Benchmarking pipelines for subclonal deconvolution of bulk tumour sequencing data

Intratumour heterogeneity provides tumours with the ability to adapt and acquire treatment resistance. The development of more effective and personalised treatments for cancers, therefore, requires accurate characterisation of the clonal architecture of tumours, enabling evolutionary dynamics to be tracked. Many methods exist for achieving this from bulk tumour sequencing data, involving identifying mutations and performing subclonal deconvolution, but there is a lack of systematic benchmarking to inform researchers on which are most accurate, and how dataset characteristics impact performance. To address this, we use the most comprehensive tumour genome simulation tool available for such purposes to create 80 bulk tumour whole exome sequencing datasets of differing depths, tumour complexities, and purities, and use these to benchmark subclonal deconvolution pipelines. We conclude that i) tumour complexity does not impact accuracy, ii) increasing either purity or purity-corrected sequencing depth improves accuracy, and iii) the optimal pipeline consists of Mutect2, FACETS and PyClone-VI. We have made our benchmarking datasets publicly available for future use.

Bilineal evolution of a U2AF1-mutated clone associated with acquisition of distinct secondary mutations

Rare hematologic malignancies display evidence of both myeloid and lymphoid differentiation. Here, we describe such a novel bilineal event discovered in an adult woman with B-lymphoblastic leukemia (BLL). At the time of BLL diagnosis, the patient had a normal karyotype and a bulk sequencing panel identified pathogenic variants in BCOR, EZH2, RUNX1, and U2AF1, a genotype more typical of myeloid neoplasia. Additionally, the patient was noted to have 3-year history of cytopenias, and morphologic dyspoiesis was noted on post-treatment samples, raising the possibility of an antecedent hematologic disorder. To investigate the clonal architecture of her disease, we performed targeted sequencing on fractionated samples enriched for either B-lymphoblasts or circulating granulocytes. These studies revealed a truncal founder mutation in the spliceosome gene U2AF1 in both fractions, while distinct secondary mutations were present only in B-lymphoblasts (BCOR, NRAS) or myeloid cells (ASXL1, EZH2, RUNX1). These results indicate that both processes evolved from a common U2AF1-mutated precursor, which then acquired additional mutations during a process of divergent evolution and bilineal differentiation. Our findings highlight novel mechanisms in BLL leukemogenesis and expand the spectrum of observed bilineal neoplasms.

Whole-genome sequencing of phenotypically distinct inflammatory breast cancers reveals similar genomic alterations to non-inflammatory breast cancers

Background Inflammatory breast cancer (IBC) has a highly invasive and metastatic phenotype. However, little is known about its genetic drivers. To address this, we report the largest cohort of whole-genome sequencing (WGS) of IBC cases. Methods We performed WGS of 20 IBC samples and paired normal blood DNA to identify genomic alterations. For comparison, we used 23 matched non-IBC samples from the Cancer Genome Atlas Program (TCGA). We also validated our findings using WGS data from the International Cancer Genome Consortium (ICGC) and the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We examined a wide selection of genomic features to search for differences between IBC and conventional breast cancer. These include (i) somatic and germline single-nucleotide variants (SNVs), in both coding and non-coding regions; (ii) the mutational signature and the clonal architecture derived from these SNVs; (iii) copy number and structural variants (CNVs and SVs); and (iv) non-human sequence in the tumors (i.e., exogenous sequences of bacterial origin). Results Overall, IBC has similar genomic characteristics to non-IBC, including specific alterations, overall mutational load and signature, and tumor heterogeneity. In particular, we observed similar mutation frequencies between IBC and non-IBC, for each gene and most cancer-related pathways. Moreover, we found no exogenous sequences of infectious agents specific to IBC samples. Even though we could not find any strongly statistically distinguishing genomic features between the two groups, we did find some suggestive differences in IBC: (i) The MAST2 gene was more frequently mutated (20% IBC vs. 0% non-IBC). (ii) The TGF β pathway was more frequently disrupted by germline SNVs (50% vs. 13%). (iii) Different copy number profiles were observed in several genomic regions harboring cancer genes. (iv) Complex SVs were more frequent. (v) The clonal architecture was simpler, suggesting more homogenous tumor-evolutionary lineages. Conclusions Whole-genome sequencing of IBC manifests a similar genomic architecture to non-IBC. We found no unique genomic alterations shared in just IBCs; however, subtle genomic differences were observed including germline alterations in TGFβ pathway genes and somatic mutations in the MAST2 kinase that could represent potential therapeutic targets.

Clonal architecture in mesothelioma is prognostic and shapes the tumour microenvironment

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20–50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/−3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/−22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.

Analysis of Intratumoral Heterogeneity in Myelodysplastic Syndromes with Isolated del(5q) Using a Single Cell Approach

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Among them, the most well characterized subtype is MDS with isolated chromosome 5q deletion (MDS del(5q)), which is the only one defined by a cytogenetic abnormality that makes these patients candidates to be treated with lenalidomide. During the last decade, single cell (SC) analysis has emerged as a powerful tool to decipher clonal architecture and to further understand cancer and other diseases at higher resolution level compared to bulk sequencing techniques. In this study, a SC approach was used to analyze intratumoral heterogeneity in four patients with MDS del(5q). Single CD34+CD117+CD45+CD19- bone marrow hematopoietic stem progenitor cells were isolated using the C1 system (Fluidigm) from diagnosis or before receiving any treatment and from available follow-up samples. Selected somatic alterations were further analyzed in SC by high-throughput qPCR (Biomark HD, Fluidigm) using specific TaqMan assays. A median of 175 cells per sample were analyzed. Inferred clonal architectures were relatively simple and either linear or branching. Similar to previous studies based on bulk sequencing to infer clonal architecture, we were able to observe that an ancestral event in one patient can appear as a secondary hit in another one, thus reflecting the high intratumoral heterogeneity in MDS del(5q) and the importance of patient-specific molecular characterization.