Protocol for a comprehensive prospective cohort study of trio-based whole-genome sequencing for underlying cancer predisposition in paediatric and adolescent patients newly diagnosed with cancer: the PREDICT study.

INTRODUCTION: Identifying an underlying germline cancer predisposition (CP) in a child with cancer has potentially significant implications for both the child and biological relatives. Cohort studies indicate that 10%-15% of paediatric cancer patients carry germline pathogenic or likely pathogenic variants in cancer predisposition genes, but many of these patients do not meet current clinical criteria for genetic testing. This suggests broad tumour agnostic germline testing may benefit paediatric cancer patients. However, the utility and psychosocial impact of this approach remain unknown. We hypothesise that an approach involving trio whole-genome germline sequencing (trio WGS) will identify children and families with an underlying CP in a timely fashion, that the trio design will streamline cancer risk counselling to at-risk relatives if CP was inherited, and that trio testing will not have a negative psychosocial impact on families. METHOD AND ANALYSIS: To test this, we present the Cancer PREDisposition In Childhood by Trio sequencing study (PREDICT). This study will assess the clinical utility of trio WGS to identify CP in unselected patients with cancer 21 years or younger in New South Wales, Australia. PREDICT will perform analysis of biological parents to determine heritability and will examine the psychosocial impact of this trio sequencing approach. PREDICT also includes a broad genomics research programme to identify new candidate genes associated with childhood cancer risk. ETHICS AND DISSEMINATION: By evaluating the feasibility, utility and psychosocial impact of trio WGS to identify CP in paediatric cancer, PREDICT will inform how such comprehensive testing can be incorporated into a standard of care at diagnosis for all childhood cancer patients. TRIAL REGISTRATION NUMBER: NCT04903782.

Rare germline variants in childhood cancer patients suspected of genetic predisposition to cancer.

Identification of cancer-predisposing germline variants in childhood cancer patients is important for therapeutic decisions, disease surveillance and risk assessment for patients, and potentially, also for family members. We investigated the spectrum and prevalence of pathogenic germline variants in selected childhood cancer patients with features suggestive of genetic predisposition to cancer. Germline DNA was subjected to exome sequencing to filter variants in 1048 genes of interest including 176 known cancer predisposition genes (CPGs). An enrichment burden analysis compared rare deleterious germline CPG variants in the patient cohort with those in a healthy aged control population. A subset of predicted deleterious variants in novel candidate CPGs was investigated further by examining matched tumor samples, and the functional impact of AXIN1 variants was analyzed in cultured cells. Twenty-two pathogenic/likely pathogenic (P/LP) germline variants detected in 13 CPGs were identified in 19 of 76 patients (25.0%). Unclear association with the diagnosed cancer types was observed in 11 of 19 patients carrying P/LP CPG variants. The burden of rare deleterious germline variants in autosomal dominant CPGs was significantly higher in study patients versus healthy aged controls. A novel AXIN1 frameshift variant (Ser321fs) may impact the regulation of ß-catenin levels. Selection of childhood cancer patients for germline testing based on features suggestive of an underlying genetic predisposition could help to identify carriers of clinically relevant germline CPG variants, and streamline the integration of germline genomic testing in the pediatric oncology clinic.

Tropomyosin 2.1 collaborates with fibronectin to promote TGF-ß1-induced contraction of human lung fibroblasts.

Many lung diseases are characterized by fibrosis, leading to impaired tissue patency and reduced lung function. Development of fibrotic tissue depends on two-way interaction between the cells and the extra-cellular matrix (ECM). Concentration-dependent increased stiffening of the ECM is sensed by the cells, which in turn increases intracellular contraction and pulling on the matrix causing matrix reorganization and further stiffening. It is generally accepted that the inflammatory cytokine growth factor ß1 (TGF-ß1) is a major driver of lung fibrosis through the stimulation of ECM production. However, TGF-ß1 also regulates the expression of members of the tropomyosin (Tm) family of actin associating proteins that mediate ECM reorganization through intracellular-generated forces. Thus, TGF-ß1 may mediate the bi-directional signaling between cells and the ECM that promotes tissue fibrosis. Using combinations of cytokine stimulation, mRNA, protein profiling and cellular contractility assays with human lung fibroblasts, we show that concomitant induction of key Tm isoforms and ECM by TGF-ß1, significantly accelerates fibrotic phenotypes. Knocking down Tpm2.1 reduces fibroblast-mediated collagen gel contraction. Collectively, the data suggest combined ECM secretion and actin cytoskeleton contractility primes the tissue for enhanced fibrosis. Our study suggests that Tms are at the nexus of inflammation and tissue stiffening. Small molecules targeting specific Tm isoforms have recently been designed; thus targeting Tpm2.1 may represent a novel therapeutic target in lung fibrosis.

Investigation of clinically relevant germline variants detected by next-generation sequencing in patients with childhood cancer: a review of the literature.

Genetic predisposition is an important underlying cause of childhood cancer, although the proportion of patients with childhood cancer carrying predisposing pathogenic germline variants is uncertain. This review considers the pathogenic or likely pathogenic germline variants reported by six studies that used next-generation sequencing to investigate genetic predisposition in selected cohorts of patients with childhood cancer and used incompletely overlapping gene sets for analysis and interpretation. These six studies reported that 8.5%-35.5% of patients with childhood cancer carried clinically relevant germline variants. Analysis of 52 autosomal dominant cancer predisposition genes assumed common to all six studies showed that 5.5%-25.8% of patients with childhood cancer carried pathogenic or likely pathogenic germline variants in at least one of these genes. When only non-central nervous system solid tumours (excluding adrenocortical carcinomas) were considered, 8.5%-10.3% of the patients carried pathogenic or likely pathogenic germline variants in at least one of 52 autosomal dominant cancer predisposition genes. There was a lack of concordance between the genotype and phenotype in 33.3%-57.1% of the patients reported with pathogenic or likely pathogenic germline variants, most of which represented variants in autosomal dominant cancer predisposition genes associated with adult onset cancers. In summary, germline genetic testing in patients with childhood cancer requires clear definition of phenotypes and genes considered for interpretation, with potential to inform and broaden childhood cancer predisposition syndromes.

Two mosaic terminal inverted duplications arising post-zygotically: Evidence for possible formation of neo-telomeres.

OBJECTIVE: To elucidate the structure of terminal inverted duplications and to investigate potential mechanisms of formation in two cases where there was mosaicism with cells of apparently normal karyotype. RESULTS: A karyotype [46,XY,inv dup(4)(p16.3p15.1)/46,XY] performed on blood lymphocytes from a patient referred for developmental delay (case 1) demonstrated a normal karyotype in 60% of cells with a terminal inverted duplication 4p in the remainder. In case 2, referred for multiple fetal anomalies on an ultrasound scan, 33% of amniocyte colonies were karyotypically normal, with a terminal inv dup 10p in the remainder [46,XX,inv dup(10)(p15.3p11)/46,XX]. Duplicated FISH signals for GATA3 and NEBL loci (in case 2), and for the Wolf-Hirschhorn locus (case 1) confirmed the inverted structure of both duplications. In the GTL banded normal cells from both cases, there was a cryptic deletion detected by FISH of one copy of the subtelomere 4p (case 1, probe GS-36P21), and subtelomere 10p (case 2, probe GS-306F7). At pter on both inv dup chromosomes there was no FISH signal present for the specific subtelomere probe. However, a positive pantelomeric probe signal was detected at 4 pter and 10 pter in both the cryptically-deleted chromosomes and the inv dup chromosomes in the respective cell lines of both cases. CONCLUSION: An inv dup structure was evident for both cases on GTL bands, and confirmed by the various FISH studies. The presence of telomere (TTAGGG repeat) sequences at pter on the inv dup chromosomes (where more proximal chromosome specific subtelomeric probes were negative) was indicated by the pantelomeric probe signals in both cases. We conclude the most likely mechanism of origin in both cases was by sub-telomeric breakage in the zygote at pter, and delayed repair/rearrangement until after one or more subsequent mitotic divisions. In these divisions, at least one breakage-fusion-bridge cycle occurred, to produce inverted duplications. It is proposed then that two differently "repaired" daughter cells proliferated in parallel. In one daughter cell line (with an overtly normal karyotype) there was deletion of the subtelomere and presumed repair through capping by a neo-telomere (i.e. "healing", as initially proposed by McClintock). This occurred in both cases presented. In the other daughter cell of each case, it is proposed that chromosome stabilization was achieved (after replication) by sister chromatid reunion to form a dicentric, which broke at a subsequent anaphase, to form an inverted duplication (with loss of the reciprocal product, and the other daughter cell line). One inv dup was repaired without an interstitial specific subtelomere (case 1) and one was repaired with a duplicated specific interstitial subtelomere (case 2). After repair TTAGGG repeats were detected by FISH at each respective new pter.

Oncolysis of human ovarian cancers by echovirus type 1.

A small number of enteroviruses possess the capacity to induce rapid and marked lytic infections in cells of various human malignancies. During screening of representative human enteroviruses for their oncolytic capacity, we observed that echovirus type 1 (EV1) displayed a high level of tropism for human ovarian cancer cells. EV1 is an enterovirus which largely causes asymptomatic infections in humans and whose tissue tropism is primarily regulated via interactions with the I domain of the alpha subunit of cell surface-expressed integrin alpha2beta1. We evaluated the capacity of wild-type EV1 to act as an oncolytic agent of ovarian cancers propagated as cell monolayers, multicellular spheroids or xenografts in SCID mice. EV1 infection of in vitro propagated ovarian cell lines expressing high levels of integrin alpha2beta1 was assessed for specific viral attachment, antibody blockade, induction of cytopathic effect and production of progeny virions. EV1 lytically infected all 8 human ovarian cancer cell lines tested (2008, DOV13, JAM, OVCA-429, OVCAR-3, OVHS-1, OAW-42 and IGROV-1) but not the immortalized normal ovarian surface epithelial cell line (HOSE) or human PBMCs. EV1 challenge was equally effective in the oncolysis of human ovarian cancer cells whether in monolayer or spheroidal environments. The therapeutic efficacy of EV1 was demonstrated by rapid reduction of tumor burden by a single viral intratumoral injection in SCID mice bearing multiple preformed s.c. xenografts. Using an in vivo i.p. human ovarian cancer xenograft model, administration of EV1 was further shown to significantly inhibit the formation and burden of ascites tumors. These findings demonstrate an important proof of principle for employing wild-type EV1 as a potential oncolytic agent in the control of human ovarian cancers.