Effect of Doxycycline-Release Anastomotic Augmentation Ring on Porcine Colorectal Anastomosis.

INTRODUCTION: Collagen degradation can lead to early postoperative weakness in colorectal anastomosis. Matrix metalloproteinase inhibitors (MMPIs) are shown to decrease collagen breakdown and enhance healing in anastomosis in animal models. Here, we evaluated the effectiveness of a novel anastomotic augmentation ring (AAR) that releases doxycycline, an MMPI, from a poly(lactic-co-glycolic) acid ring in porcine anastomoses. METHODS: Two end-to-end stapled colorectal anastomoses were performed in 20 Yorkshire-Hampshire pigs. AAR was randomly incorporated into either the proximal or distal anastomosis as treatment, while nonaugmented anastomosis served as a control. Animals were then euthanized on days 3, 4, and 5 before anastomosis explantation and burst pressure measurement. Each anastomosis site was also collected for histology, hydroxyproline content, and gene expression microarray analyses. RESULTS: No abscess or anastomotic leak was detected. Average burst pressures were not significantly different at any time point. There is no statistical difference in collagen content between the treatment group and controls. Gene expression analysis revealed no statistically significant in differentially expressed genes. However, genes related to inflammation, such as C-C motif chemokine ligand 11 (CCL11), CD70, and C-X-C motif chemokine ligand 10 (CXCL10), were upregulated (not statistically significant) in AAR compared to non-AAR anastomosis sites on days 3 and 4. CONCLUSIONS: This pilot study shows that doxycycline-release AAR is feasible and safe. While burst pressure and collagen content did not change significantly with doxycycline treatment, upregulating genes related to the inflammatory process for pathogen and debris clearance in AAR may improve the early stage of colorectal anastomotic healing.

Gene expression analysis of human prostate cell lines with and without tumor metastasis suppressor CD82.

BACKGROUND: Tetraspanin CD82 is a tumor metastasis suppressor that is known to down regulate in various metastatic cancers. However, the exact mechanism by which CD82 prevents cancer metastasis is unclear. This study aims to identify genes that are regulated by CD82 in human prostate cell lines. METHODS: We used whole human genome microarray to obtain gene expression profiles in a normal prostate epithelial cell line that expressed CD82 (PrEC-31) and a metastatic prostate cell line that does not express CD82 (PC3). Then, siRNA silencing was used to knock down CD82 expression in PrEC-31 while CD82 was re-expressed in PC3 to acquire differentially-expressed genes in the respective cell line. RESULTS: Differentially-expressed genes with a P < 0.05 were identified in 3 data sets: PrEC-31 (+CD82) vs PrEC-31(-CD82), PC3-57 (+CD82) vs. PC3-5 V (-CD82), and PC3-29 (+CD82) vs. PC3-5 V (-CD82). Top 25 gene lists did not show overlap within the data sets, except (CALB1) the calcium binding protein calbindin 1 which was significantly up-regulated (2.8 log fold change) in PrEC-31 and PC3-29 cells that expressed CD82. Other most significantly up-regulated genes included serine peptidase inhibitor kazal type 1 (SPINK1) and polypeptide N-acetyl galactosaminyl transferase 14 (GALNT14) and most down-regulated genes included C-X-C motif chemokine ligand 14 (CXCL14), urotensin 2 (UTS2D), and fibroblast growth factor 13 (FGF13). Pathways related with cell proliferation and angiogenesis, migration and invasion, cell death, cell cycle, signal transduction, and metabolism were highly enriched in cells that lack CD82 expression. Expression of two mutually inclusive genes in top 100 gene lists of all data sets, runt-related transcription factor (RUNX3) and trefoil factor 3 (TFF3), could be validated with qRT-PCR. CONCLUSION: Identification of genes and pathways regulated by CD82 in this study may provide additional insights into the role that CD82 plays in prostate tumor progression and metastasis, as well as identify potential targets for therapeutic intervention.

MTA3 Regulates Extravillous Trophoblast Invasion Through NuRD Complex.

Extravillous trophoblast (EVT) invasion is required for remodeling uterine tertiary arteries and placenta development during pregnancy. Compromised EVT invasion may contribute to the pathology of placenta-related diseases. Metastasis -associated protein 3 (MTA3) is one of the subunits of nucleosome remodeling and deacetylation (NuRD) complex that represses transcription in a histone deacetylase-dependent manner. MTA3 is reported to be down-regulated in preeclamptic placentas, suggesting its potential role in EVT invasion. Here, we investigate the role of MTA3 in EVT invasion by studying its molecular mechanisms in EVT cells. First, we confirmed MTA3 expression in the EVT cells in human placenta using immunohistochemistry. We then used lentivirus-mediated MTA3 short hairpin RNA (shRNA) to knock down MTA3 expression in EVT-derived HTR8/SVneo cells and found higher invasion capacity in MTA3 knockdown cells. Using quantitative real-time PCR, we showed higher expression of invasion-related genes matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), and transcription factor Snail in MTA3 knockdown compared with control cells. Co-immunoprecipitation-Western blot assay showed the protein-protein interaction of histone deacetylase 1 (HDAC1), a subunit of NuRD, with MTA3 in HTR8/SVneo cells. Co-immunoprecipitation-Mass spectrometry assay further identified 71 proteins interacting with MTA3, including NuRD subunits, heterochromatin proteins, epigenetics modifiers and transcription factors. This result not only indicated the involvement of NuRD complex in MTA3's function, but also demonstrated the complicated multiple co-players in MTA3 and NuRD complex mediated transcription repression in EVT. In summary, our data demonstrates that MTA3 regulates EVT invasion and related gene expression via NuRD complex in EVT.

Chromosome instability drives phenotypic switching to metastasis.

Chromosome instability (CIN) is the most striking feature of human cancers. However, how CIN drives tumor progression to metastasis remains elusive. Here we studied the role of chromosome content changes in generating the phenotypic dynamics that are required for metastasis. We isolated epithelial and mesenchymal clones from human carcinoma cell lines and showed that the epithelial clones were able to generate mesenchymal variants, which had the potential to further produce epithelial revertants autonomously. The successive acquisition of invasive mesenchymal and then epithelial phenotypes recapitulated the steps in tumor progression to metastasis. Importantly, the generation of mesenchymal variants from clonal epithelial populations was associated with subtle changes in chromosome content, which altered the chromosome transcriptome and influenced the expression of genes encoding intercellular junction (IJ) proteins, whereas the loss of chromosome 10p, which harbors the ZEB1 gene, was frequently detected in epithelial variants generated from mesenchymal clones. Knocking down these IJ genes in epithelial cells induced a mesenchymal phenotype, whereas knocking down the ZEB1 gene in mesenchymal cells induced an epithelial phenotype, demonstrating a causal role of chromosome content changes in phenotypic determination. Thus, our studies suggest a paradigm of tumor metastasis: primary epithelial carcinoma cells that lose chromosomes harboring IJ genes acquire an invasive mesenchymal phenotype, and subsequent chromosome content changes such as loss of 10p in disseminated mesenchymal cells generate epithelial variants, which can be selected for to generate epithelial tumors during metastatic colonization.

Disruption of tubular Flcn expression as a mouse model for renal tumor induction.

The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of genetically defined animal models. The FLCN gene that codes for the protein folliculin, mutated in Birt-Hogg-Dubé syndrome, presents a new target for mouse modeling of kidney cancer. Here we developed a kidney-specific knockout model by disrupting the mouse Flcn in the proximal tubules, thus avoiding homozygous embryonic lethality or neonatal mortality, and eliminating the requirement of loss of heterozygosity for tumorigenesis. This knockout develops renal cysts and early onset (6 months) of multiple histological subtypes of renal neoplasms featuring high tumor penetrance. Although the majority of the tumors were chromophobe renal cell carcinomas in affected mice under 1 year of age, papillary renal cell carcinomas predominated in the kidneys of older knockout mice. This renal neoplasia from cystic hyperplasia at 4 months to high-grade renal tumors by 16 months represented the progression of tumorigenesis. The mTOR and TGF-ß signalings were upregulated in Flcn-deficient tumors, and these two activated pathways may synergetically cause renal tumorigenesis. Treatment of knockout mice with the mTOR inhibitor rapamycin for 10 months led to the suppression of tumor growth. Thus, our model recapitulates human Birt-Hogg-Dubé kidney tumorigenesis, provides a valuable tool for further study of Flcn-deficient renal tumorigenesis, and tests new drugs/approaches to their treatment.

Overexpression of HGF Promotes HBV-Induced Hepatocellular Carcinoma Progression and Is an Effective Indicator for Met-Targeting Therapy.

Hepatitis B virus (HBV) is a well-known cause of hepatocellular carcinoma (HCC), but the regulators effectively driving virus production and HCC progression remain unclear. By using genetically engineered mouse models, we show that overexpression of hepatocyte growth factor (HGF) accelerated HCC progression, supporting the genomic analysis that an up-regulated HGF signature is associated with poor prognosis in HBV-positive HCC patients. We show that for both liver regeneration and spontaneous HCC development there is an inclusive requirement for MET expression, and when HGF induces autocrine activation the tumor displays sensitivity to a small-molecule Met inhibitor. Our results demonstrate that HGF is a driver of HBV-induced HCC progression and may serve as an effective biomarker for Met-targeted therapy. MET inhibitors are entering clinical trials against cancer, and our data provide a molecular basis for targeting the Met pathway in hepatitis B-induced HCC.

Overexpression of asparagine synthetase and matrix metalloproteinase 19 confers cisplatin sensitivity in nasopharyngeal carcinoma cells.

Platinum-based concurrent chemoradiotherapy is considered a standard treatment approach for locoregionally advanced nasopharyngeal carcinoma. However, only a minority of patients benefit from this treatment regimen compared with radiotherapy alone. Identification of a set of molecular markers predicting sensitivity of platinum-based chemotherapy may contribute to personalized treatment of patients with nasopharyngeal carcinoma for better clinical outcome with less toxicity. Previously, we generated a cisplatin-sensitive nasopharyngeal carcinoma cell line, S16, by clonal selection from CNE-2 cells and found that eIF3a is upregulated and contributes to cisplatin sensitivity by downregulating the synthesis of nucleotide excision repair proteins. In this study, we conducted a gene expression profiling analysis and found three other genes, asparagine synthetase (ASNS), choriogonadotropin α subunit (CGA), and matrix metalloproteinase 19 (MMP19), that are upregulated in the cisplatin-sensitive S16 cells compared with the CNE-2 cells. However, only ASNS and MMP19, but not CGA, contributes to cisplatin sensitivity by potentiating cisplatin-induced DNA damage and apoptosis. Thus, ASNS and MMP19, along with eIF3a, are the sensitivity factors for cisplatin treatment and may serve as potential candidate molecular markers for predicting cisplatin sensitivity of advanced nasopharyngeal carcinoma.

Roles of CDX2 and EOMES in human induced trophoblast progenitor cells.

Abnormal trophoblast lineage proliferation and differentiation in early pregnancy have been associated with the pathogenesis of placenta diseases of pregnancy. However, there is still a gap in understanding the molecular mechanisms of early placental development due to the limited primary trophoblast cultures and fidelity of immortalized trophoblast lines. Trophoblasts stem (TS) cells, an in vitro model of trophectoderm that can differentiate into syncytiotrophoblasts and extravillous trophoblasts, can be an attractive tool for early pregnancy research. TS cells are well established in mouse but not in humans due to insufficient knowledge of which trophoblast lineage-specific transcription factors are involved in human trophectoderm (TE) proliferation and differentiation. Here, we applied induced pluripotent stem cell technique to investigate the human trophoblast lineage-specific transcription factors. We established human induced trophoblast progenitor (iTP) cells by direct reprogramming the fibroblasts with a pool of mouse trophoblast lineage-specific transcription factors consisting of CDX2, EOMES, and ELF5. The human iTP cells exhibit epithelial morphology and can be maintained in vitro for more than 2 months. Gene expression profile of these cells was tightly clustered with human trophectoderm but not with human neuron progenitor cells, mesenchymal stem cells, or endoderm cells. These cells are capable of differentiating into cells with an invasive capacity, suggesting extravillous trophoblasts. They also form multi-nucleated cells which secrete human chorionic gonadotropin and estradiol, consistent with a syncytiotrophoblast phenotype. Our results provide the evidence that transcription factors CDX2 and EOMES may play critical roles in human iTP cell generation.

Serum adiponectin, resistin, and circulating soluble receptor for advanced glycation end products in colectomy patients.

AIM: Surgical trauma and associated complications are frequently related to physiological stress during colectomy. This study evaluated the response of adiponectin, resistin, and circulating soluble receptor for advanced glycation end products (sRAGE) in colectomy patients with or without an enhanced recovery protocol. METHOD: Serum samples were collected from 44 colectomy patients at 3 timframes. The surgical procedures were laparoscopic (LAP), hand-assisted laparoscopic (HALS), or open colectomy (OPEN). Adiponectin, resistin, and sRAGE levels were determined by ELISA. Repeated measures ANOVA was applied and P values < 0.05 were considered significant. RESULTS: A total of 132 (44 × 3) sera were used for analysis. Levels of adiponectin was significantly decreased between PREOP and POD3 (P < 0.001). Conversely, concentrations of resistin significantly increased from PREOP to POD1 and returned to baseline value by POD3 (P < 0.001). Serum sRAGE levels were significantly higher in LAP in comparison with HALS (P = 0.004) and OPEN (P < 0.001). sRAGE levels were significantly higher in sera of patients that underwent ERP (P < 0.001). CONCLUSIONS: Serum adiponectin, resistin, and sRAGE have the potential to develop into a panel of stress markers. Higher sRAGE levels in sera of LAP and ERP patients may be indicative of a protective and syngeristic role for colectomy recovery.

Identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory response.

The antimalaria drug chloroquine has been used as an anti-inflammatory agent for treating systemic lupus erythematosus and rheumatoid arthritis. We report that chloroquine promoted the transrepression of proinflammatory cytokines by the glucocorticoid receptor (GR). In a mouse collagen-induced arthritis model, chloroquine enhanced the therapeutic effects of glucocorticoid treatment. By inhibiting lysosome function, chloroquine synergistically activated glucocorticoid signaling. Lysosomal inhibition by either bafilomycin A1 (an inhibitor of the vacuolar adenosine triphosphatase) or knockdown of transcription factor EB (TFEB, a master activator of lysosomal biogenesis) mimicked the effects of chloroquine. The abundance of the GR, as well as that of the androgen receptor and estrogen receptor, correlated with changes in lysosomal biogenesis. Thus, we showed that glucocorticoid signaling is regulated by lysosomes, which provides a mechanistic basis for treating inflammation and autoimmune diseases with a combination of glucocorticoids and lysosomal inhibitors.

Genomic expression and single-nucleotide polymorphism profiling discriminates chromophobe renal cell carcinoma and oncocytoma.

BACKGROUND: Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but closely related entities with strong morphologic and genetic similarities. While chRCC is a malignant tumor, oncocytoma is usually regarded as a benign entity. The overlapping characteristics are best explained by a common cellular origin, and the biologic differences between chRCC and oncocytoma are therefore of considerable interest in terms of carcinogenesis, diagnosis and clinical management. Previous studies have been relatively limited in terms of examining the differences between oncocytoma and chromophobe RCC. METHODS: Gene expression profiling using the Affymetrix HGU133Plus2 platform was applied on chRCC (n = 15) and oncocytoma specimens (n = 15). Supervised analysis was applied to identify a discriminatory gene signature, as well as differentially expressed genes. High throughput single-nucleotide polymorphism (SNP) genotyping was performed on independent samples (n = 14) using Affymetrix GeneChip Mapping 100 K arrays to assess correlation between expression and gene copy number. Immunohistochemical validation was performed in an independent set of tumors. RESULTS: A novel 14 probe-set signature was developed to classify the tumors internally with 93% accuracy, and this was successfully validated on an external data-set with 94% accuracy. Pathway analysis highlighted clinically relevant dysregulated pathways of c-erbB2 and mammalian target of rapamycin (mTOR) signaling in chRCC, but no significant differences in p-AKT or extracellular HER2 expression was identified on immunohistochemistry. Loss of chromosome 1p, reflected in both cytogenetic and expression analysis, is common to both entities, implying this may be an early event in histogenesis. Multiple regional areas of cytogenetic alterations and corresponding expression biases differentiating the two entities were identified. Parafibromin, aquaporin 6, and synaptogyrin 3 were novel immunohistochemical markers effectively discriminating the two pathologic entities. CONCLUSIONS: Gene expression profiles, high-throughput SNP genotyping, and pathway analysis effectively distinguish chRCC from oncocytoma. We have generated a novel transcript predictor that is able to discriminate between the two entities accurately, and which has been validated both in an internal and an independent data-set, implying generalizability. A cytogenetic alteration, loss of chromosome 1p, common to renal oncocytoma and chRCC has been identified, providing the opportunities for identifying novel tumor suppressor genes and we have identified a series of immunohistochemical markers that are clinically useful in discriminating chRCC and oncocytoma.

Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes.

Clear cell renal cell carcinoma (ccRCC) is the most common form of adult kidney cancer, characterized by the presence of inactivating mutations in the VHL gene in most cases, and by infrequent somatic mutations in known cancer genes. To determine further the genetics of ccRCC, we have sequenced 101 cases through 3,544 protein-coding genes. Here we report the identification of inactivating mutations in two genes encoding enzymes involved in histone modification-SETD2, a histone H3 lysine 36 methyltransferase, and JARID1C (also known as KDM5C), a histone H3 lysine 4 demethylase-as well as mutations in the histone H3 lysine 27 demethylase, UTX (KMD6A), that we recently reported. The results highlight the role of mutations in components of the chromatin modification machinery in human cancer. Furthermore, NF2 mutations were found in non-VHL mutated ccRCC, and several other probable cancer genes were identified. These results indicate that substantial genetic heterogeneity exists in a cancer type dominated by mutations in a single gene, and that systematic screens will be key to fully determining the somatic genetic architecture of cancer.

Activation of the PI3K/AKT pathway induces urothelial carcinoma of the renal pelvis: identification in human tumors and confirmation in animal models.

Urothelial carcinoma of the renal pelvis is a deadly disease with an unclear tumorigenic mechanism. We conducted gene expression profiling on a set of human tumors of this type and identified a phosphatidylinositol 3-kinase (PI3K)/AKT activation expression signature in 76.9% (n = 13) of our samples. Sequence analysis found both activating mutations of PIK3CA (13.6%, n = 22) and loss of heterozygosity at the PTEN locus (25%, n = 8). In contrast, none of the other subtypes of kidney neoplasms (e.g., clear-cell renal cell carcinoma) harbored PIK3CA mutations (n = 87; P < 0.001). Immunohistochemical analysis of urothelial carcinoma samples found loss of PTEN protein expression (36.4%, n = 11) and elevation of phosphorylated mammalian target of rapamycin (mTOR; 63.6%, n = 11). To confirm the role of the PI3K/AKT pathway in urothelial carcinoma, we generated mice containing biallelic inactivation of Pten in the urogenital epithelia. These mice developed typical renal pelvic urothelial carcinomas, with an incidence of 57.1% in mice older than 1 year. Laser capture microdissection followed by PCR confirmed the deletion of Pten exons 4 and 5 in the animal tumor cells. Immunohistochemical analyses showed increased phospho-mTOR and phospho-S6K levels in the animal tumors. Renal lymph node metastases were found in 15.8% of the animals with urothelial carcinoma. In conclusion, we identified and confirmed an important role for the PI3K/AKT pathway in the development of urothelial carcinoma and suggested that inhibitors of this pathway (e.g., mTOR inhibitor) may serve as effective therapeutic agents.

Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia.

The Birt-Hogg-Dubé (BHD) disease is a genetic cancer syndrome. The responsible gene, BHD, has been identified by positional cloning and thought to be a novel tumor suppressor gene. BHD mutations cause many types of diseases including renal cell carcinomas, fibrofolliculomas, spontaneous pneumothorax, lung cysts, and colonic polyps/cancers. By combining Gateway Technology with the Ksp-Cre gene knockout system, we have developed a kidney-specific BHD knockout mouse model. BHD(flox/flox)/Ksp-Cre mice developed enlarged kidneys characterized by polycystic kidneys, hyperplasia, and cystic renal cell carcinoma. The affected BHD(flox/flox)/Ksp-Cre mice died of renal failure at approximate three weeks of age, having blood urea nitrogen levels over tenfold higher than those of BHD (flox/+)/Ksp-Cre and wild-type littermate controls. We further demonstrated that these phenotypes were caused by inactivation of BHD and subsequent activation of the mTOR pathway. Application of rapamycin, which inhibits mTOR activity, to the affected mice led to extended survival and inhibited further progression of cystogenesis. These results provide a correlation of kidney-targeted gene inactivation with renal carcinoma, and they suggest that the BHD product FLCN, functioning as a cyst and tumor suppressor, like other hamartoma syndrome-related proteins such as PTEN, LKB1, and TSC1/2, is a component of the mTOR pathway, constituting a novel FLCN-mTOR signaling branch that regulates cell growth/proliferation.

A comparison study reveals important features of agreement and disagreement between summarized DNA and RNA data obtained from renal cell carcinoma.

Cytogenetic abnormalities, such as DNA amplifications and deletions, often lead to significant changes in gene expression levels within a chromosomal region. Instead of generating additional DNA copy number data, one method to identify DNA copy number abnormalities has been to search existing gene expression data for regional perturbations in gene expression. However, it is not clear how well this surrogate method performs in the examination of individual tumors and how we can use both DNA and RNA data to identify candidate genes that may be mutated. Here we report a comparison study using summarized DNA and RNA data to identify chromosomal abnormalities in human samples. Forty-four tissue samples from patients diagnosed as having renal cell carcinoma (RCC) were collected, together with 15 normal kidney samples as controls, and for each sample the genome-wide DNA and RNA data were obtained for comparison using Affymetrix 100K SNP and HGU133plus2 gene expression chips, respectively. The DNA and RNA data was summarized by both chromosome arm and cytogenetic banding patterns and compared. The result of this analysis revealed that the two summarized data sets used to identify cytogenetic changes agreed well. However, some differences between the two were also identified. These differences of large-scale gene expression deregulation without evidence of the comparable DNA copy number alterations may be the result of known mechanisms, such as large-scale methylation or chromosome inactivation, or may be the result of some new mechanism of DNA-RNA translation. The usefulness of the combined data set for identifying regions of mutated genes is also discussed.

Identification of copy number alterations and its association with pathological features in clear cell and papillary RCC.

We report and characterize the copy number alterations (CNAs) in 35 clear cell and 12 papillary renal cell carcinomas (RCC) using Affymetrix 100K SNP arrays. Novel gain and loss regions are identified in both subtypes. In addition, statistically significant CNA are detected and associated with the pathological features: VHL mutation status, tumor grades, and sarcomatoid component in clear cell RCC and in types 1 and 2 of papillary RCC. Florescence in situ hybridization confirmed the copy number gain in the transforming growth factor, beta-induced gene (TGFBI), which is a possible oncogene for clear cell RCC.

Aryl hydrocarbon receptor-interacting protein gene mutations in familial isolated pituitary adenomas: analysis in 73 families.

CONTEXT: An association between germline aryl hydrocarbon receptor-interacting protein (AIP) gene mutations and pituitary adenomas was recently shown. OBJECTIVE: The objective of the study was to assess the frequency of AIP gene mutations in a large cohort of patients with familial isolated pituitary adenoma (FIPA). DESIGN: This was a multicenter, international, collaborative study. SETTING: The study was conducted in 34 university endocrinology and genetics departments in nine countries. PATIENTS: Affected members from each FIPA family were studied. Relatives of patients with AIP mutations underwent AIP sequence analysis. MAIN OUTCOME MEASURES: Presence/absence and description of AIP gene mutations were the main outcome measures. INTERVENTION: There was no intervention. RESULTS: Seventy-three FIPA families were identified, with 156 patients with pituitary adenomas; the FIPA cohort was evenly divided between families with homogeneous and heterogeneous tumor expression. Eleven FIPA families had 10 germline AIP mutations. Nine mutations, R16H, G47_R54del, Q142X, E174frameshift, Q217X, Q239X, K241E, R271W, and Q285frameshift, have not been described previously. Tumors were significantly larger (P = 0.0005) and diagnosed at a younger age (P = 0.0006) in AIP mutation-positive vs. mutation-negative subjects. Somatotropinomas predominated among FIPA families with AIP mutations, but mixed GH/prolactin-secreting tumors, prolactinomas, and nonsecreting adenomas were also noted. Approximately 85% of the FIPA cohort and 50% of those with familial somatotropinomas were negative for AIP mutations. CONCLUSIONS: AIP mutations, of which nine new mutations have been described here, occur in approximately 15% of FIPA families. Although pituitary tumors occurring in association with AIP mutations are predominantly somatotropinomas, other tumor types are also seen. Further study of the impact of AIP mutations on protein expression and activity is necessary to elucidate their role in pituitary tumorigenesis in FIPA.

Molecular classification of patients with unexplained hamartomatous and hyperplastic polyposis.

CONTEXT: Significant proportions of patients with hamartomatous polyposis or with hyperplastic/mixed polyposis remain without specific clinical and molecular diagnosis or present atypically. Assigning a syndromic diagnosis is important because it guides management, especially surveillance and prophylactic surgery. OBJECTIVE: To systematically classify patients with unexplained hamartomatous or hyperplastic/mixed polyposis by extensive molecular analysis in the context of central rereview of histopathology results. DESIGN, SETTING, AND PATIENTS: Prospective, referral-based study of 49 unrelated patients from outside institutions (n = 28) and at a comprehensive cancer center (n = 21), conducted from May 2, 2002, until December 15, 2004. Germline analysis of PTEN, BMPR1A, STK11 (sequence, deletion), SMAD4, and ENG (sequence), specific exon screening of BRAF, MYH, and BHD, and rereview of polyp histology results were performed. MAIN OUTCOME MEASURES: Molecular, clinical, and histopathological findings in patients with unexplained polyposis. RESULTS: Of the 49 patients, 11 (22%) had germline mutations. Of 14 patients with juvenile polyposis, 2 with early-onset disease had mutations in ENG, encoding endoglin, previously only associated with hereditary hemorrhagic telangiectasia; 1 had hemizygous deletion encompassing PTEN and BMPR1A; and 1 had an SMAD4 mutation. One individual previously classified with Peutz-Jeghers syndrome had a PTEN deletion. Among 9 individuals with an unknown hamartomatous polyposis, 4 had mutations in STK11 (1), BMPR1A (2), and SMAD4 (1). Of the 23 patients with hyperplastic/mixed polyposis, 2 had PTEN mutations. Substantial discrepancies in histopathology results were seen. CONCLUSIONS: Systematic molecular classification of 49 patients with unexplained hamartomatous or hyperplastic polyposis uncovered a potential novel susceptibility gene, ENG, for juvenile polyposis. Importantly, given the substantial proportion of patients found to have germline mutations, more extensive analysis of the known susceptibility genes is indicated. Rereview of histology results by a dedicated gastrointestinal pathologist should be considered routinely, as organ-specific surveillance rests on defining syndromic diagnosis.

Proliferation and invasion: plasticity in tumor cells.

Invasive and proliferative phenotypes are fundamental components of malignant disease, yet basic questions persist about whether tumor cells can express both phenotypes simultaneously and, if so, what are their properties. Suitable in vitro models that allow characterization of cells that are purely invasive are limited because proliferation is required for cell maintenance. Here, we describe glioblastoma cells that are highly invasive in response to hepatocyte growth factor/scatter factor (HGF/SF). From this cell population, we selected subclones that were highly proliferative or displayed both invasive and proliferative phenotypes. The biological activities of invasion, migration, urokinase-type plasminogen activation, and branching morphogenesis exclusively partitioned with the highly invasive cells, whereas the highly proliferative subcloned cells uniquely displayed anchorage independent growth in soft agar and were highly tumorigenic as xenografts in immune-compromised mice. In response to HGF/SF, the highly invasive cells signal through the MAPK pathway, whereas the selection of the highly proliferative cells coselected for signaling through Myc. Moreover, in subcloned cells displaying both invasive and proliferative phenotypes, both signaling pathways are activated by HGF/SF. These results show how the mitogen-activated protein kinase and Myc pathways can cooperate to confer both invasive and proliferative phenotypes on tumor cells and provide a system for studying how transitions between invasion and proliferation can contribute to malignant progression.

A meiotic recombination in a new isolated familial somatotropinoma kindred.

We report here the genetic findings of a new isolated familial somatotropinoma (IFS) kindred in which the mother (subject I:2) and one daughter (subject II:2) are affected; their ages at diagnosis were 25 and 14 years respectively. Additionally, patient I:2 developed virilization due to an androgen-secreting adrenocortical mass, presenting clinical and molecular features of sporadic adrenal carcinoma. To genotype this family and to narrow down the candidate interval of the putative IFS gene at 11q13, we performed haplotyping on the DNA from all five members of the family and allelotyping of one available somatotropinoma using polymorphic microsatellite markers from chromosome region 11q12.1-11q13.5. Results indicated that the disease haplotype, between markers D11S956 and D11S527, was transmitted from subject I:2 only to subject II:2. A meiotic recombination event was detected in the fraternal twin sister of II:2 (subject II:1), but her disease status is unknown. Since she is only 18 years old this genetic event cannot yet narrow down the area involved in the pathogenesis of IFS. Allelotyping of the somatotropinoma from II:2 revealed loss of the chromosome carrying the wild-type copy of the putative IFS gene inherited from her father. These results support the involvement of a tumor suppressor gene at 11q13.1-q13.3 in the pathogenesis of IFS.