 primary analysis). The following results reflect an updated analysis as of Sep 2021. Between Nov 2015 and Aug 2019, 111 patients were randomized: 54 (49%) to blinatumomab and 57 (51%) to chemotherapy at 47 centers in 13 countries. Baseline characteristics were comparable in both arms as previously reported. After a median FU of 44 months, EFS was significantly higher with blinatumomab vs chemotherapy (at 4 years: 59% vs 27%, stratified log-rank p<0.001, hazard ratio [HR]: 0.35, 95% CI: 0.20-0.61). Blinatumomab also showed a strong benefit for the secondary endpoint of OS (at 4 years: 77% vs 49%, stratified log-rank p=0.002, HR: 0.34, 95% CI: 0.17-0.69). EFS, OS, and MRD remission (<10-4 blasts) were all improved with blinatumomab, both overall and by baseline MRD subgroup (< or ≥10-3) (Table 1a-b, Figure). Further, patients with and without extramedullary disease both had a benefit with blinatumomab. AlloHSCT in second CR was performed in 51/54 patients in the blinatumomab arm and in 39/57 patients in the chemotherapy arm. No new safety signals were identified. Second relapses occurred in 16/54 (30%) patients in the blinatumomab arm and 34/57 (60%) patients in the chemotherapy arm. In the blinatumomab arm, these second relapses were extramedullary in 5/54 patients, CNS in 5 (9.3%), 2 of which were solely CNS. In the chemotherapy arm, these second relapses were extramedullary in 8/57 patients, CNS in 3 (5.3%), 2 of which were solely CNS. CD19-negative relapse was seen in 3 patients in the blinatumomab arm (3/54) and 1 patient in the chemotherapy arm (1/57) prior to any subsequent CD19-directed therapy (ie, after study therapy). Image:Summary/Conclusion: In children with high-risk first-relapse B-ALL, treatment with 1 cycle of blinatumomab vs chemotherapy before alloHSCT resulted in superior EFS and improved OS and MRD response rates, all independent of baseline MRD. The incidence of CD19-negative relapse after blinatumomab was low.

Patients with high-risk neuroblastoma show in about 85% a bone marrow infiltration. Even though most of them achieve remission, minimal residual disease (MRD) frequently persists on submicroscopic level and causes a relapse. Our aim was to detect MRD cells in bone marrow aspirates taken during treatment, by employing a novel sensitive multiplex PCR chemistry, the mediator probe PCR (MP-PCR), which consists of a target-specific, unlabeled mediator probe and sequence-universal reporter conjugated with a fluorescent dye. Previously, the MP-PCR was successfully applied by Kipf et al. to determine MRD levels of acute lymphoblastic leukemia. Comparably to leukemia, high-risk neuroblastoma harbor multiple genetic alterations, which in addition appear on extrachromosomal DNA, including MYCN breakpoints, TERT rearrangements and ALK mutations. Our patient cohort comprises 18 patients with MYCN amplified neuroblastoma with multiple MYCN breakpoints and other mutations. To capture this tumor heterogeneity, we combined up to four MRD markers in one multiplex assay. Sensitive quantification of MRD benefits for the reconstruction of the clonal biology and personalization of neuroblastoma therapy.

Background: Relapse is the main cause of death from pediatric acute precursor T-cell leukemia (T-ALL), but the underlying mechanisms of disease evolution from initial disease to relapse remain incompletely understood and show remarkable interpatient heterogeneity. Aims: As cross-sectional studies failed to identify unifying determinants of relapse, we adopted a longitudinal strategy and performed multi-omic analyses in 13 matched pairs of initial diagnosis and relapse samples and their matched PDXs. We extended this set by WES and methylome analyses in an additional cohort of 25 matched DNA samples from patient cells collected at initial diagnosis, remission, and relapse. Methods: Thirty-eight patients were recruited from the ALL‐BFM 2000/2009, CoALL97/03/09, and ALL‐REZ BFM 2002 trials or from Schneider Children’s Medical Center of Israel at time points of initial diagnosis, remission, and relapse. Material from 13 matched pairs of PDXs (RNA, cells) was used for multi-omic analyses, including DNA-Seq (WES), RNA-Seq, ATAC-Seq and methylation analysis with EPIC arrays. Results: Based on the profile of SNVs and InDels we distinguished 18 (47%) type-1 (derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). We observed stronger remodeling on the way to type 2 than to type 1 relapses reflected by more evident changes in methylation, chromatin accessibility and gene expression. At the time of relapse, 3/20 type 2 patients exhibited a hypermutator phenotype, probably caused by gains of mutations in TP53, BLM and BUB1B combined with PMS2. Moreover, type 2 T-ALLs were predominantly TAL1-driven (4/8) in contrary to type 1 (0/5). T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. The fraction of subclonal mutations of those T-ALLs that later developed into a type-2 relapse was significantly higher already at the time of initial diagnosis than in those T-ALLs that later developed into a type-1 relapse (p=0.0387; Fisher’s exact), a difference that became even more pronounced at the time of relapse (p<0.0001). On the other hand, relapse type 1 T-ALLs exhibited overexpression of IL7R, its ligand HGF, and repressors of cytokine signaling (SOCS1, SOCS2, SOCS3) which regulates the IL7R pathway via negative feedback loop. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. Moreover, an analysis of remission samples revealed a significant enrichment of mutations in constitutional cancer predisposition genes (CPG) in type 2 patients, thus indicating fundamental differences between these two groups of patients. In both types of relapse, we observed known and novel drivers of drug resistance including MDR1 and MVP and NT5C2. Image:Summary/Conclusion: In sum, our comprehensive analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia. Leukemias of patients with type-1 relapses were often characterized by upregulation of the IL7R pathway, whereas type-2 relapses were characterized by (i) an enrichment of TAL-1 fusion, (ii) and of constitutional mutations in CPG, (iii) divergent genetic and epigenetic remodeling, and (iv) an enrichment of somatic hypermutator phenotypes.

Background:Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite an overall favorable prognosis, 15–20% of patients suffer a relapse. In the treatment of relapsed ALL, resistance to chemotherapy is a major challenge. The purine nucleotide metabolizing cytosolic 5́‐nucleotidase II (NT5C2) harbors activating mutations in 20% of pediatric relapsed T‐ALL and in 3–10% of relapsed B‐cell precursor ALL cases, which are considered to drive relapse formation by causing resistance to purine analogue‐based maintenance treatment of ALL. However, the impact of NT5C2 mutations for second‐line treatment and long‐term outcome of relapsed patients is unknown.Aims:To elucidate the clinical significance of NT5C2 mutations in relapsed ALL, we studied the NT5C2 gene in a large cohort of 455 patients with relapsed B‐cell precursor ALL and assessed correlations with clinical presentation, genetics, response to treatment and outcome.Methods:We detected NT5C2 mutations by Sanger sequencing and amplicon‐based next generation sequencing. To detect subclonal NT5C2 mutations, we established allele‐specific quantitative real‐time PCR assays following minimal residual disease measurement guidelines. All relapsed patients were treated according to the German ALL‐REZ BFM 2002 protocol.Results:By sequencing, we found n = 36 NT5C2 mutations in n = 34 samples. About 30% of NT5C2 mutations appeared subclonal with an estimated allele frequency of less than 20%. The variants p.R39Q (8/36, 22%) and p.R367Q (14/36, 39%) were the most prevalent mutations in our cohort. Using allele‐specific quantitative real‐time PCR targeted to p.R39Q and p.R367Q, we identified additional 69 subclonal NT5C2 mutations. In total, NT5C2 mutations were present in 16% of patients with relapsed B‐cell precursor ALL (74/455), but the majority of cases (66%) showed subclonal mutations only. NT5C2 mutations most frequently occurred in relapses of so‐called B‐other‐ALL that lacks established cytogenetic changes (P < 0.001) and were correlated with CDKN2A/B loss. In addition, predominant, but not subclonal NT5C2 mutations were associated with NRAS mutations indicating that RAS signaling may be involved in NT5C2‐driven relapse formation. Both, subclonal and predominant NT5C2 mutations, were associated with inferior event‐free survival rates of relapsed patients compared to wild‐type NT5C2 (0.189±0.058 and 0.333 ± 0.096, respectively, versus 0.575 ± 0.026; P < 0.001). Response to relapse treatment was particularly poor in relapsed patients with subclonal NT5C2 mutations only. This group showed an increased rate of nonresponse of more than 30% (P < 0.001). Moreover, patients of this group who responded to relapse treatment, showed high minimal residual disease levels at the end of relapse induction treatment in 80% of cases. To investigate whether outgrowth of NT5C2 mutant subclones during relapse treatment contributes to the poor response and outcome, we analyzed follow‐up samples and found that subclonal NT5C2 mutations never grew out to predominant mutations at the time of nonresponse or second relapse. In fact, analysis of samples taken after relapse induction treatment showed that NT5C2 mutant clones were frequently eradicated early during relapse treatment (14/20, 70%), whereas NT5C2 wild‐type leukemic cells persisted at minimal residual disease levels higher than 10E‐03 (16/20, 80%).Summary/Conclusion:Subclonal NT5C2 mutations define a patient group with poor response to treatment and outcome in relapsed B‐cell precursor ALL, but due to their transient character they may not play a driving role in this clinical phenotype.

About 25% of all neuroblastoma cases show an amplification of MYCN and half of these relapse after first-line therapy, which implies the survival of neuroblasts, referred to as minimal residual disease (MRD). We show that detection of breakpoints can be used as MRD assay for MYCN-amplified neuroblastoma.

The deregulation of pro- and anti-survival signalling pathways leads to escape from cell death and contributes to a poor response to chemotherapy and relapse in malignancies. Exploiting recently identified novel cell death mechanisms such as necroptosis represents an attractive strategy to eradicate such resistant tumor cells. Necroptosis occurs without caspase activation and relies on distinct protein-protein interactions regulated by receptor-interacting protein kinase 1, RIP1. RIP1 is held in check by the inhibitor of apoptosis proteins (IAPs). Depletion of IAPs using small-molecule SMAC mimetics (SM) can potently induce a switch from RIP1-controlled survival to cell death.

In the search for genes that define critical steps of relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) and can serve as prognostic markers, we performed targeted sequencing of 313 leukemia-related genes in 214 patients: 67 samples collected at the time of relapse and 147 at initial diagnosis. As relapse-specific genetic events, we identified activating mutations in NT5C2 (P=0.0001, Fisher's exact test), inactivation of TP53 (P=0.0007, Fisher's exact test) and duplication of chr17:q11.2-24.3 (P=0.0068, Fisher's exact test) in 32/67 of T-ALL relapse samples. Alterations of TP53 were frequently homozygous events, which significantly correlated with higher rates of copy number alterations in other genes compared with wild-type TP53 (P=0.0004, Mann-Whitney's test). We subsequently focused on mutations with prognostic impact and identified genes governing DNA integrity (TP53, n=8; USP7, n=4; MSH6, n=4), having key roles in the RAS signaling pathway (KRAS, NRAS, n=8), as well as IL7R (n=4) and CNOT3 (n=4) to be exclusively mutated in fatal relapses. These markers recognize 24/49 patients with a second event. In 17 of these patients with mostly refractory relapse and dire need for efficient treatment, we identified candidate targets for personalized therapy with p53 reactivating compounds, MEK inhibitors or JAK/STAT-inhibitors that may be incorporated in future treatment strategies.

Children with P2RY8-CRLF2-positive acute lymphoblastic leukemia have an increased relapse risk. Their mutational and transcriptional landscape, as well as the respective patterns at relapse remain largely elusive. We, therefore, performed an integrated analysis of whole-exome and RNA sequencing in 41 major clone fusion-positive cases including 19 matched diagnosis/relapse pairs. We detected a variety of frequently subclonal and highly instable JAK/STAT but also RTK/Ras pathway-activating mutations in 76% of cases at diagnosis and virtually all relapses. Unlike P2RY8-CRLF2 that was lost in 32% of relapses, all other genomic alterations affecting lymphoid development (58%) and cell cycle (39%) remained stable. Only IKZF1 alterations predominated in relapsing cases (P=0.001) and increased from initially 36 to 58% in matched cases. IKZF1's critical role is further corroborated by its specific transcriptional signature comprising stem cell features with signs of impaired lymphoid differentiation, enhanced focal adhesion, activated hypoxia pathway, deregulated cell cycle and increased drug resistance. Our findings support the notion that P2RY8-CRLF2 is dispensable for relapse development and instead highlight the prominent rank of IKZF1 for relapse development by mediating self-renewal and homing to the bone marrow niche. Consequently, reverting aberrant IKAROS signaling or its disparate programs emerges as an attractive potential treatment option in these leukemias.