The expression of hematopoietic progenitor cell antigen CD34 is regulated by DNA methylation in a site-dependent manner in gastrointestinal stromal tumours.

The anatomic site-dependent expression of hematopoietic progenitor cell antigen CD34 is a feature of gastrointestinal stromal tumours (GISTs). The basis for the differential CD34 expression is only incompletely understood. This study aimed at understanding the regulation of CD34 in GISTs and clarification of its site-dependent expression. Two sample sets of primary GISTs were interrogated including 52 fresh-frozen and 134 paraffin-embedded and formalin-fixed specimens. DNA methylation analysis was performed by HumanMethylation450 BeadChip array in three cell lines derived from gastric and intestinal GISTs, and differentially methylated CpG sites were established upstream of CD34. The methylation degree was further quantified by pyrosequencing, and inverse correlation with CD34 mRNA and protein abundance was revealed. The gene's expression could be activated upon induction of DNA hypomethylation with 5-aza-2'-deoxycytidine in GIST-T1 cells. In patient samples, a strong inverse correlation of DNA methylation degree with immunohistochemically evaluated CD34 expression was documented. Both CD34 expression and DNA methylation levels were specific to the tumours' anatomic location and mutation status. A constant decrease in methylation levels was observed ranging from almost 100% hypermethylation in intestinal GISTs from duodenum to hypomethylation in rectum. CD34 was heavily methylated in gastric PDGFRA-mutant GISTs in comparison to hypomethylated KIT-mutant counterparts. Next to CD34 hypermethylation, miR-665 was predicted and experimentally confirmed to target CD34 mRNA in GIST-T1 cells. Our results suggest that CD34 expression in GISTs may undergo a complex control by DNA methylation and miR-665. Differential methylation and expression of CD34 in GISTs along the gastrointestinal tract axis and in tumours that harbour different gain-of-function mutations suggest the origin from different cell populations in the gastrointestinal tract.

Low densities of immune cells indicate unfavourable overall survival in patients suffering from squamous cell carcinoma of the lung.

Carcinogenesis and tumor proliferation are characterized by a complex interaction of cancer cells with the tumor microenvironment. In particular, a tumor-promoting effect can be assumed for the stroma and its fibroblasts. An influence of the immune system on non small cell lung cancer (NSCLC) is now also suspected. In our study, we examined 309 sections of squamous cell carcinoma (SCC), a subtype of NSCLC. We determined the cell densities and areas of the different tissues in SCC using the software QuPath. Spearman rank correlation showed a significant positive correlation between the different tumor cell densities and stromal cell densities, and between tumor cell densities and immune cell densities. Overall survival curves by the Kaplan-Meier method revealed a prominent negative curve in cases of low immune cell density. Based on our results, we can assume a positive influence of the tumor microenvironment, especially the stromal cells, on tumor proliferation in SCC. We have also revealed that low density of immune cells is prognostically unfavorable.

CC-Chemokine Ligand 18 Is an Independent Prognostic Marker in Lymph Node-positive Non-small Cell Lung Cancer.

BACKGROUND/AIM: Tumor-associated macrophages (TAMs) are key players in the immune response in non-small cell lung cancer (NSCLC) and the main producers of CC-chemokine ligand 18 (CCL18). Our study aimed to analyze the clinical significance of CCL18 expression by TAMs in NSCLC. MATERIAL AND METHODS: Tissue multi-arrays from 243 non-selected patients with NSCLC were constructed. Immunohistochemical double staining for CD68 and CCL18 was performed and the number of CD68+, as well as CCL18+/CD68+ macrophages determined. RESULTS: Comparison of early to advanced lung adenocarcinoma showed significantly more frequent CD68+ as well as CD68/CCL18 double-positive macrophages in advanced disease (p=0.03 and p=0.04). Multivariate analysis revealed a higher proportion of double-positive macrophages to be an independent prognosticator in lymph node-positive NSCLC (hazard ratio(HR)=0.6, 95% confidence interval(CI)=0.35-0.86, p=0.009). CONCLUSION: In advanced lung adenocarcinoma, infiltration of CCL18+ TAMs was increased and higher expression of CCL18 by TAMs was associated with a favorable prognosis in lymph-node positive NSCLC.

Overexpression of SLC1a5 in lymph node metastases outperforms assessment in the primary as a negative prognosticator in non-small cell lung cancer.

Despite recent advances in therapeutic options, lung cancer is the leading cause of death among malignant diseases worldwide. Glutamine-dependence is an established attribute in cancer tissue with emerging importance as a diagnostic and therapeutic target. We analysed the expression of SLC1a5, a major glutamine transporter, in the primary tumour and corresponding nodal metastasis of non-small cell lung cancer (NSCLC) to investigate its biological impact. Expression of SLC1a5 was analysed by immunohistochemistry in 259 NSCLC and in 142 nodal metastases and correlated with clinicopathological parameters including overall survival. SLC1a5 expression in the primary tumour and in the corresponding lymph node metastasis revealed a positive correlation (p = 0.005). Moreover, overexpression of SLC1a5 was found to be an independent prognostic factor (p = 0.027) if assessed in lymph node metastases only. SLC1A5 expression was studied for the first time in both primary NSCLC and its corresponding nodal metastasis. Our results indicate that overexpression of SLC1a5 is associated with shorter overall survival. This proved to be an independent prognosticator if assessed in the lymph node metastases. Thus, diagnostics in lymph node metastasis provide superior prognostic information for SLC1a5 overexpression and may open target prediction for future therapeutic options.

Carcinosarcoma of the Pancreas: Case Report With Comprehensive Literature Review.

Carcinosarcomas are rare biphasic neoplasms with distinct malignant epithelial and mesenchymal components. Most commonly, carcinosarcomas arise in the uterus as malignant mixed müllerian tumors, but also infrequently appear in other organs such as the ovaries and breast, the prostate and urinary tract, the lungs, or in the gastrointestinal system, among others. Pancreatic carcinosarcomas are exceedingly rare; only a few cases are reported in the English literature. Their pathogenesis remains to be fully clarified. We present here the case of a pancreatic carcinosarcoma with evidence for monoclonality via determination of Kras mutational status after microdissection and suggest a common origin of the 2 tumor components. Comprehensive review of the available literature allows the conclusion that most pancreatic carcinosarcomas appear to be of monoclonal origin and seem to have arisen from a carcinoma via metaplastic transformation of 1 part or subclone of the tumor, probably by epithelial-mesenchymal transition. All reported patients were treated with surgery. Adjuvant therapy, if administered, consisted predominantly of gemcitabine. Prognosis for this neoplasm occurs to be similar or even worse compared with classic pancreatic ductal adenocarcinoma. Despite the lack of evidence-based recommendations for its treatment, resection should be performed, if possible.

Epigenetic Regulation of CD133 in Gastrointestinal Stromal Tumors.

OBJECTIVES: This study ascertained the regulation of the stem cell marker CD133 and its potential applicability for prognostication of gastrointestinal stromal tumors (GISTs). METHODS: A total of 95 resected GISTs were included in the study. CD133 protein expression was assessed immunohistochemically on tissue microarrays. Methylation percentage was quantified by pyrosequencing. Gene expression in cell lines GIST48b and GIST882 upon treatment with DNA demethylation agent 5-aza-2'-deoxycytidine was analyzed by quantitative polymerase chain reaction. RESULTS: The expression of hypermethylated CD133 could be reactivated in the GIST cell line upon hypomethylation with the drug. Similarly, in patient material, CD133 methylation percentage correlated inversely with the protein expression and reflected tumor size with hypermethylation in small (<2 cm) tumors and virtually no methylation in large (>10 cm) GISTs. The gene's methylation percentage and expression level were clearly specific to anatomic sites and distinct driver mutations. KIT -mutant gastric GISTs exhibited significantly lower methylation degrees and concomitant high CD133 protein abundance compared with KIT -mutant GISTs from the small intestine. CD133 hypermethylation was documented in PDGFRA -mutant gastric GISTs along with low CD133 expression compared with KIT -mutant gastric GISTs. High CD133 expression was a prognosticator of shorter disease-free survival in all patients. In a subgroup of KIT -mutant gastric GISTs, low CD133 methylation degree was correlated with a shorter disease-free survival. CONCLUSIONS: Our results strongly suggest epigenetic regulation of CD133 expression by promoter methylation in GISTs. Pending further validation studies, high abundance of the protein can serve as a marker for malignant GISTs.

Inactivating Mutations of RB1 and TP53 Correlate With Sarcomatous Histomorphology and Metastasis/Recurrence in Gastrointestinal Stromal Tumors.

OBJECTIVES: Loss-of-function mutations in TP53 and CDKN2A have been found at varying frequencies in gastrointestinal stromal tumors (GISTs), while no mutations of RB1 have been reported to date. The aim of the current study was to determine the mutation frequency of TP53, RB1, and CDKN2A in GISTs. METHODS: A cohort of 83 primary untreated GISTs was analyzed for mutations in TP53, RB1, and CDKN2A by massive parallel sequencing. Tumors with mutations in TP53 and RB1 were analyzed by fluorescence in situ hybridization for the corresponding gene loci. RESULTS: Two GISTs harbored inactivating mutations in RB1, and two other GISTs displayed inactivating mutations in TP53 All four tumors were KIT mutant high-risk tumors with highly cellular sarcomatous histomorphology and variable combinations of plump spindle cells to epithelioid highly atypical cells and high mitotic activity. Three of these patients developed recurrent or metastatic disease, while the fourth patient showed tumor rupture intraoperatively. The combined overall frequency of TP53 and RB1 mutations was 13% considering high-risk or malignant GISTs. CONCLUSIONS: TP53 and RB1 mutations seem to be restricted to high-risk/malignant GISTs and occur at an equal although relatively low frequency.

Amendment of biosolids with waste materials and lime: Effect on geoenvironmental properties and leachate production.

Residuals from wastewater treatment operations (biosolids) were mixed with lime, fly ash, lime kiln dust, or two smelter slags to assess their efficacy as potential stabilisation agents by assessing their effects on the shear strength, compressibility, and solids content of mixtures. In addition, the minerals formed and leachate produced during stabilisation were determined. Tests were performed to explore the change of the geoenvironmental properties of the amended biosolids, while under pressure, at different scales using laboratory, pilot and field scale tests. The settlement characteristics of the amended biosolids under a range of applied pressures were determined using a consolidometer. All amended biosolids mixtures showed higher strength than the unamended biosolids, with mixtures containing a combination of 20% fly ash and 20% lime giving the highest (up to eightfold) increase in strength, and that with lime kiln dust and the smelter slags showing the lowest (up to twofold). The biosolids mixtures with only lime gave the second highest increase in strength (up to fourfold), but produced the largest amount of leachate, with higher level of dissolved calcium. The increase in strength correlated with availability of calcium oxide in the mixtures which lead to calcium carbonate formation, accompanied with higher leachate production and settlement during consolidation. Copper, nickel and zinc concentrations increased with alkaline additives and corresponded to higher pH and DOC levels. Nonetheless, concentrations were within the New Zealand regulatory limits for Class A landfills.

Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly.

BACKGROUND: Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient's prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules '(i.e. nucleotides, aminoacids, fatty acids). In cancer, Malic enzyme (ME) and ATP-citrate lyase (ACLY) are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biological and prognostic significance in non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: ME and ACLY expression was analyzed in 258 NSCLC in correlation with clinico-pathological parameters including patient's survival. RESULTS: Though, overall expression of both enzymes correlated positively, ACLY was associated with local tumor stage, whereas ME correlated with occurrence of mediastinal lymph node metastases. Young patients overexpressing ACLY and/or ME had a significantly longer overall survival. This proved to be an independent prognostic factor. This contrasts older NSCLC patients, in whom overexpression of ACLY and/or ME appears to predict the opposite. CONCLUSION: In NSCLC, ME and ACLY show different enzyme expressions relating to local and mediastinal spread. Most important, we detected an inverse prognostic impact of ACLY and/or ME overexpression in young and elderly patients. It can therefore be expected, that treatment of NSCLC especially, if targeting metabolic pathways, requires different strategies in different age groups.