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Genetic and epigenetic variation, together with transcriptional plasticity, contribute to intratumour heterogeneity 1 . The interplay of these biological processes and their respective contributions to tumour evolution remain unknown. Here we show that intratumour genetic ancestry only infrequently affects gene expression traits and subclonal evolution in colorectal cancer (CRC). Using spatially resolved paired whole-genome and transcriptome sequencing, we find that the majority of intratumour variation in gene expression is not strongly heritable but rather ‘plastic’. Somatic expression quantitative trait loci analysis identified a number of putative genetic controls of expression by cis -acting coding and non-coding mutations, the majority of which were clonal within a tumour, alongside frequent structural alterations. Consistently, computational inference on the spatial patterning of tumour phylogenies finds that a considerable proportion of CRCs did not show evidence of subclonal selection, with only a subset of putative genetic drivers associated with subclone expansions. Spatial intermixing of clones is common, with some tumours growing exponentially and others only at the periphery. Together, our data suggest that most genetic intratumour variation in CRC has no major phenotypic consequence and that transcriptional plasticity is, instead, widespread within a tumour. Intratumour genetic ancestry only infrequently affects gene expression traits and subclonal evolution in colorectal cancer, with most genetic intratumour variation having no detected phenotypic consequence and transcriptional plasticity being widespread within a tumour.

Colorectal malignancies are a leading cause of cancer-related death 1 and have undergone extensive genomic study 2 , 3 . However, DNA mutations alone do not fully explain malignant transformation 4 – 7 . Here we investigate the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,370 samples from 30 primary cancers and 8 concomitant adenomas and generated 1,207 chromatin accessibility profiles, 527 whole genomes and 297 whole transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent somatic chromatin accessibility alterations, including in regulatory regions of cancer driver genes that were otherwise devoid of genetic mutations. Genome-wide alterations in accessibility for transcription factor binding involved CTCF, downregulation of interferon and increased accessibility for SOX and HOX transcription factor families, suggesting the involvement of developmental genes during tumourigenesis. Somatic chromatin accessibility alterations were heritable and distinguished adenomas from cancers. Mutational signature analysis showed that the epigenome in turn influences the accumulation of DNA mutations. This study provides a map of genetic and epigenetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology. A study maps genetic and epigenetic heterogeneity of primary colorectal adenomas and cancers at single-clone resolution through spatial multi-omic profiling of individual glands and adjacent normal tissue.

The bone marrow (BM) stroma in myeloid neoplasms is altered and it is hypothesized that this cell compartment may also harbor clonal somatically acquired mutations. By exome sequencing of in vitro expanded mesenchymal stromal cells (MSCs) from n = 98 patients with myelodysplastic syndrome (MDS) and n = 28 healthy controls we show that these cells accumulate recurrent mutations in genes such as ZFX (n = 8/98), RANK (n = 5/98), and others. MDS derived MSCs display higher mutational burdens, increased replicative stress, senescence, inflammatory gene expression, and distinct mutational signatures as compared to healthy MSCs. However, validation experiments in serial culture passages, chronological BM aspirations and backtracking of high confidence mutations by re-sequencing primary sorted MDS MSCs indicate that the discovered mutations are secondary to in vitro expansion but not present in primary BM. Thus, we here report that there is no evidence for clonal mutations in the BM stroma of MDS patients. Bone marrow-derived mesenchymal stroma cells (MSCs) in myeloid neoplasia have been hypothesized to carry somatic mutations and contribute to pathogenesis. Here the authors analyse ex-vivo cultures and primary MSCs derived from patients with myelodysplastic syndromes, finding functional alterations but no evidence of clonal mutations.

Cancer treatment frequently fails due to the evolution of drug-resistant cell phenotypes driven by genetic or non-genetic changes. The origin, timing, and rate of spread of these adaptations are critical for understanding drug resistance mechanisms but remain challenging to observe directly. We present a mathematical framework to infer drug resistance dynamics from genetic lineage tracing and population size data without direct measurement of resistance phenotypes. Simulation experiments demonstrate that the framework accurately recovers ground-truth evolutionary dynamics. Experimental evolution to 5-Fu chemotherapy in colorectal cancer cell lines SW620 and HCT116 validates the framework. In SW620 cells, a stable pre-existing resistant subpopulation was inferred, whereas in HCT116 cells, resistance emerged through phenotypic switching into a slow-growing resistant state with stochastic progression to full resistance. Functional assays, including scRNA-seq and scDNA-seq, validate these distinct evolutionary routes. This framework facilitates rapid characterisation of resistance mechanisms across diverse experimental settings. Understanding the dynamics of how drug resistance originates in cancer remains crucial, but it is not possible to observe them directly. Here, the authors construct a mathematical framework to infer drug resistance dynamics in cancer using lineage tracing and population size data, which is confirmed with experimental evidence and single-cell sequencing.

The important role of insulin‐like growth factor binding protein 7 (IGFBP7) as a tumor suppressor in solid tumors has been revealed in several studies. Interestingly, in a recent study IGFBP7 was also shown to be aberrantly expressed in acute leukemia. Moreover, in acute T‐lymphoblastic leukemia (T‐ALL), high IGFBP7 expression predicts primary therapy resistance. In order to elucidate the mechanisms underlying aberrant IGFBP7 expression, we used pyrosequencing technology to investigate the DNA methylation of IGFBP7 in 109 T‐ALL patient samples. Aberrant methylation was shown and hypomethylation was associated with an early immunophenotype and co‐expression of the stem cell markers CD117 (P < 0.001) and CD34 (P < 0.001). In concordance, gene expression profiles of 86 T‐ALL patients revealed upregulation of stem cell markers (CD34 and CD133) as well as genes associated with poor outcome and pathogenesis of leukemia (MN1, BAALC, FLT3) in the high IGFBP7 expression group. In conclusion, aberrant IGFBP7 expression is regulated by DNA methylation in acute leukemia. Hypomethylation of the gene is likely to characterize an immature and a more malignant subtype of the disease. (Cancer Sci 2011; 102: 253–259)

To infect cells, human immunodeficiency virus (HIV) binds the CD4 receptor and a chemokine coreceptor, CCR5 or CXCR4. In an HIV-infected patient with acute myelogenous leukemia, an allogeneic stem-cell transplantation was performed using a matched donor with a CCR5 variant that is resistant to HIV acquisition. Twenty months after successful transplantation, HIV remains undetectable in this patient, who has not been receiving antiretroviral therapy. In an HIV-infected patient with acute myelogenous leukemia, an allogeneic stem-cell transplantation was performed using a matched donor with a CCR5 variant that is resistant to HIV acquisition. Twenty months after successful transplantation, HIV remains undetectable in this patient. HIV-1 enters host cells by binding to a CD4 receptor and then interacting with either CCR5 or the CXC chemokine receptor (CXCR4). Homozygosity for a 32-bp deletion (delta32/delta32) in the CCR5 allele results in an inactive CCR5 gene product and consequently confers high resistance against HIV-1 acquisition. 1 Allogeneic stem-cell transplantation from an HLA-matched donor is a feasible option for patients with hematologic neoplasms, but it has not been established as a therapeutic option for patients who are also infected with HIV. 2 Survival of patients with HIV infection has improved considerably since the introduction of highly active antiretroviral therapy (HAART), 3 and . . .

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness. Analysis of live-cell imaging and single-cell genome sequencing data of colorectal cancer organoids identifies temporal dynamics of sub-chromosomal copy-number amplifications.

BackgroundThe risk of developing advanced neoplasia (AN; colorectal cancer and/or high-grade dysplasia) in ulcerative colitis (UC) patients with a low-grade dysplasia (LGD) lesion is variable and difficult to predict. This is a major challenge for effective clinical management.ObjectiveWe aimed to provide accurate AN risk stratification in UC patients with LGD. We hypothesised that the pattern and burden of somatic genomic copy number alterations (CNAs) in LGD lesions could predict future AN risk.DesignWe performed a retrospective multicentre validated case–control study using n=270 LGD samples from n=122 patients with UC. Patients were designated progressors (n=40) if they had a diagnosis of AN in the ~5 years following LGD diagnosis or non-progressors (n=82) if they remained AN-free during follow-up. DNA was extracted from the baseline LGD lesion, low-coverage whole genome sequencing performed and data processed to detect CNAs. Survival analysis was used to evaluate CNAs as predictors of future AN risk.ResultsCNA burden was significantly higher in progressors than non-progressors (p=2×10−6 in discovery cohort) and was a very significant predictor of AN risk in univariate analysis (OR=36; p=9×10−7), outperforming existing clinical risk factors such as lesion size, shape and focality. Optimal risk prediction was achieved with a multivariate model combining CNA burden with the known clinical risk factor of incomplete LGD resection. Within-LGD lesion genetic heterogeneity did not confound risk prediction.ConclusionMeasurement of CNAs in LGD is an accurate predictor of AN risk in inflammatory bowel disease and is likely to support clinical management.

IntroductionIncreased lymphocyte infiltration at the invasive margin or in lymph node metastases in colorectal cancers (CRCs) may lead to increased immune selection pressure for additional immune escape mechanisms here. We aimed to identify whether immune escape mechanisms are selectively enriched in invasive CRC or node metastases.MethodsTargeted sequencing (130x) of the immunopeptidome (genomic regions that generate neoantigens binding to the ten most common HLA alleles) was performed on 82 superficial, invasive and lymph node deposits (region of interest (ROI)) from 11 stage III microsatellite-stable CRCs. Cyclic immunofluorescence using 22 antibodies was performed on 34 matching cancer ROIs and 10 normal ROIs. Values were compared using unpaired Wilcoxon rank sum tests unless specified otherwise.ResultsNo significant within-tumour differences in cytotoxic T lymphocyte fraction were seen, suggesting no increase in immune selection pressure in invasive or metastatic CRC. Consistent with this, HLA mutations/LOH HLA were not specifically enriched in superficial, invasive or lymph node metastases.Fraction of non-epithelial cells expressing PDL1 was elevated in invasive compared to superficial ROIs (p=0.0001). However, no significant difference in fraction of PDL1-expressing cancer cells or cells expressing PD1, CTLA4, VISTA or IDO1 were noted across superficial, invasive and node deposits.ROIs with increased non-epithelial PDL1+ fraction did not show increased subclonal neoantigen burden (Spearman correlation, missense: mutations: rho=0.01, p=0.93, indels- mutations: rho=0.02, p=0.91), suggesting no differential immune escape in these ROIs, leading to an accrual of neoantigens.ConclusionsNo evidence of functional difference in immune escape was noted, suggesting that additional immune escape mechanisms are not gained at the point of invasion or regional metastasis to lymph nodes.

In low-risk Myelodysplastic Neoplasms (MDS), increased activity of apoptosis-promoting factors such as tumor necrosis factor (TNFα) and pro-apoptotic Fas ligand (CD95L) have been described as possible pathomechanisms leading to impaired erythropoiesis. Asunercept (APG101) is a novel therapeutic fusion protein blocking CD95, which has previously shown partial efficacy in reducing transfusion requirement in a clinical phase I trial for low-risk MDS patients (NCT01736436; 2012-11-26). In the current study we aimed to evaluate the effect of Asunercept therapy on the clonal bone marrow composition to identify potential biomarkers to predict response. Bone marrow samples of n = 12 low-risk MDS patients from the above referenced clinical trial were analyzed by serial deep whole exome sequencing in a total of n = 58 time points. We could distinguish a mean of 3.5 molecularly defined subclones per patient (range 2–6). We observed a molecular response defined as reductions of dominant clone sizes by a variant allele frequency (VAF) decrease of at least 10% (mean 20%, range: 10.5–39.2%) in dependency of Asunercept treatment in 9 of 12 (75%) patients. Most of this decline in clonal populations was observed after completion of 12 weeks treatment. Particularly early and pronounced reductions of clone sizes were found in subclones driven by mutations in genes involved in regulation of methylation (n = 1 DNMT3A, n = 1 IDH2, n = 1 TET2). Our results suggest that APG101 could be efficacious in reducing clone sizes of mutated hematopoietic cells in the bone marrow of Myelodysplastic Neoplasms, which warrants further investigation.