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PubMed2001

Gene expression profiles of pancreatic cancer and stromal desmoplasia.

Gene expression studies were undertaken in normal pancreas and pancreatic adenocarcinomas to determine new candidate genes that can potentially be used as markers of the disease. The characteristic desmoplastic stromal reaction of pancreatic adenocarcinoma greatly hampers expression studies in this tumour type, and usually necessitates time-consuming tissue microdissection for enrichment of the tumour cell population. We show that fine needle aspiration of cancer provides a fast and efficient way of obtaining samples highly enriched in tumour cells with sufficient yields of RNA. Using Atlas cancer cDNA arrays with 588 cancer-related genes, we describe gene expression profiles of normal pancreas, bulk pancreatic tumour tissues and pancreatic tumour aspirates containing more than 95% tumour cells. Analysis of bulk tissue specimens revealed differentially expressed genes belonging predominantly to the stromal component of the tumour. This contrasted with the results obtained from tumour-cell enriched samples. Several genes already described in pancreatic cancer (caspase 8, TIMP1, CD9, IL-13) were also differentially expressed in our study. Furthermore, we found dysregulated expression of genes not previously associated with pancreatic adenocarcinoma, such as Rac 1, GLG1, NEDD5, RPL-13a, RPS9 and members of the Wnt5A gene family. In summary, we present a panel of genes newly identified in the pathogenesis of pancreatic adenocarcinoma and demonstrate that fine needle aspirates of the tumour mass are a convenient source of material for gene expression studies in tumours accompanied by desmoplastic reactions.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Direct comparison of microarray gene expression profiles between non-amplification and a modified cDNA amplification procedure applicable for needle biopsy tissues.

Global gene expression profiling by cDNA microarray analysis has been used to discover the biomarkers for early diagnosis of various cancers, subclassing cancer type, and prediction of patient's treatment outcome. The information provided by gene expression profiling may contribute to the design of molecular mechanism-based strategies for cancer prevention and/or treatment. However, the standard procedure for cDNA microarray analysis requires 5mug of good quality total RNA as starting material for each target preparation reaction. Thus, there is a limit for needle biopsy samples, laser capture microdissected tissues, or flow-sorted cells to successfully utilize the microarray technology. In order to profile the gene expression of needle biopsy tissue, we have modified the standard protocol by carrying out cDNA amplification after cDNA synthesis. We compared percentage present calls, absent calls, reproducibility, and concordance in needle biopsy samples processed by standard microarray protocol (cDNA non-amplification method) and our modified protocol (cDNA amplification method). The results showed that cDNA amplification method provided high reproducibility, representation, and concordance with the standard cDNA non-amplification method. We have successfully analyzed the gene expression profiles of needle biopsy tissues using the modified method without significantly changing the expression profiles. These results suggest that the global gene expression profiles of small biopsy samples can be achieved by our modified method to facilitate the analysis of gene expression profiles for clinical application.

Biopsy, Needle↗
PubMed2004

Regulation of apoptosis by peroxisome proliferators.

Peroxisome proliferators (PPs) constitute a large and chemically diverse family of non-genotoxic rodent hepatocarcinogens that activate the PP-activated receptor alpha (PPARalpha). In order to investigate the hypothesis that PPs elicit their carcinogenic effects through the suppression of apoptosis, we established an in vitro assay for apoptosis using both primary rat hepatocytes and the FaO rat hepatoma cell line. Apoptosis was induced by transforming growth factor beta1 (TGFbeta1), the physiological negative regulator of liver growth. In this system, PPs could suppress both spontaneous and TGFbeta1-induced apoptosis. In order to understand the mechanisms of this regulation of apoptosis, we conducted microarray analysis followed by pathway-specific gene clustering in TGFbeta1-treated cells. After treatment, 76 genes were up-regulated and 185 were down-regulated more than 1.5-fold. Cluster analysis of up-regulated genes revealed three clusters, A-C. Cluster A (4h) was associated with 12% apoptosis and consisted of genes mainly of the cytoskeleton and extracellular matrix such as troponin and the proteoglycan SDC4. In cluster B (8h; 25% apoptosis), there were many pro- and anti-apoptotic genes such as XIAP, BAK1 and BAD, whereas at 16h (40% apoptosis) the regulated genes were mainly those of the cellular stress pathways such as the genes implicated in the activation of the transcription factor NFkappab. Genes found down-regulated in response to TGFbeta1 were mainly those associated with oxidative stress and several genes implicated in glutathione production and maintenance. Thus, TGFbeta1 may induce apoptosis via a down regulation of oxidant defence leading to the generation of reactive oxygen species. The ability of PPs to impact on these apoptosis pathways remains to be determined. To approach this question, we have developed a technique using laser capture microdissection of livers treated with the PP, clofibric acid coupled with gene expression array analysis. Results show that some of the key steps of the LCM process had an impact on the gene profiles generated. However, this did not preclude accurate determination of a PP-specific molecular signature. Thus, the choice of appropriate controls will ensure that meaningful gene expression analyses can be performed on tissue microdissected from the foci generated in clofibric acid treated livers. These data will allow the identification of specific genes that are regulated by PPs leading to changes in apoptosis and ultimately to tumours.

Animals↗
PubMed1999

VHL gene deletion and enhanced VEGF gene expression detected in the stromal cells of retinal angioma.

OBJECTIVE: Retinal angioma frequently occurs in von Hippel-Lindau (VHL) disease. However, VHL gene alterations have not been documented in retinal angiomas. METHODS: Using tissue microdissection and polymerase chain reaction amplification, we have analyzed 7 retinal angiomas associated with VHL disease for loss of heterozygosity of the VHL gene. In addition, vascular endothelial growth factor expression was evaluated in these tumors by immunohistochemistry and in situ hybridization. RESULTS: All 6 informative retinal angiomas showed loss of heterozygosity of the VHL gene. Loss of heterozygosity was detected in vacuolated "stromal" cells, but not in vascular cells or reactive glial tissue. Vascular endothelial growth factor protein and messenger RNA were also present in vacuolated "stromal" cells. CONCLUSIONS: These findings suggest that vacuolated "stromal" cells represent the true neoplastic component in retinal angioma. These cells express vascular endothelial growth factor and therefore may be responsible for abundant neovascularization of retinal angioma.

Adult↗
PubMed2004

A technical triade for proteomic identification and characterization of cancer biomarkers.

Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 x 10(-5) and 4.6 x 10(-5)) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.

Biomarkers, Tumor↗
PubMed2005

Alteration of RPL14 in squamous cell carcinomas and preneoplastic lesions of the esophagus.

Allelic loss on chromosome 3p occurs frequently in esophageal cancer. The human ribosomal protein L14 gene (RPL14) is located on chromosome 3p21.3. In the present study, we investigated alteration of RPL14 at both the genomic DNA and RNA levels in 129 Chinese esophageal squamous cell carcinomas (ESCC) and 17 dysplasia adjacent to tumor tissues by a combination of tissue microdissection, microsatellite analysis of the intragenic marker, reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing. In the tested informative cases, loss of heterozygosity (LOH) of RPL14 was observed in 29 out of 68 (43%) tumors. Decreased expression of the gene was detected in 31 out of 49 (63%) carcinomas. No mutation was found in the remaining RPL14 allele of the tumors with LOH. We examined subsequently the allelic status of RPL14 in the dysplasia (preneoplastic lesions) between malignant tissues and histologically normal epithelia. Of 17 tested dysplasia in which the tumors showed LOH, eight (47%) displayed the same allelic loss as their corresponding tumors, seven (41%) exhibited microsatellite instability (MSI), and only two retained both the RPL14 alleles. The data suggest that alteration of RPL14 occurred frequently in ESCC and might be an earlier event in the tumorigenesis of the esophagus. Analysis to RPL14 gene may contribute to the early detection of ESCC as a potential molecular marker.

Adult↗
PubMed2001

Gastrointestinal cell proliferation and crypt fission are separate but complementary means of increasing tissue mass following infusion of epidermal growth factor in rats.

BACKGROUND AND AIMS: Epidermal growth factor (EGF) is a potent mitogen for the gastrointestinal tract and also influences the number of new crypts formed by crypt fission. The time course of these events and possible linkage between these two complementary mechanisms is however poorly understood. We therefore examined the temporal relationship of proliferation and fission in rats treated with EGF. METHODS: Osmotic minipumps were implanted subcutaneously into male Wistar rats to infuse EGF continuously (60 microg/rat/day) for periods of 1-14 days. Proliferation and crypt branching were quantified following vincristine induced metaphase arrest and morphometric assessment of microdissected tissue. RESULTS: In the small intestine, EGF significantly increased epithelial cell proliferation and crypt and villus area after 24 hours of EGF, although maximal effects were only reached following six days of infusion. EGF also resulted in an approximate 30% reduction in crypt fission in the small bowel. In the colon, EGF caused a twofold increase in epithelial cell proliferation one day after infusion, from 15.3 (2.3) to 29.6 (3.5) metaphases per crypt (p<0.01). Maximal effects were seen in rats receiving EGF for seven days. For all time points, colonic crypt size increased in response to EGF. The amount of branching increased following one day of infusion with EGF (from 15.3 (1.9) to 32.4 (5.5)%; p<0.001) but was significantly lower (approximately 25% of control values) following longer periods of infusion. Crypt fission did not correlate with crypt area. CONCLUSION: EGF has profound effects on cell proliferation and also altered crypt fission, with its actions on crypt fission most pronounced in the colon where it first increased and then decreased fission. EGF can thus be a potent stimulus for crypt fission during short term infusion and may reduce the number of branched crypts present in a resting or quiescent stage. Growth factors can alter cell mass by two separate but linked mechanisms, namely altered cell production and crypt fission.

Animals↗
PubMed2006

[Value of combinational detection by IgH and IgL primers in improving detection rate of lymphoma gene in paraffin-embedded tissue].

BACKGROUND & OBJECTIVE: Clonality detection through amplifying immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) is a useful tool in diagnosis of lymphoma, but the false negative rate is high, especially in paraffin-embedded tissues. This study was to explore the value of tumor tissue microdissection and combinational detection of IgH and immunoglobulin light chain (Ig kappa or Ig lambda) in diagnosis of non-Hodgkin's lymphoma (NHL). METHODS: Two pairs of conventional primers for IgH and T-cell receptor gamma (TCRgamma), 2 novel designed pairs of primers for Ig kappa and Ig lambda were used to detect 58 paraffin-embedded blocks, which had been diagnosed by pathology and histochemistry. Of the 58 cases of lymph node tissues, 39 were B-cell lymphoma, 16 were T-cell lymphoma, and 3 were reactive proliferative lymph node tissue. Lymphoma cell lines DG75 and Jurkat were used as control. RESULTS: The positive rates of IgH primers (P1) and IgL primers (P kappa/P lambda) were 79.5% and 71.8% in the 39 cases of B-cell lymphoma (P>0.05), 6.3% and 12.5% in the 16 cases of T-cell lymphoma, respectively. The positive rate was greatly increased to 92.3% in combinationally detecting the primers for IgH and P kappa/P lambda. There was no positive detection among the reactive proliferative lymph node tissues. CONCLUSION: B-cell lymphoma detection rate can be significantly improved by the combination of IgH and IgL gene rearrangement primers, which provides efficient assistant method for the diagnosis and differential diagnosis of lymphoma.

DNA Primers↗
PubMed2004

Endometrial glandular dysplasia: a putative precursor lesion of uterine papillary serous carcinoma. Part II: molecular features.

Endometrial glandular dysplasia (EmGD) may be a newly defined precursor lesion of uterine papillary serous carcinoma (UPSC) by morphology. In this report, we studied molecular changes present in EmGD by the loss of heterozygosity (LOH) approach using laser capture microdissected tissue samples. Nineteen uteri showing at least 1 focus of EmGD by morphology were selected. These cases were 12 UPSC, 2 clear cell carcinomas, 1 mixed uterine papillary serous and endometrioid carcinoma, 1 uterine carcinosarcoma, 1 serous endometrial intraepithelial carcinoma (EIC), and 2 EmGD involving endometrial polyps. Seven microsatellite polymorphic DNA markers (TP53 at 17p, D1S211, and D1S162 at 1p32, D17S1323 at 17q21, D17S1330 at 17q25, D5S346 at 5q, and D2S123 at 2p) were utilized. A total of 123 laser-captured microdissection samples from 19 cases was studied with LOH method. The frequencies and patterns of LOH were analyzed and compared among benign resting endometrium (RE), EmGD, serous EIC, and UPSC. LOH was observed for at least 1 of the 7 markers in all categories of lesions, EmGD, serous EIC, and UPSC. The frequency of LOH for EmGD ranged from 4.2% to 31.3%; the range for serous EIC was 5.9% to 78.6%; and that for UPSC was 7.7% to 62.5%. The most frequent LOH in the 3 above-cited categories of lesions was identified at 17p (TP53) and 1p (D1S162). The frequency of LOH in EmGD with markers of TP53 and D1S162 was significantly higher than in RE (p < 0.05). With markers of D1S211 and D2S123, LOH in EmGD was higher than RE, approaching to a statistically significant level. Compared with foci of serous EIC and UPSC, however, the rate of LOH in EmGD was significantly lower only with TP53 locus (31.3% vs more than 60%, p < 0.05). The difference of LOH frequency with other chromosomal markers between EmGD and serous EIC/UPSC did not reach a statistically significant level. A significantly high concordant LOH pattern was found between foci of EmGD and serous EIC/UPSC (p = 0.05). We conclude that EmGD frequently shows LOH at multiple chromosomal loci, particularly at 17p and 1p. Significantly high concordant LOH frequency between EmGD and paired serous EIC or UPSC strongly suggests that EmGD is a noncancerous precursor lesion of UPSC, probably also of serous EIC. The clinical significance of EmGD needs further studies.

Aged↗
PubMed2004

Expression of PTEN in malignant and non-malignant human prostate tissues: comparison with p27 protein expression.

The role of the putative tumour suppressor PTEN in prostate carcinogenesis is controversial. There are conflicting data regarding the rate of its gene inactivation, the role of transcriptional and post-transcriptional factors, as well as its relationship to tumour progression and to the potential downstream regulator, the cell-cycle inhibitor p27. The present study has assessed the in situ expression of PTEN mRNA and protein in 26 prostate intraepithelial neoplasias (PINs), 58 primary prostate carcinomas, and 15 metastases. Although there was a correlation between PTEN mRNA and protein expression, mRNA detection exceeded detection of protein in 19% of PINs and 30% of all invasive tumours. Using RT-PCR and western blotting on microdissected tissue, this discrepancy was attributed, at least in part, to transcription of the PTEN pseudo-gene, which lacks introns. Total or partial loss of PTEN protein occurred with tumour progression but this association was not statistically significant. Analysing the relationship between PTEN and p27 protein expression on consecutive sections by immunohistochemistry, the results do not support a direct link between the two oncosuppressors, other than an associated loss of expression in advanced tumour stages. However, in the basal cells of prostate glands and in most PINs, an inverse relationship was observed between PTEN and p27. This may reflect the existence of a functional balance that controls the cell cycle in prostatic epithelium and that is probably disturbed in invasive tumour cells.

Adenocarcinoma↗
PubMed2005

Laser capture microdissection and colorectal cancer proteomics.

The ability to define protein profiles of normal and diseased cells is important in understanding cell function. Laser capture microdissection permits the isolation of specific cell types for subsequent molecular analysis. In this study we have established conditions for obtaining proteomic information from laser capture microdissected colorectal cancer cells. Laser capture microdissection was performed on toluidine blue-stained frozen sections of colorectal cancer. Proteins were solubilized from microdissected cells and the solubilized proteins were separated by two-dimensional gel electrophoresis: protein spots were characterized by peptide mass mapping using matrix assisted laser desorption ionization-time of flight mass spectrometry. Proteins isolated from laser capture microdissected tissue retained their expected electrophoretic mobility and peptide mass mapping was also unaffected. The ability to study the protein expression profile of specific cell types will allow for the identification of novel disease markers and therapeutic targets and also provide for the enhanced understanding of pathogenetic mechanisms.

Animals↗
PubMed2006

Concentration of nucleosides and related compounds in cerebral and cerebellar cortical areas and white matter of the human brain.

1. Nucleosides potentially participate in the neuronal functions of the brain. However, their distribution and changes in their concentrations in the human brain is not known. For better understanding of nucleoside functions, changes of nucleoside concentrations by age and a complete map of nucleoside levels in the human brain are actual requirements. 2. We used post mortem human brain samples in the experiments and applied a recently modified HPLC method for the measurement of nucleosides. To estimate concentrations and patterns of nucleosides in alive human brain we used a recently developed reverse extrapolation method and multivariate statistical analyses. 3. We analyzed four nucleosides and three nucleobases in human cerebellar, cerebral cortices and in white matter in young and old adults. Average concentrations of the 308 samples investigated (mean+/-SEM) were the following (pmol/mg wet tissue weight): adenosine 10.3+/-0.6, inosine 69.5+/-1.7, guanosine 13.5+/-0.4, uridine 52.4+/-1.2, uracil 8.4+/-0.3, hypoxanthine 108.6+/-2.0 and xanthine 54.8+/-1.3. We also demonstrated that concentrations of inosine and adenosine in the cerebral cortex and guanosine in the cerebral white matter are age-dependent. 4. Using multivariate statistical analyses and degradation coefficients, we present an uneven regional distribution of nucleosides in the human brain. The methods presented here allow to creation of a nucleoside map of the human brain by measuring the concentration of nucleosides in microdissected tissue samples. Our data support a functional role for nucleosides in the brain.

Adult↗
PubMed1995

Upregulation of the 72-kDa type IV collagenase in epithelial and stromal cells during rat tracheal gland morphogenesis.

Submucosal glands secrete most of the mucus that lubricates the tracheal surface and protects it from irritants and infection. These glands develop postnatally in the rat, permitting convenient study of the mechanisms controlling this process. One such mechanism involves degradation of the supportive connective tissue matrix at the front of the growing glands. We recently showed that tracheal gland cell invasion of collagen gels in vitro is dependent on secretion of a 72-kDa type IV collagenase. In the present study, we show that the activity of this enzyme (also referred to as matrix metalloproteinase-2 or gelatinase A) is elevated at the time of gland development in vivo. That this increase is at least partly mediated at the level of steady-state mRNA was indicated by semiquantitative PCR analysis of gland-enriched, microdissected tissue samples. Immunohistochemistry revealed that the enzyme was present at the interface between the glands and extracellular matrix. In situ hybridization revealed that the cognate mRNA was present in epithelial cells of glands undergoing morphogenesis (particularly Postnatal Day 7) but not in those of adult glands or the surface epithelium. At all ages, stromal cells below the surface epithelium were labeled; labeling intensity was highest at the time and location of gland morphogenesis. These findings suggest that the 72-kDa type IV collagenase is developmentally regulated in gland and stromal cells at the level of steady-state mRNA and plays a role in the degradation of extracellular matrix during tracheobronchial gland morphogenesis.

Animals↗
PubMed1982

Activity of 3 beta-hydroxysteroid dehydrogenase delta 4-5 isomerase in the human skin.

The activity of 3 beta-hydroxysteroid dehydrogenase delta 4-5 isomerase (3 beta-HSD) was assayed in various tissues microdissected from the freeze-dried human skin of 13 subjects. The sebaceous gland possessed the highest activity of 3 beta-HSD in the skin, while the apocrine sweat gland showed only one fourth of that activity. The vellus hair follicle showed nearly one half of the activity of the sebaceous gland, whereas the terminal hair follicle exhibited much lower activity. The activity of the epidermis and of the dermis were negligible. Incubation of fresh whole skin of the forehead with 7 alpha-3H-DHA and subsequent isolation of various tissues revealed that the metabolites identified in the sebaceous gland were androstanedione and androstenedione, whereas testosterone and/or dihydrotestosterone were not detected.

3-Hydroxysteroid Dehydrogenases↗
PubMed2006

Microdissection genotyping analysis of the effect of intraarterial cytoreductive chemotherapy in the treatment of lacrimal gland adenoid cystic carcinoma.

PURPOSE: To investigate the feasibility of integrating molecular analysis into standard histopathology for lacrimal gland adenoid cystic carcinoma (ACC), and to gain insights into the molecular pathogenesis of this tumor and its response to intraarterial cytoreductive chemotherapy (IACC) that is of clinical use. DESIGN: A retrospective, comparative case series. METHODS: setting: Institutional. patient population: Nine consecutive patients with lacrimal gland ACC were treated with IACC, followed by orbital exenteration and chemoradiotherapy. This case series was compared with a series of seven patients treated by conventional local therapies. intervention procedure: Gene analysis was performed on microdissected tissue samples. Mutational allelotyping targeting nine genomic loci was performed with 15 polymorphic microsatellite markers situated in proximity to known tumor suppressor genes serving as markers for the presence of gene deletion. main outcome measure: A fractional mutation index was used to compare the acquired mutational load between different tumors having nonidentical patterns of microsatellite informativeness. RESULTS: Allelic imbalance (loss of heterozygosity [LOH]) for microsatellite markers at 1p36 was the single most common site affected by imbalance in this series, followed by LOH in temporal sequence involving 9p21, 22q12, 10q23, and 9q22. CONCLUSIONS: Microdissection genotyping holds promise as a clinical tool in integrating molecular analysis into standard histopathology to advance the understanding of lacrimal gland ACC tumorigenesis. A unique time course for temporal mutation acquisition in ACC is proposed, consisting of 1p36 loss first. Allelic loss for microsatellite markers at 1p36 may be a common as well as an early event in ACC formation and progression.

Adult↗
PubMed2006

Differential gene expression profiling in aggressive bladder transitional cell carcinoma compared to the adjacent microscopically normal urothelium by microdissection-SMART cDNA PCR-SSH.

Identifying novel and known genes that are differentially expressed in aggressive bladder transitional cell carcinoma (BTCC) has important implications in understanding the biology of bladder tumorigenesis and developing new diagnostic and therapeutic agents. In this study we identified the differential gene expression profiles comparing tumor to the adjacent microscopically normal mucosa by manual microdissection on frozen sections. The RNAs extracted from microdissected tissues were amplified by SMART cDNA PCR technology to generate forward subtractive cDNA library by suppressive subtractive hybridization (SSH). We obtained 376 positive clones, one hundred clones of aggressive BTCC subtracted cDNA library were selected at random and inserts were reamplified by PCR. After differential screening by reverse dot blotting, 73 positive clones, that contend inserts putatively upregulated in aggressive BTCC, were further analysed by DNA sequencing, GenBank and EST database searching. Sequencing results showed that 66 clones stand for 23 known genes and 7 clones for three new EST (Genbank number: DN236875, DN236874 and DN236873). In conclusion, microdissection-SMART cDNA PCR-SSH allowed for an efficient way to identify aggressive BTCC-specific differential expressed genes that may potentially be involved in the carcinogenesis and/or progression of aggressive BTCC. These differentially expressed genes may be of potential utility as therapeutic and diagnostic targets for aggressive BTCC.

Carcinoma, Transitional Cell↗
PubMed2006

Gene expression in tumor-adjacent normal tissue is associated with recurrence in patients with rectal cancer treated with adjuvant chemoradiation.

Recurrence is a significant clinical problem for patients with rectal cancer treated with adjuvant chemoradiation. Previous studies have suggested that determining intratumoral gene expression of key genes may be helpful in predicting clinical outcome of patients with gastrointestinal malignancies undergoing chemotherapy. The role of molecular predictors for prediction of recurrence in the setting of adjuvant chemoradiotherapy is not well established. The present study was designed to identify a genetic profile that would be associated with recurrence in patients with rectal cancer treated with adjuvant chemoradiation therapy. A retrospective study with a longitudinal cohort and a cross-sectional cohort of 67 patients with locally advanced rectal cancer who underwent cancer resection, followed by 5-fluorouracil (5-FU) plus pelvic radiation was conducted. Total RNA was extracted from formalin-fixed, paraffin-embedded, laser-captured-microdissected tissue. We determined mRNA levels of genes involved in the 5-FU pathway (thymidylate synthase, dihydropyrimidine dehydrogenase), DNA-repair (excision-repair cross-complementing factor 1, Rad51), angiogenesis/radiation sensitivity [vascular endothelial growth factor (VEGF)] and radio-sensitivity [epidermal growth factor receptor (EGFR)] in tumor tissue and tumor-adjacent normal tissue by quantitative reverse transcriptase-polymerase chain reaction. In univariate analysis, only intratumoral gene expression level of VEGF (P = 0.055) was associated with recurrence, whereas elevated mRNA expression levels of thymidylate synthase (P = 0.008), VEGF (P = 0.023) and EGFR (P = 0.004) in tumor-adjacent normal tissue were significantly associated with recurrence. Multivariate analysis using recursive partitioning indicated that distinct groups of recurrence could be defined by elevated mRNA expression levels of VEGF, EGFR in tumor-adjacent normal tissue, and Rad51 in tumor tissue. These data suggest that the genetic profile of the tumor-adjacent normal tissue may be associated with treatment failure, indicating that tumor microenvironment may be more important in the development of recurrence of rectal tumors than formerly expected.

Adult↗
PubMed2000

Frequent c-myc and Int-2 overrepresentations in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a commonly occurring tumor in southern China. Although several causative factors have now been recognized, the genetic basis underlying its tumorigenesis is still unclear. To identify potential chromosomal aberrations for further investigations, comparative genomic hybridization (CGH) analysis was applied to the study of genomic imbalances in 10 NPC biopsy specimens. Before CGH analysis, the tumor cell content within the biopsy specimens was enriched by tissue microdissection, and universal genome amplification was performed on the recovered DNA. Recurrent chromosomal gains were detected on 1q (6 of 10 cases), 2q (5 of 10 cases), 3q (7 of 10 cases), 6p (8 of 10 cases), 6q (5 of 10 cases), 7q11.2 (4 of 10 cases), 8q (6 of 10 cases), 11q13, 12, and 15q (8 of 10 cases each), 17q (6 of 10 cases), and 20q (5 of 10 cases). Common losses were identified on 3p (5 of 10 cases), 9p (5 of 10 cases), 11q14-qter (8 of 10 cases), and 14q (5 of 10 cases). Among these aberrations, 7, 8, and 11 gains were further investigated on a series of NPC tissue samples, by interphase fluorescent in situ hybridization (FISH), for the incidence of alpha-satellites: 7, 8, and 11 c-myc and Int-2. Low-level increases of alpha-satellite 7 (9 of 34 cases; 26.5%), alpha-satellite 8 (15 of 34 cases; 44%), and alpha-satellite 11 (8 of 32 cases; 25%) were detected, whereas high-level copy gains of c-myc (21 of 34 cases; 62%) and Int-2 (26 of 34 cases; 76.5%) were more frequently found. Our series is the first to identify genomic overrepresentations of c-myc and Int-2 in NPC. The high incidence of Int-2 amplifications strongly suggests a role of this proto-oncogene in the pathogenesis of NPC.

Adult↗