Molecular and Cellular Pathobiology Biallelic DICER1 Mutations in Sporadic Pleuropulmonary Blastoma

Pleuropulmonary blastoma (PPB) is a rare pediatric malignancy whose pathogens are poorly understood. Recent reports suggest that germline mutations in the microRNA-processing enzyme DICER1 may contribute to PPB development. To investigate the genetic basis of this cancer, we performed whole-exome sequencing or targeted deep sequencing of multiple cases of PPB. We found biallelic DICER1 mutations to be very common, more common than TP53 mutations also found in many tumors. Somatic ribonuclease III (RNase IIIb) domain mutations were identified in all evaluable cases, either in the presence or absence of nonsense/frameshift mutations. Most cases had mutated DICER1 alleles in the germline with or without an additional somatic mutation in the remaining allele, whereas other cases displayed somatic mutations exclusively where the RNase IIIb domainwas invariably affected. Our results highlight the role of RNase IIIb domainmutations inDICER1 along with TP53 inactivation in PPB pathogenesis. Cancer Res; 74(10); 2742–9. 2014 AACR. Introduction Pleuropulmonary blastoma (PPB) is an extremely rare and highly aggressive pulmonary malignancy occurring in early childhood. It is characterized histologically by a primitive blastoma and a malignant mesenchymal stroma in the lung that often shows multidirectional differentiation (1). PPB may be sporadic or hereditary and may also present as a part of a familial tumor syndrome (2) consisting of cystic nephroma and other tumor types, such as ovarian tumor, embryonal rhabdomyosarcoma, and malignant germ cell tumors (2). Recently, germline DICER1 mutations have been demonstrated in majority of patients with PPB and DICER1 syndrome (2, 3). DICER1 is a member of the ribonuclease III (RNase III) protein family that is involved in the generation of microRNAs (miRNA), modulating gene expression at the posttranscriptional level (4). The DICER1 protein contains RNase IIIa and RNase IIIb domains, which are considered to dimerize intramolecularly with Mg2þ/Mn2þ to form the active site of the enzyme (5). In PPB, almost all mutations are reported to be heterozygous frameshift or nonsense mutations of germline origin, suggesting an important role of DICER1 haploinsufficiency in PPB pathogenesis (2, 3). However, most obligate carriers of DICER1 mutations and heterozygous Dicer1-deficient mice did not develop PPB or other types of tumors, suggesting that DICER1 haploinsufficiency alone is insufficient for tumor development but requires additional genetic alterations (3, 6). To identify a complete set of genetic alterations underlying PPB pathogenesis, we performed whole-exome sequencing of paired tumor and normal DNA from seven cases with sporadic PPB, of which two cases were analyzed for samples obtained at both initial presentation and relapse. Mutations in DICER1 and other genes were examined by targeted deep sequencing in 16 samples from 12 sporadic PPB cases, including three analyzed by whole-exome sequencing. Materials and Methods Specimens Genomic DNA for 11 cases was extracted from fresh-frozen samples stored at 80 C and obtained approximately 2 to 15 years previously. Paraffin-embedded samples were used as tumor samples for cases 10 (at relapse) and 11 (at diagnosis). These samples were stored for approximately 1 year. For Authors' Affiliations: Department of Pediatrics; Cancer Genomics Project, Graduate School of Medicine; Laboratory of DNA Information Analysis and SequenceDataAnalysis, HumanGenomeCenter, Institute of Medical Science; Department of Cell Therapy and Transplantation Medicine, TheUniversity of Tokyo, Tokyo; Department ofPathology andTumor Biology,GraduateSchool ofMedicine,KyotoUniversity, Kyoto; Divisionof Pediatric Hematology and Oncology, Ibaraki Children's Hospital, Mito, Ibaraki; Department of Hematology/Oncology, Saitama Children's Medical Center, Saitama, Saitama; Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka; Gunma Children's Medical Center, Shibukawa, Gunma; Department of Hematology and Oncology, Hyogo Prefectural Kobe Children's Hospital, Kobe, Hyogo; and National Center for Child Health and Development, Tokyo, Japan Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Authors: Junko Takita, Department of Pediatrics, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 81-3-3815-5411; Fax: 81-3-3816-4108; E-mail: [email protected]; and Seishi Ogawa, Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, YoshidaKonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-4300; Fax: 81-75-753-9282; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-247