Proteomic analyses identify HK1 and ATP5A to be overexpressed in distant metastases of lung adenocarcinomas compared to matched primary tumors.

Lung cancer is the leading cause of cancer-related deaths worldwide with lung adenocarcinoma (LUAD) being the most common type. Genomic studies of LUAD have advanced our understanding of its tumor biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD are still insufficiently explored. The prognosis for lung cancer patients is still mostly determined by the stage of disease at the time of diagnosis. Focusing on late-stage metastatic LUAD with poor prognosis, we compared the proteomic profiles of primary tumors and matched distant metastases to identify relevant and potentially druggable differences. We performed high-performance liquid chromatography (HPLC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a total of 38 FFPE (formalin-fixed and paraffin-embedded) samples. Using differential expression analysis and unsupervised clustering we identified several proteins that were differentially regulated in metastases compared to matched primary tumors. Selected proteins (HK1, ATP5A, SRI and ARHGDIB) were subjected to validation by immunoblotting. Thereby, significant differential expression could be confirmed for HK1 and ATP5A, both upregulated in metastases compared to matched primary tumors. Our findings give a better understanding of tumor progression and metastatic spreads in LUAD but also demonstrate considerable inter-individual heterogeneity on the proteomic level.

Histone Demethylase KDM5C Drives Prostate Cancer Progression by Promoting EMT.

Prostate cancer (PCa) poses a major public health problem in men. Metastatic PCa is incurable, and ultimately threatens the life of many patients. Mutations in tumor suppressor genes and oncogenes are important for PCa progression, whereas the role of epigenetic factors in prostate carcinogenesis is insufficiently examined. The histone demethylase KDM5C exerts important roles in tumorigenesis. KDM5C has been reported to be highly expressed in various cancer cell types, particularly in primary PCa. Here, we could show that KDM5C is highly upregulated in metastatic PCa. Functionally, in KDM5C knockdown cells migratory and invasion capacity was reduced. Interestingly, modulation of KDM5C expression influences several EMT signaling pathways (e.g., Akt/mTOR), expression of EMT transcription factors, epigenetic modifiers, and miR-205, resulting in increased expression of E-cadherin and reduced expression of N-cadherin. Mouse xenografts of KDM5C knockdown cells showed reduced tumor growth. In addition, the Akt/mTOR pathway is one of the classic signaling pathways to mediate tumor metabolic homeostasis, which is beneficial for tumor growth and metastasis. Taken together, our findings indicate that a combination of a selective KDM5C- and Akt/mTOR-inhibitor might be a new promising therapeutic strategy to reduce metastatic burden in PCa.

PD-L1 expression in carcinoma of the esophagogastric junction is positively correlated with T-cell infiltration and overall survival.

INTRODUCTION: In recent years antitumor immunity and inhibition of checkpoint molecules, such as PD-1 and PD-L1, have emerged as potential therapeutic strategies in advanced stages of various malignancies. We investigated PD-L1 expression in adenocarcinomas of the esophagogastric junction and correlated the results with densitiy of intratumoral T-lymphocytes. METHODS: Immunohistochemical staining for PD-L1 was carried out on 135 samples using a tissue microarray. Scoring was done according to the combined positivity score. RESULTS: 48.1% of tumors (65 cases) showed PD-L1 positivity with a score ≥ 1 while 51.9% were PD-L1 negative (70 cases). A positive correlation between PD-L1 negativity and mucinous and poorly cohesive carcinoma could be shown (p = 0.043), while no association existed for either gender, T-stage, N-stage, grading, surgical resection status, neoadjuvant therapy, distant metastases, lymphovascular or perineural invasion. No correlation of PD-L1 expression and overall survival could be detected (p = 0.497). Again, when stratified according to presence or absence of neoadjuvant therapy, no survival differences could be shown for either group (p = 0.540 and p = 0.736). When PD-L1 expression was correlated with density of tumor-infiltrating T-lymphocytes a positive correlation between PD-L1 positivity and denser T-cell infiltration could be shown (p = 0.001). Concerning overall survival, in PD-L1 negative cases, denser CD8-positive T-cell infiltrates were associated with prolonged survival times (p = 0.045). No differences could be shown for PD-L1 positive cases or CD103-positive T-cells. CONCLUSION: PD-L1 expression is frequent in esophagogastric adenocarcinoma and - when combined with dense CD8 infiltration - PD-L1 negativity correlates with prolonged overall survival.

Density of CD8-positive tumor-infiltrating T-lymphocytes is an independent prognostic factor in adenocarcinoma of the esophagogastric junction.

Tumor-infiltrating lymphocytes (TILs) have commonly been associated with markedly improved prognosis in a variety of human cancers, including carcinomas of the upper and lower gastrointestinal tract. Especially the presence of T-cells (cytotoxic as well as helper cells) seems to define a subgroup of patients with prolonged overall and event-free survival. The density of TILs was assessed via immunohistochemistry for CD8 and CD103 in a population of 228 adenocarcinomas of the esophagogastric junction. Density of CD8+ T-lymphocytes was inversely correlated with depth of tumor infiltration (p=0.013) while no correlation with any of the analyzed clinicopathologic factors could be established for CD103-density. High density of CD8-positive T-cells additionally showed significantly longer overall survival (OS) with a p-value of 0.024 while density of CD103+ cells was associated with prolonged tumor free survival (p-value 0.011). Independence could be demonstrated applying Cox proportional hazard analysis (Hazard Ratio 0.742; 95%-Confidence Interval 0.579-0.951; p=0.019). High density of CD8-positive T-lymphocytes identifies a patient subgroup with significantly prolonged overall survival, is correlated with tumor stage and might open up new therapeutic possibilities via immunomodulating drugs.

MicroRNAs as Potential Biomarkers for Chemoresistance in Adenocarcinomas of the Esophagogastric Junction.

Concerning adenocarcinomas of the esophagogastric junction, neoadjuvant chemotherapy is regularly implemented, but patients' response varies greatly, with some cases showing no therapeutic effect, being deemed as chemoresistant. Small, noncoding RNAs (miRNAs) have evolved as key players in biological processes, including malignant diseases, often promoting tumor growth and expansion. In addition, specific miRNAs have been implicated in the development of chemoresistance through evasion of apoptosis, cell cycle alterations, and drug target modification. We performed a retrospective study of 33 patients receiving neoadjuvant chemotherapy by measuring their miRNA expression profiles. Histologic tumor regression was evaluated using resection specimens, while miRNA profiles were prepared using preoperative biopsies without prior therapy. A preselected panel of 96 miRNAs, known to be of importance in various malignancies, was used to test for significant differences between responsive (chemosensitive) and nonresponsive (chemoresistant) cases. The cohort consisted of 12 nonresponsive and 21 responsive cases with the following 4 miRNAs differentially expressed between both the groups: hsa-let-7f-5p, hsa-miRNA-221-3p, hsa-miRNA-31-5p, and hsa-miRNA-191-5p. The former 3 showed upregulation in chemoresistant cases, while the latter showed upregulation in chemosensitive cases. In addition, significant correlation between high expression of hsa-miRNA-194-5p and prolonged survival could be demonstrated (p value <0.0001). In conclusion, we identified a panel of 3 miRNAs predicting chemoresistance and a single miRNA contributing to chemosensitivity. These miRNAs might function as prognostic biomarkers and enable clinicians to better predict the effect of one or more reliably select patients benefitting from (neoadjuvant) chemotherapy.

MicroRNA analysis of gastroenteropancreatic neuroendocrine tumors and metastases.

The incidence of neuroendocrine neoplasias (NEN) continues to increase. Since the primary tumor cannot be diagnosed in some cases of metastatic disease, new biomarkers are clearly needed to find the most probable site of origin. Tissue samples from 79 patients were analyzed and microRNA profiles were generated from a total of 76 primary tumors, 31 lymph node and 14 solid organ metastases. NEN metastases were associated with elevated levels of miR-30a-5p, miR-210, miR-339-3p, miR-345 and miR-660. Three microRNAs showed a strong correlation between proliferation index and metastatic disease in general (miR-150, miR-21 and miR-660). Further, each anatomic location (primary or metastatic) had one or more site-specific microRNAs more highly expressed in these tissues. Comparison between primary tumors and metastases revealed an overlap only in pancreatic (miR-127) and ileal tumors (let-7g, miR-200a and miR-331). This thorough analysis of gastroenteropancreatic neuroendocrine tumors demonstrates site-specific microRNA profiles, correlation with proliferation indices as well as corresponding nodal and distant metastases. Using microRNA profiling might improve NEN diagnostics by linking metastases to a most probable site of origin.

Expression Pattern of CDX2, Estrogen and Progesterone Receptors in Primary Gastroenteropancreatic Neuroendocrine Tumors and Metastases.

BACKGROUND: A significant number of patients with gastroenteropancreatic neuroendocrine tumors (GEP NETs) present with metastatic disease and with unknown primary in about 15% of cases. MATERIALS AND METHODS: We analyzed 163 primaries of GEP NET and 115 metastases for expression of caudal type homebox 2 (CDX2), estrogen receptor (ER), progesterone receptor (PR), somatostatin receptor 2a (SSTR2a) and Ki67. RESULTS: PR was most often positive in pancreatic NET and only rarely in non-pancreatic NET (p<0.001). ER was more frequently expressed in non-pancreatic NET (p<0.001) and was more often positive in females than males (p=0.019). CDX2 was positive in all primaries of the duodenum, ileum and appendix, but was also detected in 24% of metastases with pancreatic primary. SSTR2a and Ki67 did not differ significantly between primaries and metastases. CONCLUSION: Our data substantiate the value of PR, ER and CDX2 in GEP NET, and steroid hormone receptors, being differentially expressed in male and female patients. Differences between primaries and metastases were small but potentially relevant.

Generation and Characterization of Vascular Smooth Muscle Cell Lines Derived from a Patient with a Bicuspid Aortic Valve.

Thoracic aortic dilation is the most common malformation of the proximal aorta and is responsible for 1%-2% of all deaths in industrialized countries. In approximately 50% of patients with a bicuspid aortic valve (BAV), dilation of any or all segments of the aorta occurs. BAV patients with aortic dilation show an increased incidence of cultured vascular smooth muscle cell (VSMC) loss. In this study, VSMC, isolated from the ascending aorta of BAV, was treated with Simian virus 40 to generate a BAV-originated VSMC cell line. To exclude any genomic DNA or cross-contamination, highly polymorphic short tandem repeats of the cells were profiled. The cells were then characterized using flow cytometry and karyotyping. The WG-59 cell line created is the first reported VSMC cell line isolated from a BAV patient. Using an RT² Profiler PCR Array, genes within the TGFß/BMP family that are dependent on losartan treatment were identified. Endoglin was found to be among the regulated genes and was downregulated in WG-59 cells following treatment with different losartan concentrations, when compared to untreated WG-59 cells.

Erratum: Lazar-Karsten, P., et al. Generation and Characterization of Vascular Smooth Muscle Cell Lines Derived from a Patient with a Bicuspid Aortic Valve. Cells 2016, 5, 19.

The authors wish to make the following erratum to this paper [1].[...].

In vitro proliferation and differentiation of human CD34+ cells from peripheral blood into mature red blood cells with two different cell culture systems.

BACKGROUND: An in vitro erythropoiesis assay is a powerful tool for investigating red blood cell (RBC) development and diseases of the erythroid lineage. Most assays, however, failed in either proliferation or terminal differentiation. Here two liquid cultures (LCs) for in vitro generation of RBCs from peripheral blood CD34+ cells were compared. STUDY DESIGN AND METHODS: Granulocyte-colony-stimulating factor-mobilized CD34+ cells were cultured for 16 days in a two-phase LC (2P-LC; Days 1-8, stem cell factor [SCF], erythropoietin [EPO], insulinlike growth factor [IGF]-1, and steroids; Days 9-16, EPO and insulin) and for 21 days in a three-phase LC (3P-LC; Days 1-7, SCF, thrombopoetin, and Flt3-ligand; Days 8-14, SCF, EPO, and IGF-1; Days 15-21, EPO and IGF-1). Maturation was analyzed by flow cytometry (CD36, CD71, glycophorin A [GPA]) and microscopy. RESULTS: In the 2P-LC, cell numbers increased from 0.5 x 10(6) to 25.7 x 10(6) +/- 15.1 x 10(6) cells per mL. More than 95 percent were GPA+ and showed morphologic characteristics of normoblasts (52 +/- 15%) and enucleated reticulocytes (43 +/- 18%). In the 3P-LC, a higher overall proliferation to 55.7 x 10(6) +/- 37.7 x 10(6) cells per mL was achieved (p < 0.05). This was also accompanied by a high degree of normoblasts (36 +/- 16%) and reticulocytes (48 +/- 24%). The amount of GPA+ cells was slightly lower (88.4 +/- 16.4%), associated with a significantly higher contamination by nonerythroid cells (15.8 +/- 19.3% vs. 3.9 +/- 2.9%, p < 0.05). CONCLUSION: Both LCs were able to generate fully matured RBCs and represent powerful tools for fundamental research in erythroid development and diseases targeting the erythroid lineage. A slightly higher proliferation was achieved in the 3P-LC. This was associated with a limited homogeneity due to more nonerythroid cells, however. Therefore the 2P-LC is favored, also saving additional culture days and growth factors.