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This case report describes a preterm baby with juvenile extracutaneous xanthogranuloma that rapidly expanded. The tumor was excised because of rapid enlargement and extension into the thoracic inlet. To our knowledge, this is the first case of a preterm baby with extracutaneous juvenile xanthogranuloma. The case illustrates the need for careful surveillance of this lesion because it cannot always be managed conservatively.

Segmental mediolytic arteriopathy (SMA) is a rare condition. It was first defined in 1976 and has been well described in the literature, although to date the aetiology of the condition is unknown. In most case reports SMA is diagnosed retrospectively once tissue has undergone histological examination. We present the first known case of SMA of the colic, mid-jejunal, common hepatic, intrahepatic and gastric arteries to be diagnosed at angiography after multiple episodes of undiagnosed intraperitoneal bleeding, and, perhaps related to this, one of the few reported patients with SMA involving multiple intra-abdominal arteries to have survived.

We demonstrate an optically sectioned fluorescence lifetime imaging microscope with a wide-field detector, using a convenient, continuously tunable (435-1150 nm) ultrafast source for fluorescence imaging applications that is derived from a visible supercontinuum generated in a microstructured fiber.

Multidimensional fluorescence imaging is a powerful molecular imaging modality that is emerging as an important tool in the study of biological tissues. Due to the large volume of multi-spectral data associated with the technique, it is often difficult to find the best combination of parameters to maximize the contrast between different tissue types. This paper presents a novel framework for the characterization of tissue compositions based on the use of time resolved fluorescence imaging without the explicit modeling of the decays. The composition is characterized through soft clustering based on manifold embedding for reducing the dimensionality of the datasets and obtaining a consistent differentiation scheme for determining intrinsic constituents of the tissue. The proposed technique has the benefit of being fully automatic, which could have significant advantages for automated histopathology and increasing the speed of intraoperative decisions. Validation of the technique is carried out with both phantom data and tissue samples of the human pancreas.

Following the considerable impact of the application of convenient ultrafast lasers to multiphoton microscopy on biomedical imaging, it seems to us that FLIM and MDFI continue the trend in which advances in instrumentation will facilitate new discoveries — and modes of discovery — in biology and medicine. We hope we have shown the reader that fluorescence lifetime can provide intrinsic molecular contrast in unstained tissue and that the prospects for in vivo application are exciting. We believe that the capability to excite fluorophores at almost any excitation wavelength and the opportunities to extract more information from fluorescence signals by resolving with respect to lifetime, excitation and emission spectrum and also polarisation, will have a major impact on the ability to identify and exploit intrinsic contrast and on investigations of molecular biology. There the combination of new fluorescence probe technology, including genetically-expressed labels and nano-engineered devices, with new modes of interrogation and analysis, will continue to fuel the astounding advances in this field. There is a real prospect that our ability to ask and test biological questions will cease to be limited by the availability of suitable instrumentation. Rather it is likely to be limited by our ability to analyse and comprehend the (rapidly increasing volume of) data that we collect.

Background Myolipoma is a benign tumour in which smooth muscle cells are mixed with adipocytes. Case presentation A 34-year old lady presented with a mass in the right iliac fossa detected on computerised tomographic (CT) scan. Wide excision of the retroperitoneal mass was done. Histopathology showed features of myolipoma. There was no recurrence or metastasis at three years. Conclusion Myolipoma is a rare benign entity; hence a benign course and good prognosis are expected.

Background & Aims: Fibroblast growth factors (Fgfs) and their receptors (Fgfrs) are important intercellular signaling molecules that are essential to mammalian embryonic development. The signaling pathways between endoderm-derived gastric epithelium and the surrounding mesenchyme are largely unknown; however, the developmental expression profile of the IIIb isoform of Fgfr2 ( Fgfr2b ) and its main ligand, Fgf10, suggest that they may be strong candidates. Mice lacking either component ( Fgfr2b −/− or Fgf10 −/− ) were examined to determine the role of Fgfr2b-mediated signaling during gastric organogenesis. Methods: Stomachs from embryonic day 13.5–18.5 Fgfr2b −/− , Fgf10 −/− , and wild-type littermates were collected and analyzed by conventional histology, immunohistochemistry, in situ hybridization, and electron microscopy. Results: Fgfr2b −/− and Fgf10 −/− fetuses had stomachs smaller than wild-type, consisting of relatively proportionate forestomach but disproportionately reduced glandular stomach, the mucosa of which has low cytoarchitectural complexity with a spiral arrangement of large mucosal folds. During mid to late fetal stages (embryonic day 15.5–18.5), epithelial differentiation to mucous and chief cell lineages was rudimentary, with no expression of several early cytodifferentiation markers including GATA4 , GATA6 , and H + /K + -adenosine triphosphatase and abnormal expression of members of the hedgehog family of signaling molecules. Conclusions: Fgfr2b and Fgf10 are part of a signaling network with Sonic hedgehog and Indian hedgehog that are essential to anterior-posterior and radial patterning in gastric development.

We report real-time (video-rate) fluorescence lifetime imaging and its application to tissue autofluorescence and endoscopy, demonstrating FLIM of unstained ex vivo tissue at update rates of 5.5 Hz through a flexible endoscope.

Expression of hepatocyte growth factor (HGF) and its receptor, c-met is up-regulated in various forms of liver injury. This study evaluated the relationship between HGF and c-met expression and pathological changes in experimental alcoholic liver disease. Rats (5 per group) were fed ethanol and a diet containing saturated fat corn oil or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in controls. In a second set of experiments, Kupffer cells, endothelial cells and hepatocytes were isolated from rats in each group. Pathological evaluation and analysis of HGF and c-met expression were performed in liver and the different cell types. Increased expression of HGF and c-met expression was detected in the liver of rats showing necroinflammatory changes. The Kupffer and endothelial cells were primarily responsible for the increase in HGF, c-met expression was seen only in hepatocytes. Thus, up-regulation of HGF and c-met occurred in the presence of the necrosis and inflammation suggesting that HGF may be acting to protect against liver injury or accelerate the regenerative process.

Background Growth factor, cytokine and chemokine-induced activation of PI3K enzymes constitutes the start of a complex signalling cascade, which ultimately mediates cellular activities such as proliferation, differentiation, chemotaxis, survival, trafficking, and glucose homeostasis. The PI3K enzyme family is divided into 3 classes; class I (subdivided into IA and IB), class II (PI3K-C2α, PI3K-C2β and PI3K-C2γ) and class III PI3K. Expression of these enzymes in human tissue has not been clearly defined. Methods In this study, we analysed the immunohistochemical topographical expression profile of class IA (anti-p85 adaptor) and class II PI3K (PI3K-C2α and PI3K-C2β) enzymes in 104 formalin-fixed, paraffin embedded normal adult human (age 33–71 years, median 44 years) tissue specimens including those from the gastrointestinal, genitourinary, hepatobiliary, endocrine, integument and lymphoid systems. Antibody specificity was verified by Western blotting of cell lysates and peptide blocking studies. Immunohistochemistry intensity was scored from undetectable to strong. Results PI3K enzymes were expressed in selected cell populations of epithelial or mesenchymal origin. Columnar epithelium and transitional epithelia were reactive but mucous secreting and stratified squamous epithelia were not. Mesenchymal elements (smooth muscle and endothelial cells) and glomerular epithelium were only expressed PI3K-C2α while ganglion cells expressed p85 and PI3K-C2β. All three enzymes were detected in macrophages, which served as an internal positive control. None of the three PI3K isozymes was detected in the stem cell/progenitor compartments or in B lymphocyte aggregates. Conclusions Taken together, these data suggest that PI3K enzyme distribution is not ubiquitous but expressed selectively in fully differentiated, non-proliferating cells. Identification of the normal in vivo expression pattern of class IA and class II PI3K paves the way for further analyses which will clarify the role played by these enzymes in inflammatory, neoplastic and other human disease conditions.

The acute phase response is strictly connected with modulation of gene expression. Transcriptional control of many genes is mediated by binding of diverse transcription factors to cis-acting DNA motifs in the respective promoter sequence. We now describe such regulatory elements for the TFF3 gene coding for a peptide involved in response to gut irritation. TNF-alpha stimulation, which induces NF-kappaB activation, evoked up to 10-fold reduction of TFF3 expression in the colon tumour cell line HT-29. Several consensus binding sites for members of the NF-kappaB transcription factor subunits are located in the 5'-flanking region of TFF3. Mutating these sites was aimed at discovering which one is responsible for NF-kappaB binding and thus regulation of TFF3 expression.

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance. © 2003 Optical Society of America.

Of 14 chemokine receptors investigated, only CXCR4 was expressed on ovarian cancer cells [C. J. Scotton et al., Cancer Res., 61: 4961-4965, 2001]. To further understand the role of this chemokine receptor in ovarian tumor biology, we studied the action of its ligand, CXCL12 (stromal cell-derived factor 1), on the CXCR4-expressing ovarian cancer cell lines IGROV. Ligand stimulation of the CXCR4 receptor resulted in sustained activation of Akt/protein kinase B and biphasic phosphorylation of p44/42 mitogen-activated protein kinase in IGROV. When IGROV cells were cultured under suboptimal conditions, CXCL12 stimulated their in vitro growth, an effect that was abrogated by neutralizing antibodies to CXCR4. This increase in cell number was attributable to stimulation of DNA synthesis, not protection from apoptosis. CXCL12 treatment of IGROV cells also induced mRNA and protein for tumor necrosis factor alpha, a cytokine that is expressed by tumor cells in ovarian cancer biopsies. IGROV cells invaded through Matrigel toward a CXCL12 gradient. Invasion was abrogated by the broad spectrum matrix metalloproteinase and TNFalpha converting enzyme inhibitor Marimastat and was partially inhibited by neutralizing antitumor necrosis factor alpha antibodies. These effects were not limited to the IGROV cell line. They could also be demonstrated in the CAOV-3 ovarian cancer cell line and primary ovarian tumor cells isolated from ovarian ascites. These biological effects of CXCL12 on IGROV cells were also inhibited by the small molecular weight CXCR4 antagonist AMD3100. Finally, we found abundant intracellular CXCL12 protein in tumor cells in 15 of 18 ovarian cancer biopsies but not in epithelial cells from normal ovary or borderline disease. The chemokine CXCL12 may have multiple biological effects in ovarian cancer, stimulating cell migration and invasion through extracellular matrix, as well as DNA synthesis and establishment of a cytokine network in situations that are suboptimal for tumor cell growth.

MUC1 mucin is transcriptionally regulated by estrogen, progesterone, and glucocorticoids. Our objective was to determine whether androgen receptor. (20AR) activation regulates expression of MUC1. The following breast and prostatic cell lines were phenotyped and grouped according to AR and MUC1protein expression: 1) AR+MUCi + [DAR17+19. (20AR transfectants of DU-145), ZR-75-1, MDA-MB-453, and T47D]; 2) AR-MUCi+ [DZeoi. (20AR- vector control), DU-145, BT20, MDA-MB231, and MCF7]; 3) AIR +MUCi -. (20LNCaP and LNCaP-r). Cell proliferation was determined using the MTT assay in the presence of synthetic androgen R1881, 0.1 pM to 1 µM. Cell surface MUC1expression was determined by flow cytometry in the presence or absence of oestradiol, medroxy progesterone acetate or R1881, with and without 4 hydroxy-flutamide. (204-OH), a nonsteroidal AR antagonist. The functional significance of MUC1expression was investigated with a cell-cell aggregation assay. Only AR+ MUC1 + cell lines showed a significant increase in MUC1expression with AR activation. (20P. (20range) =.01 to .0001), reversed in the presence of 4-OHF. Cell proliferation was unaffected. Increased expression of MUC1was associated with a significant. (20P. (20range) =.002 to .001) reduction in cell-cell adhesion. To our knowledge, this is the first description of androgen-dependent regulation of MUC1mucin. This is also functionally associated with decreased cell-cell adhesion, a recognised feature of progressive malignancy. These findings have important implications for physiological and pathological processes.

We present an ex vivo study of temporally and spectrally resolved autofluorescence in a total of 47 endoscopic excision biopsy/resection specimens from colon, using pulsed excitation laser sources operating at wavelengths of 375 nm and 435 nm. A paired analysis of normal and neoplastic (adenomatous polyp) tissue specimens obtained from the same patient yielded a significant difference in the mean spectrally averaged autofluorescence lifetime −570 ± 740 ps (p = 0.021, n = 12). We also investigated the fluorescence signature of non-neoplastic polyps (n = 6) and inflammatory bowel disease (n = 4) compared to normal tissue in a small number of specimens.

DNA ligase IV is the most recently identified member of a family of enzymes joining DNA strand breaks in mammalian cell nuclei [1] [2]. The enzyme occurs in a complex with the XRCC4 gene product [3], an interaction mediated via its unique carboxyl terminus [4] [5]. Cells lacking XRCC4 are hypersensitive to ionising radiation and defective in V(D)J recombination [3] [6], implicating DNA ligase IV in the pathway of nonhomologous end-joining (NHEJ) of DNA double-strand breaks mediated by XRCC4, the Ku70/80 heterodimer and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in mammalian cells (reviewed in [7]). The phenotype of a null mutant of the Saccharomyces cerevisiae DNA ligase IV homologue indicates that the enzyme is non-essential and functions in yeast NHEJ [8] [9] [10]. Unlike other mammalian DNA ligases for which cDNAs have been characterised, DNA ligase IV is encoded by an intronless gene (LIG4). Here, we show that targeted disruption of LIG4 in the mouse leads to lethality associated with extensive apoptotic cell death in the embryonic central nervous system. Thus, unlike Ku70/80 and DNA-PKcs [11] [12] [13] [14], DNA ligase IV has an essential function in early mammalian development.

Expression of activated MMP-2 (72 kDa type IV collagenase) is highly associated with the malignant phenotype in adenocarcinomas, but predominant expression of the mRNA appears to be in stromal cells. MT1-MMP (membrane type 1-matrix metalloproteinase) is implicated in tumor-epithelial cell surface activation of latent pro-MMP-2, indicating a mechanism for tumor-stromal interaction in invasion. We determined the relative mRNA distribution of these MMPs in human ovarian tumors with a view to analyzing potential variations in the epithelial-mesenchymal interactions dictating ovarian tumor cell spread. In situ hybridization using 35S-labeled riboprobes was used to analyze 33 human ovarian tumors and mouse xenografts of human ovarian (DOV 13, SKOV3) and breast (MCF 7) tumor cell lines known to express MT1-MMP and MMP-2. MMP-2 mRNA was expressed in 31 of 33 and MT1-MMP mRNA was expressed in 29 of 33 tumor cases. MMP-2 mRNA was predominantly expressed in desmoplastic fibroblasts and in the subepithelial stroma. MT1-MMP mRNA showed some colocalization with MMP-2 in stromal cells. Neoplastic epithelial cell labeling for MT1-MMP mRNA was present in borderline and malignant tumors but not in benign tumors, and was invariably less than stromal labeling. Xenografts of DOV 13, SKOV 3, and MCF 7 cells showed some stromal localization of MMP-2 mRNA and weak labeling of DOV 13 cells. There was variable labeling for MT1-MMP mRNA in the neoplastic cells only. The colocalization of MT1-MMP and MMP-2 mRNAs in ovarian carcinoma stroma supports the view that MT1-MMP is closely associated with MMP-2 expression and function. It suggests that either additional mechanisms are involved in regulating MMP-2 activation at the tumor cell surface, or more intriguingly, that desmoplastic fibroblasts may be the primary mediators of extracellular matrix remodeling with respect to this system.

It is now recognised that epithelial-stromal interactions are important in a wide range of disease processes including neoplasia and inflammation. Metalloproteinases are central to matrix degradation and remodelling, which are key events in tumour invasion and metastasis and may also be involved in tissue changes occurring in chronic inflammation. Immunohistochemistry was performed on sections from 50 patients with pancreatic cancer (n = 27), ampullary cancer (n = 12), low bile duct cancer (n = 3), neuroendocrine tumours (n = 3) and chronic pancreatitis (n = 5), using antibodies raised against collagenase (MMP2), stromelysin (MMP3) and tissue inhibitor of metalloproteinase (TIMP1) and developed using the avidin-biotin complex method. Abundance of MMP2, MMP3 and TIMP1 was greater in pancreatic and ampullary cancer than any other pathology and immunoreactivity in the malignant epithelial cells in pancreatic and ampullary cancer was greater than in the stromal tissues (in pancreatic cancer: MMP2 100% vs 37%, MMP3 93% vs 15%, TIMP1 93% vs 4%, P < 0.0001). There were strong correlations between the immunoreactivity of the two antibodies for MMP2 (P < 0.0001), between MMP2 and TIMP1 (P < 0.0001) and between MMP3 and TIMP1 (P < 0.0001). The immunoreactivity for TIMP1 in pancreatic and ampullary cancers with lymph node metastases was significantly less compared with those cases without lymph node metastases (P < 0.02) and there was an association between increased immunoreactivity for MMP2 and the degree of tumour differentiation (P < 0.01). The results implicate MMP2, MMP3 and TIMP1 in the invasive phenotype of pancreatic and ampullary cancer. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

Aims-To establish two cell lines from a primary duodenal adenocarcinoma; to describe the morphological, growth, ploidy, and immunophenotypic characteristics of these cell lines.Methods-The cell lines, designated DAC/S and DAC/E, were characterised using both in vitro and in vivo cell culture techniques, light and electron microscopy, immunocytochemistry, and FACS analyses.Results-Both cell lines have an epithelial origin, are aneuploid and display characteristics of transformed cells. The cell lines differ from each other in morphology, doubling time and serum requirements. These cell lines are anchorage dependent and do not grow in nude mice.Conclusions-DAC/S and DAC/E cell lines are derived from neoplastic epithelium and could provide in vitro model systems for future investigations of the cell and molecular biology of duodenal neoplasia.

Polyacrylamide gel electrophoresis (PAGE) of proteins with subsequent western blotting has become a routine technique for the analysis of proteins from both cultured cells and fresh whole tissue. We have developed a method to extract proteins from methacarn‐fixed tissue which renders them suitable for SDS‐PAGE and western blotting. With a panel of antibodies to specific intermediate filaments, transforming growth factor‐α (TGF‐α), and albumin, immunohistochemistry was performed in parallel with western blotting on sections cut from methacarn‐fixed samples of normal rat liver and liver from rats treated under a regime which induces oval cell proliferation. Immunohistochemistry enabled the determination of changes in tissue distribution and abundance of the target proteins, which was mirrored by the corresponding western blot data. This technique can be especially effective when used in conjunction with immunohistochemistry. Tissue samples are easy to prepare, avoiding the precautions which need to be taken when handling fresh tissue (Abstract: J Pathol 1994; 173S: No. 41). Copyright © 1995 John Wiley & Sons, Ltd.

HECD-1 monoclonal antibody has been used to localize E-cadherin, a calcium-dependent cell-cell adhesion molecule, in microwave-treated, paraffin-embedded sections from 53 cases of cervical intraepithelial neoplasia (CIN) (11 CIN I, 22 CIN II, and 20 CIN III), 16 invasive cervical squamous cell carcinomas, and seven metastases. In normal cervix, E-cadherin was expressed on the cell membrane of basal and parabasal cells. Cytoplasmic staining was present in occasional basal cells only. In CIN, the presence and localization of cytoplasmic E-cadherin were found to be significantly correlated with the grade of the CIN lesion. In squamous cell carcinomas, reduced membranous and increased cytoplasmic staining was seen with worsening differentiation. Loss of membranous E-cadherin expression was also detected in 4/7 metastatic deposits. E-cadherin expression (120 kD form on Western blotting) was seen in human cervical carcinoma cell lines (HT3, ME180, C4I, Caski) that maintained the ability to aggregate in a homotypic adhesion assay and showed a typical epithelial morphology. E-cadherin-negative cell lines (Hela, SiHa, C33A) did not show adhesion. HOG-1 was the only E-cadherin-negative cell line which showed a significant degree of cell-cell aggregation. These data indicate that loss of membranous E-cadherin expression may represent one of the abnormalities underlying loss of cell polarity and differentiation which characterize CIN and invasive cervical cancer.

Chemokines may control the macrophage infiltrate found in many solid tumors. In human ovarian cancer, in situ hybridization detected mRNA for the macrophage chemokine monocyte chemoattractant protein-1 (MCP-1) in 16/17 serous carcinomas, 4/4 mucinous carcinomas, 2/2 endometrioid carcinomas, and 1/3 borderline tumors. In serous tumors, mRNA expression mainly localized to the epithelial areas, as did immunoreactive MCP-1 protein. In the other tumors, both stromal and epithelial expression were seen. All tumors contained variable numbers of cells positive for the macrophage marker CD68. MCP-1 mRNA was also detected in the stroma of 5/5 normal ovaries. RT-PCR demonstrated mRNA for MCP-1 in 7/7 serous carcinomas and 6/6 ovarian cancer cell lines. MCP-1 protein was detected by ELISA in ascites from patients with ovarian cancer (mean 4.28 ng/ml) and was produced primarily by the cancer cells. Human MCP-1 protein was also detected in culture supernatants from cell lines and in ascites from human ovarian tumor xenografts which induce a peritoneal monocytosis in nude mice. We conclude that the macrophage chemoattractant MCP-1 is produced by epithelial ovarian cancer and that the tumor cells themselves are probably a major source. MCP-1 may contribute to the accumulation of tumor-associated macrophages, which may subsequently influence tumor behavior.

We have used an avidin-biotin immunoperoxidase technique to localise epithelial cadherin (E-cadherin), a calcium-dependent cell-cell adhesion molecule, in 107 paraffin-embedded sections from 93 patients consisting of 24 with colorectal adenoma, 55 with rectal carcinoma and 14 with liver metastases. The corresponding primary colorectal tumours were also studied in these cases. E-cadherin was expressed by normal colorectal epithelial cells with typical membranous staining at the intercellular junctions. Loss of normal membranous E-cadherin expression and presence of cytoplasmic staining were found frequently in adenomas larger than 1 cm (P

Background: Genetic intra-tumor heterogeneity (ITH) may foster tumor adaptation by providing selectable phenotypes for Darwinian evolution. Extensive genetic heterogeneity may also hinder personalized medicine strategies that rely on the portrayal of the mutational landscape from single tumor biopsies. Methods: To examine ITH, we have subjected multiple biopsies from primary renal cell carcinomas and associated metastatic sites to exome capture sequencing (n=2), SNP-array based chromosomal aberration and ploidy profiling analysis (n=4). Phylogenetic relationships of tumor regions were reconstructed by clonal ordering of non-synonymous somatic mutations. The phenotypic consequences of genetic ITH were characterised by immunohistochemistry, mutation functional analysis and mRNA expression profiling. Results: Phylogenetic reconstruction revealed branched evolutionary tumor growth with 63-69% of somatic mutations identified from single biopsies not detectable across all sequenced tumor regions. Independent tumor suppressor gene loss of function mutations with distinct regional distributions were detected within individual tumors: SETD2 harbored 5 different mutations in 2 tumors and PTEN and KDM5C two different mutations in one tumor, each. Thus, despite genetic divergence during tumor progression, phenotypic convergent evolution occurred, indicating a high degree of early mutational diversity. ITH was observed for a mutation in the kinase domain of mTOR, correlating with S6 and 4EBP phosphorylation specifically in cancer regions carrying the mutation and constitutive activation of mTOR kinase activity in vitro. Expression of a renal cancer-specific prognostic signature differed between tumor regions. Chromosomal aberration analysis revealed extensive ITH with 26/30 tumor biopsies from four tumors harboring divergent allelic imbalance profiles. Ploidy profiling revealed heterogeneity in two out of four tumors and identified an aneuploid tumor cell population in a metastasis that probably evolved from a tetraploid intermediate detectable in the primary tumor. Conclusions: Genetic ITH was present in all tumors and occurs through spatially separated heterogeneous somatic mutations and chromosomal and ploidy aberrations leading to both, phenotypic intra-tumor diversity (mTOR activating mutation) and convergent loss of function(SETD2, PTEN and KDM5C). ITH can lead to underestimation of the tumor genomic landscape portrayed from single biopsies and may present significant challenges to personalized medicine and biomarker development. ITH, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. Targeting mutations commonly located on the trunks, rather than the branches of phylogenetic trees, may improve therapeutic outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 964. doi:1538-7445.AM2012-964

603 Background: MMR deficiency (dMMR) has been reported in 15% of CRC, but with a lower frequency in advanced disease. Most cases are due to sporadic MLH1 promoter hypermethylation (often with BRAF mutations), with a minority reflecting germline mutations in MLH1, MSH2, PMS2, or MSH6 (Lynch Syndrome [LS]). The Revised Bethesda Guidelines (RBG) are one means of selecting individuals at risk of LS for further assessment, but will miss a proportion of cases. Methods: We screened all consenting patients for eligibility for CRC trials recruiting specific genetic aberrations, which included MMR assessment. Results: Of 314 patients, immunohistochemistry (IHC) for MMR protein expression is complete on 171. Staining was reduced/absent in 19.3% of tests, and heterogeneous in 12.1%. The dMMR rate was 6.4%. 2 dMMR patients* were identified as at risk of LS, and referred to genetics by their treating clinician before IHC results were known. However 4 other cases† were not referred, and an underlying predisposition would have been missed without this unbiased approach. 4 patients developed metastatic disease, with none experiencing a partial response to chemotherapy thus far. (Table.) Conclusions: This data is representative of a practice with a high proportion of metastatic disease. It suggests that within oncology, an unbiased screening approach for LS is preferable. Whilst the RBG detect the majority of cases, they may be underutilised as other management issues take precedence in oncology clinics. A cost-effective alternative may be the introduction of a nurse-led programme to identify cases at risk, as is being introduced at our centre. A spectrum of clinical behavior exists amongst metastatic dMMR CRC, and larger numbers will reveal if this affects therapeutic response. [Table: see text]

Pancreatic malignancies account for 3% of all cancer diagnoses in the UK and prognosis is poor with overall 1-year survival rates at 20% and 5-year survival rates at 5%. The majority of these cancers (75%-95%) arise from the exocrine part of the gland and are almost all invasive ductal adenocarcinomas. One per cent of all pancreatic tumours are endocrine tumours. There is limited data regarding the management of such rare neoplasms of the pancreas and some evidence suggests that prognoses and risk factors may be different. Therefore, it is important to report experience of this type of malignancy in order to build a knowledge base to guide the practice of future clinicians. The authors report a case of an intraductal tubulopapillary neoplasm of the pancreas. This is very unusual form of intraductal pancreatic tumour, which is now thought to occupy a distinct histological subcategory and has arisen within a previously irradiated field.

TPS229 Background: Recent studies have indicated that KIT mutations occur in approximately 20% of acral and mucosal melanomas (Curtin, al. 2006; Beadling, et al. 2008). Several case reports have sh...

Frequent loss of heterozygosity (LOH) on both arms of chromosome 17 has been described in ovarian carcinoma (OC) by a number of groups, and the recent fine mapping of an inherited breast-ovarian cancer gene (brcAI) to a small region at 17q12-21 has focused interest on this area. We studied 28 sporadic OCs with 21 markers at 18 loci on chromosome 17 (5 on 17p and 13 on 17q). LOH on 17p was 78%, and always involved p53. In 86% of cases showing LOH, all informative markers on chromosome 17 showed reduction to homozygosity. Using 6 markers flanking the brcAI locus on 17q, LOH was 75%, but only one tumour showed LOH with telomeric retention. The data therefore suggest that small deletions on chromosome 17 are very uncommon in sporadic OC. No genomic rearrangements by Southern blotting were seen in the brcAI candidate gene estradiol 17 beta dehydrogenase 2 (17hsd2), or in erbB2, prohibition (phb) and nmeI (previously nm23-HI). LOH on chromosome 17 was more common in high-grade, late-stage carcinomas, and no LOH was seen in any benign or borderline tumour. This study has clearly demonstrated that LOH at any one site on chromosome 17 is most commonly explained by LOH over the whole of the chromosome. We consider possible reasons for the absence of small deletions on chromosome 17 in OC.

In the past 5 years a number of cytokines have been identified that control B cell development, proliferation, and maturation. The role of such cytokines in the evolution, pathophysiology, and treatment of B cell malignancies is an area of great interest. The in vitro response of freshly isolated peripheral blood mononuclear cells from patients with B cell chronic lymphocytic leukemia (B-CLL) and a high white cell count, to four cytokines, IFN-alpha, IFN-gamma, IL-2 and TNF, was studied. No culture condition or cytokine resulted in a significant increase in cell number over 4 days, cells survived better in autologous serum than in heat inactivated foetal calf serum, and a small but significant increase in blast cells was seen when the cells were cultured in IL-2. There was a discrepancy between uptake of thymidine and increase in cell number which can be explained by the low labelling index of these cultures and the fact that cells can incorporate [3H]-thymidine without going into mitosis. The majority of cultures produced biologically active TNF at levels ranging from 1 to 40 pg/ml. In IFN-alpha treated cultures TNF levels were decreased. Cultures contained biologically active IL-6 at levels ranging from 2 to 2800 U/ml. IL-6 production was not influenced by other cytokines. Thirteen of 28 patients had detectable IL-6 in their serum, but in cells lysed no more than 2 h after removal from the patient, message for IL-6 could not be detected by Northern blotting. Cells also failed to express IL-1 beta mRNA but seven of eight patients had low levels of TNF message. Preliminary data using in situ hybridization techniques revealed that whilst no mRNA for IL-6 was detected, TNF and IL-1 beta mRNA were detected in a minority of mononuclear cells.

Expansion of Scientific knowledge and techniques has put pressure on researchers to narrow their interest to quite specific topics. Apart from the clinical benefits that it is bringing, work on Helicobacter pylori is refreshing because it forces us to think laterally and consider interactions between changes observed in different disciplines such as biochemistry, physiology, immunology and bacteriology.

The European Union multi-disciplinary Personalised RNA interference to Enhance the Delivery of Individualised Cytotoxic and Targeted therapeutics (PREDICT) consortium has recently initiated a framework to accelerate the development of predictive biomarkers of individual patient response to anti-cancer agents. The consortium focuses on the identification of reliable predictive biomarkers to approved agents with anti-angiogenic activity for which no reliable predictive biomarkers exist: sunitinib, a multi-targeted tyrosine kinase inhibitor and everolimus, a mammalian target of rapamycin (mTOR) pathway inhibitor. Through the analysis of tumor tissue derived from pre-operative renal cell carcinoma (RCC) clinical trials, the PREDICT consortium will use established and novel methods to integrate comprehensive tumor-derived genomic data with personalized tumor-derived small hairpin RNA and high-throughput small interfering RNA screens to identify and validate functionally important genomic or transcriptomic predictive biomarkers of individual drug response in patients. PREDICT's approach to predictive biomarker discovery differs from conventional associative learning approaches, which can be susceptible to the detection of chance associations that lead to overestimation of true clinical accuracy. These methods will identify molecular pathways important for survival and growth of RCC cells and particular targets suitable for therapeutic development. Importantly, our results may enable individualized treatment of RCC, reducing ineffective therapy in drug-resistant disease, leading to improved quality of life and higher cost efficiency, which in turn should broaden patient access to beneficial therapeutics, thereby enhancing clinical outcome and cancer survival. The consortium will also establish and consolidate a European network providing the technological and clinical platform for large-scale functional genomic biomarker discovery. Here we review our current understanding of molecular mechanisms driving resistance to anti-angiogenesis agents, the current limitations of laboratory and clinical trial strategies and how the PREDICT consortium will endeavor to identify a new generation of predictive biomarkers.

Background Kisspeptin is a critical regulator of normal reproductive function. A single injection of kisspeptin in healthy human volunteers potently stimulates gonadotropin release. However, the effects of kisspeptin on gonadotropin release in women with hypothalamic amenorrhea (HA) and the effects of repeated administration of kisspeptin to humans are unknown. Aim The aim of this study was to determine the effects of acute and chronic kisspeptin administration on gonadotropin release in women with HA. Methods We performed a prospective, randomized, double-blinded, parallel design study. Women with HA received twice-daily sc injections of kisspeptin (6.4 nmol/kg) or 0.9% saline (n = 5 per group) for 2 wk. Changes in serum gonadotropin and estradiol levels, LH pulsatility, and ultrasound measurements of reproductive activity were assessed. Results On the first injection day, potent increases in serum LH and FSH were observed after sc kisspeptin injection in women with HA (mean maximal increment from baseline within 4 h after injection: LH, 24.0 ± 3.5 IU/liter; FSH, 9.1 ± 2.5 IU/liter). These responses were significantly reduced on the 14th injection day (mean maximal increment from baseline within 4 h postinjection: LH, 2.5 ± 2.2 IU/liter, P < 0.05; FSH, 0.5 ± 0.5 IU/liter, P < 0.05). Subjects remained responsive to GnRH after kisspeptin treatment. No significant changes in LH pulsatility or ultrasound measurements of reproductive activity were observed. Conclusion Acute administration of kisspeptin to women with infertility due to HA potently stimulates gonadotropin release, but chronic administration of kisspeptin results in desensitization to its effects on gonadotropin release. These data have important implications for the development of kisspeptin as a novel therapy for reproductive disorders in humans.

To find out whether non-steroidial anti-inflammatory drugs (NSAIDs) inhibit the proliferation of mucosal cells that normally leads to healing of gastric ulcers a microdissection technique was used to quantify mitosis in gastric glands at the ulcer edge in relation to that in the adjacent mucosa. The regeneration index thus obtained of the ulcer edge was greater in the 9 subjects with gastric ulcers not taking NSAIDs (mean index 3.1 [SEM 0.61]) than that in the 8 patients taking NSAIDs (index 1.49 [0.16]). In rats in which gastric ulcers were produced with a cryoprobe, the ulcers were larger and slower to heal in those receiving indomethacin than in controls; also, immunohistochemical staining indicated significantly fewer mitotic cells in glands adjacent to the ulcer in indomethacin-treated rats (8 mitoses [SEM 3]) than in control animals (25 [5]). The prostaglandin E1 analogue misoprostol reversed the inhibition of healing and substantially restored the proliferative rate in the animals. Inhibition of epithelial cell division normally involved in gastric ulcer healing would contribute to the high prevalence of gastric ulcer during NSAID therapy.

High-speed (video-rate) fluorescence lifetime imaging (FLIM) through a flexible endoscope is reported based on gated optical image intensifier technology. The optimization and potential application of FLIM to tissue autofluorescence for clinical applications are discussed.

High-speed (video-rate) fluorescence lifetime imaging (FLIM) is reported using two different time-domain approaches based on gated optical image intensifier technology. The first approach utilizes a rapidly switchable variable delay generator with sequential image acquisition, while the second employs a novel segmented gated optical imager to acquire lifetime maps in a single shot. Lifetimes are fitted using both a non-linear least-squares fit analysis and the rapid lifetime determination method. Monte Carlo simulations were used to optimize the acquisition parameters and a comparison between theory and experiment is presented. The importance of single-shot imaging to minimize the deleterious impact of sample movements is highlighted. Real-time FLIM movies of multi-well plate samples and tissue autofluorescence are presented.

We report the development of a high-speed wide-field fluorescence-lifetime imaging (FLIM) system that provides fluorescence-lifetime images at rates of as many as 29 frames/s. A FLIM multiwell plate reader and a potentially portable FLIM endoscopic system operating at 355-nm excitation have been demonstrated.

Nature Med. 5, 828– 831 (1999). On page 829, the legend for Fig 1 was incorrect. The corrected figure and legend are shown here. We regret this error.