Identification of protein signatures of genomic stability/instability in epithelial cancers

Aneuploidy is a consistent genetic alteration of the cancer genome. At early steps in the sequence of malignant transformation during human tumorigenesis, chromosomal aneuploidies can be the first detectable genetic aberrations found. Since late diagnosis results in a significant reduction of average survival times, it is of high interest to elucidate proteins and pathways of genomic instability or stability. This may also give new insights into tumorigenesis and reveal clinically relevant targets for improved diagnostics and therapeutics. The high variability of protein expression complicates the clinical application of ‘proteomics’, but continuous improvements in instrumentation, analytical methodologies, and labeling chemistries nowadays allow the thorough study and detection of tissue protein biomarkers. In addition, the need of strict quality controls of samples and clinical validation in large patient cohorts is important to transfer novel biomarkers into clinical use. In this manner, we detected genomic instability-specific protein alterations in colorectal and endometrial malignancies as detailed in this thesis. A comprehensive proteomic analysis of diploid and aneuploid colorectal cell lines and clinical tissues was carried out. We found that two proteins, TXNL1 and HDAC2, were not only significantly expressed in our 2-DE analysis and validated by Western blotting, but showed expression differences also in clinical samples discerning aneuploid from diploid carcinomas. We then analyzed protein expression patterns between normal endometrium and endometrial carcinomas that profoundly differed in their degree of genomic instability and their histopathologic subtype. We detected 121 ploidy-associated proteins to be differentially expressed. Interestingly, one protein, TXNL1, was expressed at low level in both aneuploid colorectal and endometrial malignancies. In order to identify the impact of chromosomal aberrations onto the protein expression in the clinical setting, we further mapped genomic imbalances with associated gene and protein expression changes of the endometrial cancer patients. AKR7A2 and ANXA2 were identified to show similar trends of changes at the gene and protein levels. We conclude that the grade of genomic instability correlates with a recurrent pattern of chromosomal imbalances and dominates specific gene and protein expression changes, irrespective of the histopathological subtypes in endometrial cancers. To further elucidate the relevance of highly conserved chromosomal aberrations in colorectal cancer, we used specific chromosomal aneuploidies in cancer cells and dissected the consequences of genomic imbalances on the proteome: three artificial trisomic clones of the diploid colorectal cancer cell line DLD1 were analyzed. Chromosomal aneuploidies resulted in a significant increase in the average translational activity of proteins encoded by genes not located on the trisomic chromosome, indicating pathway-related regulation of the cellular proteome equilibrium. A malignancy related protein-profile could also be identified for colorectal cancer: 31 proteins showed differential expression between normal mucosa and colorectal carcinomas in general, while 39 polypeptides were distinctly expressed between diploid and aneuploid carcinomas only. Overall, distinct genes and proteins not known to be associated with colorectal and endometrial cancer or genomic (in)stability will likely reveal novel targets involved in personalized medicine

Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuickTM Isolation

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuickTM in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuickTM. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuickTM compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs' protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached

PD-L1 is expressed on human platelets and is affected by immune checkpoint therapy

Cancer immunotherapy has been revolutionised by drugs that enhance the ability of the immune system to detect and fight tumors. Immune checkpoint therapies that target the programmed death-1 receptor (PD-1), or its ligand (PD-L1) have shown unprecedented rates of durable clinical responses in patients with various cancer types. However, there is still a large fraction of patients that do not respond to checkpoint inhibitors, and the challenge remains to find cellular and molecular cues that could predict which patients would benefit from these therapies. Using a series of qualitative and quantitative methods we show here that PBMCs and platelets from smokers and patients with head and neck squamous cell carcinoma (HNSCC) or lung cancer express and up-regulate PD-L1 independently of tumor stage. Furthermore, treatment with Atezolizumab, a fully humanised monoclonal antibody against PD-L1, in 4 patients with lung cancer caused a decrease in PD-L1 expression in platelets, which was restored over 20 days. Altogether, our findings reveal the expression of the main therapeutic target in current checkpoint therapies in human platelets and highlight their potential as biomarkers to predict successful therapeutic outcomes

PD-L1 is expressed on human platelets and is affected by immune checkpoint therapy

Cancer immunotherapy has been revolutionised by drugs that enhance the ability of the immune system to detect and fight tumors. Immune checkpoint therapies that target the programmed death-1 receptor (PD-1), or its ligand (PD-L1) have shown unprecedented rates of durable clinical responses in patients with various cancer types. However, there is still a large fraction of patients that do not respond to checkpoint inhibitors, and the challenge remains to find cellular and molecular cues that could predict which patients would benefit from these therapies. Using a series of qualitative and quantitative methods we show here that PBMCs and platelets from smokers and patients with head and neck squamous cell carcinoma (HNSCC) or lung cancer express and up-regulate PD-L1 independently of tumor stage. Furthermore, treatment with Atezolizumab, a fully humanised monoclonal antibody against PD-L1, in 4 patients with lung cancer caused a decrease in PD-L1 expression in platelets, which was restored over 20 days. Altogether, our findings reveal the expression of the main therapeutic target in current checkpoint therapies in human platelets and highlight their potential as biomarkers to predict successful therapeutic outcomes

Biomarkers in Liquid Biopsies for Prediction of Early Liver Metastases in Pancreatic Cancer

Individualized diagnostics approaches in modern cancer therapy require predictive and prognostic biomarkers that are easily accessible and stratify patients for optimal and individualized treatment. Pancreatic ductal adenocarcinoma (PDAC) is still a life-threatening disease mainly because of its late diagnosis in advanced stages or rapid progress even in patients with curative resection of the primary tumor. Moreover, patients with liver metastases exhibit an even worse prognosis. Hence, this retrospective multi-center study aims to identify biomarkers in perioperative serum of PDAC patients predicting early liver metastasis. A highly sensitive biomarker analysis was performed using two different methodological approaches. Olink® analysis, which was also used to validate LEGENDplexTM results, identified significant differences in proteins involved in chemotaxis and migration of immune cells as well as cell growth in serum of patients with early versus late onset of liver metastasis. Further studies with larger cohorts are required to validate these findings for clinical translation

SATB1, genomic instability and Gleason grading constitute a novel risk score for prostate cancer

Current prostate cancer risk classifications rely on clinicopathological parameters resulting in uncertainties for prognostication. To improve individual risk stratification, we examined the predictive value of selected proteins with respect to tumor heterogeneity and genomic instability. We assessed the degree of genomic instability in 50 radical prostatectomy specimens by DNA-Image-Cytometry and evaluated protein expression in related 199 tissue-microarray (TMA) cores. Immunohistochemical data of SATB1, SPIN1, TPM4, VIME and TBB5 were correlated with the degree of genomic instability, established clinical risk factors and overall survival. Genomic instability was associated with a GS >= 7 (p = 0.001) and worse overall survival (p = 0.008). A positive SATB1 expression was associated with a GS = 7 were identified as markers for poor prognosis. Their combination overcomes current clinical risk stratification regimes.Functional Genomics of Muscle, Nerve and Brain Disorder

Genomic instability influences the transcriptome and proteome in endometrial cancer subtypes

Peer reviewe

Israeliten und Hyksos : der historische Kern der Sage vom Aufenthalte Israels in Ägypten

von Martin GemollAus der Sammlung des Leo Baeck Institute, digitalisiert in Kooperation mit dem Center for Jewish History, N

Jahresbericht / Bemerkungen zu Xenophons Anabasis

von Wilhelm Gemol