Germline Loss Of Function Mutations Research Articles

Circulating Plasma miRNA Homologs in Mice and Humans Reflect Familial Cerebral Cavernous Malformation Disease.

Patients with familial cerebral cavernous malformation (CCM) inherit germline loss of function mutations and are susceptible to progressive development of brain lesions and neurological sequelae during their lifetime. To date, no homologous circulating molecules have been identified that can reflect the presence of germ line pathogenetic CCM mutations, either in animal models or patients. We hypothesize that homologous differentially expressed (DE) plasma miRNAs can reflect the CCM germline mutation in preclinical murine models and patients. Herein, homologous DE plasma miRNAs with mechanistic putative gene targets within the transcriptome of preclinical and human CCM lesions were identified. Several of these gene targets were additionally found to be associated with CCM-enriched pathways identified using the Kyoto Encyclopedia of Genes and Genomes. DE miRNAs were also identified in familial-CCM patients who developed new brain lesions within the year following blood sample collection. The miRNome results were then validated in an independent cohort of human subjects with real-time-qPCR quantification, a technique facilitating plasma assays. Finally, a Bayesian-informed machine learning approach showed that a combination of plasma levels of miRNAs and circulating proteins improves the association with familial-CCM disease in human subjects to 95% accuracy. These findings act as an important proof of concept for the future development of translatable circulating biomarkers to be tested in preclinical studies and human trials aimed at monitoring and restoring gene function in CCM and other diseases.

SAT-140 Determining the Undetermined: The Role of Tumor Tissue Staining for Interpretation of Inconclusive Genetic Testing Results in Patients with Pheochromocytomas and Paragangliomas

Background:Paragangliomas and pheochromocytomas are neuroendocrine tumors that can occur in several hereditary tumor syndromes. While these are generally rare, individuals with germline loss of function mutations in the succinate dehydrogenase (SDH) genes are at high risk of developing these tumours, with a penetrance of 70% by age 50. Functional SDHB acts as a tumor suppressor. Consequently, pathogenic mutations in the SDHB gene predispose to familial paraganglioma syndrome type 4, with high incidence of extra adrenal paragangliomas and high rates of metastasis. SDHB mutation carriers are also predisposed to developing tumors in other sites such as renal cell cancer, gastrointestinal stromal tumors and pituitary adenomas. Genetic testing for hereditary syndromes is recommended in patients who present with paragangliomas and pheochromocytomas, especially in those with aggressive tumours or who present at a young age. It is recommended that mutation carriers are monitored with routine clinical and imaging surveillance, and effort is made to identify and screen at-risk family members. In some cases, genetic testing can identify variants that are not clearly pathogenic or benign. In such “variants of undetermined significance”, immunohistochemistry or family history can be a helpful tool in discriminating between SDHB related and non-SDH-related pheochromocytomas and paragangliomas.Clinical case:We report on three families who presented with manifestations of paraganglioma syndrome and were found to have Variants of Uncertain Significance (VUSs) in the SDHB gene. Absence of SDHB staining was seen on tumour histopathology in two of the families; staining was not performed in the third. The proband in the third case initially presented at the age of 22 with a cardiac pheochromocytoma. Subsequently, her son was diagnosed with metastatic renal cancer at the age of 37. Genetic test results from both these patients identified a heterozygous VUS in SDHB. The son passed away from complications of his aggressive cancer shortly after diagnosis. Had familial screening and surveillance been initiated sooner in this family, this poor outcome may have been prevented.Conclusion:Our case highlights the important diagnostic dilemma that can arise in patients with VUSs in risk genes for hereditary pheochromocytomas and paragangliomas. While universally treating these VUSs as pathogenic would be costly, low-yield and potentially harmful, the incorporation of family history and tumour tissue staining for SDHB should be considered in all individuals with pheochromocytomas and paragangliomas to help guide interpretation of inconclusive genetic testing results, inform subsequent management and help predict risk for inheritance and recurrence.

Loss of Janus Associated Kinase 1 Alters Urothelial Cell Function and Facilitates the Development of Bladder Cancer.

Inherited Primary Immunodeficiency (PID) disorders are associated with increased risk of malignancy that may relate to impaired antitumor immune responses or a direct role for PID germline mutations in tumorigenesis. We recently identified germline loss of function mutations in Janus Associated Kinase 1 (JAK1) causing primary immunodeficiency characterized by infections and associated with early onset, fatal high-grade bladder carcinoma. Somatic mutations in JAK1, required for immune cell signaling in response to interferon gamma (IFNγ), have been associated with several non-hematopoietic and hematopoietic cancer cell types but pathogenic mechanisms remain largely unexplored. Here we demonstrate that JAK1 is required for the intrinsic IFNγ response of urothelial cells impacting immunogenicity and cell survival. Specifically, JAK1-deficient urothelial cells showed reduced surface expression of major histocompatibility complex class II (MHC II), intercellular adhesion molecule-1 (ICAM-1) and programmed death-ligand-1 (PD-L1) after IFNγ stimulation and were resistant to IFNγ-induced apoptosis and lymphocyte-mediated killing. In addition, we identify a previously unknown role for IFNγ signaling in modulating urothelial differentiation. Together, our findings support a role for urothelial cell JAK1 in immune surveillance and development of bladder cancer. Our results have implications for patients with rare JAK1 PID and, more broadly, inform development of biomarker and targeted therapies for urothelial carcinoma.

BRCA2 loss-of-function germline mutations are associated with esophageal squamous cell carcinoma risk in Chinese.

Esophageal squamous cell carcinoma (ESCC) occurs with highest frequency in China with over 90% mortality, highlighting the need for early detection and improved treatment strategies. We aimed to identify ESCC cancer predisposition gene(s). Our study included 4,517 individuals. The discovery phase using whole-exome sequencing (WES) included 186 familial ESCC patients from high-risk China. Targeted gene sequencing validation of 598 genes included 3,289 Henan and 1,228 moderate-risk Hong Kong Chinese. A WES approach identified BRCA2 loss-of-function (LOF) mutations in 3.23% (6/186) familial ESCC patients compared to 0.21% (9/4300) in the ExAC East Asians (odds ratio [OR] = 15.89, p = 2.48 × 10-10 ). BRCA2 LOF mutation frequency in the combined Henan cohort has significantly higher prevalence (OR = 10.55, p = 0.0035). Results were independently validated in an ESCC Hong Kong cohort (OR = 10.64, p = 0.022). One Hong Kong pedigree was identified to carry a BRCA2 LOF mutation. BRCA2 inactivation in ESCC was via germline LOF mutations and wild-type somatic allelic loss via loss of heterozygosity. Gene-based association analysis, including LOF mutations and rare deleterious missense variants defined with combined annotation dependent depletion score ≥30, confirmed the genetic predisposition role of BRCA2 (OR = 9.50, p = 3.44 × 10-5 ), and provided new evidence for potential association of ESCC risk with DNA repair genes (POLQ and MSH2), inflammation (TTC39B) and angiogenesis (KDR). Our findings are the first to provide compelling evidence of the role of BRCA2 in ESCC genetic susceptibility in Chinese, suggesting defective homologous recombination is an underlying cause in ESCC pathogenesis, which is amenable to therapeutic options based on synthetic lethality approaches such as targeting BRCA2 with PARP1 inhibitors in ESCC.

Abstract 2574: Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability

Abstract Werner Syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss of function mutations in WRN lead to premature aging and pre-disposition to cancer. We evaluated synthetic lethality (SL) between not only of WRN, but also of another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines. We found that WRN exhibited SL with a DNA mismatch repair protein MutL homolog 1 (MLH1), loss of which is associated with high microsatellite instability (MSI-H). We then demonstrated that MSI-H cells exhibited increased double-stranded DNA breaks, altered cell cycles and decreased viability in response to WRN knockdown. Although WRN is the only human RecQ enzyme with a distinct exonuclease domain, only loss of helicase activity drives the MSI SL interaction. This SL interaction in MSI cancer cells positions WRN as a relevant therapeutic target in patients with MSI-H tumors. Citation Format: Lorn Kategaya, Senthil K. Perumal, Jeffrey H. Hager, Lisa D. Belmont. Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2574.

PF342 COMPENSATORY MUTATIONS IN PROMOTERS OF TELOMERASE GENES ARE RARE IN TELOMERE BIOLOGY DISORDERS WITH BONE MARROW FAILURE

Background:Dyskeratosis congenita, also referred to as telomere biology disorder (TBD), causes premature telomere shortening and manifests with progressive bone marrow failure (BMF). In addition to BMF, classical features include mucocutaneous triad (leukoplakia, nail dystrophy and skin dyspigmentation) and pulmonary fibrosis. To date, constitutional loss‐of‐function (LOF) mutations of 13 genes (DKC1, TERT, TERC, TINF2, TCAB2, RTEL1, NHP2, NOP10, CTC1, USB1, PARN, ACD, NAF1) involved in telomere maintenance and elongation have been associated with TBD. Human cancers can reactivate telomerase activity by acquiring gain‐of‐function (GOF) mutations in TERT gene promoter leading to increased telomerase activity (Vinagre, Nat Commun 2013). Moreover, GOF mutations at TERT promoter hotspots c.‐124C>T and c.‐146C>T were found in 5% of patients with idiopathic lung fibrosis who had an underlying germline LOF mutation in TERT or TERC (Maryoung, JCI 2017). These mutations counterbalanced the LOF germline variants and the patients had normal blood counts.Aims:We hypothesized that GOF mutations in promoters of telomerase genes can be acquired in pediatric TBD and might explain the heterogeneity of hematologic manifestations.Methods:For this purpose, we investigated a total of 64 TBD patients (52 with classical DC phenotype and 12 DC‐like with BMF and short telomeres) and 9 mutation carriers (family members), all referred for diagnostics within the framework of the EWOG‐MDS and the Aachen Telomeropathy Registries. Two amplicon‐based deep sequencing panels were designed in order to screen for I) constitutional variants consisting of the 13 TBD genes in addition to SAMD9 and SAMD9L, and II) somatic mutations in the promoter regions of TERT, TERC, DKC1, RTEL1, TCAB1 and TINF2.Results:Constitutional pathogenic mutations were found in TERT (20.9%), TERC (17.7%), DKC1 (16.1%), TINF2 (12.9%), RTEL1 (10%), TCAB1 (6.4%) and SAMD9/SAMD9L (3.6%), while no mutations were found in 8 patients (14.8%). Previously reported hotspot mutations in the TERT and TERC gene promoters were not detected in our patients. With a median depth of 13283 reads even the presence of very small clones could be excluded. Conversely, germline variants of uncertain significance (VUS) were identified in the 5́UTR/promoter regions of TCAB1 at c.‐499dupG in three patients (P36, P16, P11; variant allele frequency (VAF) 43%, minor allele frequency in gnomAD healthy database (MAF) 0.003%) and in DKC1 at c.‐519C>T (P15: VAF94%, MAF 0.004%). Another variant, a common polymorphism in the TERT promoter region at c.‐245T>C, which was previously shown disrupt a preexisting ETS2 binding site, was observed in 8 patients (VAF50–100%, MAF 23%).Summary/Conclusion:In summary, activating mutations in promoters of telomerase genes previously found in cancers and idiopathic pulmonary fibrosis are absent in DC with BMF. However, we identified TCAB1 promoter variant in 3 patients, and DKC1 promoter in 1 patient. These variants might potentially act as phenotype modifiers and warrant functional studies.

Werner Syndrome Helicase Is Required for the Survival of Cancer Cells with Microsatellite Instability

Werner Syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss of function mutations in WRN lead to premature aging and pre-disposition to cancer. We evaluated synthetic lethality (SL) between not only of WRN, but also of another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines. We found that WRN exhibited SL with a DNA mismatch repair protein MutL homolog 1 (MLH1), loss of which is associated with high microsatellite instability (MSI-H). We then demonstrated that MSI-H cells exhibited increased double-stranded DNA breaks, altered cell cycles and decreased viability in response to WRN knockdown. Although WRN is the only human RecQ enzyme with a distinct exonuclease domain, only loss of helicase activity drives the MSI SL interaction. This SL interaction in MSI cancer cells positions WRN as a relevant therapeutic target in patients with MSI-H tumors.

Germline mutations and somatic inactivation of TRIM28 in Wilms tumour.

Wilms tumour is a childhood tumour that arises as a consequence of somatic and rare germline mutations, the characterisation of which has refined our understanding of nephrogenesis and carcinogenesis. Here we report that germline loss of function mutations in TRIM28 predispose children to Wilms tumour. Loss of function of this transcriptional co-repressor, which has a role in nephrogenesis, has not previously been associated with cancer. Inactivation of TRIM28, either germline or somatic, occurred through inactivating mutations, loss of heterozygosity or epigenetic silencing. TRIM28-mutated tumours had a monomorphic epithelial histology that is uncommon for Wilms tumour. Critically, these tumours were negative for TRIM28 immunohistochemical staining whereas the epithelial component in normal tissue and other Wilms tumours stained positively. These data, together with a characteristic gene expression profile, suggest that inactivation of TRIM28 provides the molecular basis for defining a previously described subtype of Wilms tumour, that has early age of onset and excellent prognosis.

Involvement of glycogen debranching enzyme in bladder cancer.

Bladder cancer is the most common malignancy of the urinary system, however the molecular pathways underlying this disease are incompletely understood. To understand new regulators of bladder cancer progression, the authors carried out a functional genomic screen which identified glycogen debranching enzyme (AGL) as a novel regulator of bladder cancer growth. Glycogen debranching enzyme is involved in glycogen breakdown and germline loss of function mutation of this gene leads to glycogen storage disease type III. To the best of the authors' knowledge, the present study is the first to demonstrate that loss of AGL leads to aggressive bladder tumor growth. AGL mRNA and protein expression in bladder tumors serve as a prognostic marker for patients. Interestingly, AGL's participation in regulating tumor growth is independent of its enzymatic function and involvement with glycogen metabolism in general. Detailed metabolomics and transcriptomic analysis indicated that increases in glucose metabolism, glycine synthesis driven by serine hydroxymethyltransferase 2 and increases in hyaluronic acid synthase 2-driven HA synthesis are major contributors of aggressive bladder tumor growth with loss of AGL. However, the detailed mechanism of how AGL regulates the above mentioned metabolic and genetic pathways is unknown and is being investigated. The present review focuses on AGL's involvement in bladder cancer.

Somatic overgrowth disorders of the PI3K/AKT/mTOR pathway & therapeutic strategies.

The phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway plays an essential role in regulation of normal cell growth, metabolism, and survival. Somatic activating mutations in the PI3K/AKT/mTOR pathway are among the most common mutations identified in cancer, and have been shown to cause a spectrum of overgrowth syndromes including PIK3CA-Related Overgrowth Spectrum, Proteus syndrome, and brain overgrowth conditions. Clinical findings in these disorders may be isolated or multiple, including sporadic or mosaic overgrowth (adipose, skeletal, muscle, brain, vascular, or lymphatic), and skin abnormalities (including epidermal nevi, hyper-, and hypopigmented lesions), and have the potential risk of tumorigenesis. Key negative regulators of the PI3K-AKT signaling pathway include PTEN and TSC1/TSC2 and germline loss-of function mutations of these genes are established to cause PTEN Hamartoma Tumor Syndrome and Tuberous Sclerosis Complex. Mosaic forms of these conditions lead to increased activation of PI3K and mTOR at affected sites and there is phenotypic overlap between these conditions. All are associated with significant morbidity with limited options for treatment other than symptomatic therapies and surgeries. As dysregulation of the PI3K/AKT/mTOR pathway has been implicated in cancer, several small molecule inhibitors targeting different components of the PI3K/AKT/mTOR signaling pathway are under clinical investigation. The development of these therapies brings closer the prospect of targeting treatment for somatic PI3K/AKT/mTOR-related overgrowth syndromes. This review describes the clinical findings, gene function and pathogenesis of these mosaic overgrowth syndromes, and presents existing and future treatment strategies to reduce or prevent associated complications of these disorders. © 2016 Wiley Periodicals, Inc.

Abstract 611: Exploratory analyses suggest ovarian tumors with somatic or germline loss of function mutations in BRCA1 or BRCA2 are biologically similar and sensitive to PARP inhibition

Abstract The PARP inhibitor olaparib (LynparzaTM) elicits responses in ovarian cancer patients with inherited (germline) loss of function mutations in the BRCA1 or BRCA2 genes (gBRCAm). In the tumors of such patients the unmutated (wt) copy of the relevant BRCA gene is usually lost. Many patients are aware of their BRCA mutation status due to the wide availability of blood testing. However, in around a third of ovarian tumors with BRCA mutations the mutation has not been inherited but is tumor-specific (sBRCAm). Given the difference in origin of mutation, it is important to determine whether there are genotypic and phenotypic differences between sBRCAm and gBRCAm ovarian tumors. In a randomized, Phase II trial of 265 patients with platinum-sensitive relapsed serous ovarian cancer (NCT00753545; Study 19), maintenance treatment with olaparib (400 mg bid; capsules) led to a significant increase in progression-free survival (PFS) vs placebo in patients with a BRCA mutation (HR 0.18; 95% CI 0.10-0.31; P<0.00001). Diagnostic tumor samples from the study were sequenced by Foundation Medicine. After filtering for common polymorphisms, BRCA gene variants were classified using American College of Medical Genetics standards as mutant, wild type or variant of unknown significance (VUS). VUS were further classified on the basis of information from the Breast Information Core database. Mutation origin (g vs s) and zygosity were determined by tumor allele frequency analysis and/or by comparison to germline sequencing, and heterogeneity was determined using the Foundation Medicine SGZ algorithm, which compares allele frequency to model expectations, taking into account tumor purity, ploidy and local copy number. BRCA mutations were found in 53% of the 209 samples with data available. Known germline BRCA mutations were correctly classified as germline by the SGZ algorithm. Tumor BRCA mutations were identified in 40 patients without confirmation of a mutation in a blood sample. Twenty of these patients were classified as sBRCAm (12 BRCA1 and 8 BRCA2) by the algorithm. Only one case was called without loss of the wt BRCA allele. It has been hypothesized that TP53 mutation is an early event and was found to be consistent between tumors and precursor lesions. Allele frequencies/clonality for BRCA and TP53 mutations were comparable and there was no evidence of systematic subclonality in the sBRCAm cases. An exploratory analysis of PFS and overall survival (OS) was performed on the 20 patients (10 received treatment and 10 placebo). Consistent with results from the gBRCAm population, a trend in event rate in favor of treatment over placebo was observed (3/10 vs 8/10 events for PFS and 3/10 vs 7/10 for OS). Although from a small number of patients, data are consistent with sBRCA mutation as an early event in tumorigenesis and support phenotypic similarity of gBRCA and sBRCA mutated tumors. Citation Format: Brian Dougherty, Zhongwu Lai, Jonathan A. Ledermann, Jane D. Robertson, Tony W. Ho, Wenting Wu, Darren R. Hodgson, Maria C M Orr, Mark J. O'Connor, Matthew J. Hawryluk, Timothy A. Brennan, Roman Yelensky, James X. Sun, J Carl Barrett. Exploratory analyses suggest ovarian tumors with somatic or germline loss of function mutations in BRCA1 or BRCA2 are biologically similar and sensitive to PARP inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 611. doi:10.1158/1538-7445.AM2015-611

Abstract 2426: Exacerbation of oxidative DNA damage as a synthetic lethal approach in mismatch repair deficient tumors

Abstract The post-replicative DNA mismatch repair (MMR) pathway plays a key role in the maintenance of genomic stability. MMR corrects mismatches within the DNA helix that arise due to errors in nucleotide incorporation during either semi-conservative replication or synthesis associated with DNA repair. The importance of this system is testified by the fact that mutations affecting components of this repair pathway cause a “mutator” phenotype and predisposition to a wide variety of malignancies including colorectal, gastric, endometrial, and urothelial cancers. For example, the hereditable disorder Lynch syndrome (hereditary nonpolyposis colon cancer) is associated with germline loss of function mutations in the MMR tumor suppressor genes MLH1, MSH2, PMS2, and MSH6. In addition to this familial syndrome, MMR deficiency is present in 15% to 17% of all colorectal cancer cases. Synthetic lethal relationships have been identified that involve specific MMR genes, suggesting that synthetic lethal treatments could be applied to MMR deficient tumors. Previously, efforts to functionally identify genetic DNA mismatch repair synthetic lethalities and potential therapeutic targets have focused on the analysis of either candidate genes or relatively small gene subsets such as protein kinases. In order to define additional SL targets in MMR-deficient genetic backgrounds, we describe here the application of an shRNA screen in matched isogenic dMMR/pMMR (MMR-deficient/MMR-proficient) model cell lines. Using this screening approach, we identify new synthetic lethal interactions between MMR deficiency and components of the base excision repair and of the nucleotide sanitization systems, which restrict the extent of oxidative DNA damage. We propose that inhibition of the processes that minimize the oxidized DNA load could constitute a promising therapeutic approach in mismatch repair deficient cancers. Citation Format: Yari Fontebasso, Madeleine Hewish, Chris J. Lord, Alan Ashworth. Exacerbation of oxidative DNA damage as a synthetic lethal approach in mismatch repair deficient tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2426. doi:10.1158/1538-7445.AM2014-2426

Multiple congenital anomalies‐intellectual disability (MCA‐ID) and neuroblastoma in a patient harboring a de novo 14q23.1q23.3 deletion

Neuroblastoma is the most frequent extra cranial solid tumor in infants and children. Genetic predisposition to neuroblastoma has been suspected previously due to familial cases of sporadic NB and predisposition to NB in several syndromes. Here, we report on a de novo 14q23.1-q23.3 microdeletion in a male presenting with a neuroblastoma diagnosed at 9 months, and spherocytosis, congenital heart defect, cryptorchidism, hypoplasia of corpus callosum, epilepsy, and developmental delay. Myc-associated-factor X (MAX) haploinsufficiency could be regarded as the predisposing factor to NB. Indeed 14q deletion is a recurrent somatic rearrangement in NB and MAX somatic and germline loss of function mutation have recently been described in pheochromocytoma and paraganglioma. However, MAX was expressed in the tumor of the patient we report on and, accordingly, loss of heterozygosity, mutation, or promoter methylation were excluded. In addition, we discuss the potential involvement in the clinical spectrum presented by the patient of five of the deleted genes, namely DAAM1, PLEKHG3, SPTB, AKAP5, and ARID4A.

Somatic CTNNB1 mutation in hepatoblastoma from a patient with Simpson–Golabi–Behmel syndrome and germline GPC3 mutation

Simpson-Golabi-Behmel syndrome is a rare overgrowth syndrome caused by the GPC3 mutation at Xq26 and is clinically characterized by multiple congenital abnormalities, intellectual disability, pre/postnatal overgrowth, distinctive craniofacial features, macrocephaly, and organomegaly. Although this syndrome is known to be associated with a risk for embryonal tumors, similar to other overgrowth syndromes, the pathogenetic basis of this mode of tumorigenesis remains largely unknown. Here, we report a boy with Simpson-Golabi-Behmel syndrome who had a germline loss-of function mutation in GPC3. At 9 months of age, he developed hepatoblastoma. A comparison of exome analysis results for the germline genome and for the tumor genome revealed a somatic mutation, p.Ile35Ser, within the degradation targeting box of β-catenin. The same somatic mutation in CTNNB1 has been repeatedly reported in hepatoblastoma and other cancers. This finding suggested that the CTNNB1 mutation in the tumor tissue represents a driver mutation and that both the GPC3 and the CTNNB1 mutations contributed to tumorigenesis in a clearly defined sequential manner in the propositus. The current observation of a somatic CTNNB1 mutation in a hepatoblastoma from a patient with a germline GPC3 mutation supports the notion that the mutation in GPC3 may influence one of the initial steps in tumorigenesis and the progression to hepatoblastoma.

Lhermitte-Duclos disease: A case report

A 43-year-old female with a past history of breast carcinoma presented with headaches and dizziness concerning for metastatic disease. An MRI showed a large non-enhancing right cerebellar mass. Microscopic examination of the subsequent biopsy showed fragments of cerebellar parenchyma with disorganised collections of atypical ganglion cells of variable size consistent with dysplastic gangliocytoma/Lhermitte-Duclos disease (LDD). LDD is a rare, slow growing benign lesion of the cerebellum. The histogenesis remains uncertain as it is not clear whether it represents a hamartomatous or neoplastic process. Grossly there is enlargement and coarsening of the cerebellar folia. Histologically there is relative preservation of architecture with diffuse enlargement of the molecular and internal granular layers which contain atypical ganglion cells of various size and reduced numbers of purkinje cells. Adult onset of LLD is pathognomonic of Cowden syndrome a hamartoma/cancer syndrome associated with a germline loss of function mutation of the PTEN gene resulting in an increased risk of breast, thyroid, endometrial, colorectal and renal cell carcinoma. While LDD is a slow growing lesion without reported malignant potential, the association with Cowden syndrome and subsequent increased risk of epithelial malignancy warrants referral for genetic counselling.

Abstract A09: Induced aggravation of oxidative DNA damage as a targeted synthetic lethal approach in mismatch repair-deficient tumors

Abstract The post-replicative DNA mismatch repair (MMR) pathway plays a key role in the maintenance of genomic stability. MMR corrects mismatches within the DNA helix that arise due to errors in nucleotide incorporation during either semi-conservative replication or synthesis associated with DNA repair. The importance of this system is testified by the fact that mutations affecting components of this repair pathway cause a “mutator” phenotype and predisposition to a wide variety of malignancies including colorectal, gastric, endometrial, and urothelial cancers. For example, the hereditable disorder Lynch syndrome (hereditary nonpolyposis colon cancer) is associated with germline loss of function mutations in the MMR tumor suppressor genes MLH1, MSH2, PMS2 and MSH6. In addition to this familial syndrome, MMR deficiency is present in 15% to 17% of all colorectal cancer cases and a common cause of nonfamilial MMR deficiency is biallelic hypermethylation of the MLH1 promoter that results in a reduction of MLH1 expression. Synthetic lethal relationships have been identified that involve specific MMR genes, suggesting that the synthetic lethal paradigm could be applied to MMR deficiency. Previously, efforts to functionally identify genetic DNA mismatch repair synthetic lethalities and potential therapeutic targets have focused on the analysis of either candidate genes or relatively small gene subsets such as protein kinases. In order to define additional SL targets in MMR-deficient genetic backgrounds, we describe here the application of an shRNA screen in matched isogenic dMMR/pMMR (MMR-deficient/MMR-proficient) model cell lines. Using this screening approach, we identify new synthetic lethal interactions between MMR deficiency and components of the base excision repair and of the nucleotide sanitization systems. We propose that selective aggravation of oxidative damage constitutes a promising therapeutic approach in mismatch repair - deficient cancer cells. Citation Format: Yari Fontebasso, Christopher J. Lord, Alan Ashworth. Induced aggravation of oxidative DNA damage as a targeted synthetic lethal approach in mismatch repair-deficient tumors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A09.

Trilateral Retinoblastoma in a Patient With Peutz-Jeghers Syndrome

Germline loss of function mutations in tumor suppressor genes RB1 and LKB1/STK11 are associated with the autosomal dominant cancer predisposing syndromes familial retinoblastoma and Peutz-Jeghers syndrome (PJS), respectively. We present a rare case of a young woman with trilateral retinoblastoma diagnosed as an infant who survived and was then diagnosed with PJS as a teenager. There was no family history of either disorder. Analysis of the LKB1/STK11 gene sequence identified a germline frameshift mutation (c.107del) leading to a nonsense mutation near the N-terminus of the protein, confirming a clinical diagnosis of Peutz-Jeghers syndrome. Extensive RB1 gene analysis failed to detect germline mutations or deletions, and immunohistochemical analysis of her ocular tumors demonstrated nuclear staining of immunoreactive pRB. This result suggests that the RB1 gene is intact. We estimate the chance of trilateral retinoblastoma and PJS occurring in the same individual at approximately 1 in 134 billion live births, and we discuss the possibility that this case could be explained by a putative modifier of pRB action that is associated with the LKB1/STK11 pathway.

More epigenetic hits than meets the eye: microRNAs and genes associated with the tumorigenesis of retinoblastoma

Retinoblastoma (RB), a childhood neoplasia of the retinoblasts, can occur unilaterally or bilaterally, with one or multiple foci per eye. RB is associated with somatic loss of function of both alleles of the tumor suppressor gene RB1. Hereditary forms emerge due to germline loss of function mutations in RB1 alleles. RB has long been the prototypic “model” cancer ever since Knudson's “two-hit” hypothesis. However, a simple two-hit model for RB is challenged by an increasing number of studies documenting additional hits that contribute to RB development. Here we review the genetics and epigenetics of RB with a focus on the role of small non-coding RNAs (microRNAs) and on novel findings indicating the relevance of DNA methylation in the development and prognosis of this neoplasia. Studies point to an elaborated landscape of genetic and epigenetic complexity, in which a number of events and pahtways play crucial roles in the origin and prognosis of RB. These include roles for microRNAs, inprinted loci, and parent-of-origin contributions to RB1 regulation and RB progression. This complexity is also manifested in the structure of the RB1 locus itself: it includes numerous repetitive DNA segments and retrotransposon insertion elements, some of which are actively transcribed from the RB1 locus. Altogether, we conclude that RB1 loss of function represents the tip of an iceberg of events that determine RB development, progression, severity, and disease risk. Comprehensive assessment of personalized RB risk will require genetic and epigenetic evaluations beyond RB1 protein coding sequences.

Deletion of the Tumor Suppressor Gene NF1 Is An Alternative Mechanism for Aberrant Activation of the RAS Pathway and Is Found in 11% of Acute Myeloid Leukemia.

Abstract 401The NF1 gene encodes neurofibromin 1, a Ras-specific guanosine triphosphatase-activating protein (GAP) that negatively regulates the p21ras (Ras) family of signaling proteins by accelerating GTP hydrolysis. Germline loss of function mutations of NF1 lead to Neurofibromatosis type I. Carriers of the mutations develop benign neurofibromas and are predisposed to neuronal tumors, but also to juvenile myelomonocytic leukemia (JMML), MDS, and AML. In addition, in children suffering from CMML without germline NF1 mutations, acquired loss of function mutations of NF1 have been reported. In contrast, the role of NF1 in adult myeloid malignancies has not been studied in detail. Therefore, we first evaluated NF1 gene expression in 272 AML cases which were analyzed by Affymetrix HG-U133 Plus 2.0 microarrays. These included cases with t(15;17)(q22;q12) (n=15), t(8;21)(q22;q22) (n=16), inv(16)(p13q22) (n=7), t(11q23)/MLL-rearrangement (n=10), complex aberrant karyotype (n=47), normal karyotype (n=97), and with various other genetic abnormalities (n=80). The median NF1 expression intensity was 131.6 (range 35.2 - 457.5). 68 cases showed an expression intensity of NF1 below 98.6 (first quartile). In this cohort cases with t(8;21) (n=10) or complex karyotype (n=18) were over-represented (Chi-square: p<0.0001 and p=0.021, respectively), while cases with normal karyotype (n=16) were under-represented (Chi-square: p=0.016). In 54/68 of the latter cases with low expression material was availabel for FISH analysis with a probe spanning the NF1 locus. Remarkably, in 11/54 of these cases (20.4%) a NF1 deletion was observed by interphase FISH (% of cells with NF1 deletion median 90% (range: 60-99%). The mean±SD NF1 expression intensity in cases with NF1 deletion was 60.4±17.7 as compared to 75.5±18.0 in cases with 2 NF1 copies (p=0.023). Chromosome banding analysis in these 11 cases revealed a complex karyotype (n=7), a normal karyotype (n=2), an inv(3)(q21q26), and a 5q-deletion accompanied by trisomy 21, respectively. To further investigate the incidence of NF1 deletion in myeloid malignancies 425 additional patients were analyzed by FISH for NF1 deletion using a 420 kb probe spanning the NF1 gene. A heterozygous NF1 deletion was observed in 30/425 (7.1%) patients: de novo AML: 13/142 (9.2%), s-AML: 5/39 (12.8%), t-AML: 4/13 (31%), CMML: 5/99 (5.6%), MDS: 0/122 (0%), MPN: 3/10 (30%). Chromosome banding analysis in the NF1-deleted cases revealed a normal karyotype (n=4), an inv(16)(p13q22) (n=6), an inv(3)(q21q26) (n=6), a complex aberrant karyotype (n=5) or other abnormalities (n=9). In conclusion, NF1 deletions occur in 11% of AML, 5% of CMML and 3/10 MPN and therefore are a frequent and important alternative genetic mechanism for activating the RAS pathway in adult myeloid malignancies. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Use of PRKO mice to study the role of progesterone in mammary gland development.

To better understand the distinct physiological roles played by progesterone and estrogen receptors (PR and ER) as well as to study directly PR function in an in vivo context, a novel mutant mouse strain, the PR knockout (PRKO) mouse, was generated carrying a germline loss of function mutation at the PR locus. Mouse mammary gland development has been examined in PRKO mice using reciprocal transplantation experiments to investigate the effects of the stromal and epithelial PRs on ductal and lobuloalveolar development. The absence of PR in transplanted donor epithelium, but not in recipient stroma, prevented normal lobuloalveolar development in response to estrogen and progesterone treatment. Conversely, the presence of PR in the transplanted donor epithelium, but not in the recipient stroma, revealed that PR in the stroma may be necessary for ductal development. Stimulation of ductal development by the PR may, therefore, be mediated by an unknown secondary signaling molecule, possibly a growth factor. The continued stimulation of the stromal PR appears to be dependent on reciprocal signal(s) from the epithelium. Thus, the combination of gene knockout and reciprocal transplantation technologies has provided some new insights into the role of stromal-epithelial interactions and steroid hormones in mammary gland development.