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Supplementary Figure from Targeted Analysis of Cell-free Circulating Tumor DNA is Suitable for Early Relapse and Actionable Target Detection in Patients with Neuroblastoma

Supplementary Data from Targeted Analysis of Cell-free Circulating Tumor DNA is Suitable for Early Relapse and Actionable Target Detection in Patients with Neuroblastoma

Purpose:Treating refractory or relapsed neuroblastoma remains challenging. Monitoring body fluids for tumor-derived molecular information indicating minimal residual disease supports more frequent diagnostic surveillance and may have the power to detect resistant subclones before they give rise to relapses. If actionable targets are identified from liquid biopsies, targeted treatment options can be considered earlier.Experimental Design:Droplet digital PCR assays assessing MYCN and ALK copy numbers and allelic frequencies of ALK p.F1174L and ALK p.R1275Q mutations were applied to longitudinally collected liquid biopsies and matched tumor tissue samples from 31 patients with high-risk neuroblastoma. Total cell-free DNA (cfDNA) levels and marker detection were compared with data from routine clinical diagnostics.Results:Total cfDNA concentrations in blood plasma from patients with high-risk neuroblastoma were higher than in healthy controls and consistently correlated with neuron-specific enolase levels and lactate dehydrogenase activity but not with 123I-meta-iodobenzylguanidine scores at relapse diagnosis. Targeted cfDNA diagnostics proved superior for early relapse detection to all current diagnostics in 2 patients. Marker analysis in cfDNA indicated intratumor heterogeneity for cell clones harboring MYCN amplifications and druggable ALK alterations that were not detectable in matched tumor tissue samples in 17 patients from our cohort. Proof of concept is provided for molecular target detection in cerebrospinal fluid from patients with isolated central nervous system relapses.Conclusions:Tumor-specific alterations can be identified and monitored during disease course in liquid biopsies from pediatric patients with high-risk neuroblastoma. This approach to cfDNA surveillance warrants further prospective validation and exploitation for diagnostic purposes and to guide therapeutic decisions.

Neuroblastoma is characterised by extensive inter- and intra-tumour genetic heterogeneity and varying clinical outcomes. One possible driver for this heterogeneity are extrachromosomal DNAs (ecDNA), which segregate independently to the daughter cells during cell division and can lead to rapid amplification of oncogenes. While ecDNA-mediated oncogene amplification has been shown to be associated with poor prognosis in many cancer entities, the effects of ecDNA copy number heterogeneity on intermediate phenotypes are still poorly understood.Here, we leverage DNA and RNA sequencing data from the same single cells in cell lines and neuroblastoma patients to investigate these effects. We utilise ecDNA amplicon structures to determine precise ecDNA copy numbers and reveal extensive intercellular ecDNA copy number heterogeneity. We further provide direct evidence for the effects of this heterogeneity on gene expression of cargo genes, includingMYCNand its downstream targets, and the overall transcriptional state of neuroblastoma cells.These results highlight the potential for rapid adaptability of cellular states within a tumour cell population mediated by ecDNA copy number, emphasising the need for ecDNA-specific treatment strategies to tackle tumour formation and adaptation.

Activating mutations in cytosolic 5′-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2 mutations in relapsed ALL, we analyzed NT5C2 in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2 mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2 mutations compared with those without (19% and 25% vs 53%, P < .001). However, subclonal, but not clonal, NT5C2 mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P = .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P < .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2 mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2 mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2 mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2 mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.

Neuroblastoma is a pediatric solid tumor characterized by strong clinical heterogeneity. Although certain complex genomic alterations, such as extrachromosomal DNA amplifications (ecDNA), have been recurrently detected in neuroblastomas, the mutational processes involved in their generation remain largely unclear. By examining the topography of complex rearrangements along with mutational signatures derived from all variant classes, we identify previously unrecognized co-occurring mutational footprints, which we termed mutational scenarios. We demonstrate that clinical neuroblastoma heterogeneity is linked to differences in the processes driving these mutational scenarios. Whereas high-risk MYCN-amplified neuroblastoma genomes were characterized by signs of replication slippage and stress, homologous recombination-associated signatures defined high-risk non-MYCN-amplified patients. Non-high-risk neuroblastomas, on the other hand, were marked by footprints of chromosome missegregation. This analysis provides a systematic perspective on the repertoire of mutational patterns that contribute to clinical neuroblastoma heterogeneity.

Purpose: Treating refractory or relapsed neuroblastoma remains challenging. Monitoring body fluids for tumor-derived molecular information indicating minimal residual disease supports more frequent diagnostic surveillance and may have the power to detect resistant subclones before they give rise to relapses. If actionable targets are identified from liquid biopsies, targeted treatment options can be considered earlier. Experimental Design: Droplet digital PCR assays assessing MYCN and ALK copy numbers and allelic frequencies of ALK p.F1174L and ALK p.R1275Q mutations were applied to longitudinally collected liquid biopsies and matched tumor tissue samples from 31 patients with high-risk neuroblastoma. Total cell-free DNA (cfDNA) levels and marker detection were compared with data from routine clinical diagnostics. Results: Total cfDNA concentrations in blood plasma from patients with high-risk neuroblastoma were higher than in healthy controls and consistently correlated with neuron-specific enolase levels and lactate dehydrogenase activity but not with 123I-meta-iodobenzylguanidine scores at relapse diagnosis. Targeted cfDNA diagnostics proved superior for early relapse detection to all current diagnostics in 2 patients. Marker analysis in cfDNA indicated intratumor heterogeneity for cell clones harboring MYCN amplifications and druggable ALK alterations that were not detectable in matched tumor tissue samples in 17 patients from our cohort. Proof of concept is provided for molecular target detection in cerebrospinal fluid from patients with isolated central nervous system relapses. Conclusions: Tumor-specific alterations can be identified and monitored during disease course in liquid biopsies from pediatric patients with high-risk neuroblastoma. This approach to cfDNA surveillance warrants further prospective validation and exploitation for diagnostic purposes and to guide therapeutic decisions.

Extrachromosomal circularization of DNA is an important genomic feature in cancer. The structure, composition and genome-wide frequency of extrachromosomal circular DNA, however, have not yet been extensively profiled. Here, we combined genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multi-hit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer.

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA. The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyzed the MYCN amplicon structure and its chromatin landscape. This revealed two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplified a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons was characterized by high structural complexity, lacked key local enhancers, and instead contained distal chromosomal fragments, which harbored CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.

Simultaneous inhibition of multiple molecular targets is an established strategy to improve the continuance of clinical response to therapy. Here, we screened 49 molecules with dual nanomolar inhibitory activity against BRD4 and PLK1, best classified as dual kinase-bromodomain inhibitors, in pediatric tumor cell lines for their antitumor activity. We identified two candidate dual kinase-bromodomain inhibitors with strong and tumor-specific activity against neuroblastoma, medulloblastoma, and rhabdomyosarcoma tumor cells. Dual PLK1 and BRD4 inhibitor treatment suppressed proliferation and induced apoptosis in pediatric tumor cell lines at low nanomolar concentrations. This was associated with reduced MYCN-driven gene expression as assessed by RNA sequencing. Treatment of patient-derived xenografts with dual inhibitor UMB103 led to significant tumor regression. We demonstrate that concurrent inhibition of two central regulators of MYC protein family of protooncogenes, BRD4, and PLK1, with single small molecules has strong and specific antitumor effects in preclinical pediatric cancer models.

Contains fulltext : 207610.pdf (Publisher’s version ) (Closed access) BACKGROUND: Assessment of minimal residual disease (MRD) is an integral component for response monitoring and treatment stratification in acute lymphoblastic leukemia (ALL). We aimed to evaluate the genomic ETV6-RUNX1 fusion sites as a single marker for MRD quantification. PROCEDURE: In a representative, uniformly treated cohort of pediatric relapsed ALL patients (n = 52), ETV6-RUNX1 fusion sites were compared to the current gold standard, immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements. RESULTS: Primer/probe sets designed to ETV6-RUNX1 fusions achieved significantly more frequent a sensitivity and a quantitative range of at least 10(-4) compared to the gold standard with 100% and 73% versus 76% and 47%, respectively. The breakpoint sequence was identical at diagnosis and relapse in all tested cases. There was a high degree of concordance between quantitative MRD results assessed using ETV6-RUNX1 and the highest Ig/TCR marker (Spearman's 0.899, P < .01) with differences >(1/2) log-step in only 6% of patients. A high proportion of ETV6-RUNX1-positive ALL relapses (40%) in our cohort showed a poor response to induction treatment at relapse, and therefore had an indication for hematopoietic stem cell transplantation, demonstrating the need of accurate identification of this subgroup. CONCLUSIONS: ETV6-RUNX1 fusion sites are highly sensitive and reliable MRD markers. Our data confirm that they are unaffected by clonal evolution and selection during front-line and second-line chemotherapy in contrast to Ig/TCR rearrangements, which require several markers per patient to compensate for the observed loss of target clones. In future studies, the genomic ETV6-RUNX1 fusion can be used as single MRD marker.

Neuroblastoma is the most common extracranial tumor in children. Despite aggressive multimodal treatment, high-risk neuroblastoma remains a clinical challenge with survival rates below 50%. Adding targeted drugs to first-line therapy regimens is a promising approach to improve survival in these patients. TACR1 activation by substance P has been reported to be mitogenic in cancer cell lines. Tachykinin receptor (TACR1) antagonists are approved for clinical use as an antiemetic remedy since 2003. Tachykinin receptor inhibition has recently been shown to effectively reduce growth of several tumor types. Here, we report that neuroblastoma cell lines express TACR1, and that targeting TACR1 activity significantly reduced cell viability and induced apoptosis in neuroblastoma cell lines. Gene expression profiling revealed that TACR1 inhibition repressed E2F2 and induced TP53 signaling. Treating mice harboring established neuroblastoma xenograft tumors with Aprepitant also significantly reduced tumor burden. Thus, we provide evidence that the targeted inhibition of tachykinin receptor signaling shows therapeutic efficacy in preclinical models for high-risk neuroblastoma.

Concomitant inhibition of multiple therapeutic targets is an established strategy to improve the durability of clinical responses to targeted therapies. Rhabdomyosarcoma is the most common childhood soft tissue solid tumor and cure rates for patients with high-risk rhabdomyosarcoma remain dismal. Resistance to intensive chemotherapy is common, and few molecular therapies have been introduced clinically. High-risk rhabdomyosarcoma is driven by expression of the oncogenic fusion protein PAX3-FOXO1, which acts as a transcription factor. Recently, many efforts have been made to identify pharmacological means of inhibiting PAX3-FOXO1, given its central role in rhabdomyosarcoma cell line survival. The BET protein BRD4 has been shown to be required for transcription of PAX3-FOXO1 and its target genes and pharmacological inhibition via BET protein inhibitors exhibits anti-rhabdomyosarcoma effects. Phosphorylation of PAX3-FOXO1 by the polo like kinase 1 (PLK1) increases the stability of the oncoprotein. Based on these previous observations, we hypothesized that simultaneous inhibition of BRD4 and PLK1 could synergistically target PAX3-FOXO1 stability and activity, leading to increased anti-rhabdomyosarcoma effects. Indeed, combination treatment with PLK1 and BRD4 inhibitors exhibited synergistic effects specifically against PAX3-FOXO1-driven rhabdomyosarcoma cell lines. Dual PLK1-BRD4 inhibition suppressed proliferation and induced apoptosis in rhabdomyosarcoma cell lines at low nanomolar concentrations. Our results demonstrate a new strategy towards achieving dual synergistic targeting of BRD4 and PLK1 with a single agent which may lead to clinical translation for therapy of refractory rhabdomyosarcoma. Citation Format: Natalie Timme, Shuai Liu, Hailemichael Yosief, Heathcliff Dorado García, Ian MacArthur, Annabell Szymansky, Constantin Dohna, Georg Seifert, Patrick Hundsdörfer, Andrej Lissat, Angelika Eggert, Johannes Schulte, Wei Zhang, Anton Henssen. Dual PLK1 and BRD4 inhibition has synergistic therapeutic effects against high-risk rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2624.

Synthetic lethal dependencies have recently emerged as tumor-specific vulnerabilities which provide broad therapeutic windows and have successfully been used for rational therapeutic target discovery. Despite intense international efforts over many decades, cure rates for patients with high-risk rhabdomyosarcoma remain dismal. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. Telomere maintenance is a hallmark of cancer and requires either activation of telomerase (TERT) or alternative lengthening of telomeres (ALT). ALT is active in a large subset of rhabdomyosarcomas and not active in healthy tissues suggesting it is a unique, tumor-specific process. ALT has recently been shown to depend on DNA damage response activation through the ATR kinase, and studies in osteosarcoma have revealed that ALT renders cancer cells hypersensitive to ATR inhibition. This defines ALT as a tumor-specific synthetic lethal determinant for susceptibility to ATR inhibition. Consistent with previous reports, we observed that ALT-dependent rhabdomyosarcoma cells are hypersensitive to ATR inhibition via AZD6738, a small molecule ATR inhibitor currently being tested in clinical trials. Intriguingly, we found that treatment with ATR inhibitors not only induced DNA damage but also resulted in aneuploidy and subsequent apoptosis in ALT-dependent rhabdomyosarcoma cells, suggesting loss of telomere protection and inadequate chromosomal segregation occur during mitosis upon ATR inhibition. To test this, we measured unrepaired DNA double-strand break formation using TUNEL. ALT-dependent cells, but not TERT-dependent cells, showed increased TUNEL labeling, consistent with an increase in double-stranded DNA breaks and unprotected DNA ends. Consistent with reduced telomere maintenance, ATR inhibition significantly reduced the number of C-circles in rhabdomyosarcoma cells relying on ALT. Furthermore, AZD6738 treatment reduced phosphorylation of known ATR targets, and differentially phosphorylated peptides were enriched for proteins involved in alternative lengthening of telomeres as measured using SILAC labeling and LC-MS/MS phospho-proteomic analysis. Finally, treatment with AZD6738 reduced tumor burden in patient-derived primary rhabdomyosarcoma xenografts, which was potentiated by combined treatment with cisplatin. Our findings delineate a therapeutically actionable DNA repair dependency induced at least in part by ALT and may lead directly to clinical translation of ATR inhibitors for therapy of refractory rhabdomyosarcoma. Citation Format: Heathcliff Dorado García, Jennifer von Stebut, Ian MacArthur, Koshi Imami, Natalie Timme, Kerstin Schoenbeck, Annabell Szymansky, Georg Seifert, Patrick Hundsdoerfer, Andrej Lissat, Matthias Selbach, Angelika Eggert, Johannes Schulte, Anton Henssen. Synthetic lethal targeting of ATR in alternative lengthening of telomeres-dependent rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2628.

The ubiquitous cytosolic 5´nucleotidase II (NT5C2) dephosphorylates purine nucleotide monophosphates and has an important role in cellular purine metabolism. Increased levels of nucleotidase activity have been correlated with resistance to nucleoside analog drugs that are commonly used in the treatment of children with acute lymphoblastic leukemia (ALL). Recently, activating mutations of NT5C2 have been identified in relapsed childhood ALL. NT5C2 mutations were present in 20% and 10% of children with relapsed T-cell ALL and with relapsed B-cell precursor ALL, respectively. In vitro studies showed that NT5C2 mutations conferred an increased resistance to purine analog drugs to ALL cell lines. However, the predictive and prognostic value of NT5C2 mutations for response and outcome of patients has remained elusive and systematic studies are warranted. Therefore, we studied the presence of NT5C2 mutations in 259 children with first relapse of B-cell precursor ALL. The patient cohort was a representative subset the German relapse trial ALL-REZ BFM 2002. NT5C2 exons 9, 13, 15 and 16 were sequenced according to Sanger in leukemic samples taken at diagnosis of first relapse. The clinical significance of NT5C2 mutations was assessed by comparing clinical parameters and survival differences between patients with and without NT5C2 mutation, and by multivariate Cox regression modelling. We identified NT5C2 mutations in 5.8% (15/259) of patients with first relapse of B-cell precursor ALL. This confirms the overall lower NT5C2 mutation rate in children with relapsed B-cell precursor ALL compared to those with relapsed T-cell ALL. Sixty percent (9/15) of the patients with NT5C2 mutation showed a G to A transition in codon 367 in exon 13. This mutation substitutes arginine with glutamine (p.R367Q) and represents a mutation hot spot in relapsed T-cell ALL. Our study reveals that p.R367Q is the predominant site of mutation also in relapsed ALL of B-cell precursor lineage. Patients with NT5C2 mutation significantly more often presented with a very early relapse within 18 months after initial diagnosis (P We conclude that mutation of NT5C2 can serve as predictor for the occurrence of a second relapse independent of response to relapse treatment in children with relapsed B-cell precursor ALL. Disclosures No relevant conflicts of interest to declare.