Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR’s pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.

One caveat of next-generation sequencing (NGS)-based clinical oncology testing is the high amount of input DNA required. We sought to develop a focused NGS panel that could capture hotspot regions in relevant genes requiring 0.5-10 ng input DNA. The resulting Penn Precision Panel (PPP) targeted 20 genes containing clinically significant variants relevant to many cancers. One hundred twenty-three samples were analyzed, including 83 solid tumor specimens derived from FFPE. Various input quantities of DNA (0.5-10 ng) were amplified with content-specific PCR primer pools, then sequenced on a MiSeq instrument (Illumine, Inc.) via paired-end, 2 x 186 base pair reads to an average read depth of greater than 6500x. Variants were detected using an in-house analysis pipeline. Clinical sensitivity and specificity were assessed using results from our previously validated solid tumor NGS panel; sensitivity of the PPP is 96.75% (387/400 variants) and specificity is 99.9% (8427/8428 base pairs). Variant allele frequencies (VAFs) are highly concordant across both assays (r = 0.98 p < 0.0001). The PPP is a robust, clinically validated test optimized for low-yield solid tumor specimens, capturing a high percentage of clinically relevant variants found by larger commercially available NGS panels while using only 0.5-10 ng of input DNA.

Abstract Germline mutations in BRCA1 or BRCA 2 can lead to increased genomic instability and mutational burden in mutation-associated breast and ovarian cancer. Given the association of tumor neoepitope load and clinical response to immune checkpoint therapy, we used whole exome sequencing (WES) to predict missense mutational burden and potential neoepitope expression in 34 breast and 71 ovarian tumors with germline BRCA1/2 mutations (combining Penn and TCGA cohorts). Analysis of WES data found that tumors from patients with BRCA1/2 mutations had significantly higher mutational burden than non-BRCA1/2 tumors (p<0.01). Based on a novel bioinformatics pipeline for human leukocyte antigen (HLA) genotyping and MHC class I neoantigen prediction, we found that neoantigen load (predicted IC50 <500 nM) varied across patients (breast: mean - 147, std. dev. – 127; ovarian: mean – 126, std. dev. – 96). While higher neoantigen load promotes cytotoxic CD8+ T-cell responses in a number of cancers, we found among the BRCA1/2 subset that neoantigen load did not correlate with cytolytic signature, an RNA-based metric of anti-tumor immune response. From TCGA RNA-seq data, immune pathways (q<0.01) including PD-1 signaling, reflective of counter-regulatory responses often observed in response to immune pressure, positively correlated with cytolytic signature. Neoantigen load, however, correlated only with GPCR signaling and extracellular matrix (ECM) proteins (q<0.01). By contrast, the expression of genes involved in ECM and angiogenesis, which are involved in tumor immune escape and suppression of immune cell infiltration, negatively correlated with cytolytic signature (q<0.01). Increased PDL1 and PDL2 (p<0.05) expression associated with copy number loss in PTEN. These data were confirmed by tissue microarray analysis, which found that levels of T cell infiltration did not associate with neoantigen load, whereas PTEN loss was associated with decreased T cell infiltration (p<0.05). Our work demonstrates that neoantigen load alone is not sufficient to induce T-cell infiltration and cytotoxicity in tumors from patients with BRCA1/2 mutations, but rather points to tumor intrinsic pathways involving PTEN copy alterations and a dense extracellular matrix as determinants of tumor immunity, both of which may lead to novel opportunities for combination immune therapy in these cancers. Citation Format: Adam Kraya, Kara N. Maxwell, Brandon M. Wenz, Bradley Wubbenhorst, Daniel De Sloover, Nicole Lunceford, Amanda Barrett, Jennifer D. Morrissette, Michael Feldman, Robert H. Vonderheide, Susan M. Domcheck, Katherine L. Nathanson. Analysis of tumor immunogenicity in germline BRCA1/2 mutation associated breast and ovarian cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A12.

Complete loss of BRCA1 or BRCA2 function is associated with sensitivity to DNA damaging agents. However, not all BRCA1 and BRCA2 germline mutation-associated tumors respond. Herein we report analyses of 160 BRCA1 and BRCA2 germline mutation-associated breast and ovarian tumors. Retention of the normal BRCA1 or BRCA2 allele (absence of locus-specific loss of heterozygosity (LOH)) is observed in 7% of BRCA1 ovarian, 16% of BRCA2 ovarian, 10% of BRCA1 breast, and 46% of BRCA2 breast tumors. These tumors have equivalent homologous recombination deficiency scores to sporadic tumors, significantly lower than scores in tumors with locus-specific LOH (ovarian, P = 0.0004; breast P < 0.0001, two-tailed Student’s t -test). Absence of locus-specific LOH is associated with decreased overall survival in ovarian cancer patients treated with platinum chemotherapy ( P = 0.01, log-rank test). Locus-specific LOH may be a clinically useful biomarker to predict primary resistance to DNA damaging agents in patients with germline BRCA1 and BRCA2 mutations.

Background Next-generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low-input DNA available from rare CTCs. Moreover, whole genome amplification (WGA) approaches used to generate sufficient input DNA are often incompatible with blood collection tube preservatives used to facilitate clinical sample batching.Methods To address this, we have developed a novel approach combining tumor cell isolation from preserved blood with Repli-G WGA and Illumina TruSeq Amplicon Cancer Panel-based NGS. We purified cell pools ranging from 10 to 1000 cells from three different cell lines, and quantitatively demonstrate comparable quality of DNA extracted from preserved versus unpreserved samples.Results Preservation and WGA were compatible with the generation of high-quality libraries. Known point mutations and gene amplification were detected for libraries that had been prepared from amplified DNA from preserved blood.Conclusion These spiking experiments provide proof of concept of a clinically applicable work-flow for real-time monitoring of patient tumor using noninvasive liquid biopsies.

Abstract Tumor neoantigens are peptides unique to the cancer genome that can induce endogenous T-cell activity and contribute to the efficacy of immunotherapy. We sought to characterize the neoantigen repertoire in 20 breast and 19 ovarian tumors from patients with germline BRCA1/2 mutations. Loss of BRCA1/2 function leads to genomic instability and increased mutational burden, a key correlate with clinical benefit from immunotherapy. To determine patient class I Human Leukocyte Antigen (HLA) haplotypes, whole exome sequencing data were analyzed using OptiType. Class I HLA alleles were typed to four-digit accuracy and were used in the prediction of tumor neoantigens using NetCTLpan 1.1. Briefly, non-synonymous variants from tumor exome data were translated to peptides of length 15, 17, and 19 amino acids centered on the variant of interest, where NetCTLpan analyzed all 8-, 9-, and 10-mer amino acid substrings, respectively, within the peptide sequences to predict HLA allele-specific binding affinities. IC50 values, predicted probability of binding to the TAP transporter, and probability of C-terminal cleavage were used to prioritize candidate neoepitopes of interest. When considering peptides with a predicted affinity less than 500nM, the minimum criterion for a likely binding occurrence in vivo, neoantigen loads varied extensively across both breast and ovarian patients, ranging from a minimum of 10 predicted binders in patients with the lowest neo-antigen load to greater than 300 predicted binders in patients with the highest load (mean 87, std. dev. 71). 10 of 20 breast cancer patients and 13 of 19 ovarian cancer patients harbored a moderate neoantigen load of 50-150 predicted neoantigens. In comparison, tumor types with low neoantigen load (e.g. acute myeloid leukemia) and high neoantigen load (e.g. melanoma) exhibit 8 and 480 predicted neoantigens on average, respectively, consistent with their missense mutational burden. Positive correlates of neoantigen load in our studies included mutational burden (p<0.01), genome gain/loss, tumor ploidy, and BRCA1/2 loss of heterozygosity (p<0.05), all related markers of genomic instability. These relationships will be further validated in an independent sample set of germline BRCA1/2 mutation carriers from the TCGA, and expression levels of these predicted neoantigens will be investigated by RNASeq. Further, additional neoepitope prediction tools will be used to generate a consensus list of top-ranking candidate neoepitopes with high predicted immunogenic potential, which will be functionally evaluated for T-cell reactivity. Our work demonstrates that breast and ovarian cancer patients with BRCA1/2 mutations vary substantially in terms of predicted immunogenicity and suggests that a subset of such patients may be responsive to immunotherapy. Citation Format: Adam A. Kraya, Kara N. Maxwell, Brandon M. Wenz, Bradley Wubbenhorst, Daniel DeSloover, Nicole Lunceford, Jennifer D. Morrissette, Michael Feldman, Robert H. Vonderheide, Susan M. Domchek, Katherine L. Nathanson. Characterization of tumor neoantigen repertoire in patients with germline BRCA1/2 mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4511.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAGermline mutations in the tumor suppressors BRCA1 and BRCA2 lead to increased risks of breast and ovarian cancers at least in part due to their roles in homologous recombination based double stranded DNA repair. Genetic alterations including loss of the wild-type BRCA1/2 allele, PTEN loss and TP53 mutations are thought to contribute to genomic instability in susceptible tissues and therefore tumor formation. In order to further investigate the mechanisms of tumorigenesis in BRCA1/2 carriers, we performed whole exome sequencing of 39 breast and ovarian tumors and matched blood germline DNA. Sequencing data were analyzed using GATK and MuTECT for variant calls and ngCGH and Sequenza for copy number analysis. Using cellularity corrected tumor versus germline allele frequency calculations, we found no evidence of loss of heterozygosity (LOH) in 17% of BRCA1 tumors (n = 4 of 24) and 40% of BRCA2 tumors (n = 6 of 15). Genomic instability as measured by percentage of genome in the diploid state showed that BRCA1/2 tumors with no evidence of LOH had decreased genomic instability (average% genome diploid 76+27% vs 57+16%, p = 0.01) with no difference in the overall somatic mutation rate (average 50+71 vs 83+156 single nucleotide variants per megabase, p = NS). While there were also no significant differences in the average number of predicted deleterious somatic mutations in BRCA1/2 tumors with no LOH versus tumors with LOH (n = 13+15 vs 13+6, p = NS), tumors without LOH were significantly less likely to have mutations or loss in TP53 or loss of PTEN (50% versus 93%, p = 0.007). Of the ten tumors with no LOH, five were from patients who had received prior cytotoxic chemotherapy. Other tumors with no LOH were from patients with atypical presentations including a mixed histology epithelial ovarian tumor in a BRCA1 carrier, and a triple negative breast cancer in a 70 year old BRCA2 carrier. Of the 29 tumors with evidence of LOH, no patient had received cytotoxic chemotherapy for a prior malignancy or in the neoadjuvant setting. No recurrent driver mutations other than TP53 mutations were identified in the BRCA1/2 tumors with or without LOH. Twenty-six pathogenic likely driver mutations were identified in 25 cancer genes outside of TP53, including NRAS, PIK3CA, and ABL2. Our results indicate that approximately 75% of tumors in BRCA1/2 germline mutation carriers arise via a classic pathway involving LOH of the wildtype allele, loss of function of TP53 and/or PTEN and significant genomic instability. However, approximately 25% of tumors may arise via an alternative pathway, possibly related to prior chemotherapy in some cases. Further studies are needed to determine the molecular and clinical factors associated with this proposed classic versus atypical pathway of tumorigenesis in BRCA1/2 carriers and whether these characteristics are associated with outcomes such as survival and platinum and/or PARP inhibitor sensitivity.Citation Format: Kara N. Maxwell, Daniel De Sloover, Bradley Wubbenhorst, Brandon Wenz, Nicole Lunceford, Lyndsey Emery, Kurt Du0027Andrea, Robert D. Daber, Michael D. Feldman, Susan M. Domchek, Katherine L. Nathanson. Evidence for diverse mechanisms of tumorigenesis in breast and ovarian tumors of BRCA1/2 carriers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2990. doi:10.1158/1538-7445.AM2015-2990

Transgene stacking in trait development process through genetic engineering is becoming complex with increased number of desired traits and multiple modes of action for each trait. We demonstrate here a novel gene stacking strategy by combining bidirectional promoter (BDP) and bicistronic approaches to drive coordinated expression of multi-genes in corn. A unidirectional promoter, Ubiquitin-1 (ZMUbi1), from Zea mays was first converted into a synthetic BDP, such that a single promoter can direct the expression of two genes from each end of the promoter. The BDP system was then combined with a bicistronic organization of genes at both ends of the promoter by using a Thosea asigna virus 2A auto-cleaving domain. With this gene stacking configuration, we have successfully obtained expression in transgenic corn of four transgenes; three transgenes conferring insect (cry34Ab1 and cry35Ab1) and herbicide (aad1) resistance, and a phiyfp reporter gene using a single ZMUbi1 bidirectional promoter. Gene expression analyses of transgenic corn plants confirmed better coordinated expression of the four genes compared to constructs driving each gene by independent unidirectional ZmUbi1 promoter. To our knowledge, this is the first report that demonstrates application of a single promoter for co-regulation of multiple genes in a crop plant. This stacking technology would be useful for engineering metabolic pathways both for basic and applied research.

Pheochromocytomas and paragangliomas (PCC/PGL) are the solid tumour type most commonly associated with an inherited susceptibility syndrome. However, very little is known about the somatic genetic changes leading to tumorigenesis or malignant transformation. Here we perform whole-exome sequencing on a discovery set of 21 PCC/PGL and identify somatic ATRX mutations in two SDHB-associated tumours. Targeted sequencing of a separate validation set of 103 PCC/PGL identifies somatic ATRX mutations in 12.6% of PCC/PGL. PCC/PGL with somatic ATRX mutations are associated with alternative lengthening of telomeres and clinically aggressive behaviour. This finding suggests that loss of ATRX, an SWI/SNF chromatin remodelling protein, is important in the development of clinically aggressive PCC/PGL.

1510 Background: Individuals who carry one mutated copy of the BRCA1 or BRCA2 genes have elevated lifetime risks of breast and ovarian cancer. A number of studies have investigated the somatic mutational spectra of breast and ovarian tumors; however, BRCA1/2mutated tumors are underrepresented. Methods: Sixty-eight formalin-fixed paraffin embedded samples from BRCA1/2patients have been identified. Massively parallel sequencing using 48 gene capture is in process, whole exome sequencing of tumor and matched germline DNA is planned. Data are analyzed using a custom bioinformatics pipeline. Results: In analysis of data from the first 26 breast (4 BRCA1, 6 BRCA2) and ovarian (8 BRCA1, 8 BRCA2) tumors, the majority (23/26, 88%) had 0-2 variants in 48 cancer genes. Known deleterious TP53 mutations were the only variants identified in 2/4 BRCA1 and 2/6 BRCA2 breast tumors. Of those remaining, 2 BRCA1 and 1 BRCA2 breast tumors had no identified deleterious mutations. Two BRCA2 breast tumors with no TP53 mutations had known deleterious mutations in a single gene each - FGFR2 and PI3KCA. One BRCA2 breast tumor with no TP53 mutation had a variant of uncertain significance in FLT3. Finally, one BRCA2 breast tumor had a very high mutational rate, with one deleterious TP53 mutation and 7 other small deletion and single nucleotide variants. For the ovarian tumors, 15/16 BRCA1 and BRCA2 tumors had known deleterious TP53 mutations; the ovarian tumor with no TP53 mutation had no other variants. TP53 mutations were the sole identified mutations in 8 ovarian tumors. One ovarian tumor carried a known JAK3 activating mutation and 4 ovarian tumors carried one variant of uncertain significance in a single gene - SMO, PDGFRA, GNA11 and NRAS. Finally, two ovarian tumors were found to have high mutational rates. Conclusions: Using a targeted resequencing panel, we confirmed the high rate of TP53 mutations in BRCA1/2 breast tumors and observed a higher than expected rate in BRCA1/2 ovarian tumors. Importantly, we have identified mutations in other known driver genes using FFPE samples, allowing generalizability to other sites. These analyses may uncover novel mutations that could be exploited in the development of targeted therapeutic agents for BRCA1/2 carriers.

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola β-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species.

Chloroplast movement in response to changing light conditions optimizes photosynthetic light absorption [1]. This repositioning is stimulated by blue light perceived via the phototropin photoreceptors [2-4] and is transduced to the actin cytoskeleton [5]. Some actin-based motility systems use filament reorganizations rather than myosin-based translocations [6]. Recent research favors the hypothesis that chloroplast movement is driven by actin reorganization at the plasma membrane [7, 8], but no proteins affecting chloroplast movements have been shown to associate with both the plasma membrane and actin filaments in vivo. Here we identified THRUMIN1 as, a critical link between phototropin photoreceptor activity at the plasma membrane and actin-dependent chloroplast movements. THRUMIN1 bundles filamentous actin in vitro, and it localizes to the plasma membrane and displays light- and phototropin-dependent localization to microfilaments in vivo. These results suggest that phototropin-induced actin bundling via THRUMIN1 is important for chloroplast movement. A mammalian homolog of THRUMIN1, GRXCR1, has been implicated in auditory responses and hair cell stereocilia development as a regulator of actin architecture [9, 10]. Studies of THRUMIN1 will help elucidate the function of this family of eukaryotic proteins.