Predicting Response to Radical Chemoradiotherapy with Circulating HPV DNA (cHPV-DNA) in Locally Advanced Uterine Cervix Cancer.

BACKGROUND: The majority of locally advanced cervical cancers (LaCC) are causally related to HPV. We sought to investigate the utility of an ultra-sensitive HPV-DNA next generation sequencing (NGS) assay-panHPV-detect-in LaCC treated with chemoradiotherapy, as a marker of treatment response and persistent disease. METHOD: Serial blood samples were collected from 22 patients with LaCC before, during and after chemoradiation. The presence of circulating HPV-DNA was correlated with clinical and radiological outcomes. RESULTS: The panHPV-detect test demonstrated a sensitivity and specificity of 88% (95% CI-70-99%) and 100% (95% CI-30-100%), respectively, and correctly identified the HPV-subtype (16, 18, 45, 58). After a median follow up of 16 months, and three relapses all had detectable cHPV-DNA at 3 months post-CRT despite complete response on imaging. Another four patients with radiological partial or equivocal response and undetectable cHPV-DNA at the 3-month time point did not go on to develop relapse. All patients with radiological CR and undetectable cHPV-DNA at 3-months remained disease free. CONCLUSIONS: These results demonstrate that the panHPV-detect test shows high sensitivity and specificity for detecting cHPV-DNA in plasma. The test has potential applications in assessment of the response to CRT and in monitoring for relapse, and these initial findings warrant validation in a larger cohort.

Accumulation of copy number alterations and clinical progression across advanced prostate cancer.

BACKGROUND: Genomic copy number alterations commonly occur in prostate cancer and are one measure of genomic instability. The clinical implication of copy number change in advanced prostate cancer, which defines a wide spectrum of disease from high-risk localised to metastatic, is unknown. METHODS: We performed copy number profiling on 688 tumour regions from 300 patients, who presented with advanced prostate cancer prior to the start of long-term androgen deprivation therapy (ADT), in the control arm of the prospective randomised STAMPEDE trial. Patients were categorised into metastatic states as follows; high-risk non-metastatic with or without local lymph node involvement, or metastatic low/high volume. We followed up patients for a median of 7 years. Univariable and multivariable Cox survival models were fitted to estimate the association between the burden of copy number alteration as a continuous variable and the hazard of death or disease progression. RESULTS: The burden of copy number alterations positively associated with radiologically evident distant metastases at diagnosis (P=0.00006) and showed a non-linear relationship with clinical outcome on univariable and multivariable analysis, characterised by a sharp increase in the relative risk of progression (P=0.003) and death (P=0.045) for each unit increase, stabilising into more modest increases with higher copy number burdens. This association between copy number burden and outcome was similar in each metastatic state. Copy number loss occurred significantly more frequently than gain at the lowest copy number burden quartile (q=4.1 × 10-6). Loss of segments in chromosome 5q21-22 and gains at 8q21-24, respectively including CHD1 and cMYC occurred more frequently in cases with higher copy number alteration (for either region: Kolmogorov-Smirnov distance, 0.5; adjusted P<0.0001). Copy number alterations showed variability across tumour regions in the same prostate. This variance associated with increased risk of distant metastases (Kruskal-Wallis test P=0.037). CONCLUSIONS: Copy number alteration in advanced prostate cancer associates with increased risk of metastases at diagnosis. Accumulation of a limited number of copy number alterations associates with most of the increased risk of disease progression and death. The increased likelihood of involvement of specific segments in high copy number alteration burden cancers may suggest an order underlying the accumulation of copy number changes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00268476 , registered on December 22, 2005. EudraCT  2004-000193-31 , registered on October 4, 2004.

Quantitative Assessment and Prognostic Associations of the Immune Landscape in Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer characterised by a high frequency of loss-of-function ARID1A mutations and a poor response to chemotherapy. Despite their generally low mutational burden, an intratumoural T cell response has been reported in a subset of OCCC, with ARID1A purported to be a biomarker for the response to the immune checkpoint blockade independent of micro-satellite instability (MSI). However, assessment of the different immune cell types and spatial distribution specifically within OCCC patients has not been described to date. Here, we characterised the immune landscape of OCCC by profiling a cohort of 33 microsatellite stable OCCCs at the genomic, gene expression and histological level using targeted sequencing, gene expression profiling using the NanoString targeted immune panel, and multiplex immunofluorescence to assess the spatial distribution and abundance of immune cell populations at the protein level. Analysis of these tumours and subsequent independent validation identified an immune-related gene expression signature associated with risk of recurrence of OCCC. Whilst histological quantification of tumour-infiltrating lymphocytes (TIL, Salgado scoring) showed no association with the risk of recurrence or ARID1A mutational status, the characterisation of TILs via multiplexed immunofluorescence identified spatial differences in immunosuppressive cell populations in OCCC. Tumour-associated macrophages (TAM) and regulatory T cells were excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A-mutated OCCCs compared to ARID1A wild-type tumours, suggesting that the exclusion of these immune effectors could determine the host response of ARID1A-mutant OCCCs to therapy. Overall, our study has provided new insights into the immune landscape and prognostic associations in OCCC and suggest that tailored immunotherapeutic approaches may be warranted for different subgroups of OCCC patients.

Mutational signatures impact the evolution of anti-EGFR antibody resistance in colorectal cancer.

Anti-EGFR antibodies such as cetuximab are active against KRAS/NRAS wild-type colorectal cancers (CRCs), but acquired resistance invariably evolves. It is unknown which mutational mechanisms enable resistance evolution and whether adaptive mutagenesis (a transient cetuximab-induced increase in mutation generation) contributes in patients. Here, we investigate these questions in exome sequencing data from 42 baseline and progression biopsies from cetuximab-treated CRCs. Mutation loads did not increase from baseline to progression, and evidence for a contribution of adaptive mutagenesis was limited. However, the chemotherapy-induced mutational signature SBS17b was the main contributor of specific KRAS/NRAS and EGFR driver mutations that are enriched at acquired resistance. Detectable SBS17b activity before treatment predicted shorter progression-free survival and the evolution of these specific mutations during subsequent cetuximab treatment. This result suggests that chemotherapy mutagenesis can accelerate resistance evolution. Mutational signatures may be a new class of cancer evolution predictor.

The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing.

1. BACKGROUND: The application of massively parallel sequencing has led to the identification of aberrant druggable pathways and somatic mutations within therapeutically relevant genes in gastro-oesophageal cancer. Given the widespread use of formalin-fixed paraffin-embedded (FFPE) samples in the study of this disease, it would be beneficial, especially for the purposes of biomarker evaluation, to assess the concordance between comprehensive exome-wide sequencing data from archival FFPE samples originating from a prospective clinical study and those derived from fresh-frozen material. 2. METHODS: We analysed whole-exome sequencing data to define the mutational concordance of 16 matched fresh-frozen and FFPE gastro-oesophageal tumours (N = 32) from a prospective clinical study. We assessed DNA integrity prior to sequencing and then identified coding mutations in genes that have previously been implicated in other cancers. In addition, we calculated the mutant-allele heterogeneity (MATH) for these samples. 3. RESULTS: Although there was increased degradation of DNA in FFPE samples compared with frozen samples, sequencing data from only two FFPE samples failed to reach an adequate mapping quality threshold. Using a filtering threshold of mutant read counts of at least ten and a minimum of 5% variant allele frequency (VAF) we found that there was a high median mutational concordance of 97% (range 80.1-98.68%) between fresh-frozen and FFPE gastro-oesophageal tumour-derived exomes. However, the majority of FFPE tumours had higher mutant-allele heterogeneity (MATH) scores when compared with corresponding frozen tumours (p < 0.001), suggesting that FFPE-based exome sequencing is likely to over-represent tumour heterogeneity in FFPE samples compared to fresh-frozen samples. Furthermore, we identified coding mutations in 120 cancer-related genes, including those associated with chromatin remodelling and Wnt/ß-catenin and Receptor Tyrosine Kinase signalling. 4. CONCLUSIONS: These data suggest that comprehensive genomic data can be generated from exome sequencing of selected DNA samples extracted from archival FFPE gastro-oesophageal tumour tissues within the context of prospective clinical trials.

Next Generation Sequencing Assay for Detection of Circulating HPV DNA (cHPV-DNA) in Patients Undergoing Radical (Chemo)Radiotherapy in Anal Squamous Cell Carcinoma (ASCC).

Background: Following chemo-radiotherapy (CRT) for human papilloma virus positive (HPV+) anal squamous cell carcinoma (ASCC), detection of residual/recurrent disease is challenging. Patients frequently undergo unnecessary repeated biopsies for abnormal MRI/clinical findings. In a pilot study we assessed the role of circulating HPV-DNA in identifying "true" residual disease. Methods: We prospectively collected plasma samples at baseline (n = 21) and 12 weeks post-CRT (n = 17). Circulating HPV-DNA (cHPV DNA) was measured using a novel next generation sequencing (NGS) assay, panHPV-detect, comprising of two primer pools covering distinct regions of eight high-risk HPV genomes (16, 18, 31, 33, 35, 45, 52, and 58) to detect circulating HPV-DNA (cHPV DNA). cHPV-DNA levels post-CRT were correlated to disease response. Results: In pre-CRT samples, panHPV-detect demonstrated 100% sensitivity and specificity for HPV associated ASCC. PanHPV-detect was able to demonstrate cHPV-DNA in 100% (9/9) patients with T1/T2N0 cancers. cHPV-DNA was detectable 12 weeks post CRT in just 2/17 patients, both of whom relapsed. 1/16 patients who had a clinical complete response (CR) at 3 months post-CRT but relapsed at 9 months and 1/1 patient with a partial response (PR). PanHPV-detect demonstrated 100% sensitivity and specificity in predicting response to CRT. Conclusion: We demonstrate that panHPV-detect, an NSG assay is a highly sensitive and specific test for the identification of cHPV-DNA in plasma at diagnosis. cHPV-DNA post-treatment may predict clinical response to CRT.

Extreme intratumour heterogeneity and driver evolution in mismatch repair deficient gastro-oesophageal cancer.

Mismatch repair deficient (dMMR) gastro-oesophageal adenocarcinomas (GOAs) show better outcomes than their MMR-proficient counterparts and high immunotherapy sensitivity. The hypermutator-phenotype of dMMR tumours theoretically enables high evolvability but their evolution has not been investigated. Here we apply multi-region exome sequencing (MSeq) to four treatment-naive dMMR GOAs. This reveals extreme intratumour heterogeneity (ITH), exceeding ITH in other cancer types >20-fold, but also long phylogenetic trunks which may explain the exquisite immunotherapy sensitivity of dMMR tumours. Subclonal driver mutations are common and parallel evolution occurs in RAS, PIK3CA, SWI/SNF-complex genes and in immune evasion regulators. MSeq data and evolution analysis of single region-data from 64 MSI GOAs show that chromosome 8 gains are early genetic events and that the hypermutator-phenotype remains active during progression. MSeq may be necessary for biomarker development in these heterogeneous cancers. Comparison with other MSeq-analysed tumour types reveals mutation rates and their timing to determine phylogenetic tree morphologies.

Author Correction: Extreme intratumour heterogeneity and driver evolution in mismatch repair deficient gastro-oesophageal cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunopeptidomics of colorectal cancer organoids reveals a sparse HLA class I neoantigen landscape and no increase in neoantigens with interferon or MEK-inhibitor treatment.

BACKGROUND: Patient derived organoids (PDOs) can be established from colorectal cancers (CRCs) as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFNγ) or MEK-inhibitor treatment. METHODS: Four microsatellite stable PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analyzed by MS. RESULTS: We identified an average of 9936 unique peptides per PDO which compares favorably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. In contrast, computational HLA binding prediction estimated that 304 mutations could generate neoantigens. One hundred ninety-six of these were located in expressed genes, still exceeding the number of MS-detected neoantigens 65-fold. Treatment of four PDOs with IFNγ upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFNγ-inducible genes. HLA class II presented peptides increased dramatically with IFNγ treatment. MEK-inhibitor treatment showed no consistent effect on HLA class I or II expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment. CONCLUSIONS: Only 3 out of 612 non-silent mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

Correction: Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Detecting and Tracking Circulating Tumour DNA Copy Number Profiles during First Line Chemotherapy in Oesophagogastric Adenocarcinoma.

DNA somatic copy number aberrations (SCNAs) are key drivers in oesophagogastric adenocarcinoma (OGA). Whether minimally invasive SCNA analysis of circulating tumour (ct)DNA can predict treatment outcomes and reveal how SCNAs evolve during chemotherapy is unknown. We investigated this by low-coverage whole genome sequencing (lcWGS) of ctDNA from 30 patients with advanced OGA prior to first-line chemotherapy and on progression. SCNA profiles were detectable pretreatment in 23/30 (76.7%) patients. The presence of liver metastases, primary tumour in situ, or of oesophageal or junctional tumour location predicted for a high ctDNA fraction. A low ctDNA concentration associated with significantly longer overall survival. Neither chromosomal instability metrics nor ploidy correlated with chemotherapy outcome. Chromosome 2q and 8p gains before treatment were associated with chemotherapy responses. lcWGS identified all amplifications found by prior targeted tumour tissue sequencing in cases with detectable ctDNA as well as finding additional changes. SCNA profiles changed during chemotherapy, indicating that cancer cell populations evolved during treatment; however, no recurrent SCNA changes were acquired at progression. Tracking the evolution of OGA cancer cell populations in ctDNA is feasible during chemotherapy. The observation of genetic evolution warrants investigation in larger series and with higher resolution techniques to reveal potential genetic predictors of response and drivers of chemotherapy resistance. The presence of liver metastasis is a potential biomarker for the selection of patients with high ctDNA content for such studies.

CEA expression heterogeneity and plasticity confer resistance to the CEA-targeting bispecific immunotherapy antibody cibisatamab (CEA-TCB) in patient-derived colorectal cancer organoids.

BACKGROUND: The T cell bispecific antibody cibisatamab (CEA-TCB) binds Carcino-Embryonic Antigen (CEA) on cancer cells and CD3 on T cells, which triggers T cell killing of cancer cell lines expressing moderate to high levels of CEA at the cell surface. Patient derived colorectal cancer organoids (PDOs) may more accurately represent patient tumors than established cell lines which potentially enables more detailed insights into mechanisms of cibisatamab resistance and sensitivity. METHODS: We established PDOs from multidrug-resistant metastatic CRCs. CEA expression of PDOs was determined by FACS and sensitivity to cibisatamab immunotherapy was assessed by co-culture of PDOs and allogeneic CD8 T cells. RESULTS: PDOs could be categorized into 3 groups based on CEA cell-surface expression: CEAhi (n = 3), CEAlo (n = 1) and CEAmixed PDOs (n = 4), that stably maintained populations of CEAhi and CEAlo cells, which has not previously been described in CRC cell lines. CEAhi PDOs were sensitive whereas CEAlo PDOs showed resistance to cibisatamab. PDOs with mixed expression showed low sensitivity to cibisatamab, suggesting that CEAlo cells maintain cancer cell growth. Culture of FACS-sorted CEAhi and CEAlo cells from PDOs with mixed CEA expression demonstrated high plasticity of CEA expression, contributing to resistance acquisition through CEA antigen loss. RNA-sequencing revealed increased WNT/ß-catenin pathway activity in CEAlo cells. Cell surface CEA expression was up-regulated by inhibitors of the WNT/ß-catenin pathway. CONCLUSIONS: Based on these preclinical findings, heterogeneity and plasticity of CEA expression appear to confer low cibisatamab sensitivity in PDOs, supporting further clinical evaluation of their predictive effect in CRC. Pharmacological inhibition of the WNT/ß-catenin pathway may be a rational combination to sensitize CRCs to cibisatamab. Our novel PDO and T cell co-culture immunotherapy models enable pre-clinical discovery of candidate biomarkers and combination therapies that may inform and accelerate the development of immuno-oncology agents in the clinic.

Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer.

Despite showing clinical activity in BRAF-mutant melanoma, the MEK inhibitor (MEKi) trametinib has failed to show clinical benefit in KRAS-mutant colorectal cancer. To identify mechanisms of resistance to MEKi, we employed a pharmacogenomic analysis of MEKi-sensitive versus MEKi-resistant colorectal cancer cell lines. Strikingly, interferon- and inflammatory-related gene sets were enriched in cell lines exhibiting intrinsic and acquired resistance to MEK inhibition. The bromodomain inhibitor JQ1 suppressed interferon-stimulated gene (ISG) expression and in combination with MEK inhibitors displayed synergistic effects and induced apoptosis in MEKi-resistant colorectal cancer cell lines. ISG expression was confirmed in patient-derived organoid models, which displayed resistance to trametinib and were resensitized by JQ1 co-treatment. In in vivo models of colorectal cancer, combination treatment significantly suppressed tumor growth. Our findings provide a novel explanation for the limited response to MEK inhibitors in KRAS-mutant colorectal cancer, known for its inflammatory nature. Moreover, the high expression of ISGs was associated with significantly reduced survival of colorectal cancer patients. Excitingly, we have identified novel therapeutic opportunities to overcome intrinsic and acquired resistance to MEK inhibition in colorectal cancer.

Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing.

BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies. METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction. RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing. CONCLUSIONS: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution. CLINICALTRIALSGOV IDENTIFIER: NCT02112357.

Deterministic Evolutionary Trajectories Influence Primary Tumor Growth: TRACERx Renal.

The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with >10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance.

Corrigendum: Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

This corrects the article DOI: 10.1038/nature22364.

Tracking the Evolution of Non-Small-Cell Lung Cancer.

BACKGROUND: Among patients with non-small-cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early-stage NSCLC. METHODS: In this prospective cohort study, we performed multiregion whole-exome sequencing on 100 early-stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence-free survival. RESULTS: We observed widespread intratumor heterogeneity for both somatic copy-number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy-number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy-number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10-4), which remained significant in multivariate analysis. CONCLUSIONS: Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601 .).

Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies.

ERK-Induced Activation of TCF Family of SRF Cofactors Initiates a Chromatin Modification Cascade Associated with Transcription.

We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription.

Distinct modes of SMAD2 chromatin binding and remodeling shape the transcriptional response to NODAL/Activin signaling.

NODAL/Activin signaling orchestrates key processes during embryonic development via SMAD2. How SMAD2 activates programs of gene expression that are modulated over time however, is not known. Here we delineate the sequence of events that occur from SMAD2 binding to transcriptional activation, and the mechanisms underlying them. NODAL/Activin signaling induces dramatic chromatin landscape changes, and a dynamic transcriptional network regulated by SMAD2, acting via multiple mechanisms. Crucially we have discovered two modes of SMAD2 binding. SMAD2 can bind pre-acetylated nucleosome-depleted sites. However, it also binds to unacetylated, closed chromatin, independently of pioneer factors, where it induces nucleosome displacement and histone acetylation. For a subset of genes, this requires SMARCA4. We find that long term modulation of the transcriptional responses requires continued NODAL/Activin signaling. Thus SMAD2 binding does not linearly equate with transcriptional kinetics, and our data suggest that SMAD2 recruits multiple co-factors during sustained signaling to shape the downstream transcriptional program.