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The histone lysine demethylase 4 (KDM4) family plays an important role in regulating gene transcription, DNA repair, and metabolism. The dysregulation of KDM4 functions is associated with many human disorders, including cancer, obesity, and cardiovascular diseases. Selective and potent KDM4 inhibitors may help not only to understand the role of KDM4 in these disorders but also to provide potential therapeutic opportunities. Here, we provide an overview of the field and discuss current status, challenges, and opportunities lying ahead in the development of KDM4-based anticancer therapeutics.

Histone lysine demethylases facilitate the activity of oncogenic transcription factors, including possibly MYC. Here we show that multiple histone demethylases influence the viability and poor prognosis of neuroblastoma cells, where MYC is often overexpressed. We also identified the approved small-molecule antifungal agent ciclopirox as a novel pan-histone demethylase inhibitor. Ciclopirox targeted several histone demethylases, including KDM4B implicated in MYC function. Accordingly, ciclopirox inhibited Myc signaling in parallel with mitochondrial oxidative phosphorylation, resulting in suppression of neuroblastoma cell viability and inhibition of tumor growth associated with an induction of differentiation. Our findings provide new insights into epigenetic regulation of MYC function and suggest a novel pharmacologic basis to target histone demethylases as an indirect MYC-targeting approach for cancer therapy. Cancer Res; 77(17); 4626–38. ©2017 AACR.

Figure S1 shows siRNA screening in neuroblastoma cells. Figure S2 shows the dentification of ciclopirox that targets KDM4B. Figure S3 shows the CPX effect on other histone methyl marks. Figure S4 shows that CPX targets the Myc pathway. Figure S5 shows that CPX inhibits oxidative phosphorylation. Figure S6 shows the CPX effect on BE2C xenografts with KDM4B knockdown. Table S1 shows the sequences of the focused KDM siRNA library. Table S2 shows the primers for real time PCR.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4–11 cells at low picomolar concentrations (IC(50) = 3 pM; BRD4 DC(50) = 0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Background The sensitivity to endocrine therapy assay (SET2,3) predicts treatment outcomes in Stage II-III breast cancer. SET2,3 measures transcription related to estrogen and progesterone receptors (SETER/PR index) and the molecular subtype (RNA4: ESR1, PGR, ERBB2, AURKA) from formalin-fixed paraffin-embedded (FFPE) tissue sections. Methods We designed a nested study across 3 pathology laboratories, each testing 60 breast cancers twice in controlled batches. Laboratories macrodissected and directly homogenized the unstained FFPE tumor sections, then performed the QuantiGene Plex bead-based hybridization assay. SET2,3 was calculated centrally using predefined statistical R-scripts and applying pre-defined cutpoints. Concordance correlation coefficient (CCC) was calculated from continuous measurements and Kappa statistic from categorical results. A mixed-effects model estimated contributions to bias (fixed effects) and variance (random effects) from the replicated design. Results Intralaboratory (CCC 0.96–0.99) and interlaboratory (CCC 0.98–0.99) SET2,3 results were concordant, with rates of agreement for high/low categorization within (Kappa 0.83–0.93) and between laboratories (Kappa 0.87–0.88). The relative contributions to overall variance of SET2,3 measurements were 96.90% from biological differences between cancers, 0.67% from interlaboratory variability, and 2.44% from residual causes including intralaboratory replicates. Similar results were obtained with SETER/PR, the baseline prognostic index calculated using pathological or clinical tumor and nodal staging information, and the 4 individual genes (ESR1, PGR, ERBB2, and AURKA). Conclusion Intra- and interpathology laboratory measurements of SET2,3 and its components were highly reproducible when tested from FFPE tumor sections.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.

Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. Significance: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

Isotopically labeled amino acids are widely used to study the structure and dynamics of proteins by NMR. Herein we describe a facile, gram-scale synthesis of compounds 1b and 2b under standard laboratory conditions from the common intermediate 7. 2b is obtained via simple deprotection, while 1b is accessed through a reductive deoxygenation/deuteration sequence and deprotection. 1b and 2b provide improved signal intensity using lower amounts of labeled precursor and are alternatives to existing labeling approaches.

In an effort to better understand mental health and enrichment differences between veteran women and men in a veteran service organization (VSO), a research team conducted secondary analysis of assessment data from Team Red, White, and Blue (Team RWB) veteran members who joined between 2014 and 2016. In this secondary analysis of the 2014–2016 Team RWB initial member survey data, frequencies and crosstab analyses were conducted for veteran respondents (N = 10,015), a portion of whom (31.5%) identified as former servicewomen (n = 3,152). Women were statistically overrepresented by a factor of 2–3 times in organizational membership. Gender-correlated differences were found with levels of enrichment; at baseline, women reported lower levels of social support as described by the subcategory of belonging (p < .001) and lower levels of sense of purpose as measured by two subcategories (p < .002 and p = .03). Primary findings from this study indicate an opportunity for future research on mental health and enrichment when a member joins a service or social networking organization. is indicated. Lower levels of mental health and enrichment among new Team RWB members who are female veterans indicates an opportunity for the organization to actively focus programs and resources on this fast-growing subpopulation of veterans to enhance retention and the overall participant experience.

Background: Translational cancer research is dependent on well annotated human samples, but scarcity of specimen DNA and RNA hinders research and novel diagnostic testing. We demonstrate a novel nucleic acid isolation and anchoring approach that facilitates uniform, consistent replication of the isolated and captured DNA and RNA. This approach allows for expanded investigations of precious samples and increases the value of biobanked material. Methods: Our simplSEQ replication methodology enzymatically adds homopolymeric tails to the 3’ end of DNA and RNA, with a terminal dideoxy biotin-modified NTP, to attach the nucleic acids to streptavidin beads. The 3’ immobilized nucleic acids are copied using complementary homopolymers. The simplSEQ advantage is that molecules of all sizes are attached and copied from their 3’ ends yielding cDNA copies that are full-length representations of the input. Various DNA samples were investigated to determine the characteristics of cDNA copies as well as stability of bound nucleic acids. Samples evaluated included Human 1,000 genome project, bacterial genomes, commercial DNA controls, and synthesized DNA molecules. The samples represent a range of DNA sizes, AT/GC content and Variant Allele Frequencies (VAF). NGS assays quantified the presence of full-length genomic sequences. Variant analysis determined accuracy of copies based on comparison with published data (NA12878). Synthetic DNA oligonucleotides representing regions in the human genome and constructed to enable molecular counting independent of adapter ligation efficiency. All sample types were replicated, sequenced, and analyzed for coverage and uniformity. This replication process was repeated multiple times. Results: Three replicates of each DNA type were attached to streptavidin beads and subjected to multiple rounds of the simplSEQ protocol. Analysis of overall coverage and variant calling for human NA12878 replicates compared to the platinum standard NA12878 genome show an average coverage ranging from 27x-32x with >99% correlation between replicates. Sensitivity ranging from 98.6%-99.5%, with a precision range from 96.0% to 98.0% for 6 replicates. Bacterial samples with coverage over 100x and >99% correlation. Synthetic DNA oligonucleotide pools with coverage over 10,000x and >99% correlation. Conclusion: Our novel enzymatic tailing methodology produces highly accurate copies of full-length nucleic acids. This technology obviates sample scarcity for genomic evaluations and allows for expanded hypothesis testing in translational research. This methodology also provides an option for extended storage of nucleic acids from which replicates can be repeatedly generated to allow for reinterrogation. Citation Format: John Christopher Spinosa, Brandon Young, Vincent Aguilar, Baiju Parikh. Novel sample isolation and immortalization method for DNA and RNA to extend translational research capabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 496.

Summary Histone lysine demethylases (KDMs) play critical roles in oncogenesis and therefore may be effective targets for anticancer therapy. Using a time-resolved fluorescence resonance energy transfer demethylation screen assay, in combination with multiple orthogonal validation approaches, we identified geldanamycin and its analog 17-DMAG as KDM inhibitors. In addition, we found that these Hsp90 inhibitors increase degradation of the alveolar rhabdomyosarcoma (aRMS) driver oncoprotein PAX3-FOXO1 and induce the repressive epigenetic mark H3K9me3 and H3K36me3 at genomic loci of PAX3-FOXO1 targets. We found that as monotherapy 17-DMAG significantly inhibits expression of PAX3-FOXO1 target genes and multiple oncogenic pathways, induces a muscle differentiation signature, delays tumor growth and extends survival in aRMS xenograft mouse models. The combination of 17-DMAG with conventional chemotherapy significantly enhances therapeutic efficacy, indicating that targeting KDM in combination with chemotherapy may serve as a therapeutic approach to PAX3-FOXO1-positive aRMS., Graphical Abstract, Highlights • Identification of geldanamycin/17-DMAG as histone lysine demethylase inhibitors • Geldanamycin/17-DMAG causes degradation of PAX3-FOXO1, an Hsp90 client • Geldanamycin/17-DMAG induces epigenetic changes and targets PAX3-FOXO1 pathway • 17-DMAG alone or combined with chemotherapy show potency to PAX3-FOXO1 xenografts, Molecular Biology; Cancer; Omics

Purpose The Herceptin Adjuvant study is an international multicenter randomized trial that compared 1 or 2 years of trastuzumab given every 3 weeks with observation in women with human epidermal growth factor 2–positive (HER2+) breast cancer after chemotherapy. Identification of biomarkers predictive of a benefit from trastuzumab will minimize overtreatment and lower health care costs. Methods To identify possible single-gene biomarkers, an exploratory analysis of 3,669 gene probes not expected to be expressed in normal breast tissue was conducted. Disease-free survival (DFS) was used as the end point in a Cox regression model, with the interaction term between C8A mRNA and treatment as a categorical variable split on the cohort mean. Results A significant interaction between C8A mRNA and treatment was detected ( P < .001), indicating a predictive response to trastuzumab treatment. For the C8A-low subgroup (mRNA expression lower than the cohort mean), no significant treatment benefit was observed ( P = .73). In the C8A-high subgroup, patients receiving trastuzumab experienced a lower hazard of a DFS event by approximately 75% compared with those in the observation arm (hazard ratio [HR], 0.25; P < .001). A significant prognostic effect of C8A mRNA also was seen ( P < .001) in the observation arm, where the C8A-high group hazard of a DFS event was three times the respective hazard of the C8A-low group (HR, 3.27; P < .001). C8A mRNA is highly prognostic in the Hungarian Academy of Science HER2+ gastric cancer cohort (HR, 1.72; P < .001). Conclusion C8A as a single-gene biomarker prognostic of DFS and predictive of a benefit from trastuzumab has the potential to improve the standard of care in HER2+ breast cancer if validated by additional studies. Understanding the advantage of overexpression of C8A related to the innate immune response can give insight into the mechanisms that drive cancer.

The Bromodomain and Extra-Terminal domain family (BET) family of proteins are involved in the regulation of gene transcription, and their dysregulation is implicated in several diseases including cancer. BET proteins contain two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine residues and appear to have distinct biological roles. We compared several published co-crystal structures and found five positions near the substrate binding pocket that vary between BET bromodomains. One position located in the ZA loop has unique properties. In BRD2-4, this residue is glutamine in BD1 and lysine in BD2; in BRDT, this residue is arginine in BD1 and asparagine in BD2. Using molecular modeling, we identified differences in the water-mediated network at this position between bromodomains. Molecular dynamics simulations helped rationalize the observed bromodomain selectivity for exemplar BET inhibitors and a congeneric series of tetrahydroquinolines (THQ) that differed by a single heteroatom near the ZA channel. The 2-furan SJ830599, the most BD2-selective THQ analog, did not disrupt the water-mediated networks in either domain, but was electrostatically-repulsed by the specific arrangement of the W5 water dipole in BD1. Our work underscores the value of exploring water-mediated interactions to study ligand binding, and highlights the difficulty of optimizing polar interactions due to high desolvation penalties. Finally, we suggest further modifications to THQ-based BET inhibitors that would increase BD2-selectivity in BRD2-4, while minimizing affinity for one or both bromodomains of BRDT.

Background Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287–395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3′ partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Conclusions Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6–7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.

Background: RNA-Seq from total RNA in FFPE tissue can be more challenging due to limited capture of partially degraded RNA. Exome-capture based RNA-Seq may circumvent such problems and allow reproducible complete molecular characterization of low-quality RNA from small clinical samples. Methods: HER2 negative patients within the GeparQuinto trial were treated with neoadjuvant anthracycline-taxane-based chemotherapy +/- bevacizumab. Patients with bevacizumab therapy had a significantly higher pCR rate, especially within the triple negative subgroup. We performed exome-capture RNA-Seq on 5µm FFPE sections from pre-therapeutic cores of 400 HER2 negative samples from this trial. In a prospectively planned, blinded study we correlated molecular subtypes and metagenes for proliferation, stroma, MHC2, and VEGFA with clinical and histopathological data. Molecular subtypes were defined using the AIMS methods. Metagenes were calculated as mean values corresponding to previously described gene clusters after platform transfer (Rody et al. 2011 PMID 21978456, Hu et al. 2009 PMID 19291283) and then z-transformed. Results: 296 samples with RNA-Seq data were classified as either of high (n=226) or of limited quality (n=70). For 22 samples RNA yield was insufficient and 82 did not pass initial QC. 121 (41%), 63 (21%), 34 (11.5%), 46 (15.5%), and 32 (11%) samples were defined as basal-like, HER2-enriched, luminal A, luminal B, and normal-like, respectively. Subtyping was robust with regard to gene filtering, normalization, and sample quality. ER and PR status from local IHC strongly correlated with gene expression (overall correctness 84% and 80% for ER, and 85% and 74% for PR, in samples with high and limited quality, respectively) and luminal subtypes (95% ER positive). Proliferation metagene correlated with histological grade (median -0.73, -0.39, and 0.53 in G1, G2, and G3, respectively; P Conclusions: Exome-capture RNA-Seq allows robust genomic characterization of clinical samples with limited FFPE material from core biopsies, and molecular subtypes and immune metagenes are predictive for pCR. The VEGFA metagene is a specific predictor for response to neoadjuvant bevacizumab treatment. Citation Format: Karn T, Meissner T, Weber K, Sinn B, Denkert C, Budczies J, Nekljudova V, Fasching PA, Holtrich U, Schem C, Solbach C, Hartmann A, Röcken C, Untch M, Young BM, Willis S, Leyland-Jones B, von Minckwitz G, Loibl S. Blinded molecular subtyping analysis from RNA-Seq of FFPE samples in the GeparQuinto trial reveals predictive value of VEGFA metagene for bevacizumab treatment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-02.

Within the last decade, the Bromodomain and Extra-Terminal domain family (BET) of proteins have emerged as promising drug targets in diverse clinical indications including oncology, auto-immune disease, heart failure, and male contraception. The BET family consists of four isoforms (BRD2, BRD3, BRD4, and BRDT/BRDT6) which are distinguished by the presence of two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine (KAc) residues and appear to have distinct biological roles. BET BD1 and BD2 bromodomains differ at five positions near the substrate binding pocket: the variation in the ZA channel induces different water networks nearby. We designed a set of congeneric 2- and 3-heteroaryl substituted tetrahydroquinolines (THQ) to differentially engage bound waters in the ZA channel with the goal of achieving bromodomain selectivity. SJ830599 (9) showed modest, but consistent, selectivity for BRD2-BD2. Using isothermal titration calorimetry, we showed that the binding of all THQ analogs in our study to either of the two bromodomains was enthalpy driven. Remarkably, the binding of 9 to BRD2-BD2 was marked by negative entropy and was entirely driven by enthalpy, consistent with significant restriction of conformational flexibility and/or engagement with bound waters. Co-crystallography studies confirmed that 9 did indeed stabilize a water-mediated hydrogen bond network. Finally, we report that 9 retained cytotoxicity against several pediatric cancer cell lines with EC50 values comparable to BET inhibitor (BETi) clinical candidates.

Purpose Identification of single-gene biomarkers that are prognostic of outcome can shed new insights on the molecular mechanisms that drive breast cancer and other cancers. Methods Exploratory analysis of 20,464 single-gene messenger RNAs (mRNAs) in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) discovery cohort indicates that low expression of FGD3 mRNA is prognostic for poor outcome. Prognostic significance of faciogenital dysplasia 3 (FGD3), SUSD3, and other single-gene proliferation markers was evaluated in breast cancer and The Cancer Genome Atlas (TCGA) cohorts. Results A meta-analysis of Cox regression of FGD3 mRNA as a continuous variable for overall survival of estrogen receptor (ER)–positive samples in METABRIC discovery, METABRIC validation, TCGA breast cancer, and Combination Chemotherapy in Treating Women With Breast Cancer (E2197) cohorts resulted in a combined hazard ratio (HR) of 0.69 (95% CI, 0.63 to 0.75), indicating better outcome with high expression. In the ER-negative samples, the combined meta-analysis HR was 0.72 (95% CI, 0.63 to 0.82), suggesting that FGD3 is prognostic regardless of ER status. The potential of FGD3 as a biomarker for freedom from recurrence was evaluated in the Breast International Group 1-98 (BIG 1-98; Letrozole or Tamoxifen in Treating Postmenopausal Women With Breast Cancer) study (HR, 0.85; 95% CI, 0.76 to 0.93) for breast cancer–free interval. In the Hungarian Academy of Science (HAS) breast cancer cohort, splitting on the median had an HR of 0.49 (95% CI, 0.42 to 0.58) for recurrence-free survival. A comparison of the Stouffer P value in five ER-positive cohorts showed that FGD3 ( P = 3.8E-14) outperformed MKI67 ( P = 1.06E-8) and AURKA ( P = 2.61E-5). A comparison of the Stouffer P value in four ER-negative cohorts showed that FGD3 ( P = 3.88E-5) outperformed MKI67 ( P = .477) and AURKA ( P = .820). Conclusion FGD3 was previously shown to inhibit cell migration. FGD3 mRNA is regulated by ESR1 and is associated with favorable outcome in six distinct breast cancer cohorts and four TCGA cancer cohorts. This suggests that FGD3 is an important clinical biomarker.

Introduction Next generation sequencing has facilitated the understanding of pathogenesis and molecular heterogeneity of breast cancer (BC) as well as accelerated the path towards precision medicine. DNA sequencing (DNA-Seq) based assays for the detection of mutations and alterations in solid and hematologic cancers are finding their way into clinical practice and are readily available as clinical products. RNA sequencing (RNA-Seq), so far being vastly applied in the research context, promises to expand the diagnostic, prognostic and therapeutic use of this technology in cancer. Beyond mutational status, RNA-Seq enables the detection of fusions, quantification of gene expression level, detection of differentially expressed genes, molecular based subtyping, and risk-stratification. In this study we analyzed RNA-Seq and copy number data from BC patients that had undergone DNA-Seq based diagnostics through commercial providers with the goal to detect additional actionable targets. Materials and Methods We included 18 BC patients (5/18 triple negative) that had previously undergone DNA-based targeted (321 genes) sequencing. RNA-Seq to a minimum of 75M reads (75pb) was performed using 100 ng of total RNA on the Illumina NextSeq 500 platform. STAR was used for alignment (hg19) and gene expression quantification (RefSeq). Fusions were detected using STAR-Fusion. DESeq2 was utilized to identify patient specific differentially expressed genes by analyzing samples individually against a set of 13 controls from healthy breast tissue generated in-house. Copy number variations (CNVs) were detected using the Nanostring CNV Cancer panel (89 genes) on the Nanostring nCounter platform. Differentially upregulated or amplified genes were queried against DGIdb and Gene Drug Knowledge database for suitable drug matches, limiting the queries to clinically actionable antineoplastic drugs. Results Analyzing the cohort of 18 BC patients, we detected on average 26 BC relevant genes (526 total, log2 FC > 2) to be upregulated per patient. Querying the upregulated genes against DGIdb, we found a total of 18 genes that had drug matches and fulfilled the criteria of being actionable antineoplastic drugs, with 17/18 samples having a minimum of two gene targets (avg: 4). Most frequent upregulated genes were TOP2A (83%), AURKA (61%), AURKB (56%), RET (39%)and FGFR3 (28%). In the case of CNVs, 12/18 patients showed at least one gene target with clinically actionable drugs associated. This was observed across 12 gene targets that were amplified (avg: 3) and 4 gene targets that underwent deletions (avg: 1). Most frequent CNVs included MYC (14%) and CCND1 (12%). 4/7 patients having an AURKA overexpression also showed an AURKA amplification on the CNV assay. 10/18 patients had fusions events, with an average of three fusions per patient, including GAB2-WNT11, PAK1-TENM4 and FGFR2-CEP55 fusions. Conclusions We show that RNA-Seq and copy number assays provide additional clinical value by detecting suitable drug targets beyond traditional DNA-based approaches. We are conducting further analysis on how these additionally derived drug targets could improve the current treatment schedule of those patients. Citation Format: Meissner T, Amallraja A, Mark A, Andrews A, Connolly C, Young B, De P, Williams C, Leyland-Jones B. The value of RNA-Seq for the detection of clinically actionable targets in breast cancer - A small cohort analysis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-22.

Introduction Variant calling based on DNA samples has been the gold standard of clinical testing since the advent of Sanger sequencing. The use of DNA variants has proved a great value to guide treatment in cancer patients. However, DNA based analysis will not inform about expression status of the gene harboring a particular variant. RNA has long been used to monitor expression. To this point RNA assays and analysis are confined to the research laboratory and rarely used clinically except in specifically defined gene signatures such as PAM50 and OncoType Dx. Beyond expression, RNA has the ability to confirm expression of DNA variants and identify fusion events. We hypothesize that the combination of DNA and RNA based data will allow the determination of variant specific expression status and improve clinical diagnostics. It has been previously shown that RNA sequencing (RNA-Seq) based variant calls are highly accurate and confirm DNA based variant calls. In this study we investigated the utility of RNA-Seq as a diagnostic assay integrated with DNA based sequencing data. Materials and Methods Targeted DNA sequencing of 321 genes was performed on 37 patient samples (FFPE), including 22 breast cancer samples by a commercial vendor. RNA-Seq on the same patient samples was performed using 100ng of total RNA. Libraries were run on the Illumina NextSeq 500 with a minimum of 75M paired 75bp reads. To evaluate RNA-seq expression reproducibility, replicates of 6 normal ovarian tissue samples (min. 50M reads) were run in sets of triplicates. STAR was used for alignment (hg19) and gene expression quantification (RefSeq). RNA-Seq based variant calling was performed using the SNPiR pipeline. Based on the results of the commercial assay, DNA based variants were examined for expression of the corresponding genes and ability to confirm variants in the RNA-Seq data. Results RNA expression data showed no corresponding gene expression for at least one single nucleotide variant (SNV) in 9/37 patients analyzed (24.3%). In 18/37 patients (48.6%) SNV corresponding expression was in the lowest quartile of expression values. Variant calls could be confirmed by RNA-Seq for 95/455 SNVs, with adequate coverage in 263 of the remaining 360 variant locations (median coverage: 34). Of these, a homozygous reference call was made in 166/263 SNVs. Concordance for RNA-Seq gene level expression data between replicates was > 0.995. Conclusions These findings suggest that RNA-Seq based data can provide clinical value when using gene expression values in combination with DNA based variant calls. We found gene level expression to be highly reproducible and will further investigate the use of spike in controls to determine clinically usable expression ranges and lower limit of expression values. To our knowledge, it has not been shown that RNA-Seq based variant calls are reproducible which is the focus of our current research as this will be one requirement for usage in a regulated environment. While our use of RNA Seq is currently limited to gene expression level data, we have demonstrated a clinically relevant benefit to using RNA Seq data as an additive feature to the current standard of DNA variant calling. Citation Format: Young B, Mark A, Meissner T, Amallraja A, Andrews A, Connolly C, Williams C, Leyland-Jones B. Utilities and challenges of RNA-Seq based expression and variant calling in a clinical setting [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-23.

Histone lysine demethylases (KDMs) are emerging as therapeutic targets in cancer. Development of potent KDM inhibitors may provide additional options for epigenomics-oriented therapies. Using a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) functional demethylation assay, in combination with a high-content immunofluorescence imaging phenotypic screen, Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance mass spectrometry (MALDI-FTICR MS) and Amplified Luminescent Proximity Homogeneous Assay (ALPHA), we identified geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), as a novel inhibitor of JmjC-domain containing demethylases such as KDM4B. We further found that geldanamycin can destabilize the PAX3-FOXO1 fusion oncoprotein, an Hsp90 client, which is a driver of clinically unfavorable alveolar rhabdomyosarcoma (aRMS). We then hypothesized that dual inhibition of PAX3-FOXO1 and epigenetic modifiers of aRMS would have synergistic antitumor activity. We repurposed the geldanamycin analog 17-DMAG to target aRMS and found that 17-DMAG significantly delays tumor growth, extends survival in xenograft mouse models, and inhibits expression of PAX3-FOXO1 targets and multiple oncogenic pathways including MYC, E2F and NOTCH. In addition, the combination of 17-DMAG with conventional chemotherapy or the bromodomain inhibitor JQ1 significantly enhances therapeutic efficacy. In summary, we have identified geldanamycin and 17-DMAG as dual KDM/Hsp90 inhibitors and 17-DMAG is efficacious against PAX3-FOXO1-driven rhabdomyosarcoma.

Purpose: In breast cancer, bevacizumab increased pCR rate but not long-term survival and no predictive markers are available to identify patients with long-term benefit from the drug. Experimental Design: We profiled 289 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) biopsies of HER2-negative patients from the GeparQuinto trial of neoadjuvant chemotherapy ± bevacizumab by exome-capture RNA-sequencing (RNA-seq). In a prospectively planned study, we tested molecular signatures for response prediction. IHC validation was performed using tissue microarrays. Results: We found strong agreement of molecular and pathologic parameters as hormone receptors, grading, and lymphocyte infiltration in 221 high-quality samples. Response rates (49.3% pCR overall) were higher in basal-like (68.9%) and HER2-enriched (45.5%) than in luminal B (35.7%), luminal A (17.9%), and normal-like (20.0%) subtypes. T-cell (OR = 1.60; 95% confidence interval, 1.21–2.12; P = 0.001), proliferation (OR = 2.88; 95% CI, 2.00–4.15; P < 0.001), and hypoxia signatures (OR = 1.92; 95% CI, 1.41–2.60; P < 0.001) significantly predicted pCR in univariate analysis. In a prespecified multivariate logistic regression, a small hypoxia signature predicted pCR (OR = 2.40; 95% CI, 1.28–4.51; P = 0.006) with a significant interaction with bevacizumab treatment (P = 0.020). IHC validation using NDRG1 as marker revealed highly heterogenous expression within tissue leading to profound loss of sensitivity in TMA analysis, still a significant predictive value for pCR was detected (P = 0.025). Conclusions: Exome-capture RNA-seq characterizes small FFPE core biopsies by reliably detecting factors as for example ER status, grade, and tumor-infiltrating lymphocytes levels. Beside molecular subtypes and immune signatures, a small hypoxia signature predicted pCR to bevacizumab, which could be validated by IHC. The signature can have important applications for bevacizumab treatment in different cancer types and might also have a role for novel combination therapies of bevacizumab with immune checkpoint inhibition.

Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon–carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

Whole-transcriptome microarray analysis has become a popular strategy to study gene-expression in cancer, exploiting the largely available formalin-fixed paraffin-embedded (FFPE) sample resources. However, there have been relatively few comparative studies evaluating the performance of the different gene-expression array platforms. We compared two commonly used whole-transcriptome microarray platforms: Illumina human whole genome cDNA-mediated annealing, selection extension and ligation (DASL) beadchip and Affymetrix U133 Plus2 GeneChip (Affymetrix). Gene expression data based on both platforms were collected on the same total RNA extracted from FFPE tissue samples of 221 advanced breast cancer patients. Correlations between two platforms were assessed using Pearson and Spearman correlation coefficients (CCs). For both platforms we also assessed coefficient of variation, which measures relative dispersion. Finally, we compared the applicability of DASL and Affymetrix for classification of breast cancer molecular subtypes using the PAM50 classifiers. Overall, there was a statistically significant, positive gene- and patient-wise correlation between the two platforms, with stronger relationship for patient-wise CC. The relative dispersion was smaller in DASL compared to Affymetrix. The consistency in subtype classification for both microarray platforms was weak (63 % ). We observed weak, yet positive correlation between two platforms and different magnitudes of correlations were observed according to the metrics used.

Background: The prognostic significance of increased levels of CD8+ tumor infiltrating lymphocytes(TILs) in ER- breast cancer has been described. We sought to identify possible immune-related biomarkers for predicting benefit from letrozol(LET) or tamoxifen(TAM) for recurrence in ER+ breast cancer. Patient and Methods: We used Illumina DASL Assay to measure gene expression in FFPE primary breast cancers from a subset of postmenopausal patients enrolled in the BIG 1-98 randomized phase 3 trial comparing 5 years LET (n=344) vs TAM (n=381) as adjuvant endocrine therapy. Gene sets (n=1910) that represent cell states and perturbations within the immune system from the Human Immunology Project Consortium were used in an exploratory analysis to identify possible predictive signatures. Results: We identified five distinct gene signatures from previously reported laboratory experiments associated with immune cell differentiation that are highly predictive of benefit (reduced breast cancer recurrence risk) of LET over TAM, each with gene signature p-values Conclusion: The role of selective estrogen receptor modulators on immune response has been well described, where TAM has been shown to prevent differentiation and activation of dendritic cells (Naibandian 2005). Similarly, it has been shown that MET inhibitors negatively regulate neutrophils suggesting that anti-MET drugs in cancer could impact immune response (Finisquerra 2015). These findings suggest that if TAM is a negative regulator of immune response why in the ATAC clinical trial, the combination therapy of anastrozole plus TAM were not significantly different from TAM alone were anastrozole was superior. With the increasing importance of understanding the role of immune response on outcome and the use of combination therapies the assessment of TILs in the neoadjuvant setting will be critical for guiding therapy. Citation Format: Willis S, Gray KP, Regan MM, Rae JM, Kammler R, Young B, Ditzel HJ, Lyng MB, Colleoni M, Viale G, Leyland-Jones B. Immune related gene expression signatures predict benefit of letrozole over tamoxifen in BIG 1-98. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-36.

We previously reported a novel inhibitor of the ataxia-telangiectasia mutated (ATM) kinase, which is a target for novel radiosensitizing drugs. While our initial lead, compound 4, was relatively potent and nontoxic, it exhibited poor stability to oxidative metabolism and relatively poor selectivity against other kinases. The current study focused on balancing potency and selectivity with metabolic stability through structural modification to the metabolized site on the quinazoline core. We performed extensive structure-activity and structure-property relationship studies on this quinazoline ATM kinase inhibitor in order to identify structural variants with enhanced selectivity and metabolic stability. We show that, while the C-7-methoxy group is essential for potency, replacing the C-6-methoxy group considerably improves metabolic stability without affecting potency. Promising analogues 20, 27g, and 27n were selected based on in vitro pharmacology and evaluated in murine pharmacokinetic and tolerability studies. Compound 27g possessed significantly improve pharmacokinetics relative to that of 4. Compound 27g was also significantly more selective against other kinases than 4. Therefore, 27g is a good candidate for further development as a potential radiosensitizer.

The precision medicine (PM) revolution is well underway, and next-generation sequencing (NGS) is helping take cancer treatment to new levels. Finding actionable targets in real time is now a reality, and data from several clinical trials based on identified molecular alterations can be assessed and can help address the question of whether personalized treatment based on genomics produces superior survival outcomes compared with unselected treatment. Targeted treatment-based clinical trials have shown benefit in molecular subgroups of patients. Of further interest, it appears that genomics and immunotherapy are coupled to each other, since the immune system recognizes neo-antigens produced by the mutanome. Hence, some of the most important markers predicting response to immunotherapy are genomic markers, PDL1 amplification (for PD-1/PD-L1 checkpoint inhibitors), and tumor mutational burden. However, a number of challenges remain to be addressed if the use of molecular profiling-guided therapy in PM-designed clinical trials is to become the standard on which cancer treatment is based. In this chapter, we explore what it will take for PM trials that use molecular profiling as part of the eligibility criteria to be successful and the remaining hurdles that need to be overcome.

// Michael Forster 1 , Adam Mark 1, 2 , Friederike Egberts 3 , Elisa Rosati 1 , Elke Rodriguez 3 , Martin Stanulla 4 , Dirk Bauerschlag 5 , Christian Schem 6 , Nicolai Maass 5 , Anu Amallraja 7 , Karla K. Murphy 8 , Bruce R. Prouse 8 , Raed A. Sulaiman 8 , Brandon M. Young 7 , Micaela Mathiak 9 , Georg Hemmrich-Stanisak 1 , David Ellinghaus 1 , Stephan Weidinger 3 , Philip Rosenstiel 1 , Norbert Arnold 1, 5 , Brian Leyland-Jones 7 , Casey B. Williams 7 , Andre Franke 1, * and Tobias Meisner 7, * 1 Institute of Clinical Molecular Biology, Kiel University, Kiel, Germany 2 Current address: Center for Computational Biology and Bioinformatics, Department of Medicine, University of California, San Diego, CA, USA 3 Department of Dermatology, Schleswig-Holstein University Hospital, Kiel, Germany 4 Department of Pediatric Haematology and Oncology, Hannover Medical School, Hannover, Germany 5 Department of Gynaecology and Obstetrics, Schleswig-Holstein University Hospital, Kiel, Germany 6 Mammazentrum Hamburg, Hamburg, Germany 7 Department of Molecular and Experimental Medicine, Avera Cancer Institute, Sioux Falls, SD, USA 8 Pathology, Avera McKennan, Sioux Falls, SD, USA 9 Department of Pathology, Schleswig-Holstein University Hospital, Kiel, Germany * These authors share last authorship Correspondence to: Michael Forster, email: m.forster@ikmb.uni-kiel.de Keywords: breast cancer sequencing; normal RNA expression; drug selection; formalin fixed paraffin embedded; fresh frozen Received: March 07, 2018 Accepted: August 04, 2018 Published: August 17, 2018 ABSTRACT Background: While standard RNA expression tests stratify patients into risk groups, RNA-Seq can guide personalized drug selection based on expressed mutations, fusion genes, and differential expression (DE) between tumor and normal tissue. However, patient-matched normal tissue may be unavailable. Additionally, biological variability in normal tissue and technological biases may confound results. Therefore, we present normal expression reference data for two sequencing methods that are suitable for breast biopsies. Results: We identified breast cancer related and drug related genes that are expressed uniformly across our normal samples. Large subsets of these genes are identical for formalin fixed paraffin embedded samples and fresh frozen samples. Adipocyte signatures were detected in frozen compared to formalin samples, prepared by surgeons and pathologists, respectively. Gene expression confounded by adipocytes was identified using fat tissue samples. Finally, immune repertoire statistics were obtained for healthy breast, tumor and fat tissues. Conclusions: Our reference data can be used with patient tumor samples that are asservated and sequenced with a matching aforementioned method. Coefficients of variation are given for normal gene expression. Thus, potential drug selection can be based on confidently overexpressed genes and immune repertoire statistics. Materials and Methods: Normal expression from formalin and frozen healthy breast tissue samples using Roche Kapa RiboErase (total RNA) (19 formalin, 9 frozen) and Illumina TruSeq RNA Access (targeted RNA-Seq, aka TruSeq RNA Exome) (11 formalin, 1 frozen), and fat tissue (6 frozen Access). Tumor DE using 10 formalin total RNA tumor samples and 1 frozen targeted RNA tumor sample.

Purpose Triple negative (TN) breast cancers which lack expression of the estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors convey a poor prognosis due in part to a lack of targeted therapies. Methods To identify viable targets for the treatment of TN disease, we have conducted a gene set enrichment analysis (GSEA) on seven different breast cancer whole genome gene expression cohorts comparing TN vs. ER + HER2 − to identify consistently enriched genes that share a common promoter motif. The seven cohorts were profiled on three different genome expression platforms (Affymetrix, Illumina and RNAseq) consisting in total of 2088 samples with IHC metadata. Results GSEA identified enriched gene expression patterns in TN samples that share common promoter motifs associated with SOX9, E2F1, HIF1A, HMGA1, MYC BACH2, CEBPB, and GCNF/NR6A1. Unexpectedly, NR6A1 an orphan nuclear receptor normally expressed in germ cells of gonads is highly expressed in TN and ER + HER2 − samples making it an ideal drug target. Conclusion With the increasing number of large sample size breast cancer cohorts, an exploratory analysis of genes that are consistently enriched in TN sharing common promoter motifs allows for the identification of possible therapeutic targets with extensive validation in patient derived data sets.

Background: The optimal treatment strategy for patients with metastatic breast cancer (MBC) is currently unknown. Resistance to standard therapies like anthracyclines and taxanes limit the number of treatment options in many patients to a small number of non-cross-resistant regimens. We hypothesized that genomic and proteomic profiling of clinical MBC samples would identify genomic alterations that are linked to targeted therapies, and that this could facilitate a personalized approach to therapy for our patients. Methods: We retrospectively analyzed 31 consecutive metastatic breast cancer patients that had genomic and/or proteomic studies sent over a 4 month period from February to May 2014. All patients were seen in our Genomic Oncology Clinic and subsequently underwent a rebiopsy of a metastatic site. Formalin-fixed, paraffin-embedded (FFPE) tissue was sent to either Foundation Medicine for genomic profiling, Theranostics for reverse-phase protein microarray, or both. Standard immunohistochemistry for ER, PR, and HER2 was also performed on all patients. Results: Genomic or proteomic alterations were identified in all 31 patients. All patients harbored at least one actionable target and a treatment recommendation for a currently available FDA approved drug or drug combination was able to be suggested in all but one patient. The most commonly observed genomic alterations were within PIK3CA (26%), TP53 (23%), CCND1 (19%), and MYC (16%). Over 30 distinct genomic alterations were identified. Proteomic results were available from 16 patients. Activation of the AKT/mTOR pathway was evident in a majority of patients. A change in HER2 status was also found in 26% of patients. 16% of cases underwent a negative to positive conversion in HER2 status while 10% of cases underwent a positive to negative conversion. It is notable that all 5 patients that underwent a negative to positive conversion in HER2 status had biopsies taken from metastatic disease in the liver. Conclusions: All patients in this retrospective study harbored genomic or proteomic alterations that are associated with targeted therapies. Treatment recommendations were suggested in all but one patient and a majority of patients are receiving the suggested therapy. Our data demonstrate that routine genomic and proteomic analysis in a clinical setting makes a significant positive impact for patients. Citation Format: Casey B Williams, Pradip De, Nandini Dey, Kirstin A Williams, Jessica Klein, Michelle King, Misty Small, Brigitte Cyr, Scooter Willis, Yuliang Sun, Jennifer Carlson, Brandon M Young, Amy Krie, Brian Leyland-Jones. New treatment options for metastatic breast cancer revealed by reverse-phase protein microarray and genomic profiling [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-14.

HERA is an international multi-center randomized trial comparing 1 or 2 years trastuzumab, given every 3 weeks, with observation in women with HER2+ breast cancer after standard neoadjuvant or adjuvant chemotherapy. From December 2001 to June 2005, 5102 patients were randomized. Comparing 1 year trastuzumab with observation at 8 years of follow-up, statistically significant differences in disease-free survival (DFS) and overall survival, despite crossover to trastuzumab of observation arm patients, were found. No benefit in DFS of 2 years compared to 1 year trastuzumab was found[Goldhirsch 2013]. To determine possible predictive single-gene biomarkers, an exploratory ITT analysis, with DFS as the primary endpoint, was conducted using mRNA expression data from 610 TransHERA FFPE samplesprofiled on Illumina Whole-Genome DASL cubic spline normalization was applied to the data. Outcome was obtained from the HERA database with 8 years median follow-up and clinical cut-off date April 12, 2012. Characteristics were well balanced between treatment groups. The exploratory analysis of 20,464 genes using DFS as the endpoint identified C8A as a possible biomarker that is prognostic and predictive of response to treatment. Cox regression was used to model DFS, with the interaction term between treatment and C8A as a continuous and a categorical variable split on the cohort mean. The observation arm consists of 199 samples with 66 events and the trastuzumab arm(1&2-year combined) of 411 samples with 108 events. A statistically significant interaction between C8A mRNA and treatment was detected (p C8A is a member of the membrane attack complex and is part of the innate immune system. C8A inserts into the membrane of the target cell and binds with multiple copies of the pore-forming C9 leading to cell lysis. From the GeneAtlas, C8A is highly expressed in liver tissue suggesting an advantage for tumors with high expression of C8A and innate immune response. The Cancer Cell Line Encyclopedia indicates a wide range of C8A mRNA expression. C8A as a single gene biomarker that is prognostic of DFS and predictive of benefit from trastuzumab has the potential to improve the standard of care in HER2+ breast cancer. Understanding the advantage of over expression of C8A related to the innate immune response can give insight into the mechanisms that drive cancer. We note with caution that this finding is the result of an exploratory analysis and is being pursued in additional trastuzumab cohorts for further validation. Citation Format: Scooter Willis, Varvara Polydoropoulou, Zoi Tsourti, Urania Dafni, Brandon Young, Bradley Long, Pradip De, Nandini Dey, Casey Williams, Mitch Dowsett, Brian Leyland-Jones. Exploratory analysis of single gene predictive biomarkers in TransHERA DASL cohort reveals that C8A mRNA expression is prognostic of outcome and predictive of benefit of trastuzumab [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-02.

Histone lysine demethylases facilitate the activity of oncogenic transcription factors, including possibly MYC. Here we show that multiple histone demethylases influence the viability and poor prognosis of neuroblastoma cells, where MYC is often overexpressed. We also identified the approved small-molecule antifungal agent ciclopirox as a novel pan-histone demethylase inhibitor. Ciclopirox targeted several histone demethylases, including KDM4B implicated in MYC function. Accordingly, ciclopirox inhibited Myc signaling in parallel with mitochondrial oxidative phosphorylation, resulting in suppression of neuroblastoma cell viability and inhibition of tumor growth associated with an induction of differentiation. Our findings provide new insights into epigenetic regulation of MYC function and suggest a novel pharmacologic basis to target histone demethylases as an indirect MYC-targeting approach for cancer therapy. Cancer Res; 77(17); 4626–38. ©2017 AACR.

Background In prior published work, the Worldwide Innovative Network (WIN) Consortium described the Simplified Interventional Mapping System (SIMS) algorithm, based on the hypothesis that both genomic and transcriptomic data are important for matching to a tri-therapy regimen. To account for variation in transcription levels between different tissues, gene by gene tumor RNA levels are normalized against RNA levels in analogous biopsied normal tissue. SPRING is the first trial to assess a SIMS-based tri-therapy regimen in advanced NSCLC. Methods Patients with metastatic NSCLC (no EGFR or ALK alterations; no ROS1 alteration if tested; PD-L1 unrestricted; ≤2 prior therapy lines) were treated with avelumab, palbociclib, and axitinib (3 + 3 dose escalation design). After consent, biopsies of tumor and normal endobronchial mucosa were obtained on all patients for analysis on a central genomics/transcriptomics platform and retrospective SIMS algorithm validation. A safety monitoring committee reviews study conduct at least weekly. Results Twelve patients have been treated (3 at dose level 1; 6, dose level 2; 3, dose level 3). Three dose-limiting toxicities (DLTs) at least possibly drug related occurred: 1 DLT at dose level 2 (Grade 3 (G3) infusion reaction); 2 DLTs at dose level 3 (G3 hand/foot syndrome and a G5 respiratory failure). The partial response (PR) rate was 44% (4/9 patients who have reached restaging including 2/3 patients at dose level 1; two PRs are in patients who failed prior pembrolizumab). The maximum tolerated dose was avelumab 10 mg/kg IV q2weeks, axitinib 5 mg po bid, palbociclib 75 mg po daily (7 days off; 21 days on) (dose level 2). Conclusions The tri-therapy combination of avelumab, axitinib, and palbociclib is tolerable with early evidence of activity including in NSCLC patients who failed a prior checkpoint inhibitor. Expansion cohorts are being added to further explore safety of a recommended phase II dose. Transcriptomic and genomic correlates of response are being assessed. Clinical trial identification NCT03386929; First posted: December 29, 2017. Legal entity responsible for the study Worldwide Innovative Network (WIN) Association - WIN Consortium. Funding ARC Foundation for Cancer Research and Pfizer. Disclosure B. Solomon: Travel / Accommodation / Expenses: Elekta. A. Callejo: Speaker Bureau / Expert testimony: Roche; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Boerhinger; Speaker Bureau / Expert testimony: Sanofi; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: MSD; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: BMS; Speaker Bureau / Expert testimony: KYOWA KIRIN ; Travel / Accommodation / Expenses: Pfizer; Travel / Accommodation / Expenses: Celgene. J. Bar: Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy: Genentech; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: AbbVie; Advisory / Consultancy, Research grant / Funding (institution): MSD; Advisory / Consultancy: Takeda; Advisory / Consultancy: Vascular Biogenics; Research grant / Funding (institution): MedImmune. G. Berchem: Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Celgene. L. Bazhenova: Advisory / Consultancy: Takeda; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: AbbVie; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Loxo Oncology; Shareholder / Stockholder / Stock options: Epic Sciences; Research grant / Funding (institution): Beyondspring Pharma. P. Saintigny: Honoraria (self), Research grant / Funding (self), Travel / Accommodation / Expenses: BMS; Honoraria (self), Research grant / Funding (self), Travel / Accommodation / Expenses: Roche; Honoraria (self), Research grant / Funding (self), Travel / Accommodation / Expenses: AstraZeneca; Advisory / Consultancy, Research grant / Funding (self): HTG Diagnostics. E. Raymond: Full / Part-time employment: Genoscience; Full / Part-time employment: Scor; Advisory / Consultancy: Pharmaengine; Travel / Accommodation / Expenses: Merck. N. Girard: Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Hoffmann-La Roche; Advisory / Consultancy: Lilly; Advisory / Consultancy, Research grant / Funding (institution): Boehringer Ingelheim; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: AstraZeneca; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pfizer; Advisory / Consultancy, Travel / Accommodation / Expenses: Bristol-Myers Squibb; Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Advisory / Consultancy: Takeda; Advisory / Consultancy: GSK; Advisory / Consultancy: AbbVie. R. Sulaiman: Full / Part-time employment: Physicians Laboratory . E. Rubin: Travel / Accommodation / Expenses: Roche. V. Lazar: Licensing / Royalties: Worldwide Innovative Network Association. E. Felip: Advisory / Consultancy, Speaker Bureau / Expert testimony: ABBVIE; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy: Blue Print Medicines; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony: Bristol-Myers Squibb; Advisory / Consultancy: Celgene; Advisory / Consultancy, Speaker Bureau / Expert testimony: Eli Lilly; Advisory / Consultancy: Guardant Health; Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck MGaA; Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck Sharp & Domme; Advisory / Consultancy, Speaker Bureau / Expert testimony: NOVARTIS; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Takeda; Advisory / Consultancy: JANSSEN; Research grant / Funding (institution): Fundation Merck Salud; Research Grant / Funding (Institution): Grant For Oncology Innovation Emd Serono. A. Onn: Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca. B. Leyland-Jones: Speaker Bureau / Expert testimony: Bayer; Speaker Bureau / Expert testimony: Exelixis; Speaker Bureau / Expert testimony: Genentech; Speaker Bureau / Expert testimony: Puma; Travel / Accommodation / Expenses: NFCR; Travel / Accommodation / Expenses: AkesoGen. R. Kurzrock: Shareholder / Stockholder / Stock options, Co-founder: CureMatch; Shareholder / Stockholder / Stock options: IDbyDNA; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Soluventis; Advisory / Consultancy: Gaido; Advisory / Consultancy: Loxo Oncology; Advisory / Consultancy: Xbiotech; Advisory / Consultancy: Acurate Therapeutics; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy: NeoMed; Research grant / Funding (institution): uardant Health; Research grant / Funding (institution): Grifols; Research grant / Funding (institution): Konica Minolta; Research grant / Funding (institution): OmniSeq; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Merck Serono; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Sequenom; Research grant / Funding (institution): Foundation Medicine. All other authors have declared no conflicts of interest.

Background: NSCLC is the leading cause of cancer death. Recently, much progress has been made to identify and target specific oncogenic drivers such as EGFR mutations and ALK translocations. Unfortunately, the majority of NSCLCs do not have a single driver, but instead harbor complex systems of molecular alterations. The advent of immunooncology has opened newer avenues for treating these patients, but still, many tumors are resistant. Therefore, selection of precise therapies targeting the appropriate pathways in these tumors is an important area of research. In previously published work, the Worldwide Innovative Network (WIN) Consortium has developed the Simplified Interventional Mapping System (SIMS) algorithm to match patients to a targeted drug triplet. The SIMS algorithm is based on the hypothesis that the use of both genomic as well as transcriptomic data is important in selecting an ideal tri-therapy regimen. Since the transcriptome varies widely between different types of normal tissue, the relative level of transcription of any given gene in a tumor must be normalized against the expected level of transcription of that gene in the analogous normal tissue. The SPRING trial is the first trial assessing a rational, SIMS-based tri-therapy regimen in advanced NSCLC. The drugs utilized for SPRING–avelumab (anti-PDL1), axitinib (VEGFR inhibitor) and palbociclib (CDK4/6 inhibitor)–were chosen because the SIMS genomic/transcriptomic analysis demonstrated that ~10% of NSCLCs have a combination of these targets. Methods: Patients with locally advanced or metastatic NSCLC without EGFR or ALK alterations are being enrolled and treated with avelumab, palbociclib, and axitinib after informed consent. During phase I, any prior treatment is allowed. SPRING will assess the safety of the tri-therapy combination and determine the recommended phase II dose (RP2D) (3+3 dose escalation). Once the RP2D is found, the study will proceed directly to the phase II portion, which will permit only previously untreated patients in the metastatic setting. A biopsy of both tumor and normal tissue (by endobronchial mucosal biopsy) is obtained on all patients, both of which are analysed in a central genomics and transcriptomics platform. The SIMS algorithm is being validated retrospectively by determining the degree of genomic and transcriptomic matching and correlating with outcome. Results: The phase I study is open in USA, Israel, Spain and Luxembourg and has enrolled 12 patients to date and has progressed to cohort 3. The phase II portion is expected to open later in 2019. This research is supported by the EMD Serono/Pfizer alliance and ARC Foundation for cancer research. Citation Format: Benjamin Solomon, Enriqueta Felip, Jair Bar, Guy Berchem, Lyudmila Bazhenova, Pierre Saintigny, Nicolas Girard, Raed Sulaiman, Catherine Bresson, Fanny Wunder, J Jack Lee, Jacques Raynaud, Eitan Rubin, Brandon Young, Vladimir Lazar, Amir Onn, Brian Leyland Jones, Razelle Kurzrock. Survival Prolongation by Rationale INnovative Genomics (SPRING): An international WIN Consortium Phase I/II proof-of-concept study to explore the safety and efficacy of a tri-therapy approach using avelumab, palbociclib and axitinib in advanced/metastatic non-small cell lung cancer (NSCLC) with integrated genomic and transcriptomic correlates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT223.

Trisomy 21 in humans causes cognitive impairment, craniofacial dysmorphology, and heart defects collectively referred to as Down syndrome. Yet, the pathophysiology of these phenotypes is not well understood. Craniofacial alterations may lead to complications in breathing, eating, and communication. Ts65Dn mice exhibit craniofacial alterations that model Down syndrome including a small mandible. We show that Ts65Dn embryos at 13.5 days gestation (E13.5) have a smaller mandibular precursor but a normal sized tongue as compared to euploid embryos, suggesting a relative instead of actual macroglossia originates during development. Neurological tissues were also altered in E13.5 trisomic embryos. Our array analysis found 155 differentially expressed non-trisomic genes in the trisomic E13.5 mandible, including 20 genes containing a homeobox DNA binding domain. Additionally, Sox9, important in skeletal formation and cell proliferation, was upregulated in Ts65Dn mandible precursors. Our results suggest trisomy causes altered expression of non-trisomic genes in development leading to structural changes associated with DS. Identification of genetic pathways disrupted by trisomy is an important step in proposing rational therapies at relevant time points to ameliorate craniofacial abnormalities in DS and other congenital disorders.

A number of studies suggest that response to antihuman epidermal growth factor receptor-2 (currently known as ERBB2, butreferred to asHER2 in this study) agents differs by estrogen receptor (ER) level status. The clinical relevance of this is unknown.To determine the magnitude of trastuzumab benefit according to quantitative levels of ER and HER2 in the HERceptin Adjuvant (HERA) trial.The HERA trial was an international, multicenter, randomized trial that included 5099 patients with early-stage HER2-positive breast cancer, randomized between 2001 and 2005 to receive either no trastuzumab or trastuzumab, after adjuvant chemotherapy. This is a secondary analysis of the HERA study. Local ER immunohistochemical (IHC) analyses, HER2 fluorescence in situ hybridization (FISH) ratio, and copy number results were available for 3037 patients (59.6%) randomized to observation and trastuzumab (1 or 2 years) (cohort 1). Transcript levels of ESR1 and HER2 genes were available for 615 patients (12.1%) (cohort 2).Patients were randomized to receive either no trastuzumab or 1 year vs 2 years of trastuzumab. Endocrine therapy was given to patients with hormone receptor-positive disease as per local guidelines.Disease-free survival (DFS) and overall survival (OS) were the primary and secondary end points in the intent-to-treat population (ITT). Analyses adjusting for crossover (censored and inverse probability weighted [IPW]) were also performed. Interactions among treatment, ER status, and HER2 amplification using predefined cutoffs were assessed in Cox proportional hazards regression models.Median follow-up time was 8 years. Levels of FISH and HER2 copy numbers were significantly higher in ER-negative patients (P .001). In cohort 1, for DFS and OS, a significant treatment effect was found for all ER, IHC, and FISH levels, except for the ER-positive/HER2 low FISH ratio (≥2 to5) group (DFS: 3-way ITT Pvalue for interaction = .07; censored = .02; IPW = .03; OS ITT Pvalue for interaction = .007; censored = .04; IPW = .03). In cohort 2, consistent with cohort 1, a significant predictive effect of the ESR1 gene for both end points was also observed (DFS Pvalue for interaction = .06; OS = .02), indicating that breast cancers with higher ESR1 levels also derive less benefit from trastuzumab.Patients with HER2-positive breast cancers that are ER-positive by IHC analyses with low FISH ratio (≥2 to5), or with higher ESR1 levels derive significantly less benefit from adjuvant trastuzumab after chemotherapy. These data may explain heterogeneity in response to anti-HER2 agents in HER2-positive, ER-positive breast cancers as some may be more luminal-like than HER2 driven.clinicaltrials.gov Identifier: NCT00045032.

Background: Delivery of premature infants outside tertiary care centers is not always preventable. The aim of this study was to compare rates of survival and common morbidities in extremely premature babies transported to a level III facility versus those born at the level III center. Methods: Retrospective chart review was performed on all neonates born at ≤ 28 weeks of gestation with birthweight ≤1500 g who were admitted to the Newborn Intensive Care Unit at Kapi‘olani Medical Center for Women and Children (KMCWC) between 1 January 2000 and 31 December 2005. Infants were divided into two groups, those born at KMCWC (Inborn) and those born at level I institutions and subsequently transported (Transport) to KMCWC. Results: A total of 394 neonates met the study criteria; 349 were inborn while 45 were transported. Survival rates were identical for both groups. However, the Transport group survivors displayed a significantly longer mean length of stay and higher rate of severe retinopathy of prematurity than those in the Inborn group (P≤ 0.01). Conclusion: Identical rates of survival in both groups suggest that community medical professionals are providing satisfactory care to stabilize critical neonates without reducing their chances of survival. However, increased length of stay and higher rate of retinopathy of prematurity in the Transport group suggest that differences in medical management during the first few hours of life may adversely affect outcomes.

Background: Formalin-fixed, paraffin-embedded (FFPE) tumour tissue represents an immense but mainly untapped resource with respect to molecular profiling. The DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay is a recently described, RT–PCR-based, highly multiplexed high-throughput gene expression platform developed by Illumina specifically for fragmented RNA typically obtained from FFPE specimens, which enables expression profiling. In order to extend the utility of the DASL assay for breast cancer, we have custom designed and validated a 512-gene human breast cancer panel. Methods: The RNA from FFPE breast tumour specimens were analysed using the DASL assay. Breast cancer subtype was defined from pathology immunohistochemical (IHC) staining. Differentially expressed genes between the IHC-defined subtypes were assessed by prediction analysis of microarrays (PAM) and then used in the analysis of two published data sets with clinical outcome data. Results: Gene expression signatures on our custom breast cancer panel were very reproducible between replicates (average Pearson's R2=0.962) and the 152 genes common to both the standard cancer DASL panel (Illumina) and our breast cancer DASL panel were similarly expressed for samples run on both panels (average R2=0.877). Moreover, expression of ESR1, PGR and ERBB2 corresponded well with their respective pathology-defined IHC status. A 30-gene set indicative of IHC-defined breast cancer subtypes was found to segregate samples based on their subtype in our data sets and published data sets. Furthermore, several of these genes were significantly associated with overall survival (OS) and relapse-free survival (RFS) in these previously published data sets, indicating that they are biomarkers of the different breast cancer subtypes and the prognostic outcomes associated with these subtypes. Conclusion: We have demonstrated the ability to expression profile degraded RNA transcripts derived from FFPE tissues on the DASL platform. Importantly, we have identified a 30-biomarker gene set that can classify breast cancer into subtypes and have shown that a subset of these markers is prognostic of OS and RFS.

The melanocortin-3 receptor (MC3R) gene is pleiotropic, influencing body composition, natriuresis, immune function, and entrainment of circadian rhythms to nutrient intake. MC3Rs are expressed in hypothalamic and limbic regions of the brain and in peripheral tissues. To investigate the roles of central MC3Rs, we inserted a "lox-stop-lox" (LoxTB) 5' of the translation initiation codon of the mouse Mc3r gene and reactivated transcription using neuron-specific Cre transgenic mice. As predicted based on earlier observations of Mc3r knock-out mice, Mc3r(TB/TB) mice displayed reduced lean mass, increased fat mass, and accelerated diet-induced obesity. Surprisingly, rescuing Mc3r expression in the nervous system using the Nestin-Cre transgene only partially rescued obesity in chow-fed conditions and had no impact on the accelerated diet-induced obesity phenotype. The ventromedial hypothalamus (VMH), a critical node in the neural networks regulating feeding-related behaviors and metabolic homeostasis, exhibits dense Mc3r expression relative to other brain regions. To target VMH MC3R expression, we used the steroidogenic factor-1 Cre transgenic mouse. Although restoring VMH MC3R signaling also had a modest impact on obesity, marked improvements in metabolic homeostasis were observed. VMH MC3R signaling was not sufficient to rescue the lean mass phenotype or the regulation of behaviors anticipating food anticipation. These results suggest that actions of MC3Rs impacting on energy homeostasis involve both central and peripheral sites of action. The impact of central MC3Rs on behavior and metabolism involves divergent pathways; VMH MC3R signaling improves metabolic homeostasis but does not significantly impact on the expression of behaviors anticipating nutrient availability.

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is a highly aggressive tumor with a 5-year survival of We and others document that inhibiting the function of BET bromodomain proteins suppresses PDAC tumor growth in preclinical models. The BET family of proteins is characterized by two N-terminal bromodomains, BD1 and BD2, which bind to acetylated lysine residues on specific histones. The binding of BET proteins to acetylated lysines promotes the formation of transcriptional complexes, and impacts gene transcription and chromatin structure. The literature suggests the potential specific roles for BD1 vs BD2 using pharmacological inhibitors and dominant negative mutational analyses. However, the relative importance of each bromodomain with respect to transcriptional regulation compared to chromatin structure is not well understood. Further, there is suggestion in the literature that each bromodomain function may differ in specific tumor cell types. Functional differences between BD1 and BD2 in PDAC cells have not been reported. The goal of the current study was to compare the efficacy of the BD2-specific BET inhibitor (BETi) SJ018 with the efficacy of the pan-BET inhibitor I-BET762 in PDAC cells. We exposed four PDAC cell lines (Panc1, BxPC3, MiaPaCa2, and CFPAC) to a range of concentrations of SJ018 or I-BET762 for 96 hours, and assessed cell viability using alamarBlue. Data, analyzed using GraphPad Prism 7, demonstrated that SJ018 exerted up to ~60-fold lower IC50 values across all cell lines tested compared to the pan-BET inhibitor I-BET762. Further, we recently made a novel observation that the pan-BETi JQ1 induced DNA damage and inhibits expression of the DNA repair protein Rad51 in PDAC cells. Immunoblot analysis for phosphorylated Ser-139 H2AX (γH2AX), a marker of DNA damage, demonstrated that SJ018 and I-BET762 also increased DNA damage, and that the BD2-specific SJ018 induces DNA damage to a greater extent and at a lower dose than the BD1/BD2 inhibitor I-BET762 in BxPC3 cells. The data also showed that both SJ018 and I-BET762 inhibited expression of the homologous recombination protein Rad51 in vitro. We conclude that SJ018 is more potent than I-BET762 in PDAC cells as a single agent, and that degree of potency is reflected by higher levels of DNA damage. Citation Format: Aubrey Lynn Miller, Peter J. Slavish, Brandon M. Young, Philip M. Potter, Anang A. Shelat, Karina J. Yoon. Targeting the BD2 BET bromodomain to inhibit proliferation of pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5794.

Elevation of intraocular pressure (IOP) following injection of intravitreal triamcinolone acetonide (IVTA) is an important clinical problem. The etiology of the steroid response is poorly understood, although a genetic determinant has long been suspected. We performed a pharmacogenomic association study with glucocorticoid receptor polymorphisms.Fifty-two patients (56 eyes) who underwent treatment with IVTA for various retinal diseases were genotyped for six well-studied glucocorticoid receptor polymorphisms (ER22/23EK, N363S, BclI, N766N, and single nucleotide polymorphisms (SNPs) within introns 3 and 4).Three polymorphisms (ER22/23EK, N363S, and the intron 3 SNP) were essentially nonpolymorphic within this population sample and excluded from further analysis. The remaining three polymorphisms (BclI, N766N, and within intron 4) passed the Hardy-Weinberg Equilibrium test, indicating good genotyping quality and normal population distribution of allelic frequency. No statistically significant correlations were found between these three polymorphisms and magnitude of IOP elevation following IVTA, using single point association and haplotype analyses.In this small, pilot study, we found no statistically significant relationship between glucocorticoid receptor polymorphisms and IOP elevation following IVTA. The precise etiology of the steroid response remains obscure. To our knowledge, this is the first published pharmacogenomic study of this common clinical entity.

Estrogen receptor 1 (ESR1) and ESR2 gene polymorphisms have been associated with endocrine-mediated physiological mechanisms, and inconsistently with breast cancer risk and outcomes, bone mineral density changes, and hot flushes/night sweats. DNA was isolated and genotyped for six ESR1 and two ESR2 single-nucleotide polymorphisms (SNPs) from tumor specimens from 3691 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Associations with recurrence and adverse events (AEs) were assessed using Cox proportional hazards models. 3401 samples were successfully genotyped for five SNPs. ESR1 rs9340799(XbaI) (T>C) variants CC or TC were associated with reduced breast cancer risk (HR = 0.82,95% CI = 0.67-1.0), and ESR1 rs2077647 (T>C) variants CC or TC was associated with reduced distant recurrence risk (HR = 0.69, 95% CI = 0.53-0.90), both regardless of the treatments. No differential treatment effects (letrozole vs. tamoxifen) were observed for the association of outcome with any of the SNPs. Letrozole-treated patients with rs2077647 (T>C) variants CC and TC had a reduced risk of bone AE (HR = 0.75, 95% CI = 0.58-0.98, P interaction = 0.08), whereas patients with rs4986938 (G>A) genotype variants AA and AG had an increased risk of bone AE (HR = 1.37, 95% CI = 1.01-1.84, P interaction = 0.07). We observed that (1) rare ESR1 homozygous polymorphisms were associated with lower recurrence, and (2) ESR1 and ESR2 SNPs were associated with bone AEs in letrozole-treated patients. Genes that are involved in estrogen signaling and synthesis have the potential to affect both breast cancer recurrence and side effects, suggesting that individual treatment strategies can incorporate not only oncogenic drivers but also SNPs related to estrogen activity.

To determine whether CYP19A1 polymorphisms are associated with abnormal activity of aromatase and with musculoskeletal and bone side effects of aromatase inhibitors. DNA was isolated from tumor specimens of 4861 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Tumors were genotyped for six CYP19A1 polymorphisms using PCR-based methods. Associations with breast cancer-free interval (BCFI), distant recurrence-free interval (DRFI), musculoskeletal and bone adverse events (AEs) were assessed using Cox proportional hazards models. All statistical tests were two-sided. No association between the CYP19A1 genotypes and BCFI or DRFI was observed overall. A reduced risk of a breast cancer event for tamoxifen-treated patients with rs700518 variants was observed (BCFI CC/TC vs. TT: HR 0.53, 95 % CI 0.34-0.82, interaction P = 0.08), but not observed for letrozole-treated patients. There was an increased risk of musculoskeletal AEs for patients with rs700518 variants CC/TC versus TT (HR 1.22, 95 % CI 1.03-1.45, P = 0.02), regardless of treatment. Tamoxifen-treated patients with rs4646 variants had a reduced risk of bone AEs (AA/CA vs. CC: HR 0.76, 95 % CI 0.59-0.98), whereas an increase of minor allele (C) of rs10046 was associated with an increased risk of bone AEs (HR 1.28, 95 % CI 1.07-1.52). rs936308 variants were associated with a reduced risk of bone AEs in letrozole-treated patients (GG/GC vs. CC: HR 0.73, 95 % CI 0.54-0.99), different from in tamoxifen-treated patients (GG/GC vs. CC: HR 1.32, 95 % CI 0.92-1.90, interaction P = 0.01). CYP19A1 rs700518 variants showed associations with BCFI, DRFI, in tamoxifen treated patients and musculoskeletal AEs regardless of treatment. SNPs rs4646, rs10046, and rs936308 were associated with bone AEs.