Frozen Cancer Tissues Research Articles

Proteomic Discovery of Plasma Protein Biomarkers and Development of Models Predicting Prognosis of High-Grade Serous Ovarian Carcinoma

Ovarian cancer is one of the most lethal female cancers. For accurate prognosis prediction, this study aimed to investigate novel, blood-based prognostic biomarkers for high-grade serous ovarian carcinoma (HGSOC) using mass spectrometry–based proteomics methods. We conducted label-free liquid chromatography–tandem mass spectrometry using frozen plasma samples obtained from patients with newly diagnosed HGSOC (n = 20). Based on progression-free survival (PFS), the samples were divided into two groups: good (PFS ≥18 months) and poor prognosis groups (PFS <18 months). Proteomic profiles were compared between the two groups. Referring to proteomics data that we previously obtained using frozen cancer tissues from chemotherapy-naïve patients with HGSOC, overlapping protein biomarkers were selected as candidate biomarkers. Biomarkers were validated using an independent set of HGSOC plasma samples (n = 202) via enzyme-linked immunosorbent assay (ELISA). To construct models predicting the 18-month PFS rate, we performed stepwise selection based on the area under the receiver operating characteristic curve (AUC) with 5-fold cross-validation. Analysis of differentially expressed proteins in plasma samples revealed that 35 and 61 proteins were upregulated in the good and poor prognosis groups, respectively. Through hierarchical clustering and bioinformatic analyses, GSN, VCAN, SND1, SIGLEC14, CD163, and PRMT1 were selected as candidate biomarkers and were subjected to ELISA. In multivariate analysis, plasma GSN was identified as an independent poor prognostic biomarker for PFS (adjusted hazard ratio, 1.556; 95% confidence interval, 1.073–2.256; p = 0.020). By combining clinical factors and ELISA results, we constructed several models to predict the 18-month PFS rate. A model consisting of four predictors (FIGO stage, residual tumor after surgery, and plasma levels of GSN and VCAN) showed the best predictive performance (mean validated AUC, 0.779). The newly developed model was converted to a nomogram for clinical use. Our study results provided insights into protein biomarkers, which might offer clues for developing therapeutic targets.

Whole-exome sequencing in unresectable pancreatic cancer patients with long-term survival.

260 Background: Advances in technology of genomic sequencing have revealed the mutational landscape of pancreatic cancer. However, little is known with regard to molecular mechanisms underlying clinical diversity in “unresectable” advanced pancreatic cancer. Methods: We performed whole-exome sequencing using frozen cancer tissues prospectively obtained by EUS-FNA from primary tumors before chemotherapy in patients with metastatic or locally advanced pancreatic cancer (n = 35). All patients had pathological confirmation (adenocarcinoma, n = 34; carcinoma, n = 1). Somatic alterations in the cancer genome were compared with clinical outcomes. Results: Seventeen patients had metastatic disease and 18 had locally advanced disease. Thirty-four patients received chemotherapy with or without radiotherapy and one patient was treated with supportive care alone because of rapidly progressive disease. With a median follow-up time of 60.6 months (range 41.9–94.0) for censored cases, the median survival for all patients was 10.8 months. Nine patients survived more than 2 years ( &gt; 6 years, n = 2; &gt; 3 years, n = 3; &gt; 2 years, n = 4), while 11 patients died within 4 months. We show mutational landscape of unresectable advanced pancreatic cancer and identified somatic mutations in known cancer related genes including KRAS (89%), TP53 (71%), SMAD4 (20%), CDKN2A (17%) and ARID1A (14%). We found that ARID1A mutation was mutually exclusive with TP53 mutation except for one tumor and had a significant correlation with survival outcomes. Among 9 patients who survived more than 2 years, 5 patients (56%) had ARID1A somatic mutation, whereas none (0%) had mutations in the remaining 26 patients who died within 2 years ( P = 0.0004). The median overall survival was 47.7 months for 5 patients with ARID1A-mutated tumors and 8.9 months for 30 patients with ARID1A wild-type tumors ( P = 0.0101). Conclusions: This is the first study to perform whole-exome sequencing in unresectable pancreatic cancer patients including very long-term survivors. We found that ARID1A mutations were associated with longer survival.

TEM8 functions as a receptor for uPA and mediates uPA-stimulated EGFR phosphorylation

BackgroundTEM8 is a cell membrane protein predominantly expressed in tumor endothelium, which serves as a receptor for the protective antigen (PA) of anthrax toxin. However, the physiological ligands for TEM8 remain unknown.ResultsHere we identified uPA as an interacting partner of TEM8. Binding of uPA stimulated the phosphorylation of TEM8 and augmented phosphorylation of EGFR and ERK1/2. Finally, TEM8-Fc, a recombinant fusion protein comprising the extracellular domain of human TEM8 linked to the Fc portion of human IgG1, efficiently abrogated the interaction between uPA and TEM8, blocked uPA-induced migration of HepG2 cells in vitro and inhibited the growth and metastasis of human MCF-7 xenografts in vivo. uPA, TEM8 and EGFR overexpression and ERK1/2 phosphorylation were found co-located on frozen cancer tissue sections.ConclusionsTaken together, our data provide evidence that TEM8 is a novel receptor for uPA, which may play a significant role in the regulation of tumor growth and metastasis.

PO-302 The role of SPARCL1 in upper urinary tract cancer of Taiwan

IntroductionUpper urinary tract urothelial carcinoma (UTUC) in Taiwan is a relatively high prevalent cancer and locally advanced UTUC often carries a poor prognosis. This study is to analyse role of SPARCL1 in UTUC of Taiwan and analyse if there were any association between SPARCL1 presentation and clinical oncologic outcome.Material and methodsWe initially got fresh frozen cancer tissue from three high grade locally advanced UTUC (stage >2) and three fresh frozen normal urothelium tissue from low grade and stage 0 disease in Kaohsiung Chang Gung Memorial Hospital. In this study, we used DNA methylation assay and SPARCL1 was identified as most significant different DNA methylation in locally advanced cancer compared with normal urothelium tissue. From 2005 to 2012, this study included 78 patients with pT3N0M0 UTUC. Perioperative factors, pathological features, and SPARCL1 immunostaining were reviewed and prognostic factors were examined by multivariate analysis. Cell line study with BFTC909 and BFTC909-SPARCL1 overexpression was used for cancer behaviour observation. The sensitivity of both cell line to cisplatin and radiation was tested.Results and discussionsSPARCL1 was revealed as 20 fold change of methylation in locally advanced UTUC compared with normal urothelium. The patient demography revealed that all pT3N0M0 patient is each group have similar clinical and pathological factors. Systemic UTUC recurrence was significant more in UTUC with negative SPARCL1 presentation (p=0.042). Multivariate analysis found that negative SPARCL1 and non-papillary tumour architexture were the two significant prognostic factors associated with systemic UTUC recurrence (p=0.011 and 0.008 respectively). Cell line study revealed that BFTC 909-SPARCL1 overexpression is associated with less aggressive cancer cell migration/invasion and more sensitivity to cisplatin or radiation treatment.ConclusionSPARCL1 is the most significant methylated gene in locally advanced tissue compared with normal urothelium. For those patients with pT3N0M0 UTUC, SPARCL1 is an independent prognostic marker. The presence of SPARCL1 interferes with UTUC cell line behaviour and sensitivity to cisplatin or radiation.

RUNX3 inhibits glioma survival and invasion via suppression of the β-catenin/TCF-4 signaling pathway.

Runt-related transcription factor 3 (RUNX3) exerts a tumor suppressor gene associated with gastric and other cancers, including glioma. However, how its anti-tumor mechanism in functions glioma is unclear. We assayed expression of RUNX3 with a tissue microarray (TMA), frozen cancer tissues and malignant glioma cell lines using immunohistochemistry, qRT-PCR and Western bolt analysis. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm the effect of RUNX3 medicated malignant phenotype. TOP/FOP experiment was used to detect the β-catenin/Tcf-4 transcription activity by RUNX3. Enforced RUNX3 expression inhibited proliferation and invasion, induced cell cycle arrest and promoted apoptosis in vitro and in vivo, Bim siRNA partically reversed the effect of RUNX3-induced apoptosis in LN229 and U87 cells, suggesting a dependent role of Bim-caspase pathway. Moreover, Mechanism investigations revealed that restoration of RUNX3 suppressed β-catenin/Tcf-4 transcription activity. RUNX3 plays a pivotal role in glioma initiation and progression as a tumor suppressor via attenuation of Wnt signaling, highlighting it as a potential therapeutic target for glioma.

Abstract 1792: Immune system pathway activation for prediction of the response to neoadjuvant chemotherapy in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer patients (TNBCs) who receive neoadjuvant chemotherapy (NAC) generally show a response to NAC, but about 5% experience progressive disease (PD). Although TNBCs that respond to NAC have been well-studied, reports about TNBCs with PD during NAC are absent. We aimed to compare kinase activity profiles of TNBCs of both groups to identify a biological predictor of NAC non-responders. Methods: Tyrosine kinase activity profiles of lysates of fresh frozen cancer tissues from NAC non-responders and responders, the latter represented by TNBCs who did not receive NAC, were determined with a peptide microarray system (Cancer Res. 2009; 69(14):5987-95). Tumor infiltrating lymphocytes (TILs) were counted in these tissues. Results: Among 740 TNBCs, 20 NAC non-responders were identified. For the 7 samples from PD confirmed patients and 10 TNBCs not receiving NAC, no correlation was observed between NAC response and age, menopausal status, tumor size or axillary lymph node status. Kinase activity measurements showed large differences in overall tyrosine kinase activity between responders and non-responders. 35 peptides had significantly (p &amp;lt; 0.05) higher phosphorylation in the responder group. Kinases that phosphorylate these peptides (upstream kinases) were identified from literature. Among others, the immune related kinases Zap70, Syk and JAK2 were identified as upstream kinases. Pathway analysis yielded evidence for increased activity in the responder group in immune-related pathways and in pathways involved in development and signal transduction. Responders had a significantly higher (p = 0.0053) TILs count. Conclusion: Kinases related to the immune system are less activated in NAC non-responders. The low TILs count in the non-responders confirmed that activation of the immune system in non-responders is lower than in NAC responders. These data merit a further investigation of the use of kinase activity profiling either alone or in combination with TIL scoring for the prediction of the response to NAC in TNBC with a larger patient cohort. Citation Format: Takeshi Sawada, Riet Hilhorst, Savithri Rangarajan, Masayuki Yoshida, Kenji Tamura, Rinie van Beuningen, Rob Ruijtenbeek, Hitoshi Tsuda, Fumiaki Koizumi. Immune system pathway activation for prediction of the response to neoadjuvant chemotherapy in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1792. doi:10.1158/1538-7445.AM2017-1792

Prognostic value of NK and T-lymphocytes markers in operable non-small cell lung cancer (NSCLC).

11556 Background: Therapies aimed at activation of T and NK cells are developed to expand NSCLC treatments options. It is conceivable that markers of ‘immune ignorant’, ‘immune excluding’ or ‘inflamed’ tumor phenotypes could be prognostic or predictive of benefit from specific immune-targeting therapies. Aim: To assess the prognostic value of expression of T and NK cells mRNA markers and immune-related genes in early stage NSCLC. Methods: qRT-PCR was used to assess 48 mRNAs levels in frozen cancer tissue sections and matched normal lung parenchyma from 56 surgically treated stage I-IIIA NSCLC patients. The mRNA expression (normalized vs. 4 reference genes) was compared between the groups that did (44%) or did not relapse, as well as clinicopathological features (33% never-smokers, 75% lung adenocarcinoma). Results: Low expression of FAS-L (p.adj. = 0.048) , TIGIT and LAG3 was correlated with shorter distant metastasis free survival (DMFS) (p &lt; 0.04). Expression of PD-1 (p = 0.024) and CTLA4 (p = 0.04) was significantly lower in relapsed vs. non-relapsed NSCLCs, whereas there was no difference for PDL-1 and PDL-2. Expression of NK activation markers: NCR3 and NCR1, but not NCR3-ligand 1 was significantly lower in relapsed vs. not relapsed NSCLCs. Other NK cell markers: CD96 and NKG2D were expressed at lower levels (p = 0.02) in relapsed vs. not relapsed NSCLCs, whereas there was no difference for NKG2C and NKG2A. Expression of CXCR3 was lower in relapsed NSCLCs (p = 0.03), however, the expression of its ligands (chemoattractants for lymphocytes) - CXCL9, CXCL10 or CXCL11 or endothelin receptor type B was not different according to metastatic status. GITR and FOXP3 expression was significantly higher in cancers vs. normal lung parenchyma (p.adj. &lt; 0.003). There were no differences in expression according to gender, smoking or NSCLC histological types. Conclusions: Non-inflamed NSCLC phenotype is associated with higher risk of dissemination after primary resection. Neoplastic tissue is characterized by higher level of immune tolerance in comparison to normal lung tissue.

An 'activatable' aptamer-based fluorescence probe for the detection of HepG2 cells.

It is significant to develop a probe with sensitivity and specificity for the detection of cancer cells. The present study aimed to develop an ‘activatable’ aptamer-based fluorescence probe (AAFP) to detect cancer cells and frozen cancer tissue. This AAFP consisted of two fragments: aptamer TLS11a that targets HepG2 cells, and two short extending complementary DNA sequences with a 5′- and 3′-terminus that make the aptamer in hairpin structure a capable quencher to fluorophore. The ability of the AAFP to bind specifically to cancer cells was assessed using flow cytometry, fluorescence spectroscopy and fluorescence microscopy. Its ability to bind to frozen cancer tissue was assessed using fluorescence microscopy. As a result, in the absence of cancer cells, AAFP showed minimal fluorescence, reflecting auto-quenching. In the presence of cancer cells, however, AAFP showed a strong fluorescent signal. Therefore, this AAFP may be a promising tool for sensitive and specific detection of cancer.

Abstract LB-251: Comparison of MicroRNA deep sequencing of matched formalin-fixed paraffin-embedded and fresh frozen cancer tissues.

Abstract Purpose/Objective(s): MicroRNAs regulate several aspects of tumorigenesis and cancer progression. Most cancer tissues are archived formalin-fixed and paraffin-embedded (FFPE). While microRNAs are a more stable form of RNA thought to withstand FFPE-processing and degradation there is only limited evidence for the latter assumption. We examined whether microRNA profiling can be successfully conducted on FFPE cancer tissues using SOLiD ligation based sequencing. Materials/Methods: Nine paired human malignant tissue samples were received. Remnant surgical tissues were taken after diagnostic samples were secured from patients (five males and four females; median age 58 years, range 39-78 years) with breast invasive ductal carcinoma (2), renal clear cell carcinoma (2), lung squamous cell carcinoma (1) and adenocarcinoma (1), prostate adenocarcinoma (1), metastatic melanoma (1), and sarcoma of thigh (1). RNA from FFPE samples were extracted using Recover All Total Nucleic Acid Isolation Kit (Life Technologies Corporation, Carlsbad, CA). Small RNAs from FFPE and fresh frozen samples were prepared for SOLiD (Version 4) system, and reads data were analyzed by using the SOLiD™ small RNA Analysis pipeline (Life Technologies), then normalized as reads per million (RPM). Results: Tissue storage times (2-9 years) appeared to not affect the number of detected microRNAs in FFPE samples compared to matched frozen samples (paired t-test p&amp;gt;0.7). Correlations of microRNA expression values were very high across microRNAs in a given sample (Pearson's r=0.74-0.94). Higher variance of expression values among samples was associated with higher correlation coefficients between FFPE and frozen tissues. One of the FFPE samples in this study was degraded for unknown reasons with a peak read length of 17 nucleotides compared to 21 in all other samples. The number of detected microRNAs in this sample was within the range of microRNAs detected in all other samples. Conclusion: Ligation-based microRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed in our study appears to not affect the number of microRNAs that can be quantified. Citation Format: Wei Meng, Joseph McElroy, Stefano Volinia, Jeff Palatini, Sarah Warner, Leona Ayers, Kamalakannan Palanichamy, Arnab Chakravarti, Tim Lautenschlaeger. Comparison of MicroRNA deep sequencing of matched formalin-fixed paraffin-embedded and fresh frozen cancer tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-251. doi:10.1158/1538-7445.AM2013-LB-251

Vimentin expression is associated with decreased survival in gastric cancer

Gastric cancer rich in stromal tissue, such as diffuse-type disease, carries a poor prognosis. In some cancers, expression of vimentin, a mesenchymal maker, is associated with poor survival. The expression of mesenchymal markers such as vimentin is observed after epithelial-mesenchymal transition (EMT), an important initial behavioral change related to the adhesion and migration properties of tumor cells that is required for local tumor invasion. A hallmark of EMT is the loss of E-cadherin. EMT-inducing regulators, including SIP1, Slug, and Twist, repress E-cadherin transcription by interacting with E-cadherin promoter. We investigated the expression of vimentin and EMT-related genes, including SIP1, Slug, and Twist, in frozen cancer tissues and normal tissues by real-time quantitative reverse-transcriptase polymerase chain reaction. Tumor samples were obtained from 106 patients with gastric adenocarcinomas who underwent a gastrectomy. The relation of the expression of these genes to clinicopathological factors and outcomes was studied. Vimentin mRNA was significantly higher in diffuse type compared to intestinal type according to Lauren's classification (p=0.048) and was significantly elevated in patients with recurrent or distant metastatic disease (p=0.049). Immunohistochemically, however, vimentin was detected only in cancer stroma. Twist mRNA expression significantly correlated with tumor depth (p=0.042) and advanced tumor stage (I-II vs. III-IV, p=0.030). E-cadherin immunohistochemical expression was significantly associated with Lauren's histopathological type (p<0.001). Univariate analysis of relapse-free survival showed that tumor depth, lymph node metastasis, Lauren's histopathological type, and vimentin mRNA expression were significant prognostic factors (p<0.001, p=0.013, p=0.011, and p=0.019). On multivariate analysis, vimentin mRNA expression was an independent prognostic factor [hazard ratio (HR)=2.1; 95% confidence interval (CI), 1.0-4.4; p=0.036], coming after tumor depth (HR=9.7; 95%CI, 3.7-24; p<0.001). Vimentin mRNA expression is associated with recurrence or distant metastasis and decreased survival in gastric cancer.