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Locally advanced oesophageal adenocarcinoma (EAC) remains difficult to treat because of common resistance to neoadjuvant therapy and high recurrence rates. The ecological and evolutionary dynamics responsible for treatment failure are incompletely understood. Here, we performed a comprehensive multi-omic analysis of samples collected from EAC patients in the MEMORI clinical trial, revealing major changes in gene expression profiles and immune microenvironment composition that did not appear to be driven by changes in clonal composition. Multi-region multi-timepoint whole exome (300x depth) and paired transcriptome sequencing was performed on 27 patients pre-, during and after neoadjuvant treatment. EAC showed major transcriptomic changes during treatment with upregulation of immune and stromal pathways and oncogenic pathways such as KRAS, Hedgehog and WNT. However, genetic data revealed that clonal sweeps were rare, suggesting that gene expression changes were not clonally driven. Additional longitudinal image mass cytometry was performed in a subset of 15 patients and T-cell receptor sequencing in 10 patients, revealing remodelling of the T-cell compartment during treatment and other shifts in microenvironment composition. The presence of immune escape mechanisms and a lack of clonal T-cell expansions were linked to poor clinical treatment response. This study identifies profound transcriptional changes during treatment with limited evidence that clonal replacement is the cause, suggesting phenotypic plasticity and immune dynamics as mechanisms for therapy resistance with pharmacological relevance.

Introduction: Locally advanced esophageal adenocarcinoma (EAC) remains difficult to treat, and resistance is common. The ecological and evolutionary dynamics responsible for treatment failure are incompletely understood. Aim & Methods: We performed a multi-omic study with a multi-timepoint strategy to examine neoadjuvant treatment response at clonal resolution and in the surrounding tumor microenvironment. EAC samples from chemotherapy responding (REs) and chemotherapy non-responding (NRs) patients with locally advanced EAC were collected at three time points (prior, during, and after neoadjuvant therapy) within the multicenter MEMORI trial. Whole exome sequencing (mean depth 300x) was performed on 47 samples from 17 REs and on 24 samples from 10 NRs. Matched RNA sequencing was performed on 53 samples from 17 REs and on 26 samples from 10 NRs. To characterize immune response we performed imaging mass cytometry (IMC) with a 18-marker panel on 26 samples from 9 REs and 16 samples from 6 NRs and T-cell receptor sequencing in 18 RE-samples and 9 NR-samples. Results: We observed profound changes in mutation signatures over time, characterized by C>A transitions after exposure to neoadjuvant chemotherapy (FOLFOX), which is consistent with earlier observed oxaliplatin induced mutational signatures. Phylogenetic analysis showed no major changes in the clonal make-up during treatment, suggesting phenotypic plasticity rather than clonal evolution caused treatment resistance. EAC samples displayed widespread copy number alterations (CNAs), as previously described. CNAs arising during treatment were significantly more likely to be small, focal alterations rather than large alterations. At the transcriptome level neoadjuvant treatment led to significant changes with significant upregulation of immune and stromal pathways and oncogenic pathways such as KRAS, Hedgehog and WNT. The presence of immune escape mechanisms (defined as LOH or mutations in HLA, B2M mutations or PDL-1 overexpression) and a lack of clonal T-cell expansions were linked to poor clinical treatment response. Moreover, IMC analyses showed a less activated T-cell phenotype in NRs than in REs throughout treatment. Conclusion: Using a multi-timepoint approach, we integrated genetic analyses with transcriptomic analyses and analyses of the tumor immune microenvironment for a holistic understanding of treatment resistance. This study identifies profound transcriptional changes during treatment with limited evidence that clonal replacement is the cause, suggesting phenotypic plasticity as a mechanism for therapy resistance. The presence of immune escape, a less activated T-cell phenotype and a lack of clonal T-cell expansions in patients with poor clinical treatment response has high pharmacological relevance and could be exploited via combined immune-chemotherapy treatments. Citation Format: Melissa Barroux, Jacob Househam, Eszter Lakatos, Tahel Ronel, Ann-Marie Baker, Henrike Salié, Max Mossner, Kane Smith, Chris Kimberley, Salpie Nowinski, Alison Berner, Vinaya Gunasri, Marnix Jansen, Giulio Caravagna, Julia Slotta-Huspenina, Wilko Weichert, Markus Alberstmeier, Benny Chain, Helmut Friess, Bertram Bengsch, Roland M. Schmid, Jens T. Siveke, Michael Quante, Trevor A. Graham. Tumor evolution and immune microenvironment dynamics define response to neoadjuvant treatment of esophageal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4539.

Cancer genomic medicine relies on targeting driver genes. However, current catalogues of cancer drivers are mostly based on indirect measurements of mutation frequencies, positions or types, rather than their effect on clonal expansionsin vivo. Moreover, non-genetic drivers are largely unknown, as are the epigenetic and transcriptomic effects of genetic drivers. Here we perform spatial computational inference on multiomic data with matched whole-genome sequencing, ATAC-seq and RNA-seq. Using 436 samples, we directly quantify the contribution, or lack thereof, of putative driver genes to subclonal expansionsin vivoin 30 colorectal carcinomas (4-33 samples per patient, median=15). Although subclonal neutral evolution was widespread (13/26 cases with sufficient data), there were cases with clear evidence of subclonal selection (6/26) in which we measured epigenetic and transcriptomic differences between subclonesin vivo. In 7/26 cases we could not distinguish between neutral or selective evolution with the available data. We identified expanding subclones that were not driven by known genetic alterations, and propose candidate epigenetic drivers. We identified the distinguishing patterns of genomic heterogeneity produced in fast, exponentially growing tumours (7/26) versus neoplasms growing only at the periphery (19/26), as well as identifying clonally intermixed (16/28 cases with sufficient data) versus segregated malignancies (10/28). Our model-based approach measures genetic and non-genetic subclonal selection, or lack thereof, in space and time and allowsin vivocomparisons of the emergent phenotypic properties of subclones within human tumours.

Colorectal malignancies are a leading cause of cancer death. Despite large-scale genomic efforts, DNA mutations do not fully explain malignant evolution. Here we study 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 1,373 samples from 30 primary cancers and 9 concomitant adenomas and generated 1,212 chromatin accessibility profiles, 527 whole-genomes and 297 whole-transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent chromatin changes in regulatory regions of cancer drivers with otherwise no mutation. Genome-wide alterations in transcription factor binding accessibility involved CTCF, downregulation of interferon, and increased accessibility for SOX and HOX, indicating developmental genes reactivation. Epigenetic aberrations were heritable, distinguishing adenomas from cancers. Mutational signature analysis showed the epigenome influencing DNA mutation accumulation. This study provides a map of (epi)genetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology.

Expression of ERG is of prognostic significance in acute myeloid leukemia (AML) and T-lymphoblastic leukemia (T-ALL) pointing to its role in leukemogenesis. To unravel its transcriptional regulation we analyzed the expression of ERG specific isoforms. Expression of the two main isoforms ERG2 and ERG3 was found in AML and normal CD34+ cells, whereas T-ALL blasts only expressed ERG isoforms harboring exon 5 (ERG3) lacking expression of ERG2. Bisulfite sequencing revealed hypermethylation of a CpG island within the ERG2 promoter region in T-ALL. Treatment of the T-lymphoblastic cell line BE13 with decitabine led to re-expression of ERG2 and pyrosequencing showed concordant DNA hypomethylation, thus confirming a methylation regulated expression of ERG2. Moreover, the identification of a new ERG isoform (ERG3Deltaex12) suggests the association with different interaction partners and adds to the complexity of downstream pathways mediated by the expression of specific ERG transcripts in acute leukemia.

e18552Background: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis resulting in peripheral cytopenia. In low risk MDS, a pro-apoptotic milieu was found with increased ...

Activating point mutations in CBL have recently been identified in diverse subtypes of myeloid neoplasms. Because detailed clinical and hematological characteristics of CBL-mutated cases is lacking, we screened 156 BCR-ABL and JAK2 V617F negative patients with myeloproliferative neoplasms (MPN) and overlap syndromes between myelodysplastic syndrome (MDS) and MPN (MPS/MPN) for mutations in exons 8 and 9 of CBL by denaturing high-performance liquid chromatography and direct sequencing. CBL mutations were identified in 16/156 patients (10 %), of which five also carried mutations in EZH2 (n = 3) and TET2 (n = 2). Comprehensive clinical and hematological characteristics were available from 13/16 patients (81 %). In addition to splenomegaly (77 %), striking common hematological features were CML-like left-shifted leukocytosis (85 %) with monocytosis (85 %), anemia (100 %), and thrombocytopenia (62 %). Thrombocytosis was not observed in any patient. Relevant bone marrow features (n = 12) included hypercellularity (92 %) with marked granulopoiesis (92 %), nonclustered microlobulated megakaryocytes (83 %), and marrow fibrosis (83 %). Nine deaths (progression to secondary acute myeloid leukemia/blast phase, n = 7; cytopenia complications, n = 2) were recorded. Three-year survival rate was 27 %, possibly indicating poor prognosis of CBL mutated MDS/MPN patients.

Background NOTCH1 mutations have been associated with a favorable outcome in pediatric acute T-lymphoblastic leukemia. However, the results of studies on the prognostic significance of NOTCH1 mutations in adult T-lymphoblastic leukemia remain controversial. Design and Methods Here we have investigated the prognostic impact of mutations in the NOTCH1 pathway, in particular, the NOTCH1 and FBXW7 genes, in a large cohort of adult patients with T-lymphoblastic leukemia (n=126). We determined the occurrence of mutations in NOTCH1 and FBXW7 by DNA amplification and direct sequencing of polymerase chain reaction products. Results Mutations were identified in 57% and 12% of the NOTCH1 and FBXW7 genes, respectively. The characteristics of patients carrying NOTCH1 and/or FBXW7 ( NOTCH1-FBXW7 ) mutations were similar to those with wild-type genes. Patients with NOTCH1-FBXW7 mutations more often showed a thymic immunophenotype ( p =0.001). In the overall cohort, no significant differences were seen in the complete remission or event-free survival rates between patients with mutated or wild-type NOTCH1 - FBXW7 ( p =0.39). Conclusions NOTCH1 and FBXW7 mutations were not predictive of outcome in the overall cohort of adult patients with T-lymphoblastic leukemia, but there was a trend towards a favorable prognostic impact of NOTCH1-FBXW7 mutations in the small subgroup of patients with low-risk ERG/BAALC expression status. Our findings further confirm the high frequency of NOTCH1 mutations in adult T-lymphoblastic leukemia.