Tissue Microarray Study Research Articles

Abstract PL04-02: Oral iloprost for the chemoprevention of lung cancer: A multicenter, SPORE-initiated trial

Abstract Background: Tissue microarray studies have shown that the majority of NSCLC have decreased expression of prostacyclin synthase (PGIS) and prostacyclin supplementation (genetic overexpression and the oral prostacyclin analogue iloprost) chemoprevent lung cancer in a variety of murine models, including cigarette smoke exposure. Based on these promising results, a multi-center, double-blind, placebo controlled, phase II trial of iloprost in subjects at increased risk for lung cancer conducted at SPORE in lung cancer sites. Methods: Subjects were selected for the trial if they meet the following criteria: current or former smoker (> 20 pack years); at least mild cytologic atypia on sputum cytology; no previous history of cancer. Fluorescent bronchoscopy was performed with 6 standard endobronchial sites biopsied, along with all other abnormally appearing areas. Subjects were then randomized to oral iloprost (in escalating doses) or placebo for 6 months and then a repeat bronchoscopy with biopsy of all the central airway areas sampled on the first bronchoscopy. The primary endpoints for the study are bronchial histology and Ki-67 labeling index. Results: For the iloprost study, 152 subjects were randomized and 125 completed both bronchoscopies (60/75 iloprost, 65/77 placebo). Treatment groups were well matched for age, tobacco exposure and baseline histology. Endobronchial histology was summarized within patients using three separate measures: worst biopsy score (Max), dysplasia index (DI - defined as the percentage of biopsies with a score of at least 4 (mild dysplasia) or worse), and average of all biopsy scores (Avg). 74% of subjects had at least one biopsy displaying mild dysplasia (score 4.0) or worse on the initial bronchoscopy. A reproducibility study with two independent pathologists demonstrated that 85% of readings were within one histologic grade. Baseline histology was significantly worse for current smokers (Avg 3.0) than former smokers (Avg 2.1). Former smokers receiving oral iloprost exhibited a significant improvement in Avg (0.41 better, p=0.010), Max (1.10 units, p=0.002), and an improvement in DI (12.45%, p=0.006). No histologic improvement occurred in current smokers. Proliferation by Ki-67 index was a secondary endpoint and demonstrated no change with iloprost administration. There were no differences in dropout rates or SAEs between treatment groups. Analyses for other biomarkers in iloprost samples (including urinary prostaglandin metabolites, miRNAs, and EMT markers) are in progress. Conclusions: Oral iloprost significantly improves endobronchial dysplasia in former smokers and deserves further study to determine if it can prevent the development of lung cancer. Citation Information: Cancer Prev Res 2011;4(10 Suppl):PL04-02.

The impact of the number of cores on tissue microarray studies investigating prostate cancer biomarkers

Most tissue microarray studies have used a single 0.6-mm tissue core per donor tissue. It has been suggested that multiple cores per donor can increase the representativity of tissue microarray studies. To estimate the potential benefit of multiple cores, we analyzed Ki67 and p53 in triplet cores taken from three different areas of 3,261 prostate cancer tissue blocks. Both p53 and Ki67 labeling index were linked to advanced tumor stage (p<0.0001 each), Gleason score (p<0.0001), and early PSA recurrence (p<0.0001) independently of whether the 3 tissue spots were analyzed separately or combined for a consensus result. The rate of positive findings increased with the amount of analyzed tissue. The average Ki67 labeling index was higher in tumors with 3 interpretable spots (5.3±5.6) as compared to two (4.1±4.7) or one interpretable spot (4.1±4.2, p<0.0001). For p53, tumors with three interpretable spots were positive in 3.8% of cases, and tumors with 1 or 2 interpretable spots in 1.9% only (p=0.003). These data demonstrate that using multiple cores in a tissue microarray does not necessarily increase the ability to identify associations of biomarkers with tumor phenotype and prognosis but has always the disadvantage of additional work and tissue requirements. Multiple cores may even lead to statistical problems if unequal amounts of tissue are analyzed per tumor.

Abstract B33: Transcriptional control of genome surveillance and repair by a metabolic switch

Abstract A hallmark of primitive and more aggressive breast cancer is increased genome instability due to deficits in DNA repair. Recently we described a molecular mechanism through which the NADH regulated protein C-terminal binding protein (CtBP) represses the expression of the Breast Cancer One tumor suppressor gene BRCA1. These initial observations provided a molecular mechanism through which specific aspects of genome repair and stability, mediated by BRCA1, could be controlled by cellular metabolic status. By profiling the global interactions of CtBP with the breast cancer genome via chromatin immuno-precipitation combined with deep sequencing (ChlP-seq), we now reveal that CtBP acts both as a co-transcriptional repressor and activator to integrate distinct pathways that control genome stability, epithelial to mesenchymal transition, and stem cell transcriptional programming. Breast cancer cells depleted of CtBP show increased DNA repair as detected by comet and gamma H2AX foci formation assays. Over-expression of CtBP shows loss of the CD24 luminal surface marker while CtBP depletion increases CD24 expression. Kaplan-Meier survival analysis, based on our CtBP targeted gene lists, distinguishes breast cancer patients with worst outcome/survival in several published gene expression studies. Finally, tissue micro-array studies reveal significantly higher staining of CtBP predominantly in patients with poorly differentiated, triple negative and basal-like morphologic and molecular phenotypes. We propose a model in which CtBP acts at multiple levels to enhance dedifferentiation and genomic instability in malignancies of the breast. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B33.

The expression of c-Met pathway components in unclassified pleomorphic sarcoma/malignant fibrous histiocytoma (UPS/MFH): a tissue microarray study

Subclassification of undifferentiated pleomorphic sarcoma/malignant fibrous histiocytoma (UPS/MFH) into distinct biological cohorts based on the expression patterns of molecular markers can identify patient subsets with especially unfavourable clinical outcomes. Identification of molecular prognosticators amenable for drug targeting can facilitate rational development of UPS/MFH tailored therapies. The aim was to evaluate expression of c-Met pathway components in a large cohort of UPS/MFH samples. An immunohistochemical analysis for hepatocyte growth factor (HGF), c-Met, phospho-c-Met (pc-Met), phospho-mitogen-activated protein kinase kinase (MAPKK) also known as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (p-MEK) and phospho-protein kinase B (p-AKT) was performed on a clinically annotated tissue microarray of 158 UPS/MFH samples. Univariable and multivariable analyses were conducted to evaluate the correlation of molecular variables with UPS/MFH disease specific survival. All evaluated markers were expressed in UPS/MFH to varying levels. Most importantly, strong HGF, pc-Met, p-MEK and p-AKT expression correlated significantly with dismal patient outcome on univariable statistical analysis. Expression of p-MEK and p-AKT remained statistically significant independent prognosticators on multivariable analysis. c-Met pathway components and especially p-MEK and p-AKT are potential prognostic biomarkers for UPS/MFH; their inclusion in future molecular-based staging systems should be evaluated. Furthermore, novel approaches targeting HGF, c-Met, MEK/extracellular-regulated kinase (ERK) and/or AKT should be considered for a subset of UPS/MFH patients.

Abstract B41: Molecular linkages between metabolic imbalance, genome stability, and triple-negative breast cancer

Abstract Breast cancer remains a worldwide threat to public health with over one million new cases, and nearly half a million deaths each year. Defining the mechanisms underlying the causes and risks of breast cancer remains a major challenge to global health. Though numerous population-based studies have demonstrated a indisputable link between obesity and breast cancer, the underlying molecular mechanisms and their association with elevated circulating hormones and inflammatory cytokines remain poorly defined. Similarly, the reasons why young women of African descent show a nearly two fold higher frequency of the more aggressive forms of breast cancer including the Triple Negative and basal-like phenotypes, are not understood. Recently we have defined a role for the transcriptional repressor protein CtBP in the control of proteins important for DNA repair including the early onset breast cancer gene BRCA1. In response to elevated levels of the high energy metabolic intermediate, NADH (nicotinamide adenine dinucleotide), nuclear levels of CtBP are stabilized and target a transcriptional program that impairs DNA repair in breast cancer cells. Loss of CtBP or growth under calorie restricted conditions increases the ability of breast cancer cells to repair DNA damage. Genome-wide location analysis by chromatin immuno-precipitation combined with deep sequencing (ChIP-Seq) reveals that CtBP targets gene classes that play a significant role in epithelial-to-mesenchymal transition, genome stability and stem cell transcriptional programs. Moreover, by Kaplan-Meier survival analysis of several published gene expression studies, CtBP targeted gene lists distinguish breast cancer patients with worst outcome/survival. Finally, profiling of CtBP protein expression in tissue micro-array studies of breast cancer in African American and Nigerian populations demonstrates elevated levels of CtBP expression in patients with more aggressive disease phenotypes. We explore the role of CtBP and its gene regulatory network (the “CtBP Web”) in the population-based association of breast cancer with obesity and racial/ethnic groups at higher risk for triple negative breast cancer. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B41.

MTOR–RAPTOR and 14-3-3σ immunohistochemical expression in high grade prostatic intraepithelial neoplasia and prostatic adenocarcinomas: a tissue microarray study

BackgroundThe mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase which associates with regulatory-associated protein of TOR (RAPTOR), forming the mTORC1 complex, which is necessary for activation of the...

STAT6 expression in glioblastoma promotes invasive growth

BackgroundGlioblastoma (GBM) is a highly aggressive malignant primary brain tumor, characterized by rapid growth, diffuse infiltration of cells into both adjacent and remote brain regions, and a generalized resistance to currently available treatment modalities. Recent reports in the literature suggest that Signal Transducers and Activators of Transcription (STATs) play important roles in the regulation of GBM pathophysiology.MethodsSTAT6 protein expression was analyzed by Western blotting in GBM cell lines and by immunohistochemistry in a tissue microarray (TMA) of glioma patient tissues. We utilized shRNA against STAT6 to investigate the effects of prolonged STAT6 depletion on the growth and invasion of two STAT6-positive GBM cell lines. Cell proliferation was assessed by measuring 3H-Thymidine uptake over time. Invasion was measured using an in vitro transwell assay in which cells invade through a type IV collagen matrix toward a chemoattractant (Fetal Bovine Serum). Cells were then stained and counted. Kaplan-Meyer survival curves were generated to show the correlation between STAT6 gene expression and patient survival in 343 glioma patients and in a subset of patients with only GBM. Gene expression microarray and clinical data were acquired from the Rembrandt [1] public data depository (https://caintegrator.nci.nih.gov/rembrandt/). Lastly, a genome-wide expression microarray analysis was performed to compare gene expression in wild-type GBM cells to expression in stable STAT6 knockdown clones.ResultsSTAT6 was expressed in 2 GBM cell lines, U-1242MG and U-87MG, and in normal astrocytes (NHA) but not in the U-251MG GBM cell line. In our TMA study, STAT6 immunostaining was visible in the majority of astrocytomas of all grades (I-IV) but not in normal brain tissue. In positive cells, STAT6 was localized exclusively in the nuclei over 95% of the time.STAT6-deficient GBM cells showed a reduction in 3H-Thymidine uptake compared to the wild-type. There was some variation among the different shRNA- silenced clones, but all had a reduction in 3H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt [1] gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance.ConclusionsTaken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.

Abstract 780: Immune escape in colorectal carcinoma: role of the IFN-γ pathway

Abstract The presence of a Th1 adaptive immune response in colorectal carcinoma (CRC) has been associated with improved clinical outcome and survival of patients. The Th1 adaptive immune response is mediated by IFN-γ, a known anti-proliferative, pro-apoptotic and pro-immunogenic cytokine. We have previously identified the guanylate-binding protein 1 (GBP-1), one of the proteins the most highly induced by IFN-γ, as a sensitive marker for the detection of this immune response in CRC in vivo. In a tissue microarray study with 388 CRC tumors, 30% showed GBP-1 expression. Interestingly, in these tumors the expression of GBP-1 was only induced in the cells of the desmoplastic stroma and not observed in adjacent tumor cells. This suggested that CRC tumor cells might undergo an immune escape from the Th1 adaptive immune response, characterized by the loss of IFN-γ responsiveness. Therefore, we investigated the responsiveness to IFN-γ in sixth different CRC cell lines and in normal colon epithelial cells. After treatment with IFN-γ, three of the sixth CRC cell lines (DLD-1, SW480 and HCT116) failed to express GBP-1 as well as other IFN-induced proteins such as caspase-1 and MxA. On the contrary, a robust GBP-1 induction was present in normal colon epithelial cells, in T84, WiDr and HT29 cells. We then investigated whether the loss of expression of IFN-γ target genes was associated with phenotypic changes in CRC cells. In fact, we found that DLD-1, HCT116 and SW480 were resistant to IFN-γ-induced apoptosis. Furthermore, the loss of IFN-γ responsiveness correlated in SW480 and HCT116 with the absence of expression of the IFN-γ receptor alpha chain (IFNγRα). Tumor xenotransplant experiments using DLD-1 cells with reconstituted GBP-1 expression resulted in significantly reduced tumor growth as compared to xentotransplants with control vector transfected DLD-1 cells. This indicated that GBP-1 expression may mediate the anti-tumorigenic effects of the Th1 response. Overall, these data suggested that loss of IFN-γ responsiveness may be a common event in CRC and may protect tumor cells against the anti-tumorigenic (e.g. pro-apoptotic) effects of IFN-γ. The presence of an immune escape from the Th1 adaptive immune response may have important repercussions for prognosis and patients recruitment for immune-based therapy in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 780. doi:10.1158/1538-7445.AM2011-780

Enhancement Expression of bFGF in Chinese Patients with Moyamoya Disease

Objective To detect the content of the basic fibroblast growth factor in blood samples of patients with Moyamoya disease, and investigate the relationship between Moyamoya disease and the basic fibroblast growth factor. Methods This tissue microarray study included 24 cases of superficial temporal artery samples, 15 cases of Moyamoya disease, and 9 cases of normal arteries as control, and bFGF immunofluorescence assay was applied to test the samples. The number of positive cells and total cells of the muscular layer and the endothelium layer were counted separately in every picture, the positive rates were calculated, and the experimental data were analyzed statistically. Results The bFGF immunofluorescence staining of smooth muscular layer cells, intima cells and endothelial cells from the moyamoya disease group were obviously stronger than that from the control group ( P<0.01). Conclusion The enhancement expression of bFGF in the Moyamaya disease group implicates that bFGF plays an important part in the pathogenesis of Moyamoya disease.

Abrogated expression of DEC1 during oesophageal squamous cell carcinoma progression is age- and family history-related and significantly associated with lymph node metastasis

Background:Oesophageal squamous cell carcinoma (SCC) causes the highest number of cancer deaths in some regions of Northern China. Previously, we narrowed down a critical region at 9q33-34, identified to be associated with tumour-suppressive function of deleted in oesophageal cancer 1 (DEC1) in oesophageal SCC.Methods:We generated DEC1 antibody and constructed tissue microarrays (TMAs) utilising tissue specimens from Henan, a high-risk region for oesophageal SCC, to investigate the importance of DEC1 expression in this cancer.Results:Tissue microarray immunohistochemical staining reveals significant loss of DEC1 from hyperplasia, to tumour, and to lymph node metastasis. In addition, the loss of DEC1 in tumour is age-dependent. Interestingly, there is significant abrogation of DEC1 expression in patients with a family history of oesophageal SCC. Deleted in oesophageal cancer 1 localises to both the cytoplasm and nucleus. The vesicular pattern of DEC1 in the cytoplasm appears to localise at the Golgi and Golgi–endoplasmic reticulum intermediate compartment.Conclusion:This is the first TMA study to suggest a clinical association of DEC1 in lymph node metastatic oesophageal SCC, early onset oesophageal SCC and familial oesophageal SCC development. Subcellular localisation of DEC1 and its expression in oesophageal SCC tissues provide important insight for further deciphering the molecular mechanism of DEC1 in oesophageal SCC development.

Expression and significance of FOXM1 in human cervical cancer: A tissue micro-array study

This study was designed to investigate the expression and significance of the Forkhead Box M1 (FOXM1) transcription factor in human cervical cancer. The expression of FOXM1 protein was assessed in tissue microarrays containing 102 cervical cancer tissues by the Streptavidin-Peroxidase (SP) immunohistochemistry technique. The relationship between FOXM1 protein and clinico-pathological features (pathological stages, pathological types, TNM stage) was analyzed. FOXM1 protein was located in the cytoplasma and/or nucleus. The overall expression of FOXM1 in the cytoplasm and nucleus was not associated with T stages (P=0.217) or lymph node status (P=0.313). The nuclear expression of FOXM1 protein was not associated with T stage (P=0.508) or lymph node status (P=0.345). Elevated translocation and activity of FOXM1 were discovered with a secondary analysis that showed that the differences of the nuclear expression of FOXM1, among different pathological stages, were statistically significant (P < 0.05). The nuclear expression of FOXM1 in low differential cervical cancer tissues was significantly higher than in high differential cervical cancer tissues (P < 0.05). The overexpression of FOXM1 protein in cervical cancer maybe associated with the progression of cervical cancer, and could be a potentially novel tumor marker useful for diagnosis and therapy of cervical cancer.

Role of the mTOR Pathway in the Progression and Recurrence of Bladder Cancer: An Immunohistochemical Tissue Microarray Study

PurposeNumerous trials have been conducted to develop new treatment regimens for superficial and invasive bladder cancer, because there is an urgent need to identify novel agents to prevent the recurrence and progression of these cancers. We evaluated the prognostic and biological significance of mTOR pathway-related markers in patients with bladder cancer who had undergone transurethral resection of their bladder tumors and radical cystectomy.Materials and MethodsWe retrieved 208 bladder cancer specimens collected from patients between 1989 and 2007 and constructed a tissue microarray comprising 208 tumor samples and 25 benign urothelium samples. Immunohistochemical staining was performed for mTOR, phosphorylated (phos) S6, and phos4E-BP1. The pattern, percentage, and intensity of staining for all three markers were evaluated.ResultsThe median age at diagnosis of the patient cohort was 67 years (range: 29-87 years), and the median follow-up was 72 months (range: 1-257 months). The expression of phos4E-BP1 was higher in the bladder cancer cohort than in the benign cohort, whereas phosS6 expression was lower in the bladder cancer cohort than in the benign cohort. The expression of phosS6 was significantly higher in high-grade bladder cancer (p<0.01). There was a significant positive correlation between the H-scores of mTOR and phos4E-BP1 (coefficient of correlation, r=0.37, p<0.01) as well as between the H-scores of mTOR and phosS6 (r=0.17, p<0.05). In the multivariate analysis, strong phosS6 expression predicted shorter progression (p<0.01; hazard ratio [HR], 2.516) and disease-specific survival (p<0.01; HR, 2.396) but not overall survival (p=0.112), whereas strong phos4E-BP1 expression was a predictor of disease-specific survival (p<0.05; HR, 2.105). Moreover, strong phosS6 expression predicted shorter recurrence-free (p<0.05) and progression-free (p<0.05) survival in the superficial bladder cancer cohort.ConclusionsOur results demonstrate that mTOR pathway activation, as assessed by phos4E-BP1 phosphorylation, is related to bladder cancer tumorigenesis and that S6 protein phosphorylation is associated with a high level of disease recurrence and progression and poor cancer-specific survival.

Determining the Optimal Numbers of Cores Based on Tissue Microarray Antibody Assessment in Non-Small Cell Lung Cancer

Background: Tissue microarrays (TMAs) have been commonly utilized in translational research to rapidly screen numerous biomarkers in large samples. One major concern has been the adequate assessment of biomarkers affected by within-tumor heterogeneity and by molecular targets. Methods: Our study was designed to answer a fundamental question: How do researchers define the optimal cores to sample when designing a TMA study to minimize sampling bias and core artifact? We compared the staining results from a full-section tissue slide to the virtual TMA and from the actual TMA to the virtual TMA. Results: Three cores were demonstrated as optimal for markers such as TTF-1 and p53, but no optimal core number could be determined for markers such as Ki-67 due to the poor TMA representation of the entire tissue. Conclusion: We propose that before using TMAs to analyze large samples, particularly with significant withinsample heterogeneity, a preliminary investigation using a virtual TMA could help decide target markers to be tested for valid and valued results.

XB130, a Novel Adaptor Protein, Promotes Thyroid Tumor Growth

Adaptor proteins with multimodular structures can participate in the regulation of various cellular functions. We have cloned a novel adaptor protein, XB130, which binds the p85α subunit of phosphatidyl inositol 3-kinase and subsequently mediates signaling through RET/PTC in TPC-1 thyroid cancer cells. In the present study, we sought to determine the role of XB130 in the tumorigenesis in vivo and in related molecular mechanisms. In WRO thyroid cancer cells, knockdown of XB130 using small interfering RNA inhibited G(1)-S phase progression, induced spontaneous apoptosis, and enhanced intrinsic and extrinsic apoptotic stimulus-induced cell death. Growth of tumors in nude mice formed from XB130 shRNA stably transfected WRO cells were significantly reduced, with decreased cell proliferation and increased apoptosis. Microarray analysis identified 246 genes significantly changed in XB130 shRNA transfected cells. Among them, 57 genes are related to cell proliferation or survival, including many transcription regulators. Ingenuity Pathway Analysis showed that the top-ranked disease related to XB130 is cancer, and the top molecular and cellular functions are cellular growth and proliferation and cell cycle. A human thyroid tissue microarray study identified expression of XB130 in normal thyroid tissue as well as in human thyroid carcinomas. These observations suggest that the expression of XB130 in these cancer cells may affect cell proliferation and survival by controlling the expression of multiple genes, especially transcription regulators.

Abstract A56: Induction of spermine oxidase by enterotoxigenic Bacteroides fragilis contributes to tumorigenesis in a model of colitis-associated colorectal cancer

Abstract It is estimated that the etiology of 20-30% of epithelial cancers is directly associated with inflammation, though the molecular events linking inflammation and the necessary carcinogenic mutations remain to be clarified. In the context of gastrointestinal disease, the bacterial pathogens Helicobacter pylori and enterotoxigenic Bacteroides fragilis (ETBF) are significant sources of chronic inflammation and have been implicated as risk factors for the development of gastric and colorectal cancer, respectively. We have previously demonstrated that H. pylori and the cytokine tumor necrosis factor-α (TNF-α) rapidly induce spermine oxidase (SMO), resulting in increased cellular reactive oxygen species (ROS) and DNA damage, in gastric and lung epithelial cells. In addition, tissue microarray studies revealed that increased SMO expression was associated with prostatic intraepithelial neoplasia and prostate cancer lesions and that benign prostate epithelial tissue from men with these diseases exhibits higher SMO expression than normal controls. We now demonstrate that purified B. fragilis toxin (BFT) upregulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in wild-type or APC+/− Min C57Bl/6 mice is associated with increased SMO expression. Inhibition of SMO activity by MDL 72,527 in vivo (20 mg/kg i.p. 3 days per week) significantly reduces ETBF-induced chronic inflammation, proliferation, and induction of Th17-associated cytokines. Most importantly, in Min mice, treatment with the SMO inhibitor reduces ETBF-induced colon tumorigenesis by 69% (p&amp;lt;.001). Our studies suggest that SMO is a novel target for chemopreventive or chemotherapeutic drug development. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A56.

The HER2 gene copies per tumor cell either before or after correction for chromosome-17 correlated significantly with HER2 IHC results in epithelial ovarian cancer in a tissue microarray study

HER2 gene amplification and HER2 protein overexpression are important factors in predicting clinical sensitivity to anti-HER2 monoclonal antibody therapy in breast cancer patients. The purpose of this study is to evaluate the correlation between HER2 protein expressions and the HER2 gene copies per tumor cell either before or after chromosome-17 correction in epithelial ovarian cancer (EOC). Adopting 2007 ASCO/CAP guideline recommendations for HER2 testing, 27 tissue microarray (TMA) samples from EOC patients were analyzed by immunohistochemistry (IHC) using Dako, c-erb-B2 antibody and subsequently examined by fluorescence in situ hybridization (FISH) using Abbott/Vysis, PathVysion HER2 DNA Probe Kit. The overall concordance revealed 81.5% between HER2 IHC and HER2 FISH results. Additionally, HER2 gene copies prior to chromosome-17 correction increased significantly in a stepwise order through the negative, equivocal, and positive IHC result categories (P = 0.026), as did the HER2 gene copies after chromosome-17 correction (P = 0.028). On the other hand, HER2 IHC results correlated significantly with both chromosome-17 uncorrected HER2 gene copy numbers (ρ = 0.430, P = 0.025) and chromosome-17 corrected HER2 gene copy numbers (ρ = 0.524, P = 0.027). We demonstrated that both chromosome-17 corrected and uncorrected HER2 gene copies correlated significantly with HER2 IHC result categories; and tests for the HER2 gene copies per tumor cell either before or after correction for chromosome-17 can be applied as a potentially valuable tool in analyzing the HER2 status in EOC.

A Tissue Microarray Study of Osteosarcoma: Histopathologic and Immunohistochemical Validation of Xenotransplanted Tumors as Preclinical Models

Osteosarcomas (OS) are aggressive neoplasms with a wide range of morphologic patterns. OS cases (primary and xenotransplanted) with paraffin blocks available were collected and included in tissue microarrays (TMAs). A morphologic evaluation including the different passages in mice was carried out according to the new WHO criteria. In addition, TMAs were analyzed with a wide panel of immunohistochemical (IHC) markers (osteonectin, osteocalcin,cytokeratin, S100, Sox-9, Ki-67, Bcl-2, p53, p16, survivin, CD99, and caveolin-1). A total of 61 cases were collected. The distribution of the cases according to the histopathologic pattern was: 38 osteogenic OS, 8 primary chondrogenic OS, 2 primary telangiectatic OS, 6 parosteal OS, 2 primary small cell OS, 2 primary poorly differentiated OS, 1 primary dedifferentiated OS, and 3 primary pleomorphic MFH-like OS. The tumor morphology in xenotransplants was similar to the primary or metastatic tumor of origin and was generally maintained over the passages. The IHC results were heterogeneous and osteonectin and osteocalcin were the most expressed in original tumor and xenografts. S100 and Sox-9 were expressed in chondrogenic areas. Caveolin and survivin showed significant IHC variation between the subsequent passages. p16 displayed heterogenic expression. p53 expression increased over the passages, and Ki-67 expression was not associated with a more undifferentiated pattern, but increased over the passages. An accurate morphologic evaluation using TMAs in original tumor is essential for the OS diagnosis; hence there is no IHC marker that alone distinguishes the OS subtypes. Xenografts in OS allow the study of tumor progression in this type of aggressive neoplasm.

BRCA1 Basal-like Breast Cancers Originate from Luminal Epithelial Progenitors and Not from Basal Stem Cells

Breast cancers in BRCA1 mutation carriers frequently have a distinctive basal-like phenotype. It has been suggested that this results from an origin in basal breast epithelial stem cells. Here, we demonstrate that deleting Brca1 in mouse mammary epithelial luminal progenitors produces tumors that phenocopy human BRCA1 breast cancers. They also resemble the majority of sporadic basal-like breast tumors. However, directing Brca1 deficiency to basal cells generates tumors that express molecular markers of basal breast cancers but do not histologically resemble either human BRCA1 or the majority of sporadic basal-like breast tumors. These findings support a derivation of the majority of human BRCA1-associated and sporadic basal-like tumors from luminal progenitors rather than from basal stem cells. They also demonstrate that when target cells for transformation have the potential for phenotypic plasticity, tumor phenotypes may not directly reflect histogenesis. This has important implications for cancer prevention strategies.

Immunohistochemical examination of the mTORC1 pathway in high grade prostatic intraepithelial neoplasia (HGPIN) and prostatic adenocarcinomas (PCa): A tissue microarray study (TMA)

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and a key regulator of protein synthesis and growth and is upregulated in many cancers. mTOR is activated by AKT phosphorylation (p-mTOR). p-mTOR associates with regulatory-associated protein of TOR (RAPTOR), forming the mTORC1 complex. mTORC1 promotes the activation of p70 ribosomal protein s6 kinase 1 (p70(S6K1)) and ribosomal protein s6 (RPS6). Upregulation of this pathway can lead to an aberrant increase in cell growth and metabolism characteristic of malignant transformation. This study presents the immunohistochemical (IHC) expression of the mTORC1 pathway in prostate neoplasia. The expression of p-mTOR and RAPTOR and p-p70(S6K1) and p-RPS6 were examined in HGPIN and PCa using tissue microarrays (TMA). Since each case in our TMAs was represented by three tissue cores, we quantified the IHC intratumoral heterogeneity of mTOR expression. This extensive analysis is the first detailed assessment documenting the IHC heterogeneity of mTOR expression in HGPIN and prostate cancer and represents the first IHC description of the mTORC1 pathway in HGPIN and PCa. A Cochran-Armitage analysis demonstrated decreasing p-mTOR activity progressing from PIN through GL6 and GL7 to HG PCa. There was considerable intratumoral IHC heterogeneity within an individual patient. However, a statistically significant correlation was observed between p-mTOR, p-p70(S6K1), and p-RPS6 in each representative core. mTOR inhibitors may be an effective treatment for HGPIN and PCa. The extent of mTOR expression in an individual patient would determine the effective use of mTOR inhibitors as a potential therapeutic strategy.

The immunohistochemical expression of BNIP3 protein in non‐small‐cell lung cancer: a tissue microarray study

Drug resistance is one of the reasons for chemotherapy failure in non-small-cell lung carcinoma (NSCLC). One of the major mechanisms of drug resistance is the inhibition of chemotherapy-induced apoptosis. Therefore, the study of novel cell death pathways could possibly enable us to overcome resistance to apoptosis in NSCLC. One of the non-caspase types of cell death is autophagy. BNIP3 protein, a Bcl-2 family member, highly expressed in some tumours, plays a key role in the induction of autophagy. In the present study, we investigated the immunohistochemical expression and subcellular localization of BNIP3 in a series of early- and late-stage non-small-cell lung carcinomas and normal bronchial tissues, and correlated this expression with the occurrence of metastasis and survival. BNIP3 was strongly expressed in the nucleus of cancer cells in 16/79 (20.3%) cases. This BNIP3 positivity did not correlate with histological grade, stage, histology type, metastatic potential, or expression of BNIP3 according to median values. No significant correlation was observed between the expression of BNIP3 and the overall survival of NSCLC patients (p = 0.55). Nor did we find any significant correlation between BNIP3 expression and the occurrence of site-specific metastasis (p = 0.85).