The pathogenesis of cystic nephroma of the kidney has interested pathologists for over 50 years. Emerging from its initial designation as a type of unilateral multilocular cyst, cystic nephroma has been considered as either a developmental abnormality or a neoplasm or both. Many have viewed cystic nephroma as the benign end of the pathologic spectrum with cystic partially differentiated nephroblastoma and Wilms tumor, whereas others have considered it a mixed epithelial and stromal tumor. We hypothesize that cystic nephroma, like the pleuropulmonary blastoma in the lung, represents a spectrum of abnormal renal organogenesis with risk for malignant transformation. Here we studied DICER1 mutations in a cohort of 20 cystic nephromas and 6 cystic partially differentiated nephroblastomas, selected independently of a familial association with pleuropulmonary blastoma and describe four cases of sarcoma arising in cystic nephroma, which have a similarity to the solid areas of type II or III pleuropulmonary blastoma. The genetic analyses presented here confirm that DICER1 mutations are the major genetic event in the development of cystic nephroma. Further, cystic nephroma and pleuropulmonary blastoma have similar DICER1 loss of function and 'hotspot' missense mutation rates, which involve specific amino acids in the RNase IIIb domain. We propose an alternative pathway with the genetic pathogenesis of cystic nephroma and DICER1-renal sarcoma paralleling that of type I to type II/III malignant progression of pleuropulmonary blastoma.

Poorly differentiated thyroid carcinomas (PDTC) in young individuals are rare and their clinical and histopathologic features, genetic mechanisms, and outcomes remain largely unknown. Here, we report a detailed characterization of a series of six PDTC in patients ≤21 years old defined by Turin diagnostic criteria studied for mutations and gene fusions characteristic of thyroid cancer using targeted next-generation sequencing (NGS) and whole-exome sequencing (WES). All tumors had solid, insular, or trabecular growth pattern and high mitotic rate, and five out of six tumors showed tumor necrosis. Targeted NGS assay identified somatic mutations in the DICER1 gene in five of six (83%) tumors, all of which were "hotspot" mutations encoding the metal-ion binding sites of the RNase IIIb domain of DICER1. WES was performed in five cases which confirmed all hotspot mutations and detected two tumors with additional inactivating DICER1 alterations. Of these two, one was a germline pathogenic DICER1 variant and the other had loss of heterozygosity for DICER1. No other mutations or gene fusions characteristic of adult well-differentiated thyroid cancer and PDTC (BRAF, RAS, TERT, RET/PTC, and other) were detected. On follow-up, available for five patients, three patients died of disease 8-24 months after diagnosis, whereas two were alive with no disease. The results of our study demonstrate that childhood- and adolescent-onset PDTC are genetically distinct from adult-onset PDTC in that they are strongly associated with DICER1 mutations and may herald DICER1 syndrome in a minority. As such, all young persons with PDTC may benefit from genetic counseling. Furthermore, their clinically aggressive behavior contrasts sharply with the indolent nature of the great majority of thyroid tumors with DICER1 mutations reported to date.

Genetic disorders are historically defined through phenotype-first approaches. However, risk estimates derived from phenotype-linked ascertainment may overestimate severity and penetrance. Pathogenic variants in DICER1 are associated with increased risks of rare and common neoplasms and thyroid disease in adults and children. This study explored how effectively a genome-first approach could characterize the clinical traits associated with germline DICER1 putative loss-of-function (pLOF) variants in an unselected clinical cohort.

The risk of thyroid cancer and multinodular goiter (MNG) in DICER1 syndrome, a rare tumor-predisposition disorder, is unknown.

Variants of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including alpha, beta, gamma, delta, and omicron, threaten to prolong the pandemic, leading to more global morbidity and mortality. Genome sequencing is the mainstay of tracking the evolution of the virus, but is costly, slow, and not easily accessible. Multiplex quantitative RT-PCR assays for SARS-CoV-2 have been developed that identify all VOCs as well as other mutations of interest in the viral genome, nine mutations in total, using single-nucleotide discriminating molecular beacons. The presented variant molecular beacon assays showed a limit of detection of 50 copies of viral RNA, with 100% specificity. Twenty-six SARS-CoV-2-positive patient samples were blinded and tested using a two-tube assay. When testing patient samples, the assay was in full agreement with results from deep sequencing with a sensitivity and specificity of 100% (26 of 26). We have used our design methodology to rapidly design an assay that detects the new omicron variant. This omicron assay was used to accurately identify this variant in 17 of 33 additional patient samples. These quantitative RT-PCR assays identify all currently circulating VOCs of SARS-CoV-2, as well as other important mutations in the spike protein coding sequence. These assays can be easily implemented on broadly available five-color thermal cyclers and will help track the spread of these variants.

Germline pathogenic variation in DICER1 underlies a tumor-predisposition disorder with increased risk for cervical embryonal rhabdomyosarcoma and ovarian sex-cord stromal tumors, particularly Sertoli-Leydig cell tumors. The gynecologic and reproductive health of these females has not yet been described.

Pleuropulmonary Blastoma (PPB) is the primary neoplastic manifestation of a pediatric cancer predisposition syndrome that is associated with several diseases including cystic nephroma, Wilms tumor, neuroblastoma, rhabdomyosarcoma, medulloblastoma, and ovarian Sertoli-Leydig cell tumor. The primary pathology of PPB, epithelial cysts with stromal hyperplasia and risk for progression to a complex primitive sarcoma, is associated with familial heterozygosity and lesion-associated epithelial loss-of-heterozygosity of DICER1. It has been hypothesized that loss of heterozygosity of DICER1 in lung epithelium is a non-cell autonomous etiology of PPB and a critical pathway that regulates lung development; however, there are no known direct targets of epithelial microRNAs (miRNAs) in the lung. Fibroblast Growth Factor 9 (FGF9) is expressed in the mesothelium and epithelium during lung development and primarily functions to regulate lung mesenchyme; however, there are no known mechanisms that regulate FGF9 expression during lung development. Using mouse genetics and molecular phenotyping of human PPB tissue, we show that FGF9 is overexpressed in lung epithelium in the initial multicystic stage of Type I PPB and that in mice lacking epithelial Dicer1, or induced to overexpress epithelial Fgf9, increased Fgf9 expression results in pulmonary mesenchymal hyperplasia and a multicystic architecture that is histologically and molecularly indistinguishable from Type I PPB. We further show that miR-140 is expressed in lung epithelium, regulates epithelial Fgf9 expression, and regulates pseudoglandular stages of lung development. These studies identify an essential miRNA-FGF9 pathway for lung development and a non-cell autonomous signaling mechanism that contributes to the mesenchymal hyperplasia that is characteristic of Type I PPB.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.

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.

We report two malignant sacrococcygeal tumors in infants that were associated with pathogenic DICER1 variation. These tumors were composed of primitive neuroepithelium, embryonal rhabdomyosarcoma, and cartilage and initially diagnosed as immature teratomas. One child developed intracranial metastasis and died. The second child underwent surgery and chemotherapy and achieved complete remission. This child subsequently developed five additional DICER1-associated neoplasms by age nine. Genetic analysis revealed that both tumors harbored biallelic pathogenic DICER1 variation. We believe these cases represent another novel subtype of DICER1-associated tumor. This new entity, which we propose to call DICER1-associated presacral malignant teratoid neoplasm, may be difficult initially to distinguish from immature teratoma, but recognizing it as an entity can prompt appropriate classification as an aggressive malignancy and facilitate appropriate genetic counseling, DICER1 germline variant testing, screening, and education.

Data on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence in the United States are still emerging.