High-throughput Tissue Microarray Research Articles

Pictilisib Enhances the Antitumor Effect of Doxorubicin and Prevents Tumor-Mediated Bone Destruction by Blockade of PI3K/AKT Pathway.

Despite advances in neoadjuvant chemotherapy, outcomes for patients with osteosarcoma resistant to first-line chemotherapy have been dismal for decades. There is thus an urgent need to develop novel targeted drugs to effectively treat refractory osteosarcoma. Dysregulation in the PI3K/AKT pathway has been observed during the development of osteosarcoma. Herein, we first evaluated p-AKT (Ser473) expression levels in osteosarcoma tissue using high-throughput tissue microarrays. Then, we demonstrated the role of pictilisib, a novel potent PI3K inhibitor, in osteosarcoma and related osteolysis. Functional studies of pictilisib in osteosarcoma cell lines and bone marrow-derived macrophages were performed in vitro. Patient-derived xenografts and orthotopic mouse models were used to assess the effects of pictilisib in vivo. The results showed that positive p-AKT expression levels after neoadjuvant chemotherapy were significantly associated with tumor cell necrosis rate. Pictilisib effectively inhibited the proliferation of osteosarcoma through G0/G1-S phase cell cycle arrest, and enhanced the sensitivity of osteosarcoma to doxorubicin, although it failed to induce cell apoptosis alone. In addition, pictilisib inhibited differentiation of osteoclasts and bone resorption in vitro and tumor-related osteolysis in vivo via inhibition of the PI3K/AKT/GSK3β and NF-κB pathways. Pictilisib combined with conventional chemotherapy drugs represents a potential treatment strategy to suppress tumor growth and bone destruction in p-AKT-positive patients.

Classification of colorectal tissue images from high throughput tissue microarrays by ensemble deep learning methods

Tissue microarray (TMA) core images are a treasure trove for artificial intelligence applications. However, a common problem of TMAs is multiple sectioning, which can change the content of the intended tissue core and requires re-labelling. Here, we investigate different ensemble methods for colorectal tissue classification using high-throughput TMAs. Hematoxylin and Eosin (H&E) core images of 0.6 mm or 1.0 mm diameter from three international cohorts were extracted from 54 digital slides (n = 15,150 cores). After TMA core extraction and color enhancement, five different flows of independent and ensemble deep learning were applied. Training and testing data with 2144 and 13,006 cores included three classes: tumor, normal or “other” tissue. Ground-truth data were collected from 30 ngTMA slides (n = 8689 cores). A test augmentation is applied to reduce the uncertain prediction. Predictive accuracy of the best method, namely Soft Voting Ensemble of one VGG and one CapsNet models was 0.982, 0.947 and 0.939 for normal, “other” and tumor, which outperformed to independent or ensemble learning with one base-estimator. Our high-accuracy algorithm for colorectal tissue classification in high-throughput TMAs is amenable to images from different institutions, core sizes and stain intensity. It helps to reduce error in TMA core evaluations with previously given labels.

Cancer-testis antigen lactate dehydrogenase C4 in hepatocellular carcinoma: a promising biomarker for early diagnosis, efficacy evaluation and prognosis prediction.

Expressions and clinical implications of cancer-testis antigen (CTA) lactate dehydrogenase (LDH)-C4 in hepatocellular carcinoma (HCC) have not been fully elucidated. Herein, expressions of LDHC mRNA in the serum and serum-derived exosomes of early-stage HCC patients were determined using qRT-PCR, and the expression of LDH-C4 protein in HCC tissues was detected using high-throughput tissue microarray analysis. It was found that positive rates of LDHC mRNA expressions in the serum and serum exosomes of HCC patients were 68% and 60%, respectively. The AUCs of serum and exosomal LDHC in differentiating HCC patients from healthy controls were 0.8382 and 0.9451, respectively. The serum and exosomal LDHC levels in HCC patients in the treatment group were higher than the levels in the preliminary diagnosis group, but lower than those in the recurrence group. Survival analysis showed that the expression of LDH-C4 was negatively correlated with the prognosis of HCC. The Cox regression analysis showed that an LDH-C4 level was an independent risk factor for the prognosis of HCC patients. Therefore, serum and exosomal LDHC can be used as a biomarker for early diagnosis, efficacy evaluation and recurrence prediction of HCC. Moreover, LDH-C4 can be used as an important reference indicator for monitoring the prognosis of HCC.

Diagnostic and prognostic value of the cancer-testis antigen lactate dehydrogenase C4 in breast cancer

BackgroundLactate dehydrogenase C4 (LDH-C4) as a cancer/testis antigen (CTA) is abnormally expressed in some malignant tumors. However, the expression and clinical significance of LDH-C4 in breast cancer (BC) has not been characterized. MethodsWe determined LDHC mRNA expression in serum and serum-derived exosomes of BC patients by quantitative RT-PCR. We also evaluated the protein expression of LDH-C4 in BC tissues using high-throughput tissue microarray analysis and immunohistochemistry. ResultsOur results showed high mRNA expression level of LDHC in serum and serum-derived exosomes of BC patients. The LDHC level in serum and exosomes could distinguish BC cases from healthy individuals based on their AUCs of 0.9587 and 0.9464, respectively. Besides, the LDHC level in exosomes of BC patients associated with tumor size, and positively correlated with HER2 and Ki-67 expressions (all with P < 0.05). Serum and exosomal level of LDHC negatively correlated with medical treatment and positively with the recurrence of BC. Survival analysis showed that LDH-C4 expression negatively correlated with BC prognosis. ConclusionSerum and exosomal LDHC may be an effective indicator for the diagnosis, efficacy evaluation, and monitoring the recurrence of BC. LDH-C4 may act as a biomarker that predicts BC prognosis.

Immunohistochemical Study of NR2C2, BTG2, TBX19, and CDK2 Expression in 31 Paired Primary/Recurrent Nonfunctioning Pituitary Adenomas

This study investigated potential markers for predicting nonfunctioning pituitary adenoma (NFPA) invasion and recurrence by high-throughput tissue microarray analyses. We retrospectively studied two groups of patients: 60 nonrecurrent NFPA cases that included noninvasion and invasion subtypes and 43 recurrent cases that included primary NFPA. A total of 31 paired patient samples were evaluated (12 patients with one surgery and 31 who had undergone two operations, with both tumors analyzed). Expressions of nuclear receptor subfamily 2 group C member 2 (NR2C2), B cell translocation gene 2, T-box-19 (TBX19), and cyclin-dependent kinase 2 (CDK2) in surgically resected specimens were assessed by immunohistochemistry. The relationships between marker expression and clinical characteristics including age, sex, tumor volume, and follow-up time were analyzed. Tumor volume and invasion as well as follow-up time were significantly associated with invasion and recurrence (P < 0.01). Of the 60 nonrecurrent samples, 15/41 and 13/19 showed high NR2C2 expression in the noninvasion and invasion groups, respectively (χ2 =5.287, P = 0.021). NR2C2 was also overexpressed in 43 primary recurrent cases (χ2 =5.433, P = 0.02), whereas CDK2 (χ2 = 11.242, P = 0.001) and TBX19 (χ2 = 4.875, P = 0.027) were downregulated. In the 31 paired samples, NR2C2 was more highly expressed in the recurrent as compared to the primary tumor. High NR2C2 expression was associated with NFPA invasion, recurrence, and progression, while TBX19 and CDK2 were associated with NFPA recurrence.

The correlation of clinicopathological features and prognosis in extranodal natural killer/T cell lymphoma: a report of 42 cases in the early stage.

This study aimed to explore the clinicopathological features and prognostic correlation of extranodal natural killer (NK)/T cell lymphoma (ENKTCL) in the early stage, screen out the prognostic markers of ENKTCL, and to establish the molecular model of ENKTCL prognosis. A retrospective study was conducted in 88 patients from May 1999 to Dec 2013 in Chinese Academy of Medical Sciences Cancer Hospital, who were diagnosed with ENKTCL according to WHO lymphoid hematopoietic tumor classification (published in 2008). The clinical data and paraffin-embedded tissue blocks were collected. The expressions of CD56, MLH1, PDGFRA, VEGF, PD-L1, PD-1, CyclinD1, p53, and Ki-67 were detected by high-throughput tissue microarray and immunohistochemistry (IHC) staining. The relationship between nine protein expressions and the clinicopathological features and prognosis of patients with ENKTCL were analyzed. The survival time of the 42 patients with complete clinical and follow-up data was 0~153months. The average survival time was 60.1months. The survival rates of 1year, 2years, and 3year were 85.7%, 78.6%, and 71.4%, respectively. Single factor survival analysis showed that the increase of serum lactate dehydrogenase (LDH ≥ 240UI/L) before treatment was associated with poor prognosis, and there was a significant difference in survival rate (P = 0.006). Different therapy methods were related with prognosis (P = 0.011); in specifically, radiotherapy alone had the best treatment effect, followed by concurrent chemoradiotherapy, and the worst was chemotherapy alone. But, multivariate statistics indicated that the LDH level and the treatment approach were not independent prognostic factors of ENKTCL. There was no statistical difference between epidemiological factors such as gender, age, and other clinicopathological factors including tumor location, B symptoms, β2-microglobulin levels before treatment, and prognosis. Survival analysis of single factor showed that the positive expression of PDGFRA and PD-L1 was, respectively, related to the poor prognosis of patients with ENKTCL (P = 0.040, 0.007). The patients with Ki-67 overexpression (≥ 50%) had a worse prognosis than those with lower expression (< 50%), and the difference of survival rate between the two groups has statistical significance (P = 0.038). The expression of CD56, MLH1, VEGF, PD-1, CyclinD1, and p53 has no effect on survival rate (P > 0.05). Multivariate survival analysis showed that the expression levels of PDGFRA, PD-L1, and Ki-67 were independent factors in the prognosis of patients with ENKTCL. And the positive expressions of these three proteins were risk factors for prognosis of patients with ENKTCL (PDGFRA: P = 0.045, HR = 8.265, 95% CI: 1.050-65.054; PD-L1: P = 0.005, HR = 9.369, 95% CI: 1.950-45.003; Ki-67: P = 0.023, HR = 3.545, 95% CI: 1.187-10.585). The elevation of serum lactate dehydrogenase (LDH ≥ 240UI/L) before treatment and the treatment approach were associated with poor prognosis, which could be used as adjunct indexes to the prognosis. However, they were not independent factors for the prognosis of patients with ENKTCL. The expressions of PDGFRA, PD-L1, and Ki-67 were independent factors in the prognosis of patients with ENKTCL and these three proteins were risk factors of prognosis. The above markers combined with clinical factors may establish the prognosis model of ENKTCL.

Association of TGF-β1 and WIF1 Expression with 36 Paired Primary/Recurrent Nonfunctioning Pituitary Adenomas: A High-Throughput Tissue Microarrays Immunohistochemical Study

This study was undertaken primarily to research transforming growth factor β1 (TGF-β1) and Wnt inhibitory factor 1 (WIF1) for the prediction of nonfunctioning pituitary adenoma (NFPAs) invasion and recurrence of tumor samples and the relations between quantitatively determined markers and clinical characters. We studied 104 patients, including 59 patients without recurrence and 45 patients with recurrence (9 patients with one surgery and 36 patients operated twice, both tumors being studied). All tissues were immunostained for TGF-β1 and WIF1 using tissue microarrays and confirmed with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. We found that invasion, TGF-β1, and WIF1 were significantly associated with recurrence and that age was associated with low expression of TGF-β1 and WIF1 (P < 0.001). There were no statistically significant differences in the expression of the 2 proteins between the noninvasive and the invasive groups. The expression of TGF-β1 and WIF1 in primary tumors in the recurrence group was lower than in the nonrecurrence group (P < 0.001). In the 36 paired primary or recurrent tumors, the expression of TGF-β1 and WIF1 in recurrent tumors was higher than the expression of primary tumors, which was confirmed with qRT-PCR and Western blot. Therefore, TGF-β1 and WIF1 seem to be related to recurrence or progression of pituitary adenomas. The expression of TGF-β1 and WIF1 in NFPAs correlated with cell proliferation and recurrence potential. They may be good markers of progressive behavior in NFPAs; however, the biologic mechanism needs further study.

NKX2.2, PDX-1 and CDX-2 as potential biomarkers to differentiate well-differentiated neuroendocrine tumors

BackgroundWell-differentiated neuroendocrine tumors (NET) most frequently arise from the gastrointestinal tract (GI), pancreas, and lung. Patients often present as metastasis with an unknown primary, and the clinical management and outcome depend on multiple factors, including the accurate diagnosis with the tumor primary site. Determining the site of the NET with unknown primary remains challenging. Many biomarkers have been investigated in primary NETs and metastatic NETs, with heterogeneous sensitivity and specificity observed.MethodsWe used high-throughput tissue microarray (TMA) and immunohistochemistry (IHC) with antibodies against a panel of transcriptional factors including NKX2.2, PDX-1, PTF1A, and CDX-2 on archived formalin-fixed paraffin-embedded NETs, and investigated the protein expression pattern of these transcription factors in 109 primary GI (N = 81), pancreatic (N = 17), and lung (N = 11) NETs.ResultsDifferential expression pattern of these markers was observed. In the GI and pancreatic NETs (N = 98), NKX2.2, PDX-1, and CDX-2 were immunoreactive in 82 (84%), 14 (14%), and 52 (52%) cases, respectively. PDX-1 was expressed mainly in the small intestinal and appendiceal NETs, occasionally in the pancreatic NETs, and not in the colorectal NETs. All three biomarkers including NKX2.2, PDX-1, and CDX-2 were completely negative in lung NETs. PTF1A was expressed in all normal and neuroendocrine tumor cells.ConclusionsOur findings suggest that NKX2.2 was a sensitive and specific biomarker for the GI and pancreatic neuroendocrine tumors. We proposed that a panel of immunostains including NKX2.2, PDX-1, and CDX-2 may show diagnostic utility for the most common NETs.

Molecular similarities and differences from human pulmonary fibrosis and corresponding mouse model: MALDI imaging mass spectrometry in comparative medicine

Animal models can reproduce some model-specific aspects of human diseases, but some animal models translate poorly or fail to translate to the corresponding human disease. Here, we develop a strategy to systematically compare human and mouse tissues, and conduct a proof-of-concept experiment to identify molecular similarities and differences using patients with idiopathic pulmonary fibrosis and a bleomycin-induced fibrosis mouse model. Our novel approach employs high-throughput tissue microarrays (TMAs) of humans and mice, high-resolution matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance-mass spectrometry imaging (MALDI-FT-ICR-MSI) to spatially resolve mass spectra at the level of specific metabolites, and hierarchical clustering and pathway enrichment analysis to identify functionally similar/different molecular patterns and pathways in pathological lesions of humans and mice. We identified a large number of common molecules (n=1366) and fewer exclusive molecules in humans (n=83) and mice (n=54). Among the common molecules, the ‘ascorbate and aldarate metabolism' pathway had the highest similarity in human and mouse lesions. This proof-of-concept study demonstrates that our novel strategy employing a reliable and easy-to-perform experimental design accurately identifies pathways and factors that can be directly compared between animal models and human diseases.

Evaluation of P-glycoprotein (Pgp) expression in human osteosarcoma by high-throughput tissue microarray.

Survival of osteosarcoma patients is currently limited by the development of metastases and multidrug resistance (MDR). A well-established cause of MDR involves overexpression of P-glycoprotein (Pgp) in tumor cells. However, some discrepancies still exist as to the clinical significance of Pgp in osteosarcoma. We sought to elucidate further whether the Pgp expression correlated with clinical behavior in a series of patients with osteosarcoma via high-throughput tissue microarray (TMA) analysis. Immunohistochemical analysis of Pgp expression in a TMA of 114 specimens with a retrospective review of 70 osteosarcoma patients admitted to the Massachusetts General Hospital (MGH) was performed. High Pgp expression was correlated with metastasis development and poor response to pre-operative chemotherapy in osteosarcoma patients. Eighteen of the fifty-seven patients initially admitted with primary osteosarcoma showed high Pgp expression. Among these 18 patients with high Pgp expression, 13 of 18 (72%) patients eventually developed metastases. There was no significant clinical relevance between Pgp expression and osteosarcoma survival. These results support that high expression of Pgp is important, but cannot be assigned as, an individual predictor in the development of human osteosarcoma. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1606-1612, 2016.

Immunohistochemical expression of chemokine receptor CXCR3 and its ligand CXCL10 in low-grade astrocytomas and glioblastoma multiforme: A tissue microarray-based comparison.

Glioblastoma multiforme (GBM) and diffuse astrocytoma (DA) are the most frequently encountered gliomas. Due to poor prognosis and limited success of the currently available treatment modalities there is a need to identify new therapeutic targets. Chemokines (CKs) regulate cellular functions like chemotaxis, angiogenesis, apoptosis, and cell cycle progression that play role in tumor growth. To study comparative immunoexpression of CXCR3 and CXCL10 in DA and GBM using a high-throughput tissue microarray (TMA). A TMA of 1.0 mm core diameter was made from formalin-fixed, paraffin-embedded donor blocks of 25 pilocytic astrocytomas (PA), 45 DA, and 75 GBM. Immunohistochemical staining for CXCR3 and CXCL10 was performed. Out of 145, 129 cores were suitable for immunohistochemical evaluation after processing and immunohistochemistry. Strong CXCR3 immunoexpression was observed in 72.7% cases of GBM as compared to 31.8% cases of DA. 50.7% of GBM and 24.5% of DA showed strong immunoexpression of CXCL10. Overall comparisons between DA and GBM for CXCR3 and CXCL10 showed statistically significant correlation between the two with P < 0.001 and P = 0.02, respectively. A positive correlation was observed between CXCR3 and MIB-1. Pearson's correlation coefficient r = 0.548 and 0.330 for DA and GBM, respectively with P < 0.01. GBM shows overexpression of CXCR3 and CXCL10 in comparison to DA, indicating that they play an important role in tumor growth and progression. Inhibition of this receptor-ligand axis may be a potential therapeutic target for arresting tumor growth and development of a glioblastoma.

Expression of metallothionein protein relating to proliferative cell index in malignant feline mammary tumors using high throughput tissue microarray technique

The present study aimed to examine the relationship between proliferation markers by detecting Ki-67 and proliferative cell nuclear antigen (PCNA) indices, and metallothionein expression, in different histological groups and nuclear grades of malignant feline mammary tumors by using immunohistochemistry and tissue microarray technique. Seventy feline mammary carcinoma (FMC) biopsy specimens were collected to perform tissue microarray (TMA) slide. The immunohistochemical staining was performed on the TMA slides against pancytokeratin (CK19), PCNA, Ki-67 clone MIB, and metallothionein 1 (MT-1) antibodies. The PCNA index showed no significant differences among histological types and nuclear grades, whereas the Ki-67 index showed statistical difference among histological types and malignancy grades (p < 0.05). The pattern of cytoplasmic MT expression was observed in glandular carcinoma cells. The percentage ± SD of MT expression was 44.13 ± 30.24 in tubular adenocarcinomas, 39.29 ± 26.21 in papillary adenocarcinomas, 22.66 ± 17.81 in cribriform carcinomas, and 38.12 ± 30.33 in solid carcinomas. The percentage ± SD of MT expression was 32.68 ± 27.59 in grade I, 37.34 ± 26.37 in grade II, and 43.54 ± 32.97 in grade III. Our study showed that the expression of MT increased with nuclear grade rather than histological subtype. It is suggested that the role of MT in carcinogenesis and tumor progression could be linked to involvement of MT in processes of cell proliferation and differentiation of feline mammary carcinomas.

Fucosyltransferase 8 expression in breast cancer patients: A high throughput tissue microarray analysis.

The aim of this study was to compare the expression of fucosyltransferase 8 (FUT8) in breast cancer tissue and to investigate the relationship between this marker with tumor progression and its applicability to differential diagnosis. An immunohistochemical study was performed for FUT8 using the tissue microarray technique. In addition, the mRNA and protein levels of FUT8 in the tissue were also tested by real-time PCR and Western blot. There was a significant difference in cytoplasmic expression of FUT8 between breast cancer tissue and matched normal tissue (p<0.001). The percent of FUT8 staining in breast cancer tissues ranging from negative, weak positive, positive and strong positive were 2.7%, 40.2%, 54% and 3.2%, respectively. High FUT8 protein expression correlated with lymphatic metastasis (p=0.008) and with stage status (p=0.039). We detected that reduced FUT8 expression correlated with disease-free survival (p=0.02) and overall survival (p=0.04) of breast cancer patients. Expression of FUT8 can stratify breast cancer tissue and may be considered a prognostic marker for breast cancer patients.

Novel biomarker candidates for the diagnosis of ovarian clear cell carcinoma.

Ovarian clear cell carcinoma can arise from endometriosis; however, it is distinct from other types of epithelial ovarian carcinoma in terms of its clinicopathological and molecular features. Cancer antigen 125 lacks the sensitivity and specificity required for accurate clinical diagnosis of clear cell carcinoma. Therefore, the aim of the current review was to identify novel biomarker candidates for the immunohistochemical and serological diagnosis of clear cell carcinoma. A search of the relevant English language literature published between 1966 and 2014 was conducted using the PubMed MEDLINE online database. High-throughput tissue microarray technology and proteomic screening combined with mass spectrometry may provide additional information regarding diagnostic biomarker candidates for ovarian clear cell carcinoma. The present review summarizes the characteristics of potential genomic alterations that activate cancer signaling pathways and, thus, contribute to carcinogenesis. The major signaling pathways activated in clear cell carcinoma are associated with cell cycle regulation (hepatitis A virus cellular receptor 1 and tumor protein D52), growth factor signaling (insulin-like growth factor binding protein 1; KiSS-1 metastasis-suppressor; erb-b2 receptor tyrosine kinase 2; and fibroblast growth factor receptor 2), anti-apoptosis and survival pathways [sialidase 3 (membrane sialidase)], metabolism (γ-glutamyltransferase 1), chemoresistance (napsin A aspartic peptidase, glutathione peroxidase 3; and aldehyde dehydrogenase 1 family, member A1), coagulation [coagulation factor III (thromboplastin, tissue factor); and tissue factor pathway inhibitor 2], signaling (lectin, galactoside-binding and soluble, 3), and adhesion and the extracellular matrix [cadherin 1, type 1, E-cadherin (epithelial); versican; and laminin, α 5]. The present review of the relevant literature may provide a basis for additional clinical investigation of the ovarian clear cell carcinoma serum biomarker candidate proteins identified herein.

Automated Quantitative Analysis of Tissue Microarray of 443 Patients with Colorectal Adenocarcinoma: Low Expression of Bcl-2 Predicts Poor Survival

Bcl-2 has been implicated in the development and progression of a number of cancers including colorectal cancer. Reports of Bcl-2 expression in colorectal cancer and patient outcomes have been inconsistent due to small cohorts and semi-quantitative grading methods. We used a high throughput tissue microarray system (automated quantitative analysis [AQUA]), analyzing colorectal adenocarcinoma samples from 443 patients resected during the period of 1967 to 1986. This system uses fully quantitative, automated fluorescent microscopy to accurately assess Bcl-2 expression in colorectal cancer samples. Clinicopathologic variables were collected prospectively and were assessed using log-rank tests and Cox proportional hazards models. At a median follow-up of 54 months, the 5- and 10-year disease-specific survivals for all patients were 59.2% and 52.1%, respectively. Loss of Bcl-2 expression was seen in 70.4% of tumors and was associated with a decreased 5-year disease-specific survival (55.8% vs 75.6%, p= 0.001 and relative risk [RR] 1.8) and decreased 5-year overall survival (45.8% vs 56.5%, p= 0.046 and RR 1.2). On univariate analysis, T stage, N stage, and loss of Bcl-2 expression predicted poor disease-specific survival. On multivariate analysis, Bcl-2 expression was an independent prognostic factor for disease-specific survival (p= 0.034). Our results indicate that loss of Bcl-2 expression in colorectal cancer is associated with decreased disease-specific and overall survival. This finding may help identify a subset of patients with a more aggressive phenotype and guide adjuvant chemotherapy choices.

Identification of candidate biomarkers for the early detection of nasopharyngeal carcinoma by quantitative proteomic analysis.

NPC is a lethal malignancy that is most prevalent in Southeast Asia, and early detection and treatment are essential for the survival and good prognosis of NPC patients. In the present work, we identified 26 differentially expressed proteins in NNET, AH and different stages of NPC tissues by using 2D-DIGE combined with MALDI-TOF/TOF analysis. Of these proteins, the down-regulation of ENO1 and over-expression of CYPA were confirmed with a high-throughput tissue microarray that included various stages of NPC tissues via an IHC assay, and the results indicated that the combination of ENO1 and CYPA can serve as a potential molecular marker for the early detection of NPC.

Potential Significance of EMP3 in Patients with Upper Urinary Tract Urothelial Carcinoma: Crosstalk with ErbB2-PI3K-Akt Pathway

Upper urinary tract (pyelocalyceal cavities and ureter) urothelial carcinoma is a relatively rare neoplastic disease. Although diagnosis and treatment of this tumor variant have improved significantly, accurate risk stratification remains a challenge. To identify the putative oncogene involved in urothelial carcinoma progression we performed bioinformatics guided experimental investigation targeting chromosome 19q13. We investigated the effects of EMP3 on cancer cell growth, migration and adhesion in transfection and siRNA experiments in vitro. Crosstalk of integrins or ErbB2 with EMP3 was examined by reverse transcriptase-polymerase chain reaction and immunoblot. The potential involvement of epigenetic alterations of EMP3 in vitro and in vivo was analyzed by methylation specific polymerase chain reaction. To validate clinical relevance we measured EMP3 expression at the mRNA and protein levels in a cohort of 77 patients with upper urinary tract urothelial carcinoma and compared prognostic significance in relation to that of ErbB2 expression. We noted functional crosstalk between ErbB2 and EMP3 in vitro. EMP3 over expression promoted cancer cell proliferation and migration but suppressed cell adhesion in vitro. EMP3 activated the ErbB2-PI3K-AKT pathway to increase cell growth in vitro. In the clinical cohort Kaplan-Meier survival estimates showed that ErbB2 and EMP3 co-expression was the most important indicator of progression-free and metastasis-free survival in patients with upper urinary tract urothelial carcinoma (log rank test p = 0.018 and 0.04, respectively). EMP3 is an important prognostic indicator for selecting patients with upper urinary tract urothelial carcinoma for more intensive therapy. EMP3 is an innovative co-targeting candidate for designing ErbB2 based cancer therapy.

Proteins involved in regulating bone invasion in skull base meningiomas

BackgroundBone invasive skull base meningiomas are a subset of meningiomas that present a unique clinical challenge due to brain and neural structure involvement and limitations in complete surgical resection, resulting in higher recurrence and need for repeat surgery. To date, the pathogenesis of meningioma bone invasion has not been investigated. We investigated immunoexpression of proteins implicated in bone invasion in other tumor types to establish their involvement in meningioma bone invasion.MethodsRetrospective review of our database identified bone invasive meningiomas operated on at our institution over the past 20 years. Using high-throughput tissue microarray (TMA), we established the expression profile of osteopontin (OPN), matrix metalloproteinase-2 (MMP2), and integrin beta-1 (ITGB1). Differential expression in tumor cell and vasculature was evaluated and comparisons were made between meningioma anatomical locations.ResultsMMP2, OPN, and ITGB1 immunoreactivity was cytoplasmic in tumor and/or endothelial cells. Noninvasive transbasal meningiomas exhibited higher vascular endothelial cell MMP2 immunoexpression compared to invasive meningiomas. We found higher expression levels of OPN and ITGB1 in bone invasive transbasal compared to noninvasive meningiomas. Strong vascular ITGB1 expression extending from the endothelium through the media and into the adventitia was found in a subset of meningiomas.ConclusionsWe have demonstrated that key proteins are differentially expressed in bone invasive meningiomas and that the anatomical location of bone invasion is a key determinant of expression pattern of MMP1, OPN, and ITGB1. This data provides initial insights into the pathophysiology of bone invasion in meningiomas and identifies factors that can be pursued as potential therapeutic targets.

Detection of Long Non-Coding RNA in Archival Tissue: Correlation with Polycomb Protein Expression in Primary and Metastatic Breast Carcinoma

A major function of long non-coding RNAs (lncRNAs) is regulating gene expression through changes in chromatin state. Experimental evidence suggests that in cancer, they can influence Polycomb Repressive Complexes (PRC) to retarget to an occupancy pattern resembling that of the embryonic state. We have previously demonstrated that the expression level of lncRNA in the HOX locus, including HOTAIR, is a predictor of breast cancer metastasis. In this current project, RNA in situ hybridization of probes to three different lncRNAs (HOTAIR, ncHoxA1, and ncHoxD4), as well a immunohistochemical staining of EZH2, is undertaken in formalin-fixed paraffin-embedded breast cancer tissues in a high throughput tissue microarray format to correlate expression with clinicopathologic features. Though overall EZH2 and HOTAIR expression levels were highly correlated, the subset of cases with strong HOTAIR expression correlated with ER and PR positivity, while the subset of cases with strong EZH2 expression correlated with an increased proliferation rate, ER and PR negativity, HER2 underexpression, and triple negativity. Co-expression of HOTAIR and EZH2 trended with a worse outcome. In matched primary and metastatic cancers, both HOTAIR and EZH2 had increased expression in the metastatic carcinomas. This is the first study to show that RNA in situ hybridization of formalin fixed paraffin-embedded clinical material can be used to measure levels of long non-coding RNAs. This approach offers a method to make observations on lncRNAs that may influence the cancer epigenome in a tissue-based technique.

Imaging the Clear Cell Renal Cell Carcinoma Proteome

A key barrier to identifying tissue biomarkers of clear cell renal cell carcinoma is the heterogeneity of protein expression in tissue. However, by providing spectra for every 0.05 mm(2) area of tissue, imaging mass spectrometry reveals the spatial distribution of peptides. We determined whether this approach could be used to identify and map protein signatures of clear cell renal cell carcinoma. We constructed 2 tissue microarrays with 2 cores each of matched tumor and normal tissue from the nephrectomy specimens of 70 patients with clear cell renal cell carcinoma. Samples were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In each tissue microarray peptide signatures were identified that differentiated cancer from normal tissue. The signatures were then cross validated. Mass spectrometry/mass spectrometry sequencing was performed to determine the identity of select, differentially expressed peptides. Immunohistochemistry was used for validation. In each tissue microarray peptide signatures were identified that had 94.7% to 98.5% classification accuracy for each 0.05 mm(2) spot (spectrum) and 96.9% to 100% accuracy for each tissue core. Cross validation across tissue microarrays revealed a classification accuracy of 82.6% to 84.7% for each spot and 88.9% to 92.4% for each core. We identified vimentin, histone 2A.X and α-enolase as proteins with greater expression in cancer tissue. This was validated by immunohistochemistry. Imaging mass spectrometry identified and mapped specific peptides that accurately distinguished malignant from normal renal tissue. This demonstrates its potential as a novel, high throughput approach to clear cell renal cell carcinoma biomarker discovery. Given the multiple pathways and known heterogeneity involved in tumors such as clear cell renal cell carcinoma, multiple peptide signatures that maintain their spatial relationships may outperform traditional protein biomarkers.