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Central diagnostics, genomics and biobanking

Subject Area Hematology, Oncology
Term from 2016 to 2021
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 226262100
 

Final Report Abstract

This central project is to provide diagnostics, biobanking, and genomics for the CRU. In the first funding period CLL patients have been phenotypically and molecularly characterized by means of an advanced diagnostic work-up. These investigations included cytological, immunophenotypical, cytogenetic and molecular genetics techniques. Through cytological investigations CLL cases could be clearly separated from B cell prolymphocytic leukemia (B-PLL) and other related B cell malignancies. For flow cytometry a broad panel of different antigens has been analyzed which included standard antigens for the diagnosis of CLL such as CD5, CD19 and CD23, antigens for the diagnosis of related diseases such as CD10, CD11c and FMC7, as well as new antigens, such as CD43, CD200 and ROR1. Furthermore, we performed conventional cytogenetics (karyotyping/banding analyses) at initial diagnosis as well as during relapsing disease in order to search for recurrent or non-recurrent chromosomal aberrations. As we could previously show that complex karyotypes correlate with a significantly adverse prognosis in CLL first line treatment in comorbid patients we extended these investigations also to younger patients and relapses. In given cases multicolor-FISH (mFISH, “chromosomal painting”) has been be performed in order to uncover difficult structural lesions. We performed IgVH sequencing by using IG specific primers for site specific PCR reactions, i.e. according to the Biomed-2 recommendations, followed by Sanger sequencing. Similarly, we have fully established a PCR-based mutational analysis of exon 4 to 10 of the TP53 gene, as well as a NGS technique. Samples from CLL patients who agreed to it have been -after obtaining written consent- characterized by above mentioned approaches and stored at our central biobank and were distributed to the various RPs. In total, another 1,500 samples have been diagnostically characterized, banked and distributed. In the second funding period, we have extended our aims to provide support for genomic analyses and bioinformatics. To this end, we have implemented and established an automated platform to analyze tumor sequencing data. This platform is built on the open-source metadata management system iRODS to securely, consistently, transparently, and reproducibly process large amount of sequencing data. To make this system accessible to non-computational biologists, we also provided a graphical userinterface that allows the management and retrieval of metadata provided and obtained during data processing. As part of our computational pipeline, we have implemented a new method to analyze copy number changes and to infer subclonal populations from whole exome or genome data. Our method combines copy number analysis with mutational clustering (to infer the subclonal populations) and requires significantly less computational resources than alternative approaches. Within the Pan-Cancer Analysis of Whole Genomes conducted by the ICGC, we were able to validate and apply our method on large datasets. In addition, we applied our method to whole exome sequencing data from venetoclax relapsed patients to reconstruct the clonal dynamics towards resistance. Having also collected samples before the venetoclax treatment, we found a highly diverse clonal dynamics leading to venetoclax resistance, including linear, divergent, and convergent evolution. Furthermore, we found recurrent alterations at relapse affecting BTG1 and CDKN2A/B. In addition, functional analyses showed that oncogenic BRAF mutations is substantially lowering the treatment efficacy of venetoclax. Recently, Blombery et al. found a resistance conferring mutation in BCL2 (p.G101V) that remained subclonal after relapse in most cases. Using digital droplet PCR, we found the presence of this mutation in three of our cases that have undergone whole exome sequencing17. However, the allele frequency was far below the detection limit of whole exome sequencing and the pronounced subclonality of the mutations challenging the notion that G101V mutation of BCL2 is solely driving venetoclax resistance in these samples.

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