Immunohistochemistry Of Tissue Arrays Research Articles

Identification of FKBP10 prognostic value in lung adenocarcinoma patients with surgical resection of brain metastases: A retrospective single-institution cohort study

ObjectiveTo explore the expression levels and clinical value of FKBP10 in lung adenocarcinoma brain metastases. DesignA retrospective single-institution cohort study. PatientsThe perioperative records of 71 patients with lung adenocarcinoma brain metastases who underwent surgical resection at the authors’ institution between November 2012 and June 2019 were retrospectively analyzed. MethodsThe authors evaluated FKBP10 expression levels using immunohistochemistry in tissue arrays of these patients. Kaplan-Meier survival curves were constructed, and a Cox proportional hazards regression model was used to identify independent prognostic biomarkers. A public database was used to detect FKBP10 expression and its clinical value in primary lung adenocarcinoma. ResultsThe authors found that the FKBP10 protein was selectively expressed in lung adenocarcinoma brain metastases. Survival analysis showed that FKBP10 expression (p = 0.02, HR = 2.472, 95% CI [1.156, 5.289]), target therapy (p < 0.01, HR = 0.186, 95% CI [0.073, 0.477]), and radiotherapy (p = 0.006, HR = 0.330, 95% CI [0.149, 0.731]) were independent prognostic factors for survival in lung adenocarcinoma patients with brain metastases. The authors also detected FKBP10 expression in primary lung adenocarcinoma using a public database, found that FKBP10 is also selectively expressed in primary lung adenocarcinoma, and affects the overall survival and disease-free survival of patients. LimitationsThe number of enrolled patients was relatively small and patients’ treatment options varied. ConclusionsA combination of surgical resection, adjuvant radiotherapy, and precise target therapy may benefit the survival of selected patients with lung adenocarcinoma brain metastases. FKBP10 is a novel biomarker for lung adenocarcinoma brain metastases, which is closely associated with survival time and may serve as a potential therapeutic target.

Improved Survival in Hepatocellular Carcinoma Patients with Cardiac Arrhythmia by Amiodarone Treatment through Autophagy.

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. In basic studies, the regulation of autophagy has offered promising results for HCC treatment. This study aimed to address the question of whether amiodarone can improve survival rates in HCC patients associated with autophagy. Using datasets from the National Health Insurance Research Database, we enrolled patients over 18 years of age that had been diagnosed with HCC between January 1997 and December 2010. Amiodarone and non-amiodarone users were matched at a 1:1 frequency, according to all variables. Additionally, HepG2 cells treated with amiodarone were evaluated by cell viability and autophagic change. Autophagic signaling was examined by immunoblotting and tissue array immunohistochemistry. Of the 10,946 patients diagnosed with HCC, each cohort included 221 patients after 1:1 propensity score matching. The median survival was 36.70 months for the amiodarone users, and 24.48 months for the non-amiodarone users. After adjusting for age, gender, comorbidities and treatment, amiodarone users had a significantly lower risk of mortality. Amiodarone users also demonstrated an improved 3-year survival rate. Furthermore, amiodarone treatment-induced autophagy in HepG2 cells was demonstrated by autophagosome formation associated with increasing LC3B-II, P62, and Beclin-1 expression. Autophagic flux also increased following amiodarone treatment with bafilomycin A1. SiRNA of LC3B knocked down endogenous LC3B formation and restored HepG2 cell viability. This study provides epidemiologic evidence that amiodarone via autophagic degradation machinery may offer survival benefits for HCC patients with a history of arrhythmia. Further randomized, blinded, and placebo-controlled trials are warranted for patients with HCC.

Mesenchymal Stem Cell Marker Expression in Gastric Cancer Stroma.

Spindle-shaped stromal cells of tumors are derived from various cellular origins, including mesenchymal stem cells (MSCs). MSCs express CD73, CD90 and CD105 antigens. Herein, the aim was to investigate the association between the expression of specific MSC markers in gastric cancer stromal cells and the clinicopathological features of the disease. The expression of CD73, CD90 and CD105 in spindle-shaped cancer stromal cells was studied by immunohistochemistry in tissue arrays containing 546 gastric cancer cases. Univariate and multivariate analyses were performed to evaluate the association of MSC marker expression with clinicopathological variables. Spindle-shaped cancer stromal cells expressing the MSC markers CD73, CD90 or CD105 were associated with larger tumor size, advanced cancer, venous infiltration, lymphatic infiltration, and lymph node metastasis. Statistical analysis demonstrated that the presence of CD105-positive spindle cells was an independent prognostic factor of advanced cancer, lymph node metastasis and EBV infection in multivariate analysis. Spindle-shaped gastric cancer stromal cells expressing CD73, CD90 or CD105 are involved in disease progression, and among them, CD105-positive cells are strongly associated with poor prognosis.

Circulating microRNA-99 family as liquid biopsy marker in pancreatic adenocarcinoma.

Recently, we identified the microRNA-99 family as unfavorable prognostic factor in patients with pancreatic ductal adenocarcinoma (PDAC). The aim of this study is to evaluate its value as circulating biomarker for PDAC. Tissue and corresponding preoperative blood samples of 181 patients with PDAC UICC Stages I-IV (n = 90), intraductal papillary mucinous neoplasm (IPMN, n = 11), chronic pancreatitis (n = 40), pancreatic cystadenoma (n = 20), and age-matched healthy blood serum controls (n = 20) were collected between 2014 and 2017 prospectively. Expression of microRNA-21 as confirmatory marker and the microRNA-99 family, consisting of microRNA-99a, -99b, and -100, was analyzed by qRT-PCR. Target analysis of insulin-like growth factor 1 receptor (IGF1R) was performed using tissue array immunohistochemistry and Western blotting. Expression of microRNA-99 family members was significantly increased in macrodissected tumor tissue and corresponding blood serum samples (p < 0.05) of patients with PDAC of all stages. Correspondingly, its target protein IGF1R was upregulated (p < 0.001) in carcinoma tissue. Circulating and tissue-related microRNA-100 could well discriminate PDAC from healthy samples with area under the receiver operating characteristic (ROC) curve (AUC) values of 0.81 and 0.85, respectively. Low expression of circulating microRNA-100 was associated with significantly improved overall survival (p = 0.004) and recurrence-free survival (p = 0.03) in multivariate analyses. Circulating microRNA-21 was overexpressed in PDAC with fair discrimination between PDAC and healthy controls (AUC = 0.71) and decreased overall survival (p = 0.046) and recurrence-free survival (p = 0.03) in PDAC patients. Multivariate survival and ROC analyses identified circulating microRNA-100 as potential diagnostic and prognostic marker in PDAC patients.

PSIP1/p75 promotes tumorigenicity in breast cancer cells by promoting the transcription of cell cycle genes.

Breast cancer (BC) is a highly heterogeneous disease, both at the pathological and molecular level, and several chromatin-associated proteins play crucial roles in BC initiation and progression. Here, we demonstrate the role of PSIP1 (PC4 and SF2 interacting protein)/p75 (LEDGF) in BC progression. PSIP1/p75, previously identified as a chromatin-adaptor protein, is found to be upregulated in basal-like/triple negative breast cancer (TNBC) patient samples and cell lines. Immunohistochemistry in tissue arrays showed elevated levels of PSIP1 in metastatic invasive ductal carcinoma. Survival data analyses revealed that the levels of PSIP1 showed a negative association with TNBC patient survival. Depletion of PSIP1/p75 significantly reduced the tumorigenicity and metastatic properties of TNBC cell lines while its over-expression promoted tumorigenicity. Further, gene expression studies revealed that PSIP1 regulates the expression of genes controlling cell-cycle progression, cell migration and invasion. Finally, by interacting with RNA polymerase II, PSIP1/p75 facilitates the association of RNA pol II to the promoter of cell cycle genes and thereby regulates their transcription. Our findings demonstrate an important role of PSIP1/p75 in TNBC tumorigenicity by promoting the expression of genes that control the cell cycle and tumor metastasis.

Abstract 471: Transcriptional coactivator CBP regulates hTERT expression through the cooperation with Sp1/AP-2β and predicts poor prognosis in human lung cancers

Abstract Human telomerase reverse transcriptase (hTERT) is linked to tumorigenesis and development. However, how hTERT is upregulated in tumor cells remains poorly understood. In this study, we identified CBP as a transcriptional coactivator that specifically binds to hTERT promoter through co-anchoring with transcriptional factors Sp1 and AP-2β and promotes hTERT expression in human lung cancer cells. Streptavidin-agarose pulldown and chromatin immunoprecipitation assays revealed the tumor-specific binding of CBP, Sp1 and AP-2β on the hTERT promoter. The colocalization and interaction between CBP and Sp1 or AP-2β in lung cancer cells were detected. High expression levels of CBP and hTERT in lung cancer cells and tissues were also detected compared to normal lung cells and adjacent normal lung tissues. Tissue array immunohistochemistry of lung adenocarcinomas showed a strong postive correlation between CBP and hTERT expression, and demonstrated the patients with high CBP and hTERT expression had a significantly shorter overall survival than those with low CBP and hTERT expression. Ectopic expression of CBP activated hTERT promoter-driven luciferase gene and endogenous hTERT gene expression in lung cancer cells. Inhibition of CBP expression by CBP-specific siRNA or chemical inhibitor attenuated hTERT promoter-driven luciferase expression and the endogenous hTERT activity, and abrogated the binding of Sp1 and AP-2β on hTERT promoter region. Moreover, CBP knockdown by siRNA significantly inhibited lung cancer cell proliferation, whereas CBP overexpression promoted cell proliferation. These results therefore indicate overexpression of CBP contributes to hTERT upregulation through its cooperation with transactivators Sp1 and AP-2β and predicts poor prognosis in human lung cancers. Note: This abstract was not presented at the meeting. Citation Format: Wei Guo. Transcriptional coactivator CBP regulates hTERT expression through the cooperation with Sp1/AP-2β and predicts poor prognosis in human lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 471. doi:10.1158/1538-7445.AM2014-471

The potential contributions of a Y-located protooncogene and its X homologue in sexual dimorphisms in hepatocellular carcinoma.

There is a significant sex disparity favoring males among hepatocellular carcinoma (HCC) patients. Although various risk factors have been identified, the exact etiology of such sexual dimorphism(s) in HCC is uncertain. Previous studies showed that overexpression of the Y-located protooncogene, testis-specific protein Y encoded (TSPY), promotes cell proliferation and oncogenesis whereas its X-located homologue, TSPYhomologue X (TSPX), retards cell cycle and oncogenic progression. Furthermore, TSPX promotes proteasomal degradation of hepatitis B virus-encoded X oncoprotein and hence could serve as a tumor suppressor in virus-associated HCC. Using immunohistochemistry and reverse-transcription polymerase chain reaction analysis, we had examined the expression of TSPY and TSPX with reference to other established biomarkers in HCC and related liver cancers. Our results demonstrated that 55 (19.2%) of 287 male cases were TSPY positive in immunohistochemistry of tissue arrays, and 15 (46.9%) of 32 male cases were TSPY positive in reverse-transcription polymerase chain reaction analysis of clinical samples. TSPY expression was closely associated with the expression of HCC biomarkers, such as glypican 3. In contrast, TSPX expression was down-regulated in 54.5% of total tumor/nontumorous paired samples (18/33) and negatively associated with those of TSPY, glypican 3, and forkhead box M1 (FOXM1) and was positively associated with that of a tumor suppressor, insulin-like growth factor binding protein 3. The present findings support the hypothesis that the oncogenic events leading to an ectopic activation of the Y-located protooncogene TSPY and/or inactivating mutation/epigenetic silencing of the X-located tumor suppressor gene TSPX could collectively contribute to the sexual dimorphism(s) in HCC and related liver cancers in male-biased manners.

Relationship of immunohistochemical biomarker expression and lymph node involvement in patients undergoing surgical treatment of NSCLC with long-term follow-up

We try to identify the relationship between immunohistochemical marker expression and lymph node involvement in a cohort of 282 patients followed for 5 years after curative resection for NSCLC. In 189 patients (67%), lymph nodes were unaffected while 93 patients (33%) showed nodal involvement. The expression of 15 molecular markers was determined from each patient by tissue-array immunohistochemistry. Univariate analysis indicated significantly higher expression of E-cadherin, γ-catenin, p27, and p53 in patients with lymph node involvement. In those with unaffected nodes, p16 and Rb were expressed. E-cadherin expression was related to a 50% mortality reduction in patients with node involvement (hazard ratio (HR) 0.5; p = 0.017). c-erbB-2 expression was correlated with a 3.4-fold increase in mortality compared to patients without expression of this marker in subjects without node involvement (HR 3.41; p = 0.017). Multivariate analysis identified c-erbB-2 (HR 2.22; p = 0.089) and p27 (HR 1.44; p = 0.019) as prognostics of mortality while Rb (HR 0.74) indicated a good prognosis. The expression of proteins encoded by oncogenes and tumor suppressor genes was different according to lymph node involvement. The increased mortality related to c-erbB-2 expression in patients with unaffected lymph nodes would suggests a need for adjuvant treatment.

Differences in the expression of five senescence markers in oral cancer, oral leukoplakia and control samples in humans

Oncogene-induced senescence (OIS) may be a response to oncogenic activation, acting as a natural barrier against carcinogenesis at a premalignant stage. Thus, numerous cells in premalignant lesions enter senescence, but none or few in malignant tumours. This event could be due to the loss of senescence pathway effectors, including p16 (INK4a)-pRb or ARF-p53. The aim of this study was to characterize and compare the expression of certain senescent markers between oral precancer and cancer tissue samples. The expression of cyclin D1, Rb, maspin, p53 and mouse double minute 2 (MDM2) was analyzed in 20 paraffin-embedded tissue samples of normal oral mucosa (NOM), 14 samples of oral leukoplakia without dysplasia (OLD-), 11 samples of leukoplakia with dysplasia (OLD+) and 15 samples of oral squamous cell carcinoma (OSCC) by immunohistochemistry in tissue arrays. The expression of p16-pRb pathway markers, cyclin D1, maspin and Rb, was more frequent in OLD+ samples than in OSCC samples, although a statistical significance was only observed for maspin (P=0.036). Cyclin D1 expression was also significantly more frequent in OLD- samples vs. NOM samples. For the ARF-p53 pathway, the expression of p53 and MDM2 was significantly more frequent in the OLD- samples compared to in the NOM ones. These findings may indicate a role for cellular senescence in oral carcinogenesis, considering maspin as a reliable senescence marker and prognostic factor in oral premalignant lesions.

Expression and clinical significance of Notch signaling genes in colorectal cancer

The objective of this study was to explore the expression and clinical significance of Notch signaling genes in colorectal cancer. Colorectal cancer samples were prospectively collected from patients post-surgery at the 3rd Affiliated Hospital, Nanjing University of Traditional Chinese Medicine. Immunohistochemistry of tissue arrays was used to analyze the samples and genes involved in the Notch signaling pathway. Microsatellite instability (MSI) was detected by fluorescence multiplex polymerase chain reaction. A total of 146 colorectal cancer samples were collected, including 84 men (57.7%) and 62 women (42.5%). The average age of the study population was 60.8 ± 10.5 years. Notch1 and Notch2 gene expression correlated with tumor pathology type and degree of differentiation. In addition, Jagged 1 (JAG1) and hairy enhancer of split 1 gene expression correlated with the degree of tumor differentiation. Delta-like 1 gene expression varied significantly with tumor location. There was a significant difference between gene expression and MSI. Of the 138 patients, 134 (91.8%) participated in on-site visits, and the average follow-up time was 42.3 ± 13.3 months. During this period, 86 patients (71.6%) were tumor-free. At 1 year post-surgery, 93% of patients were alive, 74% of patients lived for 3 years, and 67% of patients lived for 5 years. The log-rank test was used to perform univariate analysis, and the COX proportional hazards model was used for the multivariate analysis. Based on these analyses, tumor prognosis correlated with the TNM stage, pathological type, microsatellite status as well as Notch2 and JAG1 gene expression. Patients expressing high levels of Notch2 and JAG1 presented with a significantly better prognosis compared to patients expressing negative or weak levels of Notch2 and JAG1. The expression levels of genes associated with the Notch signaling pathway correlated with tumor pathology and the degree of differentiation. In addition, Notch2 and JAG1 expression levels correlated with survival; however, the underlying mechanism for these correlations remains unclear.

Inhibition of Epstein-Barr Virus Infection by Lactoferrin

Lactoferrin (LF) is a multifunctional glycoprotein that plays an important role in native immune defense against infections, including human herpetic viruses, such as cytomegalovirus and herpes simplex virus types 1 and 2. However, its anti-Epstein-Barr virus (EBV, a γ-herpesvirus) function has not been reported in the literature. EBV is widespread in all human populations and is believed to be linked to tumorigenesis, such as lymphomas and nasopharyngeal carcinoma (NPC). We previously reported that LF expressed a significantly lower level in NPC tissues and was a likely tumor suppressor. Since EBV infection is a major carcinogen of NPC development, we investigated the effect of LF on EBV infection and found that LF could protect human primary B lymphocytes and nasopharyngeal epithelial cells from EBV infection, but had no effect on EBV genome DNA replication. LF prevented EBV infection of primary B cells mediated by its direct binding to the EBV receptor (CD21) on the B-cell surface. Tissue array immunohistochemistry revealed that LF expression was significantly downregulated in NPC specimens, in which high EBV viral capsid antigen-IgA levels were observed. These data suggest that LF may inhibit EBV infection and that its downregulation could contribute to NPC development.

The First Type III Domain of Fibronectin is Associated with the Expression of Cytokines within the Lung Tumor Microenvironment

Recent studies have pointed to changes in tissue mechanics as a contributory element to the development of malignancies. Increased tissue rigidity is associated with the unfolding of the Type III domains of fibronectin within the extracellular matrix. The consequences of this unfolding on cellular functions within the lung are not well understood. In the present study, we evaluated the effect of a peptide representing a partially unfolded intermediate of the first Type III repeat of fibronectin (FnIII-1c) on inflammatory gene expression in adult human lung fibroblast cells. FnIII-1c induced expression of cytokines, CXCL1-3, IL-8 and TNF-α, by lung fibroblast cells. The increase in IL-8 expression was dependent on Toll-like receptor 2 and NFκB. Immunohistochemistry of tissue arrays representing squamous cell carcinoma of the lung revealed extensive stromal staining for IL-8 and fibronectin fibrils which were co-aligned with myofibroblasts. These data suggest a model in which unfolding of FnIII domains secondary to myofibroblast-generated tension may induce the release of cytokines by stromal fibroblasts present within the lung tumor.

Differential expression pattern of estrogen receptors, aromatase, and sulfotransferase in breast cancer tissue and corresponding lymph node metastases

Patients with hormone receptor positive breast cancer who are treated with endocrine therapy generally have a good prognosis. However, resistance to hormonal therapy and progression occurs, and the reasons for this are manifold. It has been proposed that the local estrogenic environment has a role in the process of local invasion and progression. We have determined the expression pattern of estrogen receptor α, estrogen receptor β, and the epithelial and stromal expression of the estrogen-metabolizing enzymes aromatase and sulfotransferase by immunohistochemistry in tissue arrays, containing 50 paraffin-embedded sets of tissues obtained from breast cancer and from corresponding metastatic axillary lymph nodes of the same patients. We have found statistically significant higher estrogen receptors α and β expression in primary tumors than in corresponding lymph node metastases (p = 0.0004 and p = 0.003, respectively). Aromatase was also expressed more frequently in epithelial as well as in stromal cells of the malignant tumor when compared to according lymph node metastases (p = 0.08 and p = 0.12, respectively). While in lymph node metastases only estrogen receptor α and stromal aromatase expression were correlated (p = 0.01), significant associations were seen between the estrogen receptor β and sromal aromatase, and epithelial sulfotransferase (p = 0.0006 and p = 0.03, respectively) in the primary tumor. We hypothesize that the decreased expression of local estrogens by aromatase, in combination with a decreased expression of estrogen receptors α and β in lymphatic metastases, renders these metastases hormone insensitive and could contribute to the poor response to endocrine therapy that is often seen in nodal-positive tumors.

New Monoclonal Antibody B7 Selectively Recognizes Rat Myocardium Microvascular Endothelial Cells

Myocardial microvascular endothelial cells (MMECs) play a vital role in modulating cardiomyocyte development, survival, and contraction during embryonic cardiogenesis and mature myocardium. However, specific molecular composition of MMECs is still not well known, with no special MMEC marker to distinguish microvascular endothelial cells (ECs) from macrovascular ECs. In the present study, we immunized BALB/c mice with membrane proteins of MMECs. Through screening by enzyme linked immunosorbent assay and immunohistochemistry, a new monoclonal antibody that specifically recognizes MMECs was yielded. Immunohistochemistry of tissue arrays showed that MAb B7 also selectively recognized microvascular ECs but not macrovascular ECs in other tissues. Immunofluorescence and immuno-electron microscope assay indicated that B7 antigen was a plasma membrane molecule. Interestingly, we also found that B7 antigen was dramatically decreased in diabetic rat compared with that in normal rat. In conclusion, MAb B7 not only provides a new biomarker to help us understand the molecular composition of microvascular ECs, but also indicates that B7 antigen might play an important role in the dysfunction of microvascular ECs.

Abstract 4546: Expression profiling of the major folate facilitative transporters in human tumors and normal tissues

Abstract Membrane transport of folates into mammalian cells involves major facilitative transporters including the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). Classical antifolates such as methotrexate (Mtx), pemetrexed, or raltitrexed are all substrates for RFC and PCFT such that the cytotoxic effects of these drugs are determined by levels of these transporters in tumors and normal tissues. As PCFT is a folate-proton symporter with an acidic pH optimum, extracellular pH represents another key determinant of transport, such that PCFT may provide a mechanism for targeting cytotoxic antifolates to tumors, based on their acidic microenvironments. We recently identified novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that are selective transport substrates for PCFT over RFC and that potently inhibit cell proliferation by targeting glycinamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis. To better exploit this approach for selectively targeting tumors versus normal tissues, we systematically determined expression profiles for PCFT and RFC in human malignancies and normal tissues. Real-time reverse transcription PCR (qRT-PCR) was used to determine transcript levels in 57 human solid tumor and 27 leukemia cell lines. Both RFC and PCFT transcripts were highly expressed in the majority of solid tumor cell lines whereas only RFC was expressed appreciably in the leukemia cell lines. For 10 solid tumor cell lines, PCFT and RFC transcripts were accompanied by corresponding levels of respective proteins on western blots. Functional validation of transporter expression in solid tumor cell lines was determined by transport of [3H]Mtx at pH 5.5 for PCFT, and at pH 7.2 for RFC. Tumor uptake of [3H]Mtx correlated closely with transcript and protein levels for both PCFT and RFC. qRT-PCR of cDNA arrays from 48 normal human tissues measured high levels of PCFT expression in the small intestine, kidney, liver, and the adrenal and pituitary glands. On cDNA arrays of human primary tumors, significant albeit variable levels of PCFT transcripts were detected in breast, colon, kidney, liver, lung, ovary, and prostate tumors. Patterns of transporter expression in primary tumors were confirmed by immunohistochemistry of tissue arrays probed with transporter-specific antibodies. In conclusion, we have established the expression patterns and pH-specific transport for PCFT and RFC in human solid tumor cell lines, primary tumors and select normal tissues. This study represents the first comprehensive analysis of PCFT expression in tumors and normal tissues and how this relates to RFC. Our finding of widespread PCFT expression in human solid tumors paired with our previous discovery of novel PCFT-specific antifolates offers exciting new therapeutic possibilities for selectively targeting chemotherapy to tumors based on their acidic microenvironments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4546.

IL15 Can Reverse the Unresponsiveness of Wilms' Tumor Antigen-Specific CTL in Patients with Prostate Cancer

The Wilms' tumor antigen 1 (WT1) is overexpressed in several leukemias and solid tumors, but there is currently limited information regarding its role in prostate cancer. This study aimed to investigate WT1 expression in prostate cancer, and to determine the number and function of WT1-specific T cells in the peripheral blood of patients. Immunohistochemistry was used to assess WT1 expression in cancer tissues. Human leukocyte antigen A2 (HLA-A2) tetramers served to detect WT1-specific T cells, and peptide-specific stimulation was used to assess T-cell function in vitro. Immunohistochemistry of tissue arrays comprising 36 cancer and 8 normal prostate samples revealed nuclear WT1 staining in 39% of cancer samples, but not in normal prostate tissues. Tetramer analysis revealed a low frequency of WT1-specific T cells in 20 of 38 HLA-A2-positive patients. In vitro stimulation with WT1 peptide plus interleukin 2(IL2) and interleukin 7 (IL7) did not lead to an accumulation of WT1-specific T cells in any of the patient samples, although all patients were able to generate T-cell responses against Melan-A/MART1 control peptide. Stimulation with WT1 peptide in the presence of interleukin 15 (IL15), a cytokine that was shown to reverse tolerance of murine tumor-specific T cells, was able to restore the expansion and IFNgamma production of WT1-specific T cells in a subgroup of prostate cancer patients. The observation that IL15 can restore the function of WT1-specific T cells that were unresponsive to IL2 has implications for vaccination and immunotherapeutic strategies that aim to enhance WT1-specific T cell immunity in patients.

YKL-40 tissue expression and plasma levels in patients with ovarian cancer

BackgroundYKL-40 (chitinase-3-like-1) is a member of "mammalian chitinase-like proteins". The protein is expressed in many types of cancer cells and the highest plasma YKL-40 levels have been found in patients with metastatic disease, short recurrence/progression-free intervals, and short overall survival. The aim of the study was to determine the expression of YKL-40 in tumor tissue and plasma in patients with borderline ovarian tumor or epithelial ovarian cancer (OC), and investigate prognostic value of this marker.MethodsYKL-40 protein expression was determined by immunohistochemistry in tissue arrays from 181 borderline tumors and 473 OC. Plasma YKL-40 was determined by ELISA in preoperative samples from 19 patients with borderline tumor and 76 OC patients.ResultsYKL-40 protein expression was found in cancer cells, tumor associated macrophages, neutrophils and mast cells. The tumor cell expression was higher in OC than in borderline tumors (p = 0.001), and associated with FIGO stage (p < 0.0001) and histological subtype (p = 0.0009). Positive YKL-40 expression (≥ 5% staining) was not associated with reduced survival. Plasma YKL-40 was also higher in patients with OC than in patients with borderline tumors (p < 0.0001), and it was positively correlated to serum CA-125 (p < 0.0001) and FIGO stage (p = 0.0001). Univariate Cox analysis of plasma YKL-40 showed association with overall survival (p < 0.0001). Multivariate Cox analysis, including plasma YKL-40, serum CA125, FIGO stage, age and radicality after primary surgery as variables, showed that elevated plasma YKL-40 was associated with a shorter survival (HR = 2.13, 95% CI: 1.40–3.25, p = 0.0004).ConclusionYKL-40 in OC tissue and plasma are related to stage and histology, but only plasma YKL-40 is a prognostic biomarker in patients with OC.

Expression of MMP1, 2, 9, and 11 in breast cancer and metastatic lymph node tissue

Invasiv growth requires degradation of extracellular matrix. A number of Zn 2+ -dependent matrix metalloproteinases (MMPs) are associated with this process. In most cases these enzymes are not produced by the malignant epithelium itself but by adjacent breast stroma, suggesting an important role for cell-cell interactions. Materials and methods: We determined the expression patterns of MMP1, MMP2, MMP9 and MMP11 by immunohistochemistry in tissue arrays, containing 50 paraffin-embedded sets of tissues obtained from the malignant tumor of the breast, and 50 paraffin-embedded sets of tissues from metastatic lymph nodes (purchased from Biomax, Biomax Inc, Rockville, MD). Results: MMP2S protein expression was significantly higher in lymph node tumor tissue when compared to matched breast tumor tissue (10% vs. 2%; p=0.005, Chi Square Test). Furthermore, MMP9S was also significantly up-regulated in lymph node tumor tissue when compared to breast tumor tissue (78.3% vs. 94.8%, p=0.01 Chi Square test) and MMP9E showed a borderline significance in lymph node tumor tissue (92.6% vs. 73.5%, p=0.05). While in the subgroup breast tissue significant correlations were observed between all MMPs except MMP2S this associations were less pronounced in the subgroup lymph node. Conclusions: The increased protein expression of MMP2S, MMP9S and MMP9E in lymph node tissue is in line with other studies which suggested MMP2 and MMP9 expression in primary breast cancer tissue samples was associated with breast cancer progression and invasion [20–23].

Expression of matrix metalloproteinases (MMP1, 2, 9, and 11) in metastatic lymph node tissue of breast cancer patients

21138 Background: Invasive growth requires degradation of extracellular matrix. A number of Zn 2+ -dependent matrix metalloproteinases (MMPs) is associated with this process and leads to local invasion and metastasis in malignant breast tumors. Especially the expression of MMP-2 and MMP-9 has been associated with high potential of metastasis in breast cancer. Methods: We determined the expression patterns of epithelial (E) and stromal (S) MMP-1, MMP-2, MMP-9 and MMP-11 by immunohistochemistry in tissue arrays, containing 50 paraffin-embedded sets of tissues obtained from the malignant tumor of the breast, and 50 paraffin-embedded sets of tissues from metastatic lymph nodes (purchased from Biomax, Biomax Inc, Rockville, MD). Results: MMP2-S and MMP9-S protein expression was significantly higher in lymph node tumor tissue when compared to breast tumor tissue (10% vs. 2%; p=0.005; 78.3% vs. 94.8%, p=0.01 Chi Square test, respectively). MMP9-E showed a borderline significance in lymph node tumor tissue (92.6% vs. 73.5%; p=0.05 Chi Square test). While in breast tumor tissue significant correlations were observed between all MMPs except MMP2-S, these associations were less pronounced in lymph node tumor tissue. Conclusions: We suggest that the expression of MMP-2 and MMP-9 in breast cancer tissue is associated with a high invasive and metastatic potential. This might be confirmed by our finding that MMP2-S, MMP9-S and MMP9-E are increased expressed in metastatic lymph node tissue. This is in line with other studies which showed that MMP-2 and MMP-9 expression in primary breast cancer tissue samples was associated with breast cancer progression and invasion. No significant financial relationships to disclose.

Up-regulation of ERM/ETV5 correlates with the degree of myometrial infiltration in endometrioid endometrial carcinoma

To elucidate alterations in gene expression in endometrioid endometrial carcinoma (EEC), differential gene expression profiling was previously described in both tumour and non-tumour contexts, and the up-regulation of the RUNX1/AML1 proto-oncogene in EEC was characterized. Among the set of genes found to be up-regulated significantly in EEC, the most relevant, ERM/ETV5, corresponds to the PEA3 subfamily and is a member of the Ets family of transcription factors that contain the Ets DNA-binding domain and are involved in matrix remodelling. In the present work, an attempt was made to characterize the expression of ERM/ETV5 in EEC throughout the process of tumourigenesis. Gene expression levels of ERM/ETV5 were quantified by real-time quantitative PCR (RT-Q-PCR) using a large panel of samples ranging from non-invasive IA to metastatic IIIA stages, and protein expression was characterized by tissue array immunohistochemistry (TMA). RT-Q-PCR validated ERM/ETV5 up-regulation in EEC and demonstrated a specific and significant increase restricted to those tumour stages associated with myometrial invasion. TMA showed that ERM/ETV5 up-regulation correlated mainly with the transition from atrophic endometrium to hyperplasia and carcinoma during tumour progression. Furthermore, ERM/ETV5 gene and protein expression levels were associated with low tumour grade. Finally, ERM/ETV5 up-regulation correlated with that of RUNX1/AML1. All of these results lead to the proposal of a co-operative role between ERM/ETV5 and RUNX1/AML1 during the early events of endometrial tumourigenesis, which may be associated with a switch to myometrial infiltration.