In molecular pathological diagnostics amplicon-based parallel sequencing has been used up to date. By this method, therapeutic genes and gene segments can be amplified, enriched and subsequently sequenced by means of multiplex-PCR. This method can also be used on fragmented and chemically modified DNA from formalin-fixed, paraffin-embedded (FFPE) tissue.
As an alternative method, hybridization-based parallel sequencing is used for the simultaneous detection of somatic gene mutations, gene fusions, and copy number changes. Here, DNA is shared and adapters are ligated. Custom designed, biotinylated DNA probes are hybridised to target sequences to allow for sequence enrichment using streptavidin beads. Finally, libraries containing the target sequences are sequenced on platforms like NextSeq 500 from Illumina.
Additionally on an RNA level, RNA-based parallel sequencing assay have already been developed for the detection of gene fusions. Here, specific primers are used for the detection of known fusions and previously unknown fusions can be detected by an 5 '/ 3' imbalance score. These assays can also be used for the simultaneous detection of several fusion events.
In two translational projects, performed in cooperation with clinical professions, two customized NGS panels are established for the analysis of lymphoma and pediatric tumors on FFPE material. On DNA level, a hybrid capture assay is developed for the simultaneous detection of somatic mutations and copy number variations and on RNA level a “Single Primer Extension” assay for the detection of fusions. These translational assays are not for research only but should be implemented in routine diagnostics after a careful evaluation.
The cooperation of Ruhr University Bochum with the lung cancer center Bonn / Rhein-Sieg and the Cologne Institute of Pathology has successfully applied for a follow-up application to the project FP 339 "Development of protein-analytic procedures for the identification of candidate markers to support the (early) diagnosis of asbestos-associated pulmonary and pleural tumors". The project focusses on the development and verification of the numerous results achieved in the initial project. In addition, an on the levels of genomics and transcriptomics necessary for mechanistic verification will be added. In this field, the Institute of Pathology has many years of expertise in the analysis of genomic alterations in lung tumors. In the future, not only people in the general population, but also employees will be able to benefit from the results obtained in the context of follow-up screening, if it is possible to establish early identification markers that can reliably diagnose lung tumors at a treatable stage. By 31.10.2019, material from 153 study participants had been fixed, cut, evaluated and returned to Bochum within the framework of this project. Of these, 69.5% are primary lung tumours and 11% are metastases from other entities, corresponding to 124 cases. In 19.5% of the cases non-malignant tumours were diagnosed, which are used as control tissue. In addition to the establishment of biomarkers for early detection, the mechanistic findings on the cancer development process expected in the project can lay the foundation for personalised cancer therapy and thus also provide a benefit for tertiary prevention. A long-term goal here is also the implementation of blood sample diagnostics for personalized cancer therapy of lung tumors. Within the project, an additional corresponding plasma sample was obtained from each of the 153 study participants. Of these, 55 samples could be analysed by parallel sequencing until 31.10.2019. Here, the crucial factor is whether genomic changes in cancerous tissue in early, still curatively resectable lung tumours can already be detected in the plasma. If this question can be answered positively, it is possible to significantly improve the specificity of early cancer diagnosis of suspected lung foci detected by CT screening and to use this method in the monitoring of persons at risk.
The outcome in esophageal adenocarcinoma (EAC) is still poor with only 20 % of patients in Western populations surviving for more than 5 years. Almost nothing is known about the precise composition of immune cells and their gene expression profiles in primary resected EACs and also nothing compared to neoadjuvant treated EACs.
This study analyzes and compares immune profiles of primary resected and neoadjuvant treated esophageal adenocarcinoma and unravels possible targets for immunotherapy. We analyzed 47 EAC in total considering a set of 30 primary treatment-naive EACs and 17 neoadjuvant pretreated (12 x CROSS, 5 x FLOT) using the Nanostring's panel-based gene expression platform including 770 genes being important in malignant tumors and their immune micromileu. Most of the significantly altered genes are involved in the regulation of immune responses, T-and B cell functions as well as antigen processing (CD3, CD8, MHC-I). Chemokine-receptor axes like the CXCL9, -10,-11/CXCR3- are prominent in esophageal adenocarcinoma promoting cancer cell proliferation and metastasis. ARG1, as a regulator of T-cell fate is 6-fold down-regulated in untreated primary esophageal tumors.
The influence of the currently used neoadjuvant treatment revealed a down-regulation of nearly all important checkpoint markers and inflammatory related genes in the local microenvironment. We found a higher expression of checkpoint markers like LAG3, TIM3, CTLA4 and CD276 in comparison to PD-L1/PD-1 supporting clinical trials analyzing the efficacy of a combination of different checkpoint inhibitors in EACs. We found an up-regulation of CD38 or LILRB1 as examples of additional immune escape mechanism.
Histone modifications have been shown to be key players in epigenetic alterations and are often dysregulated in cancer. Therefore, histone deacetylases (HDAC) seem to be promising therapeutic targets for HDAC inhibitor based therapy in cancer. HR23b, the UV excision repair protein Rad23 homolog B, was revealed as a potential biomarker for the sensitivity to HDAC inhibitor (HDACi) based therapy in cutaneous T-cell lymphoma. Furthermore, in hepatocellular carcinoma HR23b expression was associated with stable disease under HDAC inhibitor therapy. Therefore, we aimed at evaluating HR23b as a candidate biomarker for HDACi response in solid tumors (sarcomas and gastrointestinal stromal tumors (GIST), melanomas and adenocarcinoma of the lung).
Our previous studies have shown that sarcomas differ significantly concerning HR23b protein expression. Sensitivity to the HDACi vorinostat correlated significantly with HR23b protein expression. Immunohistochemical prevalence screening in clinical samples of relevant adult-type tumors revealed that 12.5% of sarcomas (among them malignant peripheral nerve sheath tumors, pleomorphic liposarcomas, leiomyosarcomas, dedifferentiated liposarcomas, synovial sarcomas and angiosarcomas) and 23.2% of GIST show high HR23b expression (Ihle et al., JCP, 2015).
To expand these observations to other solid tumors the expression of HR23b is analyzed in melanoma and adenocarcinoma of the lung using immunohistochemistry. Cell lines are examined for sensitivity to HDACi based therapy in relation to their HR23b expression. Furthermore, the expression of different HDACs is determined in correlation with the inhibitory effect of HDACi in all tumor entities. To address the question of the HR23b function in the context of HDACi sensitivity stable specific knock-down and overexpression of HR23b is conducted by CRISPR/Cas and the effects on sensitivity, viability, apoptosis and signal transduction are determined. Previous studies showed that HR23b dependent sensitivity towards HDACi is mediated by apoptosis induction via the NGFR death receptor pathway. In contrast, HR23b loss reduces apoptosis induction and shifts response to autophagy. Furthermore we observed a significant upregulation of anticancer immunotherapy targets (e.g. cancer testis antigens) enhancing the response to immunotherapy agents.
HR23b dependent sensitivity towards HDACi is mediated by apoptosis induction via the NGFR death receptor pathway. In contrast, HR23b loss reduces apoptosis induction and shifts response to autophagy. Furthermore we observed a significant upregulation of anticancer immunotherapy targets (e.g. cancer testis antigens) enhancing the response to immunotherapy agents.
