Germline DICER1 Mutations Research Articles

Abstract PR05: Dicer1 deletion: Sufficient for angiosarcoma development and a haploinsufficient tumor suppressor in rhabdomyosarcoma

Abstract In this study, the role of Dicer and microRNAs (miRNAs) is investigated in pediatric and adult soft tissue sarcomas with the hypothesis that understanding miRNAs and their biogenesis may provide biological insights into the pathogenesis of these diseases and identify novel therapeutic targets. In several mouse models of cancer, Dicer1 is thought to function as a haploinsufficient tumor suppressor where loss of a single allele promotes tumorigenesis yet loss of both alleles is detrimental to tumor formation. Heterozygous germline mutations in DICER1 in humans is associated with rare malignant and benign neoplasias including embryonal rhabdomyosarcoma (ERMS). With DICER1 syndrome mutations found in ERMS, a pediatric soft tissue sarcoma resembling skeletal muscle precursors that fail to differentiate, we sought to further understand the role of Dicer and miRNAs utilizing a transgenic mouse model of ERMS combined with conditional Dicer1Flox/Flox null mice. The ERMS mouse model results from activation of a constitutively active SmoM2 allele with Cre recombinase expressed from the adipose protein 2 (aP2) promoter resulting in unrestrained hedgehog signaling in the aP2 lineage. Heterozygous Dicer1 loss led to increased penetrance of and a decrease in latency to onset of tumors. In agreement with the haploinsufficient tumor suppressor model, homozygous Dicer1Flox/Flox mice rarely developed tumors in stark contrast to wild type and heterozygous littermates. Furthermore, tumors that did develop in Dicer1Flox/Flox mice escaped Cre recombination and retained a flox allele indicating functional Dicer1 activity. Serendipitously, aP2-Cre; SmoM2; Dicer1Flox/Flox mice that did not develop ERMS all developed multifocal angiosarcomas. This finding prompted the analysis of Dicer loss alone in the aP2 lineage and remarkably Dicer1Flox/Flox mice similarly developed angiosarcoma whereas Dicer1Flox/+ do not, providing evidence for Dicer loss independent of a cooperating oncogene driving tumorigenesis in a mouse model. This study provides evidence for the role of Dicer and miRNAs in rhabdomyosarcoma and angiosarcomas. Further understanding the molecular mechanisms and genetic drivers of soft tissue sarcomas is critical for the development of novel targeted therapeutics. This is true especially for soft tissue sarcomas where treatment strategies and survival outcomes have not changed in decades despite numerous clinical trials. This abstract is also presented as Poster B06. Citation Format: Jason A. Hanna, Matthew R. Garcia, Jerold Rehg, Mark E. Hatley. Dicer1 deletion: Sufficient for angiosarcoma development and a haploinsufficient tumor suppressor in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR05.

DICER1Mutations and Differentiated Thyroid Carcinoma: Evidence of a Direct Association

DICER1 germline mutation carriers have an increased predisposition to cancer, such as pleuropulmonary blastoma (PPB) and Sertoli-Leydig cell tumor (SLCT), and a high prevalence of multinodular goiter (MNG). Although differentiated thyroid carcinoma (DTC) has been reported in some DICER1 mutation carriers with PPB treated with chemotherapy, the association of DTC with DICER1 mutations is not well established. We report a family with DICER1 mutation and familial DTC without a history of chemotherapy. A 12-year-old female (patient A) and her 14-year-old sister (patient B) presented with MNG. Family history was notable for a maternal history of DTC and bilateral ovarian SLCT. Both sisters underwent total thyroidectomy. Pathological examination showed nodular hyperplasia and focal papillary thyroid carcinoma within hyperplastic nodules. Subsequently, patient A developed virilization secondary to a unilateral ovarian SLCT. During her evaluation, an incidental cystic nephroma was also found. Three other siblings had MNG on surveillance ultrasound examination; two had thyroidectomies, and one had two microscopic foci of papillary carcinoma. Patient A, her mother, and four affected siblings had a germline heterozygous pathogenic DICER1 mutation c.5441C>T in exon 25, resulting in an amino acid change from p.Ser1814Leu of DICER1. Somatic DICER1 RNase IIIb missense mutations were identified in thyroid nodules from three of the four siblings. This family provides novel insight into an emerging phenotype for DICER1 syndrome, with evidence that germline DICER1 mutations are associated with an increased risk of developing familial DTC, even in the absence of prior treatment with chemotherapy.

Pleuropulmonary Blastoma: Evolution of an Entity as an Entry into a Familial Tumor Predisposition Syndrome.

Pleuropulmonary blastoma (PPB) is the most common primary malignant neoplasm of the lung in children. Like other solid dysontogenic neoplasms, this tumor typically presents before 7 years of age. The earliest manifestation is the presence of a lung cyst(s), which is usually recognized in the first year of life and is difficult to differentiate on the basis of imaging studies from non-neoplastic cysts of early childhood. From a multilocular cyst, PPB has the potential to progress to a high-grade multipatterned primitive sarcoma. More than 65% of all affected children have a heterozygous germline mutation in DICER1. The DICER1 PPB familial tumor predisposition syndrome is initially recognized in most cases on the basis of PPB alone but also by several other unique and characteristic extrapulmonary tumors, including pediatric cystic nephroma, nasal chondromesenchymal hamartoma, nodular lesions of the thyroid, embryonal rhabdomyosarcoma of the cervix, and ciliary body medulloepithelioma.

High-sensitivity sequencing reveals multi-organ somatic mosaicism causing DICER1 syndrome

BackgroundSomatic mosaicism is being increasingly recognised as an important cause of non-Mendelian presentations of hereditary syndromes. A previous whole-exome sequencing study using DNA derived from peripheral blood identified mosaic mutations...

Abstract 4934: Mosaic RNase IIIb domain DICER1 mutations in children with multiple primary tumors

Abstract The DICER1 syndrome or pleuropulmonary blastoma (PPB) familial tumor and dysplasia syndrome (PPB FTDS) (OMIM #601200) is caused by heterozygous germ-line mutations in the microRNA maturation pathway gene, DICER1. Several rare phenotypes constitute the syndrome including PPB, cystic nephroma (CN), ovarian Sertoli-Leydig cell tumors (SLCT), intra-ocular medulloepithelioma, nasal chondromesenchymal hamartoma (NCMH), pineoblastoma, pituitary blastoma, multinodular goitre (MNG) and other rare childhood sarcomas and dysplasias. Highly characteristic second somatic mutations have been identified in DICER1-associated tumors, affecting amino acid residues central to the catalytic activity of the RNase IIIb domain. We describe four children with multiple primary tumours associated with the DICER1 syndrome. Sanger sequencing of constitutional DNA obtained from peripheral blood lymphocytes and/or saliva revealed no likely deleterious germ-line DICER1 mutations. We subsequently sequenced the region encoding the DICER1 RNase IIIa and RNase IIIb domains in gDNA extracted from the tumor samples, and noted the presence of the same RNase IIIb missense mutation in multiple tumors from each patient (Patient A: c.5437G>C; Patient B: c.5125G>A; Patient C: c.5439G>C; Patient D: c.5425G>A). We performed targeted capture followed by deep sequencing on DNA extracted from both normal and tumor tissue, which revealed the presence of the respective RNase IIIb mutations in a low percentage of sequencing reads (0.2 - 13%) in constitutional DNA from three of the four patients (Patients A, B and C). The relative abundance of the allele harboring the DICER1 RNase IIIb mutation was significantly higher in the tumors compared to normal tissue from the surrounding organ and/or distant sites. Taken together, these findings indicate a mosaic origin of the DICER1 RNase IIIb missense mutations. The mosaic origin of Patient D's mutation remains to be unequivocally established. We further hypothesized that, in the setting of a mosaic DICER1 RNase IIIb mutations, we might discover second somatic mutations outside of the RNase IIIb domain which initiate two-hit tumorigenesis as seen in most DICER1-related tumors. Sequencing data identified individually distinct second somatic, likely-deleterious DICER1 mutations in Patient A's left ovary SLCT and sinonasal inflammatory polyp, in Patient C's NCMH, and in Patient D's Type II PPB which arose in a pre-existing lung cyst. Each of these second somatic mutations are predicted to prematurely truncate the DICER1 protein. We demonstrate that mosaic DICER1 RNase IIIb missense mutations are an occasional and important genetic cause of the DICER1 syndrome in patients presenting with multiple primary tumors associated with the syndrome, and that for tumor initiation, they appear to be accompanied by second somatic truncating non-RNase IIIb DICER1 mutations. Citation Format: Leanne de Kock, Barbara Rivera Polo, Mona Wu, Evan Weber, Claudio Sandoval, Saskia M. J. Hopman, J. Hans M. Merks, Annet van Hagen, D. A. Plager, Nelly Sabbaghian, Nancy Hamel, Dorothée Bouron-Dal Soglio, John R. Priest, William D. Foulkes. Mosaic RNase IIIb domain DICER1 mutations in children with multiple primary tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4934. doi:10.1158/1538-7445.AM2015-4934

Ovarian sex cord-stromal tumors in patients with probable or confirmed germline DICER1 mutations.

The DICER1 gene encodes an endoribonuclease involved in the production of mature microRNAs which regulates gene expression through several mechanisms. Recent studies have demonstrated somatic mutations in DICER1 in approximately 60% of ovarian Sertoli-Leydig cell tumors. Furthermore, patients with germline mutations in DICER1 are predisposed to developing a range of rare neoplasms including ovarian sex cord-stromal tumors most of which have been classified as Sertoli-Leydig cell tumor. However, the histologic features of these tumors have not been reported in detail. We describe the morphologic and immunophenotypic findings of 4 sex cord-stromal tumors arising in patients with proven or likely germline DICER1 mutations including 3 individuals from 1 family. Three tumors showed similar appearances characterized by marked architectural and cytologic heterogeneity including sertoliform, juvenile granulosa cell tumor-like, and unclassifiable elements. The remaining case mainly showed heterologous mucinous epithelial and neuroendocrine differentiation with only a minor intermediate-grade Sertoli cell component. This tumor and one of the 3 former cases arose in related patients with identical germline DICER1 mutations indicating that additional factors influence tumor morphology. All tumors were positive for steroidogenic factor-1 and FOXL2 on immunohistochemical analysis, whereas there was more variable expression of inhibin, calretinin, CD56, CD99, and hormone receptors. The present small series suggests that some ovarian Sertoli-Leydig cell tumor associated with germline DICER1 mutations may show distinctive histologic features in particular admixed Sertoli cell and juvenile granulosa cell tumor-like features. Larger studies are required to establish whether heterologous elements are also a more common feature of these tumors.

Abstract IA03: DICER1: From ontogenesis to oncogenesis

Abstract The DICER1 syndrome, also known as the pleuropulmonary blastoma familial tumor dysplasia syndrome (PPB-FTDS) (OMIM #601200), is a recently described entity comprising a number of rare to ultra-rare tumors arising mainly in childhood or adolescence. The most frequent and characteristic disorders are pleuropulmonary blastoma, cystic nephroma and ovarian Sertoli-Leydig cell tumors. Some aspects of the syndrome were identified in the 1970s and 1990s, but discovery in 2009 by Hill et al of heterozygous disease-associated germ-line DICER1 mutations in affected kindred brought the syndrome into focus. Several studies since 2009 have extended the phenotypes to include more common conditions such as multinodular goiter and Wilms tumor, as well as much rarer entities such as cervical embryonal rhabdomyosarcoma, pineoblastoma and pituitary blastoma. The critical molecular defect appears to be impairment of DICER1's RNase III endonuclease function, which normally would cleave precursor microRNAs to their final mature length. These microRNAs function by targeted silencing and/or degradation of specific messenger RNAs. DICER1 may be considered an unusual type of tumor suppressor gene, in that the first inherited “hit” usually cripples one allele completely, whereas a second somatic “hit” is nearly always limited to the RNase III domains (and is in fact often even more focused on the metal-binding domains of RNase IIIb). These second hits are most commonly a single base substitution leading to an amino acid change, which functionally impairs the protein without overall protein loss. In this presentation I will summarize the current knowledge on the role of DICER1 mutations in cancer and will describe the edges of the known associated phenotypes. Citation Format: William D. Foulkes, Mona Wu, Leanne De Kock, Leora Witkowski, Nelly Sabbaghian, Evan Weber, Nancy Hamel, John R. Priest. DICER1: From ontogenesis to oncogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA03. doi:10.1158/1538-7445.CANSUSC14-IA03

DICER1 mutations in a patient with an ovarian Sertoli-Leydig tumor, well-differentiated fetal adenocarcinoma of the lung, and familial multinodular goiter

DICER1 syndrome, a recently described tumor-predisposition syndrome, often involves multiple organs and is characterized by pleuropulmonary blastoma (PPB), cystic nephroma, ovarian Sertoli-Leydig tumors, familial multinodular goiter, etc. Germline DICER1 mutations have been identified in individuals with a variety of malignant conditions. However, in a review of the reported DICER1 syndrome cases that feature an unusual array of neoplastic and hyperplastic phenotypes, no mentions are made of these patients also presenting well-differentiated fetal adenocarcinoma of the lung.Here, we present a 16-year-old Chinese adolescent suffering from an ovarian Sertoli-Leydig cell tumor, well-differentiated fetal adenocarcinoma of the lung, and familial multinodular goiter with a nonsense mutation (c.3540C > A; p.Tyr1180*) in exon 21 of DICER1. This report presents the first case in which the clinical features of DICER1 syndrome appear in combination with well-differentiated fetal adenocarcinoma of the lung. We hypothesize that this case may suggest that well-differentiated fetal adenocarcinoma of the lung falls within the wide spectrum of manifestations of the DICER1 syndrome. Remarkably, this mutation is reported in a patient from The International PPB Registry.

Pleuropulmonary blastoma: a report on 350 central pathology-confirmed pleuropulmonary blastoma cases by the International Pleuropulmonary Blastoma Registry.

Pleuropulmonary blastoma (PPB) has 3 subtypes on a tumor progression pathway ranging from type I (cystic) to type II (cystic/solid) and type III (completely solid). A germline mutation in DICER1 is the genetic cause in the majority of PPB cases. Patients confirmed to have PPB by central pathology review were included, and their clinical characteristics and outcomes were reported. Germline DICER1 mutations were sought with Sanger sequencing. There were 435 cases, and a central review confirmed 350 cases to be PPB; 85 cases (20%) were another entity. Thirty-three percent of the 350 PPB cases were type I or type I regressed (type Ir), 35% were type II, and 32% were type III or type II/III. The median ages at diagnosis for type I, type II, and type III patients were 8, 35, and 41 months, respectively. The 5-year overall survival (OS) rate for type I/Ir patients was 91%; all deaths in this group were due to progression to type II or III. OS was significantly better for type II versus type III (P = .0061); the 5-year OS rates were 71% and 53%, respectively. Disease-free survival (DFS) was also significantly better for type II versus type III (P = .0002); the 5-year DFS rates were 59% and 37%, respectively. The PPB type was the strongest predictor of outcome. Metastatic disease at the diagnosis of types II and III was also an independent unfavorable prognostic factor. Sixty-six percent of the 97 patients tested had a heterozygous germline DICER1 mutation. In this subset, the DICER1 germline mutation status was not related to the outcome. Cystic type I/Ir PPB has a better prognosis than type II, and type II has a better outcome than type III. Surveillance of DICER1 carriers may allow the earlier detection of cystic PPB before its progression to type II or III PPB and thereby improve outcomes.

Nasal chondromesenchymal hamartomas arise secondary to germline and somatic mutations of DICER1 in the pleuropulmonary blastoma tumor predisposition disorder.

Nasal chondromesenchymal hamartoma (NCMH) is a rare nasal tumor that typically presents in young children. We previously reported on NCMH occurrence in children with pleuropulmonary blastoma (PPB), a rare pulmonary dysembryonic sarcoma that is the hallmark neoplasm in the PPB-associated DICER1 tumor predisposition disorder. Original pathologic materials from individuals with a PPB, PPB-associated tumor and/or a DICER1 mutation were centrally reviewed by the International PPB Registry. Paraffin-embedded NCMH tumor tissue was available in three cases. Laser-capture microdissection was used to isolate mesenchymal spindle cells and cartilage in one case for Sanger sequencing of DICER1. Nine patients (5F/4M) had PPB and NCMH. NCMH was diagnosed at a median age of 10 years (range 6-21 years). NCMH developed 4.5-13 years after PPB. Presenting NCMH symptoms included chronic sinusitis and nasal congestion. Five patients had bilateral tumors. Local NCMH recurrences required several surgical resections in two patients, but all nine patients were alive at 0-16 years of follow-up. Pathogenic germline DICER1 mutations were found in 6/8 NCMH patients tested. In 2 of the patients with germline DICER1 mutations, somatic DICER1 missense mutations were also identified in their NCMH (E1813D; n = 2). Three additional PPB patients developed other nasal lesions seen in the general population (a Schneiderian papilloma, chronic sinusitis with cysts, and allergic nasal polyps with eosinophils). Two of these patients had germline DICER1 mutations. Pathogenic germline and somatic mutations of DICER1 in NCMH establishes that the genetic etiology of NCMH is similar to PPB, despite the disparate biological potential of these neoplasms.

Germ-line and somatic DICER1 mutations in pineoblastoma.

Germ-line RB-1 mutations predispose to pineoblastoma (PinB), but other predisposing genetic factors are not well established. We recently identified a germ-line DICER1 mutation in a child with a PinB. This was accompanied by loss of heterozygosity (LOH) of the wild-type allele within the tumour. We set out to establish the prevalence of DICER1 mutations in an opportunistically ascertained series of PinBs. Twenty-one PinB cases were studied: Eighteen cases had not undergone previous testing for DICER1 mutations; three patients were known carriers of germ-line DICER1 mutations. The eighteen PinBs were sequenced by Sanger and/or Fluidigm-based next-generation sequencing to identify DICER1 mutations in blood gDNA and/or tumour gDNA. Testing for somatic DICER1 mutations was also conducted on one case with a known germ-line DICER1 mutation. From the eighteen PinBs, we identified four deleterious DICER1 mutations, three of which were germ line in origin, and one for which a germ line versus somatic origin could not be determined; in all four, the second allele was also inactivated leading to complete loss of DICER1 protein. No somatic DICER1 RNase IIIb mutations were identified. One PinB arising in a germ-line DICER1 mutation carrier was found to have LOH. This study suggests that germ-line DICER1 mutations make a clinically significant contribution to PinB, establishing DICER1 as an important susceptibility gene for PinB and demonstrates PinB to be a manifestation of a germ-line DICER1 mutation. The means by which the second allele is inactivated may differ from other DICER1-related tumours.

A novel DICER1 mutation identified in a female with ovarian Sertoli-Leydig cell tumor and multinodular goiter: a case report

IntroductionGerm-line mutations in the micro-ribonucleic acid processing gene DICER1 have been shown to predispose to a subset of benign tumors susceptible to malignant transformation, including ovarian Sertoli-Leydig cell tumor, nontoxic multinodular goiter, multilocular cystic nephroma and pleuropulmonary blastoma, which can occur in children and young adults. This may be due to reduced Dcr-1 homolog expression in carriers of germline mutations, which causes impairment of micro-ribonucleic acid processing and deregulates the growth and differentiation of target cells, leading to an increased risk of tumorigenesis. Many carriers of germ-line DICER1 mutations remain unaffected, but development of tumors within carriers is associated with varying prognoses.Case presentationDespite the Dcr-1 homolog syndrome phenotype being incompletely defined, a DICER1 mutation was suspected when a girl (case 1 patient) of Danish ethnicity presented with both an ovarian Sertoli-Leydig cell tumor and a multinodular goiter at the age of 13 years. In addition, family history included a male sibling (case 2 patient) who also had a multinodular goiter and had undergone a hemithyroidectomy at the age of 14 years. Subsequent DICER1 screening of the girl identified two novel mutations in exon 21 - a nonsense (c.3647C>A, p.Ser1216*) and a missense (c.3649T>A, p.Tyr1217Asn) mutation. The siblings had inherited the mutations from their father and paternal grandfather, which both currently were asymptomatic, indicating reduced penetrance of the nonsense mutation. Analysis of the parents revealed that the mutations were present in cis, making the contribution of the missense mutation less significant.ConclusionWe report a novel pathogenic DICER1 mutation (p.Ser1216*) in a Danish family associated with ovarian Sertoli-Leydig cell tumor and a multinodular goiter. A multinodular goiter was diagnosed in the siblings during childhood. Clinicians should be aware of a potential germ-line DICER1 mutation when evaluating multinodular goiter in young patients with or without a family history of thyroid diseases.

Exploring the Association BetweenDICER1Mutations and Differentiated Thyroid Carcinoma

Carriers of germline DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 syndrome. Thyroid abnormalities are a common finding in DICER1 syndrome with multinodular goiter frequently present in many families in which a germline DICER1 mutation is segregating. Differentiated thyroid carcinoma (DTC) is infrequently seen in such pedigrees. In addition to germline DICER1 mutations, specific somatic mutations have been identified in the DICER1 ribonuclease IIIb catalytic domain in several tumor types. We aimed to determine whether such characteristic somatic DICER1 mutations are present in DTCs that arise within germline DICER1 mutation carriers. The study involved an opportunistic collection of 3 cases of DTC arising in individuals suspected to have DICER1 syndrome and hospital-based ascertainment and testing was implemented. We identified somatic DICER1 mutations in 3 DTCs arising in unrelated germline DICER1 mutation carriers, all of whom had been diagnosed in infancy with pleuropulmonary blastoma (PPB), were treated with chemotherapy, exposed frequently to diagnostic radiation, and subsequently developed DTC. The somatic mutations occurred within the DICER1 ribonuclease IIIb domain, affecting highly conserved amino acid residues central to the catalytic activity of the domain. This report of somatic DICER1 mutations in DTC strengthens the association between DTC and the DICER1 syndrome. The possible association between germline DICER1 mutations, PPB treatment, and the risk of subsequent DTC must be considered by clinicians when treating PPB.

Serum levels of mature microRNAs in DICER1-mutated pleuropulmonary blastoma

DICER1 is a critical gene in the biogenesis of mature microRNAs, short non-coding RNAs that derive from either -3p or -5p precursor microRNA strands. Germline mutations of DICER1 are associated with a range of human malignancies, including pleuropulmonary blastoma (PPB). Additional somatic ‘hotspot' mutations in the microRNA processing ribonuclease IIIb (RNase IIIb) domain of DICER1 are reported in cancer, and which affect microRNA biogenesis, resulting in a -3p mature microRNA strand bias. Here, in a germline (exon11 c.1806_1810insATTGA) DICER1-mutated PPB, we first confirmed the presence of an additional somatic RNase IIIb hotspot mutation (exon25 c.5425G>A [p.G1809R]) by conventional sequencing. Second, we investigated serum levels of mature microRNAs at the time of PPB diagnosis, and compared the findings with serum results from a comprehensive range of pediatric cancer patients and controls (n=52). We identified a panel of 45 microRNAs that were present at elevated levels in the serum at the time of PPB diagnosis, with a significant majority noted be derived from the -3p strand (P=0.013). In addition, we identified a subset of 10 serum microRNAs (namely miR-125a-3p, miR-125b-2-3p, miR-380-5p, miR-125b-1-3p, let-7f-2-3p, let-7a-3p, let-7b-3p, miR-708-3p, miR-138-1-3p and miR-532-3p) that were most abundant in the PPB case. Serum levels of two representative microRNAs, miR-125a-3p and miR-125b-2-3p, were not elevated in DICER1 germline-mutated relatives. In the PPB case, serum levels of miR-125a-3p and miR-125b-2-3p increased before chemotherapy, and then showed an early reduction following treatment. These microRNAs may offer future utility as serum biomarkers for screening patients with known germline DICER1 mutations for early detection of PPB, and for potential disease-monitoring in cases with confirmed PPB.

Germline mutations of DICER1 in Chinese women with BRCA1/BRCA2-negative familial breast cancer.

Germline mutations in identified breast cancer susceptibility genes account for less than 20% of Chinese familial breast cancers. Dicer is an essential component of the microRNA-producing machinery; germline mutations of DICER1 have been confirmed in familial pleuropulmonary blastoma, ovarian sex cord-stromal tumors, and other cancers. Low expression of DICER1 is frequently detected in breast cancer. However, whether germline mutations of DICER1 occur in familial breast cancers remain unknown. Sixty-five breast cancer probands from BRCA1/BRCA2-negative Chinese breast cancer families were screened for germline mutations in DICER1. In addition, 100 unrelated healthy females were enrolled as controls. A polymerase chain reaction sequencing assay was used to screen for mutations in coding regions and at the exon-intron boundaries of DICER1. All variants in introns were evaluated using the NNSplice software to determine the potential splicing effect. A total of 12 germline variants were found, including 11 variants in introns and 1 variant in the 3'-non-coding region. Four variants (IVS8-205 C>T, IVS11+131 delGAAA, IVS16+42 delTA, and IVS19+160 T>C) were novel. Three variants (IVS11+105 C>T, IVS16+42 delTA, and 6095 T>A) may affect splice sites. None of the observed variants appeared to be disease-related, suggesting that germline mutations in DICER1 are rare or absent in familial breast cancer patients.

DICER1-pleuropulmonary blastoma familial tumor predisposition syndrome: a unique constellation of neoplastic conditions.

Germline mutations in DICER1 are associated with increased risk for a wide variety of neoplastic conditions, including pleuropulmonary blastoma (PPB), cystic nephroma, nasal chondromesenchymal hamartoma, ovarian Sertoli-Leydig cell tumors, botryoid embryonal rhabdomyosarcoma of the uterine cervix, ciliary body medulloepithelioma, pineoblastoma, pituitary blastoma and nodular thyroid hyperplasia or thyroid carcinoma. These tumors may be seen in isolation or in constellation with other characteristic tumor types in individuals or family members. Here we describe the medical history of a child with a heterozygous, loss of function germline DICER1 mutation and multiple tumors associated with the syndrome.. Although germline mutations in DICER1 are rare, tumors of these types will be seen by practicing pathologists and should prompt consideration of an underlying DICER1 mutation.

Germ-line deletion in DICER1 revealed by a novel MLPA assay using synthetic oligonucleotides

DICER1 is an endoribonuclease responsible for the production of mature microRNAs which are small, single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to mRNA and repressing the expression of target genes. Germ-line mutations in DICER1 are responsible for a rare cancer syndrome, including tumors that can co-occur with multinodular goiter (MNG). Using Sanger sequencing, we screened all DICER1 exons and intron boundaries in 20 suspected mutation carriers: nine with ovarian sex cord-stromal tumors (including Sertoli-Leydig cell tumors (SLCTs)), five with pleuropulmonary blastoma, one with cystic nephroma, one with nasal chondromesenchymal hamartoma and four with more than one manifestation suggestive of a germ-line DICER1 mutation. All were negative for any apparently deleterious variants. We developed a Multiplex Ligation-based Probe Amplification assay for DICER1 to screen for large deletions or duplications. Synthetic oligonucleotides were designed to cover all exons in three probe-mixes. In a child with a SLCT and MNG, and in her mother and brother (both diagnosed with MNG), we identified a heterozygous germ-line deletion of approximately 3 kilobases that eliminates exon 21 of DICER1 and two-thirds of intron 21, accompanied by an insertion of a G nucleotide at the 3' end of the deletion (c.3270-6_4051-1280delinsG). This allele is expressed in the patient's cDNA, creating an out-of-frame deletion predicted to result in a truncated protein (r.3270_4050del; p.Tyr1091Ser*28). Our novel finding of a disease-causing large deletion in DICER1 emphasizes the need to include assays that can detect rearrangements, duplications and deletions in any DICER1 screening protocol.

Variations of DICER1 sequence in Chinese women with BRCA1/BRCA2 negative familial breast cancer.

e12557 Background: Germline mutations in identified breast cancer susceptibility genes account for less than 20% of Chinese familial breast cancers. Dicer is an essential component of the microRNA producing machinery and germline mutations of DICER1 gene have been confirmed in familial pleuropulmonary blastoma, ovarian sex cord-stromal tumor and so on. Moreover, low expression of DICER1 is frequently detected in breast cancer. However, whether germline mutations of DICER1 occur in familial breast cancers remains unknown. Methods: 65 breast cancer probands from BRCA1/BRCA2 negative Chinese breast cancer families were used for screening of germline mutations in DICER1. In addition, 100 unrelated healthy females were enrolled as the controls. Polymerase chain reaction (PCR)-sequencing assay was employed to screen mutations in the entire coding regions and exon-intron boundaries of DICER1. Genotype and haplotype analyses were studied by widely available programs. Results: No deleterious mutations were identified in the 65 breast cancer probands. However, we found 12 germline variations and 4 of which were novel. In addition, the G allele frequency of rs2297730 (IVS12-91 A>G) was higher in familial breast cancer patients group than that in healthy controls group (OR = 1.873, 95% CI: 1.174-2.988, P = 0.008), and the A/G genotype were significantly associated with familial breast cancer (OR = 3.133; 95% CI: 1.562-6.287, P = 0.004). According to the minimum value of Akaike’s Information Criterion (AIC), the best genetic model was dominant. In addition, the haplotype GCGGAT was significantly associated with familial breast cancer (OR = 2.17, 95% CI: 1.20-3.91, P = 0.011). Conclusions: Although none of deleterious mutations were identified in the DICER1 gene, the current study provided a haplotype analysis of the DICER1 gene polymorphisms. We showed a significantly increased risk of BRCA1/BRCA2 negative familial breast cancer for DICER1 IVS12-91 A>G variant. It might be a potential risk marker for breast cancer in thus population.

Pleuropulmonary blastoma: The causative role of germ-line DICER1 mutations.

10024 Background: Pleuropulmonary blastoma (PPB) is a rare, aggressive childhood lung cancer that is often the first manifestation of the PPB-DICER1 familial tumor predisposition which includes other benign and malignant conditions. The initial genetic mutation is inherited by a germ-line loss of function of DICER1 gene which was discovered by Hill et al. It is proposed that loss of DICER1 expression alters miRNA regulation of key regulatory cellular mechanisms which promote tumor growth. PPB can progress from the purely cystic, curable Type I to a high-grade Type III having a dismal prognosis. We sought to determine the frequency of DICER1mutation in the largest cohort of PPB patients to date. Methods: We obtained germ-line DNA from saliva or blood samples from 113 PPB patients collected from 2005-2012. DNA was extracted using the Maxwell 16 Research System (Promega Corporation, Madison WI). Sample quality and quantity was checked using the Nanodrop 2000 (Thermo Scientific, Wilmington, DE). Sequencing was performed using the Sanger sequencer. For a subset of cases we used targeted sequencing services or full gene sequence analysis (Ambry Genetics, Aliso Viejo, CA). Results: Seventy-four (65.5%) PPB patients were found to have deleterious DICER1germ-line mutations. The most common mutation type found was small insertion/deletions. These 74 samples were composed of 7 (9.5%)Type Ir PPBs, 22 (29.7%)Type I PPBs, 24 (32.4%) Type II PPBs, and 21 (28.4%)Type III PPBs. The following Table shows a summary of mutation types identified. Conclusions: Our results confirm that DICER1 germline mutations are the most common genetic alterations in PPB making it critical for genetic testing to be performed at diagnosis. Additionally, this underscores the need for important correlative studies to describe the genotype-phenotype relationship in order for appropriate screening guidelines to be developed and implemented to allow for early detection. While a majority of cases are explained by germline DICER1 mutations using current sequencing methods, further investigation is warranted to elucidate other possible mechanisms in the development of PPB. [Table: see text]

Biallelic DICER1 mutations occur in Wilms tumours

DICER1 is an endoribonuclease central to the generation of microRNAs (miRNAs) and short interfering RNAs (siRNAs). Germline mutations in DICER1 have been associated with a pleiotropic tumour predisposition syndrome and Wilms tumour (WT) is a rare manifestation of this syndrome. Three WTs, each in a child with a deleterious germline DICER1 mutation, were screened for somatic DICER1 mutations and were found to bear specific mutations in either the RNase IIIa (n = 1) or the RNase IIIb domain (n = 2). In the two latter cases, we demonstrate that the germline and somatic DICER1 mutations were in trans, suggesting that the two-hit hypothesis of tumour formation applies for these examples of WT. Among 191 apparently sporadic WTs, we identified five different missense or deletion somatic DICER1 mutations (2.6%) in four individual WTs; one tumour had two very likely deleterious somatic mutations in trans in the RNase IIIb domain (c.5438A>G and c.5452G>A). In vitro studies of two somatic single-base substitutions (c.5429A>G and c.5438A>G) demonstrated exon 25 skipping from the transcript, a phenomenon not previously reported in DICER1. Further we show that DICER1 transcripts lacking exon 25 can be translated in vitro. This study has demonstrated that a subset of WTs exhibits two 'hits' in DICER1, suggesting that these mutations could be key events in the pathogenesis of these tumours.