Karen W. Eldin

Diagnostic Yield of Clinical Tumor and Germline Whole-Exome Sequencing for Children With Solid Tumors.

Importance Whole-exome sequencing (WES) has the potential to reveal tumor and germline mutations of clinical relevance, but the diagnostic yield for pediatric patients with solid tumors is unknown. Objective To characterize the diagnostic yield of combined tumor and germline WES for children with solid tumors. Design Unselected children with newly diagnosed and previously untreated central nervous system (CNS) and non-CNS solid tumors were prospectively enrolled in the BASIC3 study at a large academic childrens hospital during a 23-month period from August 2012 through June 2014. Blood and tumor samples underwent WES in a certified clinical laboratory with genetic results categorized on the basis of perceived clinical relevance and entered in the electronic health record. Main Outcomes and Measures Clinical categorization of somatic mutations; frequencies of deleterious germline mutations related to patient phenotype and incidental medically-actionable mutations. Results Of the first 150 participants (80 boys and 70 girls, mean age, 7.4 years), tumor samples adequate for WES were available from 121 patients (81%). Somatic mutations of established clinical utility (category I) were reported in 4 (3%) of 121 patients, with mutations of potential utility (category II) detected in an additional 29 (24%) of 121 patients. CTNNB1 was the gene most frequently mutated, with recurrent mutations in KIT, TSC2, and MAPK pathway genes (BRAF, KRAS, and NRAS) also identified. Mutations in consensus cancer genes (category III) were found in an additional 24 (20%) of 121 tumors. Fewer than half of somatic mutations identified were in genes known to be recurrently mutated in the tumor type tested. Diagnostic germline findings related to patient phenotype were discovered in 15 (10%) of 150 cases: 13 pathogenic or likely pathogenic dominant mutations in adult and pediatric cancer susceptibility genes (including 2 each in TP53, VHL, and BRCA1), 1 recessive liver disorder with hepatocellular carcinoma (TJP2), and 1 renal diagnosis (CLCN5). Incidental findings were reported in 8 (5%) of 150 patients. Most patients harbored germline uncertain variants in cancer genes (98%), pharmacogenetic variants (89%), and recessive carrier mutations (85%). Conclusions and Relevance Tumor and germline WES revealed mutations in a broad spectrum of genes previously implicated in both adult and pediatric cancers. Combined reporting of tumor and germline WES identified diagnostic and/or potentially actionable findings in nearly 40% of newly diagnosed pediatric patients with solid tumors.

Smad1-Smad5 Ovarian Conditional Knockout Mice Develop a Disease Profile Similar to the Juvenile Form of Human Granulosa Cell Tumors

Granulosa cell tumors (GCTs) of the ovary are rare sex cord stromal tumors. Although generally indolent, GCTs recur, and if not diagnosed and treated in early stages, survival rates are significantly shortened. Very little is known regarding GCT etiology. Because of the low incidence of cases and lack of standard diagnostics, mouse models for granulosa cell tumors are a valuable tool for studying GCTs and provide models for developing diagnostic and treatment strategies. We recently developed a novel mouse model of metastatic granulosa cell tumors by genetic deletion of the bone morphogenetic protein signaling transcription factors (SMADs) in granulosa cells of the ovary. Histological and serum hormone analyses reveal that this mouse model most closely resembles the juvenile form of GCT. We further analyzed samples of human juvenile GCT (JGCT) for expression of anti-Müllerian hormone and activation of two major signaling pathways: TGFbeta/SMAD2/3 and wingless-related mouse mammary tumor virus integration site (Wnt)/beta-catenin. The TGFbeta family is active in mouse Smad1-Smad5 double knockout tumors, and here we show that this pathway, but not the beta-catenin pathway, is activated in samples of human JGCT. These data suggest that the SMAD family, possibly through disruption of SMAD1/5 or activation of SMAD2/3 may contribute to the pathogenesis of JGCT in humans.

A Recurrent PDGFRB Mutation Causes Familial Infantile Myofibromatosis

Infantile myofibromatosis (IM) is the most common benign fibrous tumor of soft tissues affecting young children. By using whole-exome sequencing, RNA sequencing, and targeted sequencing, we investigated germline and tumor DNA in individuals from four distinct families with the familial form of IM and in five simplex IM cases with no previous family history of this disease. We identified a germline mutation c.1681C>T (p.Arg561Cys) in platelet-derived growth factor receptor β (PDGFRB) in all 11 affected individuals with familial IM, although none of the five individuals with nonfamilial IM had mutations in this gene. We further identified a second heterozygous mutation in PDGFRB in two myofibromas from one of the affected familial cases, indicative of a potential second hit in this gene in the tumor. PDGFR-β promotes growth of mesenchymal cells, including blood vessels and smooth muscles, which are affected in IM. Our findings indicate p.Arg561Cys substitution in PDGFR-β as a cause of the dominant form of this disease. They provide a rationale for further investigations of this specific mutation and gene to assess the benefits of targeted therapies against PDGFR-β in aggressive life-threatening familial forms of the disease.

Detection of polyomavirus SV40 in tonsils from immunocompetent children.

BACKGROUND BK virus (BKV), JC virus (JCV) and simian virus 40 (SV40) are nonenveloped DNA viruses, members of the family Polyomaviridae. BK and JC viruses establish persistent infections in humans, and evidence suggests that SV40 can infect humans, as well. Whether persistence occurs in the lymphoid system is unknown. METHODS Paraffin-embedded tonsils from 220 immunocompetent children (mean age 9.3 years) were examined by polymerase chain reaction (PCR) to detect viral DNA of BKV, JCV, SV40, and Epstein-Barr virus (EBV). RESULTS Polyomavirus-specific DNA sequences were detected in 8.3% (29/351) of specimens collected from 220 children. Twenty-one (9.5%) children had polyomavirus DNA present in at least one tonsil, with sequences identified as SV40 (n=20) and BKV (n=1). Polyomavirus JCV was not detected. Among patients positive for SV40, 8 of 14 (57%) contained viral DNA in both available tonsils. EBV DNA was detected in 99 (28.2%) samples from 67 (30.5%) patients. Eleven samples (3.1%) from 8 (3.6%) children were positive for both polyomavirus and EBV. SV40-positive children were significantly older than the SV40-negative subjects (P<0.001). T-antigen expression was detected in an SV40 DNA-positive tonsil by immunohistochemistry. CONCLUSIONS These results suggest that SV40 can infect tonsils, that lymphoid tissue may represent a site for polyomavirus persistence, and that immunohistochemistry is not a useful detection assay when there are very few virus-infected cells in a tissue.

Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney

The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR–CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.

Correlating surgical and pathological diagnoses in pediatric appendicitis

BACKGROUND The stratification of appendicitis into simple and complex variants has far-reaching implications. While the operative diagnosis made by the surgeon dictates clinical management, the pathologic diagnosis often differs and is frequently used for coding and reimbursement. The purpose of this study was to examine discrepancies between the operative and pathologic diagnoses with subsequent correlation to clinical outcomes. METHODS Patients with acute appendicitis from July 2011 to July 2012 were identified. Diagnoses included simple (normal, acute, and suppurative) and complex (gangrenous and perforated). We evaluated the inter-rater reliability between pathologic and operative diagnoses in the five appendicitis categories. Clinical outcomes of deep and superficial surgical site infections were evaluated according to the pathologic and surgical diagnosis. RESULTS During the study period, we identified 1166 patients with acute appendicitis. The surgeon and pathologist agreed on the specific diagnosis (acute, suppurative, gangrenous, perforated, normal) in 48% of patients (kappa 0.289, 95% CI 0.259-0.324, p=0.001). Agreement on disease severity (simple vs. complex) improved to 82%. The operative diagnosis more accurately predicted infectious complications than the pathologic diagnoses. CONCLUSION Significant discordance exists between surgical and pathologic diagnoses. While the relevance of this discordance to clinical outcomes is still not clear, a potential for incorrect hospital coding and subsequent reimbursement exists. Future quality improvement projects should focus on standardizing the surgical and pathologic diagnoses.

Minimal change disease with IgM+ immunofluorescence: a subtype of nephrotic syndrome

Immunoglobulin (Ig) M nephropathy is defined by electron-dense mesangial deposits and mesangial IgM visible by immunofluorescence (IF) without other histopathologic and immunofluorescent microscopic abnormalities. Certain patients have only immuno-positive (IgM+) IF. Children presenting with steroid-dependent or steroid-resistant nephrotic syndrome have a high prevalence of IgM+ IF with or without electron-dense deposits. We reviewed the clinical course of children with steroid-dependent or steroid-resistant nephrotic syndrome who underwent renal biopsy at Texas Children‘s Hospital from 1989 to 2006 to further characterize IgM+ IF in children with nephrotic syndrome. Of the 55 children with steroid-resistant or -dependent minimal change disease (MCD), 23 had IgM+ IF. Of these 23 children, 61% had microscopic hematuria at presentation, 48% (11/23) were steroid-dependent, and 48% (11/23) steroid-resistant (one underwent biopsy prior to steroid therapy). We compared the efficacy of adjuvant treatment with cyclophosphamide and cyclosporine: 18% initially treated with cyclophosphamide obtained remission, while 55% had no response; 83% obtained subsequent remission with cyclosporine. Of those initially treated with cyclosporine, 88% obtained complete or partial remission. IgM+ IF may be surrogate marker for the severity of MCD. Based on our results, children with MCD and IgM+ IF have a better response to cyclosporine than cyclophosphamide.

BMP- specific SMADs function as novel repressors of PDGFA and modulate its expression in ovarian granulosa cells and tumors

Platelet-derived growth factor alpha (PDGFA) is frequently upregulated in various cancers and thought to function as a key player in the development and progression of tumor growth by regulating aspects of cell proliferation, angiogenesis and metastasis. However, the mechanism by which it is upregulated is not fully understood. Previously, we demonstrated that conditional deletion of two transcription factors that signal for the bone morphogenetic proteins (Smad1 and Smad5) in ovarian granulosa cells causes metastatic granulosa cell tumors (GCTs) in female mice and phenocopies human juvenile GCTs (JGCTs). Smad1/5 double conditional knockout tumors, as well as human JGCTs, are highly vascularized, hemorrhagic and mitotically active. Expression analysis of these tumors and their metastases revealed a significant upregulation of key proliferation and pro-angiogenic factors such as Pdgfa, Pdgfb and Vegf. We examined whether these genes were direct targets of SMAD1 and SMAD5. Knockdown of SMAD1 and SMAD5 in mouse primary granulosa cells and a human GCT-derived cell line (COV434) resulted in upregulation of PDGFA, but not PDGFB nor VEGF. We identified several putative SMAD1/5-binding sites in the PDGFA promoter, and chromatin immunoprecipitation and reporter assays demonstrated that SMAD1/5 interact with the PDGFA promoter to regulate its activity. Further, SMAD1/5 antagonize the activity of the transcription factor Sp1, a well-known positive regulator of PDGFA, by inhibiting its occupancy at a key regulatory site on the proximal PDGFA promoter. Collectively, our studies establish that loss of SMAD1/5 leads to upregulation of PDGFA in ovarian granulosa cells, and that a novel regulatory interaction exists between the BR-SMADs and Sp1 in controlling PDGFA expression during granulosa cell tumorigenesis.

Copa Syndrome: a Novel Autosomal Dominant Immune Dysregulatory Disease

Inherently defective immunity typically results in either ineffective host defense, immune regulation, or both. As a category of primary immunodeficiency diseases, those that impair immune regulation can lead to autoimmunity and/or autoinflammation. In this review we focus on one of the most recently discovered primary immunodeficiencies that leads to immune dysregulation: “Copa syndrome”. Copa syndrome is named for the gene mutated in the disease, which encodes the alpha subunit of the coatomer complex-I that, in aggregate, is devoted to transiting molecular cargo from the Golgi complex to the endoplasmic reticulum (ER). Copa syndrome is autosomal dominant with variable expressivity and results from mutations affecting a narrow amino acid stretch in the COPA gene-encoding COPα protein. Patients with these mutations typically develop arthritis and interstitial lung disease with pulmonary hemorrhage representing a striking feature. Immunologically Copa syndrome is associated with autoantibody development, increased Th17 cells and pro-inflammatory cytokine expression including IL-1β and IL-6. Insights have also been gained into the underlying mechanism of Copa syndrome, which include excessive ER stress owing to the impaired return of proteins from the Golgi, and presumably resulting aberrant cellular autophagy. As such it represents a novel cellular disorder of intracellular trafficking associated with a specific clinical presentation and phenotype.

TGFβ Signaling Promotes Juvenile Granulosa Cell Tumorigenesis by Suppressing Apoptosis

Molecular changes that give rise to granulosa cell tumors of the ovary are not well understood. Previously, we showed that deletion in granulosa cells of the bone morphogenetic protein receptor-signaling transcription factors, Smad1 and Smad5, causes development of metastatic granulosa cell tumors that phenocopy the juvenile form of granulosa cell tumors (JGCTs) in humans. The TGFβ-SMAD2/3 pathway is active in JGCTs, but its role is unknown. We tested the in vivo contribution of TGFβ-SMAD signaling to JGCT development by genetically deleting the common Smad4 from Smad1/5 double knockout mice. Smad1/5/4 triple knockout mice were sterile and had significantly increased survival and delayed tumor development compared to those for the Smad1/5 double knockout mice. The few tumors that did develop were smaller, showed no evidence of metastasis, and had increased apoptosis. In the human JGCT cell line COV434, TGFβ1 increased viability by inhibiting apoptosis through a TGFβ type I receptor-dependent repression of caspase activity and inhibition of poly(ADP-ribose) polymerase cleavage. These data support a tumor-promoting function of TGFβ in JGCTs through its ability to repress apoptosis.