Characteristics of the Patients

The PCGP cohort included 588 children and adolescents with leukemia (52.5%), 245 with central nervous system (CNS) tumors (21.9%), and 287 with non-CNS solid tumors (25.6%) (Figure 1, and Table S1 in Supplementary Appendix 1). The median age of the patients was 6.9 years (range, 8 days to 19.7 years). The cancers that were selected for sequencing included those that have been associated with a poor clinical outcome (e.g., hypodiploid leukemia)14 and those without a clearly defined oncogenic cause (e.g., diffuse intrinsic pontine glioma).15 Our cohort included more patients with leukemia and adrenocortical tumors than was expected on the basis of the population in the Surveillance, Epidemiology, and End Results (SEER) program (http://seer.cancer.gov/iccc) (Figure 1). Lymphoma, Wilms’ tumor, germ-cell tumors, non-rhabdomyosarcoma soft-tissue sarcoma, and hepatoblastoma were not included because of an inadequate number of samples for high-risk subtypes.

Germline Mutations in Cancer-Predisposition Genes

In the 60 genes that have been associated with autosomal dominant cancer-predisposition syndromes, we identified 633 nonsilent germline variants, of which 78 (12%) were deemed to be pathogenic, 17 (3%) probably pathogenic, 226 (36%) of uncertain significance, 273 (43%) probably benign, and 39 (6%) benign (Table S4A in Supplementary Appendix 2). The 95 variants that were deemed to be pathogenic or probably pathogenic included 54 missense mutations, 14 nonsense mutations, 12 frameshift mutations, 9 splice-site mutations, and 1 in-frame deletion, as well as 5 copy-number alterations (Fig. S2 in Supplementary Appendix 1).

Figure 3. Figure 3. Distribution of Germline Mutations in Different Gene Categories and Cancer Subtypes. Panels A and B include only mutations that were deemed to be pathogenic or probably pathogenic and that affect genes that have been associated with autosomal dominant cancer-predisposition syndromes, according to tumor subtype. Panel A shows the distribution of mutations in each gene among patients with various cancers included in the PCGP cohort. Panel B shows the prevalence of the mutations in each cancer subtype. Five patients with melanoma without mutations are not shown, and one patient (HGG027) who had a CNS tumor with biallelic mutation in an autosomal recessive gene (ATM) is not included in the summary. Panel C shows the number of patients who had germline mutations considered to be pathogenic or probably pathogenic in genes that have been associated with autosomal dominant (60 genes) and autosomal recessive (29) cancer susceptibility, according to cancer subtype. Panel D shows the total number of patients who had truncation mutations in tumor-suppressor genes, tyrosine kinase genes, and other cancer genes, according to cancer subtype.

The 95 variants that were deemed to be pathogenic or probably pathogenic were detected in 21 of the 60 genes in 94 patients (Figure 3A, and Fig. S3 in Supplementary Appendix 1). TP53 was most commonly involved (in 50 patients), followed by APC (in 6), BRCA2 (in 6), NF1 (in 4), PMS2 (in 4), RB1 (in 3), and RUNX1 (in 3). One patient (Patient HGG111) with café au lait spots and a high-grade glioma had 2 distinct PMS2 truncation mutations, which indicated a diagnosis of biallelic mismatch-repair deficiency that was corroborated by the somatic hypermutation observed in the genome of the high-grade glioma.15 The most common cancer types that were associated with germline TP53 mutations included adrenocortical tumors (in 27 of 39 patients [69%]), hypodiploid acute lymphoblastic leukemia (in 9 of 47 [19%]), and choroid plexus carcinoma (in 1 of 4 [25%]) — findings that were consistent with those in previous reports.14 As anticipated, the tumors from all 37 of these patients had a loss of heterozygosity at the TP53 locus (Table S4 in Supplementary Appendix 2), including 1 patient who had a germline deletion of 8.7 kb that removed TP53 exons 2 through 5 (Fig. S2 in Supplementary Appendix 1).

Figure 4. Figure 4. Distinguishing Mosaicism from Tumor Contamination. Panel A shows that in the tumor-contaminated germline sample of Patient 1 (E2A019), most somatic mutations were observed at a lower frequency in the germline than in the tumor. Nine genes were selected to show this point. Panel B shows that in the case of mosaicism in Patient 2 (HYPO055), only one TP53 mutation was observed at a lower frequency in the germline than in the tumor. Other somatic mutations in the tumor were absent in the matched germline sample. Panel C shows that MiSeq sequencing confirmed that the mutant allele fraction (MAF) of TP53 c.C374G (coding for p.T125R) in the germline sample of Patient 2 was still low (0.20; only 487 reads of 2383 reads had the mutation), a finding that is consistent with germline mosaicism. Two minor peaks supporting C and G alleles (arrows) were seen in the Sanger-sequencing chromatograph.

Four germline mutations were mosaic, with the detected level of the mutant allele less than a single copy (mutant allele fraction, 0.08 to 0.30). One patient with retinoblastoma had a mosaic RB1 mutation, and three patients with hypodiploid acute lymphoblastic leukemia had a mosaic TP53 mutation (Table S4A in Supplementary Appendix 2). The mutant allele fraction in matching tumor specimens ranged from 0.76 to 0.90, a finding that is consistent with the presence of a second hit within the tumors. Validation by means of deep sequencing at more than 2000× coverage verified the mutant allele fraction within the germline and tumor samples in each patient (Figure 4).

In the first control data set, from the 1000 Genomes Project, we identified 11 pathogenic or probably pathogenic mutations in the 60 genes that have been associated with autosomal dominant cancer-predisposition syndromes; mutations were found in APC (in one person), BRCA1 (in one), BRCA2 (in four), MSH6 (in one), SDHA (in one), SDHB (in one), and TP53 (in two) (Table S6 in Supplementary Appendix 1). The prevalence of mutations was 1.1%, which was significantly lower than the 8.4% prevalence observed in the PCGP cohort (P=5.9×10−16 by Fisher’s exact test). A similar trend was observed in the second control set, which involved participants from the autism study (frequency, 0.6%; P=7.4×10−16 for the comparison with the PCGP cohort) (Table S7 in Supplementary Appendix 1).

The PCGP cohort included a greater-than-expected proportion of patients with hypodiploid acute lymphoblastic leukemia and those with adrenocortical tumors (Figure 1). However, after these two subtypes were excluded, the prevalence of germline mutations of 5.6% was still significantly higher than the prevalence in the two control cohorts (P<10−7 by Fisher’s exact test for both comparisons).

In our analysis of 29 autosomal recessive cancer-predisposition genes, we observed only one instance of biallelic pathogenic mutations in 1 patient (Table S8 in Supplementary Appendix 1). Combining data from this single patient, who had ataxia telangiectasia caused by biallelic mutations in ATM (Fig. S4 in Supplementary Appendix 1), with data from the 94 patients who had pathogenic mutations in the 60 autosomal dominant cancer-predisposition genes, we observed an 8.5% prevalence (95 of 1120 patients) of germline mutations that were pathogenic or probably pathogenic in the sample we analyzed. A total of 61 of the 93 patients (66%) with monoallelic germline mutations had a second hit within the tumor genome (Table S4 in Supplementary Appendix 2), as shown by loss of heterozygosity (in 57 patients) or mutational inactivation of the second allele (in 4). These data are available on our pediatric cancer data portal (http://pecan.stjude.org) (Figs. S5 and S6 in Supplementary Appendix 1)

Prevalence of Germline Mutations across Tumor Types

The prevalence of germline mutations that were pathogenic or probably pathogenic was greatest among patients with non-CNS solid tumors (48 of 287 patients [16.7%]), followed by those with CNS tumors (21 of 245 [8.6%], including the patient with biallelic loss of ATM) or leukemia (26 of 588 [4.4%]) (Figure 3B). The prevalence of germline mutations varied among patients with different subtypes of non-CNS solid tumors, such as adrenocortical tumor (69.2%), osteosarcoma (17.9%), retinoblastoma (13.3%), Ewing’s sarcoma (10.9%), rhabdomyosarcoma (7.0%), and neuroblastoma (4.0%) (Figure 3B). The histologic subtypes of CNS tumor that were most often associated with germline mutations included choroid plexus carcinoma (in 1 of 4 patients [25%]), medulloblastoma (in 5 of 37 [13.5%]), high-grade glioma (in 9 of 99 [9.1%]), low-grade glioma (in 3 of 38 [7.9%]), and ependymoma (in 4 of 67 [6.0%]). Overall, patients with leukemia had the lowest prevalence of germline mutations (4.4%), despite the inclusion of patients with hypodiploid acute lymphoblastic leukemia, a subtype with a high frequency of germline mutation.14

Correlation between Germline Genotype and Tumor Phenotype

The correlation of patient genotype with tumor phenotype revealed several known associations as well as some new ones. The known associations included the association of TP53 mutations with classic Li–Fraumeni syndrome–associated component cancers such as rhabdomyosarcomas, osteosarcomas, adrenocortical tumors, CNS tumors, and leukemia; NF1 mutations with CNS tumors; RB1 mutations with retinoblastoma and osteosarcoma; and ALK mutations with neuroblastoma (Figure 3A). New associations included the association of germline TP53, PMS2, and RET mutations with Ewing’s sarcoma; APC and SDHB mutations with neuroblastoma; and a variety of mutations (APC, VHL, CDH1, PTCH1, or SDHA) with leukemia.

A total of eight children had germline mutations in the adult-onset cancer–predisposition genes BRCA1, BRCA2, and PALB2. The spectrum of cancers observed in these children included leukemia, CNS tumors, neuroblastoma, osteosarcoma, and rhabdomyosarcoma. Although biallelic mutations of BRCA1/2 and PALB2 are known to cause Fanconi’s anemia,16-19 there were no germline mutations or deletions involving the second alleles of these genes in any of the affected patients.

Medical and Family History

Medical records were available for review for 75 of the 95 patients with mutations that were deemed to be pathogenic or probably pathogenic. The records showed that only 12 patients had undergone clinical genetic testing previously. Clinical testing did not identify the predisposing genetic lesions in 2 patients. Of these 2 patients, 1 had an adrenocortical tumor tested for TP53 (TP53 p.I332F in Patient ACT001) and 1 had retinoblastoma that was tested for RB1 (mosaic RB1 p.R445* in Patient RB002); both lesions were identified by means of the next-generation sequencing approaches used in this study.

A total of 58 of the 75 records (77%) contained information regarding family history, and only 23 of 58 records (40%) indicated a family history of cancer (defined here as one or more first- or second-degree relatives with cancer) (Fig. S7 in Supplementary Appendix 1). Furthermore, among these 23 patients, only 13 (57%) had a history that was consistent with the underlying genetic syndrome, including 8 patients with TP53 mutations (and thus the Li–Fraumeni syndrome), 2 with APC mutations (familial adenomatous polyposis), 2 with BRCA2 mutations (hereditary breast and ovarian cancer; the pedigrees are shown in Fig. S8 in Supplementary Appendix 1), and 1 with PMS2 mutations (hereditary nonpolyposis colorectal cancer, also known as the Lynch syndrome). The 8 patients with the Li–Fraumeni syndrome all met the revised Chompret criteria regarding family history.20

We completed a similar analysis of a comparison cohort of 100 randomly selected patients who did not have germline mutations in the 60 autosomal dominant cancer-predisposition genes. We observed that the percentage of patients with a family history of cancer (42%; 18 of 43 records with family-history information) was similar to that observed among persons with germline mutations (40%; 23 of 58 records).

Germline Mutations in Other Cancer-Associated Genes

We identified 4348 nonsilent coding mutations in the remaining 476 genes that were analyzed. These included 114 heterozygous truncation mutations, in 109 patients, that involved tumor-suppressor genes, tyrosine kinase genes, or other cancer genes (Figure 3D, and Table S5 in Supplementary Appendix 1 and Table S4 in Supplementary Appendix 2). The most commonly affected tumor-suppressor genes included CHEK2 (in 4 patients), PML (in 4), and BUB1B (in 3). A total of 18 patients who did not have pathogenic mutations in genes that have been associated with cancer-predisposition syndromes had protein-truncating mutations in tumor-suppressor genes. Two known hotspots of somatic activating mutations in EGFR, T790 and H773, were identified once each in the germline of 2 patients with leukemia (Fig. S6 in Supplementary Appendix 1).