Ataxia-Telangiectasia Mutated Loss-of-Function Displays Variant and Tissue-Specific Differences across Tumor Types.

PURPOSE: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. EXPERIMENTAL DESIGN: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. RESULTS: ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. CONCLUSIONS: These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

Cell-free Circulating Tumor DNA Variant Allele Frequency Associates with Survival in Metastatic Cancer.

PURPOSE: Physicians are expected to assess prognosis both for patient counseling and for determining suitability for clinical trials. Increasingly, cell-free circulating tumor DNA (cfDNA) sequencing is being performed for clinical decision making. We sought to determine whether variant allele frequency (VAF) in cfDNA is associated with prognosis. EXPERIMENTAL DESIGN: We performed a retrospective analysis of 298 patients with metastatic disease who underwent clinical comprehensive cfDNA analysis and assessed association between VAF and overall survival. RESULTS: cfDNA mutations were detected in 240 patients (80.5%). Median overall survival (OS) was 11.5 months. cfDNA mutation detection and number of nonsynonymous mutations (NSM) significantly differed between tumor types, being lowest in appendiceal cancer and highest in colon cancer. Having more than one NSM detected was associated with significantly worse OS (HR = 2.3; P < 0.0001). VAF was classified by quartiles, Q1 lowest, Q4 highest VAF. Higher VAF levels were associated with a significantly worse overall survival (VAF Q3 HR 2.3, P = 0.0069; VAF Q4 HR = 3.8, P < 0.0001) on univariate analysis. On multivariate analysis, VAF Q4, male sex, albumin level <3.5 g/dL, number of nonvisceral metastatic sites >0 and number of prior therapies >4 were independent predictors of worse OS. CONCLUSIONS: Higher levels of cfDNA VAF and a higher number of NSMs were associated with worse OS in patients with metastatic disease. Further study is needed to determine optimal VAF thresholds for clinical decision making and the utility of cfDNA VAF as a prognostic marker in different tumor types.

Molecular Profiling of Hepatocellular Carcinoma Using Circulating Cell-Free DNA.

PURPOSE: Molecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies. EXPERIMENTAL DESIGN: We analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA). RESULTS: A total of 153/206 (74.3%) were men; median age, 62 years (range, 18-91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1-13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%-55.03%). TP53 was the common altered gene [>120 alterations (non-unique)] followed by EGFR, MET, ARID1A, MYC, NF1, BRAF, and ERBB2 [20-38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in MYC, EGFR, ERBB2, BRAF, CCNE1, MET, PIK3CA, ARID1A, CDK6, and KRAS. In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have ERBB2 alterations, 35.7% (5/14) versus 8.8% HBV-negative (P = 0.04). CONCLUSIONS: This study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology.

Identification of Actionable Genomic Alterations Using Circulating Cell-Free DNA.

PURPOSE: Cell-free DNA (cfDNA) next-generation sequencing is a noninvasive approach for genomic testing. We report the frequency of identifying alterations and their clinical actionability in patients with advanced/metastatic cancer. PATIENTS AND METHODS: Prospectively consented patients had cfDNA testing performed. Alterations were assessed for therapeutic implications. RESULTS: We enrolled 575 patients with 37 tumor types. Of these patients, 438 (76.2%) had at least one alteration detected, and 205 (35.7%) had one or more alterations of high potential for clinical action. In diseases with 10 or more patients enrolled, 50% or more had at least one alteration deemed of high potential for clinical action. Trials were identified in 80% of patients (286 of 357) with any alteration and in 92% of patients (188 of 205) with one or more alterations of high potential for clinical action of whom 57.6% (118 of 205) had 6 or more months of follow-up available. Of these patients, 10% (12 of 118) had received genomically matched therapy through enrollment in clinical trials (n = 8), off-label drug use (n = 3), or standard of care (n = 1). Although 88.6% of all patients had a performance status of 0 or 1 upon enrollment, the primary reason for not acting on alterations was poor performance status at next treatment change (28.1%; 27 of 96). CONCLUSION: cfDNA testing represents a readily accessible method for genomic testing and allows for detection of genomic alterations in most patients with advanced disease. Utility may be higher in patients interested in investigational therapeutics with adequate performance status. Additional study is needed to determine whether utility is enhanced by testing earlier in the treatment course.

Precision Oncology Decision Support: Current Approaches and Strategies for the Future.

With the increasing availability of genomics, routine analysis of advanced cancers is now feasible. Treatment selection is frequently guided by the molecular characteristics of a patient's tumor, and an increasing number of trials are genomically selected. Furthermore, multiple studies have demonstrated the benefit of therapies that are chosen based upon the molecular profile of a tumor. However, the rapid evolution of genomic testing platforms and emergence of new technologies make interpreting molecular testing reports more challenging. More sophisticated precision oncology decision support services are essential. This review outlines existing tools available for health care providers and precision oncology teams and highlights strategies for optimizing decision support. Specific attention is given to the assays currently available for molecular testing, as well as considerations for interpreting alteration information. This article also discusses strategies for identifying and matching patients to clinical trials, current challenges, and proposals for future development of precision oncology decision support. Clin Cancer Res; 24(12); 2719-31. ©2018 AACR.

Physician interpretation of genomic test results and treatment selection.

BACKGROUND: Genomic testing is increasingly performed in oncology, but concerns remain regarding the clinician's ability to interpret results. In the current study, the authors sought to determine the agreement between physicians and genomic annotators from the Precision Oncology Decision Support (PODS) team at The University of Texas MD Anderson Cancer Center in Houston regarding actionability and the clinical use of test results. METHODS: On a prospective protocol, patients underwent clinical genomic testing for hotspot mutations in 46 or 50 genes. Six months after sequencing, physicians received questionnaires for patients who demonstrated a variant in an actionable gene, investigating their perceptions regarding the actionability of alterations and clinical use of these findings. Genomic annotators independently classified these variants as actionable, potentially actionable, unknown, or not actionable. RESULTS: Physicians completed 250 of 288 questionnaires (87% response rate). Physicians considered 168 of 250 patients (67%) as having an actionable alteration; of these, 165 patients (98%) were considered to have an actionable alteration by the PODS team and 3 were of unknown significance. Physicians were aware of genotype-matched therapy available for 119 patients (71%) and 48 of these 119 patients (40%) received matched therapy. Approximately 46% of patients in whom physicians regarded alterations as not actionable (36 of 79 patients) were classified as having an actionable/potentially actionable mutation by the PODS team. However, many of these were only theoretically actionable due to limited trials and/or therapies (eg, KRAS). CONCLUSIONS: Physicians are aware of recurrent mutations in actionable genes on "hotspot" panels. As larger genomic panels are used, there may be a growing need for annotation of actionability. Decision support to increase awareness of genomically relevant trials and novel treatment options for recurrent mutations (eg, KRAS) also are needed. Cancer 2018;124:966-72. © 2017 American Cancer Society.

"Personalized Cancer Therapy": A Publicly Available Precision Oncology Resource.

High-throughput genomic and molecular profiling of tumors is emerging as an important clinical approach. Molecular profiling is increasingly being used to guide cancer patient care, especially in advanced and incurable cancers. However, navigating the scientific literature to make evidence-based clinical decisions based on molecular profiling results is overwhelming for many oncology clinicians and researchers. The Personalized Cancer Therapy website (www.personalizedcancertherapy.org) was created to provide an online resource for clinicians and researchers to facilitate navigation of available data. Specifically, this resource can be used to help identify potential therapy options for patients harboring oncogenic genomic alterations. Herein, we describe how content on www.personalizedcancertherapy.org is generated and maintained. We end with case scenarios to illustrate the clinical utility of the website. The goal of this publicly available resource is to provide easily accessible information to a broad oncology audience, as this may help ease the information retrieval burden facing participants in the precision oncology field. Cancer Res; 77(21); e123-6. ©2017 AACR.

Precision oncology: neither a silver bullet nor a dream.

Precision oncology is not an illusion, nor is it the magic bullet that will eradicate all cancers. Precision oncology is simply another weapon in our growing armament against cancer. Rather than honing in on the failures of a relatively young field, one should advocate for integrating its successes into widespread clinical practice, especially for indications, such as: ABL, ALK, BRAF, BRCA1, BRCA2, EGFR, KIT, KRAS, PDGFRA, PDGFRB, ROS1, BCR-ABL, FLT3 and ROS1, where aberrations have been shown to alter responses to US FDA approved drugs - that is, level 1 data. Moreover, to truly assess the promise of precision oncology, we must first begin by defining our expectations for this field. Importantly, we must recognize that the conception of precision oncology arose as an antithesis of the 'one-size fits all' cancer therapeutics approach. Consequently, tools used for evaluating these conventional, large-scale trials, are not directly transferable for assessing nonconventional, smaller-scale trials needed for evaluating precision oncology. Hence, a thorough vetting of precision oncology as another tool of the trade, must first begin by reassessing our expectations for this field, as well as current clinical trial designs and end point measurements. Importantly, we must recognize that most targeted therapy approaches are in their infancy, with only monotherapy approaches being assessed and combination therapies likely being necessary to fulfill the promise of precision oncology.

ALK: a tyrosine kinase target for cancer therapy.

The anaplastic lymphoma kinase (ALK) gene plays an important physiologic role in the development of the brain and can be oncogenically altered in several malignancies, including non-small-cell lung cancer (NSCLC) and anaplastic large cell lymphomas (ALCL). Most prevalent ALK alterations are chromosomal rearrangements resulting in fusion genes, as seen in ALCL and NSCLC. In other tumors, ALK copy-number gains and activating ALK mutations have been described. Dramatic and often prolonged responses are seen in patients with ALK alterations when treated with ALK inhibitors. Three of these-crizotinib, ceritinib, and alectinib-are now FDA approved for the treatment of metastatic NSCLC positive for ALK fusions. However, the emergence of resistance is universal. Newer ALK inhibitors and other targeting strategies are being developed to counteract the newly emergent mechanism(s) of ALK inhibitor resistance. This review outlines the recent developments in our understanding and treatment of tumors with ALK alterations.

Automated identification of molecular effects of drugs (AIMED).

INTRODUCTION: Genomic profiling information is frequently available to oncologists, enabling targeted cancer therapy. Because clinically relevant information is rapidly emerging in the literature and elsewhere, there is a need for informatics technologies to support targeted therapies. To this end, we have developed a system for Automated Identification of Molecular Effects of Drugs, to help biomedical scientists curate this literature to facilitate decision support. OBJECTIVES: To create an automated system to identify assertions in the literature concerning drugs targeting genes with therapeutic implications and characterize the challenges inherent in automating this process in rapidly evolving domains. METHODS: We used subject-predicate-object triples (semantic predications) and co-occurrence relations generated by applying the SemRep Natural Language Processing system to MEDLINE abstracts and ClinicalTrials.gov descriptions. We applied customized semantic queries to find drugs targeting genes of interest. The results were manually reviewed by a team of experts. RESULTS: Compared to a manually curated set of relationships, recall, precision, and F2 were 0.39, 0.21, and 0.33, respectively, which represents a 3- to 4-fold improvement over a publically available set of predications (SemMedDB) alone. Upon review of ostensibly false positive results, 26% were considered relevant additions to the reference set, and an additional 61% were considered to be relevant for review. Adding co-occurrence data improved results for drugs in early development, but not their better-established counterparts. CONCLUSIONS: Precision medicine poses unique challenges for biomedical informatics systems that help domain experts find answers to their research questions. Further research is required to improve the performance of such systems, particularly for drugs in development.

Common pathways regulate Type III TGFß receptor-dependent cell invasion in epicardial and endocardial cells.

Epithelial-Mesenchymal Transformation (EMT) and the subsequent invasion of epicardial and endocardial cells during cardiac development is critical to the development of the coronary vessels and heart valves. The transformed cells give rise to cardiac fibroblasts and vascular smooth muscle cells or valvular interstitial cells, respectively. The Type III Transforming Growth Factor ß (TGFßR3) receptor regulates EMT and cell invasion in both cell types, but the signaling mechanisms downstream of TGFßR3 are not well understood. Here we use epicardial and endocardial cells in in vitro cell invasion assays to identify common mechanisms downstream of TGFßR3 that regulate cell invasion. Inhibition of NF-κB activity blocked cell invasion in epicardial and endocardial cells. NF-κB signaling was found to be dysregulated in Tgfbr3(-/-) epicardial cells which also show impaired cell invasion in response to ligand. TGFßR3-dependent cell invasion is also dependent upon Activin Receptor-Like Kinase (ALK) 2, ALK3, and ALK5 activity. A TGFßR3 mutant that contains a threonine to alanine substitution at residue 841 (TGFßR3-T841A) induces ligand-independent cell invasion in both epicardial and endocardial cells in vitro. These findings reveal a role for NF-κB signaling in the regulation of epicardial and endocardial cell invasion and identify a mutation in TGFßR3 which stimulates ligand-independent signaling.

BMP2 signals loss of epithelial character in epicardial cells but requires the Type III TGFß receptor to promote invasion.

Coronary vessel development depends on a subpopulation of epicardial cells that undergo epithelial to mesenchymal transformation (EMT) and invade the subepicardial space and myocardium. These cells form the smooth muscle of the vessels and fibroblasts, but the mechanisms that regulate these processes are poorly understood. Mice lacking the Type III Transforming Growth Factor ß Receptor (TGFßR3) die by E14.5 due to failed coronary vessel development accompanied by reduced epicardial cell invasion. BMP2 signals via TGFßR3 emphasizing the importance of determining the relative contributions of the canonical BMP signaling pathway and TGFßR3-dependent signaling to BMP2 responsiveness. Here we examined the role of TGFßR3 in BMP2 signaling in epicardial cells. Whereas TGFß induced loss of epithelial character and smooth muscle differentiation, BMP2 induced an ALK3-dependent loss of epithelial character and modestly inhibited TGFß-stimulated differentiation. Tgfbr3(-/-) cells respond to BMP2 indicating that TGFßR3 is not required. However, Tgfbr3(-/-) cells show decreased invasion in response to BMP2 and overexpression of TGFßR3 in Tgfbr3(-/-) cells rescued invasion. Invasion was dependent on ALK5, ALK2, ALK3, and Smad4. Expression of TGFßR3 lacking the 3 C-terminal amino acids required to interact with the scaffolding protein GIPC (GAIP-interacting protein, C terminus) did not rescue. Knockdown of GIPC in Tgfbr3(+/+) or Tgfbr3(-/-) cells rescued with TGFßR3 decreased BMP2-stimulated invasion confirming a requirement for TGFßR3/GIPC interaction. Our results reveal the relative roles of TGFßR3-dependent and TGFßR3-independent signaling in the actions of BMP2 on epicardial cell behavior and demonstrate the critical role of TGFßR3 in mediating BMP2-stimulated invasion.

TGFß and BMP-2 regulate epicardial cell invasion via TGFßR3 activation of the Par6/Smurf1/RhoA pathway.

Coronary vessel development requires transfer of mesothelial cells to the heart surface to form the epicardium where some cells subsequently undergo epithelial-mesenchymal transformation (EMT) and invade the subepicardial matrix. Tgfbr3(-/-) mice die due to failed coronary vessel formation associated with decreased epicardial cell invasion but the mediators downstream of TGFßR3 are not well described. TGFßR3-dependent endocardial EMT stimulated by either TGFß2 or BMP-2 requires activation of the Par6/Smurf1/RhoA 1pathway where Activin Receptor Like Kinase (ALK5) signals Par6 to act downstream of TGFß to recruit Smurf1 to target RhoA for degradation to regulate apical-basal polarity and tight junction dissolution. Here we asked if this pathway was operant in epicardial cells and if TGFßR3 was required to access this pathway. Targeting of ALK5 in Tgfbr3(+/+) cells inhibited loss of epithelial character and invasion. Overexpression of wild-type (wt) Par6, but not dominant negative (dn) Par6, induced EMT and invasion while targeting Par6 by siRNA inhibited EMT and invasion. Overexpression of Smurf1 and dnRhoA induced loss of epithelial character and invasion. Targeting of Smurf1 by siRNA or overexpression of constitutively active (ca) RhoA inhibited EMT and invasion. In Tgfbr3(-/-) epicardial cells which have a decreased ability to invade collagen gels in response to TGFß2, overexpression of wtPar6, Smurf1, or dnRhoA had a diminished ability to induce invasion. Overexpression of TGFßR3 in Tgfbr3(-/-) cells, followed by siRNA targeting of Par6 or Smurf1, diminished the ability of TGFßR3 to rescue invasion demonstrating that the Par6/Smurf1/RhoA pathway is activated downstream of TGFßR3 in epicardial cells.

The cytoplasmic domain of TGFßR3 through its interaction with the scaffolding protein, GIPC, directs epicardial cell behavior.

The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor ß Receptor (TGFßR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFßR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFßR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3(-/-) hearts. Tgfbr3(-/-) epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFß1 and TGFß2. Unexpectedly, loss of TGFßR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFßR3 in Tgfbr3(-/-) cells rescued deficits in invasion in vitro in response TGFß1 and TGFß2 as well as FGF2 and HMW-HA. Expression of TGFßR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3(-/-) cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3(+/+) cells decreased invasion in response to TGFß2, FGF2, and HMW-HA. We conclude that TGFßR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3(-/-) mice.

Increased level of cellular Bip critically determines estrogenic potency for a xenoestrogen kepone in the mouse uterus.

Xenoestrogen mimics estrogen-like activities primarily based on alterations of gene expression and interactions with estrogen receptor (ER)-alpha and -beta. However, the requirement for large concentrations to induce estrogenic phenotypes and low affinity for ERs has challenged the notion that prevailing xenoestrogens are significant health hazards. Here in this study, we show that under certain conditions, exposure of xenoestrogen could be potentially harmful in respect to enhanced uterine estrogenicity. Previously, we have demonstrated that estradiol-17beta up-regulates uterine Bip, a stress-related endoplasmic reticulum protein, via an ER-independent mechanism in mice. Moreover, this protein essentially involves in estradiol-17beta-mediated uterine growth response and ERalpha-dependent gene transcription. Here, we demonstrate that among three tested xenoestrogens, only kepone (>15-30 mg/kg) exerts sustained inductive response for uterine Bip expression. Interestingly, this kepone-induced Bip strongly correlates with ERalpha-dependent growth and gene expressional responses in the mouse uterus. Furthermore, these effects were strongly suppressed after knockdown of uterine Bip, via the adenovirus approach. Although kepone at 7.5 mg/kg was not effective, it was strongly stimulatory by the adenovirus-driven forced expression of uterine Bip. In contrast, the control green fluorescence protein virus was not effective in the aforementioned responses. Furthermore, the induction of uterine Bip by stress-related signals also revealed the onset of uterine growth in mice when exposed to a sublethal dose of kepone. Collectively, studies provide novel molecular evidence that Bip acts as a critical regulator to amplify estrogenic potency for a weak xenoestrogen kepone.