Barbara Rivera

Germline and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.

Germline RECQL mutations are associated with breast cancer susceptibility

Several moderate- and high-risk breast cancer susceptibility genes have been discovered, but more are likely to exist. To discover new breast cancer susceptibility genes, we used 2 populations (from Poland and Quebec, Canada) and applied whole-exome sequencing in a discovery phase (n = 195), followed by validation. We identified rare recurrent RECQL mutations in each population. In Quebec, 7 of 1,013 higher-risk breast cancer cases and 1 of 7,136 newborns carried the c.643C>T (p.Arg215*) variant (P = 0.00004). In Poland, 30 of 13,136 unselected breast cancer cases and 2 of 4,702 controls carried the c.1667_1667+3delAGTA (p.K555delinsMYKLIHYSFR) variant (P = 0.008). RECQL is implicated in resolving stalled DNA replication forks to prevent double-stranded DNA (dsDNA) breaks. This function is related to that of other known breast cancer susceptibility genes, many of which are involved in repairing dsDNA breaks. We conclude that RECQL is a breast cancer susceptibility gene.

Biallelic NTHL1 Mutations in a Woman with Multiple Primary Tumors

A patient is described with multiple cancers and compound heterozygous mutations in NTHL1, a recently described polyposis gene. The involvement of a second causative mutation is reported.

Age at Onset Should Be a Major Criterion for Subclassification of Colorectal Cancer

An important proportion of early-onset colorectal cancer (CRC) does not show a hereditary component with limited knowledge about its molecular basis and features. We analyzed a subset of patients with early-onset CRC and compared them with patients with late-onset CRC. We analyzed the microsatellite instability and CpG island methylator phenotype (CIMP) in both populations and classified them into four molecular subtypes. We analyzed the differential features between groups. Only 12 of 81 early-onset cases (15%) showed microsatellite instability, 10 of which (83%) were Lynch syndrome cases; microsatellite instability cases in elderly patients were sporadic. Early-onset microsatellite-stable cases showed different tumor locations and more family history of cancer than the elderly. Microsatellite instability/CIMP-high early-onset CRC was associated with Lynch syndrome, whereas the elderly cases were associated with BRAF mutations. Early-onset microsatellite-stable/CIMP-high CRCs were more frequently mucinous and right sided than elderly cases, with a high incidence of Lynch syndrome neoplasms; early-onset microsatellite stable/CIMP-low/0 differed from elderly cases in location, stages, incidence of multiple primary neoplasms, and the familial component. The clinical and familial differences observed between early- and late-onset CRC when considering the different carcinogenetic pathways underline that the age at onset criterion should be considered when classifying CRC.

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.

Multiple DICER1-related tumors in a child with a large interstitial 14q32 deletion

Germ‐line interstitial deletions involving the 14q32 chromosomal region, resulting in 14q32 deletion syndrome, are rare. DICER1 is a recently described cancer‐predisposition gene located at 14q32.13. We report the case of a male child with a ∼5.8 Mbp 14q32.13q32.2 germ‐line deletion, which included the full DICER1 locus. We reviewed available clinical and pathological material, and conducted genetic analyses. In addition to having congenital dysmorphic features, the child developed multiple DICER1 syndrome‐related tumors before age 5 y: a pediatric cystic nephroma (pCN), a ciliary body medulloepithelioma (CBME), and a small lung cyst (consistent with occult pleuropulmonary blastoma Type I/Ir cysts seen in DICER1 mutation carriers). He also developed a cerebral spindle‐cell sarcoma with myogenous differentiation. Our investigations revealed that the deletion encompassed 31 protein‐coding genes. In addition to the germ‐line DICER1 deletion, somatic DICER1 RNase IIIb mutations were found in the CBME (c.5437G > A, p.E1813K), pCN (c.5425G > A, p.G1809R), and sarcoma (c.5125G > A, p.D1709N). The sarcoma also harbored a somatic TP53 mutation: c.844C > T, p.R282W. Additional copy number alterations were identified in the CBME and sarcoma using an OncoScan array. Among the 8 cases with molecularly‐defined 14q32 deletions involving DICER1 and for whom phenotypic information is available, our patient and one other developed DICER1‐related tumors. Biallelic DICER1 mutations have not previously been reported to cause cerebral sarcoma, which now may be considered a rare manifestation of the DICER1 syndrome. Our study shows that DICER1‐related tumors can occur in children with 14q32 deletions and suggests surveillance for such tumors may be warranted.

Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma

RAD51D is a key player in DNA repair by homologous recombination (HR), and RAD51D truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating RAD51D variants to cancer predisposition remains uncertain. Using deep sequencing and case-control genotyping studies, we show that in French Canadians, the missense RAD51D variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D-XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D-XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic RAD51D missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense RAD51D variants. Cancer Res; 77(16); 4517-29. ©2017 AACR.

Sequencing of DICER1 in sarcomas identifies biallelic somatic DICER1 mutations in an adult-onset embryonal rhabdomyosarcoma

Background:Sarcomas are rare and heterogeneous cancers. We assessed the contribution of DICER1 mutations to sarcoma development.Methods:The coding region of DICER1 was sequenced in 67 sarcomas using a custom Fluidigm Access Array. The RNase III domains were Sanger sequenced in six additional sarcomas to identify hotspot DICER1 variants.Results:The median age of sarcoma diagnosis was 45.7 years (range: 3 months to 87.4 years). A recurrent embryonal rhabdomyosarcoma (ERMS) of the broad ligament, first diagnosed at age 23 years, harboured biallelic pathogenic somatic DICER1 variants (1 truncating and 1 RNase IIIb missense). We identified nine other DICER1 variants. One somatic variant (p.L1070V) identified in a pleomorphic sarcoma and one germline variant (c.2257-7A>G) may be pathogenic, but the others are considered to be benign.Conclusions:We show that deleterious DICER1 mutations underlie the genetic basis of only a small fraction of sarcomas, in particular ERMS of the urogenital tract.

Genome-wide Association Study: Not so benign

Susceptibility to uterine fibroids, benign tumors that affect the health of many women, is linked to genes that are responsible for preserving genome integrity and promoting genitourinary development.

Revisiting pleuropulmonary blastoma and atypical choroid plexus papilloma in a young child: DICER1 syndrome or not?

To the editor: In 2016, Liu et al. reported a child who developed Type I pleuropulmonary blastoma (PPB) at age 1 year and later developed atypical choroid plexus papilloma (aCPP).1 A germline pathogenic variant (PV) in DICER1 was not identified. Because PPB is the sentinel disease of DICER1 syndrome, a childhood tumor predisposition syndromewhose phenotypic range continues to evolve, we set out to confirm absence of a germline PV and explore alternative abnormalities in DICER1 to explain the tumors in this child. A Fluidigm array covering the entire DICER1 coding sequence and exon boundarieswas performed inDNAderived fromperipheral blood mononuclear cells (PBMC) from the patient and parents, and multiplex ligation-dependent probe amplification to investigate large gene rearrangements was performed.2 Both assays gave negative results. Sanger sequencing of PPB tumor tissue revealed two mutations in DICER1: a hotspot mutation in exon 25 (c.5438A >G, p.[E1813G]) and a frameshift mutation in exon 15 (c.2392_2393insA, p.(T798Nfs*33)). Investigating the possible relationship between the patients aCPP and the PPB, we performed Sanger sequencing of the patients aCPP; no DICER1 variants were found. Because mosaicism is described in patients with DICER1 syndrome,3,4 we performed digital droplet polymerase chain reaction in PBMC-derived DNA, aCPP, PPB, and normal lung from the patient. Six blood DNAs from healthy individuals were used as controls. The absence of the mutations in the patients aCPP confirms that mosaicism for aDICER1 PV as a cause of the two tumors is ruled out (Figures 1A and 1B) and (SupplementaryMaterial S1). To determine the phase of the two mutations found and to investigate whether any full-length DICER1 protein is likely to be present in the lung tumor, we performed a cloning experiment in the cDNA from the patients PPB (methods described in Supplementary Material S1). Twenty-three of the 24 clones had only the c.5438A > G, p.E1813G variant, likely because the c.2392_2393insA, p.T798Nfs*33 allele is subject to nonsense-mediated decay. We conclude that the two variants are in trans (Figure 1C). To investigate other plausible genetic causes, whole exome sequencing (WES) was done on gDNA from the patient, his parents, and aCPP tumor tissue.We analyzedWESdata focusing on 114 known cancer susceptibility genes5 (our analysis did not include copy number variants; methods are described in Supplementary Material S1). The only PV found by WES was c.263dupT, p.(I88*fs) in SBDS, present in both the patient and his father. However, SBDS is not a plausible cause of the two phenotypes.6 The cause of the patients aCPP remains enigmatic. We conclude that: (a) the PPB results from somatically acquired, tumor-restricted mutations in DICER1, a mechanism previously reported,4 and (b) the patients aCPP is genetically unrelated to his PPB. Based on current understandings of DICER1 disease, we believe that the child has no heritable defect in DICER1 and no generalized susceptibility to DICER1 phenotypes. The child does not have DICER1 syndrome. Further, aCPP is not a phenotype of DICER1 syndrome. The childs family appears not to be at increased cancer risk. When traditionalmolecularmechanismsofDICER1disease arenotpresent, intensive investigations can be of great benefit to the patient and family.