Leanne de Kock

Exploring the Association Between DICER1 Mutations and Differentiated Thyroid Carcinoma

CONTEXT 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. OBJECTIVE We aimed to determine whether such characteristic somatic DICER1 mutations are present in DTCs that arise within germline DICER1 mutation carriers. DESIGN AND SETTING 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. RESULTS 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. CONCLUSION 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.

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.

Germ-line and somatic DICER1 mutations in a pleuropulmonary blastoma.

To the Editor: DICER1 is a non-coding small RNA processing enzyme that cleaves microRNA (miRNA) precursors into mature miRNAs, which regulate mRNA expression [1]. Germ-line DICER1 mutations are associated with the pleiotropic cancer predisposition syndrome known variously as pleuropulmonary blastoma (PPB)-familial tumor and dysplasia syndrome, and DICER1 syndrome [2–4]. This syndrome comprises several very rare entities, including PPB, cystic nephroma, Sertoli-Leydig cell tumors (SLCT), multinodular goiter, embryonal rhabdomyosarcomas (ERMS), and Wilms tumors (WT). Specific somatic mutations affecting the DICER1 RNase III domains (that affect metal ionbinding capacity) occur in trans with these germ-line mutations in persons diagnosed with SLCT, ERMS or WT [5–6]. These somatic mutations significantly reduce the production of miRNAs derived from the 5’arm (5p) of precursor miRNA, altering the function of these post-transcriptional regulators [7]. PPB is a malignant tumor of the lungs and pleura that presents in early childhood, sometimes accompanied by a family history of pediatric neoplasia that fall within the DICER1 syndrome. As stated, germ-lineDICER1mutations have been observed in patients harboring PPBs, but neither loss of heterozygosity, nor somatic mutations were reported [2,3]. The phenotypically normal presentation of obligate carriers in families in which a germ-line DICER1mutation is segregating suggests a second somatic “hit” is necessary to initiate tumor development. We now report a case of a child with a PPB that harbors two DICER1 mutations. A 3-year-old, previously healthy female of Polish descent (Fig. 1A) presented with acute dyspnea and leftsided chest pain. Chest computed tomography (CT) revealed a large heterogeneous mass occupying the majority of the left hemithorax, with mass effect shifting the cardiac and mediastinal structures. Pathology report of the initial biopsy detailed features of an embryonal rhabdomyosarcoma, but was later classified as a type II PPB, because a non-malignant cyst lined by respiratory epithelium was identified in the resection specimen. At 13 years old, the patient is now cancer-free. A deleterious germ-line mutation, c.4555delG, was observed in exon 23 of DICER1 (Ambry Genetics, Aliso Viejo, CA). This encodes a protein in which the RNase IIIa domain is preserved, but both the RNase IIIb and double-stranded RNA-binding (dsRBD) domains are lost. We subsequently identified an acquired somatic mutation, c.5438A>G, predicted to result in p.Glu1813Gly at the protein level, in the formalin-fixed paraffin-embedded (FFPE) PPB (Fig. 1B). Sequencing of cDNA synthesized from tumor RNA confirmed the expression of this mutation (Fig. 1C). It affects a highly conserved amino acid residuewithin the RNase IIIb catalytic domain, and is predicted to be damaging by both SIFT and Polyphen2 with scores of 0 and 1 respectively. The essential role of DICER1 in normal lung development is well-characterized. The acquisition of a second “hit” in the lung tissue is therefore strongly suggestive of a key mutational event capable of initiating tumorigenesis. Clinically, the potentially fatal nature of PPB argues that screening of DICER1 mutation carriers should be offered during the years of susceptibility. Identification of all at-risk children is therefore an important part of care of families of children diagnosed with PPB.

DICER1 mutations in an adolescent with cervical embryonal rhabdomyosarcoma (cERMS)

To the Editor: Embryonal rhabdomyosarcomas of the cervix (cERMS) (“cervical sarcoma botryoides”) are rare embryonal tumors, usually diagnosed in childhood or adolescence, with a world literature of uterine/cervical ERMS limited to 115 cases [1]. The association of cERMSwith germlineDICER1mutations was first established in three families by Foulkes in 2011, and subsequently reported by Dehner et al., although the coexistence of cERMS and ovarian Sertoli-Leydig cell tumors (OSLCT) had previously been recognized [2–4]. In 2012, Doros et al. [5] reported germline DICER1 mutations in 2/52 (3.8%) sporadic ERMS, including 7 uterine/vaginal/pelvic cases.

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

Background Somatic 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 in DICER1 in two children with overgrowth and developmental delay as well as more typical phenotypes of germline DICER1 mutation. However, very-low-frequency mosaicism is difficult to detect, and thus, causal mutations can go unnoticed. Highly sensitive, cost-effective approaches are needed to molecularly diagnose these persons. We studied four children with multiple primary tumours known to be associated with the DICER1 syndrome, but in whom germline DICER1 mutations were not detected by conventional mutation detection techniques. Methods and results We observed the same missense mutation within the DICER1 RNase IIIb domain in multiple tumours from different sites in each patient, raising suspicion of somatic mosaicism. We implemented three different targeted-capture technologies, including the novel HaloPlexHS (Agilent Technologies), followed by deep sequencing, and confirmed that the identified mutations are mosaic in origin in three patients, detectable in 0.24–31% of sequencing reads in constitutional DNA. The mosaic origin of patient 4s mutation remains to be unequivocally established. We also discovered likely pathogenic second somatic mutations or loss of heterozygosity (LOH) in tumours from all four patients. Conclusions Mosaic DICER1 mutations are an important cause of the DICER1 syndrome in patients with severe phenotypes and often appear to be accompanied by second somatic truncating mutations or LOH in the associated tumours. Furthermore, the molecular barcode-containing HaloPlexHS provides the sensitivity required for detection of such low-level mosaic mutations and could have general applicability.

DICER1 Mutations Are Consistently Present in Moderately and Poorly Differentiated Sertoli-Leydig Cell Tumors

Ovarian Sertoli-Leydig cell tumors (SLCTs) are uncommon sex cord-stromal tumors associated with both germ-line and somatic DICER1 mutations, the frequency of which has varied widely in different studies (0% to 62.5%). The current World Health Organization Classification includes 3 histologic types of SLCTs (well-differentiated, moderately differentiated, and poorly differentiated); heterologous elements and/or retiform patterns may be present in moderately and poorly differentiated neoplasms. We investigated the frequency of DICER1 mutations in a series of 38 ovarian tumors initially diagnosed as SLCTs, and explored whether identified mutations were associated with specific morphologic features. Specialist pathology review performed blinded to molecular results confirmed 34 tumors to be SLCTs (22 moderately differentiated, 8 poorly differentiated; 4 well-differentiated), while the remaining 4 neoplasms were considered not to represent SLCTs. Of the 34 cases diagnosed as SLCTs, 30 (88%) harbored ≥1 DICER1 mutation. All 30 moderately differentiated/poorly differentiated SLCTs contained mutations, but we did not find deleterious DICER1 mutations in the 4 well-differentiated SLCTs. Our study reports the highest DICER1 mutation frequency to date in SLCTs, with 100% of moderately differentiated and poorly differentiated tumors being DICER1-mutated. This suggests that DICER1 mutation may be a defining feature of these neoplasms. Although the number of cases is limited, well-differentiated SLCTs appear to be DICER1-independent. Moderately differentiated and poorly differentiated SLCT components often coexist with each other and form part of a spectrum, while well-differentiated SLCTs usually occur in pure form, suggesting that fundamentally, these represent 2 separate and independent tumor types with a different pathogenesis. We suggest that all patients with ovarian SLCTs undergo germ-line DICER1 mutation testing.

Uterine Tumor Resembling Ovarian Sex Cord Tumor (UTROSCT) Commonly Exhibits Positivity With Sex Cord Markers FOXL2 and SF-1 but Lacks FOXL2 and DICER1 Mutations.

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare neoplasm which morphologically and immunohistochemically exhibits overlap with an ovarian sex cord tumor. Although many of these neoplasms are positive with markers of ovarian sex cord-stromal tumors, staining is often limited and the pathogenesis of UTROSCT is unknown. To further explore the sex cord lineage of UTROSCT, we studied 19 of these neoplasms and examined the expression of 2 recently described markers of ovarian sex cord-stromal tumors, FOXL2, and steroidogenic factor-1. We also undertook FOXL2 and DICER1 mutation analysis in these cases; a somatic missense mutation in codon C134W (402C→G) of FOXL2 gene has been demonstrated in the vast majority (>95%) of ovarian adult granulosa cell tumors and somatic DICER1 mutations are found in approximately 60% of ovarian Sertoli-Leydig cell tumors. Ten of 19 cases (53%) exhibited nuclear immunoreactivity with FOXL2 and 11 of 19 (58%) exhibited nuclear staining with steroidogenic factor-1. Neither FOXL2 nor DICER1 mutations were identified in any case where there was sufficient tumor tissue for analysis (18 and 9 cases, respectively). Despite exhibiting an immunophenotype characteristic of a sex cord-stromal tumor, mutations in FOXL2 and DICER1, the 2 most common mutations hitherto reported in ovarian sex cord-stromal tumors, are not a feature of UTROSCT.

Somatic DICER1 mutations in adult-onset pulmonary blastoma

Several rare lung tumours morphologically mimic embryonal structures of the developing human lung. Historically, these blastomatous tumours were described under the umbrella term of pulmonary blastoma. Subsequently, distinct entities were recognised, such as childhood pleuropulmonary blastoma (PPB) [1] (International Classification of Diseases for Oncology (ICD-O-3) code 8973/3). Later, adult-onset pulmonary blastoma was separated into well-differentiated fetal adenocarcinoma (WDFA) (ICD 8333/3) and pulmonary blastoma (ICD 8972/3) [2]. Pulmonary blastoma is a biphasic epithelial and mesenchymal malignancy, whereas PPB is purely sarcomatous and WDFA is characterised by a monophasic immature epithelium. Since 1988, the childhood PPB has received particular attention because of 1) its unique developmental progression from relatively indolent neonatal-onset lung cysts, to aggressive cystic-solid and solid sarcomas by age 72 months; 2) PPBs status heralding a newly recognised familial tumour predisposition syndrome and 3) its strong association with both germ-line and somatic DICER1 mutations, which is not only true for PPB, but also for many other tumours in pleiotropic predisposition syndrome (now referred to as DICER1 syndrome). Until recently, neither WDFA nor pulmonary blastoma had been observed in families manifesting DICER1 syndrome [3]. However, in 2015, we identified a second somatic DICER1 RNase IIIb mutation [4] in a WDFA that arose in a 16-year-old germ-line DICER1 mutation carrier [5]. In addition to DICER1 mutations in PPB and WDFA, somatic CTNNB1 mutations (encoding β-catenin) appear to characterise WDFA and pulmonary blastoma [6], and are far less frequent in PPB [7, 8]. In contrast, TP53 mutations are found in both PPB [8] and pulmonary blastoma [9], but not WDFA [9]. We identified two somatic DICER1 mutations in each of two adult pulmonary blastomas, implicating DICER1 in causation http://ow.ly/10aM9V

Infantile Pulmonary Teratoid Tumor

Infantile Pulmonary Teratoid Tumor Pleuropulmonary blastoma is a childhood tumor related to DICER mutations. The authors identify a new cancer, infantile pulmonary teratoid tumor, that has some his...

A case of neuroblastoma in DICER1 syndrome: Chance finding or noncanonical causation?

DICER1 syndrome is an inherited disorder associated with at least a dozen rare, mainly pediatric‐onset tumors. Its characterization remains incomplete. Some studies suggested that neuroblastoma (NB) may be involved in this syndrome. Here, we describe the case of a 14‐year‐old female presenting with a multinodular goiter (MNG) and a collision tumor composed of NB and cystic nephroma (CN). She is a carrier of a deleterious germline mutation in exon 23 of DICER1 and harbored different somatic mutations in the CN and MNG. However, no second hit was found in the NB, questioning its status as a DICER1‐related tumor.