New strategies in parallel sequencing – alternative enrichment technologies and RNA sequencing

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.

The disadvantage of the amplicon-based parallel sequencing is that this method does not routinely detect chromosomal aberrations such as gene fusions and translocations on DNA level. These aberrations are still widely analysed by FISH and RT-PCR or immunohistochemistry. The main disadvantage of these methods is that a specific assay has to be performed for each individual chromosomal aberration. As a result, these methods are time-consuming, a lot of starting material is needed and in particular FISH is very cost-intensive. The development of new parallel sequencing approaches to detect all therapeutically relevant genomic changes in a single assay is an ongoing process. Currently, various technologies are being evaluated for the detection of chromosomal aberrations.

On a DNA level, 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.

On a 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. However, it is not clear whether somatic gene mutations, such as point mutations, small deletions, insertions or duplications, are reliably detected with this approach. For detection of somatic gene mutations, an additional DNA-based assay is currently required. In addition, the extraction of intact RNA from FFPE material is challenging.

The aim of this work is to evaluate and compare the new parallel sequencing strategies for molecular pathological diagnostics and to implement the most suitable methods into routine diagnostics.

Establishment of new sequencing technologies for detection of chromosomal aberrations and gene mutations in sarcomas

In this research project, a new hybridization-based parallel sequencing approach will be established for the detection of genomic aberrations in sarcomas on FFPE material. This assay will be used to address research questions in the field of targeted therapies. Additionally, this assay will be implemented into the routine molecular pathological diagnostics in order to promote, among other things, the establishment of targeted therapeutic strategies within the university hospital in the field of sarcomas. Our long-term goal is to strengthen the infrastructure for molecular targeted therapies in clinical oncology. By publishing the approach in a peer-reviewed journal, the sarcoma panel and the experience gained will be available for other molecular pathological departments.

nCounter Vantage 3D assays – simultaneous profiling of DNA, RNA and protein targets

NanoString's nCounter analysis technology is based on the marking of specific target regions using color-coded hybridization probes. This results in a wide variety of possible applications, e.g. 1) gene expression analyses, 2) SNV determination and 3) detections of chromosomal fusions. The analysis is performed on RNA, extracted from fresh tissue, cell lysate or FFPE-fixed tumor tissue. A custom-designed  panel is available for the detection of fusion genes in lung tumors, which can detect chromosomal aberrations in the genes ALK-, RET-, ROS1 and NTRK1, -2, -3. On the one hand specific fusion events and their associated translocation partners can be identified (specific probes). On the other hand, the detection of fusions with an unknown translocation partner is also possible.

(Lung-) Fusions Panel
Specific Lung Fusion probes detect the following fusions:

ALKRETROS1NTRK1
EML4-ALKCCDC6-RETCD74-ROS1CD74-NTRK1
HIP1-ALKKIF5B-RETEZR-ROS1MPRIP-NTRK1
KIF5B-ALKGOPC-ROS1
TFG-ALKLRIG3-ROS1
TPR-ALKSDC4-ROS1
SLC34A2-ROS1
TPM-ROS1

5´/3´ Expression of imbalanced  probe pairs can detect fusion events with unknown translocation partners:

ALKRETROS1NTRK1NTRK2NTRK3
8 imbalanced probes 8 imbalanced probes 8 imbalanced probes 8 imbalanced probes 8 imbalanced probes 8 imbalanced probes
ProSigna

Therapy of patients with breast cancer at an early stage starts with an accurate assessment of the Risk of Recurrence (ROR). The Prosigna assay was developed on the basis of the PAM50 gene signature and allows the stratification of tumors into different intrinsic subtypes, carrying different biological and clinical features. In addition, the test determines a proliferation value, i.e. the expression of different genes of the proliferative signaling pathway. In combination with clinical data on tumor size and number of positive lymph nodes, prognostic information for therapy decision can be made. The risk-of-recurrence score (ROR score) is given on a scale from 0-100 and has been proven to be prognostically significant for the period of endocrine treatment over 5 years. You will find detailed information on the test and costs on the manufacturer's homepage

Development of protein-analytic procedures for the identification of candidate markers to support the (early) diagnosis of asbestos-associated pulmonary and pleural tumors

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 grant for 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 is focusing on the development and verification of the numerous results achieved in the initial project. In addition, an extension necessary for mechanistic verification is added at the levels of genomics and transcriptomics. The Institute of Pathology has many years of expertise in the analysis of genomic aberrations in lung tumors. In the future, not only people from the general population, but above all also employees, will be able to benefit from the results obtained in the course of the subsequent screening if it is possible to establish early identification markers that can reliably diagnose lung tumors in a treatable stage. Tissues from 150 operative pulmonary carcinomas are to be cryopreserved every year, and whole blood, plasma and saliva samples are collected from the same patients. In surgical specimens, tumor-free marginal tissue should also be considered. Further, lymph node biopsies from 50 additional patients will be obtained. In addition to the establishment of biomarkers for early detection, the basis for personalized cancer therapy can be laid by the mechanistic findings to be expected in the project, thus also benefiting from tertiary prevention. A long-term goal is the implementation of the blood test diagnostics for personalized cancer therapy in lung tumors. In particular, it is crucial whether genomic changes in the cancerous tissue can be detected in the plasma in early, curatively resectable lung tumors. If this question can be answered in a positive way, it is possible to significantly improve the specificity of cancer early diagnosis of suspicious pulmonary arteries detected by CT screening, and to use this methodology in the monitoring of risk persons. 

Biomarker Research

HR23b as a predictive biomarker for HDAC inhibitor based therapy in soft tissue sarcomas

HR23b as a predictive biomarker for HDAC inhibitor based therapy in soft tissue sarcomas
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. Murine mesenchymal tumors are induced to investigate the effect of functional HR23b depletion or overexpression on the sensitivity towards HDACi treatment in vivo.

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