An imbalance between proliferation and differentiation underlies the development of microRNA-defective pineoblastoma.

Mutations in the microRNA processing genes DICER1 and DROSHA drive several cancers that resemble embryonic progenitors. To understand how microRNAs regulate tumorigenesis, we ablated Drosha or Dicer1 in the developing pineal gland to emulate the pathogenesis of pineoblastoma, a brain tumor that resembles undifferentiated precursors of the pineal gland. Accordingly, these mice develop pineal tumors marked by loss of microRNAs, including the let-7/miR-98-5p family, and de-repression of microRNA target genes. Pineal tumors driven by loss of Drosha or Dicer1 mimic tumors driven by Rb1 loss, as they exhibit upregulation of S-phase genes and homeobox transcription factors that regulate pineal development. Blocking proliferation of these tumors facilitates expression of pinealocyte maturation markers, with a concomitant reduction in embryonic markers. Select embryonic markers remain elevated, however, as the microRNAs that normally repress these target genes remain absent. One such microRNA target gene is the oncofetal transcription factor Plagl2 , which regulates expression of pro-growth genes, and inhibiting their signaling impairs tumor growth. Thus, we demonstrate that tumors driven by loss of microRNA processing may be therapeutically targeted by inhibiting downstream drivers of proliferation.

DROSHA regulates mesenchymal gene expression in Wilms tumor.

Wilms tumor, the most common pediatric kidney cancer, resembles embryonic renal progenitors. Currently, there are no ways to therapeutically target Wilms tumor driver mutations, such as in the microRNA processing gene DROSHA. Here we used a "multi-omics" approach to define the effects of DROSHA mutation in Wilms tumor. We categorized Wilms tumor mutations into four mutational subclasses with unique transcriptional effects: microRNA processing, MYCN activation, chromatin remodeling, and kidney developmental factors. In particular, we find that DROSHA mutations are correlated with de-repressing microRNA target genes that regulate differentiation and proliferation and a self-renewing, mesenchymal state. We model these findings by inhibiting DROSHA expression in a Wilms tumor cell line, which led to upregulation of the cell cycle regulator cyclin D2 (CCND2). Furthermore, we observed that DROSHA mutations in Wilms tumor and DROSHA silencing in vitro were associated with a mesenchymal state with aberrations in redox metabolism. Accordingly, we demonstrate that Wilms tumor cells lacking microRNAs are sensitized to ferroptotic cell death through inhibition of glutathione peroxidase 4 (GPX4), the enzyme that detoxifies lipid peroxides. Implications: This study reveals genotype-transcriptome relationships in Wilms tumor and points to ferroptosis as a potentially therapeutic vulnerability in one subset of Wilms tumor.

Outcomes in ovarian Sertoli-Leydig cell tumor: A report from the International Pleuropulmonary Blastoma/DICER1 and Ovarian and Testicular Stromal Tumor Registries.

OBJECTIVE: Sertoli-Leydig cell tumors (SLCTs) are rare sex cord-stromal tumors, representing <0.5% of all ovarian tumors. We sought to describe prognostic factors, treatment and outcomes for individuals with ovarian SLCT. METHODS: Individuals with SLCT were enrolled in the International Pleuropulmonary Blastoma/DICER1 Registry and/or the International Ovarian and Testicular Stromal Tumor Registry. Medical records were systematically abstracted, and pathology was centrally reviewed when available. RESULTS: In total, 191 participants with ovarian SLCT enrolled, with most (92%, 175/191) presenting with FIGO stage I disease. Germline DICER1 results were available for 156 patients; of these 58% had a pathogenic or likely pathogenic germline variant. Somatic (tumor) DICER1 testing showed RNase IIIb hotspot variants in 97% (88/91) of intermediately and poorly differentiated tumors. Adjuvant chemotherapy was administered in 40% (77/191) of cases, and among these, nearly all patients received platinum-based regimens (95%, 73/77), and 30% (23/77) received regimens that included an alkylating agent. Three-year recurrence-free survival for patients with stage IA tumors was 93.6% (95% CI: 88.2-99.3%) compared to 67.1% (95% CI: 55.2-81.6%) for all stage IC and 60.6% (95% CI: 40.3-91.0%) for stage II-IV (p < .001) tumors. Among patients with FIGO stage I tumors, those with mesenchymal heterologous elements treated with surgery alone were at higher risk for recurrence (HR: 74.18, 95% CI: 17.99-305.85). CONCLUSION: Most individuals with SLCT fare well, though specific risk factors such as mesenchymal heterologous elements are associated with poor prognosis. We also highlight the role of DICER1 surveillance in early detection of SLCT, facilitating stage IA resection.

Specifications of the ACMG/AMP Variant Classification Guidelines for Germline DICER1 Variant Curation.

Germline pathogenic variants in DICER1 predispose individuals to develop a variety of benign and malignant tumors. Accurate variant curation and classification is essential for reliable diagnosis of DICER1-related tumor predisposition and identification of individuals who may benefit from surveillance. Since 2015, most labs have followed the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) sequence variant classification guidelines for DICER1 germline variant curation. However, these general guidelines lack gene-specific nuances and leave room for subjectivity. Consequently, a group of DICER1 experts joined ClinGen to form the DICER1 and miRNA-Processing Genes Variant Curation Expert Panel (VCEP), to create DICER1- specific ACMG/AMP guidelines for germline variant curation. The VCEP followed the FDA-approved ClinGen protocol for adapting and piloting these guidelines. A diverse set of 40 DICER1 variants were selected for piloting, including 14 known Pathogenic/Likely Pathogenic (P/LP) variants, 12 known Benign/Likely Benign (B/LB) variants, and 14 variants classified as variants of uncertain significance (VUS) or with conflicting interpretations in ClinVar. Clinically meaningful classifications (i.e., P, LP, LB, or B) were achieved for 82.5% (33/40) of the pilot variants, with 100% concordance among the known P/LP and known B/LB variants. Half of the VUS or conflicting variants were resolved with four variants classified as LB and three as LP. These results demonstrate that the DICER1-specific guidelines for germline variant curation effectively classify known pathogenic and benign variants while reducing the frequency of uncertain classifications. Individuals and labs curating DICER1 variants should consider adopting this classification framework to encourage consistency and improve objectivity.

Intronic Germline DICER1 Variants in Patients With Sertoli-Leydig Cell Tumor.

Germline pathogenic loss-of-function (pLOF) variants in DICER1 are associated with a predisposition for a variety of solid neoplasms, including pleuropulmonary blastoma and Sertoli-Leydig cell tumor (SLCT). The most common DICER1 pLOF variants include small insertions or deletions leading to frameshifts, and base substitutions leading to nonsense codons or altered splice sites. Larger deletions and pathogenic missense variants occur less frequently. Identifying these variants can trigger surveillance algorithms with potential for early detection of DICER1-related cancers and cascade testing of family members. However, some patients with DICER1-associated tumors have no pLOF variants detected by germline or tumor testing. Here, we present two patients with SLCT whose tumor sequencing showed only a somatic missense DICER1 RNase IIIb variant. Conventional exon-directed germline sequencing revealed no pLOF variants. Using a custom capture panel, we discovered novel intronic variants, ENST00000343455.7: c.1752+213A>G and c.1509+16A>G, that appear to interfere with normal splicing. We suggest that when no DICER1 pLOF variants or large deletions are discovered in exonic regions despite strong clinical suspicion, intron sequencing and splicing analysis should be performed.

Rare Tumors: Opportunities and challenges from the Children's Oncology Group perspective.

While all childhood cancers are rare, tumors that are particularly infrequent or underrepresented within pediatrics are studied under the umbrella of the Children's Oncology Group Rare Tumor committee, divided into the Retinoblastoma and Infrequent Tumor subcommittees. The Infrequent Tumor subcommittee has traditionally included an emphasis on globally rare tumors such as adrenocortical carcinoma, nasopharyngeal carcinoma, or those tumors that are rare in young children, despite being common in adolescents and young adults, such as colorectal carcinoma, thyroid carcinoma, and melanoma. Pleuropulmonary blastoma, gonadal stromal tumors, pancreatic tumors including pancreatoblastoma, gastrointestinal stromal tumor, nonmelanoma skin cancers, neuroendocrine tumors, and desmoplastic small round cell tumors, as well as other carcinomas are also included under the heading of the Children's Oncology Group Rare Tumor committee. While substantial challenges exist in rare cancers, inclusion and global collaboration remain key priorities to ensure high quality research to advance care.

Pan-cancer structurome reveals overrepresentation of beta sandwiches and underrepresentation of alpha helical domains.

The recent progress in the prediction of protein structures marked a historical milestone. AlphaFold predicted 200 million protein models with an accuracy comparable to experimental methods. Protein structures are widely used to understand evolution and to identify potential drug targets for the treatment of various diseases, including cancer. Thus, these recently predicted structures might convey previously unavailable information about cancer biology. Evolutionary classification of protein domains is challenging and different approaches exist. Recently our team presented a classification of domains from human protein models released by AlphaFold. Here we evaluated the pan-cancer structurome, domains from over and under expressed proteins in 21 cancer types, using the broadest levels of the ECOD classification: the architecture (A-groups) and possible homology (X-groups) levels. Our analysis reveals that AlphaFold has greatly increased the three-dimensional structural landscape for proteins that are differentially expressed in these 21 cancer types. We show that beta sandwich domains are significantly overrepresented and alpha helical domains are significantly underrepresented in the majority of cancer types. Our data suggest that the prevalence of the beta sandwiches is due to the high levels of immunoglobulins and immunoglobulin-like domains that arise during tumor development-related inflammation. On the other hand, proteins with exclusively alpha domains are important elements of homeostasis, apoptosis and transmembrane transport. Therefore cancer cells tend to reduce representation of these proteins to promote successful oncogeneses.

Children's Oncology Group's 2023 blueprint for research: Rare tumors.

The Children's Oncology Group (COG) Rare Tumor Committee includes the Infrequent Tumor and Retinoblastoma subcommittees, encompassing a wide range of extracranial solid tumors that do not fall within another COG disease committee. Current therapeutic trial development focuses on nasopharyngeal carcinoma, adrenocortical carcinoma, pleuropulmonary blastoma, colorectal carcinoma, melanoma, and thyroid carcinoma. Given the rarity of these tumors, novel strategies and international collaborative efforts are necessary to advance research and improve outcomes.

Integrated genomic analysis reveals aberrations in WNT signaling in germ cell tumors of childhood and adolescence.

Germ cell tumors (GCTs) are neoplasms of the testis, ovary and extragonadal sites that occur in infants, children, adolescents and adults. Post-pubertal (type II) malignant GCTs may present as seminoma, non-seminoma or mixed histologies. In contrast, pre-pubertal (type I) GCTs are limited to (benign) teratoma and (malignant) yolk sac tumor (YST). Epidemiologic and molecular data have shown that pre- and post-pubertal GCTs arise by distinct mechanisms. Dedicated studies of the genomic landscape of type I and II GCT in children and adolescents are lacking. Here we present an integrated genomic analysis of extracranial GCTs across the age spectrum from 0-24 years. Activation of the WNT pathway by somatic mutation, copy-number alteration, and differential promoter methylation is a prominent feature of GCTs in children, adolescents and young adults, and is associated with poor clinical outcomes. Significantly, we find that small molecule WNT inhibitors can suppress GCT cells both in vitro and in vivo. These results highlight the importance of WNT pathway signaling in GCTs across all ages and provide a foundation for future efforts to develop targeted therapies for these cancers.

WILMS TUMOR MUTATIONAL SUBCLASSES CONVERGE TO DRIVE CCND2 OVEREXPRESSION.

Wilms tumor, the most common kidney cancer in pediatrics, arises from embryonic renal progenitors. Although many patients are cured with multimodal therapy, outcomes remain poor for those with high-risk features. Recent sequencing efforts have provided few biological or clinically actionable insights. Here, we performed DNA and RNA sequencing on 94 Wilms tumors to understand how Wilms tumor mutations transform the transcriptome to arrest differentiation and drive proliferation. We show that most Wilms tumor mutations fall into four classes, each with unique transcriptional signatures: microRNA processing, MYCN activation, chromatin remodeling, and kidney development. In particular, the microRNA processing enzyme DROSHA is one of the most commonly mutated genes in Wilms tumor. We show that DROSHA mutations impair pri-microRNA cleavage, de-repress microRNA target genes, halt differentiation, and overexpress cyclin D2 (CCND2). Several mutational classes converge to drive CCND2 overexpression, which could render them susceptible to cell-cycle inhibitors.

Integrated Molecular Analysis Reveals 2 Distinct Subtypes of Pure Seminoma of the Testis.

Objective: Testicular germ cell tumors (TGCT) are the most common solid malignancy in adolescent and young men, with a rising incidence over the past 20 years. Overall, TGCTs are second in terms of the average life years lost per person dying of cancer, and clinical therapeutics without adverse long-term side effects are lacking. Platinum-based regimens for TGCTs have heterogeneous outcomes even within the same histotype that frequently leads to under- and over-treatment. Understanding of molecular differences that lead to diverse outcomes of TGCT patients may improve current treatment approaches. Seminoma is the most common subtype of TGCTs, which can either be pure or present in combination with other histotypes. Methods: Here we conducted a computational study of 64 pure seminoma samples from The Cancer Genome Atlas, applied consensus clustering approach to their transcriptomic data and revealed 2 clinically relevant seminoma subtypes: seminoma subtype 1 and 2. Results: Our analysis identified significant differences in pluripotency stage, activity of double stranded DNA breaks repair mechanisms, rates of loss of heterozygosity, and expression of lncRNA responsible for cisplatin resistance between the subtypes. Seminoma subtype 1 is characterized by higher pluripotency state, while subtype 2 showed attributes of reprograming into non-seminomatous TGCT. The seminoma subtypes we identified may provide a molecular underpinning for variable responses to chemotherapy and radiation. Conclusion: Translating our findings into clinical care may help improve risk stratification of seminoma, decrease overtreatment rates, and increase long-term quality of life for TGCT survivors.

Extrarenal Anaplastic Wilms Tumor: A Case Report With Genomic Analysis and Tumor Models.

Primary extrarenal Wilms tumors are rare neoplasms that are presumed to arise from metanephric or mesonephric remnants outside of the kidney. Their pathogenesis is debated but has not been studied, and there are no reports of genomic descriptions of extrarenal Wilms tumors. We describe a diffusely anaplastic extrarenal Wilms tumor that occurred in the lower abdomen and upper pelvis of a 10-year-old boy. In addition to the clinical, histopathologic, and radiologic features, we describe the cytogenetic changes and exomic profile of the tumor. The tumor showed loss of the tumor suppressor AMER1, loss of chromosome regions 1p, 16q, and 22q, gain of chromosome 8, and loss of function TP53 mutation-findings known to occur in renal Wilms tumors. This is the first description of the exomic profile of a primary extrarenal Wilms tumor. Our data indicate that primary extrarenal Wilms tumors may follow the same pathogenetic pathways that are seen in renal Wilms tumors. Finally, we describe the establishment of first ever tumor models (primary cell line and patient-derived xenograft) from an extrarenal Wilms tumor.

Pitfalls in the diagnosis of yolk sac tumor: Lessons from a clinical trial.

Though outcomes for patients with recurrent/refractory malignant germ cell tumors (mGCTs) are poor, therapies targeting mTOR and EGFR inhibition have shown promise in vitro. We hypothesized that the combination of sirolimus and erlotinib will show activity in patients with recurrent/refractory mGCTs. Patients were enrolled in a prospective phase II clinical trial; central review of existing pathology specimens was performed. Of the five patients evaluated, two had their diagnoses revised to pancreatic acinar cell carcinoma and alpha-fetoprotein (AFP)-secreting gastric adenocarcinoma, respectively. Although mGCTs are common AFP-secreting neoplasms, recurrence or refractoriness to standard regimens should prompt histologic reevaluation for other diagnoses.

Mutations in microRNA processing genes in Wilms tumors derepress the IGF2 regulator PLAG1.

Many childhood Wilms tumors are driven by mutations in the microRNA biogenesis machinery, but the mechanism by which these mutations drive tumorigenesis is unknown. Here we show that the transcription factor pleomorphic adenoma gene 1 (PLAG1) is a microRNA target gene that is overexpressed in Wilms tumors with mutations in microRNA processing genes. Wilms tumors can also overexpress PLAG1 through copy number alterations, and PLAG1 expression correlates with prognosis in Wilms tumors. PLAG1 overexpression accelerates growth of Wilms tumor cells in vitro and induces neoplastic growth in the developing mouse kidney in vivo. In both settings, PLAG1 transactivates insulin-like growth factor 2 (IGF2), a key Wilms tumor oncogene, and drives mammalian target of rapamycin complex 1 (mTORC1) signaling. These data link microRNA impairment to the PLAG1-IGF2 pathway, providing new insight into the manner in which common Wilms tumor mutations drive disease pathogenesis.

EGF Receptor and mTORC1 Are Novel Therapeutic Targets in Nonseminomatous Germ Cell Tumors.

Germ cell tumors (GCT) are malignant tumors that arise from pluripotent embryonic germ cells and occur in children and young adults. GCTs are treated with cisplatin-based regimens which, while overall effective, fail to cure all patients and cause significant adverse late effects. The seminoma and nonseminoma forms of GCT exhibit distinct differentiation states, clinical behavior, and response to treatment; however, the molecular mechanisms of GCT differentiation are not fully understood. We tested whether the activity of the mTORC1 and MAPK pathways were differentially active in the two classes of GCT. Here we show that nonseminomatous germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma) from both children and adults display activation of the mTORC1 pathway, while seminomas do not. In seminomas, high levels of REDD1 may negatively regulate mTORC1 activity. In NSGCTs, on the other hand, EGF and FGF2 ligands can stimulate mTORC1 and MAPK signaling, and members of the EGF and FGF receptor families are more highly expressed. Finally, proliferation of NSGCT cells in vitro and in vivo is significantly inhibited by combined treatment with the clinically available agents erlotinib and rapamycin, which target EGFR and mTORC1 signaling, respectively. These results provide an understanding of the signaling network that drives GCT growth and a rationale for therapeutic targeting of GCTs with agents that antagonize the EGFR and mTORC1 pathways. Mol Cancer Ther; 17(5); 1079-89. ©2018 AACR.

Erratum: Publisher Correction: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

[This corrects the article DOI: 10.1038/ncomms5802.].

A novel TP53-KPNA3 translocation defines a de novo treatment-resistant clone in osteosarcoma.

Osteosarcoma is the most common primary bone cancer. It can be cured by aggressive surgery and chemotherapy, but outcomes for metastatic or chemoresistant disease remain dismal. Cancer sequencing studies have shown that the p53 pathway is dysregulated in nearly every case, often by translocation; however, no studies of osteosarcoma evolution or intratumor heterogeneity have been done to date. We studied a patient with chemoresistant, metastatic disease over the course of 3 years. We performed exome sequencing on germline DNA and DNA collected from tumor at three separate time points. We compared variant calls and variant allele frequencies between different samples. We identified subclonal mutations in several different genes in the primary tumor sample and found that one particular subclone dominated subsequent tumor samples at relapse. This clone was marked by a novel TP53-KPNA3 translocation and loss of the opposite-strand wild-type TP53 allele. Future research must focus on the functional significance of such clones and strategies to eliminate them.

p53 genes function to restrain mobile elements.

Throughout the animal kingdom, p53 genes govern stress response networks by specifying adaptive transcriptional responses. The human member of this gene family is mutated in most cancers, but precisely how p53 functions to mediate tumor suppression is not well understood. Using Drosophila and zebrafish models, we show that p53 restricts retrotransposon activity and genetically interacts with components of the piRNA (piwi-interacting RNA) pathway. Furthermore, transposon eruptions occurring in the p53(-) germline were incited by meiotic recombination, and transcripts produced from these mobile elements accumulated in the germ plasm. In gene complementation studies, normal human p53 alleles suppressed transposons, but mutant p53 alleles from cancer patients could not. Consistent with these observations, we also found patterns of unrestrained retrotransposons in p53-driven mouse and human cancers. Furthermore, p53 status correlated with repressive chromatin marks in the 5' sequence of a synthetic LINE-1 element. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to contain retrotransposons. Since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease in part by restricting transposon mobility.

Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

Wilms tumour is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumours, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1, and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumour miRNA expression, in vitro processing assays and genomic editing in human cells demonstrates that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations preferentially impair processing of miRNAs deriving from the 5'-arm of pre-miRNA hairpins, while DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Both DROSHA and DICER1 mutations impair expression of tumour-suppressing miRNAs, including the let-7 family, important regulators of MYCN, LIN28 and other Wilms tumour oncogenes. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogramme miRNA expression in human cancer and suggest that these defects define a distinct subclass of Wilms tumours.