Immune reconstitution after transplantation of autologous peripheral stem cells in children: a comparison between CD34+ selected and nonmanipulated grafts.

BACKGROUND AND AIMS: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) improves the prognosis in pediatric patients with several solid tumors and lymphomas. Little is known about the reconstitution of the immune system after ASCT and the influence of CD34+ cell selection on the reconstitution in pediatric patients. METHODS: Between 1990 and 2001, 94 pediatric patients with solid tumors and lymphomas received autologous CD34+ selected or unmanipulated peripheral stem cells after HDC. CD34+ selection was carried out with magnetic microbeads. The absolute numbers of T cells, B cells and natural killer (NK) cells were measured and compared in both groups at various time points post-transplant. RESULTS: Recovery of T cells was significantly faster in the unmanipulated group at day 30, with no significant difference later on. Reconstitution of B and NK cells was similar in both groups without significant differences at any time. The CD34+-selected group was divided into patients receiving less or more than 5.385 × 106/kg CD34+ cells. Patients in the CD34+ high-dose group displayed significantly faster reconstitutions of neutrophiles and lymphocyte subsets than the CD34+ low-dose group. CONCLUSIONS: Engraftment and reconstitution of leukocytes, B cells and NK cells after transplantation of CD34+ selected stem cells were comparable to that in patients receiving unmanipulated grafts. T-cell recovery was faster in the unmanipulated group only within the first month. However, this delay could be compensated by transplantation of >5.385 × 106 CD34+ cells/kg. Especially for patients receiving immunotherapy after HDC large numbers of immune effector cells such as NK and T cells are necessary to mediate antibody-dependent cellular cytotoxicity. Therefore, in patients receiving autologous CD34+-selected grafts, our data emphasize the need to administer high stem cell counts.

A novel approach to guide GD2-targeted therapy in pediatric tumors by PET and [64Cu]Cu-NOTA-ch14.18/CHO.

Background: The tumor-associated disialoganglioside GD2 is a bona fide immunotherapy target in neuroblastoma and other childhood tumors, including Ewing sarcoma and osteosarcoma. GD2-targeting antibodies proved to be effective in neuroblastoma and GD2-targeting chimeric antigen receptors (CAR)- expressing T cells as well as natural killer T cells (NKTs) are emerging. However, assessment of intra- and intertumoral heterogeneity has been complicated by ineffective immunohistochemistry as well as sampling bias in disseminated disease. Therefore, a non-invasive approach for the assessment and visualization of GD2 expression in-vivo is of upmost interest and might enable a more appropriate treatment stratification. Methods: Recently, [64Cu]Cu-NOTA-ch14.18/CHO (64Cu-GD2), a radiolabeled GD2-antibody for imaging with Positron-Emission-Tomography (PET) was developed. We here report our first clinical patients' series (n = 11) in different pediatric tumors assessed with 64Cu-GD2 PET/MRI. GD2-expression in tumors and tissue uptake in organs was evaluated by semiquantitative measurements of standardized uptake values (SUV) with PET/MRI on day 1 p.i. (n = 11) as well as on day 2 p.i. (n = 6). Results: In 8 of 9 patients with suspicious tumor lesions on PET/MRI at least one metastasis showed an increased 64Cu-GD2 uptake and a high tracer uptake (SUVmax > 10) was measured in 4 of those 8 patients. Of note, sufficient image quality with high tumor to background contrast was readily achieved on day 1. In case of 64Cu-GD2-positive lesions, an excellent tumor to background ratio (at least 6:1) was observed in bones, muscles or lungs, while lower tumor to background contrast was seen in the spleen, liver and kidneys. Furthermore, we demonstrated extensive tumor heterogeneity between patients as well as among different metastatic sites in individual patients. Dosimetry assessment revealed a whole-body dose of only 0.03 mGy/MBq (range 0.02-0.04). Conclusion: 64Cu-GD2 PET/MRI enables the non-invasive assessment of individual heterogeneity of GD2 expression, which challenges our current clinical practice of patient selection, stratification and immunotherapy application scheme for treatment with anti-GD2 directed therapies.

Precise CRISPR-Cas9 gene repair in autologous memory T cells to treat familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity. Here, we used an adeno-associated virus-based CRISPR-Cas9 system with an inhibitor of nonhomologous end joining to repair such mutations in potentially long-lived T cells ex vivo. Repaired CD8 memory T cells efficiently cured lethal hyperinflammation in a mouse model of Epstein-Barr virus-triggered FHL2, a subtype caused by perforin-1 (Prf1) deficiency. Furthermore, repair of PRF1 and Munc13-4 (UNC13D)-whose deficiency causes the FHL subtype FHL3-in mutant memory T cells from two critically ill patients with FHL restored T cell cytotoxicity. These results provide a starting point for the treatment of genetic T cell immune dysregulation syndromes with repaired autologous T cells.

Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer.

DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.

Pharmacokinetics, Safety, and Efficacy of Letermovir for Cytomegalovirus Prophylaxis in Adolescent Hematopoietic Cell Transplantation Recipients.

INTRODUCTION: Letermovir is a cytomegalovirus (CMV) terminase complex inhibitor approved for prophylaxis of CMV infection and disease in adult CMV-seropositive allogeneic hematopoietic cell transplantation (allo-HCT) recipients (R+). We report pharmacokinetics (PK), safety, and efficacy of letermovir in adolescent (12-18 years) allogeneic HCT recipients from an ongoing clinical study. METHODS: In this phase 2b, multicenter, open-label study (NCT03940586), 28 adolescents received 480 mg letermovir [240 mg with cyclosporin A (CsA)] once daily orally or intravenously. Blood was collected for intensive (n = 14) plasma concentrations of letermovir. Intensive PK data were used for dose confirmation. Target exposure range 34,400-100,000 h × ng/mL for pediatric median exposures was based on model-predicted phase 3 population PK simulations in adult HCT recipients. RESULTS: All participants were CMV-seropositive (body weight 28.7-95.0 kg). Of 12 PK-evaluable participants, 8 receiving 480 mg letermovir without CsA and 4 receiving 240 mg letermovir with CsA achieved exposures comparable to the adult exposure range. Exposure above the target but below the adult clinical program maximum was observed in 1 patient. Safety was consistent with previously described safety in adults. The proportion of participants with clinically significant CMV infection through week 24 post-HCT was comparable (24%) to that in the pivotal phase 3 study in adults (37.5%). CONCLUSIONS: Administration of adult letermovir doses in this adolescent cohort resulted in exposures within adult clinical program margins and was associated with safety and efficacy similar to adults. Results support a letermovir dose of 480 mg (240 mg with CsA) in adolescent allo-HCT recipients.

Elimusertib has Antitumor Activity in Preclinical Patient-Derived Pediatric Solid Tumor Models.

The small-molecule inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), elimusertib, is currently being tested clinically in various cancer entities in adults and children. Its preclinical antitumor activity in pediatric malignancies, however, is largely unknown. We here assessed the preclinical activity of elimusertib in 38 cell lines and 32 patient-derived xenograft (PDX) models derived from common pediatric solid tumor entities. Detailed in vitro and in vivo molecular characterization of the treated models enabled the evaluation of response biomarkers. Pronounced objective response rates were observed for elimusertib monotherapy in PDX, when treated with a regimen currently used in clinical trials. Strikingly, elimusertib showed stronger antitumor effects than some standard-of-care chemotherapies, particularly in alveolar rhabdomysarcoma PDX. Thus, elimusertib has strong preclinical antitumor activity in pediatric solid tumor models, which may translate to clinically meaningful responses in patients.

Impact of a booster dose on SARS-CoV2 mRNA vaccine-specific humoral-, B- and T cell immunity in pediatric stem cell transplant recipients.

Stem cell transplant recipients (SCTR) are imperiled to increased risks after SARS-CoV2 infection, supporting the need for effective vaccination strategies for this vulnerable group. With respect to pediatric patients, data on immunogenicity of SARS-CoV2 mRNA-based vaccination is limited. We therefore comprehensively examined specific humoral, B- and T cell responses in a cohort of 2-19 year old SCTR after the second and third vaccine dose. Only after booster vaccination, transplant recipients reached similar levels of vaccine-specific IgG, IgA and neutralizing antibodies against omicron variant as age-matched controls. Although frequencies of SARS-CoV2 specific B cells increased after the third dose, they were still fourfold reduced in patients compared to controls. Overall, the majority of individuals enrolled mounted SARS-CoV2 Spike protein-specific CD4+ T helper cell responses with patients showing significantly higher portions than controls after the third dose. With respect to functionality, however, SCTR were characterized by reduced frequencies of specific interferon gamma producing CD4+ T cells, along with an increase in IL-2 producers. In summary, our data identify distinct quantitative and qualitative impairments within the SARS-CoV2 vaccination specific B- and CD4+ T cell compartments. More importantly, humoral analyses highlight the need for a booster vaccination of SCTR particularly for development of neutralizing antibodies.

Mutational topography reflects clinical neuroblastoma heterogeneity.

Neuroblastoma is a pediatric solid tumor characterized by strong clinical heterogeneity. Although clinical risk-defining genomic alterations exist in neuroblastomas, the mutational processes involved in their generation remain largely unclear. By examining the topography and mutational signatures derived from all variant classes, we identified co-occurring mutational footprints, which we termed mutational scenarios. We demonstrate that clinical neuroblastoma heterogeneity is associated with differences in the mutational processes driving these scenarios, linking risk-defining pathognomonic variants to distinct molecular processes. Whereas high-risk MYCN-amplified neuroblastomas were characterized by signs of replication slippage and stress, homologous recombination-associated signatures defined high-risk non-MYCN-amplified patients. Non-high-risk neuroblastomas were marked by footprints of chromosome mis-segregation and TOP1 mutational activity. Furthermore, analysis of subclonal mutations uncovered differential activity of these processes through neuroblastoma evolution. Thus, clinical heterogeneity of neuroblastoma patients can be linked to differences in the mutational processes that are active in their tumors.

Defining the landscape of circular RNAs in neuroblastoma unveils a global suppressive function of MYCN.

Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis.

IGF2BP1 induces neuroblastoma via a druggable feedforward loop with MYCN promoting 17q oncogene expression.

BACKGROUND: Neuroblastoma is the most common solid tumor in infants accounting for approximately 15% of all cancer-related deaths. Over 50% of high-risk neuroblastoma relapse, emphasizing the need of novel drug targets and therapeutic strategies. In neuroblastoma, chromosomal gains at chromosome 17q, including IGF2BP1, and MYCN amplification at chromosome 2p are associated with adverse outcome. Recent, pre-clinical evidence indicates the feasibility of direct and indirect targeting of IGF2BP1 and MYCN in cancer treatment. METHODS: Candidate oncogenes on 17q were identified by profiling the transcriptomic/genomic landscape of 100 human neuroblastoma samples and public gene essentiality data. Molecular mechanisms and gene expression profiles underlying the oncogenic and therapeutic target potential of the 17q oncogene IGF2BP1 and its cross-talk with MYCN were characterized and validated in human neuroblastoma cells, xenografts and PDX as well as novel IGF2BP1/MYCN transgene mouse models. RESULTS: We reveal a novel, druggable feedforward loop of IGF2BP1 (17q) and MYCN (2p) in high-risk neuroblastoma. This promotes 2p/17q chromosomal gains and unleashes an oncogene storm resulting in fostered expression of 17q oncogenes like BIRC5 (survivin). Conditional, sympatho-adrenal transgene expression of IGF2BP1 induces neuroblastoma at a 100% incidence. IGF2BP1-driven malignancies are reminiscent to human high-risk neuroblastoma, including 2p/17q-syntenic chromosomal gains and upregulation of Mycn, Birc5, as well as key neuroblastoma circuit factors like Phox2b. Co-expression of IGF2BP1/MYCN reduces disease latency and survival probability by fostering oncogene expression. Combined inhibition of IGF2BP1 by BTYNB, MYCN by BRD inhibitors or BIRC5 by YM-155 is beneficial in vitro and, for BTYNB, also. CONCLUSION: We reveal a novel, druggable neuroblastoma oncogene circuit settling on strong, transcriptional/post-transcriptional synergy of MYCN and IGF2BP1. MYCN/IGF2BP1 feedforward regulation promotes an oncogene storm harboring high therapeutic potential for combined, targeted inhibition of IGF2BP1, MYCN expression and MYCN/IGF2BP1-effectors like BIRC5.

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells.

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond.

Molecular characterization of the circadian clock in paediatric leukaemia patients: a prospective study protocol.

BACKGROUND: In many organisms, including humans, the timing of cellular processes is regulated by the circadian clock. At the molecular level the core-clock consists of transcriptional-translational-feedback loops including several genes such as BMAL1, CLOCK, PERs and CRYs generating circa 24-h rhythms in the expression of about 40% of our genes across all tissues. Previously these core-clock genes have been shown to be differentially expressed in various cancers. Albeit a significant effect in treatment optimization of chemotherapy timing in paediatric acute lymphoblastic leukaemia has previously been reported, the mechanistic role played by the molecular circadian clock in acute paediatric leukaemia remains elusive. METHODS: To characterize the circadian clock, we will recruit patients with newly diagnosed leukaemia and collect time course saliva and blood samples, as well as a single bone marrow sample. From the blood and bone marrow samples nucleated cells will be isolated and further undergo separation into CD19+ and CD19- cells. qPCR is performed on all samples targeting the core-clock genes including BMAL1, CLOCK, PER2 and CRY1. Resulting data will be analysed for circadian rhythmicity using the RAIN algorithm and harmonic regression. DISCUSSION: To the best of our knowledge this is the first study aiming to characterize the circadian clock in a cohort of paediatric patients with acute leukaemia. In the future we hope to contribute to uncovering further vulnerabilities of cancers associated with the molecular circadian clock and in particular adjust chemotherapy accordingly, leading to more targeted toxicity, and hence decreased systemic toxicities.

Anti-GD2 Antibody Dinutuximab Beta and Low-Dose Interleukin 2 After Haploidentical Stem-Cell Transplantation in Patients With Relapsed Neuroblastoma: A Multicenter, Phase I/II Trial.

PURPOSE: Patients with relapsed high-risk neuroblastoma (rHR-NB) have a poor prognosis. We hypothesized that graft-versus-neuroblastoma effects could be elicited by transplantation of haploidentical stem cells (haplo-SCT) exploiting cytotoxic functions of natural killer cells and their activation by the anti-GD2 antibody dinutuximab beta (DB). This phase I/II trial assessed safety, feasibility, and outcomes of immunotherapy with DB plus subcutaneous interleukin-2 (scIL2) after haplo-SCT in patients with rHR-NB. METHODS: Patients age 1-21 years underwent T-/B-cell-depleted haplo-SCT followed by DB and scIL2. The primary end point 'success of treatment' encompassed patients receiving six cycles, being alive 180 days after end of trial treatment without progressive disease, unacceptable toxicity, acute graft-versus-host-disease (GvHD) ≥grade 3, or extensive chronic GvHD. RESULTS: Seventy patients were screened, and 68 were eligible for immunotherapy. Median number of DB cycles was 6 (range, 1-9). Median number of scIL2 cycles was 3 (1-6). The primary end point was met by 37 patients (54.4%). Median observation time was 7.8 years. Five-year event-free survival (EFS) and overall survival from start of trial treatment were 43% (95% CI, 31 to 55) and 53% (95% CI, 41 to 65), respectively. Five-year EFS among patients in complete remission (CR; 52%; 95% CI, 31 to 69) or partial remission (44%; 95% CI, 27 to 60) before immunotherapy were significantly better compared with patients with nonresponse/mixed response/progressive disease (13%; 95% CI, 1 to 42; P = .026). Overall response rate in 43 patients with evidence of disease after haplo-SCT was 51% (22 patients), with 15 achieving CR (35%). Two patients developed GvHD grade 2 and 3 each. No unexpected adverse events occurred. CONCLUSION: DB therapy after haplo-SCT in patients with rHR-NB is feasible, with low risk of inducing GvHD, and results in long-term remissions likely attributable to increased antineuroblastoma activity by donor-derived effector cells.

Genomic ALK alterations in primary and relapsed neuroblastoma.

BACKGROUND: Genomic alterations of the anaplastic lymphoma kinase gene (ALK) occur recurrently in neuroblastoma, a pediatric malignancy of the sympathetic nervous system. However, information on their development over time has remained sparse. METHODS: ALK alterations were assessed in neuroblastomas at diagnosis and/or relapse from a total of 943 patients, covering all stages of disease. Longitudinal information on diagnostic and relapsed samples from individual patients was available in 101 and 102 cases for mutation and amplification status, respectively. RESULTS: At diagnosis, ALK point mutations occurred in 10.5% of all cases, with highest frequencies in stage 4 patients <18 months. At relapse, ALK alteration frequency increased by 70%, both in high-risk and non-high-risk cases. The increase was most likely due to de novo mutations, frequently leading to R1275Q substitutions, which are sensitive to pharmacological ALK inhibition. By contrast, the frequency of ALK amplifications did not change over the course of the disease. ALK amplifications, but not mutations, were associated with poor patient outcome. CONCLUSIONS: The considerably increased frequency of ALK mutations at relapse and their high prevalence in young stage 4 patients suggest surveying the genomic ALK status regularly in these patient cohorts, and to evaluate ALK-targeted treatment also in intermediate-risk patients.

Automated production of specific T cells for treatment of refractory viral infections after allogeneic stem cell transplantation.

Therapy-resistant viral reactivations contribute significantly to mortality after hematopoietic stem cell transplantation. Adoptive cellular therapy with virus-specific T cells (VST) has shown efficacy in various single-center trials. However, the scalability of this therapy is hampered by laborious production methods. In this study we describe the in-house production of VST in a closed system (CliniMACS Prodigy® system, Miltenyi Biotec). In addition, we report the efficacy in 26 patients with viral disease following hematopoietic stem cell transplantation in a retrospective analysis (adenovirus, n=7; cytomegalovirus, n=8; Epstein-Barr virus, n=4; multi-viral, n=7). The production of VST was successful in 100% of cases. The safety profile of VST therapy was favorable (n=2 grade 3 and n=1 grade 4 adverse events; all three were reversible). A response was seen in 20 of 26 patients (77%). Responding patients had a significantly better overall survival than patients who did not respond (P<0.001). Virus-specific symptoms were reduced or resolved in 47% of patients. The overall survival of the whole cohort was 28% after 6 months. This study shows the feasibility of automated VST production and safety of application. The scalability of the CliniMACS Prodigy® device increases the accessibility of VST treatment.

Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencing.

Circular RNA (circRNA) is a noncoding RNA class with important implications for gene expression regulation, mostly by interaction with other RNA species or RNA-binding proteins. While the commonly applied short-read Illumina RNA-sequencing techniques can be used to detect circRNAs, their full sequence is not revealed. However, the complete sequence information is needed to analyze potential interactions and thus the mechanism of action of circRNAs. Here, we present an improved protocol to enrich and sequence full-length circRNAs by using the Oxford Nanopore long-read sequencing platform. The protocol involves an enrichment of lowly abundant circRNAs by exonuclease treatment and negative selection of linear RNAs. Then, a cDNA library is created and amplified by PCR. This protocol provides enough material for several sequencing runs. The library is used as input for ligation-based sequencing together with native barcoding. Stringent quality control of the libraries is ensured by a combination of Qubit, Fragment Analyzer and qRT-PCR. Multiplexing of up to 4 libraries yields in total more than 1-2 Million reads per library, of which 1-2% are circRNA-specific reads with >99% of them full-length. The protocol works well with human cancer cell lines. We further provide suggestions for the bioinformatic analysis of the created data, as well as the limitations of our approach together with recommendations for troubleshooting and interpretation. Taken together, this protocol enables reliable full-length analysis of circRNAs, a noncoding RNA type involved in a growing number of physiologic and pathologic conditions. Metadata Associated content. https://dx.doi.org/10.17504/protocols.io.rm7vzy8r4lx1/v2.

Therapeutic targeting of ATR in alveolar rhabdomyosarcoma.

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.

Case Report: Rubella Virus-Induced Cutaneous Granulomas in Two Pediatric Patients With DNA Double Strand Breakage Repair Disorders - Outcome After Hematopoietic Stem Cell Transplantation.

We report two patients with DNA repair disorders (Artemis deficiency, Ataxia telangiectasia) with destructive skin granulomas, presumably triggered by live-attenuated rubella vaccinations. Both patients showed reduced naïve T cells. Rapid resolution of skin lesions was observed following hematopoietic stem cell transplantation. However, the patient with AT died due to complications of severe hepatic veno-occlusive disease 6 month after HSCT. Dried blood spots obtained after birth were available from this patient and showed absent T-cell receptor excision circles (TRECs). Therefore, newborn screening may help to prevent patients with moderate T-cell deficiency from receiving live-attenuated rubella vaccine potentially causing granulomas.

Circulating Cell-Free DNA Assessment in Biofluids from Children with Neuroblastoma Demonstrates Feasibility and Potential for Minimally Invasive Molecular Diagnostics.

Liquid biopsy strategies in pediatric patients are challenging due to low body weight. This study investigated cfDNA size distribution and concentration in blood, bone marrow, cerebrospinal fluid, and urine from 84 patients with neuroblastoma classified as low (n = 28), intermediate (n = 6), or high risk (n = 50) to provide key data for liquid biopsy biobanking strategies. The average volume of blood and bone marrow plasma provided ranged between 1 and 2 mL. Analysis of 637 DNA electropherograms obtained by Agilent TapeStation measurement revealed five different major profiles and characteristic DNA size distribution patterns for each of the biofluids. The proportion of samples containing primarily cfDNA was, at 85.5%, the highest for blood plasma. The median cfDNA concentration amounted to 6.28 ng/mL (blood plasma), 58.2 ng/mL (bone marrow plasma), 0.08 ng/mL (cerebrospinal fluid), and 0.49 ng/mL (urine) in samples. Meta-analysis of the dataset demonstrated that multiple cfDNA-based assays employing the same biofluid sample optimally require sampling volumes of 1 mL for blood and bone marrow plasma, 2 mL for cerebrospinal fluid, and as large as possible for urine samples. A favorable response to treatment was associated with a rapid decrease in blood-based cfDNA concentration in patients with high-risk neuroblastoma. Blood-based cfDNA concentration was not sufficient as a single parameter to indicate high-risk disease recurrence. We provide proof of concept that monitoring neuroblastoma-specific markers in very small blood volumes from infants is feasible.