Two-hit Hypothesis Research Articles

Analysis of ARMC5 expression in human tissues

Mutations in ARMC5 gene have been recently identified as the main cause of Primary Macronodular Adrenocortical Hyperplasia (PMAH). PMAH patients have an ARMC5 germline mutation and, in addition, somatic tissue-specific mutations. This is consistent with the two-hit hypothesis of tumorigenesis and suggests that ARMC5 may be a tumor suppressor gene. As its function is still unclear, we analyzed the expression of the four ARMC5 isoforms in 46 normal human tissues. This showed that at least one ARMC5 isoform is ubiquitously expressed throughout the body; however, only 7 tissues expressed all isoforms, including the adrenal gland and the brain. Interestingly, the highest expression for ARMC5 in the brain is in the pituitary gland. The isoform ARMC5-003 was present in most endocrine tissues including the pituitary, adrenal glands and the pancreas. In this report, we present new data about the ARMC5 expression pattern in human tissues; its wide expression in brain, pituitary gland and other tissues suggest that mutations may be responsible for additional pathologies, beyond what is already known in PMAH and meningiomas.

Phylogeny of Wilms tumor?

Phylogeny of Wilms tumor?

Morphine paradoxically prolongs neuropathic pain in rats by amplifying spinal NLRP3 inflammasome activation

Opioid use for pain management has dramatically increased, with little assessment of potential pathophysiological consequences for the primary pain condition. Here, a short course of morphine, starting 10 d after injury in male rats, paradoxically and remarkably doubled the duration of chronic constriction injury (CCI)-allodynia, months after morphine ceased. No such effect of opioids on neuropathic pain has previously been reported. Using pharmacologic and genetic approaches, we discovered that the initiation and maintenance of this multimonth prolongation of neuropathic pain was mediated by a previously unidentified mechanism for spinal cord and pain-namely, morphine-induced spinal NOD-like receptor protein 3 (NLRP3) inflammasomes and associated release of interleukin-1β (IL-1β). As spinal dorsal horn microglia expressed this signaling platform, these cells were selectively inhibited in vivo after transfection with a novel Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Multiday treatment with the DREADD-specific ligand clozapine-N-oxide prevented and enduringly reversed morphine-induced persistent sensitization for weeks to months after cessation of clozapine-N-oxide. These data demonstrate both the critical importance of microglia and that maintenance of chronic pain created by early exposure to opioids can be disrupted, resetting pain to normal. These data also provide strong support for the recent "two-hit hypothesis" of microglial priming, leading to exaggerated reactivity after the second challenge, documented here in the context of nerve injury followed by morphine. This study predicts that prolonged pain is an unrealized and clinically concerning consequence of the abundant use of opioids in chronic pain.

Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.

The review outlines the diagnosis, prevention strategies, and possible treatment options for acute and chronic antibody-mediated rejection (AMR). Although rare, severe acute AMR (aAMR) usually occurs in patients with high mean fluorescence intensity despite serial dilutions or high-titer preformed class I donor-specific alloantibodies (DSA). The diagnosis is suspected when allograft dysfunction occurs with DSA, diffuse C4d staining, and a microvascular injury, and may be aided by the aAMR score. However, the incidence of and treatment approach to combined T-cell-mediated rejection (TCMR) with DSA present and some but not all features of AMR is yet to be determined. Chronic liver allograft AMR is characterized by low-grade chronic inflammation and progressive fibrosis with DSA, the chronic AMR (cAMR) score may facilitate diagnosis. The 'two-hit' hypothesis, whereby a coexistent insult upregulates human leukocyte antigen class II target antigens on the microvascular endothelium, may explain why suboptimal donors with lower sensitization levels might suffer from acute AMR and those with chronic complications (e.g., recurrent original disease) might be more susceptible to chronic AMR. Although treatment algorithms are needed, prevention is preferable and at a minimum includes transfusion minimization, and medication adherence. Severe acute AMR is rare but diagnosable, and there is need to determine the incidence of and optimal therapy for less severe combined AMR and TCMR. Chronic AMR is likely more common and of significant relevance to long-term allograft survival improvement. The two-hit hypothesis may help to explain the rarity of both findings and shed insight onto future prevention and treatment strategies.

Stress and neuroinflammation: a systematic review of the effects of stress on microglia and the implications for mental illness.

RationalePsychosocial stressors are a well-documented risk factor for mental illness. Neuroinflammation, in particular elevated microglial activity, has been proposed to mediate this association. A number of preclinical studies have investigated the effect of stress on microglial activity. However, these have not been systematically reviewed before.ObjectivesThis study aims to systematically review the effects of stress on microglia, as indexed by the histological microglial marker ionised calcium binding adaptor molecule 1 (Iba-1), and consider the implications of these for the role of stress in the development of mental disorders.MethodsA systematic review was undertaken using pre-defined search criteria on PubMed and EMBASE. Inclusion and data extraction was agreed by two independent researchers after review of abstracts and full text.ResultsEighteen studies met the inclusion criteria. These used seven different psychosocial stressors, including chronic restraint, social isolation and repeated social defeat in gerbils, mice and/or rats. The hippocampus (11/18 studies) and prefrontal cortex (13/18 studies) were the most frequently studied areas. Within the hippocampus, increased Iba-1 levels of between 20 and 200 % were reported by all 11 studies; however, one study found this to be a duration-dependent effect. Of those examining the prefrontal cortex, ∼75 % found psychosocial stress resulted in elevated Iba-1 activity. Elevations were also consistently seen in the nucleus accumbens, and under some stress conditions in the amygdala and paraventricular nucleus.ConclusionsThere is consistent evidence that a range of psychosocial stressors lead to elevated microglial activity in the hippocampus and good evidence that this is also the case in other brain regions. These effects were seen with early-life/prenatal stress, as well as stressors in adulthood. We consider these findings in terms of the two-hit hypothesis, which proposes that early-life stress primes microglia, leading to a potentiated response to subsequent stress. The implications for understanding the pathoaetiology of mental disorders and the development of new treatments are also considered.

Genetic influences in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a metabolic liver disease with widely variable phenotypes extending from simple steatosis, through nonalcoholic steatohepatitis (NASH) to cirrhosis and hepatocellular carcinoma. Inevitably, this reflects the interplay of well-recognized environmental factors and disease associations such as obesity and insulin resistance with host genetic factors, which are polygenic or complex in nature. Most of the observed phenotypic variability will probably be explained by variations in single nucleotide polymorphism frequency, although knowledge of the effect of most polymorphisms on biologic function is currently limited. Several observational studies of kindred with NASH suggest a genetic contribution. Most data characterizing genetic variation in different NAFLD phenotypes is derived from case-control association studies involving putative candidate genes. These candidate genes have been selected largely based upon the "two-hit hypothesis" of the pathogenesis of NAFLD, although other hypothesis-independent approaches can also be informative in gene selection. Thus far, candidate gene association studies have had significant limitations such as small cohort sizes and poor reproducibility. Rapid technologic developments are increasing the capability of detecting genetic variation. Identification of the genetic contribution to NAFLD will inform theories of disease pathogenesis and progression and ultimately improve management.

An autoregulatory network between menin and pri-miR-24-1 is required for the processing of its specific modulator miR-24-1 in BON1 cells.

Multiple endocrine neoplasia type 1 (MEN1) is a rare hereditary cancer complex syndrome manifesting a variety of endocrine and non-endocrine neoplasms and lesions. MEN1 is characterized by tumours of the parathyroids, of the neuroendocrine cells of the gastroenteropancreatic tract, and of the anterior pituitary. The MEN1 gene, a tumour suppressor gene, encodes the menin protein. Loss of heterozygosity (LOH) at 11q13 is typical of MEN1 tumours in agreement with Knudson's two-hit hypothesis. We previously showed that the MEN1 parathyroid tumorigenesis is under the control of an "incoherent feedback loop" between miR-24-1 and the menin protein that generates a "Gene Regulatory Network" (GRN) that mimics the second hit of Knudson's hypothesis and that could buffer the effect of the stochastic factors that contribute to the onset and progression of this disease. Here we show, in the BON1 cell line derived from lymphnode metastasis of a human carcinoid tumour of the pancreas, that menin binds specifically to the primary RNA sequence pri-miR-24-1 by promoting the miR-24-1 biogenesis. Network simulation showed a new feed-forward loop between menin, microRNA-24-1 and Musashi-1 proteins. This result shows a novel mechanism whereby menin, a RNA-binding protein, facilitates the processing of its specific miRNA by regulating the dynamics of the menin-miR-24 Gene Regulatory Network at the level of pri-miRNA processing.

Subclinical Keratoconus Detection in Identical Twins

ABSTRACT Purpose To report clinical keratoconus in only one eye of two identical female twins, along with subclinical disease in the fellow eyes, elaborating on the two-hit hypothesis of ectasia development, which relates to the combination of genetics and the impact of environment. Methods Case report and literature review. Results Two identical 48-year-old female twins were presented for clinical evaluation. Mild keratoconus was detected in the right eye of twin 1, characterized by classic slit-lamp findings (exacerbated corneal nerves and incomplete Fleisher's ring) and front surface curvature abnormalities, including asymmetry of the inferior–superior curvature at 6 mm (I–S value) of 2.78 D and a grade 1 Topographic Keratoconus Classification pattern. Topographic Keratoconus Classification was possible in the fellow eye of twin 1 (I–S value = 1.22 D) and negative in both eyes of twin 2 (I–S value = −0.46 OD and −0.13 OS). Ambrósio's Relational Thickness for the maximal progression meridian was 209 and 354 in twin 1 and 360 and 392 in twin 2 in the right and left eyes respectively. The final deviation value of the Belin–Ambrósio Enhanced Ectasia Display was 4.54 and 1.47 in twin 1 and 1.7 and 1.35 in twin 2. Conclusion Corneal tomography data provide a better representation of corneal genotype in detecting mild, subclinical, or forme fruste keratoconus in the fellow eyes with normal topography of these twins. These cases present high risk or susceptibility for ectasia progression if environmental factors are associated (second hit). How to cite this article Guerra G, de Oliveira VB, Ferreira I, Ramos I, Belin MW, Ambrósio R Jr. Subclinical Keratoconus Detection in Identical Twins. Int J Kerat Ect Cor Dis 2016;5(1):35-39.

Abstract A1-32: The Stanford-TCGA portal: An interactive web/mobile interface for exploring the clinical phenotypic relevance of specific cancer drivers

Abstract The Stanford-TCGA Portal (http://genomeportal.stanford.edu/pan-tcga) enables users to easily navigate cancer genomic/proteomic data of cancer patients with their clinical information from the Cancer Genome Atlas (TCGA) project. As of 2014 January, the TCGA processed thousands of samples from over 20 cancer types and the data is publically available. This huge data set can offer many valuable insights about cancer research. However, exploring data from TCGA remains a challenge, particularly for researchers and others who lack formal bioinformatics training. With the Stanford-TCGA Portal, anyone can use a personal device such as laptops or smart phones to explore the TCGA data in regards to clinically relevant questions. For example, “What genes are associated with advanced breast cancer?”, “What is the difference in the frequency of TP53 mutations between diffuse and intestinal gastric cancers?”, or “What is the frequency of copy number deletion of APC for samples with/without PIK3CA mutations?”. While several websites such as cBio portal or UCSC cancer genomics browser was developed in order to make TCGA data more accessible to a community, there are few interactive features that enable querying of clinically-relevant phenotypic associations with drivers and modifier genes. The Stanford-TCGA portal allows users to navigate TCGA data in three different ways; i) search for clinically relevant genes/micro RNAs (miRs)/proteins by names, cancer types or clinical parameters, ii) profile genomic/proteomic changes by clinical parameters in a cancer type, or iii) test two-hit hypothesis. Thus, one can address specific questions regarding the association of cancers drivers with clinical parameters and outcome. We identify clinically relevant genes/miRs/proteins by our rigorous statistical analysis, which has been previously published, from 17 cancer types with 19 clinical parameters such as clinical stage or smoking history. Basically, elastic-net method estimates an optimal multiple linear regularized regression of the clinical parameters on the space of genomic/proteomic features. This analysis identified the set of top gene predictors of each clinical parameter in each cancer type respectively. Users can easily access the lists of these identified genes through the Stanford-TCGA Portal. In summary, the Stanford-TCGA Portal enables the cancer research community and others to fully utilize TCGA data. It provides simple yet clinically relevant information for web and mobile interface, thus one can examine queries and test hypothesis regarding genomic/proteomic alterations in cancers from any time and place. This will be an important step towards translation of genomic/proteomic data into clinics. Citation Format: HoJoon Lee, Jennifer Palm, Hanlee Ji. The Stanford-TCGA portal: An interactive web/mobile interface for exploring the clinical phenotypic relevance of specific cancer drivers. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-32.

NGTS-04SUGGESTION OF TP53 GERMLINE GENETIC TESTING BASED ON RESULTS FROM NEXT GENERATION SEQUENCING OF TUMOR SPECIMEN: A CASE REPORT

Molecular testing provides clinicians with a unique characterization of a patient's tumor. Next generation sequencing (NGS) based genetic analysis is the most commonly used tool for multiple cancer pathologies and has implications for better understanding tumorigenesis and designing potential targeted treatments. We report the case of a 52-year-old male diagnosed with anaplastic oligodendroglioma at age 50. NGS analysis of tumor specimen detected two mutations in the tumor suppressor gene TP53. Somatic TP53 mutations are frequently identified in a variety of human cancers, including primary brain tumors. Germline TP53 mutations are associated with Li-Fraumeni Syndrome (LFS), a condition in which individuals are at high risk for developing multiple primary tumors during their lifetime, typically at earlier ages of onset than the general population. Germline genetic testing is indicated when an individual's personal and/or family history raises the clinician's suspicion for the disease. Our patient's past medical history and family history were noncontributory and his presentation of disease was consistent with an isolated lesion. However, Knudson's two-hit hypothesis states that cells with pre-existing germline mutations in key genes are at high risk to become tumor cells when a somatic mutation occurs in the second allele. Therefore, the presence of two somatic TP53 mutations raised the question of whether one was also present in the germline. Subsequent germline genetic testing did detect one of the TP53 mutations, consistent with a diagnosis of LFS. This case is an example of another application of molecular tumor testing to individualize patient care. Further investigation of patients with known LFS and other hereditary cancer syndromes revealed similar molecular results in their tumor samples. While some individuals may have two somatic TP53 mutations and negative germline testing, this molecular finding may raise the clinician's awareness that further genetic work-up is warranted.

The Cancer Genome Atlas Clinical Explorer: a web and mobile interface for identifying clinical-genomic driver associations.

BackgroundThe Cancer Genome Atlas (TCGA) project has generated genomic data sets covering over 20 malignancies. These data provide valuable insights into the underlying genetic and genomic basis of cancer. However, exploring the relationship among TCGA genomic results and clinical phenotype remains a challenge, particularly for individuals lacking formal bioinformatics training. Overcoming this hurdle is an important step toward the wider clinical translation of cancer genomic/proteomic data and implementation of precision cancer medicine. Several websites such as the cBio portal or University of California Santa Cruz genome browser make TCGA data accessible but lack interactive features for querying clinically relevant phenotypic associations with cancer drivers. To enable exploration of the clinical–genomic driver associations from TCGA data, we developed the Cancer Genome Atlas Clinical Explorer.DescriptionThe Cancer Genome Atlas Clinical Explorer interface provides a straightforward platform to query TCGA data using one of the following methods: (1) searching for clinically relevant genes, micro RNAs, and proteins by name, cancer types, or clinical parameters; (2) searching for genomic/proteomic profile changes by clinical parameters in a cancer type; or (3) testing two-hit hypotheses. SQL queries run in the background and results are displayed on our portal in an easy-to-navigate interface according to user’s input. To derive these associations, we relied on elastic-net estimates of optimal multiple linear regularized regression and clinical parameters in the space of multiple genomic/proteomic features provided by TCGA data. Moreover, we identified and ranked gene/micro RNA/protein predictors of each clinical parameter for each cancer. The robustness of the results was estimated by bootstrapping. Overall, we identify associations of potential clinical relevance among genes/micro RNAs/proteins using our statistical analysis from 25 cancer types and 18 clinical parameters that include clinical stage or smoking history.ConclusionThe Cancer Genome Atlas Clinical Explorer enables the cancer research community and others to explore clinically relevant associations inferred from TCGA data. With its accessible web and mobile interface, users can examine queries and test hypothesis regarding genomic/proteomic alterations across a broad spectrum of malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0226-3) contains supplementary material, which is available to authorized users.

Knudson's hypothesis revisited in Indian retinoblastoma patients.

Retinoblastoma (RB) is the most common primary intraocular malignancy affecting children under 5 years of age. This study aims to correlate the clinical parameters with RB1 mutation in the light of Knudson's two-hit hypothesis in Indian RB patients. We analyzed the clinical details of 73 RB patients visiting Aravind Eye Hospital, Madurai, India, between January and October 2012. Data on gender, presenting age and sign, laterality, number of tumors in each eye and family history were collected. A semi log plot was derived based on Knudson's two-hit hypothesis. Genetic analysis of RB1 was carried out to identify the two hits. The mean age at diagnosis for unilateral and bilateral cases was 24.0 ± 15.1 and 9.8 ± 11.5 months, respectively. Familial RB was seen in 13 (17.8%) patients of whom 11 were bilateral. Multiple tumors were observed more frequently in bilateral than in unilateral cases. All unilateral and bilateral patients followed the two-hit and one-hit curves, respectively, confirming Knudson's hypothesis in Indian patients. Genetic analysis identified two somatic mutations in tumor samples of sporadic unilateral cases. Among the two bilateral patients, one received the first hit from her father and the other patient developed a de novo germline mutation during early development. The two-hit hypothesis has been reestablished in Indian patients. Genetic analysis of tumor samples has also complemented the statistical analysis to reaffirm the two hits in tumor development.

Loss of heterozygosity: what is it good for?

BackgroundLoss of heterozygosity (LOH) is a common genetic event in cancer development, and is known to be involved in the somatic loss of wild-type alleles in many inherited cancer syndromes. The wider involvement of LOH in cancer is assumed to relate to unmasking a somatically mutated tumour suppressor gene through loss of the wild type allele.MethodsWe analysed 86 ovarian carcinomas for mutations in 980 genes selected on the basis of their location in common regions of LOH.ResultsWe identified 36 significantly mutated genes, but these could only partly account for the quanta of LOH in the samples. Using our own and TCGA data we then evaluated five possible models to explain the selection for non-random accumulation of LOH in ovarian cancer genomes: 1. Classic two-hit hypothesis: high frequency biallelic genetic inactivation of tumour suppressor genes. 2. Epigenetic two-hit hypothesis: biallelic inactivation through methylation and LOH. 3. Multiple alternate-gene biallelic inactivation: low frequency gene disruption. 4. Haplo-insufficiency: Single copy gene disruption. 5. Modified two-hit hypothesis: reduction to homozygosity of low penetrance germline predisposition alleles. We determined that while high-frequency biallelic gene inactivation under model 1 is rare, regions of LOH (particularly copy-number neutral LOH) are enriched for deleterious mutations and increased promoter methylation, while copy-number loss LOH regions are likely to contain under-expressed genes suggestive of haploinsufficiency. Reduction to homozygosity of cancer predisposition SNPs may also play a minor role.ConclusionIt is likely that selection for regions of LOH depends on its effect on multiple genes. Selection for copy number neutral LOH may better fit the classic two-hit model whereas selection for copy number loss may be attributed to its effect on multi-gene haploinsufficiency. LOH mapping alone is unlikely to be successful in identifying novel tumour suppressor genes; a combined approach may be more effective.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0123-z) contains supplementary material, which is available to authorized users.

Abstract 4857: The Stanford-Cancer Genome Atlas Portal: A web/mobile navigation interface for exploring the clinical associations of cancer drivers

Abstract The NIH Cancer Genome Atlas (TCGA) project has generated enormous data sets covering >20 malignancies and provides many valuable insights into the underlying genetic and genomic basis of cancer. However, exploring the association between TCGA results and clinical phenotype remains a challenge, particularly for individuals lacking formal bioinformatics training. This will be an important step towards the clinical translation of cancer genomic/proteomic data. Several websites such as cBio portal or UCSC genome browser make TCGA data accessible but these sites have few interactive features for querying clinically-relevant phenotypic associations with cancer drivers. To enable exploration of the clinical associations of the TCGA data, we developed the Stanford-TCGA Portal (http://genomeportal.stanford.edu/pan-tcga). Using a web/mobile interface, this website enables easy navigation of cancer genomic/proteomic/clinical data provided by the TCGA and pose clinically relevant questions. For example, “What genes are associated with advanced breast cancer?” or “What is the frequency of copy number deletion of APC for samples with/without PIK3CA mutations?”. The Stanford-TCGA portal interface provide querying of TCGA data in three different ways; i) search for clinically relevant genes/micro RNAs (miRs)/proteins by names, cancer types or clinical parameters, ii) profile genomic/proteomic changes by clinical parameters in a cancer type, or iii) test two-hit hypotheses. Any user can easily navigate the lists of these identified genes through the Stanford-TCGA Portal. SQL queries run in the background and show the results on our portal according to user's input. To derive these associations, we relied on elastic-net estimates of optimal multiple linear regularized regression; we relied on clinical parameters in the space of multiple genomic/proteomic features provided by the TCGA data. We identified the set of top gene predictors (by 10 fold cross validation) of each clinical parameter for each cancer. The robustness of results was estimated by bootstrapping with 2,000 iterations. As a proof-of-concept, we demonstrated this approach's utility in the TCGA colorectal cancer data (HoJoon Lee et al., BMC Medical Genomics 2013) and we have since expanded our analysis to include nearly all TCGA malignancies. We identify clinically relevant genes/miRs/proteins by our statistical analysis, some which has been previously described, from 17 cancer types with 19 clinical parameters such as clinical stage or smoking history. In summary, the Stanford-TCGA Portal enables the cancer research community and others to fully utilize TCGA data. With its straightforward web/mobile interface, it provides simple yet clinically relevant associations of TCGA results. Thus, one can examine queries and test hypothesis regarding genomic/proteomic alterations in cancers from any time and place. Citation Format: HoJoon Lee, Jennifer Palm, Hanlee P. Ji. The Stanford-Cancer Genome Atlas Portal: A web/mobile navigation interface for exploring the clinical associations of cancer drivers. [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 4857. doi:10.1158/1538-7445.AM2015-4857

Do immune complexes play a role in hemolytic sequelae of intravenous immune globulin?

Intravenous immune globulin (IVIG) was developed initially as an immunoglobulin replacement therapy for primary humoral immunodeficiency, but is now widely used in the treatment of autoinflammatory and autoimmune pathologies. In a small number of patients, hemolytic sequelae have been observed after IVIG administration. The lack of a simple one-to-one correlation between measurable hemagglutinins and hemolysis has led to complicated hypotheses involving coincident necessary variables (e.g., a two-hit hypothesis) and also to the positing of causal factors other than hemagglutinins. One such hypothesis is that immune complexes (ICs) contained within IVIG lead to hemolysis. IVIG-mediated hemolysis was addressed at a recent meeting sponsored by the Food and Drug Administration; the Plasma Protein Therapeutics Association; and the National Heart, Lung, and Blood Institute. The primary literature was reviewed at this meeting followed by detailed discussion. Participants concluded that there is both a theoretical basis by which ICs could contribute to hemolysis after IVIG administration and some published data in support of such a possibility. However, the reported data contain substantial caveats, and the existing evidence does not rise to a level sufficient to either confirm or reject a role for ICs. More detailed and focused human studies will be required to further assess the potential role of ICs in IVIG induced hemolysis. This paper summarizes the relevant literature and expands upon the conclusions of this workshop.

Clonally related uterine leiomyomas are common and display branched tumor evolution.

Uterine leiomyomas are extremely frequent benign smooth muscle tumors often presenting as multiple concurrent lesions and causing symptoms such as abnormal menstrual bleeding, abdominal pain and infertility. While most leiomyomas are believed to arise independently, a few studies have encountered separate lesions harboring identical genetic changes, suggesting a common clonal origin. To investigate the frequency of clonally related leiomyomas, genome-wide tools need to be utilized, and thus little is known about this phenomenon. Using MED12 sequencing and SNP arrays, we searched for clonally related uterine leiomyomas in a set of 103 tumors from 14 consecutive patients who entered hysterectomy owing to symptomatic lesions. Whole-genome sequencing was also utilized to study the genomic architecture of clonally related tumors. This revealed four patients to have two or more tumors that were clonally related, all of which lacked MED12 mutations. Furthermore, some tumors were composed of genetically distinct subclones, indicating a nonlinear, branched model of tumor evolution. DEPDC5 was discovered as a novel tumor suppressor gene playing a role in the progression of uterine leiomyomas. Perhaps counterintuitively—considering Knudson's two-hit hypothesis—a large shared deletion was followed by different truncating DEPDC5 mutations in four clonally related leiomyomas. This study provides insight into the intratumor heterogeneity of these tumors and suggests that a shared clonal origin is a common feature of leiomyomas that do not carry an MED12 mutation. These observations also offer one explanation to the common occurrence of multiple concurrent lesions.

Loss of PTPRM associates with the pathogenic development of colorectal adenoma-carcinoma sequence.

Identification and functional analysis of genes from genetically altered chromosomal regions would suggest new molecular targets for cancer diagnosis and treatment. Here we performed a genome-wide analysis of chromosomal copy number alterations (CNAs) in matching sets of colon mucosa-adenoma-carcinoma samples using high-throughput oligonucleotide microarray analysis. In silico analysis of NCBI GEO and TCGA datasets allowed us to uncover the significantly altered genes (p ≤ 0.001) associated with the identified CNAs. We performed quantitative PCR analysis of the genomic and complementary DNA derived from primary mucosa, adenoma, and carcinoma samples, and confirmed the recurrent loss and down-regulation of PTPRM in colon adenomas and carcinomas. Functional characterization demonstrated that PTPRM negatively regulates cell growth and colony formation, whereas loss of PTPRM promotes oncogenic cell growth. We further showed that, in accordance to Knudson's two-hit hypothesis, inactivation of PTPRM in colon cancer was mainly attributed to loss of heterozygosity and promoter hypermethylation. Taken together, this study demonstrates a putative tumor suppressive role for PTPRM and that genetic and epigenetic alterations of PTPRM may contribute to early step of colorectal tumorigenesis.

Lipid Emulsion Halts the Progression of Non‐Alcoholic Fatty Liver Disease (NAFLD) in Nonobese Mice Fed a Liquid High Carbohydrate Diet

A two‐hit hypothesis has been proposed for the etiology of NAFLD: first, increased lipogenesis results in triacylglycerol (TG) accumulation, then inflammatory stimuli, e.g., saturated fatty acids (SFA)‐induced lipotoxicity and inflammation, cause disease progression. Previously, we showed that NAFLD‐like symptoms developed in mice fed a liquid high carbohydrate diet (76% en‐%, HCD) for 5‐wk, while mice fed HCD containing 13.5% (en‐%) of lipid emulsion (LE, Intralipid®) exhibited less hepatic TG accumulation and inflammation. However, it is unknown whether LE can cease the first hit of NAFLD once TG accumulation has begun. To test this, male C57BL/6 mice (6 wk; n=8/group) were fed either HCD for 5 wk (HCD) or HCD for 2.5 wk followed by HCD with LE for 2.5 wk (HCD/LE). At this time, liver TG in mice fed HCD/LE was 60% lower (P<0.001) as compared to mice fed HCD. Hepatic mRNAs of PNPLA3, associated with NAFLD,and several lipogenic genes, e.g., acetyl‐CoA carboxylase 1, fatty acid synthase, and stearoyl‐CoA desaturase 1, assayed by qRT‐PCR, were decreased by 42‐93% in HCD/LE vs. HCD mice (all P<0.001). We further analyzed liver total fatty acid composition, determined as fatty acid methyl esters by GC‐MS, and found that HCD/LE mice had lower levels of C14:0, C16:0 and C18:1 (n‐9) as compared to HCD mice (all P<0.001). In conclusion, in the presence of a HCD, LE can halt the first hit of NAFLD and lower the levels of saturated FA, reducing lipotoxicity and lipogenesis.

Abstract T P231: B- Cell Depletion Decreases Lesion Burden in a Murine Model of Cerebral Cavernous Malformation Disease

Introduction: The cerebral cavernous malformation (CCM), a common cerebrovascular anomaly, predisposes patients to a lifetime risk of seizures, hemorrhagic stroke, and other neurological sequelae. Previous studies revealed robust inflammatory cell infiltration and selective synthesis of IgG within the lesions. Recently, in situ antigen-driven B- cell clonal expansion and immune complex deposits were demonstrated in CCM lesions. However, we do not know whether B cells indeed could play a role in CCM genesis and maturation. Therefore we hypothesize B-cell depletion could inhibit CCM development in a murine model of CCMs. Methods: Based on the two-hit hypothesis, we crossed Ccm3 heterozygotes into a Trp53 background, a mouse model that demonstrated 100% penetrance with severe lesion burden. Three weeks after birth, 5 CCM3+/-Trp53-/- mice were assigned into treatment group, receiving anti-mouse BR3 (Genentech) to deplete B cells with IP injection once a week (5mg/kg/week) for a total of 12-15 weeks. At 4 months of age, the mouse brains were harvested and fixed. B cell depletion was demonstrated by FACS analysis of peripheral blood and spleen cell suspensions, further confirmed in the spleens via immunohistochemistry. CCM lesion burden was assessed using either poisson regression (number) or student t test (area). Results: CD19+, CD22+, and CD45R/B220+ B cells of peripheral blood and spleen cell suspensions was significantly decreased in all 5 CCM3+/-Trp53-/- mice treated with anti-mouse BR3, as compared with those in the control group. B cell depletion was further confirmed in spleen. Anti-mouse BR3-treated mice exhibited a significantly lower CCM burden, including the number of ballooned capillaries (stage 1) and multicavernous lesions (stage 2), and the areas of stage 2 lesions per animal, as compared to 15 placebo mice. Infiltrated B cells in CCM3+/-Trp53-/- CCM lesions were also depleted. Conclusions: This represented the first report of therapeutic benefit of B- cell depletion therapy in the development and progression of CCMs and provide proof of principle that B cells is a critical step in CCM lesion genesis and maturation.

Dimerization of the PTEN Tumor Suppressor and its Structural Characterization by SAXS

The PTEN tumor suppressor is a dual-specificity phosphatase whose main target is the phosphatidylinositoltriphosphate (PI(3,4,5)P3) pool in the inner plasma membrane. It acts as the PI3K antagonist in the PI3K/Akt signaling pathway that controls cell growth and apoptosis and is the second most frequently mutated protein in human cancers. Because the propensity for tumor formation depends on PTEN dose in a way that is inconsistent with the “two-hit hypothesis”, it was postulated that PTEN multimers may form the active species of the phosphatase, and recently strong evidence has been presented for the formation of functional PTEN dimers in the cell (Papa et al., Cell 157, 2014, 595). Here, we use SAXS to investigate the multimerization of PTEN in buffer and show that it indeed forms dimers following elution from a size-exclusion column as a monomer. Electron density envelopes for the PTEN monomer and dimer obtained from SAXS could clearly be distinguished and were assigned by placing the PTEN crystal structure which was earlier determined for a truncated protein. In addition, the monomer envelope was validated by μs-long all-atom MD simulations of full-length PTEN. In these simulations, the auto-inhibitory, flexible C-terminal tail associates closely with the PTEN core domains while hopping between different bound conformations. The equilibrium ensemble of the resulting structures is in excellent agreement with the SAXS data. A structure prediction using the Rosetta docking protocol revealed a putative dimer arrangement that fits the dimer envelope derived from SAXS very well and is consistent with neutron reflectometry results for membrane-bound PTEN.