Comprehensive genomic profiling of paediatric tumours has shown promise in guiding precision oncology approaches. However, the clinical utility of targeted sequencing panels in routine practice remains unclear. The results of such testing have not been reported in Southeast Asia. We implemented the AmpliSeq Childhood Cancer Panel for prospective molecular profiling of 130 paediatric patients with various solid tumours. The panel detects single nucleotide variants, gene fusions and amplifications. Clinical impact was assessed through a molecular tumour board. Clinically significant variants were identified in 52.8% of patients, with diagnostic, therapeutic and prognostic relevance in 52%, 30% and 18% of cases, respectively. Central nervous system (CNS) tumours showed the highest yield (70%). Seven patients (5.7%) received targeted therapy based on the sequencing results. The panel was particularly useful for CNS tumours, fusion-driven sarcomas, neuroblastomas and DICER1-associated tumours. Limitations included the need for additional testing in some cases and the inability to distinguish germline from somatic variants. In conclusion, targeted panel sequencing is feasible and clinically useful for paediatric tumour profiling, with the highest yield in CNS tumours. While the proportion of patients receiving targeted therapy was relatively low, the genetic information obtained is valuable for biomarker-directed trials and advancing precision oncology in paediatric cancers.

Wilms tumors in Asian populations demonstrate uniquely favorable biological characteristics and are not appropriately stratified by current risk markers like loss of heterozygosity of 1p and 16q. We analyzed the prognostic associations of copy number changes at other genomic loci in a population-based cohort of Wilms tumor and explored genomic profiles of relapsed cases. Chromosomal aberrations of targeted loci were evaluated in 61 Wilms tumors treated with National Wilms Tumor Study regimens in Singapore hospitals from 2001-2022, and 26 tumors from a separate Asian validation set, and correlated with clinical variables. Mutational signatures of 6 relapsed patients' tumors were profiled using whole-exome sequencing. Clustering analysis identified a subgroup (65.6 %) of predominantly non-Asian patients with higher frequency of relapse, 1p and 16q loss and 1q gain. A smaller predominantly-Asian subgroup (34.4 %) with low stage and few relapses was characterized by gain of MYCN and minimal 1p, 1q and 16q aberrations. MYCN exon 2 gain was present in 9.8 % of the discovery set and 11.5 % of the validation set. Loss of IGF2 and 1p gene ABCA4, and gain of 1q genes, were associated with relapse. Notably, relapsed tumors demonstrated no recurrent somatic mutations; instead, in 2 patients with 1q gain clonal deconvolution showed persistence of chemo-refractory clones without metachronous pathogenic variants. Among Asian Wilms tumor patients, 1q gain is prognostic for relapse. Gain of MYCN features uniquely in Asian patients and may represent a favorable prognostic marker. These results identify distinct molecular subgroups associated with varying prognosis and relapse among Asian patients.

Metastasizing leiomyoma is a rare condition characterized by the development of benign-appearing smooth muscle neoplasms at extrauterine sites in patients with a history of uterine leiomyoma. These lesions occur most commonly in the lung, with the abdominopelvic and mediastinal lymph nodes being other reported sites. Malignant transformation of metastasizing leiomyoma is extremely rare, with only a few cases described in the literature. We describe a case of metastasizing leiomyoma with malignant transformation in a middle-aged Asian lady, who developed pulmonary metastatic foci 12 years after surgical excision of the original uterine leiomyomata. Molecular analysis showed a common RAB2A-PLAG1 fusion gene and identical single nucleotide variants in both tumor foci, with significantly more pronounced segmental chromosomal copy number variations in one focus showing high-grade features. A comprehensive review of the literature lends support to the hypothesis that the original leiomyomata and the metastatic foci are clonally related, with high-grade features being associated with more complex genomic signatures.

The differential diagnosis for neonatal primary lung masses includes developmental anomalies and congenital lung tumors. Fetal lung interstitial tumor (FLIT) is a rare benign mesenchymal lesion which presents either antenatally or within the first 3 months of age. FLIT is a circumscribed solid-cystic mass which histologically resembles the fetal lung during the canalicular stage at 20-24 weeks of gestation. It is composed of immature mesenchymal cells expanding the interstitium and irregular airspace-like structures. Of all published cases, only 1 identified an α2-macroglobulin (A2M)::anaplastic lymphoma kinase (ALK) fusion and all cases underwent surgical resection in the neonatal or infancy period. We present the second case of FLIT with an A2M::ALK fusion diagnosed postnatally in a neonate which partially regressed spontaneously during conservative management with interim resection at 39 months of age, and provide a review of the literature.

Introduction:. Genetic mutations have been identified in the pathogenesis of vascular anomalies. Due to overlaps in genetic variants causing vascular anomalies and cancer, we used a next-generation sequencing panel for genomic profiling of childhood cancers to detect somatic mutations in children with vascular anomalies. We aim to review the utility of an oncology panel for the molecular diagnosis of vascular anomalies. Methods:. Nine patients with histologically confirmed vascular anomalies were included. DNA was extracted from formalin-fixed paraffin-embedded tissue specimens obtained from affected tissue following diagnostic punch biopsies of the skin and core biopsies of the vascular malformation or tumor during sclerotherapy or surgical excision. Molecular analysis of the tissue samples was performed using AmpliSeq for Childhood Cancer DNA Assay Panel. Results:. Two patients had antenatally detected vascular anomalies. The median age at diagnosis for the remaining patients was 7.0 years (IQR, 0.6–10.0 years). Seven were diagnosed with vascular malformations, while 2 had vascular tumors. Pathological somatic mutations were identified in 4 patients, leading to a diagnostic yield of 44.4%. Two different PIK3CA mutations were identified in 3 cases: 1 in a case of macrocystic lymphatic malformation, the other in a case of Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevus, Spinal/Skeletal anomalies syndrome and Klippel–Trenaunay syndrome. BRAF mutation was identified in a patient with a veno-lymphatic malformation. Conclusion:. An oncology next-generation sequencing panel can be used for genetic profiling of vascular anomalies. However, a more customized and sensitive panel may be of better diagnostic yield, as detection of somatic mutations in vascular anomalies is challenging due to tissue mosaicism, low-abundant genetic variants, and specimen limitations.

Neuroblastoma is a heterogeneous paediatric malignant tumour. Telomere maintenance mechanism (TMM) by telomerase activation or alternative lengthening of telomeres (ALT) is a hallmark of high-risk neuroblastoma. However, the prior assays for telomerase, such as TERT expression by RNA sequencing or microarrays, may not be easy to perform in many histopathology laboratories in hospitals. The aims of this study are to assess the utility of ultrasensitive single-cell RNA in situ hybridisation (RNAscope), immunohistochemistry, and RT-qPCR on formalin-fixed, paraffin-embedded tumour samples as diagnostic tools for detecting TERT expression in neuroblastoma. In this study, we detected MYCN amplification in 22 of 222 cases (10%), TERT rearrangements in 18 of 220 cases (8%), and ALT activation in 39 of 222 cases (18%) using fluorescence in situ hybridisation (FISH). By RNA in situ hybridisation, 36 of 210 (17%) pretreatment neuroblastomas were found to have TERT overexpression, which was significantly associated with the high-risk group (33/78, 42%), TERT rearrangements (16/18, 89%), and MYCN amplification (13/22, 59%). None of the tumours with ALT showed TERT staining. In our study, 19 of the 55 MYCN non-amplified high-risk neuroblastomas displayed TERT mRNA expression, including 13 of the 14 TERT rearrangements, none of the 30 ALT-positive cases, and a significant proportion (6/11, 55%) that did not have the aforementioned genomic anomalies. RT-qPCR results correlated well with RNAscope levels (Spearman's rho=0.621, p<0.001, n=94). In conclusion, TERT RNA in situ hybridisation and RT-qPCR are suitable methods to evaluate TERT expression in neuroblastoma. The combination of detection of the genomic alterations and TERT mRNA expression is a powerful strategy for TMM activation detection, which can categorise neuroblastomas into multiple clinical subgroups for risk stratification in routine histopathology practice.

Supplementary Figure S7, related to Figure 5. Vincristine or topotecan works synergistically with and promotes effective dose reduction of XIAP-specific antagonist A4 in vitro.

XIAP-specific antagonist A4 works synergistically with and promotes effective dose reduction of vincristine and topotecan in vitro. A, CI demonstrating synergism between A4 with vincristine and topotecan. Increasing doses of A4 with either vincristine or topotecan were added to neuroblastoma cell lines at a fixed ratio based on the IC50 values of the individual drugs for 48 hours [A4:vincristine; BE(2)-C 1:1 (0–100 µmol/L A4: 0–100 nmol/L Vin), KELLY 1:0.5 (0–100 µmol/L A4: 0–50 nmol/L Vin) and A4:Topotecan; BE(2)-C 1:6 (0–100 µmol/L A4: 0–600 nmol/L Topo), KELLY 1:16 (0–100 µmol/L A4: 0–1,600 nmol/L Topo)], respectively. CI was generated using CompuSyn software by Chou–Talalay (25). CI60–90 represents the average CI at 60%–90% cell death. CI <1 denotes synergistic effect; CI = 1 denotes addictive effect and CI >1 denotes antagonistic effect. DRI demonstrating the fold differences of A4 effectively reducing the dose of vincristine (B) or topotecan (C) when used in combination with these agents. Increasing doses of A4 with either vincristine or topotecan were added to neuroblastoma cell lines at a fixed ratio based on the IC50 values of the individual drugs for 48 hours (A4:vincristine; BE(2)-C 1:1, KELLY 1:0.5 and A4:Topotecan; BE(2)-C 1:6, KELLY 1:16), respectively. DRI was generated using CompuSyn software by Chou–Talalay. DRI60–90 represents the average DRI at 60%–90% cell death. DRI <1 denotes unfavorable dose reduction and DRI >1 denotes favorable dose reduction. Right, Dose–response curves of vincristine(B) and topotecan (C) treated alone or in combination with A4 in BE(2)-C and KELLY neuroblastoma cells, with corresponding comparison of mean areas under the dose–response curves with versus without addition of A4 (t test).

Supplementary Table S1, related to Figure 1. Table of densitometry values calculated from western blot to generate scatter plot in Figure 1A.

AIM New histomolecular subtypes of rhabdomyosarcoma have recently been defined but their corresponding clinical characteristics are not well described. Also, these clinical phenotypes vary greatly by age and ethnicity but have not been profiled in Asian populations. Thus, we sought to determine the landscape of rhabdomyosarcoma subtypes in a national Asian cohort and compare clinical characteristics among age groups and molecular subtypes. METHODS We performed a retrospective population-based study of all rhabdomyosarcoma patients in Singapore public hospitals from 2004 to 2014 (n = 67), and assigned histomolecular subtypes according to the updated 2020 WHO classification of soft tissue tumors following central pathology review and molecular profiling. RESULTS Age-specific prevalence followed a tri-modal peak. There were significantly more embryonal and alveolar (p = 0.032) and genitourinary (non-bladder/prostate) tumors (p = 0.033) among children. Older age was associated with complete resection among spindle cell/sclerosing tumors (p = 0.027), with the omission of chemotherapy among embryonal tumors (p = 0.001), and with poorer survival among embryonal and alveolar tumors (p = 0.026, p = 0.022, respectively). Overall survival differed with stage, group, and surgical resection, adjusted for age group (p = 0.004, p = 0.001, p = 0.004, respectively). Spindle-cell/sclerosing tumors showed an indolent phenotype with a significantly lower incidence of nodal metastasis (p = 0.002), but two of 15 patients with MYOD1 mutations had a contrastingly aggressive disease. CONCLUSION Disease and treatment response profiles of rhabdomyosarcoma subtypes vary significantly between adults and children, especially surgical resectability. In our Asian population, poorer outcomes were observed in adults with embryonal and alveolar tumors, while activating mutations influence the behavior of otherwise favorable spindle cell/sclerosing tumors.

AbstractXIAP, the most potent mammalian inhibitor of apoptosis protein (IAP), critically restricts developmental culling of sympathetic neuronal progenitors, and is correspondingly overexpressed in most MYCN-amplified neuroblastoma tumors. Because apoptosis-related protein in the TGFβ signaling pathway (ARTS) is the only XIAP antagonist that directly binds and degrades XIAP, we evaluated the preclinical effectiveness and tolerability of XIAP antagonism as a novel targeting strategy for neuroblastoma. We found that antagonism of XIAP, but not other IAPs, triggered apoptotic death in neuroblastoma cells. XIAP silencing induced apoptosis while overexpression conferred protection from drug-induced apoptosis. From a screen of IAP inhibitors, first-in-class ARTS mimetic A4 was most effective against high-risk and high XIAP-expressing neuroblastoma cells, and least toxic toward normal liver- and bone marrow–derived cells, compared with pan-IAP antagonists. On target engagement assays and nuclear magnetic resonance spectroscopy, A4 was observed to degrade rather than inhibit XIAP, catalyzing rapid degradation of XIAP through the ubiquitin-proteasome pathway. In MYCN-amplified neuroblastoma patient-derived xenografts, A4 significantly prolonged survival as a single agent, and demonstrated synergism with standard-of-care agents to reduce their effective required doses 3- to 6-fold. Engagement and degradation of XIAP by ARTS mimetics is a novel targeting strategy for neuroblastoma that may be especially effective against MYCN-amplified disease with intrinsically high XIAP expression. First-in-class ARTS mimetic A4 demonstrates preclinical efficacy and warrants further development and study.Significance:XIAP degradation is sufficient to kill MYCN-amplified neuroblastoma which overexpresses and relies on XIAP as a brake against cell death, without affecting normal cells.

Supplementary Figure S4, related to Figure 3. Time course of XIAP degradation in response to XIAP-specific (A4) and pan-IAP (BV6) antagonists.

Supplementary Figure S1, related to Figure 2. Importance of XIAP expression level in mediating sensitivity of neuroblastoma cells to A4, B3 and BV6.

DICER1 mutations predispose to increased risk for various cancers, particularly pleuropulmonary blastoma (PPB), the commonest lung malignancy of childhood. There is a paucity of directly actionable molecular targets as these tumors are driven by loss-of-function mutations of DICER1. Therapeutic development for PPB is further limited by a lack of biologically and physiologically-representative disease models. Given recent evidence of Dicer's role as a haploinsufficient tumor suppressor regulating RNA polymerase I (Pol I), Pol I inhibition could abrogate mutant Dicer-mediated accumulation of stalled polymerases to trigger apoptosis. Hence, we developed a novel subpleural orthotopic PPB patient-derived xenograft (PDX) model that retained both RNase IIIa and IIIb hotspot mutations and recapitulated the cardiorespiratory physiology of intra-thoracic disease, and with it evaluated the tolerability and efficacy of first-in-class Pol I inhibitor CX-5461. In PDX tumors, CX-5461 significantly reduced H3K9 di-methylation and increased nuclear p53 expression, within 24 hours' exposure. Following treatment at the maximum tolerated dosing regimen (12 doses, 30 mg/kg), tumors were smaller and less hemorrhagic than controls, with significantly decreased cellular proliferation, and increased apoptosis. As demonstrated in a novel intrathoracic tumor model of PPB, Pol I inhibition with CX-5461 could be a tolerable and clinically-feasible therapeutic strategy for mutant Dicer tumors, inducing antitumor effects by decreasing H3K9 methylation and enhancing p53-mediated apoptosis.

Supplementary Figure S3, related to Figure 3. Generation of luciferase-tagged XIAP using CRISPR knock-in gene editing.

Supplementary Figure S6, related to Figure 4. Immunoblot analysis of tumor tissue from other sets of PDXs treated with A4.

Binding and degradation of XIAP by A4 is necessary for targeting high-risk neuroblastoma cells. A, BRET response curves which indicate direct binding/target engagement of either XIAP-specific antagonist A4 or pan-IAP antagonist BV6 to XIAP in neuroblastoma cells. Cells were transfected with XIAP NanoLuc fusions and treated with fixed NanoBRET tracer and various concentrations of unlabeled A4 or BV6 as a competitive compound. BRET signal was measured at 2 hours after treatment using a Tecan Infinite 200 Pro equipped with NanoBRET dual-filters (donor 460 nm and acceptor 618 nm) and determined via the formula (BRET ratio = values at 618 nm/values at 460 nm). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted to determine apparent intracellular affinity of A4 or BV6 to XIAP. Values were expressed mean ± SD of three independent experiments conducted in duplicates. B, Degradation profiles of endogenous XIAP in response to A4 or BV6 treatment of neuroblastoma cells, BE(2)-C (top) and KELLY (bottom). Cells were treated with increasing dose of A4 or BV6 and luminescence was measured continuously in real time for 2 hours. Luminescence values were normalized to DMSO vehicle control and degradation profiles were generated using GraphPad Prism. Values were expressed as mean ± SD of three independent experiments conducted in duplicates. C, BRET response curves which indicate the binding of ubiquitin to XIAP upon treatment with XIAP-specific antagonist A4 in neuroblastoma cells. Neuroblastoma cells with endogenous luciferase-tagged XIAP were transfected with Halo-tag ubiquitin and treated with fluorescent Halo-tag ligand followed by the addition of various concentrations of A4. BRET signal was measured every 5 minutes for 6 hours after treatment using a Tecan equipped with NanoBRET dual-filters (donor 460 nm and acceptor 618 nm) and determined via the formula (BRET ratio = values at 618 nm/values at 460 nm). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted to determine the binding of ubiquitin to XIAP. Values were expressed mean ± SD of three independent experiments. D, Immunoblot analysis of XIAP expression upon A4 treatment with (+) or without (−) 10 µmol/L proteasomal inhibitor MG-132 in neuroblastoma cells, BE(2)-C and KELLY. Cells were pretreated with or without MG-132 for 2 hours prior the addition of A4 for 4 hours. β-actin is used as internal control.