Research Areas of the AG Bullinger
We study the genomic, epigenomic and transcriptomic changes associated with hematological malignancies, with a strong focus on acute myeloid leukemia (AML).
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Our lab is committed to unravelling the genomic, epigenomic and transcriptomic changes leading up to and driving acute myeloid leukemia (AML) and other hematological malignancies. To this end, we employ massively parallelized next-generation sequencing (NGS) technologies, from Illumina and Oxford nanopore, to compare patient-derived primary tumor and matched control samples. Data analysis is developed and performed in-house by our own small team of bioinformaticians; findings of interest are followed up by functional validation in the lab. With close ties to the clinic, we are thus able to connect bench and bedside with our research in a dynamic exchange between dry in-silico and hands-on wet lab work. We are also well connected internationally and, for example, heavily involved in large cooperative projects such as SyTASC, SYNtherapy, and HARMONY.
circRNAs in cancer
Growing evidence supports a significant role of splicing pattern deregulation in the pathogenesis of AML and the role of differentially expressed linear splice isoforms. In a global evaluation of circular transcripts, we previously identified also thousands of circular RNA (circRNA) isoforms in both healthy hematopoietic cells and primary AML samples. Notably, leukemic and non-leukemic samples and even more differentiated and less differentiated healthy hematopoietic cells were each associated with distinct circRNA expression signatures. We validated circRNAs of known AML candidate genes such as NPM1 and, among its many circular isoforms, discovered hsa_circ_0075001, whose expression level was independent of NPM1 mutational status and correlated with specific gene expression signatures. We are currently performing additional functional validation studies on some other candidate genes and continuing systemic analyses of larger patient cohorts, with both Illumina and Oxford Nanopore sequencing.
Clonal evolution models
AML is a highly heterogeneous disease: Driving somatic mutations are distinct between different patients and even between different cells (“clones”) of the same sample. In addition, a patient’s clonal composition changes over time, as additional mutations are acquired and selective pressures such as cancer therapy change the relative abundance of existing clones. Thus, substantial differences can accumulate between a patient’s initial diagnostic and subsequent relapse samples. We previously studied these changes and the underlying clonal evolution of NPM1-mutated AML patients, which represent one of the largest patient subgroups. While the mutation persisted in most of the analyzed patients, who were all NPM1-mutated at diagnosis, it was lost in some. Integrating whole-exome sequencing (WES), deep targeted re-sequencing and RNA sequencing, we could show that, at relapse, NPM1-mutated patients had retained nearly none of the somatic mutations present in the respective diagnosis and instead harbored a completely different set of mutations, associated with different signaling pathways. Patients who had retained the NPM1-mutation, on the other hand, presented with highly overlapping profiles between diagnosis and relapse. Based on these findings, we postulate that relapse frequently arises from persistent leukemic clones, which drive the disease both at diagnosis and relapse, but that it may also develop as a second “de novo” or treatment-related AML (tAML).
NGS-based monitoring of minimal residual disease
One of the most common adverse-risk aberrations in AML are internal tandem duplications of the FLT3-gene (FLT3-ITDs). Early detection of residual clones persisting during and after treatment could improve patient outcome by enabling an early clinical intervention prior to any impending relapse. Respective qRT-PCR assays have been established for other recurrent aberrations, but FLT3-ITD insertion sites and lengths are so variable that patient-specific designs would be required for an analogous assay. Because this is not feasible for clinical routine, we have developed a novel NGS-based assay for residual FLT3-ITD detection and monitoring, which has since been made publicly available at https://github.com/tjblaette/getitd. To study FLT3-ITD kinetics and clonal evolution, we are currently analyzing a larger cohort of patients who carried the mutation at diagnosis and subsequently received targeted therapy with the recently approved inhibitor midostaurin.
4th generation sequencing for improved molecular diagnostics
With our recently acquired GridION, we are establishing various protocols and associated data analysis pipelines for long-read sequencing of whole genomes, epigenomes and transcriptomes. The combination will enable integrative multi-omics analyses of selected cohorts, the elucidation of the internal structure of circular and linear splice isoforms as well as different types of DNA and RNA modifications. With even direct RNA sequencing, the technology indeed offers many, and many novel possibilities.