Background Deletion of 1p is associated with poor prognosis in neuroblastoma, however selected 1p-intact patients still experience poor outcomes. Since mutations of 1p genes may mimic the deleterious ...effects of chromosomal loss, we studied the incidence, spectrum and effects of mutational variants in 1p-intact neuroblastoma. Methods We characterized the 1p status of 325 neuroblastoma patients, and correlated the mutational status of 1p tumor suppressors and neuroblastoma candidate genes with survival outcomes among 100 1p-intact cases, then performed functional validation of selected novel variants of 1p36 genes identified from our patient cohort. Results Among patients with adverse disease characteristics, those who additionally had 1p deletion had significantly worse overall survival. Among 100 tumor-normal pairs sequenced, somatic mutations of 1p tumor suppressors KIF1Bbeta and CHD5 were most frequent (2%) after ALK and ATRX (8%), and BARD1 (3%). Mutations of neuroblastoma candidate genes were associated with other synchronous mutations and concurrent 11q deletion (P = 0.045). In total, 24 of 38 variants identified were novel and predicted to be deleterious or pathogenic. Functional validation identified novel KIF1Bbeta I1355M variant as a gain-of-function mutation with increased expression and tumor suppressive activity, correlating with indolent clinical behavior; another novel variant CHD5 E43Q was a loss-of-function mutation with decreased expression and increased long-term cell viability, corresponding with aggressive disease characteristics. Conclusions Our study showed that chromosome 1 gene mutations occurred frequently in 1p-intact neuroblastoma, but may not consistently abrogate the function of bonafide 1p tumor suppressors. These findings may augment the evolving model of compounding contributions of 1p gene aberrations toward tumor suppressor inactivation in neuroblastoma. Keywords: Neuroblastoma, Chromosome 1, Tumor suppressor, Next-generation sequencing, Synchronous Mutations
Three-dimensionally printed nasopharyngeal swabs (3DP swabs) have been used to mitigate swab shortages during the coronavirus disease 2019 (COVID-19) pandemic. Clinical validation for diagnostic ...accuracy and consistency, as well as patient acceptability, is crucial to evaluate the swab's performance.
To determine the accuracy and acceptability of the 3DP swab for identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
A diagnostic study was conducted from May to July 2020 at 2 tertiary care centers in Singapore with different reference swabs (FLOQSwab COPAN Diagnostics or Dacron swab Deltalab) and swab processing techniques (wet or dry) to evaluate the performance of the 3DP swab compared with traditional, standard-of-care nasopharyngeal swabs used in health care institutions. The participants were patients with COVID-19 in the first 2 weeks of illness and controls with acute respiratory illness with negative test results for SARS-CoV-2. Paired nasopharyngeal swabs were obtained from the same nostril and tested for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction. The sequence of swabs was randomized based on odd and even participant numbers.
Primary outcome measures were overall agreement (OA), positive percentage agreement (PPA), and negative percentage agreement of the 3DP swab compared with reference swabs. Secondary outcome measures were the correlation of cycle threshold (Ct) values of both swabs.
The mean (SD) age of participants was 45.4 (13.1) years, and most participants were men (87 of 89 97.8%), in keeping with the epidemiology of the COVID-19 pandemic in Singapore. A total of 79 patients with COVID-19 and 10 controls were recruited. Among the patients with COVID-19, the overall agreement and PPA of the 3DP swab was 91.1% and 93.5%, respectively, compared with reference swabs. The PPA was 100% for patients with COVID-19 who were tested within the first week of illness. All controls tested negative. The reverse-transcriptase polymerase chain reaction Ct values for the ORF1ab and E-gene targets showed a strong correlation (intraclass correlations coefficient, 0.869-0.920) between the 3DP and reference swab on independent testing at each institution despite differences in sample processing. Discordant results for both gene targets were observed only at high Ct values.
In this diagnostic study of 79 patients with COVID-19 and 10 controls, the 3DP swab performed accurately and consistently across health care institutions and could help mitigate strained resources in the escalating COVID-19 pandemic.
Pediatric mesotheliomas are rare and their pathogenesis remains undefined. In this study, we report 5 cases of malignant mesothelioma in children, characterized by fusions involving the anaplastic ...lymphoma kinase (ALK) gene. Four cases occurred in females involving the abdominal cavity and were characterized by a pure epithelioid morphology. The fifth arose in the tunica vaginalis of a 15-year-old male and displayed a biphasic epithelioid-sarcomatoid phenotype. All cases demonstrated the classic morphologic and immunohistochemical features of malignant mesothelioma, including tubulopapillary architecture and cuboidal epithelioid cells with eosinophilic cytoplasm and uniform nuclei with vesicular chromatin. Immunohistochemically, all cases showed labeling for ALK, cytokeratins, WT1, and calretinin, while lacking expression of adenocarcinoma immunomarkers. Four cases demonstrated weak-moderate labeling for PAX8 protein, which resulted in diagnostic challenges with primary peritoneal serous carcinoma. The ALK genetic abnormalities were investigated by a combination of molecular methods. Archer FusionPlex was performed in 2 cases, showing fusions between ALK with either STRN or TPM1 genes, resulting in a transcript that retained the ALK kinase domain. One case was further studied by DNA targeted sequencing, but no additional genetic alterations were observed. In 1 case, cytogenetic analysis showed the presence of a t(2;15)(p23;q22) and fluorescence in situ hybridization confirmed the ALK gene break-apart. In the remaining 2 cases, ALK gene rearrangements were demonstrated by fluorescence in situ hybridization. Unlike adult mesotheliomas, which are tightly linked to asbestos exposure, often show loss of BAP1 expression and have complex karyotypes, ALK-rearranged mesothelioma appears to be similar to other fusion-positive mesotheliomas, such as those harboring EWSR1/FUS-ATF1 fusions, sharing significant morphologic overlap, occurring in young patients and displaying a simple, translocation-driven genetic profile.
DBA results from genetic mutations affecting ribosomal protein synthesis, leading to the apoptosis of erythroid progenitor cells. DBA is associated with an increased risk of congenital malformations, ...growth deficiency, and malignancies, including acute myelogenous leukaemia and solid tumours. Mutations of the RPL15 gene may result in hydrops fetalis, intrauterine growth restriction, and anaemia at birth, indicating a possible distinct genotype-phenotype association within DBA (appendix).
Primary paediatric epidural sarcomas are extremely rare. Overall, there remains a paucity of knowledge in paediatric epidural sarcomas owing to the infrequent number of cases. The Archer FusionPlex ...Sarcoma Kit (ArcherDX, Inc) is a next-generation sequencing assay that has been reported to be a useful technique to detect recurrent fusion in sarcomas. We report the molecular exploration of 3 primary paediatric epidural sarcomas-one in the cranium (mesenchymal chondrosarcoma) and 2 in the spine (mesenchymal chondrosarcoma and Ewing sarcoma respectively).
This is a study approved by the hospital ethics board. Clinico-pathological information from 3 consenting patients with primary epidural sarcomas was collected. These selected tumours are interrogated via Archer FusionPlex Sarcoma Kit (ArcherDX, Inc) for genomic aberrations. Results were validated with RT-PCR and Sanger sequencing. All findings are corroborated and discussed in concordance with current literature. Our findings show 2 variants of the HEY1-NCOA2 gene fusion: HEY1 (exon 4)-NCOA2 (exon 13) and HEY1 (exon 4)-NCOA2 (exon 14), in both mesenchymal chondrosarcoma patients. Next, the Ewing sarcoma tumour is found to have EWSR1 (exon 10)-FLI1 (exon 8) translocation based on NGS. This result is not detected via conventional fluorescence in situ testing.
This is a molecularly-centered study based on 3 unique primary paediatric epidural sarcomas. Our findings to add to the growing body of literature for these exceptionally rare and malignant neoplasms. The authors advocate global collaborative efforts and in-depth studies for targeted therapy to benefit affected children.
Aims
Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the ...YWHAE–NUTM2 gene fusion. A third ‘double‐negative’ (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE–NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically.
Methods and results
By next‐generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITDs, with positive BCOR immunoreactivity being found in four (36%) or eight (72%) cases, depending on the antibody clone. By reverse transcription polymerase chain reaction, none had the YWHAE–NUTM2 fusion. The DN case had a BCOR–CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative polymerase chain reaction showed markedly elevated BCOR expression in this case, whereas BCOR ITD cases had lower levels of elevated BCOR expression.
Conclusions
The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE–NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR–CCNB3 fusion sarcoma. BCOR–CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.
Ex vivo evaluation of personalized models can facilitate individualized treatment selection for patients, and advance the discovery of novel therapeutic options. However, for embryonal malignancies, ...representative primary cultures have been difficult to establish. We developed patient‐derived cell cultures (PDCs) from chemo‐naïve and post–treatment neuroblastoma tumors in a consistent and efficient manner, and characterized their in vitro growth dynamics, histomorphology, gene expression, and functional chemo‐response. From 34 neuroblastoma tumors, 22 engrafted in vitro to generate 31 individual PDC lines, with higher engraftment seen with metastatic tumors. PDCs displayed characteristic immunohistochemical staining patterns of PHOX2B, TH, and GD2 synthase. Concordance of MYCN amplification, 1p and 11q deletion between PDCs and patient tumors was 83.3%, 72.7%, and 80.0% respectively. PDCs displayed a predominantly mesenchymal‐type gene expression signature and showed upregulation of pro‐angiogenic factors that were similarly enriched in culture medium and paired patient serum samples. When tested with standard‐of‐care cytotoxics at human Cmax‐equivalent concentrations, MYCN‐amplified and non‐MYCN‐amplified PDCs showed a differential response to cyclophosphamide and topotecan, which mirrored the corresponding patients’ responses, and correlated with gene signatures of chemosensitivity. In this translational proof‐of‐concept study, early‐phase neuroblastoma PDCs enriched for the mesenchymal cell subpopulation recapitulated the individual molecular and phenotypic profile of patient tumors, and highlighted their potential as a platform for individualized ex vivo drug‐response testing.
We developed a system to efficiently and consistently generate patient‐derived cultures (PDCs) of neuroblastoma that recapitulated individual patient’s cytogenetic and immunohistochemical features, and phenotypic responses to standard‐of‐care chemotherapy. PDCs predominantly express the gene signature of the recently described neuroblastoma mesenchymal super‐enhancer state. This proof‐of concept provides translational evidence supporting the proposed role of the mesenchymal cell subpopulation in determining clinical treatment response and offers a novel platform for developing personalized ex vivo therapeutic decision‐making strategies for neuroblastoma.
Aim
Actinomycosis is a rare subacute to chronic granulomatous infection which can mimic other infectious or malignant diseases. This study examined the epidemiology and treatment outcome of ...actinomycosis in children.
Methods
A retrospective study on children admitted for actinomycosis in a tertiary paediatric hospital in Singapore, from January 2004 to December 2020. Clinical profile, therapeutic interventions and outcomes were examined.
Results
A total of 10 patients were identified; 7 were female. The median age at first presentation was 9.8 years (range 4.7–15.7). The most common presenting symptom was fever (n = 6, 60%), followed by facial or neck swelling (n = 3, 30%) and ear pain (n = 3, 30%). Actinomycosis occurred predominantly in the orocervicofacial region (n = 6, 60%). Four patients (40%) had preceding dental infections in the form of dental caries or gingivitis. One patient had poorly controlled insulin‐dependent diabetes mellitus. Actinomycosis was confirmed via culture in four patients, histopathology in four patients and both methods in two patients. All except one patient (n = 9, 90%) underwent surgical procedures. All patients received ampicillin or amoxicillin/clavulanate or other beta‐lactams, for a median duration of 6.5 months (range 1.5–14). Complications included osteomyelitis (n = 4, 40%), mastoiditis (n = 2, 20%), brain abscess (n = 1, 10%) and recurrent neck abscess (n = 1, 10%). There was no mortality and all patients achieved complete resolution.
Conclusions
Paediatric actinomycosis was rare in our 16‐year review, but had a high complication rate. It can occur in immunocompetent patients, and dental infection was the predominant risk factor identified. Prognosis was excellent after surgical intervention and appropriate antimicrobial therapy.
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