Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA ...methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation-based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAF(V600E) mutation. A CIMP-low (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent ∼7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing.
Targeted therapies against the BCR-ABL1 kinase have revolutionized treatment of chronic phase (CP) chronic myeloid leukemia (CML). In contrast, management of blast crisis (BC) CML remains challenging ...because BC cells acquire complex molecular alterations that confer stemness features to progenitor populations and resistance to BCR-ABL1 tyrosine kinase inhibitors. Comprehensive models of BC transformation have proved elusive because of the rarity and genetic heterogeneity of BC, but are important for developing biomarkers predicting BC progression and effective therapies. To better understand BC, we performed an integrated multiomics analysis of 74 CP and BC samples using whole-genome and exome sequencing, transcriptome and methylome profiling, and chromatin immunoprecipitation followed by high-throughput sequencing. Employing pathway-based analysis, we found the BC genome was significantly enriched for mutations affecting components of the polycomb repressive complex (PRC) pathway. While transcriptomically, BC progenitors were enriched and depleted for PRC1- and PRC2-related gene sets respectively. By integrating our data sets, we determined that BC progenitors undergo PRC-driven epigenetic reprogramming toward a convergent transcriptomic state. Specifically, PRC2 directs BC DNA hypermethylation, which in turn silences key genes involved in myeloid differentiation and tumor suppressor function via so-called epigenetic switching, whereas PRC1 represses an overlapping and distinct set of genes, including novel BC tumor suppressors. On the basis of these observations, we developed an integrated model of BC that facilitated the identification of combinatorial therapies capable of reversing BC reprogramming (decitabine+PRC1 inhibitors), novel PRC-silenced tumor suppressor genes (NR4A2), and gene expression signatures predictive of disease progression and drug resistance in CP.
•Genetically heterogeneous BC progenitors demonstrate molecular convergence on PRC1- and PRC2-regulated pathways.•A model of PRC-driven reprogramming identifies novel BC combination therapies, tumor suppressor genes, and biomarkers for transformation.
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Associations of sarcoma with inherited cancer syndromes implicate genetic predisposition in sarcoma development. However, due to the apparently sporadic nature of sarcomas, little attention has been ...paid to the role genetic susceptibility in sporadic sarcoma. To address this, we performed targeted-genomic sequencing to investigate the prevalence of germline mutations in known cancer-associated genes within an Asian cohort of sporadic sarcoma patients younger than 50 years old. We observed 13.6% (n = 9) amongst 66 patients harbour at least one predicted pathogenic germline mutation in 10 cancer-associated genes including ATM, BRCA2, ERCC4, FANCC, FANCE, FANCI, MSH6, POLE, SDHA and TP53. The most frequently affected genes are involved in the DNA damage repair pathway, with a germline mutation prevalence of 10.6%. Our findings suggests that genetic predisposition plays a larger role than expected in our Asian cohort of sporadic sarcoma, therefore clinicians should be aware of the possibility that young sarcoma patients may be carriers of inherited mutations in cancer genes and should be considered for genetic testing, regardless of family history. The prevalence of germline mutations in DNA damage repair genes imply that therapeutic strategies exploiting the vulnerabilities resulting from impaired DNA repair may be promising areas for translational research.
Chromatin alterations are fundamental hallmarks of cancer. To study chromatin alterations in primary gastric adenocarcinomas, we perform nanoscale chromatin immunoprecipitation sequencing of multiple ...histone modifications in five gastric cancers and matched normal tissues. We identify hundreds of somatically altered promoters and predicted enhancers. Many cancer-associated promoters localize to genomic sites lacking previously annotated transcription start sites (cryptic promoters), driving expression of nearby genes involved in gastrointestinal cancer, embryonic development and tissue specification. Cancer-associated promoters overlap with embryonic stem cell regions targeted by polycomb repressive complex 2, exhibiting promoter bivalency and DNA methylation loss. We identify somatically acquired elements exhibiting germline allelic biases and non-coding somatic mutations creating new promoters. Our findings demonstrate the feasibility of profiling chromatin from solid tumours with limited tissue to identify regulatory elements, transcriptional patterns and regulatory genetic variants associated with cancer.
Although estrogen receptor alpha (ERα) and insulin-like growth factor (IGF) signaling are important for normal mammary development and breast cancer, cross-talk between these pathways, particularly ...at the level of transcription, remains poorly understood. We performed microarray analysis on MCF-7 breast cancer cells treated with estradiol (E2) or IGF-I for 3 or 24 h. IGF-I regulated mRNA of five to tenfold more genes than E2, and many genes were co-regulated by both ligands. Importantly, expression of these co-regulated genes correlated with poor prognosis of human breast cancer. Closer examination revealed enrichment of repressed transcripts. Interestingly, a number of potential tumor suppressors, for example, B-cell linker (
BLNK
), were down-regulated by IGF-I and E2. Analysis of three down-regulated genes showed that E2-mediated repression occurred independently of IGF-IR, and IGF-I-mediated repression occurred independently of ERα. However, repression by IGF-I or E2 required common kinases, such as PI3K and MEK, suggesting downstream convergence of the two pathways. In conclusion, E2 and IGF-I co-regulate a set of genes that affect breast cancer outcome. There is enrichment of repressed transcripts, and, for some genes, the down-regulation is independent at the receptor level. This may be important clinically, as tumors with active ERα and IGF-IR signaling may require co-targeting of both pathways.
Abstract
Background: Immunotherapy has so far had limited success in colorectal cancer (CRC), with its efficacy restricted to a subset of microsatellite instability high (MSI-H) tumors. A ...comprehensive interrogation of the CRC tumor immune microenvironment (TME) is urgently needed. We present here an ongoing multi-platform study on early stage colon and rectal cancers, where immuno-genomic profiling of tumors and patient-derived cell models of tumor epithelia, cancer-associated fibroblasts and tumor-infiltrating lymphocytes (TIL) complement each other, with opportunities for mutual cross-validation between experimental and bioprofiling data.
Methods: At the time of writing, 21 of a planned 50 early stage CRC patients have been recruited. Surgically resected tumors are processed for 4 broad classes of analyses: 1) Bulk tissue profiling by RNA and whole exome sequencing; 2) High resolution protein and transcriptome analysis comprising scRNA-seq and flow cytometry/CyToF; 3) H&E analysis and multiplex immunohistochemistry for TME-specific proteins; 4) Culture of epithelial, fibroblast, and TIL lines, and generation of patient-derived xenografts for functional studies.
Results: Four tumors were MSI-H and 17 were microsatellite stable (MSS), with 1 POLE-mutant MSS patient harboring over 6000 mutations. Bulk genomics analysis revealed the most common mutations to be in TP53, APC, MUC17, and TGFBR2. The most frequently altered pathways were WNT, followed in order by p53, TGFβ, PI3K, and RAS-MAPK. scRNA-seq and flow cytometry/CyToF analyses are being performed to examine immune phenotypes, mediators of cell migration, and immune suppressive populations, which complement data on transcriptomic profile, histopathology, and spatial localization of TME cellular components. Of the 3 cultured cell types, 16 patients have lines of at least one cell compartment established in vitro. Characteristics of individual models will be reported at the meeting. Establishing the 3 cultured cell types from the same patient will enable us to develop an autologous patient-derived co-culture system to evaluate all 3 pairwise interactions, including TIL cytotoxicity toward epithelial cells, mutual modulation by fibroblast and epithelial cells in co-culture and their phenotypic alterations, and fibroblast regulation of TIL cytotoxic function. Autologous co-cultures with all 3 cell types are also planned.
Conclusions: The generation of well-annotated multi-platform profiling data from CRC tumors, complemented by matched tri-compartment patient-derived cell cultures, enables mutual cross-validation between experimental models of the TME and bioprofiling data.
Citation Format: Lindsay H. Kua, Fiona Y. Lee, Christine L. Eng, Harini Srinivasan, Rahul Nahar, Janice H. Oh, Nicole Ann L. Gunn, Kai Xian Thng, Ashley S. Yong, Adrian C. Sim, Rebecca Lim, Nicole Boo, Simeen Malik, Michael T. Wong, Tze Guan Tan, Shu Wen S. Ho, Shirleen Soh, Wan Jun Lim, Macalinao Dominique Camat, Joe P. Yeong, Clarinda W. Chua, Wei Qiang Leow, Ramanuj DasGupta, Si-Lin Koo, Lewis Hong, Brian Henry, Tony K. Lim, Iain B. Tan. Tri-compartment (epithelial, immune, fibroblast) patient-derived models of the tumor microenvironment from an immuno-genomic profiled cohort of early stage colorectal cancers abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3990.
Colorectal cancer with metastases limited to the liver (liver-limited mCRC) is a distinct clinical subset characterized by possible cure with surgery. We performed high-depth sequencing of over 750 ...cancer-associated genes and copy number profiling in matched primary, metastasis and normal tissues to characterize genomic progression in 18 patients with liver-limited mCRC.
High depth Illumina sequencing and use of three different variant callers enable comprehensive and accurate identification of somatic variants down to 2.5% variant allele frequency. We identify a median of 11 somatic single nucleotide variants (SNVs) per tumor. Across patients, a median of 79.3% of somatic SNVs present in the primary are present in the metastasis and 81.7% of all alterations present in the metastasis are present in the primary. Private alterations are found at lower allele frequencies; a different mutational signature characterized shared and private variants, suggesting distinct mutational processes. Using B-allele frequencies of heterozygous germline SNPs and copy number profiling, we find that broad regions of allelic imbalance and focal copy number changes, respectively, are generally shared between the primary tumor and metastasis.
Our analyses point to high genomic concordance of primary tumor and metastasis, with a thick common trunk and smaller genomic branches in general support of the linear progression model in most patients with liver-limited mCRC. More extensive studies are warranted to further characterize genomic progression in this important clinical population.
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