Patient characteristics

Our study included 102 children under age 18 years enrolled between January 2015 and August 2017. Patient characteristics and demographics are summarized in Table 1. According to national registry data, our cohort represents over 80% of all malignant paediatric tumours in Singapore.14,15 Fifty-two boys and 50 girls were included, with a median age at diagnosis of 4 years. Ethnic distribution was reflective of our population, comprising predominantly Chinese, followed by Indian, Malay and other ethnicities. The children presented a wide spectrum of solid tumours, broadly classified into ten histological groups (Fig. 1). Neuroblastic tumours are the most commonly observed, accounting for 19.6% (n = 20) of all solid tumours, followed by central nervous system (CNS) tumours in 11 (10.8%) children. A total of 26 (25.4%) patients presented with soft tissue and bone sarcomas of various histological classifications whereas 13 (12.7%) had Wilms tumour and 12 (11.8%) patients with extracranial germ cell tumours. The remaining children had neoplasms broadly grouped as endocrine or neuroendocrine (n = 6), ovarian (n = 5), hepatic (n = 5), and other rare tumours (n = 4), including pleuropulmonary blastoma (PPB, n = 1), nasopharyngeal carcinoma (n = 1) and Langerhans cell histiocytosis (n = 2) (Supplementary Table 1).

Table 1 Characteristics of 102 childhood tumour patients Full size table

Fig. 1 Distribution of tumour diagnoses included in this study. CNS central nervous system Full size image

Clinical checklist-aided screening for cancer predisposition syndrome

Using two published clinical checklists, we assessed 101 patients in our cohort for CPS. One patient was not evaluable due to insufficient clinical data. We found 79 (77.5%) and 66 (64.7%) patients with clinical features indicative of CPS based on the criteria proposed by Ripperger et al. or Postema et al. respectively,11,12,13 whereas ten (9.8%) patients were negative for both assessments (Table 2). Of the patients who screened positive, 54 (52.9%) met criteria in both checklists. Analysis of this group by tumour type revealed that patients with endocrine or neuroendocrine tumours are most likely to screen positive, while sarcoma patients were least likely to meet the criteria (Fig. 2). Both checklists were equally sensitive in detecting all ten patients with pathogenic germline mutations identified by sequencing (Table 2). While the use of each checklist independently was less specific, combining criteria of both checklists improved the specificity of detection to 52% without effect on sensitivity.

Table 2 Sensitivity and specificity of the two assessed clinical screening checklists Full size table

Fig. 2 Clinical checklist screening outcomes and germline mutation frequencies among evaluated patients. Proportion of checklist-positive screenings and pathogenic germline mutation carriers are indicated above each bar Full size image

Of the ten patients who screened positive and carried pathogenic germline mutations, eight fulfilled clinical criteria for CPS including Li-Fraumeni syndrome (LFS, n = 3), DICER1 syndrome (n = 3), neurofibromatosis type 1 (NF1) (n = 1) and von Hippel-Lindau (VHL, n = 1) syndrome (Table 3). Nine patients were referred by their primary oncologist for genetic counselling and testing, of which four did not follow up with the recommendation. One patient was not referred due to rapid disease progression and succumbed to complications arising from her condition.

Table 3 List of patients with identified pathogenic germline mutations Full size table

Spectrum of germline mutations in known cancer predisposition genes

We identified 12 pathogenic germline mutations in 10 children (9.8%) across six known cancer predisposition genes (Table 3). Frequency of mutations was highest in TP53 affecting five patients, followed by DICER1 mutations in three patients (Fig. 3a). Two patients harboured more than one pathogenic variant: one with diffuse astrocytoma was found to have concurrent TP53 exon 1 deletion and NF1 nonsense mutation, and a Sertoli−Leydig cell tumour (SLCT) patient harboured both DICER1 frameshift and nonsense FH mutations. In addition, two patients were found with a deleterious mutation in VHL and SDHD respectively. Pathogenic variants in autosomal recessive genes were not observed.

Fig. 3 Prevalence of pathogenic germline mutations. a Overview of identified pathogenic germline mutations across six genes in ten patients. b Lollipop diagrams visually depicting occurrence of the pathogenic germline mutations on proteins encoded by the affected genes. DA diffuse astrocytoma, ACC adrenocortical carcinoma, STS soft tissue sarcoma, RMS rhabdomyosarcoma, LPS liposarcoma, SLCT Sertoli−Leydig cell tumour, PPB pleuropulmonary blastoma, PC pheochromocytoma Full size image

All TP53 variants identified were previously seen in LFS families.16,17,18,19,20 Four of these were missense mutations that clustered within the p53 DNA-binding domain (Fig. 3b). Three (Cys141Tyr, Arg213Gln, Arg273Cys) are known to reduce p53 transactivation activity21,22, whereas Arg110Pro was demonstrated to have a dominant-negative effect on wild-type p53 function.23 Somatic loss-of-heterozygosity (LOH) was observed in all TP53 mutation carriers, including the hemizygous exon 1 deletion carrier. TP53 exon 1 encodes the 5′ untranslated-region (UTR) shown to be critical for RPL26-mediated translation of p53 mRNA upon DNA damage.24 Reported carriers of TP53 promoter or exon 1 deletion mostly presented soft tissue sarcomas and breast cancer.20,25,26,27 Somatic LOH reflected by homozygous deletion of this region in our patient’s tumour (Supplementary Figure 1) implicates deleterious effect of this variation. Interestingly, this child also harboured a truncating germline mutation in NF1 (Arg1513*) previously observed in other NF1 patients.28,29,30

The three detected DICER1 germline mutations were truncating variants, two of which are reported in ClinVar database. A second DICER1 somatic mutation was found in all three patients at the RNase IIIb domain hotspot Glu1813 residue known to inactivate DICER1 activity31,32 (Fig. 3b). A concurrent truncating FH mutation with somatic LOH was seen in one of the DICER1 germline mutation carriers (Table 3).

Amongst the tumour types, prevalence of pathogenic germline mutation was highest in endocrine or neuroendocrine tumours (n = 4/6, 67%). This is followed by ovarian tumour (n = 2/5, 40%), soft tissue sarcoma (n = 2/18, 11%) and CNS tumour (n = 1/11, 9%) (Fig. 2). Further breakdown by histological subtypes highlighted that all enrolled patients with adrenocortical carcinoma (ACC), pheochromocytoma and SLCT harboured germline mutations, implying a higher genetic susceptibility in these tumour types (Supplementary Table 2).

Association of germline mutations with clinical phenotype and family history

Overall, the detected genotypes were consistent with patient clinical phenotypes. Four among five TP53 mutation carriers presented tumours typical of LFS spectrum: ACC (n = 2) and soft tissue sarcoma (n = 2) (Fig. 3a). Moreover, their mutations were also reported in LFS families and paediatric patients with similar tumour types.3,16,17,18,33 Similarly, germline mutations in SDHD and VHL, two genes known to confer susceptibility to pheochromocytoma, were found in both patients with this diagnosis (Fig. 3a). Unsurprisingly, our DICER1 germline mutation carriers presented tumour types most frequently associated with DICER1 syndrome, namely PPB (n = 1) and SLCT with multinodular goitre (MNG) (n = 2).32

In patients harbouring more than one pathogenic germline mutation, clinical manifestations were predominantly consistent with genes in which penetrance is greater at an earlier age. For instance, the 2.6-year-old diffuse astrocytoma patient with concurrent NF1 and TP53 mutations exhibited clinical features mostly characteristic of NF1: multiple café-au-lait spots, neurofibroma and global developmental delay. This is congruent with the fact that penetrance is almost 100% by age 8 years in NF1 germline mutation carriers34 compared to penetrance of 50% by the third decade of life in individuals with germline TP53 mutations.35 However, the presence of astrocytoma unrelated to the optic pathway at this age is more consistent with the germline TP53 mutation harboured by this child, which was missed by the treating physicians given his characteristic NF1 clinical features.

Six of the ten germline mutation carriers had at least one relative with cancer (Table 3); however, only five (50%) showed a family history typical of CPS. Two are TP53 mutation carriers, who met Chompret criteria for LFS, whereas two DICER1 mutation carriers with SLCT had multiple relatives presenting MNG and thyroid cancer. The remaining VHL mutation carrier demonstrated a family history of pheochromocytoma and renal cell carcinoma consistent with VHL syndrome.

Variants of uncertain significance in known cancer predisposition genes

A total of 86 rare variants of uncertain significance (VUS) were detected, predominantly in neuroblastic tumours with an adjusted frequency of four variants, followed by soft tissue sarcoma with three VUS (Supplementary Figure 1). Amongst these, some were predicted potentially deleterious by in silico algorithms and occurred in DNA repair genes, including BRCA1, BRCA2, MLH1, PMS2 and RAD54L. Coincidentally, tumours of patients from these two histological subtypes exhibited higher incidences of karyotypic aberrations, suggesting plausible roles for DNA repair pathway deficiency.

A few potentially deleterious VUS were found in genes beyond those commonly associated with the disease. For example, a variant in MAX, an essential interacting partner of MYC, was identified in a patient with neuroblastoma. The mutation Arg100Cys, which occurred within the leucine-zipper domain of MAX important for regulation of MYC, could impair MYC activities. Additionally, we found two variants—CDH1 Pro373Leu and RAF1 Pro332Ala—individually associated with hereditary diffuse gastric cancer (HDGC)36 and childhood-onset dilated cardiomyopathy37 in two patients with hepatocellular carcinoma (HCC) and testicular germ cell tumour respectively. Although demonstrated to be functionally deleterious, association of these VUS with the clinical phenotype of our patients remains to be verified by further studies.