Validation of a clinical blood-based decision aid to guide immunotherapy treatment in patients with non-small cell lung cancer.

BACKGROUND: The widespread introduction of immunotherapy in patients with advanced non-small cell lung cancer (NSCLC) has led to durable responses but still many patients fail and are treated beyond progression. OBJECTIVE: This study investigated whether readily available blood-based tumor biomarkers allow accurate detection of early non-responsiveness, allowing a timely switch of therapy and cost reduction. METHODS: In a prospective, observational study in patients with NSCLC treated with nivolumab or pembrolizumab, five serum tumor markers were measured at baseline and every other week. Six months disease control as determined by RECIST was used as a measure of clinical response. Patients with a disease control <  6 months were deemed non-responsive. For every separate tumor marker a criterion for predicting of non-response was developed. Each marker test was defined as positive (predictive of non-response) if the value of that tumor marker increased at least 50% from the value at baseline and above a marker dependent minimum value to be determined. Also, tests based on combination of multiple markers were designed. Specificity and sensitivity for predicting non-response was calculated and results were validated in an independent cohort. The target specificity of the test for detecting non-response was set at >  95%, in order to allow its safe use for treatment decisions. RESULTS: A total of 376 patients (training cohort: 180, validation cohort: 196) were included in our analysis. Results for the specificity of the single marker tests in the validation set were CEA: 98·3% (95% CI: 90·9-100%), NSE: 96·5% (95% CI: 87·9-99·6%), SCC: 96·5% (95% CI: 88·1-99·6%), Cyfra21·1 : 91.8% (95% CI: 81·9-97·3%), and CA125 : 86·0% (95% CI: 74·2-93·7%). A test based on the combination of Cyfra21.1, CEA and NSE accurately predicted non-response in 32.3% (95% CI 22.6-43.1%) of patients 6 weeks after start of immunotherapy. Survival analysis showed a significant difference between predicted responders (Median PFS: 237 days (95% CI 184-289 days)) and non-responders (Median PFS: 58 days (95% CI 46-70 days)) (p <  0.001). CONCLUSIONS: Serum tumor marker based tests can be used for accurate detection of non-response in NSCLC, thereby allowing early and safe discontinuation of immunotherapy in a significant subset of patients.

Double somatic mutations in mismatch repair genes are frequent in colorectal cancer after Hodgkin's lymphoma treatment.

OBJECTIVE: Hodgkin's lymphoma survivors who were treated with infradiaphragmatic radiotherapy or procarbazine-containing chemotherapy have a fivefold increased risk of developing colorectal cancer (CRC). This study aims to provide insight into the development of therapy-related CRC (t-CRC) by evaluating histopathological and molecular characteristics. DESIGN: 54 t-CRCs diagnosed in a Hodgkin's lymphoma survivor cohort were analysed for mismatch repair (MMR) proteins by immunohistochemistry, microsatellite instability (MSI) and KRAS/BRAF mutations. MSI t-CRCs were evaluated for promoter methylation and mutations in MMR genes. Pathogenicity of MMR gene mutations was evaluated by in silico predictions and functional analyses. Frequencies were compared with a general population cohort of CRC (n=1111). RESULTS: KRAS and BRAF mutations were present in 41% and 15% t-CRCs, respectively. Compared with CRCs in the general population, t-CRCs had a higher MSI frequency (24% vs 11%, p=0.003) and more frequent loss of MSH2/MSH6 staining (13% vs 1%, p<0.001). Loss of MLH1/PMS2 staining and MLH1 promoter methylation were equally common in t-CRCs and the general population. In MSI CRCs without MLH1 promoter methylation, double somatic MMR gene mutations (or loss of heterozygosity as second hit) were detected in 7/10 (70%) t-CRCs and 8/36 (22%) CRCs in the general population (p=0.008). These MMR gene mutations in t-CRCs were classified as pathogenic. MSI t-CRC cases could not be ascribed to Lynch syndrome. CONCLUSIONS: We have demonstrated a higher frequency of MSI among t-CRCs, which results from somatic MMR gene mutations. This suggests a novel association of somatic MMR gene mutations with prior anticancer treatment.

Insertional mutagenesis in a HER2-positive breast cancer model reveals ERAS as a driver of cancer and therapy resistance.

Personalized medicine for cancer patients requires a deep understanding of the underlying genetics that drive cancer and the subsequent identification of predictive biomarkers. To discover new genes and pathways contributing to oncogenesis and therapy resistance in HER2+ breast cancer, we performed Mouse Mammary Tumor Virus (MMTV)-induced insertional mutagenesis screens in ErbB2/cNeu-transgenic mouse models. The screens revealed 34 common integration sites (CIS) in mammary tumors of MMTV-infected mice, highlighting loci with multiple independent MMTV integrations in which potential oncogenes are activated, most of which had never been reported as MMTV CIS. The CIS most strongly associated with the ErbB2-transgenic genotype was the locus containing Eras (ES cell-expressed Ras), a constitutively active RAS-family GTPase. We show that upon expression, Eras acts as a potent oncogenic driver through hyperactivation of the PI3K/AKT pathway, in contrast to other RAS proteins that signal primarily via the MAPK/ERK pathway and require upstream activation or activating mutations to induce signaling. We additionally show that ERAS synergistically enhances HER2-induced tumorigenesis and, in this role, can functionally replace ERBB3/HER3 by acting as a more powerful activator of PI3K/AKT signaling. Although previously reported as pseudogene in humans, we observed ERAS RNA and protein expression in a substantial subset of human primary breast carcinomas. Importantly, we show that ERAS induces primary resistance to the widely used HER2-targeting drugs Trastuzumab (Herceptin) and Lapatinib (Tykerb/Tyverb) in vivo, and is involved in acquired resistance via selective upregulation during treatment in vitro, indicating that ERAS may serve as a novel clinical biomarker for PI3K/AKT pathway hyperactivation and HER2-targeted therapy resistance.

Insertional mutagenesis identifies drivers of a novel oncogenic pathway in invasive lobular breast carcinoma.

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype and accounts for 8-14% of all cases. Although the majority of human ILCs are characterized by the functional loss of E-cadherin (encoded by CDH1), inactivation of Cdh1 does not predispose mice to develop mammary tumors, implying that mutations in additional genes are required for ILC formation in mice. To identify these genes, we performed an insertional mutagenesis screen using the Sleeping Beauty transposon system in mice with mammary-specific inactivation of Cdh1. These mice developed multiple independent mammary tumors of which the majority resembled human ILC in terms of morphology and gene expression. Recurrent and mutually exclusive transposon insertions were identified in Myh9, Ppp1r12a, Ppp1r12b and Trp53bp2, whose products have been implicated in the regulation of the actin cytoskeleton. Notably, MYH9, PPP1R12B and TP53BP2 were also frequently aberrated in human ILC, highlighting these genes as drivers of a novel oncogenic pathway underlying ILC development.

RUBIC identifies driver genes by detecting recurrent DNA copy number breaks.

The frequent recurrence of copy number aberrations across tumour samples is a reliable hallmark of certain cancer driver genes. However, state-of-the-art algorithms for detecting recurrent aberrations fail to detect several known drivers. In this study, we propose RUBIC, an approach that detects recurrent copy number breaks, rather than recurrently amplified or deleted regions. This change of perspective allows for a simplified approach as recursive peak splitting procedures and repeated re-estimation of the background model are avoided. Furthermore, we control the false discovery rate on the level of called regions, rather than at the probe level, as in competing algorithms. We benchmark RUBIC against GISTIC2 (a state-of-the-art approach) and RAIG (a recently proposed approach) on simulated copy number data and on three SNP6 and NGS copy number data sets from TCGA. We show that RUBIC calls more focal recurrent regions and identifies a much larger fraction of known cancer genes.

Pretreatment microRNA Expression Impacting on Epithelial-to-Mesenchymal Transition Predicts Intrinsic Radiosensitivity in Head and Neck Cancer Cell Lines and Patients.

PURPOSE: Predominant causes of head and neck cancer recurrence after radiotherapy are rapid repopulation, hypoxia, fraction of cancer stem cells, and intrinsic radioresistance. Currently, intrinsic radioresistance can only be assessed by ex vivo colony assays. Besides being time-consuming, colony assays do not identify causes of intrinsic resistance. We aimed to identify a biomarker for intrinsic radioresistance to be used before start of treatment and to reveal biologic processes that could be targeted to overcome intrinsic resistance. EXPERIMENTAL DESIGN: We analyzed both microRNA and mRNA expression in a large panel of head and neck squamous cell carcinoma (HNSCC) cell lines. Expression was measured on both irradiated and unirradiated samples. Results were validated using modified cell lines and a series of patients with laryngeal cancer. RESULTS: miRs, mRNAs, and gene sets that correlated with resistance could be identified from expression data of unirradiated cells. The presence of epithelial-to-mesenchymal transition (EMT) and low expression of miRs involved in the inhibition of EMT were important radioresistance determinants. This finding was validated in two independent cell line pairs, in which the induction of EMT reduced radiosensitivity. Moreover, low expression of the most important miR (miR-203) was shown to correlate with local disease recurrence after radiotherapy in a series of patients with laryngeal cancer. CONCLUSIONS: These findings indicate that EMT and low expression of EMT-inhibiting miRs, especially miR-203, measured in pretreatment material, causes intrinsic radioresistance of HNSCC, which could enable identification and treatment modification of radioresistant tumors. Clin Cancer Res; 21(24); 5630-8. ©2015 AACR.

A conditional piggyBac transposition system for genetic screening in mice identifies oncogenic networks in pancreatic cancer.

Here we describe a conditional piggyBac transposition system in mice and report the discovery of large sets of new cancer genes through a pancreatic insertional mutagenesis screen. We identify Foxp1 as an oncogenic transcription factor that drives pancreatic cancer invasion and spread in a mouse model and correlates with lymph node metastasis in human patients with pancreatic cancer. The propensity of piggyBac for open chromatin also enabled genome-wide screening for cancer-relevant noncoding DNA, which pinpointed a Cdkn2a cis-regulatory region. Histologically, we observed different tumor subentities and discovered associated genetic events, including Fign insertions in hepatoid pancreatic cancer. Our studies demonstrate the power of genetic screening to discover cancer drivers that are difficult to identify by other approaches to cancer genome analysis, such as downstream targets of commonly mutated human cancer genes. These piggyBac resources are universally applicable in any tissue context and provide unique experimental access to the genetic complexity of cancer.

Identification of distinct miRNA target regulation between breast cancer molecular subtypes using AGO2-PAR-CLIP and patient datasets.

BACKGROUND: Various microRNAs (miRNAs) are up- or downregulated in tumors. However, the repression of cognate miRNA targets responsible for the phenotypic effects of this dysregulation in patients remains largely unexplored. To define miRNA targets and associated pathways, together with their relationship to outcome in breast cancer, we integrated patient-paired miRNA-mRNA expression data with a set of validated miRNA targets and pathway inference. RESULTS: To generate a biochemically-validated set of miRNA-binding sites, we performed argonaute-2 photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (AGO2-PAR-CLIP) in MCF7 cells. We then defined putative miRNA-target interactions using a computational model, which ranked and selected additional TargetScan-predicted interactions based on features of our AGO2-PAR-CLIP binding-site data. We subselected modeled interactions according to the abundance of their constituent miRNA and mRNA transcripts in tumors, and we took advantage of the variability of miRNA expression within molecular subtypes to detect miRNA repression. Interestingly, our data suggest that miRNA families control subtype-specific pathways; for example, miR-17, miR-19a, miR-25, and miR-200b show high miRNA regulatory activity in the triple-negative, basal-like subtype, whereas miR-22 and miR-24 do so in the HER2 subtype. An independent dataset validated our findings for miR-17 and miR-25, and showed a correlation between the expression levels of miR-182 targets and overall patient survival. Pathway analysis associated miR-17, miR-19a, and miR-200b with leukocyte transendothelial migration. CONCLUSIONS: We combined PAR-CLIP data with patient expression data to predict regulatory miRNAs, revealing potential therapeutic targets and prognostic markers in breast cancer.

Analysis of tumor heterogeneity and cancer gene networks using deep sequencing of MMTV-induced mouse mammary tumors.

Cancer develops through a multistep process in which normal cells progress to malignant tumors via the evolution of their genomes as a result of the acquisition of mutations in cancer driver genes. The number, identity and mode of action of cancer driver genes, and how they contribute to tumor evolution is largely unknown. This study deployed the Mouse Mammary Tumor Virus (MMTV) as an insertional mutagen to find both the driver genes and the networks in which they function. Using deep insertion site sequencing we identified around 31000 retroviral integration sites in 604 MMTV-induced mammary tumors from mice with mammary gland-specific deletion of Trp53, Pten heterozygous knockout mice, or wildtype strains. We identified 18 known common integration sites (CISs) and 12 previously unknown CISs marking new candidate cancer genes. Members of the Wnt, Fgf, Fgfr, Rspo and Pdgfr gene families were commonly mutated in a mutually exclusive fashion. The sequence data we generated yielded also information on the clonality of insertions in individual tumors, allowing us to develop a data-driven model of MMTV-induced tumor development. Insertional mutations near Wnt and Fgf genes mark the earliest "initiating" events in MMTV induced tumorigenesis, whereas Fgfr genes are targeted later during tumor progression. Our data shows that insertional mutagenesis can be used to discover the mutational networks, the timing of mutations, and the genes that initiate and drive tumor evolution.

The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.

Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis.

The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development.

High-throughput semiquantitative analysis of insertional mutations in heterogeneous tumors.

Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data.

MicroRNA sequence and expression analysis in breast tumors by deep sequencing.

MicroRNAs (miRNA) regulate many genes critical for tumorigenesis. We profiled miRNAs from 11 normal breast tissues, 17 noninvasive, 151 invasive breast carcinomas, and 6 cell lines by in-house-developed barcoded Solexa sequencing. miRNAs were organized in genomic clusters representing promoter-controlled miRNA expression and sequence families representing seed sequence-dependent miRNA target regulation. Unsupervised clustering of samples by miRNA sequence families best reflected the clustering based on mRNA expression available for this sample set. Clustering and comparative analysis of miRNA read frequencies showed that normal breast samples were separated from most noninvasive ductal carcinoma in situ and invasive carcinomas by increased miR-21 (the most abundant miRNA in carcinomas) and multiple decreased miRNA families (including miR-98/let-7), with most miRNA changes apparent already in the noninvasive carcinomas. In addition, patients that went on to develop metastasis showed increased expression of mir-423, and triple-negative breast carcinomas were most distinct from other tumor subtypes due to upregulation of the mir~17-92 cluster. However, absolute miRNA levels between normal breast and carcinomas did not reveal any significant differences. We also discovered two polymorphic nucleotide variations among the more abundant miRNAs miR-181a (T19G) and miR-185 (T16G), but we did not identify nucleotide variations expected for classical tumor suppressor function associated with miRNAs. The differentiation of tumor subtypes and prediction of metastasis based on miRNA levels is statistically possible but is not driven by deregulation of abundant miRNAs, implicating far fewer miRNAs in tumorigenic processes than previously suggested.