Tissue Microarray-based Immunohistochemistry Research Articles

Concomitant expression patterns of CDX2 and SATB2 as prognostic factors in stage III colorectal cancers

CDX2 and SATB2 are often used as biomarkers for identification of colorectal origin in primary or metastatic adenocarcinomas. Loss of CDX2 or SATB2 expression has been associated with poor prognosis in patients with colorectal cancer (CRC). However, little is known regarding clinicopathological features, including prognosis, of CRCs with concomitant loss of CDX2 and SATB2.A total of 431 stage III CRCs were analyzed for their expression status in CDX2 and SATB2 using tissue microarray-based immunohistochemistry and expression status was correlated with clinicopathological variables, molecular alterations, and survival.CDX2-negative (CDX2–) CRCs and SATB2-negative (SATB2–) CRCs were found in 8.1 % and 17.2 % of CRCs, respectively, whereas both CDX2-negative and SATB2-negative (CDX2–/SATB2–) CRCs comprised 3.2 % of the CRCs. On survival analysis, neither CDX2–/SATB2+ nor CDX2+/SABT2– CRCs but CDX2–/SATB2– CRCs were associated with poor prognosis. CDX2–/SATB2– CRCs showed significant associations with tumor subsite of right colon, poor differentiation, decreased expression of CK20, aberrant expression of CK7, CIMP-high, MSI-high, and BRAF mutation.In summary, our results suggest that concomitant loss of CDX2 and SATB2 is a prognostic biomarker but isolated loss of CDX2 or SATB2 is not a prognostic biomarker for stage III CRCs.

Bulk and Single-Cell Transcriptome Analyses Revealed That the Pyroptosis of Glioma-Associated Macrophages Participates in Tumor Progression and Immunosuppression.

Glioma is the most common of all central nervous system (CNS) malignancies and is associated with a poor prognosis. Pyroptosis has been proven to be associated with the progression of multiple tumors and CNS diseases. However, the relationships between pyroptosis and clinical prognosis and immune cell infiltration are unclear in glioma. In this study, we conducted a comprehensive exploration of pyroptosis in glioma. First, prognosis-related genes were screened at each key regulatory locus in the pyroptosis pathway, and the prognostic ability and coexpression relationships of GSDMD and its upstream pathway genes NLRC4/CASP1/CASP4 were identified and well validated in multiple datasets. Tissue microarray-based immunohistochemistry results showed higher levels of NLRC4 and N-terminal GSDMD in high-grade gliomas, providing conclusive evidence of pyroptosis in gliomas. The robustness of the prognostic model based on these four genes was well validated in TCGA and CGGA cohorts. Bulk RNA-seq-based analysis showed that the group defined as the high-risk group according to the model showed activation of multiple inflammatory response pathways and impaired synaptic gene expression and had a higher infiltration of bone marrow-derived macrophages (BMDMs) and a hypersuppressed immune microenvironment. More importantly, three independent single-cell RNA-seq (scRNA-seq) datasets demonstrated that tumor-infiltrating macrophages, particularly BMDMs but not tissue-resident microglia, showed significant coexpression of the GSDMD and CASP genes, and BMDMs from high-grade gliomas accounted for a higher proportion of immune infiltrating cells and had higher expression of pyroptosis genes. Finally, we revealed the activation of pathways in response to LPS/bacteria and oxidative stress during BMDM development toward the pyroptosis cell fate by pseudotime trajectory analysis, suggesting potential BMDM pyroptosis initiators. The above results provide not only novel insights into the pathological mechanisms of glioma but also novel therapeutic targets for glioma, suggesting the potential application of pyroptosis inhibitors (e.g., disulfiram).

E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

BackgroundRecent studies have indicated that some members of the tripartite motif (TRIM) proteins function as important regulators for non-small cell lung cancer (NSCLC), However, the regulatory mechanism underpinning aberrant expression of TRIM in NSCLC remains unclear. Here we report that TRIM15 plays important roles in NSCLC progression through modulating Keap1-Nrf2 signaling pathway.MethodsTRIM15 expression was evaluated by western blot analysis, tissue microarray-based immunohistochemistry analysis. The interactions between TRIM15 and Keap1 were analyzed by co-immunoprecipitation (Co-IP) and immunofluorescence co-localization assay. The correlation between TRIM15 and Keap1 was measured by Co-IP and ubiquitination analysis in vitro. Gain- and lost-of-function experiments were used to detect TRIM15 promotes proliferation and invasion of NSCLC cells both in vitro and vivo.ResultsHere, we revealed that TRIM15 was frequently upregulated in NSCLC samples and associated with poor prognosis. Functionally, TRIM15 knockdown resulted in decreased cancer cell proliferation and metastasis, whereas ectopic TRIM15 expression facilitated tumor cancer cell proliferation and metastasis in vitro and in vivo. Moreover, TRIM15 promoted cell proliferation and metastasis depends on its E3 ubiquitin ligase. Mechanistically, TRIM15 directly targeted Keap1 by ubiquitination and degradation, the principal regulator of Nrf2 degradation, leading to Nrf2 escaping from Keap1-mediated degradation, subsequently promoting antioxidant response and tumor progression.ConclusionsTherefore, our study characterizes the pivotal roles of TRIM15 promotes NSCLC progression via Nrf2 stability mediated by promoting Keap1 ubiquitination and degradation and could be a valuable prognostic biomarker and a potential therapeutic target in NSCLC.44ozmiCbiGVX1qU1k16dGvVideo

FGFR3 Nuclear Translocation Contributes to Proliferative Potential and Poor Prognosis in Pancreatic Ductal Adenocarcinoma.

Fibroblast growth factor receptor 3 (FGFR3) was revealed to have divergent, even opposite roles in different neoplasms. In pancreatic ductal adenocarcinoma (PDAC), its impact on biological behavior and prognosis was not well elucidated. Fibroblast growth factor receptor 3 was downregulated by RNA interference to explore its impact on cell proliferative proclivity in PDAC cells. Furthermore, tissue microarray-based immunohistochemistry for FGFR3 was performed in 326 patients with PDAC who underwent radical resection, and its clinicopathologic and prognostic implications were then evaluated. First, successful FGFR3 knockdown remarkably decreased its expression, cell proliferation, and S-phase ratio in the cell cycle in 2 PDAC cell lines, BxPC-3 and AsPC-1. Meanwhile, alterations in p-Akt, cyclin D1, cyclin B1, and p21 were also observed. Subsequently, high nuclear FGFR3 expression, but not cytoplasmic, was significantly common in tumor tissues and positively associated with N stage and dismal overall survival in the entire cohort. In addition, nuclear FGFR3 expression was also prognostic in 10 of 14 subsets. Univariate and multivariate Cox regression analyses identified nuclear expression of FGFR3 as an independent prognosticator in the entire cohort. Our data showed that FGFR3 nuclear translocation contributes to cell proliferative potential and predicts poor long-term prognosis in PDAC after surgical resection.

Correlation of mismatch repair deficiency with clinicopathological features and programmed death-ligand 1 expression in thyroid carcinoma.

Mutations in DNA mismatch repair (MMR) genes associated with thyroid carcinoma (TC) have rarely been reported, especially in East Asian populations. We examined tumor tissue from a cohort of 241 patients diagnosed with TC between 2008 and 2020. MMR proteins were detected using tissue microarray-based immunohistochemistry in order to identify MMR-protein-deficient (MMR-D) and MMR-protein-intact (MMR-I) tumors. We retrospectively summarized the clinicopathologic characteristics of patients with MMR-D TC, measured the expression of PD-L1, and recorded overall survival (OS) and other clinical outcomes. In our cohort, there were 18 (7.5%) MMR-D (MLH1, MSH2, MSH6, and PMS2) patients, including 12 with papillary TC (PTC) (6.7%), 2 with poorly differentiated TC (PDTC) (4.7%), and 4 with anaplastic TC (ATC) (22.2%). Half of them (9/18) showed a specific deletion in MSH6, and 6 of them also carried variants in the MSH6 and PMS2 gene. Survival was significantly better in patients with MMR-D ATC than in those with MMR-I tumors (p = 0.033). Four of the 18 MMR-D patients (22%) were found to be PD-L1 positive. Their OS was much shorter than that of PD-L1-negative patients. MMR-D and PD-L1 positivity appear to be associated with clinicopathological characteristics and prognosis in TC. The results indicate that MMR status may have important prognostic significance in TC. Therefore, immune checkpoint inhibitors that target the PD-1/PD-L1 pathway may be a treatment option for TCs.

TCGA molecular classification in endometriosis-associated ovarian carcinomas: Novel data on clear cell carcinoma

BackgroundClear cell and endometrioid ovarian carcinomas (OCC and OEC, respectively) have a presumed origin in endometriosis and share molecular alterations with each other and with their endometrial counterparts. The Cancer Genome Atlas (TCGA)-based molecular classification stratifies endometrial carcinomas into four categories: POLE mutated (POLEmut), mismatch repair deficient (MMRd), p53 abnormal (p53abn) and no specific molecular profile (NSMP) with divergent prognoses. The subsequent studies are indicative that this TCGA classification has some value in OEC, but the knowledge related to OCC is limited. MethodsEndometrial carcinoma molecular classification was evaluated and compared in a large series of OCCs (n = 115) and OECs (n = 158). POLE mutation analysis and tissue microarray-based immunohistochemistry for mismatch repair and p53 proteins were performed. ResultsThe distribution to the molecular groups was as follows: POLEmut 0.9%/3.2%, MMRd 3.5%/6.3%, p53abn 20%/30%, and NSMP 76%/60% in OCCs/OECs, respectively. The proportion of NSMP tumors was the largest in both histological types and significantly higher in OCC than OEC (p = 0.009). The molecular classification correlated significantly with DSS in both OCCs and OECs (p < 0.001 and p = 0.009, respectively), and with DFS in OCCs (p = 0.001). POLEmut and MMRd OCCs carried excellent prognosis, whereas MMRd OECs presented with poorer outcome. The p53abn group was associated with the poorest prognosis in both tumor types, particularly emphasized in OCCs. ConclusionsTCGA molecular classification associated with patient outcome in both histotypes, and the difference in prognosis between the molecular groups was even more marked in OCC. The large amount of NSMP tumors highlights the need for further studies.

Knockdown of LRRN1 inhibits malignant phenotypes through the regulation of HIF-1α/Notch pathway in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most refractory and fatal human malignancies. Leucine-rich repeat neuronal protein-1 (LRRN1) plays a crucial role in the development of the nervous system. However, the clinical implications and biological functions of LRRN1 in PDAC remain unclear. We found that LRRN1 expression was upregulated in PDAC tissues compared with paracancerous tissues and normal pancreatic tissues through the different public databases, tissue microarray-based immunohistochemistry, and dimethylbenzanthracene-induced PDAC murine model. The expression level of LRRN1 was closely related to the overall survival and disease-free survival of PDAC patients. Cox multivariate analysis indicated that LRRN1 was an independent adverse prognostic factor. The small hairpin RNA (shRNA)-mediated LRRN1 knockdown remarkably restrained the proliferative, migratory, and invasive capacities, as well as promoted cell apoptosis and increased G0/G1 arrest in PDAC cells. The xenograft murine subcutaneous bearing model and metastasis model verified that silencing of LRRN1 effectively dampened tumor growth and metastasis in vivo. Specifically, LRRN1 exerted its biological functions through the HIF-1α/Notch signaling pathway, and LRRN1 knockdown could dampen Jagged 1-mediated Notch pathway activation. Therefore, LRRN1 could serve as the potential therapeutic or prognostic target for PDAC.

Upregulated CD58 is associated with clinicopathological characteristics and poor prognosis of patients with pancreatic ductal adenocarcinoma

BackgroundCD58 has been demonstrated to be abnormally expressed in multiple hematopoietic malignancies and solid tumors and plays an essential role in tumorigenesis and progression; however, its clinical significance and prognostic value in pancreatic ductal adenocarcinoma (PDAC) remain unknown.MethodsBased on diverse online public databases and 81 PDAC samples of tissue microarray-based immunohistochemistry (IHC), we evaluated CD58 expression in PDAC patients and analyzed its association with clinicopathological characteristics, clinical outcomes, and infiltration of immune cells in PDAC. Furthermore, the correlation between CD58 and the cancer stem cell (CSC)-related, epithelial–mesenchymal transition (EMT)-related, and immune-related markers were detected. Besides, the functional enrichment analysis and related pathways were analyzed and visualized.ResultsCD58 expression was elevated in pancreatitis and PDAC tissues than normal pancreas or adjacent nontumor tissues. The positive cases of CD58 (e.g. more than 50% positive cells) in PDAC account for 95.06% (77/81). Upregulated CD58 in cancer tissues was associated with worse histological grade, larger tumor size, and poorer overall survival and disease-free survival in PDAC patients. Furthermore, Cox multivariate regression analysis revealed that CD58 was an independent prognostic factor in PDAC. CD58 expression was correlated with infiltrations of neutrophils, CD8+ T cells, and dendritic cells (DCs). In addition, correlation gene analysis indicated that CD58 expression was strongly correlated with immune-related, EMT-related, and CSC-related markers. Functional enrichment analysis and KEGG pathway manifested that CD58 might be involved in PDAC initiation and progression.ConclusionsCD58 expression is upregulated in PDAC tissues and its high expression is notably related to poor survival of PDAC. Therefore, CD58 may serve as a novel and effective marker for predicting the prognosis of PDAC patients.

Prognostic significance of miR-203 and ZEB1 expression in early-stage hepatocellular carcinoma.

Background: Approximately one-quarter of patients with early-stage hepatocellular carcinoma (HCC) suffer from tumor recurrence within the first year after hepatectomy. Identification of patients at high risk of recurrence and new therapeutic approaches are crucial to improve clinical outcome. This study aimed to assess the prognostic significance of miR-203 and Zinc finger E-box binding homeobox 1 (ZEB1) in early-stage HCC and explore the association between the expression of ZEB1 and miR-203 in HCC.Methods: Tissue microarray-based immunohistochemistry (IHC) and in situ hybridization (ISH) were performed to investigate ZEB1 and miR-203 expression in 73 patients with early-stage HCC and their correlation with clinicopathological features and prognosis of patients were analyzed. The prognostic value of the two factors was also measured by public KM plotter database. Quantitative reverse transcription PCR (qRT-PCR) assays were conducted to study the relationship between miR-203 and ZEB1. Transwell assays, Cell Counting Kit-8 (CCK-8) assays were performed to detect the roles of miR-203 in migration, invasion and proliferation of HCC cells.Results: We found low expression of miR-203 was associated significantly with tumor recurrence (P<0.001) and poor survival (P=0.020) of patients with early-stage HCC. Multivariate analysis revealed that low miR-203 expression was a poor prognostic factor for both overall survival (OS) (P=0.036) and recurrence free survival (RFS) (P=0.017). ZEB1 did not show any prognostic significance in our cohort. Correlation analysis indicated that there was no significant correlation between miR-203 and ZEB1 on both mRNA and protein levels. Furthermore, functional studies indicated that miR-203 repressed migration, invasion and proliferation of HCC cells in vitro.Conclusion: Our study suggested that miR-203 could be a novel predictor in early-stage HCC and might also be a potential molecular target for HCC therapy.

Malic Enzyme 1 Indicates Worse Prognosis in Breast Cancer and Promotes Metastasis by Manipulating Reactive Oxygen Species.

PurposeMalic enzyme 1 (ME1) catalyzes malate to pyruvate and thus promotes glycolysis. Its function in breast cancer remains to be fully clarified. The aim of this work was to investigate the prognostic value of ME1 and its possible mechanism in breast cancer.MethodsWe evaluated ME1 expression in 220 early breast cancer patients with tissue microarray-based immunohistochemistry and explored the relationships between ME1 expression and clinicopathological features. Survival analyses were further performed to determine its prognostic value. The public database was used to confirm tissue microarray results. Further, cell proliferation, migration, invasion ability and reactive oxygen species (ROS) were examined in breast cancer cells.ResultsIn breast cancer tissues, high ME1 expression was significantly associated with larger tumor size, higher incidence of lymph node metastasis and higher incidence of lymph-vascular invasion. High ME1 expression significantly correlated with worse recurrence-free survival (RFS), and was an independent prognostic factor for RFS, which was confirmed by mRNA results in the public database. In vitro, upregulation of ME1 by transfecting MCF-7 cells with virus vector remarkably enhanced viability, motility and epithelial–mesenchymal transition (EMT) and decreased ROS levels, whereas knockdown in MDA-MB-468 cells produced totally opposite effects as expected. When pretreated with oxidizing agent, MCF-7 cells overexpressing ME1 lost its motility, whereas MDA-MB-468 cells with knockdown of ME1 restored its motility when pretreated with antioxidant.ConclusionTo our knowledge, these clinical and experiment works first suggested that ME1 may be a novel biomarker and potential therapeutic target for breast cancer metastasis, and its biological effect is mainly controlled by manipulating ROS.

Clinicopathological and survival characteristic of mismatch repair status in ovarian clear cell carcinoma.

We sought to explore the expression of mismatch repair (MMR) status and its correlation with clinicopathologic and survival characteristics in ovarian clear cell carcinoma (OCCC). Expression of four MMR proteins (MLH1, PMS, MSH2, and MSH6) were measured using tissue microarray-based immunohistochemistry in 120 OCCC patients. The associations of clinicopathologic parameters with recurrence-free survival (RFS) and overall survival (OS) were analyzed by the Kaplan-Meier method, and multivariate analysis was further performed by the Cox regression model. Overall, 120 OCCC patients met the entry criteria, and their MMR status was detected, consisting of 24 patients with dMMR and 96 patients with proficient MMR (pMMR). Patients with dMMR were strongly associated with platinum-sensitive disease (P = .006) and large tumor volume (P = .038). Among all the patients who have received surgery, tumors with dMMR had a better RFS and OS than those with pMMR (hazard ratio [HR] for recurrence: 0.459 [95% confidence interval {95% CI} = 0.224-0.940], P = .029; HR for death: 0.381 [95% CI = 0.170-0.853], P = .015). In subgroup analysis, dMMR patients experienced a better trend of RFS (HR = 0.273; P = .055) and OS (HR = 0.165; P = .040) than pMMR cases among early stages (I-II), but this difference was not observed in advanced stage (III-IV) patients. Meanwhile, pMMR was associated with a more favorable trend of prognosis than dMMR in platinum-resistant patients (RFS: HR = 0.317, P = .051; OS: HR = 0.370, P = .046). Multivariate analysis revealed that only advanced stages (III-IV) were adverse independent prognosticators for both RFS (HR = 5.938 [95% CI = 2.804-12.574]; P < .001) and OS (HR = 6.209 [95% CI = 2.724-14.156]; P < .001). Tumors with dMMR were related to better OS in OCCC on univariate analysis. Only the tumor stage was an independent prognosticator for both RFS and OS. MMR status is a potentially valuable prognostic index in OCCC patients, and larger prospective studies are required to validate its prognostic role.

Transducin-Like Enhancer of Split-1 Inhibits Malignant Behaviors in vitro and Predicts a Better Prognosis in Pancreatic Ductal Adenocarcinoma.

Background: Transducin-like enhancer of split-1 (TLE1), a member of the Groucho/TLE family of transcriptional corepressors, has been reported to be involved in the tumorigenesis of various cancers and function as a clinical prognostic indicator. However, the mechanisms and prognostic significance of TLE1 in pancreatic ductal adenocarcinoma (PDAC) have not been elucidated.Methods: In this study, western blot analyses and real-time polymerase chain reaction (RT-PCR) were employed to evaluate the expression of TLE1 and related proteins in PDAC cell lines. Wound healing, transwell migration and invasion, and Cell Counting Kit-8 (CCK-8) assays were used to determine cell line-specific differences in metastasis and proliferation. Flow cytometry was performed for cell cycle detection. RNA sequencing and bioinformatics were undertaken to explore the molecular mechanisms and potential targeted molecules of TLE1. TLE1 expression in tumor and para-tumor tissues was evaluated by tissue microarray-based immunohistochemistry using a semiquantitative method (H-score) in 262 patients with radical PDAC resection. Correlation, Kaplan–Meier survival, univariate, and multivariate analyses were also performed.Results: Our findings showed that TLE1 expression was common in PDAC cell lines. Upregulation of TLE1 inhibited PDAC cell migration, invasion, and proliferation in vitro by delaying the G0/G1 transition. Immunohistochemistry revealed that TLE1 was specifically expressed in the nucleus and at higher levels in tumor tissues compared with para-tumor tissues. Generally, high TLE1 expression was associated with no vascular invasion. In univariate analyses, high TLE1 expression was associated with longer disease-specific survival (DSS) in all patients and in 16 patient subgroups. In multivariate analyses, TLE1 expression was independently associated with DSS in all patients and four patient subgroups.Conclusion: In conclusion, these results suggest that TLE1 has an inhibitory role in PDAC progression and is a favorable prognostic indicator for patients with resectable PDAC.

Abstract A057: The MEK1/2 pathway as a therapeutic target in high-grade serous ovarian carcinoma

Abstract Rationale: High-grade serous ovarian carcinoma (HGSOC) is the deadliest of gynecological cancers due to high rates of recurrence and acquired chemoresistance. Recurrent tumors are suggested to originate from cancer stem-like cells (CSCs) which have been shown to have high MEK1/2 activity signaling pathway to survive and proliferate. Present work investigates the potential of selective MEK1/2 inhibition as a CSC-targeting HGSOC therapy approach. Methods: MEK1/2 pathway activity was evaluated in clinical HGSOC tissue samples and ovarian cancer cell lines using tissue microarray-based immunohistochemistry, immunoblotting and RT-qPCR. OVCAR8 and PEO4 HGSOC cell lines were used to assess the effect of MEK1/2 inhibition on cell viability, proliferation rate and stem-like characteristics in vitro. Mouse xenografts were used to investigate MEK1/2 inhibition effect on tumor growth in vivo. Drug washout experimental model was used to study the lasting effects of MEK1/2 inhibition therapy. Results: MEK1/2 signaling is active in majority of HGSOC tissue samples and cell lines and is further stimulated by cisplatin treatment. MEK1/2 inhibitor trametinib drastically inhibits activity of MEK1/2 downstream signaling, causes prominent cell cycle arrest in G1/0-phase in vitro and reduces tumor growth rate in vivo, but does not induce cell death. Cells treated with trametinib display high CD133+ fraction and increased expression of stemness-associated genes. Transient trametinib treatment causes long-term increase in high ALDH1 activity cells that possess capability of surviving and growing in non-adherent conditions. Conclusions: MEK1/2 inhibition in HGSOC cells efficiently inhibits cell proliferation and tumor growth and therefore may be promising approach to suppress ovarian cancer cell growth. As MEK1/2 inhibition promotes stem-like properties, including ALDH activity, it should be used in combination with CSC-targeting drugs, especially ALDH1 inhibitors. Citation Format: Mikhail Chesnokov, Abdelrahman Yousif, ilana Chefetz. The MEK1/2 pathway as a therapeutic target in high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A057. doi:10.1158/1535-7163.TARG-19-A057

Downregulated expression of CFHL1 is associated with unfavorable prognosis in postoperative patients with hepatocellular carcinoma.

Complement factor H-related protein 1 (CFHL1) was recently reported to be a potential biomarker in several types of cancer. CFHL1, however, has not been found to be of prognostic value in hepatocellular carcinoma (HCC) to date. In the present study, the expression levels of CFHL1 were evaluated in 8 pairs fresh frozen tissue samples using western blotting. Furthermore, the expression level of CFHL1 was evaluated in 76 pairs of formalin-fixed, paraffin-embedded (FFPE) HCC and peritumoral tissues (expression pattern cohort), and 278 FFPE HCC tissues (prognostic cohort) using tissue microarray-based immunohistochemistry. The Kaplan-Meier method, Cox regression proportional hazard model and receiver operating characteristic curve analysis were used to evaluate prognostic factors. The expression level of CFHL1 was reduced in HCC tissues in 67.1% (51/76) of the cases compared with the corresponding peritumoral tissues. Survival analyses indicated that patients with HCC with low CFHL1 expression had a worse time-to-recurrence (TTR) and overall survival (OS) compared with those with high CFHL1 expression in the prognostic cohort (P=0.002 for OS and P=0.017 for TTR). Both univariate and multivariate analyses indicated that CHFL1 was an independent prognostic factor for TTR and OS (P=0.017 and P=0.002, respectively). In addition, The Cancer Genome Atlas (TCGA) and Human Protein Atlas were used for further validation. Furthermore, a prognostic model included tumor size, tumor number, liver cirrhosis and CFHL1 expression was evaluated. The results of the present study demonstrated that CFHL1 was downregulated in HCC and its level was associated with patient prognosis; therefore, CFHL1 is a potential prognostic marker for HCC.

Abstract P2-08-45: Malic enzyme 1 is a potential metastasis-related biomarker of breast cancer

Abstract Background: Malic enzyme 1 (ME1) catalyzes malate to pyruvate and thus promotes glycolysis, playing a part in the Warburg effect. Recently, several researches have revealed its crucial role in cancer metastasis. KM-plotter, an online analysis tool, showed high ME1 mRNA level was related to poorer RFS and OS. Herein, we explore the prognostic effect of ME1 on invasive breast cancer in Chinese patients and its effect on metastasis in vitro. Methods: 220 patients with early breast cancer were included in this study. ME1 expression was evaluated semi-quantitatively with tissue microarray-based immunohistochemistry. The relationships between ME1 expression level and clinicopathological features were explored. Survival analyses were carried out by Kaplan-Meier test and COX proportional hazard model. MCF-7 and MDA-MB-468 cell lines were then used for in vitro cell migration and invasion assays. Results: High expression of ME1 was observed in 51.5% patients. The median follow-up period was 28.9 months (range 0.5-34.0). In correlation analyses, compared with ME1-low cases, ME1-high cases were significantly associated with larger tumor size (P=0.036), positive lymph nodes (P&amp;lt;0.001) and positive lymph-vascular invasion (P=0.003), and tended to be HER2 positive (P=0.094). Survival analysis by Kaplan-Meier test showed high ME1 expression was significantly correlated with poorer recurrence free survival (RFS) (P=0.015). Multivariate analysis identified high ME1 expression as an independent prognostic factor for RFS (P=0.035, HR=5.072 [1.121, 22.942]). Stratified analysis revealed high ME1 expression was related to poorer RFS among cases more than 45 years old (P=0.029, HR=9.833 [1.269, 76.164]) and among those with Ki67 index ≥20% (P=0.038, HR=3.805 [1.074, 13.486]). In vitro, HER2 positive cell line (SKBR3) and TNBC cell lines (MDA-MB-231, MDA-MB-468) showed high expression of ME1, while luminal cell line (MCF-7) showed low expression of ME1. Upregulation of ME1 in MCF-7 cell line remarkably enhanced its ability in migration and invasion, while knockdown of ME1 in MDA-MB-468 cell line had a profound inhibitory effect on migration and invasion. Conclusions: ME1 enhances migration and invasion of breast cancer cell lines and may be involved in early development of breast cancer metastasis. Thus, ME1 is a promising prognostic indicator and a potential therapeutic target. Citation Format: Liu C, Tao Z, Hu X. Malic enzyme 1 is a potential metastasis-related biomarker of breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-45.

Epigenetic deregulations of Wnt/β-catenin and transforming growth factor beta-Smad pathways in esophageal cancer: Outcome of DNA methylation.

Promoter methylation of tumor suppressor genes (TSGs) is a well-reported portent in carcinogenesis; hence, it is worthy to investigate this in high-risk Northeast population of India. The study was designed to investigate methylation status of 94 TSGs in esophageal squamous cell carcinoma (ESCC). Further, the effect of OPCML promoter methylation on gene expression was analyzed by immunohistochemistry. Moreover, in silico protein-protein interactions were examined among 8 TSGs identified in the present study and 23 epigenetically regulated genes reported previously by our group in ESCC. Methylation profiling was carried out by polymerase chain reaction array and OPCML protein expression was examined by tissue microarray-based immunohistochemistry. OPCML, NEUROG1, TERT, and WT1 genes were found hypermethylated and SCGB3A1, CDH1, THBS1, and VEGFA were hypomethylated in Grade 2 tumor. No significant change in OPCML expression was observed among control, Grade 1, and Grade 2 tumor. Conclusively, hypermethylation of the studied OPCML promoter in Grade 2 tumor produced no effect on expression. Unexpectedly, OPCML expression was downregulated in Grade 3 tumor in comparison to other groups signifying that downregulation of OPCML expression may lead to higher grade of tumor formation at the time of diagnosis of ESCC in patients. Significant interactions at protein level were found as VEGFA:PTK2, CTNNB1:CDH1, CTNNB1:VEGFA, CTNNB1:NEUROG1, CTNND2:CDH1, and CTNNB1:TERT. These interactions are pertinent to Wnt/β-catenin and TGF-β-Smad pathways. Deranged OPCML expression may lead to high-grade ESCC as well as epigenetically regulated genes, that is, CDH1, CTNNB1, CTNND2, THBS1, PTK2, WT1, OPCML, TGFB1, and SMAD4 may alter the Wnt/β-catenin and TGF-β-Smad pathways in ESCC. Further study of these genes could be useful to understand the molecular pathology of ESCC with respect to epithelial-mesenchymal transition (EMT) mediated by Wnt/β-catenin and TGF-β signaling pathways.

Clinicopathological and prognostic significance of MKK4 and MKK7 in resectable pancreatic ductal adenocarcinoma

Mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7) were shown to regulate biological behavior in many malignancies. In pancreatic ductal adenocarcinoma (PDAC), it remains controversial whether MKK4 and MKK7 have pro-oncogenic or tumor-suppressive activities. Furthermore, their clinicopathological and prognostic implications are unknown. In the present study, we detected MKK4 and MKK7 expressions in the nucleus and cytoplasm of resected PDAC tissues from 321 patients by tissue microarray-based immunohistochemistry. Cytoplasmic MKK4 and MKK7 expressions were significantly downregulated, whereas nuclear MKK4 expression was significantly upregulated in tumor tissues compared with nontumor tissues. Tumor cytoplasmic MKK4 and MKK7 expressions were significantly negatively associated with histologic grade. Cytoplasmic MKK4 expression was also negatively correlated with CA19-9 level. By univariate analysis, high cytoplasmic MKK4 expression was significantly associated with longer cancer-specific survival (hazard ratio [HR], 0.705; 95% confidence interval, 0.510-0.974), with a similar trend observed for MKK7 expression. High MKK4 and MKK7 messenger RNA expressions were significantly associated with longer overall survival in The Cancer Genome Atlas database. Although MKK4 expression was not significant in a multivariate Cox regression analysis, combination of MKK4/MKK7 and pN stage was identified as an independent prognostic indicator and had the lowest HR (HR, 0.308; 95% confidence interval, 0.126-0.752). Furthermore, combined analysis of MKK4 and MKK7 greatly increased the prognostic predictive power. In addition, downregulation of MKK4 or MKK7 increased proliferation of pancreatic cancer cells in vitro. In conclusion, high MKK4 expression and its combination with high MKK7 expression both predicted favorable prognosis in resectable PDAC.

Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through \u03b2-catenin/Erk/Akt signaling in gastric cancer

BackgroundEnzymatically inactive chitinase-like protein CHI3L1 drives inflammatory response and promotes tumor progression. However, its role in gastric cancer (GC) tumorigenesis and metastasis has not yet been fully elucidated. We determined the significance of CHI3L1 expression in patients with GC. We also explored an as-yet unknown receptor of CHI3L1 and investigated the involved signaling in GC metastasis.MethodsCHI3L1 expression was evaluated by immunoblotting, tissue microarray-based immunohistochemistry analysis (n = 100), and enzyme linked immunosorbent assay (ELISA) (n = 150). The interactions between CD44 and CHI3L1 or Interleukin-13 receptor alpha 2 (IL-13Rα2) were analyzed by co-immunoprecipitation, immunofluorescence co-localization assay, ELISA, and bio-layer interferometry. The roles of CHI3L1/CD44 axis in GC metastasis were investigated in GC cell lines and experimental animal model by gain and loss of function.ResultsCHI3L1 upregulation occurred during GC development, and positively correlated with GC invasion depth, lymph node status, and tumor staging. Mechanically, CHI3L1 binding to CD44 activated Erk and Akt, along with β-catenin signaling by phosphorylating β-catenin at Ser552 and Ser675. CD44 also interacted with IL-13Rα2 to form a complex. Notably, CD44v3 peptide and protein, but not CD44v6 peptide or CD44s protein, bound to both CHI3L1 and IL-13Rα2. Our in vivo and in vitro data further demonstrated that CHI3L1 promoted GC cell proliferation, migration, and metastasis.ConclusionsCHI3L1 binding to CD44v3 activates Erk, Akt, and β-catenin signaling, therefore enhances GC metastasis. CHI3L1 expression is a novel biomarker for the prognosis of GC, and these findings have thus identified CHI3L1/CD44 axis as a vital pathway and potential therapeutic target in GC.

Canine Gastrointestinal Spindle Cell Tumors Efficiently Diagnosed by Tissue Microarray-Based Immunohistochemistry.

Tissue microarray (TMA) is a time- and cost-saving technique allowing the simultaneous immunohistochemical evaluation of multiple tissue samples. The aim of this study was to assess the efficacy of TMA at classifying canine gastrointestinal spindle cell tumors as gastrointestinal stromal tumor (GIST), smooth muscle tumor (SMT), and non-GIST/non-SMT based on the expression of α-smooth muscle actin (α-SMA), desmin, and CD117. Thirty-four cases were investigated on TMAs, sampling 2 cores each. Immunohistochemistry was performed on TMAs and full sections, and the results were compared. Comparing full sections, TMA specificity and sensitivity were 100% and 93.8%, respectively, for α-SMA; 100% and 80.8% for desmin; and 100% and 100% for CD117. TMA allowed the identification of 6 of 6 GISTs, 25 of 26 SMTs, and 2 of 2 non-GIST/non-SMTs. One SMT was misdiagnosed as non-GIST/non-SMT. Based on these results, TMA-based immunohistochemistry is efficient at diagnosing canine gastrointestinal spindle cell tumors and might be applied on large caseloads in a research setting.

Low expression of B-Cell-Associated protein 31 is associated with unfavorable prognosis in human colorectal cancer

Colorectal cancer (CRC) is one of the most common cancers worldwide. B cell-associated protein 31 (BAP31) was shown to participate in the apoptosis, and to be an immunotherapy target and a, prognostic factor for cancer, but its role in CRC has not been elucidated. In this study, we examined the expression of BAP31 in CRC to evaluate its prognostic values. We investigated the BAP31 expression level in 142 tissues (108 CRC and 17 paired human adjacent normal mucosa, and 17 liver metastatic CRC tissues) from 108 patients, using tissue microarray-based immunohistochemistry. We further investigated the association between BAP31 expression and overall survival (OS) and disease-free survival (DFS) in 77 CRC patients using Kaplan-Meier analysis. Univariate and multivariate Cox regression analyses were applied to evaluate the potential prognostic value of BAP31 in CRC patients. BAP31 expression level was significantly increased in CRC tissues (p = 0.0014) and liver metastatic CRC tissues (p < 0.0001) compared with corresponding adjacent normal mucosa. BAP31 expression was also significantly increased in liver metastatic CRC tissues compared with corresponding primary CRC tissues (p = 0.0116). Kaplan-Meier analyses showed that CRC patients with low BAP31 expression had significantly lower survival rate (p = 0.001) and lower disease-free survival rate (P = 0.009). Furthermore, multivariate Cox analysis showed that BAP31 was an independent prognostic factor for OS (hazard ratio = 0.410, 95% confidence interval = 0.195–0.862, p = 0.019). ConclusionsOur study demonstrated that BAP31 is a potential prognostic marker for CRC patients after surgery.