Molecular profiling of clinical tissues specimens: feasibility and applications.
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Databases, Factual↗
Biomedical subjects
M R Emmert-Buck
Publications and source records attributed to M R Emmert-Buck.
High frequency allelic loss on chromosome 17p13.3-p11.1 in esophageal squamous cell carcinomas from a high incidence area in northern China.
Allelic loss on chromosome 17p has been reported frequently in esophageal squamous cell carcinoma (ESCC) and generally encompasses the p53 locus at 17p13.1. However, a good correlation between allelic loss on 17p and mutation of p53 has not been found. This suggests the possibility that unknown tumor suppressor genes near p53 may be involved in the development of ESCC. To evaluate this possibility, we analyzed 30 microsatellite markers covering the entire short arm of chromosome 17 in 56 ESCC patients from a high risk population in northern China, including 34 with a family history of upper gastrointestinal (UGI) cancer and 22 without a family history of any cancer. Cancer lifestyle risk factors and clinical/pathological characteristics were also collected. We found frequent allelic loss (>/=65%) at 28 of the 30 markers evaluated in these ESCC patients. The highest frequencies of allelic loss (> or =80%) were found in three smaller regions: deletion region I located at 17p13.3-p13.2 (between D17S849 and D17S1828); deletion region II located at 17p13.2-p13.1 (between D13S938 and TP53); deletion region III located at 17p13.1-p12 (between D17S804 and D17S799). A number of genes have already been identified in these deleted regions, including: OVCA1, OVCA2 and HIC-1 in deletion region I; p53 in deletion region II; ZNF18, ZNF29, ALDH3 and ALDH10 in deletion region III. These results will help us direct future testing of candidate genes and narrow the search region for major new tumor suppressor genes that may play a role in the pathogenesis of ESCC.
Adult↗
New approaches to molecular profiling of tissue samples.
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Alleles↗
Characterization of intracellular prostate-specific antigen from laser capture microdissected benign and malignant prostatic epithelium.
The proportion of unbound serum prostate-specific antigen (PSA; percent-free PSA) is reported to be lower in men with prostate cancer compared to men with benign prostates (U. H. Stenman et al., Cancer Res., 51: 222-226, 1991; H. Lilja et al., Clin. Chem., 37: 1618-1625, 1991; D. L. Woodrum et al., J. Urol., 159: 5-12, 1998; W. J. Catalona et al., J. Am. Med. Assoc., 279: 1542-1547, 1998). The majority of immunoreactive PSA in serum is complexed to alpha-1-antichymotrypsin (ACT). Two major mechanistic questions have previously been unknown: (a) Does PSA in human prostate cancer cells in tissue exist in a free or bound form? and (b) Is PSA produced by malignant cells in the free form because it has lost the ability to form a complex with ACT? Laser capture microdissection (LCM) enables the acquisition of pure populations of defined cell types from tissue (M. R. Emmert-Buck et al., Science, 274: 998-1001, 1996; R. F. Bonner et al., Science, 278: 1481-1483, 1997). This technology provides a unique opportunity to study intracellular protein composition and structure from human cells. In this study, we used LCM to assess the bound versus free form of intracellular PSA in both benign and malignant epithelium procured from prostate tissue. One-dimensional and two-dimensional PAGE were performed on cellular lysates from LCM-procured benign and malignant prostate epithelium from frozen tissue specimens. Western blotting analysis of one-dimensional PAGE gels revealed a strong band at M(r) 30,000 (expected molecular weight of unbound PSA) in all cases demonstrating that the vast majority of intracellular tumor and normal PSA exists within cells in the "free" form. Binding studies showed that PSA recovered from LCM-procured cells retained the full ability to bind ACT, and two-dimensional PAGE Western analysis demonstrated that the PSA/ACT complex was stable under strong reducing conditions. We conclude that intracellular PSA exists in the "free" form and that binding to ACT occurs exclusively outside of the cell.
Blotting, Western↗
An approach to proteomic analysis of human tumors.
A strategy for proteomic analysis of microdissected cells derived from human tumor specimens is described and demonstrated by using esophageal cancer as an example. Normal squamous epithelium and corresponding tumor cells from two patients were procured by laser-capture microdissection and studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Fifty thousand cells resolved approximately 675 distinct proteins (or isoforms) with molecular weights ranging between 10 and 200 kDa and isoelectric points of pH 3-10. Comparison of the microdissected protein profiles showed a high degree of similarity between the matched normal-tumor samples (98% identical). However, 17 proteins showed tumor-specific alterations, including 10 that were uniquely present in the tumors and seven that were observed only in the normal epithelium. Two of the altered proteins were characterized by mass spectrometry and immunoblot analysis and were identified as cytokeratin 1 and annexin I. Acquisition of 2D-PAGE protein profiles, visualization of disregulated proteins, and subsequent determination of the identity of selected proteins through high-sensitivity MS-MS microsequencing are possible from microdissected cell populations. These separation and analytical techniques are uniquely capable of detecting tumor-specific alterations. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms. Mol.
Electrophoresis, Gel, Two-Dimensional↗
Development of a prostate cDNA microarray and statistical gene expression analysis package.
A cDNA microarray comprising 5184 different cDNAs spotted onto nylon membrane filters was developed for prostate gene expression studies. The clones used for arraying were identified by cluster analysis of > 35 000 prostate cDNA library-derived expressed sequence tags (ESTs) present in the dbEST database maintained by the National Center for Biotechnology Information. Total RNA from two cell lines, prostate line 8.4 and melanoma line UACC903, was used to make radiolabeled probe for filter hybridizations. The absolute intensity of each individual cDNA spot was determined by phosphorimager scanning and evaluated by a bioinformatics package developed specifically for analysis of cDNA microarray experimentation. Results indicated 89% of the genes showed intensity levels above background in prostate cells compared with only 28% in melanoma cells. Replicate probe preparations yielded results with correlation values ranging from r = 0.90 to 0.93 and coefficient of variation ranging from 16 to 28%. Findings indicate that among others, the keratin 5 and vimentin genes were differentially expressed between these two divergent cell lines. Follow-up northern blot analysis verified these two expression changes, thereby demonstrating the reliability of this system. We report the development of a cDNA microarray system that is sensitive and reliable, demonstrates a low degree of variability, and is capable of determining verifiable gene expression differences between two distinct human cell lines. This system will prove useful for differential gene expression analysis in prostate-derived cells and tissue.
DNA, Complementary↗
Review of AACR meeting: new research approaches in the prevention and cure of prostate cancer, 2-6 December 1998, Indian Wells, CA.
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Animals↗
Allelic loss on chromosome arm 8p: analysis of sporadic epithelial ovarian tumors.
OBJECTIVE: Our objective was to determine the frequency of allelic loss at 8p21 in sporadic epithelial ovarian cancer. We recently described allelic loss at this locus in 7/9 ovarian cancers from patients with BRCA1 gene mutations. METHODS: We anonymously obtained and examined 40 unselected invasive epithelial ovarian cancers and 5 low-malignant-potential (LMP) ovarian tumors for loss of heterozygosity (LOH) at 8p12-22. Pure epithelial and stromal cell populations were procured selectively by laser capture microdissection and extracted DNA was amplified with polymorphic microsatellite markers spanning the region of interest. RESULTS: LOH was highest (50%) at marker D8S136 located at 8p21 with 15 of 30 informative cases exhibiting an allelic deletion. None of the LMP tumors evaluated showed LOH at 8p12-22. A trend toward more frequent LOH at 8p12-22 was identified with increasing disease aggressiveness from LMP to early stage invasive ovarian cancer to advanced stage invasive ovarian cancer (Lehman's test, P2 < 0.024). CONCLUSIONS: Fifty percent allelic loss at the distal portion of 8p21 has not been reported to date for sporadic epithelial ovarian carcinomas. The higher rate of loss in our cohort, in contrast to previous allelotyping studies, is due likely to analysis from homogenous cell populations. These results, in concert with our previous study of BRCA1 mutation-positive patients, suggest a tumor suppressor gene locus at 8p21 for epithelial ovarian cancer.
Adult↗
Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.
Microdissection of routinely stained or unstained frozen sections has been used successfully to obtain purified cell populations for the analysis of cell-specific gene expression patterns in primary tissues with a complex mixture of cell types. However, the precision and usefulness of microdissection is frequently limited by the difficulty to identify different cell types and structures by morphology alone. We therefore developed a rapid immunostaining procedure for frozen sections followed by laser capture microdissection (LCM) and RNA extraction, which allows targeted mRNA analysis of immunophenotypically defined cell populations. After fixation, frozen sections are immunostained under RNAse-free conditions using a rapid three-step streptavidin-biotin technique, dehydrated and immediately subjected to LCM. RNA is extracted from captured tissue, DNAse I treated, and reverse transcribed. Acetone-, methanol-, or ethanol/acetone-fixed sections give excellent immunostaining after 12 to 25 minutes total processing time. Specificity, precision, and speed of microdissection is markedly increased due to improved identification of desired (or undesired) cell types. The mRNA recovered from immunostained tissue is of high quality. Single-step PCR is able to amplify fragments of more than 600 bp from both housekeeping genes such as beta-actin as well as cell-specific messages such as CD4 or CD19, using cDNA derived from less than 500 immunostained, microdissected cells. Immuno-LCM allows specific mRNA analysis of cell populations isolated according to their immunophenotype or expression of function-related antigens and significantly expands our ability to investigate gene expression in heterogeneous tissues.
Cells↗
The gene for multiple endocrine neoplasia type 1: recent findings.
Multiple endocrine neoplasia type 1 (MENI) is a promising model to understand endocrine and other tumors. Its most common endocrine expressions are tumors of parathyroids, entero-pancreatic neuro-endocrine tissue, and anterior pituitary. Recently, collagenomas and multiple angiofibromas of the dermis also have been recognized as very common. MEN1 can be characterized from different perspectives: (a) as a hormone (parathyroid hormone, gastrin, prolactin, etc.) excess syndrome with excellent therapeutic options; (b) as a syndrome with sometimes lethal outcomes from malignancy of entero-pancreatic neuro-endocrine or foregut carcinoid tissues; or (c) as a disorder than can give insight about cell regulation in the endocrine, the dermal, and perhaps other tissue systems. The MEN1 gene was identified recently by positional cloning, a comprehensive strategy of narrowing the candidate interval and evaluating all or most genes in that interval. This discovery has opened new approaches to basic and clinical issues. Germline MEN1 mutations have been identified in most MEN1 families. Germline MENI mutations were generally not found in families with isolated hyperparathyroidism or with isolated pituitary tumor. Thus, studies with the MENI gene helped establish that mutation of other gene(s) is likely causative of these two MEN1 phenocopies. MEN1 proved to be the gene most frequent L4 mutated in common-variety, nonhereditary parathyroid tumor, gastrinoma, insulinoma, or bronchial carcinoid. For example, in common-variety parathyroid tumors, mutation of several other genes (such as cyclin D1 and P53) has been found, but much less frequently than MEN1 mutation. The majority of germline and somatic MEN1 mutations predicted truncation of the encoded protein (menin). Such inactivating mutations strongly supported prior predictions that MEN1 is a tumor suppressor gene insofar as stepwise mutational inactivation of both copies can release a cell from normal growth suppression. Menin is principally a nuclear protein; menin interacts with junD. Future studies, such as discovery of menin's metabolic pathway, could lead to new opportunities in cell biology and in tumor therapy.
Endocrine Gland Neoplasms↗
The genetics of cancer--a 3D model.
Gene expression microarrays hold great promise for studies of human disease states. There are significant technical issues specific to utilizing clinical tissue samples which have yet to be rigorously addressed and completely overcome. Precise, quantitative measurement of gene expression profiles from specific cell populations is at hand, offering the scientific community the first comprehensive view of the in vivo molecular anatomy of normal cells and their diseased counterparts. Here, we propose a model for integrating-in three dimensions-expression data obtained using the microarray.
Databases, Factual↗
The Cancer Genome Anatomy Project: EST sequencing and the genetics of cancer progression.
As the process of tumor progression proceeds from the normal cellular state to a preneoplastic condition and finally to the fully invasive form, the molecular characteristics of the cell change as well. These characteristics can be considered a molecular fingerprint of the cell at each stage of progression and, analogous to fingerprinting a criminal, can be used as markers of the progression process. Based on this premise, the Cancer Genome Anatomy Project was initiated with the broad goal of determining the comprehensive molecular characterization of normal, premalignant, and malignant tumor cells, thus making a reality the identification of all major cellular mechanisms leading to tumor initiation and progression ([Strausberg, R.L., Dahl, C.A., and Klausner, R.D. (1997). "New opportunities for uncovering the molecular basis of cancer." Nat. Genet., 16: 415-516.], www.ncbi.nlm.nih.gov/ncicgap/). The expectation of determining the genetic fingerprints of cancer progression will allow for 1) correlation of disease progression with therapeutic outcome; 2) improved evaluation of disease treatment; 3) stimulation of novel approaches to prevention, detection, and therapy; and 4) enhanced diagnostic tools for clinical applications. Whereas acquiring the comprehensive molecular analysis of cancer progression may take years, results from initial, short-term goals are currently being realized and are proving very fruitful.
Databases, Factual↗
Loss of heterozygosity on chromosome 13 is associated with advanced stage prostate cancer.
PURPOSE: In order to investigate the possible involvement of a tumor suppressor gene(s) on chromosome 13 in prostatic neoplasms, we performed loss of heterozygosity (LOH) analysis on normal and tumor pairs from 36 prostate cancer patients. MATERIALS AND METHODS: Pure DNA was obtained from carcinoma cells and normal epithelium by tissue microdissection. The DNA had previously been analyzed for LOH on chromosomes 8 and 16. After an initial pilot experiment to determine the region(s) of significant LOH from 9 loci on chromosome 13q, 3 loci at and near the Rb1 locus (D13S153, D13S1319, and D13S1303) were chosen for further study. RESULTS: The overall rate of LOH on chromosome 13 was 27.3%. Four tumors exhibited LOH at all 3 loci. Two tumors exhibited LOH at D13S153 but not at the other, more telomeric loci; two additional tumors had loss at D13S1303 or D13S1319 but not D13S153. These data suggest that a tumor suppressor gene involved in prostate cancer may be located just telomeric to Rb1. Analysis of clinical and pathological data from carcinomas with and without loss shows that chromosome 13q LOH is correlated with advanced stage prostate cancer. CONCLUSIONS: Our LOH data suggests that there may be a tumor suppressor gene telomeric to Rb1 that is potentially involved in prostate cancer progression. Identification of this gene may be valuable in providing diagnostic and prognostic information for prostate cancer patients.
Chromosome Mapping↗
Molecular regulation, membrane association and secretion of tumor cathepsin B.
Upregulation, membrane association and secretion of cathepsin B have been shown to occur in many types of tumors and to correlate positively with their invasive and metastatic capabilities. To further understand changes in cathepsin B activity and localization, we have been examining its regulation at many levels including transcription and trafficking. Our studies indicate that there may be three promoter regions in the cathepsin B gene. Of these, continued examination of the promoter upstream of exon 1 has indicated possible control by several regulatory factors including E-box and Sp-1 binding elements. Upregulation of cathepsin B at this level may account for some of the secretion of cathepsin B found in tumors. We have also gathered evidence that endo- and exocytosis of cathepsin B may be regulated by ras and ras-related proteins in addition to previously described trafficking systems. There is also evidence that several populations of lysosomes may exist and that trafficking to different populations may determine whether cathepsin B is secreted from the tumor cell or remains intracellular. Our results indicate that membrane association and secretion of cathepsin B is not a random process in the tumor cell, but rather part of a tightly controlled system.
Animals↗
Chromosome 16 allelic loss analysis of a large set of microdissected prostate carcinomas.
PURPOSE: To perform loss of heterozygosity (LOH) analysis on chromosome 16 in 102 highly purified DNA samples isolated from one or more adenocarcinomas, prostatic intraepithelial neoplasia (PIN), and matched benign prostatic epithelium from 95 radical prostatectomy patients. MATERIALS AND METHODS: Specimens were procured by microdissection of frozen tissue samples, thus ensuring that highly select pure populations of cells were obtained for DNA extraction and LOH analysis. Multiple microsatellite markers were used to determine allelic loss on chromosome 16q. RESULTS: Overall loss on 16q was seen in 31% of the cancers, and occurred more frequently in high stage cancers than low stage cancers. In contrast, allelic loss in PIN failed to exceed 6% at any of the loci that were examined. CONCLUSIONS: These results suggest that inactivation of a putative tumor suppressor gene on 16q may be involved in the progression of some prostate cancers.
Adenocarcinoma↗
Multiple endocrine neoplasia type 1: clinical and genetic features of the hereditary endocrine neoplasias.
MEN1 is a syndrome of parathyroid adenomas, gastrinomas, prolactinomas, and other endocrine tumors. Collagenomas and facial angiofibromas are newly recognized but common skin expressions. Many tumors in MEN1 are benign; however, many entero-pancreatic neuroendocrine tumors and foregut carcinoid tumors are malignant. MEN1 is thus the expression of a cancer gene but without available prevention or cure for malignancy. Hereditary (as compared to sporadic) endocrine tumors show early onset age and multiplicity, because each cell of the body has "one hit" by inheritance. Multiple neoplasia syndromes with endocrine tumor(s) all include nonendocrine components; their known defective genes seem mainly to disturb cell accumulation. Hereditary neoplasia/hyperplasia of one endocrine tissue reflects a defect that is tissue selective and directed at cell secretion. Though the hereditary endocrine neoplasias are rare, most of their identified genes also contribute to common sporadic endocrine neoplasms. Hereditary tumors may be caused by activation of an oncogene (e.g., RET) or, more often, by inactivation of a tumor suppressor gene (e.g., P53, MEN1). Recently, MEN1 was identified by positional cloning. This strategy included narrowing the gene candidate interval, identifying many or all genes in that interval, and testing the newly identified candidate genes for mutation in MEN1 cases. MEN1 was identified because it showed mutation in 14 of 15 MEN1 cases. NIH testing showed germline MEN1 mutations in 47 of 50 MEN1 index cases and in seven of eight cases with sporadic MEN1. Despite proven capacity to find germline MEN1 mutation, NIH testing found no MEN1 mutation among five families with isolated hyperparathyroidism, suggesting that this often arises from mutation of other gene(s). Analogous studies in Japan found that familial isolated pituitary tumors also did not show MEN1 germline mutation. MEN1 mutation testing can now be considered for cases of MEN1 and its phenocopies and for asymptomatic members of families with known MEN1 mutation. Germline MEN1 testing does not have the urgency of RET testing in MEN2a and 2b, as MEN1 testing does not commonly lead to an important intervention. Somatic MEN1 mutation was found in sporadic tumors: parathyroid adenoma (21%), gastrinoma (33%), insulinoma (17%), and bronchial carcinoid (36%). For each of these, MEN1 was the known gene most frequently mutated. MEN1 has a widely expressed mRNA that encodes a protein (menin) of 610 amino acids. The protein sequence is not informative about domains or functions. The protein was mainly nuclear. Menin binds to JunD, an AP-1 transcription factor, inhibiting JunD's activation of transcription. Most of the germline and somatic MEN1 mutations predict truncation of menin, a likely destructive change. Inactivating MEN1 mutations in germline and in sporadic neoplasms support prior predictions that MEN1 is a tumor suppressor gene. Germline MEN1 mutation underlies all or most cases of MEN1 (familial or sporadic). Somatic MEN1 mutation is the most common gene mutation in many sporadic endocrine tumor types.
Amino Acid Sequence↗
Allelic loss in esophageal squamous cell carcinoma patients with and without family history of upper gastrointestinal tract cancer.
Chromosomal regions with frequent allelic loss may point to major susceptibility genes that will assist in understanding molecular events involved in esophageal carcinogenesis. Esophageal squamous cell carcinoma samples and blood from 46 patients, including 23 patients with and 23 patients without a family history of upper gastrointestinal cancer, were screened using laser microdissected DNA and tested for loss of heterozygosity (LOH) at 18 marker loci representing 14 chromosomal regions (on 2q, 3p, 4p, 4p, 5q, 6q, 8p, 9p, 9q, 11p, 13q, 14q, 15q, and 17p) identified in an earlier genome-wide scan to have frequent LOH. Clinical/pathological and lifestyle risk factor data were also collected. For all 46 tumors combined, the lowest frequency LOH for any of the 18 markers was 37%, and 8 markers showed LOH in > or =75% of informative tumors. One marker (D13S894 on 13q) showed greater LOH in patients with a positive family history (93% versus 50%; P = 0.04), whereas two markers (D6S1027 on 6q and D9S910 on 9q) had significantly more LOH in patients with metastasis, and one marker (D4S2361 on 4p) showed significantly higher LOH in patients with a lower pathological tumor grade. No relation was seen between LOH and lifestyle risk factors. This study confirms the previously observed high frequency LOH for these 14 chromosomal regions, including a locus on 13q where LOH is more common in patients with a family history of upper gastrointestinal cancer than in those without such history, suggesting that a gene in this area may be involved in genetic susceptibility to esophageal cancer.
Adult↗
An improved method for construction of directionally cloned cDNA libraries from microdissected cells.
Here, we developed an improved method for constructing microdissected cDNA libraries, based on strand-switching properties of reverse transcriptase, followed by PCR amplification with primers to mediate unidirectional insert cloning. Using RNA from microdissected ovarian carcinoma cells, we constructed a cDNA library consisting of 1.3 x 10(6) unidirectional recombinants with an average insert size of 500 bp. Single-pass sequencing of 100 clones with the T7 primer revealed 89 inserts derived from known genes, anonymous expressed sequence tags (ESTs), or novel sequences. Among these clones were known genes and ESTs previously found in cDNA libraries from bulk ovarian tissue RNA, sequences seen for the first time in an ovarian-derived library, and novel sequences not previously seen in any cDNA library. These results demonstrate a methodology for constructing quality cDNA libraries that are cloned in a unidirectional fashion, are complex and diverse, and reflect the tissue of origin.
Actins↗