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Differential caspase-3 expression in noncancerous, premalignant and cancer tissues of stomach and its clinical implication.

BACKGROUND: Caspase-3 is a critical apoptosis-promoting element but its status during stepwise gastrocarcinogenesis needs to be further clarified. MATERIALS AND METHODS: By the use of frozen tissue microarrays constructed with the tissue spots cored from defined histological regions in tissue blocks, the pattern of caspase-3 expression in noncancerous, premalignant (atrophic gastritis and intestinal metaplasia) tissue and cancer spots were analyzed under the same experimental conditions by the methods of immunohistochemistry and mRNA-in situ hybridization. RESULTS: Caspase-3 was expressed in all 34 of the noncancerous mucosa (100%), in 16 of the 17 premalignant tissues (94.1%) and in 15 of the 48 gastric cancers (31.3%). The incidences of caspase-3 detection were significantly different (p<0.01) between noncancerous mucosa and intestinal as well as diffuse gastric cancers. CONCLUSION: Down-regulated caspase-3 is closely correlated with gastric cancer formation and would be a potential indicator of tumor formation and progression. Helicobacter pylori (H. pylori; Hp) infection is but not the only one element responsible to the enhanced caspase-3 expression in gastric epithelia.

Apoptosis↗

Tissue microarray detection of matrix metalloproteinases, in diseased tricuspid and bicuspid aortic valves with or without pathology of the ascending aorta.

OBJECTIVE: The degeneration of bicuspid aortic valve and its frequent association with ascending aortic pathology, point to a still unidentified genetic tissue defect with unknown mediators. Metalloproteinases (MMPs) are lytic enzymes that have been strongly implicated in aneurysm formation. The purpose of this study was to detect the presence of these enzymes in aortic valvular tissue in healthy and diseased aortic valves with or without the presence of synchronous ascending aortic pathology. METHODS: Aortic valve specimens from 26 aortic valve replacement patients as well as 4 healthy control tricuspid aortic valves were included. 10 patients had bicuspid aortic valves, and 16 had tricuspid aortic valves. Half of our patient population had a concomitant aortic procedure for aortic pathology. The study detected MMPs 1,2 and 9 as well as their Tissue inhibitors (TIMPs) 1 and 2. MMP and TIMP detection was accomplished with the construction of a tissue micro array and immunohistochemistry. CONCLUSIONS: MMP-9 expression was significantly higher in bicuspid aortic valves compared to normal valves (P<0.05). When compared to the tricuspid valve group, MMP-9 mean value was significantly higher in bicuspid valves (P<0.05). When the entire rest of the valve group (n=4+16, i.e. control and tricuspid valve groups) was compared to the bicuspid valve group, bicuspid valves had significantly higher MMP-2, and MMP-9 (P<0.01) expression. TIMP expression also changed in diseased valves, among different patient groups. This increased proteolytic presence in bicuspid aortic valves may attribute to the observed decreased elastin and collagen content, and their resultant functional failure.

Aortic Aneurysm↗

Cox-2 expression on tissue microarray of breast cancer.

AIM: To measure cyclooxygenase-2 (Cox-2) expression in a series of breast cancers and evaluate its potential as a predictive marker for doxorubicin chemotherapy. PATIENTS AND METHODS: Cox-2 expression was analyzed in 178 node-positive patients treated with doxorubicin-based adjuvant chemotherapy by immunohistochemistry on tissue microarray (TMA). RESULTS: Cox-2 was over-expressed in 70 out of the 178 invasive breast cancers. Cox-2 expression was significantly increased in undifferentiated tumors. There was no significant association between Cox-2 over-expression and tumor size, histologic grade, and estrogen receptor expression. Disease-free survival and overall survival of the patients having Cox-2 expressing tumor were significantly decreased when compared with the patients having Cox-2 negative tumor (p=0.009 for DFS, p=0.011 for OS). CONCLUSION: Cox-2 expression may represent an aggressive phenotype of breast cancer which is resistant to doxorubicin.

Antibiotics, Antineoplastic↗

Hypoxia-inducible factor 1alpha expression in renal cell carcinoma analyzed by tissue microarray.

OBJECTIVES: Angiogenesis is important for tumour progression and metastatic spread. Hypoxia-inducible factor 1alpha (HIF-1alpha) is a major factor regulating a number of other angiogenic factors. Renal cell carcinoma (RCC) is a malignancy with a variable clinical course, partly attributable to specific genetic alterations of the different RCC types. We therefore analysed HIF-1alpha expression using immunohistochemistry and related the results to RCC type and clinicopathologic variables. MATERIAL AND METHODS: We semiquantitatively analysed HIF-1alpha expression using immunohistological staining of a prepared tissue microarray. There were 216 patients including 176 conventional, 26 papillary, and 14 chromophobe RCCs. RESULTS: The HIF-1alpha staining was found mainly in the cytoplasm. The tumours were subdivided into HIF-1alpha(LOW) and HIF-1alpha(HIGH) on the basis of staining intensity. HIF-1alpha expression between the RCC types did not differ. Patients with conventional RCC showed a trend (p=0.055) towards a prolonged survival for those with HIF-1alpha(HIGH)-staining versus HIF-1alpha(LOW)-staining tumors. In conventional RCC there were significant differences in HIF-1alpha expression in relation to TNM stage, nuclear grade, and vein invasion. In patients with papillary RCC, difference in HIF-1alpha expression was observed only for nuclear grade. CONCLUSIONS: We studied HIF-1alpha expression in RCC using tissue microarray. In patients with conventional RCC, HIF-1alpha levels were significantly lower in locally aggressive tumors versus localized tumors, and patients with high HIF-1alpha levels tended to have a better prognosis. There seems to be a diverging regulation of angiogenesis between the different RCC types. Further studies of HIF and angiogenesis in RCC are encouraged.

Adult↗

Inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis.

Mitochondrial dysfunction is a hallmark of cancer cells. Consistent with this phenotype mutations in mitochondrial genome have been reported in all cancers examined to date. However, it is not clear whether mitochondrial genomic status in human cells affects nuclear genome stability and whether proteins involved in inter-genomic cross talk are involved in tumorigenesis. Using cell culture model and cybrid cell technology, we provide evidence that mitochondrial genetic status impacts nuclear genome stability in human cells. In particular our studies demonstrate 1) that depletion of mitochondrial genome (rho0) leads to chromosomal instability (CIN) reported to be present in variety of human tumors and 2) rho0 cells show transformed phenotype. Our study also demonstrates that mitochondrial genetic status plays a key role in regulation of a multifunctional protein APE1 (also known as Ref1 or HAP1) involved in transcription and DNA repair in the nucleus and the mitochondria. Interestingly we found that altered expression of APE1 in rho0 cells and tumorigenic phenotype can be reversed by exogenous transfer of wild type mitochondria in rho0 cells. Furthermore, we demonstrate that APE1 expression is altered in variety of primary tumors. Taken together, these studies suggest that inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis and that APE1 mediates this process.

Blotting, Western↗

Invasive colorectal micropapillary carcinoma: an aggressive variant of adenocarcinoma.

Micropapillary carcinoma (MC) has been well described in other organs, including breast, urinary bladder, lung, ovary, and salivary gland, but has not been described in the large intestine. We compared the clinicopathologic and immunohistochemical findings of MC with those of conventional adenocarcinoma in the large intestine. Fifty-five cases of adenocarcinoma with an MC component were identified among 585 consecutive cases of colorectal cancer at the Asan Medical Center between January 2003 and June 2004 and were compared with 119 cases of conventional adenocarcinoma of colorectum without an MC component. Arrayed tissue blocks were constructed and immunostained for cytokeratin 7 and 20 and CDX2. We also compared the results of MLH-1, MSH-2, p53, and carcinoembryonic antigen immunostainings between the 2 groups. The grade of both MC and conventional adenocarcinoma was mostly moderately differentiated. The proportion of MC ranged from 5% to 80%. The presence but not extent of MC in the primary tumors was associated with more frequent lymphovascular invasion and lymph node (LN) metastases, a greater mean number of positive LNs, and a higher tumor stage with more frequent distant metastases, compared with conventional adenocarcinoma (P < .05). Cytokeratin 7 staining was occasionally observed in both MC (9.1%, 5/55 cases) and conventional adenocarcinoma (13.4%, 16/119 cases). Although MLH-1 and CDX2 expression tended to be lower in conventional adenocarcinoma, none of the immunohistochemical results was significantly different between 2 groups. Recognition of MC component is important as MC appeared to be an aggressive variant of colonic adenocarcinoma and presented at a higher stage, with frequent lymphovascular invasion, LN metastasis, and distant metastasis, compared with conventional adenocarcinoma. The proportion of MC component did not impact the prognosis, and the immunoprofiles of MC were not significantly different from those of conventional adenocarcinoma.

Adenocarcinoma↗

Loss of Ku70/Ku80 expression occurs more frequently in hereditary than in sporadic colorectal tumors. Tissue microarray study.

Ku70 and Ku80 proteins take part in the repairing of DNA double-strand breaks by their function as a regulatory subunit of the DNA-dependent protein kinase. In this study, expression of both genes was analyzed in colorectal carcinoma tissue arrays applying immunohistochemistry. Expression of both genes decreased along with pT stage. Significant differences in Ku70 and Ku80 expression were found between pT3 and pT4 as well as between pT2 and pT3 tumors, respectively. Loss of Ku70/Ku80 expression was more frequently observed in hereditary than in sporadic tumors. We conclude that expression of Ku70/Ku80 genes is down-regulated in colorectal carcinoma and that defects of these genes are more frequently observed in hereditary than in sporadic tumors.

Adult↗

The up-regulation profiles of p21WAF1/CIP1 and RUNX1/AML1 correlate with myometrial infiltration in endometrioid endometrial carcinoma.

We have recently described RUNX1/AML1 up-regulation in endometrioid endometrial carcinoma (EEC), proposing that it could play a role during the initial steps of myometrial infiltration. Some cell cycle regulators, including the cyclin-dependent kinase inhibitor p21WAF1/CIP1, have been described as targets of RUNX1/AML1. In this study, we have attempted to address the question of whether RUNX1/AML1, acting both as a gene transcription activator and a repressor, depending on the context, can be correlated with the expression of p21WAF1/CIP1 in gynecologic malignancies, in particular in EEC, where the role of p21(WAF1/CIP1) remains controversial. Toward this end, we analyzed p21WAF1/CIP1 expression in a large panel of EEC samples using real-time quantitative polymerase chain reaction and tissue microarray immunohistochemistry, and evaluated the extent to which RUNX1/AML1 and p21WAF1/CIP1 interacted in the EEC samples. The strong correlation found between RUNX1/AML1 and p21WAF1/CIP1 suggested cooperation between the 2 genes in EEC, especially in those tumor samples corresponding to stage IC carcinomas, infiltrating more than 50% of the myometrium. We hypothesize that p21WAF1/CIP1 and RUNX1/AML1 interact during the initial steps of tumor dissemination in EEC, and we discuss mechanisms that could underlie myometrial infiltration and/or the promotion of an invasive phenotype.

Biomarkers, Tumor↗

Prognostic value of humbug gene overexpression in stage II colon cancer.

Overexpression of aspartyl (asparaginyl) beta-hydroxylase (AAH) has been demonstrated in hepatocellular carcinoma, cholangiocarcinoma, and pancreatic carcinoma. AAH has an important role in regulating cell motility and invasiveness. Humbug is a truncated homolog of AAH, with a role in calcium regulation. The present study examines the prognostic use of AAH and humbug gene expression in stage II colon cancer. One hundred thirty cases of TNM stage II colon carcinoma were retrieved from the Rhode Island Hospital pathology archives. Tissue microarrays were immunostained with the FB50 and 15C7 monoclonal antibodies generated to recombinant AAH. However, FB50 also recognizes humbug. In addition, AAH and humbug expression was analyzed in samples of colon cancer and adjacent normal mucosa by real-time quantitative reverse transcriptase-polymerase chain reaction. Humbug (FB50) expression was localized to the tumor cytoplasm, whereas normal colonic epithelium did not exhibit significant immunoreactivity. Humbug staining was detected in 85% of the neoplasms, 23% of which stained strongly. Strong humbug immunoreactivity positively correlated with nuclear grade (P = .006) and inversely with survival (P = .027). In contrast to humbug, AAH (15C7) immunoreactivity was seen in normal and neoplastic epithelium. There was no correlation between AAH immunoreactivity and tumor grade, or survival. Correspondingly, reverse transcriptase-polymerase chain reaction studies demonstrated up-regulation of humbug but not AAH in 95% of colon carcinomas relative to adjacent colon cancer-free mucosa (P < .0001). This study demonstrates that high levels of humbug immunoreactivity in colon carcinomas correlate with histologic grade and tumor behavior, suggesting that humbug can serve as a prognostic biomarker of TNM stage II colon cancers. In addition, molecular studies demonstrated that the increased levels of FB50 detected were due to humbug, as opposed to AAH overexpression.

Adult↗

Catenin expression in T1/2 carcinomas of the floor of the mouth.

Reduction of the expression of catenin is a crucial step in the pathogenesis, progression and prognosis of many epithelial cancers including squamous cell carcinomas (SCCs). Catenin expression in oral carcinomas was evaluated in relation to clinico-pathological features in order to determine its value as a prognostic marker. Eighty-five patients with histologically proven T1/2 squamous cell carcinoma of the oral floor who underwent surgical treatment were eligible for the study. A tissue microarray consisting of multiple representative tissue cores of each carcinoma was composed. The expression levels of alpha, beta and gamma-catenins were determined immunohistologically. Correlation between clinical features and the expression of catenin proteins was evaluated statistically using Kaplan-Meier curves, log-rank tests and chi(2)-tests. Loss of alpha-catenin expression in carcinoma of the floor of the mouth correlated significantly with poor prognosis (P=0.05). Conversely, significantly reduced rates of lymph-node metastases were observed in alpha- and beta-catenin-positive T1 and T2 SCCs. Loss of gamma-catenin expression indicated a reduced survival rate in nodal-negative tumours (P=0.02). Catenin expression in carcinomas of the floor of the mouth seems to be a predictive parameter in the prognosis of T1 and T2 SSCs.

Adult↗

Assessing the potential of immunohistochemistry for systematic gene expression profiling.

Immunohistochemistry (IHC) is a powerful technique for identifying sites of protein expression in tissues at the cellular and sub-cellular level. Here we have investigated the potential of using IHC for genome-wide expression screening by measuring the success rate and specificity of a panel of 35 monoclonal antibodies recognizing 5 well characterised CD antigens. Antibodies were pre-screened on acetone fixed frozen sections of spleen, tonsil and colon tissues. 19/35 antibodies gave staining with a success rate of 0/7 for JAM-2, 1/4 for CD99, 3/6 for CD138, 5/8 for CD45 and 10/10 for MHC-class II. 16/19 of these antibodies also gave staining on formalin fixed paraffin embedded tissue sections of tonsil and colon. All antibodies that had given staining were then profiled on tissues presented in human tissue microarrays. In the frozen microarrays 216 cores from 29 normal tissue types were present and in the formalin fixed paraffin array 344 cores from 35 normal and 4 cancers were represented. Where multiple antibodies were positive, there was evidence of consistent staining of the same tissues with several antibodies. In some cases differences in staining were observed potentially due to differential splice variants, polymorphisms or protein modification. With some antibodies there was evidence of cross-reactivity to inappropriate cells or structures. In addition the staining intensity with formalin fixation was changed quantitatively for some antibodies and in a few cases qualitatively, representing differential sensitivity of specific and non-specific epitopes to fixation. Accordingly, whilst IHC has potential for describing protein expression of unknown genes, these results emphasise a need to systematically address issues of specificity and sensitivity if appropriate profiles are to be described.

12E7 Antigen↗

SNIP1 is a candidate modifier of the transcriptional activity of c-Myc on E box-dependent target genes.

Using a yeast two-hybrid screen, we found that SNIP1 (Smad nuclear-interacting protein 1) associates with c-Myc, a key regulator of cell proliferation and transformation. We demonstrate that SNIP1 functions as an important regulator of c-Myc activity, binding the N terminus of c-Myc through its own C terminus, and that SNIP1 enhances the transcriptional activity of c-Myc both by stabilizing it against proteosomal degradation and by bridging the c-Myc/p300 complex. These effects of SNIP1 on c-Myc likely contribute to synergistic effects of SNIP1, c-Myc, and H-Ras in inducing formation of foci in an in vitro transformation assay and also in supporting anchorage-independent growth. The significant association of SNIP1 and c-Myc staining in a non-small cell lung cancer tissue array is further evidence that their activities might be linked and suggests that SNIP1 might be an important modulator of c-Myc activity in carcinogenesis.

Carcinoma, Non-Small-Cell Lung↗

A simple and economical method for the manual construction of well-aligned tissue arrays.

Tissue array technology has allowed a substantial progression of studies correlating molecular and immunohistochemical findings with clinico-pathological information. Array construction presents technical difficulties and tissue arrayers are expensive, particularly for small and medium sized laboratories. We describe a simple manual method for producing well-aligned tissue arrays using a hand-made paper mold which can successfully perform immunohistochemical staining. All 200 tissue samples were collected and constructed into blocks by the paper mold. The tissue arrays were smoothly sectioned using a standard microtome and performed for a panel of immunohistochemical study with satisfactory results. This alterative method for building custom arrays could be applied in any laboratory and is both simple and economical.

Humans↗

Altered expression of the KLF4 in colorectal cancers.

KLF4 is an important regulator of cell proliferation and is maximally expressed in epithelial cells of the gastrointestinal tract. Inactivation of the KLF4 gene by genetic and epigenetic alterations has been reported in colorectal cancers, but the expression pattern of the KLF4 protein has not been studied. Here, to investigate the roles of KLF4 in colorectal carcinogenesis, we examined the expression pattern of the KLF4 protein in 123 colorectal cancers by immunohistochemistry using tissue microarray. Moderate to strong nuclear staining for KLF4 was found in normal colonic mucosa. Interestingly, loss of KLF4 expression was observed in 30 (24.4%) of 123 colorectal cancers. Statistically, loss of KLF4 protein expression was not associated with clinocopathologic parameters, including tumor stage (Bartholomew test, P>0.05), lymph node metastasis, differentiation, tumor location, and tumor size (chi2 test, P>0.05). These results indicate that loss of the KLF4 expression might play a role in tumor development as an early event for a subset of colorectal cancers.

Adenocarcinoma↗

p21 as a prognostic factor in non-small cell lung carcinomas.

Few studies have focused on the correlation between p21 expression and survival for patients with non-small cell lung carcinoma (NSCLC), and the results are not consistent. We investigated the expression of p21 in 90 cases of NSCLC to evaluate the correlation between the p21 expression level and the clinicopathologic characteristics with patient survival. p21 was expressed in the nuclei of all the NSCLCs. The percentage of immunoreactive cells varied from 1% to 70%. All the patients were subdivided into a high and a low p21 expression group on the basis of the median percentage (17.05). There was no significant correlation between the p21 expression level and age, gender, histologic type, histologic grade, or stage. Using uni- and multivariate analyses, survival was significantly associated with gender, stage, and the p21 expression level. The survival rate for the high p21 expression group was higher than that for the low p21 expression group in the entire patient group, and especially for stage II and III patients, males, adenocarcinomas, or p53-positive tumors. Our findings showed that high p21 expression was an independent prognostic factor for NSCLC. p21 may be useful for determining the candidates for adjuvant therapies and also for selecting the appropriate chemotherapeutic agents.

Biomarkers, Tumor↗

Expression of nuclear and cytoplasmic phosphorylated FADD in gastric cancers.

Fas-associated death domain (FADD) plays a crucial role during death receptor-mediated apoptosis. In addition, FADD possesses apoptosis-independent activities, including cell-cycle regulation and cell proliferation regulated by the phosphorylation of FADD at Ser194. The aim of this study was to explore the possibility whether alteration of phosphorylated FADD (p-FADD) expression might be a characteristic of gastric cancer. We analyzed the expression of p-FADD protein in 60 gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach. In the normal gastric mucosal cells, surface and glandular epithelial cells evenly expressed p-FADD in the nuclei but not in the cytoplasm. In the cancers, p-FADD expression was detected in 38 cases (63%) of the gastric carcinomas, but there was no p-FADD immunostaining in the remaining 22 cancers (37%). Of note, p-FADD immunostaining was observed in cytoplasm/nuclei (20 cancers; 33%) and cytoplasm (18 cancers; 30%). There was no significant association of p-FADD expression with clinocopathological characteristics, including invasion, metastasis, and stage. Our data showed that the expression of p-FADD in gastric cancers was heterogenous in its location compared to the uniform nuclear expression of p-FADD in normal gastric cells. Many of the cancers (67%) were devoid of nuclear p-FADD, suggesting that p-FADD functions in the nucleus may be perturbed in the cancers. Also, p-FADD expression in the cytoplasm in a large fraction of the cancers (63%), not seen in the normal cells, suggested that the cell death functions of p-FADD could be altered in the cancer cells.

Adenocarcinoma↗

Tspy is nonfunctional in the Mongolian gerbil but functional in the Syrian hamster.

The TSPY gene is conserved in placental mammals and encodes the testis-specific protein, Y encoded. Within the testis, TSPY expression is restricted to germ cells, and it is assumed that TSPY plays a role in the proliferation of germ cells. Since it was first discovered in humans, TSPY orthologous gene families have been subsequently characterized in many mammalian lineages. In contrast to the situation in cattle and primates, in which TSPY is organized in a moderately repetitive cluster, including functional members and pseudogenes, a peculiar situation is observed in rodents, in which Tspy has been become low or single copy and degenerated to a pseudogene in some species of the subgenus Mus. We have extended this approach and investigated Tspy gene evolution in the Syrian hamster (Mesocricetus auratus) and the Mongolian gerbil (Meriones unguiculatus). Whereas the Syrian hamster Tspy is functionally conserved, organized in multiple copies, and expressed only in testis, the closely related Mongolian gerbil possesses a single-copy pseudogene that is unable to generate a functional transcript. Thus, the Tspy locus has degenerated at least twice at different points of rodent evolution, strongly supporting the hypothesis that the decay of Y-chromosomal genes is an intrinsic evolutionary process. TSPY is the first example of a Y-chromosomal tandem repetitive gene whose decay could be studied in two independent mammalian lineages.

Animals↗

Uterine carcinosarcoma: immunohistochemical studies on tissue microarrays with focus on potential therapeutic targets.

OBJECTIVE: Carcinosarcoma of the uterus is a highly aggressive tumor containing both malignant epithelial and spindle (mesenchymal) components. Because of their rarity and poor clinical outcome, investigations into the expression of potential therapeutic targets are limited. The aim of this study was to determine the expression of therapeutic targets in both the epithelial and spindle (mesenchymal) components in 30 carcinosarcomas using tissue microarrays, for potential treatment strategy. METHODS: We collected formalin-fixed, paraffin-embedded tissue blocks of carcinosarcoma of the uterine corpus resected from 30 patients who had undergone total abdominal hysterectomies at our institution between 1985 and 2005 (ages 38-83 years, mean 65.9 years). All hematoxylin-eosin stained sections from each tumor were reviewed to confirm the pathologic diagnosis. Two tissue cores from the paraffin-embedded tissue blocks were constructed into a tissue microarray. Sections were stained with monoclonal antibodies against HER-2, VEGF, c-KIT, COX-2, and EGFR. Unequivocal staining of at least 5% tumor cells was considered positive. HER-2 amplification was also examined by fluorescence in situ hybridization (FISH) in 2 cases. RESULTS: In the epithelial component, expression of HER-2, VEGF, c-KIT, COX-2, and EGFR were detected in 2 (6%), 30 (100%), 0 (0%), 21 (70%), and 9 (30%) cases, respectively, whereas these expressions in the spindle (mesenchymal) component were detected in 0 (0%), 28 (93%), 0 (0%), 5 (16%), and 20 (67%) cases, respectively. By FISH, one of the two cases with HER-2 expression showed gene amplification (2.62). CONCLUSIONS: VEGF is strongly expressed in both the epithelial and spindle (mesenchymal) components of uterine carcinosarcoma. This result warrants further study to evaluate the possible role of anti-angiogenic agents in cancer therapy for patients with uterine carcinosarcomas. The expression patterns of COX-2 and EGFR differed between the epithelial and spindle (mesenchymal) components. HER-2 and c-KIT are poor therapeutic targets for uterine carcinosarcomas.

Adult↗