Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in pleuropulmonary blastoma / DICER1 syndrome: a unique variant of the two-hit tumor suppression model

Abstract

Pleuropulmonary blastoma (PPB) is the most frequent pediatric lung tumor and often the first indication of a pleiotropic cancer predisposition, DICER1 syndrome, comprising a range of other individually rare, benign and malignant tumors of childhood and early adulthood. The genetics of DICER1-associated tumorigenesis are unusual in that tumors typically bear neomorphic missense mutations at one of five specific "hotspot" codons within the RNase IIIb domain of DICER 1, combined with complete loss of function (LOF) in the other allele. We analyzed a cohort of 124 PPB children for predisposing DICER1 mutations and sought correlations with clinical phenotypes. Over 70% have inherited or de novo germline LOF mutations, most of which truncate the DICER1 open reading frame. We identified a minority of patients who have no germline mutation, but are instead mosaic for predisposing DICER1 mutations. Mosaicism for RNase IIIb domain hotspot mutations defines a special category of DICER1 syndrome patients, clinically distinguished from those with germline or mosaic LOF mutations by earlier onsets and numerous discrete foci of neoplastic disease involving multiple syndromic organ sites. A final category of PBB patients lack predisposing germline or mosaic mutations and have sporadic (rather than syndromic) disease limited to a single PPB tumor bearing tumor-specific RNase IIIb and LOF mutations. We propose that acquisition of a neomorphic RNase IIIb domain mutation is the rate limiting event in DICER1-associated tumorigenesis, and that distinct clinical phenotypes associated with mutational categories reflect the temporal order in which LOF and RNase IIIb domain mutations are acquired during development.

Keywords: DICER1 truncation; Mosaicism; PPB; Paediatric cancer; Pleuropulmonary blastoma; RNAse IIIb.

Figure 1.. Study design – Detection and categorization of DICER1 mutations in PPB probands.

A cohort of 124 children diagnosed with pleuropulmonary blastoma (PPB) was screened for predisposing DICER1 mutations by targeted Sanger sequencing and/or low-depth, next-generation sequencing (NGS) of DNA amplified from peripheral blood cells, saliva (buccal cells) or non-neoplastic surgical specimens. Sequenced PCR amplicons covered the 26 coding exons of the DICER1 open reading frame and flanking splice signals. DICER1 coding sequence or splice site mutations detected at approximately heterozygous frequency in blood or normal tissue cells were categorized as germline mutations. For patients in whom screening revealed no germline mutation, blood and/or normal tissues were analyzed for the presence of intragenic deletions or larger genomic alterations using NanoString copy number assay and CNV array, and for coding or splice site mutations present at low allele frequencies using high-depth NGS on the Ion Torrent platform. Wherever possible, matched tumor specimens were also sequenced on the Ion Torrent platform. DICER1 mutations detected in tumor samples and at sub-heterozygous frequencies in blood or other normal tissue samples were categorized as mosaic mutations. RNase IIIb hotspot mutations detected in primary tumors of multiple organs were also categorized as mosaic mutations, even if they were not conclusively identified in blood or other normal tissues. Patients for whom both LOF and hotspot mutations were identified in a single tumor, but not found in blood or normal tissue samples, were categorized as having tumor-specific, biallelic DICER1 mutations. Cases of this last kind are considered sporadic PPB, not DICER1 syndrome.

Figure 2.. The spectrum of predisposing loss-of-function mutations in PPB/ DICER1 syndrome.

A linear schematic of the DICER1 open reading frame is shown with annotated functional domains represented to scale. Sequence changes identified as inherited or de novo germline mutations in 90 PPB/ DICER1 syndrome patients are indicated by position along the coding sequence. Mutations linked to the schematic by two, three or four fine lines are those discovered in a corresponding number of individuals from unique families.

Figure 3.. Numbers and types of disease foci in DICER1 syndrome patients with mosaic RNase IIIb domain hotspot mutations.

For each of the seven mosaic hotspot children identified in this study, an individual timeline indicates numbers of discrete foci of neoplastic disease and their histopathological types, graphed with respect to patient age at diagnosis. Across the lower portion of the chart, a single aggregate timeline (dark violet) represents the mean number of disease foci for all PPB/ DICER1 syndrome patients with predisposing loss of function (LOF) mutations identified in this study, graphed with respect to patient age at diagnosis. The shaded areas (in lighter violet) surrounding the timeline for LOF mutation patients indicates one and two standard deviations above and below the mean. The range of foci number among all LOF mutation patients was 0 to 6 in all years of age represented (not shown). Abbreviations: CN cystic nephroma; CBME ciliary body medulloepithelioma (eye); NCMH nasal chondromesenchymal hamartoma; PPB pleuropulmonary blastoma; PinB pineoblastoma; PvS pelvic sarcoma; SIP small intestinal polyp(s); SLCT Sertoli-Leydig cell tumor (ovary); TCa thyroid carcinoma; TN thyroid nodule(s).