Biomedical subjects

Francis Barany

Publications and source records attributed to Francis Barany.

At least 19 recordsLinked to original sources

PubMed2006

Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations.

Endonuclease V (endo V) recognizes a broad range of aberrations in DNA such as deaminated bases or mismatches. It nicks DNA at the second phosphodiester bond 3' to a deaminated base or a mismatch. Endonuclease V obtained from Thermotoga maritima preferentially cleaves purine mismatches in certain sequence context. Endonuclease V has been combined with a high-fidelity DNA ligase to develop an enzymatic method for mutation scanning. A biochemical screening of site-directed mutants identified mutants in motifs III and IV that altered the base preferences in mismatch cleavage. Most profoundly, a single alanine substitution at Y80 position switched the enzyme to essentially a C-specific mismatch endonuclease, which recognized and cleaved A/C, C/A, T/C, C/T and even the previously refractory C/C mismatches. Y80A can also detect the G13D mutation in K-ras oncogene, an A/C mismatch embedded in a G/C rich sequence context that was previously inaccessible using the wild-type endo V. This investigation offers insights on base recognition and active site organization. Protein engineering in endo V may translate into better tools in mutation recognition and cancer mutation scanning.

Amino Acid Substitution↗

PubMed2006

GROalpha is highly expressed in adenocarcinoma of the colon and down-regulates fibulin-1.

PURPOSE: The growth-related oncogene alpha (GROalpha) is a secreted interleukin-like molecule that interacts with the CXCR2 G-protein-coupled receptor. We found that the mRNA and protein products of GROalpha are more highly expressed in neoplastic than normal colon epithelium, and we studied potential mechanisms by which GROalpha may contribute to tumor initiation or growth. EXPERIMENTAL DESIGN: Cell lines that constitutively overexpress GROalpha were tested for growth rate, focus formation, and tumor formation in a xenograft model. GROalpha expression was determined by Affymetrix GeneChip (241 microdissected colon samples), real-time PCR (n = 32), and immunohistochemistry. Primary colon cancer samples were also employed to determine copy number changes and loss of heterozygosity related to the GROalpha and fibulin-1 genes. RESULTS: In cell cultures, GROalpha transfection transformed NIH 3T3 cells, whereas inhibition of GROalpha by inhibitory RNA was associated with apoptosis, decreased growth rate, and marked up-regulation of the matrix protein fibulin-1. Forced expression of GROalpha was associated with decreased expression of fibulin-1. Expression of GROalpha mRNA was higher in primary adenocarcinomas (n = 132), adenomas (n = 32), and metastases (n = 52) than in normal colon epithelium (P < 0.001). These results were confirmed by real-time PCR and by immunohistochemistry. Samples of primary and metastatic colon cancer showed underexpression of fibulin-1 when compared with normal samples. There were no consistent changes in gene copy number of GROalpha or fibulin-1, implying a transcriptional basis for these findings. CONCLUSION: Elevated expression of GROalpha is frequent in adenocarcinoma of the colon and is associated with down-regulation of the matrix protein fibulin-1 in experimental models and in clinical samples. GROalpha overexpression abrogates contact inhibition in cell culture models, whereas inhibition of GROalpha expression is associated with apoptosis. Importantly, coexpression of fibulin-1 with GROalpha abrogates key aspects of the transformed phenotype, including tumor formation in a murine xenograft model. Targeting GRO proteins may provide new opportunities for treatment of colon cancer.

3T3 Cells↗

PubMed2006

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations.

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.

Animals↗

PubMed2006

Relationship of gene expression and chromosomal abnormalities in colorectal cancer.

Several studies have verified the existence of multiple chromosomal abnormalities in colon cancer. However, the relationships between DNA copy number and gene expression have not been adequately explored nor globally monitored during the progression of the disease. In this work, three types of array-generated data (expression, single nucleotide polymorphism, and comparative genomic hybridization) were collected from a large set of colon cancer patients at various stages of the disease. Probes were annotated to specific chromosomal locations and coordinated alterations in DNA copy number and transcription levels were revealed at specific positions. We show that across many large regions of the genome, changes in expression level are correlated with alterations in DNA content. Often, large chromosomal segments, containing multiple genes, are transcriptionally affected in a coordinated way, and we show that the underlying mechanism is a corresponding change in DNA content. This implies that whereas specific chromosomal abnormalities may arise stochastically, the associated changes in expression of some or all of the affected genes are responsible for selecting cells bearing these abnormalities for clonal expansion. Indeed, particular chromosomal regions are frequently gained and overexpressed (e.g., 7p, 8q, 13q, and 20q) or lost and underexpressed (e.g., 1p, 4, 5q, 8p, 14q, 15q, and 18) in primary colon tumors, making it likely that these changes favor tumorigenicity. Furthermore, we show that these aberrations are absent in normal colon mucosa, appear in benign adenomas (albeit only in a small fraction of the samples), become more frequent as disease advances, and are found in the majority of metastatic samples.

Adenoma↗

PubMed2006

Harnessing asymmetrical substrate recognition by thermostable EndoV to achieve balanced linear amplification in multiplexed SNP typing.

Multiplexed amplification of specific DNA sequences, by PCR or by strand-displacement amplification, is an intrinsically biased process. The relative abundance of amplified DNA can be altered significantly from the original representation and, in extreme cases, allele dropout can occur. In this paper, we present a method of linear amplification of DNA that relies on the cooperative, sequence-dependent functioning of the DNA mismatch-repair enzyme endonuclease V (EndoV) from Thermotoga maritima (Tma) and Bacillus stearothermophilus (Bst) DNA polymerase. Tma EndoV can nick one strand of unmodified duplex DNA, allowing extension by Bst polymerase. By controlling the bases surrounding a mismatch and the mismatch itself, the efficiency of nicking by EndoV and extension by Bst polymerase can be controlled. The method currently allows 100-fold multiplexed amplification of target molecules to be performed isothermally, with an average change of <1.3-fold in their original representation. Because only a single primer is necessary, primer artefacts and nonspecific amplification products are minimized.

Base Pair Mismatch↗

PubMed2005

Multiplexed profiling of candidate genes for CpG island methylation status using a flexible PCR/LDR/Universal Array assay.

DNA methylation in CpG islands is associated with transcriptional silencing. Accurate determination of cytosine methylation status in promoter CpG dinucleotides may provide diagnostic and prognostic value for human cancers. We have developed a quantitative PCR/LDR/Universal Array assay that allows parallel evaluation of methylation status of 75 CpG dinucleotides in the promoter regions of 15 tumor suppressor genes (CDKN2B, CDKN2A, CDKN2D, CDKN1A, CDKN1B, TP53, BRCA1, TIMP3, APC, RASSF1, CDH1, MGMT, DAPK1, GSTP1, and RARB). When compared with an independent pyrosequencing method at a single promoter, the two approaches gave good correlation. In a study using 15 promoter regions and seven blinded tumor cell lines, our technology was capable of distinguishing methylation profiles that identified cancer cell lines derived from the same origins. Preliminary studies using 96 colorectal tumor samples and 73 matched normal tissues indicated CpG methylation is a gene-specific and nonrandom event in colon cancer. This new approach is suitable for clinical applications where sample quantity and purity can be limiting factors.

Cell Line, Tumor↗

PubMed2005

Fabrication of DNA microarrays onto polymer substrates using UV modification protocols with integration into microfluidic platforms for the sensing of low-abundant DNA point mutations.

We describe the microfabrication and operational characteristics of a simple flow-through biochip sensor capable of detecting low abundant point mutations in K-ras oncogenes from genomic DNA, which carry high diagnostic value for colorectal cancers. The biochip consisted of an allele-specific ligase detection reaction (LDR) coupled to a universal array for interrogating multiple mutations simultaneously from a clinical sample. The integrated sensing platform was micro-manufactured from two different polymers, polycarbonate, PC, which was used for the LDRs, and poly(methyl methacrylate), PMMA, which was used to build the microarray. Passive elements were hot embossed into the PC and PMMA microchips and then, the chips assembled into a three-dimensional architecture with the interconnect fabricated from an elastomer, poly(dimethylsiloxane), PDMS, to produce a leak-free connection between the biochips. The array in PMMA was produced using a photomodification process, which involved three steps; (1) UV (254 nm) exposure of the polymer surface; (2) EDC coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. The LDR/hybridization flow-through biochip performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time=19.1 min) and could screen multiple mutations simultaneously for high throughput applications at a level of one mutant sequence in 100 wild-type sequences.

Biosensing Techniques↗

PubMed2005

Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low-abundant DNA point mutations.

We have fabricated a flow-through biochip assembly that consisted of two different microchips: (1) a polycarbonate (PC) chip for performing an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using an universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for the LDR in a continuous-flow format, in which two primers (discriminating primer that carried the complement base to the mutation being interrogated and a common primer) that flanked the point mutation and were ligated only when the particular mutation was present in the genomic DNA. The miniaturized reactor architecture allowed enhanced reaction speed due to its high surface-to-volume ratio and efficient thermal management capabilities. A PMMA chip was employed as the microarray device, where zip code sequences (24-mers), which were complementary to sequences present on the target, were microprinted into fluidic channels embossed into the PMMA substrate. Microfluidic addressing of the array reduced the hybridization time significantly through enhanced mass transport to the surface-tethered zip code probes. The two microchips were assembled as a single integrated unit with a novel interconnect concept to produce the flow-through microfluidic biochip. A microgasket, fabricated from an elastomer poly(dimethylsiloxane) with a total volume of the interconnecting assembly of <200 nL, was used as the interconnect between the two chips to produce the three-dimensional microfluidic network. We successfully demonstrated the ability to detect one mutant DNA in 100 normal sequences with the biochip assembly. The LDR/hybridization assay using the assembly performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time 19.1 min) and could screen multiple mutations simultaneously.

Base Sequence↗

PubMed2005

Fabrication of DNA microarrays onto poly(methyl methacrylate) with ultraviolet patterning and microfluidics for the detection of low-abundant point mutations.

We have developed a simple ultraviolet (UV)-photomodification protocol using poly(methyl methacrylate) and polycarbonate to produce functional scaffolds consisting of carboxylic groups that allow covalent attachment of amine-terminated oligonucleotide probes to these surface groups through carbodiimide coupling. Use of the photomodification procedure coupled to microfluidics allowed for the rapid generation of medium-density DNA microarrays. The method reported herein involves the use of poly(dimethylsiloxane) microchannels reversibly sealed to photomodified poly(methyl methacrylate) surfaces to serve as stencils for patterning the oligonucleotide probes. After array construction, the poly(dimethylsiloxane) stencil is rotated 90 degrees to allow interrogation of the array using microfluidics. The photomodification process for array fabrication involves only three steps: (1) broadband UV exposure of the polymer surface, (2) carbodiimide coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond), and (3) washing of the surface. The density of probes attached to this activated surface was found to be approximately 41pmolcm(-2), near the steric-saturation limit for short oligonucleotide probes. We demonstrate the use of this procedure for screening multiple KRAS2 mutations possessing high diagnostic value for colorectal cancers. A ligase detection reaction/universal array assay was carried out using parallel detection of two different low-abundant DNA point mutations in KRAS2 oncogenes with the allelic composition evaluated at one locus. Four zip code probes immobilized onto the poly(methyl methacrylate) surface directed allele-specific ligation products containing mutations in the KRAS2 gene (12.2D, 12.2A, 12.2V, and 13.4D) to the appropriate address of a universal array with minimal amounts of cross-hybridization or misligation.

Adsorption↗

PubMed2005

Detection of the BRAF V600E mutation in melanocytic lesions using the ligase detection reaction.

BACKGROUND: BRAF is a member of the RAF kinase family, and it promotes signaling through the RAS-MAP kinase signal transduction cascade. Research has shown that a majority of melanomas and nevi exhibit an activating V600E (T1799A) mutation in BRAF exon 15. Additional studies of BRAF have demonstrated that the T1799A mutation is absent in uveal melanomas and Spitz nevi. METHODS: The ligase detection reaction (LDR) is a sensitive technique that can identify specific DNA mutations occurring at very low frequency within heterogeneous clinical samples, and it has previously been used to analyze mutations in paraffin-embedded tissue specimens. RESULTS: The LDR can readily detect mutations in DNA extracted from non-microdissected paraffin-embedded sections of common nevi, dysplastic nevi, and melanomas. In addition, this method demonstrated the absence of the V600E (T1799A) mutation within Spitz nevi. CONCLUSION: The LDR is a highly sensitive and specific test for detecting mutations in formalin-fixed tissue. The LDR can be easily adapted to detect any mutation associated with a cutaneous disorder. The absence of the BRAF V600E mutation in Spitz's nevi may serve as a molecular signature to distinguish these lesions from common nevi, dysplastic nevi, and some types of malignant melanoma.

Base Sequence↗

PubMed2005

Applications of the universal DNA microarray in molecular medicine.

Integration of molecular medicine into standard clinical practice will require the availability of diagnostics that are sensitive, rapid, and robust. The backbone technology underlying the diagnostic will likely serve double duty during clinical trials in order to first validate the biomarkers that contribute to both drug response and disease stratification. PCR/LDR/Universal DNA microarray is a promising technology to help drive the transition from the current paradigms of clinical decision making to the new era of personalized medicine. By uncoupling the mutation detection step from array hybridization, this technology becomes fully programmable. It exploits full use of the sensitivity that the ligase detection reaction can provide, while maintaining a rapid read out on a universal microarray. Thus, PCR/LDR/Universal DNA microarray is 50-fold more sensitive and 10-fold more rapid than conventional hybridization-only arrays. The intent of this article is to provide investigators with a perspective on current uses of this approach, as well as to serve as a practical guide to implementation.

Humans↗

PubMed2004

High sensitivity EndoV mutation scanning through real-time ligase proofreading.

The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5' terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.

Artifacts↗

PubMed2004

Unique ligation properties of eukaryotic NAD+-dependent DNA ligase from Melanoplus sanguinipes entomopoxvirus.

The eukaryotic Melanoplus sanguinipes entomopoxvirus (MsEPV) genome reveals a homologous sequence to eubacterial nicotinamide adenine dinucleotide (NAD(+))-dependent DNA ligases [J. Virol. 73 (1999) 533]. This 522-amino acid open reading frame (ORF) contains all conserved nucleotidyl transferase motifs but lacks the zinc finger motif and BRCT domain found in conventional eubacterial NAD(+) ligases. Nevertheless, cloned MsEPV ligase seals DNA nicks in a NAD(+)-dependent fashion, while adenosine 5'-monophosphate (ATP) cannot serve as an adenylation cofactor. The ligation activity of MsEPV ligase requires Mg(2+) or Mn(2+). MsEPV ligase seals sticky ends efficiently, but has little activity on 1-nucleotide gap or blunt-ended DNA substrates even in the presence of polyethylene glycol. In comparison, bacterial NAD(+)-dependent ligases seal blunt-ended DNA substrates in the presence of polyethylene glycol. MsEPV DNA ligase readily joins DNA nicks with mismatches at either side of the nick junction, except for mismatches at the nick junction containing an A base in the template strand (A/A, G/A, and C/A). MsEPV NAD(+)-dependent DNA ligase can join DNA probes on RNA templates, a unique property that distinguishes this enzyme from other conventional bacterial NAD(+) DNA ligases. T4 ATP-dependent DNA ligase shows no detectable mismatch ligation at the 3' side of the nick but substantial 5' T/G mismatch ligation on an RNA template. In contrast, MsEPV ligase joins mismatches at the 3' side of the nick more frequently than at the 5' side of the nick on an RNA template. The complementary specificities of these two enzymes suggest alternative primer design for genomic profiling approaches that use allele-specific detection directly from RNA transcripts.

Amino Acid Sequence↗

PubMed2004

Rapid and sensitive p53 alteration analysis in biopsies from lung cancer patients using a functional assay and a universal oligonucleotide array: a prospective study.

PURPOSE: Molecular profiling of alterations associated with lung cancer holds the promise to define clinical parameters such as response to treatment or survival. Because <5% of small cell lung cancers and <30% of non-small cell lung cancers are surgically resectable, molecular analysis will perforce rely on routinely available clinical samples such as biopsies. Identifying tumor mutations in such samples will require a sensitive and robust technology to overcome signal from excess amounts of normal DNA. EXPERIMENTAL DESIGN: p53 mutation status was assessed from the DNA and RNA of biopsies collected prospectively from 83 patients with lung cancer. Biopsies were obtained either by conventional bronchoscopy or computed tomography-guided percutaneous biopsy. Matched surgical specimens were available for 22 patients. Three assays were used: direct sequencing; a functional assay in yeast; and a newly developed PCR/ligase detection reaction/Universal DNA array assay. RESULTS: Using the functional assay, p53 mutation was found in 62% of biopsies and 64% of surgical specimens with a concordance of 80%. The sensitivity of the functional assay was determined to be 5%. Direct sequencing confirmed mutations in 92% of surgical specimens but in only 78% of biopsies. The DNA array confirmed 100% of mutations in both biopsies and surgical specimens. Using this newly developed DNA array, we demonstrate the feasibility of directly identifying p53 mutations in clinical samples containing <5% of tumor cells. CONCLUSIONS: The versatility and sensitivity of this new array assay should allow additional development of mutation profiling arrays that could be applied to biological samples with a low tumor cell content such as bronchial aspirates, bronchoalveolar lavage fluid, or serum.

Aged↗

PubMed2004

Capillary and microelectrophoretic separations of ligase detection reaction products produced from low-abundant point mutations in genomic DNA.

Capillary gel electrophoresis (CGE) and polymer-based microelectrophoretic platforms were investigated to analyze low-abundant point mutations in certain gene fragments with high diagnostic value for colorectal cancers. The electrophoretic separations were carried out on single-stranded DNA (ssDNA) products generated from an allele-specific ligation assay (ligase detection reaction, LDR), which was used to screen for a single base mutation at codon 12 in the K-ras oncogene. The presence of the mutation generated a ssDNA fragment that was >40 base pairs (bp) in length, while the primers used for the ligation assay were <30 bp in length. Various separation matrices were investigated, with the success of the matrix assessed by its ability to resolve the ligation product from the large molar excess of unligated primers when the mutant allele was lower in copy number compared to the wild-type allele. Using CGE, LDR product models (44 and 51 bp) could be analyzed in a cross-linked polyacrylamide gel with a 1000-fold molar excess of LDR primers (25 bp) in approximately 45 min. However, when using linear polyacrylamide gels, these same fragments could not be detected due to significant electrokinetic biasing during injection. A poly(methylmethacrylate) (PMMA) microchip of 3.5 cm effective column length was used with a 4% linear polyacrylamide gel to analyze the products generated from an LDR. When the reaction contained a 100-fold molar excess of wild-type DNA compared to a G12.2D mutant allele, the 44 bp ligation product could be effectively resolved from unligated primers in under 120 s, nearly 17 times faster than the CGE format. In addition, sample cleanup was simplified using the microchip format by not requiring desalting of the LDR prior to loading.

Acrylic Resins↗

PubMed2004

Harmonized microarray/mutation scanning analysis of TP53 mutations in undissected colorectal tumors.

Both the mutational status and the specific mutation of TP53 (p53) have been shown to impact both tumor prognosis and response to therapies. Molecular profiling of solid tumors is confounded by infiltrating wild-type cells, since normal DNA can interfere with detection of mutant sequences. Our objective was to identify TP53 mutations in 138 stage I-IV colorectal adenocarcinomas and liver metastases without first enriching for tumor cells by microdissection. To achieve this, we developed a harmonized protocol involving multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) with Universal DNA microarray analysis and endonuclease V/ligase mutation scanning. Sequences were verified using dideoxy sequencing. The harmonized protocol detected all 66 mutations. Dideoxy sequencing detected 41 out of 66 mutations (62%) using automated reading, and 59 out of 66 mutations (89%) with manual reading. Data analysis comparing colon cancer entries in the TP53 database (http://p53.curie.fr) with the results reported in this study showed that distribution of mutations and the mutational events were comparable.

Adenocarcinoma↗

PubMed2003

The presence of p53 mutations in human osteosarcomas correlates with high levels of genomic instability.

The p53 gene is a critical tumor suppressor that is inactivated in a majority of cancers. The central role of p53 in response to stresses such as DNA damage, hypoxia, and oncogene activation underlies this high frequency of negative selection during tumorigenic transformation. Mutations in p53 disrupt checkpoint responses to DNA damage and result in the potential for destabilization of the genome. Consistent with this, p53 mutant cells have been shown to accumulate genomic alterations in cell culture, mouse models, and some human tumors. The relationship between p53 mutation and genomic instability in human osteosarcoma is addressed in this report. Similar to some other primary human tumors, the mutation of p53 correlates significantly with the presence of high levels of genomic instability in osteosarcomas. Surprisingly, osteosarcomas harboring an amplification of the HDM2 oncogene, which inhibits the tumor-suppressive properties of p53, do not display high levels of genomic instability. These results demonstrate that the inactivation of p53 in osteosarcomas directly by mutation versus indirectly by HDM2 amplification may have different cellular consequences with respect to the stability of the genome.

DNA, Neoplasm↗

PubMed2003

HER 2/neu expression and gene amplification in colon cancer.

HER 2/neu is an important oncogene in breast cancer, but the prevalence and significance of HER 2/neu gene amplification in colon cancer have been poorly documented. We have evaluated HER 2/neu gene amplification and protein overexpression in a series of colon cancers to assess the frequency, concordance and clinical significance of these events. HER 2/neu gene copy number was measured in 154 primary colon tumors, 15 liver metastases and matched normal tissues using a quantitative PCR/ligase detection reaction (LDR) technique developed and validated in our laboratory. HER 2/neu copy number was confirmed by fluorescent in situ hybridization (FISH) in all tumors found to have gene amplification. In an independent and blinded fashion, HER 2/neu expression was assessed in paraffin sections from 139 of the tumor specimens using the HercepTest kit. HER 2/neu gene amplification was observed in 4 (2.4%) of the 169 tumor specimens and in none of the normal tissues. There was no apparent association with stage of disease, tumor grade or patient survival. Among 139 cases evaluated by immunohistochemistry (IHC), HER 2/neu overexpression was seen in 5 cases (3.6%). There was extremely high concordance (kappa = 0.852) between gene amplification and protein overexpression. The low prevalence of HER 2/neu gene amplification and protein overexpression suggests that this oncogene plays an infrequent role in the development and progression of colon cancer. These data indicate that the primary mechanism of dysregulated HER 2/neu expression in colon cancer, as in breast cancer, is gene amplification.

Adult↗