ABSTRACT
The consensus molecular subtype (CMS) classification of colorectal cancer is based on bulk transcriptomics. The underlying epithelial cell diversity remains unclear. We analyzed 373,058 single-cell transcriptomes from 63 patients, focusing on 49,155 epithelial cells. We identified a pervasive genetic and transcriptomic dichotomy of malignant cells, based on distinct gene expression, DNA copy number and gene regulatory network. We recapitulated these subtypes in bulk transcriptomes from 3,614 patients. The two intrinsic subtypes, iCMS2 and iCMS3, refine CMS. iCMS3 comprises microsatellite unstable (MSI-H) cancers and one-third of microsatellite-stable (MSS) tumors. iCMS3 MSS cancers are transcriptomically more similar to MSI-H cancers than to other MSS cancers. CMS4 cancers had either iCMS2 or iCMS3 epithelium; the latter had the worst prognosis. We defined the intrinsic epithelial axis of colorectal cancer and propose a refined 'IMF' classification with five subtypes, combining intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F).
Subject(s)
Colorectal Neoplasms , Neoplasms, Glandular and Epithelial , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Epithelial Cells/pathology , Humans , Microsatellite Instability , Microsatellite Repeats/genetics , Neoplasms, Glandular and Epithelial/genetics , Transcriptome/geneticsABSTRACT
PURPOSE: Despite the established role of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC, drug resistance inevitably ensues, with a paucity of treatment options especially in EGFR T790M-negative resistance. EXPERIMENTAL DESIGN: We performed whole-exome and transcriptome analysis of 59 patients with first- and second-generation EGFR TKI-resistant metastatic EGFR-mutated NSCLC to characterize and compare molecular alterations mediating resistance in T790M-positive (T790M+) and -negative (T790M-) disease. RESULTS: Transcriptomic analysis revealed ubiquitous loss of adenocarcinoma lineage gene expression in T790M- tumors, orthogonally validated using multiplex IHC. There was enrichment of genomic features such as TP53 alterations, 3q chromosomal amplifications, whole-genome doubling and nonaging mutational signatures in T790M- tumors. Almost half of resistant tumors were further classified as immunehot, with clinical outcomes conditional on immune cell-infiltration state and T790M status. Finally, using a Bayesian statistical approach, we explored how T790M- and T790M+ disease might be predicted using comprehensive genomic and transcriptomic profiles of treatment-naïve patients. CONCLUSIONS: Our results illustrate the interplay between genetic alterations, cell lineage plasticity, and immune microenvironment in shaping divergent TKI resistance and outcome trajectories in EGFR-mutated NSCLC. Genomic and transcriptomic profiling may facilitate the design of bespoke therapeutic approaches tailored to a tumor's adaptive potential.
Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , ErbB Receptors/genetics , Humans , Protein-Tyrosine Kinases/geneticsABSTRACT
Lung cancer is the world's leading cause of cancer death and shows strong ancestry disparities. By sequencing and assembling a large genomic and transcriptomic dataset of lung adenocarcinoma (LUAD) in individuals of East Asian ancestry (EAS; n = 305), we found that East Asian LUADs had more stable genomes characterized by fewer mutations and fewer copy number alterations than LUADs from individuals of European ancestry. This difference is much stronger in smokers as compared to nonsmokers. Transcriptomic clustering identified a new EAS-specific LUAD subgroup with a less complex genomic profile and upregulated immune-related genes, allowing the possibility of immunotherapy-based approaches. Integrative analysis across clinical and molecular features showed the importance of molecular phenotypes in patient prognostic stratification. EAS LUADs had better prediction accuracy than those of European ancestry, potentially due to their less complex genomic architecture. This study elucidated a comprehensive genomic landscape of EAS LUADs and highlighted important ancestry differences between the two cohorts.
Subject(s)
Adenocarcinoma of Lung/genetics , Lung Neoplasms/genetics , Mutation , Adenocarcinoma of Lung/etiology , Adenocarcinoma of Lung/mortality , Adenocarcinoma of Lung/therapy , Aged , Asian People/genetics , Cohort Studies , DNA Copy Number Variations , ErbB Receptors/genetics , Exome , Female , Gene Expression Profiling , Humans , Lung Neoplasms/etiology , Lung Neoplasms/mortality , Lung Neoplasms/therapy , Male , Middle Aged , Proto-Oncogene Proteins p21(ras)/genetics , Singapore , Tumor Suppressor Protein p53/geneticsABSTRACT
PURPOSE: Mesenchymal epithelial transition factor ( MET) activation has been implicated as an oncogenic driver in epidermal growth factor receptor ( EGFR)-mutant non-small-cell lung cancer (NSCLC) and can mediate primary and secondary resistance to EGFR tyrosine kinase inhibitors (TKI). High copy number thresholds have been suggested to enrich for response to MET inhibitors. We examined the clinical relevance of MET copy number gain (CNG) in the setting of treatment-naive metastatic EGFR-mutant-positive NSCLC. PATIENTS AND METHODS: MET fluorescence in situ hybridization was performed in 200 consecutive patients identified as metastatic treatment-naïve EGFR-mutant-positive. We defined MET-high as CNG greater than or equal to 5, with an additional criterion of MET/centromeric portion of chromosome 7 ratiο greater than or equal to 2 for amplification. Time-to-treatment failure (TTF) to EGFR TKI in patients identified as MET-high and -low was estimated by Kaplan-Meier method and compared using log-rank test. Multiregion single-nucleotide polymorphism array analysis was performed on 13 early-stage resected EGFR-mutant-positive NSCLC across 59 sectors to investigate intratumoral heterogeneity of MET CNG. RESULTS: Fifty-two (26%) of 200 patients in the metastatic cohort were MET-high at diagnosis; 46 (23%) had polysomy and six (3%) had amplification. Median TTF was 12.2 months (95% CI, 5.7 to 22.6 months) versus 13.1 months (95% CI, 10.6 to 15.0 months) for MET-high and -low, respectively ( P = .566), with no significant difference in response rate regardless of copy number thresholds. Loss of MET was observed in three of six patients identified as MET-high who underwent postprogression biopsies, which is consistent with marked intratumoral heterogeneity in MET CNG observed in early-stage tumors. Suboptimal response (TTF, 1.0 to 6.4 months) to EGFR TKI was observed in patients with coexisting MET amplification (five [3.2%] of 154). CONCLUSION: Although up to 26% of TKI-naïve EGFR-mutant-positive NSCLC harbor high MET CNG by fluorescence in situ hybridization, this did not significantly affect response to TKI, except in patients identified as MET-amplified. Our data underscore the limitations of adopting arbitrary copy number thresholds and the need for cross-assay validation to define therapeutically tractable MET pathway dysregulation in EGFR-mutant-positive NSCLC.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Drug Resistance, Neoplasm/genetics , Gene Amplification , Gene Dosage , Lung Neoplasms/genetics , Mutation , Proto-Oncogene Proteins c-met/genetics , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/secondary , Cell Line, Tumor , Databases, Factual , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Genetic Heterogeneity , Humans , In Situ Hybridization, Fluorescence , Lung Neoplasms/drug therapy , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Predictive Value of Tests , Protein Kinase Inhibitors/therapeutic use , Reproducibility of Results , Retrospective StudiesABSTRACT
Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types1-4. Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients5,6. Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control2,4,7-10, in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8+ TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein-Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8+ TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8+ TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39- CD8+ TILs. Furthermore, frequencies of CD39 expression among CD8+ TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells.
Subject(s)
Bystander Effect/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Colorectal Neoplasms/immunology , Lung Neoplasms/immunology , Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/immunology , Antigens, Neoplasm/immunology , Antigens, Viral/immunology , Apyrase/analysis , Apyrase/deficiency , Apyrase/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Separation , Colorectal Neoplasms/genetics , ErbB Receptors/genetics , Humans , Lung Neoplasms/genetics , Lymphocytes, Tumor-Infiltrating/metabolism , PhenotypeABSTRACT
EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.
Subject(s)
Adenocarcinoma/genetics , ErbB Receptors/genetics , Exome Sequencing/methods , Genomics/methods , Lung Neoplasms/genetics , Mutation , Adenocarcinoma/ethnology , Adenocarcinoma/pathology , Asian People/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Genetic Predisposition to Disease/ethnology , Genetic Predisposition to Disease/genetics , Humans , Lung Neoplasms/ethnology , Lung Neoplasms/pathology , Protein Kinase Inhibitors/pharmacologyABSTRACT
Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.
Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation, Neoplastic , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cells, B-Lymphoid/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/physiology , Clinical Trials as Topic , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Molecular Sequence Data , Phosphatidylinositol 3-Kinase/metabolism , Pre-B-Cell Leukemia Transcription Factor 1 , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-bcl-6 , Signal Transduction , Syk Kinase , Up-Regulation , src-Family Kinases/metabolismABSTRACT
Initial cell surface expression of the pre-B cell receptor induces proliferation. After 2 to 5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). The mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. The checkpoint at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling induces cell-cycle exit through up-regulation of the transcriptional repressor BCL6. Inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Hence, pre-B cell receptor-mediated activation of BCL6 limits pre-B cell proliferation and induces cellular quiescence at the small pre-BII (Fraction D) stage.
Subject(s)
Cell Cycle Checkpoints/physiology , Cell Division/physiology , DNA-Binding Proteins/biosynthesis , Pre-B Cell Receptors/metabolism , Precursor Cells, B-Lymphoid/metabolism , Proto-Oncogene Proteins c-myc/biosynthesis , Transcription, Genetic/physiology , Animals , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cyclin D2/genetics , Cyclin D2/metabolism , DNA-Binding Proteins/genetics , Gene Rearrangement, B-Lymphocyte, Light Chain/physiology , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/metabolism , Mice , Mice, Knockout , Pre-B Cell Receptors/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cells, B-Lymphoid/cytology , Proto-Oncogene Proteins c-bcl-6 , Proto-Oncogene Proteins c-myc/genetics , Signal Transduction/physiologyABSTRACT
Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.
Subject(s)
DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm , Fusion Proteins, bcr-abl/antagonists & inhibitors , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Kinase Inhibitors/pharmacology , ADP-Ribosylation Factor 1/metabolism , Animals , Cell Survival/drug effects , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-bcl-6 , Transcription, Genetic , Tumor Suppressor Protein p53/metabolismABSTRACT
BCL6 protects germinal center (GC) B cells against DNA damage-induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7-dependent B cell precursors, we report that IL-7Ralpha-Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a mu heavy chain, however, activation of pre-B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Ralpha-Stat5 signaling is attenuated. At the transition from IL-7-dependent to -independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre-B cells from DNA damage-induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre-B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.
Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/immunology , DNA-Binding Proteins/immunology , ADP-Ribosylation Factors/metabolism , Animals , Apoptosis , Base Sequence , Cell Proliferation , Cell Survival , Cells, Cultured , Cytoprotection , DNA Damage/genetics , Down-Regulation/genetics , Gene Rearrangement, B-Lymphocyte, Light Chain/genetics , Humans , Interleukin-7/metabolism , Lymphopoiesis , Mice , Molecular Sequence Data , Pre-B Cell Receptors/metabolism , Precursor Cells, B-Lymphoid/cytology , Precursor Cells, B-Lymphoid/metabolism , Proto-Oncogene Proteins c-bcl-6 , Proto-Oncogene Proteins c-myc/metabolism , Recombination, Genetic/genetics , Signal Transduction , Transcription, Genetic , Up-Regulation/geneticsABSTRACT
B cell lineage ALL represents by far the most frequent malignancy in children and is also common in adults. Despite significant advances over the past four decades, cytotoxic treatment strategies have recently reached a plateau with cure rates at 80 percent for children and 55 percent for adults. Relapse after cytotoxic drug treatment, initial drug-resistance and dose-limiting toxicity are among the most frequent complications of current therapy approaches. For this reason, pathway-specific treatment strategies in addition to cytotoxic drug treatment seem promising to further improve therapy options for ALL patients. In a recent study on 111 cases of pre-B cell-derived human ALL, we found that ALL cells carrying a BCR-ABL1-gene rearrangement lack expression of a functional pre-B cell receptor in virtually all cases. In a proof-of-principle experiment, we studied pre-B cell receptor function during progressive leukemic transformation of pre-B cells in BCR-ABL1-transgenic mice: Interestingly, signaling from the pre-B cell receptor and the oncogenic BCR-ABL1 kinase are mutually exclusive and only "crippled" pre-B cells that fail to express a functional pre-B cell receptor are permissive to transformation by BCR-ABL1.
Subject(s)
Pre-B Cell Receptors/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Humans , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-bcr/genetics , Proto-Oncogene Proteins c-bcr/metabolism , Signal TransductionABSTRACT
B cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B cell precursors that are arrested at pre-B cell receptor-dependent stages. The Philadelphia chromosome-positive (Ph(+)) subtype of ALL accounts for 25-30% of cases of adult ALL, has the most unfavorable clinical outcome among all ALL subtypes and is defined by the oncogenic BCR-ABL1 kinase and deletions of the IKAROS gene in >80% of cases. Here, we demonstrate that the pre-B cell receptor functions as a tumor suppressor upstream of IKAROS through induction of cell cycle arrest in Ph(+) ALL cells. Pre-B cell receptor-mediated cell cycle arrest in Ph(+) ALL cells critically depends on IKAROS function, and is reversed by coexpression of the dominant-negative IKAROS splice variant IK6. IKAROS also promotes tumor suppression through cooperation with downstream molecules of the pre-B cell receptor signaling pathway, even if expression of the pre-B cell receptor itself is compromised. In this case, IKAROS redirects oncogenic BCR-ABL1 tyrosine kinase signaling from SRC kinase-activation to SLP65, which functions as a critical tumor suppressor downstream of the pre-B cell receptor. These findings provide a rationale for the surprisingly high frequency of IKAROS deletions in Ph(+) ALL and identify IKAROS-mediated cell cycle exit as the endpoint of an emerging pathway of pre-B cell receptor-mediated tumor suppression.