Implementation and clinical evaluation of an Mpox virus laboratory-developed test on a fully automated random-access platform.

While Mpox virus (MPXV) diagnostics were performed in specialized laboratories only, the global emergence of Mpox cases in 2022 revealed the need for a more readily available diagnostic. Automated random-access platforms with fast nucleic acid extraction and PCR have become established in many laboratories, providing faster and more accessible testing. In this study, we adapted a previously published generic MPXV-PCR as a lab-developed test (LDT) on a NeuMoDx Molecular System and isolated MPXV clones from patient materials. To reduce the handling of infectious material, we evaluated a viral lysis buffer (VLB) for sample pretreatment. We further compared the MPXV-LDT-PCR to conventional real-time PCR, determined its sensitivity and specificity using positive swabs, and assessed its performance using external quality assessment samples. Pretreatment of samples with 50% VLB reduced MPXV infectivity by approximately 200-fold while maintaining PCR sensitivity. The assay demonstrated a sensitivity and specificity of 100% with no cross-reactivity in the samples tested and performed with a limit of detection of 262 GE/mL. In summary, the assay had a turnaround time of fewer than 2 h and can easily be transferred to other automated PCR platforms, providing a basis for developing rapid assays for upcoming pandemics.

A sensitive and scalable microsatellite instability assay to diagnose constitutional mismatch repair deficiency by sequencing of peripheral blood leukocytes.

Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low-level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low-frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically-confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at-risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome.

Genetic predisposition in children with cancer - affected families' acceptance of Trio-WES.

A considerable percentage of childhood cancers are due to cancer predisposition syndromes (CPS). The ratio of CPSs caused by inherited versus de novo germline mutations and the risk of recurrence in other children are unknown. We initiated a prospective study performing whole-exome sequencing (WES) of parent-child trios in children newly diagnosed with cancer. We initially aimed to determine the interest in and acceptance of trio WES among affected families and to systematically collect demographic, medical, and family history data to analyze whether these point to an underlying CPS. Between January 2015 and December 2016, 83 (88.3%) of 94 families participated; only 11 (11.7%) refused to participate. Five (6.0%) children presented with congenital malignancies and three (3.6%) with tumors with a high likelihood of an underlying CPS. Two (2.5%) families showed malignancies in family members < 18 years, 11 (13.8%) showed relatives < 45 years with cancer, 37 (46.3%) had a positive cancer history, and 14 (17.5%) families had > 1 relative with cancer. CONCLUSIONS: Genetic testing in pediatric oncology is of great interest to the families, and the vast majority opts for investigation into potentially underlying CPSs. Trio sequencing provides unique insights into CPS in pediatric cancers and is increasingly becoming a common approach in modern oncology, and thus, trio sequencing needs also to be integrated routinely into the practice of pediatric oncology. What is Known: • A considerable percentage of childhood cancers are due to cancer predisposition syndromes (CPS). What is New: • Knowing about an underlying CPS and, thus, the risk of recurrence in other children is of great interest to affected families.

Correction to: Genetic predisposition in children with cancer - affected families' acceptance of Trio-WES.

The original version of this article, unfortunately, contained an error. The reference citations in the original paper are incorrectly cited to its corresponding bibliographic information. The original article was corrected.

Comprehensive germline-genomic and clinical profiling in 160 unselected children and adolescents with cancer.

In childhood cancer, the frequency of cancer-associated germline variants and their inheritance patterns are not thoroughly investigated. Moreover, the identification of children carrying a genetic predisposition by clinical means remains challenging. In this single-center study, we performed trio whole-exome sequencing and comprehensive clinical evaluation of a prospectively enrolled cohort of 160 children with cancer and their parents. We identified in 11/160 patients a pathogenic germline variant predisposing to cancer and a further eleven patients carried a prioritized VUS with a strong association to the cancerogenesis of the patient. Through clinical screening, 51 patients (31.3%) were identified as suspicious for an underlying cancer predisposition syndrome (CPS), but only in ten of those patients a pathogenic variant could be identified. In contrast, one patient with a classical CPS and ten patients with prioritized VUS were classified as unremarkable in the clinical work-up. Taken together, a monogenetic causative variant was detected in 13.8% of our patients using WES. Nevertheless, the still unclarified clinical suspicious cases emphasize the need to consider other genetic mechanisms including new target genes, structural variants, or polygenic interactions not previously associated with cancer predisposition.

Family-based germline sequencing in children with cancer.

The discovery of cancer-predisposing syndromes (CPSs) using next-generation sequencing (NGS) technologies is of increasing importance in pediatric oncology with regard to diagnosis, treatment, surveillance, family counselling and research. Recent studies indicate that a considerable percentage of childhood cancers are associated with CPSs. However, the ratio of CPSs that are caused by inherited vs. de novo mutations (DNMs), the risk of recurrence, and even the total number of genes, which should be considered as a true cancer-predisposing gene, are still unknown. In contrast to sequencing only single index patients, family-based NGS of the germline is a very powerful tool for providing unique insights into inheritance patterns (e.g., DNMs, parental mosaicism) and types of aberrations (e.g., SNV, CNV, indels, SV). Furthermore, functional perturbations of key cancer pathways (e.g., TP53, FA/BRCA) by at least two co-inherited heterozygous digenic mutations from each parent and currently unrecognized rare variants and unmeasured genetic interactions between common and rare variants may be a widespread genetic phenomenon in the germline of affected children. Therefore, family-based trio sequencing has the potential to reveal a striking new landscape of inheritance in childhood cancer and to facilitate the integration and efforts of individualized treatment strategies, including personalized and preventive medicine and cancer surveillance programs. Consequently, cancer genetics is becoming an increasingly common approach in modern oncology, so trio-sequencing should also be routinely integrated into pediatric oncology.

Penetrance and Expressivity in Inherited Cancer Predisposing Syndromes.

Inherited diseases are not always expressed in the same way in every individual that carries the same variant in a disease-causing gene. This phenomenon is known as reduced or incomplete penetrance. Variable and incomplete penetrance may explain why inherited diseases are occasionally transmitted through unaffected parents, but also why clinically healthy individuals can carry potentially pathogenic variants without expressing features of the disease. Here, we will provide an overview of factors that play a fundamental role in the concept of penetrance and expressivity of cancer predisposing genes in children with malignancies. These findings are important to understand the complexity of inherited diseases and cancer development and to improve genetic counselling for the affected families.

Congenital embryonal rhabdomyosarcoma caused by heterozygous concomitant PTCH1 and PTCH2 germline mutations.

The sonic hedgehog (SHH) signaling pathway has been shown to play important roles in embryogenesis, cell proliferation as well as in cell differentiation. It is aberrantly activated in various common cancers in adults, but also in pediatric neoplasms, such as rhabdomyosarcoma (RMS) and atypical teratoid/rhabdoid tumors (AT/RTs). Dysregulation and germline mutation in PATCHED1 (PTCH1), a receptor for SHH, is responsible for the Gorlin Syndrome, a familial cancer predisposing syndrome including RMS. Here, we report a newborn diagnosed with congenital embryonal RMS. Whole-exome sequencing (WES) identified the presence of two heterozygous germline mutations in two target genes of the SHH signaling pathway. The PTCH1 mutation p.(Gly38Glu) is inherited from the mother, whereas the PTCH2 p.(His622Tyr) mutation is transmitted from the father. Quantitative RT-PCR expression analysis of GLI and SMO, key players of the SHH pathway, showed significantly increase in the tumor tissue of the patient and also enrichment in the germline sample in comparison to the parents indicating activation of the SHH pathway in the patient. These findings demonstrate that SHH pathway activity seems to play a role in eRMS as evidenced by high expression levels of GLI1 RNA transcripts. We speculate that PTCH2 modulates tumorigenesis linked to the PTCH1 mutation and is likely associated with the congenital onset of the RMS observed in our patient.

Diagnostic challenges in a child with early onset desmoplastic medulloblastoma and homozygous variants in MSH2 and MSH6.

Constitutional mismatch repair deficiency (CMMRD) is an autosomal recessively inherited childhood cancer susceptibility syndrome caused by biallelic germline mutations in one of the mismatch repair (MMR) genes. The spectrum of CMMRD-associated tumours is very broad and many CMMRD patients additionally display signposting non-neoplastic features, most frequently café-au-lait macules and other pigmentation alterations. We report on a 13-month-old girl suspected of having CMMRD due to a desmoplastic medulloblastoma and a striking skin pigmentation that included multiple café-au-lait macules, hypopigmented areas and Mongolian spots. Whole-exome sequencing revealed homozygosity for MSH2 variant p.(Leu92Val) and MSH6 variant p.(Val809del), both variants of uncertain significance (VUS). Immunohistochemical analysis of the tumour tissue showed expression of all four MMR proteins and gMSI testing was negative. However, functional assays demonstrated that the cells of the patient displayed methylation tolerance and ex vivo microsatellite instability, which unequivocally confirmed the diagnosis of CMMRD. Taken together, the results render the MSH2 variant unlikely to be responsible for the phenotype, while they are compatible with MSH6-associated CMMRD. This case illustrates the diagnostic strategy of confirming CMMRD syndrome in patients with VUS.

Autism spectrum disorder and Li-Fraumeni syndrome: purely coincidental or mechanistically associated?

BACKGROUND: Autism spectrum disorders (ASDs) are neurodevelopmental disorders with impaired social interactions and communication and restrictive, repetitive patterns of behaviors, interests, and activities. A recent epidemiological study suggests that children with ASD might have an increased cancer risk. CASE PRESENTATION: The 14.5-year-old boy, previously diagnosed with ASD, was referred with persistent bone pain. Diagnostic work-up confirmed diagnosis of acute lymphoblastic leukemia (ALL); cytogenetic analysis revealed low hypodiploid karyotype with a mutation (c.733G>A, p.Gly245Ser, rs28934575) in TP53 in the leukemic blasts. By Sanger sequencing, the presence of this mutation in the germline was subsequently confirmed and, thus, diagnosis of Li-Fraumeni syndrome (LFS) was made. His family history was remarkable with two siblings with intellectual disability and a mother who has died of premenopausal breast cancer. CONCLUSIONS: Some of the oncogenes and tumor suppressor genes causing cancer susceptibility syndromes overlap with those involved in autism. This functional overlap between autism and cancer is novel and particularly compelling. The surprising coincidence of LFS and ASD in our patient raises the question whether this is purely coincidental or mechanistically associated.