Comparative analysis of prognostic histopathologic parameters in subtypes of epithelioid pleural mesothelioma

Aims: Malignant pleural mesothelioma (MPM) is a rare malignancy with a dismal prognosis. While the epithelioid type is associated with a more favourable outcome, additional factors are needed to further stratify prognosis and to identify patients who can benefit from multimodal treatment. As epithelioid MPM shows remarkable morphological variability, the prognostic role of the five defined morphologies, the impact of the nuclear grading system and the mitosis-necrosis score were investigated in this study. Methods and results: Tumour specimens of 192 patients with epithelioid MPM from five European centres were histologically subtyped. Nuclear grading and mitosis-necrosis score were determined and correlated with clinicopathological parameters and overall survival (OS). Digital slides of 55 independent cases from The Cancer Genome Atlas (TCGA) database were evaluated for external validation. Histological subtypes were collapsed into three groups based on their overlapping survival curves. The tubulopapillary/microcystic group had a significantly longer OS than the solid/trabecular group (732 days versus 397 days, P = 0.0013). Pleomorphic tumours had the shortest OS (173 days). The solid/trabecular variants showed a significant association with high nuclear grade and mitosis-necrosis score. The mitosis-necrosis score was a robust and independent prognostic factor in our patient cohort. The prognostic significance of all three parameters was externally validated in the TCGA cohort. Patients with tubulopapillary or microcystic tumours showed a greater improvement in OS after receiving multimodal therapy than those with solid or trabecular tumours. Conclusions: Histological subtypes of epithelioid MPM have a prognostic impact, and might help to select patients for intensive multimodal treatment approaches

A comparison of sample preparation strategies for biological tissues and subsequent trace element analysis using LA-ICP-MS

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is one of the most commonly applied methods for lateral trace element distribution analysis in medical studies. Many improvements of the technique regarding quantification and achievable lateral resolution have been achieved in the last years. Nevertheless, sample preparation is also of major importance and the optimal sample preparation strategy still has not been defined. While conventional histology knows a number of sample pre-treatment strategies, little is known about the effect of these approaches on the lateral distributions of elements and/or their quantities in tissues. The technique of formalin fixation and paraffin embedding (FFPE) has emerged as the gold standard in tissue preparation. However, the potential use for elemental distribution studies is questionable due to a large number of sample preparation steps. In this work, LA-ICP-MS was used to examine the applicability of the FFPE sample preparation approach for elemental distribution studies. Qualitative elemental distributions as well as quantitative concentrations in cryo-cut tissues as well as FFPE samples were compared. Results showed that some metals (especially Na and K) are severely affected by the FFPE process, whereas others (e.g., Mn, Ni) are less influenced. Based on these results, a general recommendation can be given: FFPE samples are completely unsuitable for the analysis of alkaline metals. When analyzing transition metals, FFPE samples can give comparable results to snap-frozen tissues.180518141

Experimental Models to Study Drug Distributions in Tissue Using MALDI Mass Spectrometry

Requirements for patient safety and improved efficacy are steadily increasing in modern healthcare and are key drivers in modern drug development. New drug characterization assays are central in providing evidence of the specificity and selectivity of drugs. Meeting this need, matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) is used to study drug localization within micro-environmental tissue compartments. Thin sections of human lung tumor and rat xenograft tissues were exposed to pharmaceutical drugs by either spotting or submerging. These drugs, the epidermal growth factor receptor antagonists, erlotinib (Tarceva™) and gefitinib (Iressa™), and the acetylcholine receptor antagonist, tiotropium were characterized by microenvironment localization. Intact tissue blocks were also immersed in drug solution followed by sectioning. MALDI-MSI was then performed using a Thermo MALDI LTQ Orbitrap XL instrument to localize drug distribution patterns. We propose three MALDI-MSI models measuring drug disposition that have been used to map the selected compounds within tissue compartments of tumors isolated from lung cancer patients

Heme oxygenase-1 in macrophages controls prostate cancer progression

Innate immune cells strongly influence cancer growth and progression via multiple mechanisms including regulation of epithelial to mesenchymal transition (EMT). In this study, we investigated whether expression of the metabolic gene, heme oxygenase-1 (HO-1) in tumor microenvironment imparts significant effects on prostate cancer progression. We showed that HO-1 is expressed in MARCO-positive macrophages in prostate cancer (PCa) xenografts and human prostate cancers. We demonstrated that macrophage specific (LyzM-Cre) conditional deletion of HO-1 suppressed growth of PC3 xenografts in vivo and delayed progression of prostate intraepithelial neoplasia (PIN) in TRAMP mice. However, initiation and progression of cancer xenografts in the presence of macrophages lacking HO-1 resulted in loss of E-cadherin, a known marker of poor prognosis as well as EMT. Application of CO, a product of HO-1 catalysis, increased levels of E-cadherin in the adherens junctions between cancer cells. We further showed that HO-1-driven expression of E-cadherin in cancer cells cultured in the presence of macrophages is dependent on mitochondrial activity of cancer cells. In summary, these data suggest that HO-1-derived CO from tumor-associated macrophages influences, in part, E-cadherin expression and thus tumor initiation and progression

A protein deep sequencing evaluation of metastatic melanoma tissues.

Malignant melanoma has the highest increase of incidence of malignancies in the western world. In early stages, front line therapy is surgical excision of the primary tumor. Metastatic disease has very limited possibilities for cure. Recently, several protein kinase inhibitors and immune modifiers have shown promising clinical results but drug resistance in metastasized melanoma remains a major problem. The need for routine clinical biomarkers to follow disease progression and treatment efficacy is high. The aim of the present study was to build a protein sequence database in metastatic melanoma, searching for novel, relevant biomarkers. Ten lymph node metastases (South-Swedish Malignant Melanoma Biobank) were subjected to global protein expression analysis using two proteomics approaches (with/without orthogonal fractionation). Fractionation produced higher numbers of protein identifications (4284). Combining both methods, 5326 unique proteins were identified (2641 proteins overlapping). Deep mining proteomics may contribute to the discovery of novel biomarkers for metastatic melanoma, for example dividing the samples into two metastatic melanoma "genomic subtypes", ("pigmentation" and "high immune") revealed several proteins showing differential levels of expression. In conclusion, the present study provides an initial version of a metastatic melanoma protein sequence database producing a total of more than 5000 unique protein identifications. The raw data have been deposited to the ProteomeXchange with identifiers PXD001724 and PXD001725

Genome-wide CpG island methylation analyses in non-small cell lung cancer patients

DNA methylation is part of the epigenetic gene regulation complex, which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stages IIII non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis. Overall, we identified 2414 genomic positions differentially methylated between tumor and non-malignant lung tissue samples. Ninety-seven percent of them were found to be tumor-specifically methylated. Annotation of these genomic positions resulted in the identification of 477 tumor-specifically methylated genes of which many are involved in regulation of gene transcription and cell adhesion. Tumor-specific methylation was confirmed by a gene-specific approach. In the majority of tumors, methylation of certain genes was associated with loss of their protein expression determined by immunohistochemistry. Treatment of NSCLC cells with epigenetically active drugs resulted in upregulated expression of many tumor-specifically methylated genes analyzed by gene expression microarrays suggesting that about one-third of these genes are transcriptionally regulated by methylation. Moreover, comparison of methylation results with certain clinicopathological characteristics of the patients suggests that methylation of HOXA2 and HOXA10 may be of prognostic relevance in squamous cell carcinoma (SCC) patients. In conclusion, we identified a large number of tumor-specifically methylated genes in NSCLC patients. Expression of many of them is regulated by methylation. Moreover, HOXA2 and HOXA10 methylation may serve as prognostic parameters in SCC patients. Overall, our findings emphasize the impact of methylation on the pathogenesis of NSCLCs

Additional file 1: Table S1. of SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

Description of NSCLC cell lines used in this study. Information about histology, origin and disease stage of donors was obtained from ATCC catalogue ( https://www.lgcstandards-atcc.org ). EGFR, KRAS and TP53 mutational status and MET amplification according to supplementary references (1–3). *activating EGFR mutation in exon 19 (E746-E749 del), **activating EGFR mutation in exon 21 (L858R). N/A, not available; wt, wildtype; mut, mutated. Table S2. Clinico-pathological characteristics of 983 NSCLC patients. Overview of gender, histology, stage of disease and ethnicity of NSCLC patients obtained from TCGA database and used for mutation and copy number changes analyses of SPAG6 and L1TD1 is shown. ADC, adenocarcinoma; SCC, squamous cell carcinoma. Clinical data based on Caleydo software version 16/04/14. Table S3. Primer sequences. Summary of oligonucleotide sequences used for mRNA expression, MS-HRM, BGS analyses and construction of pCMV6-GFP expression vector. Y, random integration of C or T in fwd primer; R, random integration of G or A in rev primer. Table S4. Methylation of SPAG6 and L1TD1 in tumor cells of other tumor types. *Morphology, histology and origin of cell lines according to ATCC catalogue ( https://www.lgcstandards-atcc.org ). Percentage of methylation was calculated as described previously (4). (DOCX 33 kb

Additional file 4: Figure S3. of SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

SPAG6 and L1TD1 SNVs and deletions in NSCLC patients. TCGA LUAD and LUSC datasets were analysed with Caleydo software (version April 2014). Mutation of TP53 was used to demonstrate reliability of TCGA data analysis. ADC, adenocarcinoma patients; SCC, squamous carcinoma patients. (TIF 90 kb

Additional file 3: Figure S2. of SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

Impact of SPAG6 and L1TD1 mRNA expression on OS of NSCLC patients. (A) A shorter OS of squamous cell carcinoma patients with low SPAG6 mRNA expression (N = 155) compared to high SPAG6 mRNA expression (N = 267) was observed. (B) Adenocarcinoma patients with low L1TD1 mRNA expression (N = 138) showed a shorter OS compared to adenocarcinoma patients with high L1TD1 mRNA expression (N = 350). Gene expression microarray datasets (Affymetrix IDs 210032_s_at and 219955_at) were analysed and Kaplan-Meier plots were generated using all datasets and default settings of KM plotter. The cut-off values for “low” and “high” SPAG6 and L1TD1 mRNA expression were automatically defined by KM plotter software (Version 2013). (TIF 50 kb