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Biomedical subjects

M R Emmert-Buck

Publications and source records attributed to M R Emmert-Buck.

At least 19 recordsLinked to original sources
PubMed2003

Common genetic variants of TP53 and BRCA2 in esophageal cancer patients and healthy individuals from low and high risk areas of northern China.

TP53 and BRCA2 are frequently mutated in cancer and polymorphisms of these genes may modify cancer risk. We used SSCP and DNA sequencing to assess and compare frequencies of R72P (TP53) and 5'UTR203G>A, N372H, and K1132K (BRCA2) polymorphisms in healthy Chinese subjects at varying risk for esophageal squamous cell carcinoma (ESCC) and in ESCC patients. Suggestive overall differences in the distributions of genotypes by risk groups were seen for all genotypes except K1132K. Differences in R72P and N372H were most likely a reflection of lack of Hardy-Weinberg equilibrium (HWE), however, the difference in 203G>A was due to low prevalence of GG in ESCC patients (0.22 versus 0.36 in high risk group (P=0.047), and 0.22 versus 0.40 in low risk group (P=0.010)), consistent with a disease association. These data suggest that the 203G>A polymorphism in BRCA2 may be associated with risk of ESCC.

Adult↗
PubMed2003

Gene discovery in oral squamous cell carcinoma through the Head and Neck Cancer Genome Anatomy Project: confirmation by microarray analysis.

The near completion of the human genome project and the recent development of novel, highly sensitive high-throughput techniques have now afforded the unique opportunity to perform a comprehensive molecular characterization of normal, precancerous, and malignant cells, including those derived from squamous carcinomas of the head and neck (HNSCC). As part of these efforts, representative cDNA libraries from patient sets, comprising of normal and malignant squamous epithelium, were generated and contributed to the Head and Neck Cancer Genome Anatomy Project (HN-CGAP). Initial analysis of the sequence information indicated the existence of many novel genes in these libraries [Oral Oncol 36 (2000) 474]. In this study, we surveyed the available sequence information using bioinformatic tools and identified a number of known genes that were differentially expressed in normal and malignant epithelium. Furthermore, this effort resulted in the identification of 168 novel genes. Comparison of these clones to the human genome identified clusters in loci that were not previously recognized as being altered in HNSCC. To begin addressing which of these novel genes are frequently expressed in HNSCC, their DNA was used to construct an oral-cancer-specific microarray, which was used to hybridize alpha-(33)P dCTP labeled cDNA derived from five HNSCC patient sets. Initial assessment demonstrated 10 clones to be highly expressed (>2-fold) in the normal squamous epithelium, while 14 were highly represented in the malignant counterpart, in three of the five patient sets, thus suggesting that a subset of these newly discovered transcripts might be highly expressed in this tumor type. These efforts, together with other multi-institutional genomic and proteomic initiatives are expected to contribute to the complete understanding of the molecular pathogenesis of HNSCCs, thus helping to identify new markers for the early detection of preneoplastic lesions and novel targets for pharmacological intervention in this disease.

Aged↗
PubMed2001

Genetic progression and heterogeneity associated with the development of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma is a common fatal cancer, and Shanxi province, a region in north-central China, has some of the highest esophageal cancer rates in the world. Chromosomal regions with frequent allelic loss may point to major susceptibility genes that will assist us in understanding the molecular events involved in esophageal carcinogenesis and may serve as the basis for the development of markers for genetic susceptibility and screening for early detection of this cancer. This study was designed to identify events in the molecular progression of precursor and invasive lesions of squamous esophageal cancer. Twelve marker loci identified during our previous studies as having some of the highest rates of loss of heterozygosity (LOH) in invasive esophageal cancer were evaluated in laser-microdissected DNA obtained from low- and high-grade dysplastic lesions and invasive tumor foci from 10 fully embedded esophageal resection specimens. Each resection specimen contained a spectrum of disease, from epithelium that appeared histologically normal to invasive cancer, including a single dominant tumor surrounded by a region of precursor lesions (low- and high-grade dysplasia) and occasional "remote," nonadjacent precancerous foci. Using the 12 polymorphic markers, LOH was found in all of the three stages of disease. The frequency of LOH for all of the markers together increased with increasing disease severity. Among the informative low-grade dysplasia samples, LOH was detected with markers D3S1766 (3p), D4S2632 (4p), D9S910 (9q), and D13S1493 (13q), suggesting that LOH at these loci may be associated with early stages of tumor initiation and/or progression. LOH was detected among the informative high-grade (but not low-grade) dysplasia samples for the other eight markers tested, suggesting that LOH at these loci may occur later in the neoplastic process. In addition to the association between disease progression and these genetic changes, considerable genetic heterogeneity was found in each fully embedded resection specimen both between and within geographically separate neoplastic lesions.

Adult↗
PubMed2001

Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.

Protein arrays are described for screening of molecular markers and pathway targets in patient matched human tissue during disease progression. In contrast to previous protein arrays that immobilize the probe, our reverse phase protein array immobilizes the whole repertoire of patient proteins that represent the state of individual tissue cell populations undergoing disease transitions. A high degree of sensitivity, precision and linearity was achieved, making it possible to quantify the phosphorylated status of signal proteins in human tissue cell subpopulations. Using this novel protein microarray we have longitudinally analysed the state of pro-survival checkpoint proteins at the microscopic transition stage from patient matched histologically normal prostate epithelium to prostate intraepithelial neoplasia (PIN) and then to invasive prostate cancer. Cancer progression was associated with increased phosphorylation of Akt (P<0.04), suppression of apoptosis pathways (P<0.03), as well as decreased phosphorylation of ERK (P<0.01). At the transition from histologically normal epithelium to PIN we observed a statistically significant surge in phosphorylated Akt (P<0.03) and a concomitant suppression of downstream apoptosis pathways which proceeds the transition into invasive carcinoma.

Biomarkers, Tumor↗
PubMed2001

A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome, characterized primarily by multiple tumors in the parathyroid glands, endocrine pancreas, and anterior pituitary. Other tumors, including gastrinoma, carcinoid, adrenal cortical tumors, angiofibroma, collagenoma, and lipoma, also occur in some patients. Individuals with MEN1 almost always have loss-of-function mutations in the MEN1 gene on chromosome 11, and endocrine tumors arising in these patients usually show somatic loss of the remaining wild-type allele. To examine the role of MEN1 in tumor formation, a mouse model was generated through homologous recombination of the mouse homolog Men1. Homozygous mice die in utero at embryonic days 11.5-12.5, whereas heterozygous mice develop features remarkably similar to those of the human disorder. As early as 9 months, pancreatic islets show a range of lesions from hyperplasia to insulin-producing islet cell tumors, and parathyroid adenomas are also frequently observed. Larger, more numerous tumors involving pancreatic islets, parathyroids, thyroid, adrenal cortex, and pituitary are seen by 16 months. All of the tumors tested to date show loss of the wild-type Men1 allele, further supporting its role as a tumor suppressor gene.

Adenoma↗
PubMed2001

Allelic loss on chromosome bands 13q11-q13 in esophageal squamous cell carcinoma.

Allelic loss on chromosome 13 occurs frequently in esophageal squamous cell carcinoma. However, studies of the two known tumor suppressor genes located on 13q, RB1 and BRCA2, have shown few mutations, suggesting that other genes are likely to be involved in the development of this tumor type. To identify a minimal deletion interval, we first analyzed 42 microsatellite markers spanning chromosome bands 13q11-q13 in 56 esophageal squamous cell carcinoma patients, including 34 with a family history of upper gastrointestinal cancer and 22 without a family history of cancer. Lifestyle risk factors and clinical/pathologic characteristics were also collected. Two commonly deleted regions were identified: one was located on band 13q12.11, between markers D13S787 and D13S221; the other was located on bands 13q12.3-q13.1 from markers D13S267 to D13S219. We observed higher allelic loss frequencies for eight of the microsatellite markers in those patients with a family history of upper gastrointestinal cancer compared to patients without such a history. This study suggests that one or more unidentified tumor suppressor genes are located on chromosome arm 13q that play a role in the development of esophageal squamous cell carcinoma.

Carcinoma, Squamous Cell↗
PubMed2001

Molecular profiling of tissue samples using laser capture microdissection.

The management of cancer and other genetically based diseases is far from optimal in even our most advanced medical centers. There is still uncertainty regarding how diseases will progress in certain patients, toxicity that must be tolerated with imprecise treatment regimens and significant potential for treatment failure. As our understanding of the complexity of these diseases has increased, it has become clear that we must move toward precisely tailored approaches to treating each individual patient. To that end, a major goal of current medical research is the rapid identification of the specific molecular alterations in each patient's disease. This will enable the design of optimal diagnosis, prognosis and treatment, significantly improving survival. This review describes one important approach to the genetic analysis of disease--molecular profiling--and the tenets and technologies necessary for its success.

DNA Fingerprinting↗
PubMed2001

Expression studies and mutational analysis of the androgen regulated homeobox gene NKX3.1 in benign and malignant prostate epithelium.

PURPOSE: The NKX-3.1gene is an androgen regulated prostate specific homeobox gene that is believed to have a vital role in normal prostate development. In mice the homologue NKx3.1 is exclusively expressed in prostate epithelium. In humans NKX3.1 expression is also restricted to the prostate but to our knowledge the cellular location has not been described. Furthermore, since NKX3.1 maps to chromosomal band 8p21, a region with high loss of heterozygosity in prostate cancer, the gene has been proposed to have tumor suppressor function. In this study we demonstrate that in human prostates NKX3.1 is expressed exclusively in secretory epithelial cells and the level of NKX3.1 expression remains invariant in normal tissue and in tissue showing various grades of prostate cancer. In the 19 cases examined the DNA sequences of the NKX3.1 gene were identical and no mutation was detected. MATERIALS AND METHODS: Frozen tissue from patients who underwent radical prostatectomy was used for this study. For in situ hybridization experiments a 377 bp fragment corresponding to a portion of the 3' untranslated region of the NKX3.1 gene was amplified by polymerase chain reaction and cloned into the pCRII plasmid vector Invitrogen. Antisense or sense [33P] uridine triphosphate labeled RNA probes were generated with SP6 or T7 RNA polymerase and hybridized to the tissue sections. Slides were exposed to photographic emulsion and visualized on autoradiography. Laser capture microdissection was performed to procure pure populations of malignant epithelium. DNA was isolated by digesting samples in proteinase K buffer. Polymerase chain reaction and direct sequencing was performed using standard protocols. RESULTS: In vitro hybridization showed that NKX3.1 expression was restricted to secretory epithelial cells within benign prostate glands. No expression was detected in stroma or infiltrating lymphocytes. NKX3.1 was expressed in all grades of malignant epithelium in all 25 cases examined. Direct sequencing of the coding region of NKX3.1 revealed the wild-type sequence in all 18 microdissected cancers analyzed. CONCLUSIONS: Based on our studies we propose that NKX3.1 gene expression is restricted to benign and malignant secretory epithelium within the prostate but NKX3.1 does not appear to be a classic tumor suppressor gene responsible for prostate cancer initiation. These findings are consistent with the role of NKX3.1 in the development of normal prostate epithelium and maintenance of normal secretory function. Thus, NKX3.1 may represent a useful molecular marker for benign and malignant prostate epithelium.

DNA Mutational Analysis↗
PubMed2001

Frequent inactivation of the TP53 gene in esophageal squamous cell carcinoma from a high-risk population in China.

Esophageal squamous cell carcinoma (ESCC) is one of the most common fatal cancers worldwide, and north central China has some of the highest rates in the world. Previous studies from tumors in this area of China have shown high frequencies of allelic loss on chromosome 17p13-11, which includes the region where the TP53 gene is found. We examined 56 ESCC patients using single-strand conformation polymorphism and DNA sequencing to assess the frequency and spectrum of TP53 mutation and the association between allelic loss at microsatellite marker TP53 and TP53 mutations. Ninety-six % of cases were found to have at least one genetic alteration, including TP53 mutation (77%), allelic loss within the TP53 gene (73%), and/or loss of heterozygosity at the TP53 microsatellite marker (80%); 75% had two or more such alterations, including 59% with both a point mutation and an intragenic allelic loss ("two hits"). The majority of mutations observed were in exon 5, where the most common type of nucleotide substitution was a G:C-->A:T or C:G-->T:A transition, including half that occurred at CpG sites. Allelic loss was most commonly found in exon 4 but was very common in exon 5 as well. Taken together, the multiple genetic alterations of TP53 in this population at high risk for ESCC indicate that there is a very high degree of genetic instability in these tumors, that TP53 is a primary target for inactivation, and that this tumor suppressor gene plays a critical role in the carcinogenesis process for ESCC.

Adult↗
PubMed2000

Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma.

Annexin I protein expression was evaluated in patient-matched longitudinal study sets of laser capture microdissected normal, premalignant, and invasive epithelium from human esophageal squamous cell cancer and prostatic adenocarcinoma. In 25 esophageal cases (20 by Western blot and 5 by immunohistochemistry) and 17 prostate cases (3 by Western blot and 14 by immunohistochemistry), both tumor types showed either complete loss or a dramatic reduction in the level of annexin I protein expression compared with patient-matched normal epithelium (P < or = 0.05). Moreover, by using Western blot analysis of laser capture microdissected, patient-matched longitudinal study sets of both tumor types, the loss of protein expression occurred in premalignant lesions. Concordance of this result with immunohistochemical analysis suggests that annexin I may be an essential component for maintenance of the normal epithelial phenotype. Additional studies investigating the mechanism(s) and functional consequences of annexin I protein loss in tumor cells are warranted.

Adenocarcinoma↗
PubMed2000

Layered expression scanning: rapid molecular profiling of tumor samples.

Layered expression scanning is a new approach to comprehensive molecular analysis of tumor samples that uses a layered array of capture membranes coupled to antibodies or DNA sequences to perform multiplex protein or mRNA analysis. Cell or tissue samples are transferred through a series of individual capture layers, each linked to a separate antibody or DNA sequence. As the biomolecules traverse the membrane set, each targeted protein or mRNA is specifically captured by the layer containing its antibody or cDNA sequence. The two-dimensional relationship of the cell populations is maintained during the transfer process, thereby producing a molecular profile of each cell type present. Reduction-to-practice of the technique is demonstrated by analysis of prostate-specific antigen (PSA) protein, gelatinase A protein, and POV1 (PB39) cDNA. As layered expression scanning technology progresses, we envision a laboratory method that will have multiple applications for high-throughput molecular profiling of normal and tumor samples.

Amino Acid Transport System y+L↗
PubMed2000

Identification of novel regions of allelic loss from a genomewide scan of esophageal squamous-cell carcinoma in a high-risk Chinese population.

Esophageal cancer is one of the most common fatal cancers worldwide. Deletions of genomic regions are thought to be important in esophageal carcinogenesis. We conducted a genomewide scan for regions of allelic loss using microdissected DNA from 11 esophageal squamous-cell carcinoma patients with a family history of upper gastrointestinal tract cancer from a high-risk region in north central China. Allelic patterns of 366 fluorescently labeled microsatellite markers distributed at 10-cM intervals over the 22 autosomal chromosomes were examined. We identified 14 regions with very high frequency (>/= 75%) loss of heterozygosity (LOH), including broad regions encompassing whole chromosome arms (on 3p, 5q, 9p, 9q, and 13q), regions of intermediate size (on 2q, 4p, 11p, and 15q), and more discrete regions identified by very high frequency LOH for a single marker (on 4q, 6q, 8p, 14q, and 17p). Among these 14 regions were 7 not previously described in esophageal squamous-cell carcinoma as having very high frequency LOH (on 2q, 4p, 4q, 6q, 8p, 14q, and 15q). The very high frequency LOH regions identified here may point to major susceptibility genes, including potential tumor suppressor genes and inherited gene loci, which will assist in understanding the molecular events involved in esophageal carcinogenesis and may help in the development of markers for genetic susceptibility testing and screening for the early detection of this cancer. Genes Chromosomes Cancer 27:217-228, 2000. Published 2000 Wiley-Liss, Inc.

Adult↗
PubMed2000

MEN1 gene mutation analysis of high-grade neuroendocrine lung carcinoma.

Neuroendocrine tumors of the lung consist of a spectrum of neoplasms, including typical carcinoids, atypical carcinoids, large-cell neuroendocrine carcinomas (LCNEC), and small-cell lung carcinomas (SCLC). We previously reported frequent inactivation of the gene responsible for multiple endocrine neoplasia type 1 (MEN1) in both typical and atypical carcinoid tumors. In the present study, we extend the analysis of human NE lung tumors to include 9 primary SCLCs, 36 SCLC cell lines, and 13 primary LCNECs for MEN1 gene inactivation. In SCLC, loss of heterozygosity (LOH) at the MEN1 gene on chromosome band 11q13 was detected in one primary tumor and two cell lines. The coding sequence and splice junctions of the MEN1 gene were screened for mutations in all 44 tumors and cell lines, and no mutations were detected. Northern blot analysis of 13 SCLC cell lines showed the MEN1 transcript to be present and of normal size. In LCNECs, a somatic frameshift in the MEN1 gene (1226delC) was found in one of 13 tumors, representing the first mutation observed outside the spectrum of neoplasms associated with MEN1. Interestingly, neither a deletion nor a mutation was detected in the other allele, and wild-type mRNA sequence was expressed in the tumor, suggesting that the MEN1 gene was not inactivated by a conventional two-hit mechanism. The data support the hypothesis that SCLC and lung carcinoids develop via distinct molecular pathways; however, further investigation is necessary to determine the significance of the MEN1 gene mutation observed in a single case of LCNEC. Published 2000 Wiley-Liss, Inc.

Carcinoma, Large Cell↗
PubMed2000

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines.

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.

Blotting, Western↗
PubMed2000

Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection.

Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible to calculate the number of PSA molecules per microdissected tissue cell for case samples. In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximately 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6. 3 x 10(6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and invasive carcinoma. Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.

Calibration↗
PubMed2000

Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7-18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76-96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.

Aged↗