Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the United States and shows a rapid clinical course, with a median survival of 6 months and a 5-year survival rate of only 3%. As chemotherapy and radiotherapy have only modest benefits and surgery is only possible in 20% of patients, earliest detection that allows surgical resection offers the best hope for longer survival. The identification of novel molecular markers and the development of imaging probes for pre-neoplastic/early invasive lesions is thus a high priority. We used phage display to identify peptides that distinguish mouse and human PDAC cells from normal pancreatic duct cells in vitro. We subsequently conjugated 2 peptides with the highest affinities and specificities to magnetofluorescent nanoparticles (CLIO-VT680) and demonstrate that these agents can effectively detect emerging tumors and pre-neoplastic lesions in a relevant transgenic mouse model via intravital confocal microscopy (Olympus IV100) and optical/MR imaging (OV-100, Bruker Pharmascan). Correlative histology confirmed the specific tumoral localization of the PDAC targeted agents. Additionally, the peptide-binding partners identified from this approach represent a snapshot of the proteome in aberrant cells and also potential PDAC biomarkers. Using affinity chromatography, we identified the binding partners for several peptides and demonstrate their validity as biomarkers. These specific and sensitive probes may have clinical utility in the diagnosis and management of PDAC in humans.

To find new genes involved in esophageal squamous cell carcinogenesis, we constructed custom cDNA arrays and used the arrays to compare gene expression profiles of 12 matched normal and malignant esophageal samples including seven superficial cancer tissues. The arrays represented nearly 4000 genes, including 1728 that were specifically selected based on pilot studies to find genes that were differentially expressed in esophageal cancers. Expression values for all genes were normalized for each sample and were compared in normal versus tumor tissues. There was a marked decrease in the levels of the transcriptional elongation factor A gene in all 12 of the squamous cell cancer samples compared to matched normal samples. Because the transcription elongation factor A gene has not been previously reported to be involved in cancer development, our results suggest that further investigation of its role in esophageal carcinogenesis is warranted.

PURPOSE:Recent studies sought to refine lung cancer classification using gene expression microarrays. We evaluate the extent to which these studies agree and whether results can be integrated.EXPERIMENTAL DESIGN:We developed a practical analysis plan for cross-study comparison, validation, and integration of cancer molecular classification studies using public data. We evaluated genes for cross-platform consistency of expression patterns, using integrative correlations, which quantify cross-study reproducibility without relying on direct assimilation of expression measurements across platforms. We then compared associations of gene expression levels to differential diagnosis of squamous cell carcinoma versus adenocarcinoma via reproducibility of the gene-specific t statistics and to survival via reproducibility of Cox coefficients.RESULTS:Integrative correlation analysis revealed a large proportion of genes in which the patterns agreed across studies more than would be expected by chance. Correlation of t statistics for diagnosis of squamous cell carcinoma versus adenocarcinoma is high (0.85) and increases (0.925) when using only the most consistent genes identified by integrative correlation. Correlations of Cox coefficients ranged from 0.13 to 0.31 (0.33-0.49 with genes selected for consistency). Although we find genes that are significant in multiple studies but show discordant effects, their number is approximately that expected by chance. We report genes that are reproducible by integrative analysis, significant in all studies, and concordant in effect.CONCLUSIONS:Cross-study comparison revealed significant, albeit incomplete, agreement of gene expression patterns related to lung cancer biology and identified genes that reproducibly predict outcomes. This analysis approach is broadly applicable to cross-study comparisons of gene expression profiling projects.

Journal of the Royal Statistical Society: Series B (Statistical Methodology)Volume 64, Issue 4 p. 737-775 Discussion on the meeting on ‘Statistical modelling and analysis of genetic data’ First published: 23 October 2002 https://doi.org/10.1111/1467-9868.00359Citations: 11Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume64, Issue4October 2002Pages 737-775 RelatedInformation

SummaryGenome-wide measurement of gene expression is a promising approach to the identification of subclasses of cancer that are currently not differentiable, but potentially biologically heterogeneous. This type of molecular classification gives hope for highly individualized and more effective prognosis and treatment of cancer. Statistically, the analysis of gene expression data from unclassified tumours is a complex hypothesis-generating activity, involving data exploration, modelling and expert elicitation. We propose a modelling framework that can be used to inform and organize the development of exploratory tools for classification. Our framework uses latent categories to provide both a statistical definition of differential expression and a precise, experiment-independent, definition of a molecular profile. It also generates natural similarity measures for traditional clustering and gives probabilistic statements about the assignment of tumours to molecular profiles.

Methylation of DNA in CpG dense regions of gene promoters (CpG islands) is important for transcriptional inactivation of selective genes in normal and neoplastic cells. Here, we present a spreadsheet-based program adapted from Microsoft Excel that is useful for identifying CpG islands and for assisting in the laboratory analysis of DNA methylation of these regions. Upon execution of the program, a customized workbook analyzes an entered DNA sequence for the total number and percentage cytosine and guanine nucleotides, the total number and percentage of CpG sites, and a CpG:GpC ratio. The program also displays the distribution of CpG sites in a visual format as well as in two different graphical formats. Finally, the program assists in laboratory studies of DNA methylation that employ bisulfite modification of DNA by displaying methylation-dependent effects of bisulfite treatment on DNA sequences.

Recently, mutations of the transforming growth factor-beta receptor type I gene have been reported to occur at high frequency in breast cancer metastases, with all mutations being an identical C to A transversion at nucleotide 1160 of the gene (T. Chen et al, Cancer Res., 58: 4805-4810, 1998). This mutation would result in a serine to tyrosine substitution at codon 387 (S387Y) and would reportedly disrupt receptor function. Because this mutation reportedly occurred at high frequency in breast cancer metastases (42%) and much less frequently in primary breast cancer tumors (6%), this would seem to represent a pivotal genetic alteration in breast cancer progression. To further investigate the possible role of this specific genetic alteration in the progression of breast cancer and other forms of adenocarcinoma, we analyzed 20 breast cancer metastases, 15 lung adenocarcinoma metastases, and 13 colorectal cancer metastases for possible mutations at this site. Using both single-strand conformation polymorphism screening and sequencing, we found no mutations of this gene in any of our samples. Our results suggest the S387Y mutation of the transforming growth factor-beta receptor type I gene is not common in these types of human cancers.

In some tumors, defects in mismatch repair enzymes lead to errors in the replication of simple nucleotide repeat segments. This condition is commonly known as microsatellite instability (MSI) because of the frequent mutations of microsatellite sequences. Although the MSI phenotype is well recognized in some colon, gastric, pancreatic, and endometrial cancers, reports of MSI in breast cancer are inconsistent. We report here our experience with >10,000 amplifications of simple nucleotide repeats in noncoding genomic regions using DNA from 267 cases of breast cancer, including cases that represent all major histological types of breast cancer. We rarely (10 reactions) found unexpected bands in amplifications of tumor DNA that were not present in amplifications of normal DNA. Moreover, repeats of these reactions did not confirm microsatellite instability in a single case. We also evaluated the simple nucleotide repeats in the transforming growth factor type II receptor, insulin-like growth factor type II receptor, BAX, and E2F-4 genes, which are frequently mutated in tumors with microsatellite instability. No mutations of these genes were found in any of the 30 breast cancer cell lines and 61 primary breast cancer samples examined. These results indicate that mismatch repair errors characteristic of the MSI phenotype are uncommon in human breast cancer.

Molecular biology laboratories frequently face the challenge of aligning small overlapping DNA sequences derived from a long DNA segment. Here, we present a short program that can be used to adapt Excel spreadsheets as a tool for aligning DNA sequences, regardless of their orientation. The program runs on any Windows or Macintosh operating system computer with Excel 97 or Excel 98. The program is available for use as an Excel file, which can be downloaded from the BioTechniques Web site. Upon execution, the program opens a specially designed customized workbook and is capable of identifying overlapping regions between two sequence fragments and displaying the sequence alignment. It also performs a number of specialized functions such as recognition of restriction enzyme cutting sites and CpG island mapping without costly specialized software.

Small deletions or expansions of the simple nucleotide repeats of microsatellites (i.e., microsatellite instability [MI]) are well described in cancers from patients with the hereditary non-polyposis colorectal cancer (HNPCC) syndrome and result from mutations of mismatch repair enzyme genes such as hMSH2, hMLH1, hPMS1, hPMS2 or GTBP (reviewed by Speicher, 1995). The potential significance of mismatch mutations in carcinogenesis became apparent when HNPCC cancers were also found to have frequent frameshift mutations of simple nucleotide repeats within the coding regions of several key genes involved in the regulation of cell growth or apoptosis. These intragenic repeats include a poly (A)10 tract in the transforming growth factor-β type II receptor (RII) gene (Parsons et al., 1995), a poly (G)8 tract in the insulin-like growth factor type II receptor (IGFIIR) gene (Souza et al., 1996), a poly (G)8 tract in BAX (Rampino et al., 1997) and a poly (AGC)13 tract in the E2F-4 gene (Souza et al., 1997). Although MI is considered to be most characteristic of cancers associated with the hereditary non-polyposis colorectal cancer (HNPCC) syndrome, MI has also been described in a variety of sporadic human cancers (Speicher, 1995). For example, we have previously recognized frequent microsatellite instability in primary small cell lung cancer (SCLC), with 15 of 33 cancers displaying alterations of (CA)n repeats (Merlo et al., 1994). MI in these cases of SCLC affected only 4–44% of all tested microsatellite loci, representing a distinctly lower frequency than that found in HNPCC cancers with MI. However, the frequency of MI in at least a subset of SCLCs appears to be greater than that for many other sporadic cancers (including non-small cell lung cancer [NSCLC]), both in the number of identified cases with MI and in the frequency of alleles affected (Abe et al., 1996; Mao et al., 1994). The significance of mismatch-induced frameshift mutations in the pathogenesis of lung cancers, including SCLCs, is unknown. Previous studies of frameshift mutations in lung cancer have been limited to evaluation of the RII gene. In one study, only 1 of 203 NSCLCs was found to have MI and this tumor did not have a mutation of the RII gene (Abe et al., 1996). More recently, a set of 33 sporadic lung cancers of unspecified histology from individuals with rare H-ras1 alleles (which is frequently associated with MI [Ryberg et al., 1995]) were evaluated and no mutations of RII were found (Takenoshita et al., 1997). These previous investigations have not addressed the issues of frameshift mutations specifically in SCLC, which has a greater frequency of MI than NSCLC. Furthermore, these studies have not investigated possible frameshift mutations in genes other than RII and it is well recognized that different types of human cancers frequently have a diverse array of genetic alterations. For example, RER+ endometrial cancers frequently have mutations of IGFRII and only rarely mutations of RII, whereas RER+ colorectal cancers frequently have mutations of RII and only rarely mutations of IGFRII (Myeroff et al., 1995). The purpose of the present study, therefore, was to evaluate the 4 genes found to be commonly affected by frameshift mutations in HNPCC tumors (the RII, IGFIIR, BAX and E2F-4 genes) in cases of SCLC with MI. Adequate quantities of DNA were available from 13 of the 15 cases of SCLC previously reported to have MI. To test for mutations, polymerase chain reaction (PCR) amplification of tumor DNA was performed using the primers and conditions specified in Table I. Reactions were radiolabelled using 0.2 μCi of [32P]CTP in each 10-μl reaction mixture and products were electrophoresed on 6% polyacrylamide denaturing gels and visualized by autoradiography. Frameshift mutation was determined by visualization of bands in tumor DNA that were not present in corresponding normal. We examined all repetitive sequences in the RII, IGFIIR, BAX and E2F-4 genes for each of the 13 cases of SCLC, and found no evidence of frameshift mutations in any of these cases. Figure 1 demonstrates representative amplifications of repeats segments of the RII and IGFIIR genes. Analysis of the BAX and IGFIIR intragenic repeats in small cell lung cancer (SCLC). Polymerase chain reaction (PCR) amplified products were electrophoresed as described in text. Numbers across top correspond to the case numbers designated previously. Positive controls are DNA samples from hereditary non-polyposis colorectal cancers (HNPCCs) with known mutations. Our results demonstrate that although frameshift mutations of microsatellites are relatively common in a subset of SCLC, frameshift mutations of genes commonly mutated in HNPCC cancers (i.e., RII, IGFIIR, BAX and E2F-4) are uncommon in this type of cancer. Mutations of these four genes examined may be relatively uncommon in SCLC because the simple nucleotide repeats of these genes may not be targets of the underlying repair defects in these tumors. Alternatively, mutations of these genes may not confer a selective growth advantage to clonal precursors of SCLC. We do not know if genes other than RII, IGFIIR, BAX and E2F-4 may be affected by frameshift mutations in SCLC. Yours sincerely, Samples of DNA from HNPCC colorectal cancers were provided by Drs. S. Hamilton and A. Horii. This work was supported by award CA58184 from the National Cancer Institute, Bethesda, MD, USA. Ramaswamy Anbazhagan*, Adrian Merlo , David Sidransky* , Edward Gabrielson [email protected]* ?

Small cell lung cancer is a common type of lung cancer that is generally classified within the spectrum of neuroendocrine lung neoplasms. Using high-density cDNA arrays, we profiled gene expression of small cell lung cancers and compared these expression profiles to those of normal bronchial epithelial cells and pulmonary carcinoids, which are classified as benign neuroendocrine tumors. We found the overall expression profiles of two small cell lung cancer cell lines, two microdissected tissue samples of primary small cell lung cancer, and cultured bronchial epithelial cells to be relatively similar to one another, with an average Pearson correlation coefficient for these comparisons of 0.63. However, we found the expression profiles of small cell lung cancers (and bronchial epithelial cells) to be surprisingly dissimilar to those of two samples of pulmonary carcinoid tumors, with an average correlation coefficient for these comparisons of 0.20. We then compared the pulmonary carcinoid expression profiles to those of two samples of infiltrating astrocytic brain cancers (oligodendroglioma and high-grade astrocytoma) and found similarity of gene expression among these four samples (average correlation coefficient, 0.57). These gene expression profiles suggest that small cell lung cancers are closely related to (and possibly derived from) epithelial cells, and that pulmonary carcinoids are related to neural crest-derived brain tumors. More generally, our results suggest that broad profiles of gene expression may reveal similarities and differences between tumors that are not apparent by traditional morphological criteria.

Aims: Studies of developing human breasts are essential for understanding the organogenesis as well as molecular pathogenesis of benign and malignant breast diseases. In this study we have examined the distribution of TGF-alpha, TGF-beta 1, tenascin-C and collagen type IV with the aim of starting to build a picture of the profile of molecules that may be involved in the development of the human breast,Methods and results: Ten fetal breasts (16 to 23 weeks of gestation) and 45 infant breasts, ranging in age from newborn to 2 years, were used in this study Paraffin sections from these samples were immunostained with antibodies for these proteins and for Ki67 to elucidate the level of proliferative activity in different stages of breast development, TGF-alpha immunoreactivity was observed both in the stromal and the epithelial cells within fetal and infant breasts up to 25 days, TGF-beta 1 immunoreactivity was localized in the extracellular matrix. Tenascin-C was found around the neck of the developing breast bud and in the extracellular matrix of the infant with peaks in the newborn at 6-12 weeks. The immunoreactivity for type IV collagen was more intense in the region of the breast bud neck in the fetal breasts and reduced around the tips of lobular and terminal-end buds within the infant breasts.Conclusions: The distribution of the growth factors and extracellular matrix proteins within the developing human breast indicates that they play a significant role in different cellular compartments during morphogenesis and provides insights into breast disease.

Loss of heterozygosity (LOH) of chromosomal arm 8p has been reported to occur at high frequency for a number of common forms of human cancer, including breast cancer. The objectives of this study were to define the regions on this chromosomal arm that are likely to contain breast cancer tumor suppressor genes and to determine when loss of chromosomal arm 8p occurs during breast cancer progression. For mapping the tumor suppressor gene loci, we evaluated 60 cases of infiltrating ductal cancer for allelic loss using 14 microsatellite markers mapped to this chromosomal arm and found LOH of 8p in 36 (60%) of the tumors. Whereas most of these tumors had allelic loss at all informative markers, five tumors had partial loss of 8p affecting two nonoverlapping regions. LOH for all but one of the tumors with 8p loss involved the region between markers D8S560 and D8S518 at 8p21.3-p23.3, suggesting that this is the locus of a breast cancer tumor suppressor gene. We then studied LOH of 8p in 38 cases of ductal carcinoma in situ (DCIS) with multiple individually microdissected tumor foci evaluated for each case. LOH of 8p was found in 14 of the DCIS cases (36%), including 6 of 16 cases of low histological grade and 8 of 22 cases of intermediate or high histological grade. In four of these DCIS cases, 8p LOH was seen in some but not all of the multiple tumor foci examined. These data suggest that during the evolution of these tumors, LOH of 8p occurred after loss of other chromosomal arms that were lost in all tumor foci. Thus, LOH of 8p, particularly 8p21.3-p23, is a common genetic alteration in infiltrating and in situ breast cancer. Although 8p LOH is common even in low histological grade DCIS, this allelic loss often appears to be preceded by loss of other alleles in the evolution of breast cancer.

In order to explain the molecular events that contribute to benign and malignant breast disease, it is essential to understand the cellular context in which these are occurring. This study describes a detailed analysis of the epithelial phenotypes in the human fetal and infant breast and provides a starting point for such consideration. Using methacarn-fixed, paraffin sections from ten fetal and 45 infant breast, immunostained with a panel of antibodies to cytoskeletal proteins and kappa-casein, it has been possible to define in detail the chronological evolution of the major cell types in the human breast from 16 weeks of intrauterine life to 2 years of age, in both sexes. Cells at the tips of the lobular buds and terminal end buds have a characteristic cytoskeletal protein profile, suggesting that they may have the capacity to generate both basal cells and luminal cells. Based on the expression of cytoskeletal proteins in the developing fetal and infant breast, a model system has been proposed for mammary epithelial differentiation. (C) 1998 John Wiley & Sons, Ltd.

Integrins are widely expressed on normal tissues and their function is considered critical directly or indirectly with the control of cell growth and differentiation. Also, they are likely to play a crucial role in cell–matrix interactions during development. As the human breast develops after birth, it provides a rare opportunity in which to study human organogenesis. We have examined the distribution of integrins in the human infant breast with the aim of elucidating the possible role of these molecules in morphogenesis and differentiation. Necropsy breast specimens from six male and eight female infants, ranging in age from 1 day to 9 months, were used in this study. Cryostat sections were stained by the avidin‐biotin complex technique, using a panel of monoclonal antibodies (MAbs) which recognize β 1 , α 2 , α 6 , β 4 , α v , and α v β 3 integrin chains, which are candidate molecules for a role in mammory morphogenesis. MAbs to β 1 (DH12) and α 2 (HAS3) showed positive membrane and cytoplasmic staining of basal cells and luminal epithelial cells. In addition, positive staining for the β 1 integrin chain was found on fibroblasts. A MAb which recognizes the α 6 chain (MP4F10) showed positive staining of the basal cells and heterogeneous staining of the luminal epithelial cells, whilst β 4 chain (439‐9B) showed positive staining in the basement membrane domain of the basal cells with no staining of the luminal epithelial cells. There was a positive correlation between the intensity of expression and the structural development of the ductal system, with integrin expression reduced or absent in the end buds and lateral buds. These data provide evidence that some integrin molecules are expressed in a pattern that correlates with the morphological and functional differentiation of the normal mammary gland. Changes in the expression and function of integrins may have an inductive role in the development of the normal mammary gland.

Abstract : In this paper we describe the light and electron microscopic appearance of the embryonic type of fat in human infant breast, together with immunocytochemical findings. This fat tissue was composed of numerous capillaries surrounded by a mixed population of undifferentiated mesenchymal cells and preadipocytes at various stages of differentiation. The preadipocytes were characterised by a number of cytoplasmic processes, varying numbers of lipid droplets, and an envelope of electrondense material outside the cell membrane. Immunocytochemistry showed a characteristic distribution of collagen type IV adjacent to and vimentin and S100 protein within the preadipocytes. This is the first report of the ultrastructure of the human mammary embryonic type of fat. The possible role of the embryonic type of fat in the development and growth of the human breast is discussed. © Wiley‐Liss, Inc.

The myoepithelial cell was first identified in the breast 150 years ago when the presence of an apparent smooth muscle cell in a glandular epithelium was recognised as an unusual developmental feature. Recent knowledge of the composition of the cytoskeleton, together with ultrastructural investigations, have helped to clarify the differentiation of these cells in vivo and also to interpret their role in pathological processes. The development of new markers which identify these cells and monitor their state in culture has also raised interest in myoepithelial cell function and cell lineages within the ductal system of the organs in which they are found.