Early Detection of Aggressive Leukemia Transformation
Summary
The Richter Transformation (RT) is a notable progression of Chronic Lymphocytic Leukemia (CLL) into an extremely aggressive form of large B cell lymphoma, resulting in a poor prognosis. Despite being a well-known phenomenon, the mechanisms behind RT are yet to be fully understood. In this study, 19 cases of CLL that transformed into RT were analyzed through the characterization of their whole genome, epigenome, transcriptome, single-cell DNA/RNA sequencing, and functional experiments. By studying 54 longitudinal samples that spanned up to 19 years of disease progression, it was revealed that small subclones possessing the genomic, immunogenetic, and transcriptomic features of RT cells were present at the time of CLL diagnosis and remained dormant for up to 19 years before the transformation. Additionally, new driver alterations were identified, a new mutational signature (SBS-RT) was discovered, and an oxidative phosphorylation (OXPHOS) high-B cell receptor (BCR) low-signaling transcriptional axis was recognized in RT cells. Furthermore, it was shown that inhibiting OXPHOS reduced the proliferation of RT cells. These findings highlight the early presence of subclones that drive advanced stages of cancer evolution and provide potential therapeutic targets for RT.
Research Criteria
Cancer initiation, progression, and relapse are all driven by clonal evolution because fitter subclones are incrementally acquired and/or chosen. The examinations of bulk tumor cell populations at low resolution and at the single or limited time points of the disease course in most studies make it difficult to comprehend how tumors evolve. A deeper understanding of this process might result in anticipatory treatment plans. In individuals with CLL who have not received treatment, RT represents a paradigmatic form of cancer evolution. After receiving chemoimmunotherapy (CIT) and targeted medicines, it is discovered in 4–20% of patients. The occurrence of RT within the first few months of treatment suggests the use of pre-existing subclones. Nevertheless, little is known about the genomic and epigenomic pathways underlying RT following CIT or the use of targeted medicines. The current study's objectives were to reconstruct RT's evolutionary history and to identify the molecular mechanisms behind this change.
Fig.1 Experimental design. (Nadeu, 2022)
Sample Type
Cells from human blood samples
Result—Dormant Seeds of RT at CLL Diagnosis
In order to confirm these evolutionary histories of RT, the researchers used single-cell DNA sequencing (scDNA-seq) on 32 genes in 16 longitudinal samples from 4 individuals (12, 19, 365, and 3, 299). The RT subclone (subclone 5) at transformation (T6) contained CDKN2A/B and TP53 (p.G245D) alterations, but the primary CLL subclones causing recurrence after therapy at T4 and T5 possessed a unique TP53 mutation (patient 19; 14.4 years from diagnosis to RT) (p.I195T; subclones 3 and 4). WGS predicted that all of these subclones would be present upon CLL diagnosis (T1). Using scDNA-seq, they discovered two small populations, each comprising 0.1% of cells, at T1 with the TP53 p.I195T and p.G245D mutations, which were also identified at relapse 7.2 years later (T3). Following 3.7 years at T4 and T5, the subclone carrying TP53 p.I195T grew to dominate the second relapse but was replaced at T6 in the RT by the subclone carrying TP53 p.G245D 14.4 years after diagnosis. The original CLL subclone's SF3B1 and NOTCH1 mutations were present in all of these subclones. The scDNA-seq of three additional cases confirmed the phylogenies and the majority of the dynamics inferred from WGS. These findings are consistent with the previously described early immunogenetic and DNA methylation diversification in CLL and show that RT may originate via a selection of pre-existing subclones containing significant driver mutations rather than a de novo acquisition of leading clones. The progression of CLL to RT is also believed to be marked by an early driver diversification, which was likely created prior to diagnosis.
Fig.2 Dormant seeds of RT at diagnosis. (Nadeu, 2022)
Result—Tracking RT Subclones Using Single-Cell RNA Sequencing
Single-cell RNA sequencing (scRNA-seq) of 19 longitudinal samples from five patients tracked RT subclones across time (24,800 tumor cells passing filters, mean of 1,305 cells per sample). As expected, RT and CLL cells displayed very distinct gene expression profiles. Three primary clusters dominated the transcriptome of CLL cells among individuals, marked by varied expression of CXCR4, CD27, and MIR155HG, which may indicate CLL cell recirculation between peripheral blood and lymph nodes. RT intraclonal heterogeneity was mostly associated with different proliferative capacities, with a cluster of cells expressing high MKI67 and PCNA and having high S and G2M cell-cycle phase scores. Patients expressed CCND2, MIR155HG, and TP53INP1 in the remaining RT clusters. RT cells were seen in all CLL samples before transformation in patients 12, 19, 63, and 3,299, but not in patient 365. The transcriptome profile of these RT subclones matched WGS, scDNA-seq, and IgG results in all five patients, demonstrating they captured the same cells. Indeed, scRNA-seq identified the CNAs implicated in simple and complicated structural modifications detected at RT by WGS in quiescent RT cells at CLL diagnosis and successive periods before their final growth. RT's chromothripsis and transcriptome identity suggest early SV acquisition.
Fig.3 Tracking RT subclones using single-cell RNA sequencing. (Nadeu, 2022)
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Reference
- Nadeu, F.; et al. Detection of early seeding of Richter transformation in chronic lymphocytic leukemia. Nature Medicine. 2022, 28: 1662-1671.
