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A full list of mutations identified in SCC and MCT samples

Kaplan Meier overall survival curves by PIK3CA, CDKN2A and TP53 allelic mutation state

Genetics of recurrent PDX

Purpose: We sought to identify the genomic abnormalities in squamous cell carcinomas (SCC) arising in ovarian mature cystic teratoma (MCT), a rare gynecological malignancy of poor prognosis.Experimental design: We performed copy number, mutational state, and zygosity analysis of 151 genes in SCC arising in MCT (n = 25) using next-generation sequencing. The presence of high-/intermediate-risk HPV genotypes was assessed by quantitative PCR. Genomic events were correlated with clinical features and outcome.Results: MCT had a low mutation burden with a mean of only one mutation per case. Zygosity analyses of MCT indicated four separate patterns, suggesting that MCT can arise from errors at various stages of oogenesis. A total of 244 abnormalities were identified in 79 genes in MCT-associated SCC, and the overall mutational burden was high (mean 10.2 mutations per megabase). No SCC was positive for HPV. The most frequently altered genes in SCC were TP53 (20/25 cases, 80%), PIK3CA (13/25 cases, 52%), and CDKN2A (11/25 cases, 44%). Mutation in TP53 was associated with improved overall survival. In 8 of 20 cases with TP53 mutations, two or more variants were identified, which were bi-allelic.Conclusions: Ovarian SCC arising in MCT has a high mutational burden, with TP53 mutation the most common abnormality. The presence of TP53 mutation is a good prognostic factor. SCC arising in MCT share similar mutation profiles to other SCC. Given their rarity, they should be included in basket studies that recruit patients with SCC of other organs. Clin Cancer Res; 23(24); 7633–40. ©2017 AACR.

Supplementary Methods and legends for Supplementary tables and figures

Purpose:Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization.Experimental Design:PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models.Results:PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection.Conclusions:Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Figures S1-S8, Supplementary Methods, Supplementary Bibliography

Background & Aims Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. Methods We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient–derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. Results Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. Conclusions Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy., Graphical abstract

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3β) a key regulator of glycolysis. Pharmacological inhibition of GSK3β results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3β inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC.

Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. Experimental Design: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Background: Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer with complex genomes and dense fibrotic stroma. This study was designed to identify clinically relevant somatic aberrations in pancreatic cancer genomes of patients with primary and metastatic disease enrolled and treated in two clinical trials. Methods: Tumour nuclei were flow sorted prior to whole genome copy number variant (CNV) analysis. Targeted or whole exome sequencing was performed on most samples. We profiled biopsies from 68 patients enrolled in two Stand Up to Cancer (SU2C)-sponsored clinical trials. These included 38 resected chemoradiation naïve tumours (SU2C 20206-003) and metastases from 30 patients who progressed on prior therapies (SU2C 20206-001). Patient outcomes including progression-free survival (PFS) and overall survival (OS) were observed. Results: We defined: (a) CDKN2A homozygous deletions that included the adjacent MTAP gene, only its’ 3′ region, or excluded MTAP; (b) SMAD4 homozygous deletions that included ME2; (c) a pancreas-specific MYC super-enhancer region; (d) DNA repair-deficient genomes; and (e) copy number aberrations present in PDA patients with long-term (⩾ 40 months) and short-term (⩽ 12 months) survival after surgical resection. Conclusions: We provide a clinically relevant framework for genomic drivers of PDA and for advancing novel treatments.

Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting the DNA damage response (DDR) and replication stress. We show that patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum and PARP inhibitor therapy in vitro and in vivo. We generated a novel signature of replication stress with potential clinical utility in predicting response to ATR and WEE1 inhibitor treatment. Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy.Abstract FigureSTATEMENT OF SIGNIFICANCEWe define therapeutic strategies that target subgroups of PC using novel signatures of DNA damage response deficiency and replication stress. This potentially offers patients with DNA repair defects therapeutic options outside standard of care platinum chemotherapy and is being tested in clinical trials on the Precision-Panc platform.

Next-generation sequencing is enabling molecularly guided therapy for many cancer types, yet failure rates remain relatively high in pancreatic cancer (PC). The aim of this study is to investigate the feasibility of genomic profiling using endoscopic ultrasound (EUS) biopsy samples to facilitate personalised therapy for PC. Ninty-five patients underwent additional research biopsies at the time of diagnostic EUS. Diagnostic formalin-fixed (FFPE) and fresh frozen EUS samples underwent DNA extraction, quantification and targeted gene sequencing. Whole genome (WGS) and RNA sequencing was performed as proof of concept. Only 2 patients (2%) with a diagnosis of PC had insufficient material for targeted sequencing in both FFPE and frozen specimens. Targeted panel sequencing (n=54) revealed mutations in PC genes (KRAS, GNAS, TP53, CDKN2A, SMAD4) in patients with histological evidence of PC, including potentially actionable mutations (BRCA1, BRCA2, ATM, BRAF). WGS (n=5) of EUS samples revealed mutational signatures that are potential biomarkers of therapeutic responsiveness. RNA sequencing (n=35) segregated patients into clinically relevant molecular subtypes based on transcriptome. Integrated multi-omic analysis of PC using standard EUS guided biopsies offers clinical utility to guide personalized therapy and study the molecular pathology in all patients with PC.

The identification of molecularly defined subgroups of Pancreatic ductal Adenocarcinoma (PDAC) has the potential to transform clinical practice. There is now a growing consensus that PDAC can be divided into transcriptomic subtypes with 2 broad linages referred to as Classical (Pancreatic) and Squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal linage specification, HNF4A and GATA6, switch metabolic profiles from Classical (Pancreatic) to predominantly Squamous, with GSK3B a key regulator of glycolysis. Pharmacological inhibition of GSK3B results in selective sensitivity in the Squamous subtype, however a subset of these Squamous PDCLs acquired rapid drug tolerance. Using chromatin accessibility maps, we identify subtype specific chromatin landscapes and unique promoter usage between subpopulations of Squamous PDCLs can affect drug tolerance. Our findings demonstrate that a chromatin-based framework can be used to identify subtypes of PDAC that are indistinguishable by gene expression profiles which could refine patient selection for precision medicine.

Patient derived xenograft (PDX) models have shown great utility for pre-clinical and translational studies for a range of malignancies. We have established a comprehensive, publically available collection of 95 high-grade glioma flank PDX models. Viable PDX were derived from Glioblastoma, IDH-wildtype (n=91), Glioblastoma, IDH-mutant (n=2), Diffuse Midline Glioma, H3 K27M-mutant (n=1), and Anaplastic Oligodendroglioma (n=1) and include both primary (n=60) and recurrent (n=35) tumors. Comprehensive molecular characterization of PDX is ongoing and, to date, has included whole exome sequencing (WES, n=82), RNA-sequencing (n=40), and genome-wide methylation profiling (n=78) that included MGMT promoter, with data available in cBioPortal. PDX reflected the genetic characteristics of glioblastoma, with the majority harboring TERT promoter mutations, chromosomal gain +7, loss -10 and homozygous deletion of CKDN2A/B. EGFR alterations were frequent (~40%) and included amplification, point mutation, EGFRvIII, and other splice variants. Other common alterations, including amplifications of MET, CDK4, CDK6, MDM2, MDM4 and mutation of TP53, PTEN, NF1, RB1, PIK3CA, PIK3R1 were present at similar frequencies reported by TCGA, with the exception of PDGFRA alterations that were underrepresented in PDX. RNA-sequencing showed representation of each of the glioblastoma gene expression subtypes. To assess preservation of genetic features during xeongrafting, we performed WES on 20 matched patient tumors. The vast majority of genetic driver alterations were shared between patient and PDX, including EGFR, EGFRvIII, CDK4, MDM2, and MET amplifications. In 3 PDX, subclonal selection events were observed, including amplifications of MYCN, CDK6 and an EGFR splice variant, as well as selection against PDGFRA amplification. Overall, our large panel of PDX models reflects the genetic heterogeneity of glioblastoma and largely preserves the genetic features of the primary patient tumors. The PDX National Resource is a powerful tool for neuro-oncology research, with all PDX models and genomic data openly available (http://www.mayo.edu/research/labs/translational-neuro-oncology/mayo-clinic-brain-tumor-patient-derived-xenograft-national-resource).

Bromelain is a proteolytic enzyme from Bromeliaceae plants that can be found in pineapple (Ananas comosus (L.) Merryl). Bromelain has activity as a pharmaceutical and food. The aim of this study was to determine the bromelain content from stem bromelain extract (SBM). The stem of pineapple was extracted using a grater and then concentrated by a freeze dryer to obtain a pure concentrate. SDS-PAGE, Bradford assay and protease specific activity was used to analyze bromelain activity. The results of this study showed that bromelain from pineapple stem has a molecular weight between 19-22 kDa. Bradford assay showed 0.476 µg/mL bromelain concentration. Bromelain activity of Non freeze dried (NF) extract showed 0.317 U/mL, while freeze dried (F) extract showed 0.212 U/mL. This study concluded that the pineapple stem has bromelain activity.

Purpose: We sought to identify the genomic abnormalities in squamous cell carcinomas (SCC) arising in ovarian mature cystic teratoma (MCT), a rare gynecological malignancy of poor prognosis. Experimental design: We performed copy number, mutational state, and zygosity analysis of 151 genes in SCC arising in MCT (n = 25) using next-generation sequencing. The presence of high-/intermediate-risk HPV genotypes was assessed by quantitative PCR. Genomic events were correlated with clinical features and outcome. Results: MCT had a low mutation burden with a mean of only one mutation per case. Zygosity analyses of MCT indicated four separate patterns, suggesting that MCT can arise from errors at various stages of oogenesis. A total of 244 abnormalities were identified in 79 genes in MCT-associated SCC, and the overall mutational burden was high (mean 10.2 mutations per megabase). No SCC was positive for HPV. The most frequently altered genes in SCC were TP53 (20/25 cases, 80%), PIK3CA (13/25 cases, 52%), and CDKN2A (11/25 cases, 44%). Mutation in TP53 was associated with improved overall survival. In 8 of 20 cases with TP53 mutations, two or more variants were identified, which were bi-allelic. Conclusions: Ovarian SCC arising in MCT has a high mutational burden, with TP53 mutation the most common abnormality. The presence of TP53 mutation is a good prognostic factor. SCC arising in MCT share similar mutation profiles to other SCC. Given their rarity, they should be included in basket studies that recruit patients with SCC of other organs. Clin Cancer Res; 23(24); 7633–40. ©2017 AACR.

The aggressiveness of pancreatic cancer is associated with the acquisition of mesenchymal characteristics by a subset of pancreatic cancer cells. The factors driving the development of this subset are not well understood. In this study, we tested the hypothesis that acquisition of a mesenchymal phenotype occurs selectively in tumor cells that harbor specific enabling genetic alterations. We obtained whole-genome comparative genomic hybridization (CGH) measurements on pancreatic cancer cell lines that have either an epithelial-like (17 cell lines) or a mesenchymal-like (9 cell lines) phenotype in vitro. The total amounts of amplifications and deletions were equivalent between the epithelial and mesenchymal groups, but 20 genes showed a major difference between the groups in prevalence of alterations. All 20 alterations (18 deletions and 2 amplifications) were more prevalent in the mesenchymal group, confirming the advanced nature of this cellular subtype. CDKN2A was altered in more than 50% of both groups, but co-deletions in neighboring genes, and concomitant loss of gene expression, were more prevalent in the mesenchymal group, suggesting that the size of the loss around CDKN2A affects cell phenotype. Whole-genome CGH on 11 primary cancer tissues revealed that the 20 genes were altered at a higher prevalence (up to 55% of the cases for certain genes) than randomly selected sets of 20 genes, with the same direction of alteration as in the cell lines. These findings support the concept that specific genetic alterations enable phenotype plasticity and provide promising candidate genes for further research.

Background: Breast and other solid tumors exhibit high and varying degrees of intra-tumor heterogeneity that make the management and treatment of these diseases rather difficult. In order to study clinically relevant tumor heterogeneity in detail we performed an in-depth analysis of the clonal architecture of primary chemoradiation-naive breast cancers and their lymph node metastases. We combined DNA content-based flow cytometry and ploidy analysis with high-definition array comparative genomic hybridization (aCGH) and next-generation sequencing technologies to interrogate the genomes and transcriptomes of multiple biopsies from the tumors and involved lymph nodes. Thus rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying and defining the clonal populations and then interrogating their genomes provides a highly objective method of exploring the clinical significance of tumor heterogeneity in solid tumors. Material and methods: We used DAPI-based DNA content measurements and flow sorting to isolate nuclei from distinct clonal populations of diploid and aneuploid tumor cells in surgical tumor samples and distant lymph nodes from chemoradiation naive patients. In parallel, matching samples were processed for pathological, transcriptome, and IHC based assays. Each distinct sorted tumor cell population was then interrogated with aCGH and next generation sequencing. For each patient, the detailed profiles of ploidy, copy number aberrations and mutations were used to recreate and map the lineages present in the primary tumor and lymph. In one detailed case, we used 12 fresh frozen sections morphologically mapped from within a chemo-naïve primary invasive ductal carcinoma of histological grade 3 with lymph node involvement, 2-3 fresh frozen sections from 2 out of 5 lymph nodes with metastatic carcinoma, formalin-fixed paraffin-embedded (FFPE) tissue sections from 2 more of the involved lymph nodes and 5 morphologically mapped FFPE samples of normal tissue adjacent to the tumor. Results: We identified multiple coexisting aneuploid populations within biopsies from individual patients. Our clonal analysis determined the genomic aberrations arising in lineages that spread throughout the primary tumor and later bifurcated to distinct lymph nodes, revealing distinct therapeutic targets in the trunk and branches of the tumor. We identified driver events including high-level focal amplifications (e.g. SARC) and homozygous deletions (e.g. ROBO1 and ROBO2), that converge on distinct cellular signaling pathways. Conclusion: The approach highlighted here has broad implications in the study of tumor heterogeneity in breast and other solid tumors by providing a unique ultra-high resolution view of tumor heterogeneity that can advance more effective treatment and clinical management of patients with this disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-7.

Die Oppositionsparteien des deutschen Bundestags haben im Vorfeld der Bundestagswahlen 2013 steuerliche Reformkonzepte vorgelegt, die in erster Linie auf die Generierung zusätzlichen Steueraufkommens und die Umverteilung von Vermögen ausgerichtet sind. Im Vordergrund steht dabei die Wiederbelebung der Vermögensteuer beziehungsweise die Einführung einer einmaligen Vermögensabgabe. Allen Vorschlägen gemein ist zudem eine Erhöhung der Einkommensteuer durch eine Anhebung des Spitzensteuersatzes sowie Änderungen bei der Abgeltungsteuer. Die Umsetzung dieser Reformvorhaben würde spürbare Mehrbelastungen unternehmerischer Einkommen und Vermögen nach sich ziehen und damit die steuerliche Attraktivität Deutschlands als Unternehmensstandort sowie die Bedingungen für Investitionen in Deutschland verschlechtern. In the run-up to the 2013 election, the opposition parties of the German parliament have presented several tax reform proposals which first and foremost aim at increasing tax revenue and redistributing wealth. The focus is on reviving the wealth tax-which has not been levied since 1997-and on introducing a one-off capital levy, respectively. Furthermore, increasing the top income tax rate and withholding taxes on capital income is common to the reform programs of all three opposition parties. Implementing these reform proposals would result in a significant increase of the tax burden on business income and wealth, and thereby impair the conditions for investment in Germany and Germany's attractiveness as a location of investment.

This article traces Romania's development to a low-tax country among the member states of the European Union by analysing the major tax law changes in corporate taxation since 1992. We compute and compare the effective company tax burdens of a European model company at different points of time which constitute milestones in the development of corporate taxation in Romania. We find that the significant reduction of the corporate income tax rate from 45% in 1992 to 16% since 2005 has not been accompanied by a comprehensive broadening of the corporate income tax base as it has been in many longstanding member states of the EU and the OECD. Since 1992 the average company tax burden of the underlying model company has dropped by almost 65%. As a result, Romania holds second position among the group of Central and Eastern European EU member states.

So far, the field of cross-border taxation of dividends paid to individual investors in the European Union (EU) Member States has not been subject to harmonization. The differences in the Member States' tax systems give rise to obstacles to the internal market. As a consequence, individual investors may be discouraged from investing in shares of foreign corporations. Hence, the benefits of international risk diversification are not fully exploited. The discussion of possible solutions to these issues has recently received a new impetus as the European Commission held a consultation on the issues of cross-border dividend taxation in Spring 2011. In this article, we discuss the alternative solutions pointed out by the European Commission and present our genuine proposal. We show that dividends paid to individual investors should not be taxed at source by drawing on a set of normative criteria comprising conformity with European Law, efficiency, simplicity, and equity considerations. We furthermore argue that the abolition of withholding taxes should be accompanied by introducing automatic exchange of information. The analysis of alternative reform options is preceded by a brief, yet comprehensive summary of the taxation of dividends in the EU Member States highlighting the shortcomings of the current dividend taxation.

Translating molecular subtyping of glioblastoma (GBM) into therapeutically actionable guidance remains an unfulfilled opportunity. A central challenge is to discover therapeutic targets matched to agents (drugs or tool compounds) that are predictably present in subclasses of GBM. Transcriptional patterns in GBM cases in TCGA can identify 12 distinct molecular contexts (mC), where, across a subset of genes and tumors, conditional dependencies of gene expression (signal & echo relationships) consistently emerge. We deployed a panel of 64 patient-derived xenografts from GBM patients whose gene expression profiles allow mapping against the same 12 molecular contexts identified from TCGA cases. These clinically-relevant, preclinical models afford bioinformatics mining for actionable targets as well as chemical library screening for activity against short-term cultures derived from the PDX models. Utilizing a technique we term Chemical Biology Fingerprinting (CBF), we interrogated a series of GBM PDX models using a small chemical library (650 compounds) of clinically relevant anti-cancer agents to uncover context-specific chemovulnerabilities. Preliminary data demonstrated that, mC14 GBM (GBM59, SF7300, GBM116), characterized by mt-P53 and transcriptional similarity to GBM proneural subtype, show distinct vulnerability to Arsenic Trioxide (ATO) as compared to mC4 GBM (GBM91, GBM102), characterized by CHEK2 and NF1 mutations and transcriptional patterns similar to GBM mesenchymal subtype. To clinically-validate an ATO vulnerability signature, we acquired 22 treatment naive archival patient samples, who were part of Phase I/II clinical trial to study efficacy of ATO and Temozolomide (TMZ) in combination with radiation in treatment of high grade gliomas (NCT00275067) and which exhibited varied survival with ATO treatment (91 days to >1000 days), and determined their molecular context classification using whole transcriptome sequencing (RNAseq). In summary, we demonstrate a subclassification of GBM into novel contexts and we also show that these contexts are differentially sensitive to clinically relevant drugs. Supported by NIH U01CA168397.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

e15755 Background: Next-generation sequencing (NGS) has made genomic profiling to guide therapy a reality for many cancer types. The aim of this study is to investigate the feasibility of genomic profiling using standard clinical endoscopic ultrasound (EUS) core biopsy samples of Pancreatic Cancer (PC) to allow personalised cancer care. Methods:Patients undergoing EUS and biopsy for suspicion of PC underwent additional biopsies which was snap frozen. En-face frozen section enabled targeted macro-dissection prior to DNA extraction, quantification and targeted sequencing using a commercially available 151 gene ClearSeq Comprehensive Cancer Panel. Matching formalin-fixed (FFPE) diagnostic EUS biopsy and fresh frozen surgical resection specimens underwent genomic profiling for comparison. Whole genome sequencing (WGS) was performed in 2 patients. RNA sequencing was performed in samples with sufficient RNA yield. Results: Known PC genes ( KRAS, GNAS, TP53, CDKN2A, SMAD4) were identified in 27 out of 30 (90%) patients with histological diagnosis of PC. Potentially actionable somatic mutations (BRCA1, BRCA2, ATM, BRAF, JAK3) were found in 6 (20%) patients. In the 2 samples selected, WGS of the EUS samples confirmed point mutations identified on panel sequencing and revealed relevant mutational signatures and structural variation patterns. Targeted panel sequencing was successful in all FFPE samples. In 1 chemotherapy naïve patient, sequencing of a matching trio of fresh frozen and FFPE EUS biopsies, and resection sample revealed evidence of spatial intra-tumoral heterogeneity. In another patient, pre-treatment biopsy revealed a somatic BRCA1 mutation, and patient had a near complete pathological response to platinum containing neoadjuvant therapy in the resected specimen. RNA sequencing segregated patients into key clinically relevant molecular PC subtypes based on transcriptome as recently described. Conclusions: We demonstrate here novel multi-omic analysis of pancreatic cancer using standard clinical EUS guided fine needle biopsies. Multi-omic analysis of EUS biopsies offers potential clinical utility to guide personalized therapy of PC in the neoadjuvant and advanced settings.

Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2.

Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36 %, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1196-4) contains supplementary material, which is available to authorized users.

Tax planning with intangibles has become one of the most popular and most vividly debated topics in international taxation. We incorporate various intellectual property (IP) tax planning models into forward-looking measures of effective tax rates, namely the disposal of intangibles to low-tax subsidiaries, intra-group licensing arrangements, and intra-group contract R&D. In doing so, we draw upon the methodology put forward by Devereux and Griffith and amend this model by considering a research & development (R&D) investment which is carried out by a parent company, whereby the resulting intangible is exploited by a foreign subsidiary. We point out analytically under which conditions IP tax planning achieves the objective of reducing the effective average tax rate of the group. We find that the disposal of intangibles to low-tax subsidiaries does not achieve this tax planning objective, if the true value of the asset is subject to tax upon the disposal. We show to what extent the parent must understate the value of the intangible in order to reduce the group’s tax burden. We furthermore point out that contract R&D may generally achieve a significant lower effective tax burden. We present cost of capital and effect average tax rates to illustrate these findings.

Background: Breast tumors exhibit intratumor heterogeneity resulting in targeted therapy resistance and other challenges in disease management. To address the sources of heterogeneity, we performed a unique, in-depth analysis of clonal architecture in primary chemoradiation-naïve breast cancers. We combined DNA content-based flow cytometry and ploidy analysis with aCGH and next-generation sequencing (NGS) in multiple biopsies from the tumors and involved lymph nodes (LNs). Material and methods: We used DNA content-based flow sorting to isolate nuclei from distinct populations of diploid and aneuploid tumor cells in surgical tumor samples from two chemoradiation-naïve patients. Each sorted tumor cell population was interrogated with aCGH and exome NGS. In Patient #1, we used 12 fresh frozen sections morphologically mapped from within a HER2+, ER+, PR- primary invasive ductal carcinoma (IDC) of histological grade 3 with LN involvement and 2-3 sections from 2 out of 5 LNs. In Patient #2, 11 morphologically mapped fresh frozen sections were analyzed from a grade 2, ER+, PR+, HER2+, BRCA2 mutant LN- IDC. In parallel, matching samples were processed for IHC assays. Results: We identified multiple co-existing aneuploid populations within the biopsies. The 17 primary and LN biopsies from Patient #1 fell into 6 distinct ploidy groups albeit with aberrant but homogenous aCGH profiles, characterized by SARC amplification and homozygous deletions in ROBO1 and ROBO2. In contrast a dominant ploidy was identified throughout Patient #2 but with heterogeneous aCGH profiles. Mutation profiles obtained through exome sequencing further confirmed that ploidy was the main driver in Patient #1 whereas copy number aberrations played the key role in Patient #2 with the BRCA2 mutation (R3129X). A dendrogram based on exome variant calls of the aneuploid populations in Patient #1 strongly correlated with ploidy group and further revealed the specific clonal population characterized by a 5N ploidy and homozygous mutations in TP53 and PIK3CA as the progenitor to the ploidies present in the distant LNs. Strikngly, both patients had no HER2 amplification or mutation across their clonal populations, contradicting the initial IHC staining in a single core biopsy. Conclusions Rather than inferring the presence of distinct tumor cell populations, our novel flow-sorting based approach of first identifying the clonal populations and then interrogating their genomes, provides an objective method of exploring the sources and clinical significance of tumor heterogeneity. Our approach of clonal analysis has broad implications in the study of tumor heterogeneity and the identification of drivers in breast and other solid tumors that can advance more effective treatment and clinical management of patients with this disease. Citation Format: Princy Francis, Lucretia M Alvarez, Elizabeth Lenkiewicz, Mia Champion, Lisa Evers, Karen L Anderson, Ann E McCullough, Michael T Barrett, Barbara Pockaj. Deep clonal profiling identifies distinct mechanisms of heterogeneity and evolution in breast cancer [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 P2-03-07.

Background Pancreatic ductal adenocarcinoma (PDA) is a highly lethal cancer characterized by complex aberrant genomes. A fundamental goal of current studies is to identify those somatic events arising in the variable landscape of PDA genomes that can be exploited for improved clinical outcomes. Methods We used DNA content flow sorting to identify and purify tumor nuclei of PDA samples from 50 patients. The genome of each sorted sample was profiled by oligonucleotide comparative genomic hybridization and targeted resequencing of STAG2. Transposon insertions within STAG2 in a KRASG12D-driven genetically engineered mouse model of PDA were screened by RT-PCR. We then used a tissue microarray to survey STAG2 protein expression levels in 344 human PDA tumor samples and adjacent tissues. Univariate Kaplan Meier analysis and multivariate Cox Regression analysis were used to assess the association of STAG2 expression relative to overall survival and response to adjuvant therapy. Finally, RNAi-based assays with PDA cell lines were used to assess the potential therapeutic consequence of STAG2 expression in response to 18 therapeutic agents. Results STAG2 is targeted by somatic aberrations in a subset (4%) of human PDAs. Transposon-mediated disruption of STAG2 in a KRASG12D genetically engineered mouse model promotes the development of PDA and its progression to metastatic disease. There was a statistically significant loss of STAG2 protein expression in human tumor tissue (Wilcoxon-Rank test) with complete absence of STAG2 staining observed in 15 (4.3%) patients. In univariate Kaplan Meier analysis nearly complete STAG2 positive staining (>95% of nuclei positive) was associated with a median survival benefit of 6.41 months (P = 0.031). The survival benefit of adjuvant chemotherapy was only seen in patients with a STAG2 staining of less than 95% (median survival benefit 7.65 months; P = 0.028). Multivariate Cox Regression analysis showed that STAG2 is an independent prognostic factor for survival in pancreatic cancer patients. Finally, we show that RNAi-mediated knockdown of STAG2 selectively sensitizes human PDA cell lines to platinum-based therapy. Conclusions Based on these iterative findings we propose that STAG2 is a clinically significant tumor suppressor in PDA.

Sarcomas are cancers that arise in soft tissues or bone and make up a small percentage of malignancies. In an effort to identify potential genetic targets for therapy, this study explores the genomic landscape of a metastatic undifferentiated pleomorphic sarcoma (UPS) with spindle cell morphology. Thick sections (50 µm) of formalin-fixed, paraffin-embedded tissue from a primary, recurrent, and metastatic tumor were collected and processed from a single patient for DNA content-based flow-sorting and analyses. Nuclei of diploid and aneuploid populations were sorted from the malignant tissues and their genomes interrogated with array comparative genomic hybridization. The third sample was highly degraded and did not contain any intact ploidy peaks in our flow assays. A 2.5N aneuploid population was identified in the primary and recurrent sample. We detected a series of shared and unique genomic aberrations in the sorted aneuploid populations. The patterns of aberrations suggest that two similar but independent clonal populations arose during the clinical history of this rare tumor. None of these aberrations were detected in the matching sorted diploid samples. The targeted regions of interest might play a role in UPS and may lead to clinical significance with further investigation.

11 European countries now operate IP Box regimes that provide substantially reduced rates of corporate tax for income derived from important forms of intellectual property. We incorporate these policies into forward-looking measures of the cost of capital, effective marginal tax rates and effective average tax rates. We show that the treatment of expenses relating to IP income is particularly important in determining the effective tax burden. A key finding is that regimes that allow expenses to be deducted at the ordinary corporate income tax rate, as opposed to the IP Box tax rate, may result in negative effective average tax rates and can thereby provide a subsidy to unprofitable projects. We assess the specific design features of different regimes against the possible policy aim of improving the incentives to undertake R&D investment in a country. While some countries have tried to tie the policy to real activities, others have designed a policy targeted at the income streams associated with intellectual property. A key concern is the role that IP Boxes may play in increased, and possibly harmful, tax competition between European countries.

Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.

As part of our SU2C studies we are profiling the genomes of primary localized pancreatic ductal adenocarcinomas (PDAs) obtained from surgical samples collected at the University of Pennsylvania (SU2C-003). A fundamental challenge for the study of PDA genomes in clinical samples is the presence of varied admixtures of reactive stroma, inflammatory cells, and necrosis within the tumor. Furthermore, it is well-established that biopsies frequently contain multiple clonal populations of neoplastic cells that cannot be distinguished on the basis of morphology alone. In order to overcome the limitations of histopatholgy-based methods to study PDA genomes, we are using DNA content-based flow cytometry to identify and purify distinct clonal populations from each surgical sample. These assays are done while collecting up to 6 simultaneous sorting streams thus optimizing the use of each clinical sample. Each sorted diploid, aneuploid, and tetraploid population is interrogated with oligonucleotide CGH arrays. In addition, the epithelial and mesenchymal content of each tumor sample is assessed in flow assays with cytokeratin 19, vimentin, and ZEB1. Our unbiased approach provides clonal analysis of distinct populations in each patient’s tumor regardless of tumor cell content. We have identified a series of novel patient-specific selected aberrations, including homozygous deletions that target regulators of chromatin remodeling (SMYD3, ASXL2), and high-level focal amplicons that include mediators of oncogenic signaling pathways (FNTA, RAB3IP) and cellular metabolism (CTPS). The genes targeted by the somatic aberrations in each patient are overlaid onto a collection of 33 PDA-specific maps, containing 763 genes of interest, developed as part of the Metaminer Oncology initiative. These include signaling pathways of aberrant KRAS, DNA repair, apoptosis, epithelial mesenchymal transition (EMT), inflammation, and other mediators of PDA. We have accumulated clonal genomic data for 15 patients in the first 4 months of this study (December 2011- April 2012) and are currently profiling 3-5 new samples per month. These are providing a unique prospective data set to test hypothesis of the genomic basis of clinical outcomes in patients with localized resectable PDA. This proffered talk is also presented as Poster B19. Citation Format: Michael T. Barrett, Elizabeth Lenkiewicz, Lisa Evers, Tara Holley, Meraj Aziz, Edward Stites, Richard Posner, Amy Kramer, Jeffrey Drebin, Daniel D. Von Hoff. Clonal profiling of prospectively collected primary pancreatic ductal adenocarcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr PR7.

We trace back Romania’s development to a low-tax country among the Member States of the European Union by analysing the major tax law changes in corporate taxation since 1992. We find that the significant reduction of the corporate income tax rate from 45% in 1992 to 16% since 2005 has not been accompanied by a comprehensive broadening of the corporate income tax base as prevalent in many longstanding Member States of the EU and the OECD. Our analysis is not limited to a comprehensive description of the development of corporate taxation in Romania, but goes on with a numerical analysis of the tax burdens at different periods of time which constitute milestones in the development of corporate taxation in Romania. For this purpose, we apply the European Tax Analyzer, which is a computer-based model firm approach. We find that the average company tax burden of the underlying model company has dropped significantly by almost 65% since 1992. Furthermore, our numerical analysis does not confirm the tax base broadening policy. As a result, Romania holds position two among the group of Central and Eastern European EU Member States.

Cancers frequently arise as a result of an acquired genomic instability and the subsequent clonal evolution of neoplastic cells with variable patterns of genetic aberrations. Thus, the presence and behaviors of distinct clonal populations in each patient's tumor may underlie multiple clinical phenotypes in cancers. We applied DNA content-based flow sorting to identify and isolate the nuclei of clonal populations from tumor biopsies, which was coupled with array CGH and targeted resequencing. The results produced high-definition genomic profiles of clonal populations from 40 pancreatic adenocarcinomas and a set of prostate adenocarcinomas, including serial biopsies from a patient who progressed to androgen-independent metastatic disease. The genomes of clonal populations were found to have patient-specific aberrations of clinical relevance. Furthermore, we identified genomic aberrations specific to therapeutically responsive and resistant clones arising during the evolution of androgen-independent metastatic prostate adenocarcinoma. We also distinguished divergent clonal populations within single biopsies and mapped aberrations in multiple aneuploid populations arising in primary and metastatic pancreatic adenocarcinoma. We propose that our high-definition analyses of the genomes of distinct clonal populations of cancer cells in patients in vivo can help guide diagnoses and tailor approaches to personalized treatment.

BACKGROUND/PURPOSE The Mayo Clinic has developed a large panel of patient-derived xenografts (PDX) from patients with glioblastoma multiforme (GBM). Here we report initial molecular profiling of the Mayo PDX panel and a comparison of patient and PDX molecular profile and response to therapy. METHODS & MATERIALS Clinical data was retrieved by retrospective chart review. Animal data from all PDX investigations conducted between 2004-2014 were retrieved from experimental logs and subsequently consolidated into a database for analysis. RESULTS From 1999 to 2014, 182 patient tumor samples of varying histological grades were attempted for xenograft with 73 resulting viable PDX lines. Viable xenografts were only produced from WHO grade IV tumor specimens, yielding an overall success rate of 49% for these tumors. GBM patients that produced viable xenografts compared to those that did not exhibited a trend for decreased overall survival (P = 0.18). There was no significant association between successful xenografting and whether tissue was from newly diagnosed (45/93%) or recurrent (20/40; 50%) tumors. Patient age > 45 at diagnosis was correlated with increased PDX viability in GBMs (p = 0.05). Of the viable PDX models analyzed, EGFR mutation was identified in 17 lines, TERT mutation was found in 13 lines, IDH mutation in 1 line, and MGMT hypermethylation in 25 lines. RNAseq was performed on orthotopic tumor samples from 53 PDX models. After excluding contaminating murine sequence reads, expression analysis demonstrated 32 models with a mesenchymal phenotype. Array comparative genomic hybridization was performed on 9 patient samples and derivative early, mid and late passage PDX tumors. Using unbiased hierarchical clustering, there was a high concordance between patient and xenograft models. Within the PDX panel, fractionated radiation (RT) alone and RT combined with temozolomide (TMZ) was tested in orthotopic tumors in 38 lines. The overall median survival benefit (ratio of median survival for treated vs. placebo) in PDX lines treated with RT only was 1.6 (range: 0.9-2.5) and with RT/TMZ was 2.5 (range: 1.1 - 8.9). There was a positive association between observed patient survival and the corresponding survival benefit in the PDX for subjects treated with RT/TMZ (r = 0.2; n = 17). Response to adjuvant TMZ was evaluated in 42 tumor lines, and response to bevacizumab was tested in 33 tumor lines, and correlations with clinical treatment response are being evaluated. CONCLUSIONS: The Mayo GBM PDX panel is widely used in the neuro-oncology community. The initial molecular analysis suggests a good correlation between patient and PDX at the level of genotypic characteristics and therapeutic sensitivity. Citation Format: Dioval A. Remonde, Brett L. Carlson, Mark A. Schroeder, Brock Armstrong, Sen Peng, Lisa Evers, Paul A. Decker, Jeanette Eckel Passow, Michael E. Berens, Nhan L. Tran, Robert B. Jenkins, Jann N. Sarkaria. Characterization of patient-derived xenograft (PDX) models to evaluate clinical and therapeutic responses of glioblastoma multiforme. [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 1468. doi:10.1158/1538-7445.AM2015-1468

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Glioblastoma Multiforme (GBM) is an aggressive brain tumor with very poor prognosis and extremely limited therapeutic options. GBM is the most common malignant brain tumor and the search for novel targets and/ or the repurposing of already extant drugs to treat the disease is therefore of utmost importance. We describe here a comprehensive multidisciplinary approach to identifying said targets and ergo potential therapies. We have applied a novel analytical strategy to The Cancer Genome Atlas (TCGA) GBM expression data to stratify GBM into novel subtypes we call molecular contexts, or mCs. Subsequently, a panel of patient-derived GBM xenografts was ascribed to our novel mCs. Utilizing a technique we term Chemical Biology Fingerprinting, or CBF, short-term cultures derived from these clinically-relevant preclinical models were screened for chemosensitivity with a deeply annotated, yet clinically relevant, chemical library. Agents that were statistically more toxic to one context than another were then re-tested in true drug dose response experiments to confirm sensitivity. Preliminary data demonstrated that mC14, characterized by mutant p53 and transcriptionally similar to the GBM proneural subtype, showed distinct vulnerability to Arsenic Trioxide (ATO) as compared to mC4, enriched for NF1 mutations and with transcriptional patterns similar to the GBM mesenchymal subtype. To validate the ATO vulnerability signature in GBM, we acquired 20 treatment naive archival patient samples, that were part of a Phase I/II clinical trial to study the efficacy of ATO and Temozolomide in combination with ionizing radiation ([NCT00275067][1]). Participants in the trial exhibited varied survival with ATO treatment (91 days to >1000 days) and the clinical samples were subtyped into our molecular contexts using RNAseq data. In summary, we demonstrate a subclassification of GBM into novel molecular contexts (mCs) and show that these contexts are differentially sensitive to clinically relevant drugs. Citation Format: Darren Finlay, Harshil Dhruv, Lisa Evers, Sen Peng, Jeff Kiefer, Seungchan Kim, Jeffrey Raizer, Michael Berens, Kristiina Vuori. Identification of novel drugs for glioblastoma using chemical biology fingerprinting. [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 4671. doi:10.1158/1538-7445.AM2015-4671 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00275067&atom=%2Fcanres%2F75%2F15_Supplement%2F4671.atom

Pancreatic cancer (PC) is a heterogeneous disease. Several mutations have been reported to develop along the path of PC evolution, and it is important to identify targets for potential therapy. CGH and IHC analysis of pancreatic tumor samples may reveal prognostic and predictive markers. Methods: We evaluated tumor samples of patients (n=35) who were accrued to a phase II molecular profiling study (Ramanathan RK et al.12-LB-8953-AACR 2012). All subjects, with metastatic PC, with progression after 1st line of therapy had a core biopsy for CGH, IHC, and microarray analysis. Samples interrogated by CGH were flow sorted prior to analysis. Treatment recommendations were based on the IHC panel (Target Now®, Caris Life Sciences, Irving, TX). Results: In 30 patients, adequate tumor was available for CGH. We identified 9 long term survivors, living > 18 months from first diagnosis of mPC. Of the 9 patients, 5 had available CGH data. (1) had two distinct aneuploid tumor cell populations. There was evidence suggestive of increased WNT signaling based on amplicon targeting FZD3;. IHC identified TS and SPARC as potential targets. Recommended treatment: Capecitabine and Nab-Paclitaxel (2) had a focal amplicon targeting WNT6 and WNT10A suggestive of increased WNT signaling, and homozygous deletions targeting RB1 and PARD3B. An intragenic break within NOTCH2 was also observed. IHC identified TS and TOPO1 as potential targets. Recommended treatment: FOLFIRI (3) was identified (and confirmed to be) KRAS wild-type, but had a homozygous deletion of RASA1, the gene that codes for p120-RasGAP a known negative regulator of KRAS signaling. Commonly seen deletions of CDKN2A and SMAD4 were not observed. IHC identified TS and ERCC1 as potential targets. Recommended treatment: FOLFOX (4) had an amplification of a PARP gene (PARP8) and of a base excision enzyme (NEIL2), chromosomal copy number changes to genes involved in chromatin regulation (LCMT, EZH2, KLF13) and EMT with dedifferentiation (ZNF703). IHC identified MGMT as a potential target. Recommended treatment: Temazolamide. (5) had amplifications of PIK3CA and of KRAS, and a homozygous deletion of CDKN2A. IHC identified TOPO2 as a potential target. Recommended treatment: Doxorubicin Conclusion: Due to the heterogeneity of the genome, even in a select group with long term survival, each patient may require an individualized treatment plan. Pathway analysis of all patients treated in the study is ongoing. Treatment strategies to focus on the stroma and of methylation of the genome are being explored to circumvent the genetic heterogeneity. (Supported by a SU2C pancreatic dream team grant) Citation Format: Safi Shahda, Michael T. Barrett, Elizabeth Lenkiewicz, Lisa Evers, Richard G. Posner, Meraj Aziz, Glen J. Weiss, Gayle Jameson, Daniel D. Von Hoff, Ramesh K. Ramanathan. Analysis of the pancreatic cancer (PC) genome by immunohistochemistry (IHC) and comparative genome hybridization (CGH) in patients with long term survival with metastatic disease (mPC) treated on a phase II molecular profiling trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1911. doi:10.1158/1538-7445.AM2013-1911

It is widely accepted that many cancers arise as a result of an acquired genomic instability and the subsequent evolution of tumor cells with variable patterns of selected and background aberrations. The presence and behaviors of distinct neoplastic cell populations within a patient's tumor may underlie multiple clinical phenotypes in cancers. A goal of many current cancer genome studies is the identification of recurring selected driver events that can be advanced for the development of personalized therapies. Unfortunately, in the majority of rare tumors, this type of analysis can be particularly challenging. Large series of specimens for analysis are simply not available, allowing recurring patterns to remain hidden. In this paper, we highlight the use of DNA content-based flow sorting to identify and isolate DNA-diploid and DNA-aneuploid populations from tumor biopsies as a strategy to comprehensively study the genomic composition and behaviors of individual cancers in a series of rare solid tumors: intrahepatic cholangiocarcinoma, anal carcinoma, adrenal leiomyosarcoma, and pancreatic neuroendocrine tumors. We propose that the identification of highly selected genomic events in distinct tumor populations within each tumor can identify candidate driver events that can facilitate the development of novel, personalized treatment strategies for patients with cancer.

We have completed a second line Phase II study of therapy to be selected by molecular profiling in 35 patients with previously treated metastatic pancreatic cancer. On this study all patients have a biopsy of a metastatic lesion (usually liver). Therapeutic targets are identified by profiling the metastatic biopsies with the combined use of immunohistochemistry (IHC), comparative genomic hybridization (CGH) on sorted tumor populations, and correlating gene expression microarray data to that of a panel of xenografts for which drug sensitivity has been previously determined. The IHC assays are performed in a CLIA certified laboratory and include the following therapeutic targets: MRP1, TOPO1, MGMT, PGP, Her2/Neu, PTEN, c-kit, RRM1, EGFR, BCRP, PDGFR, TOP2A, TS, ERCC1, SPARC, ER, PR, and AR. In addition each sample is screened by PCR for the presence of the most common K-RAS mutations. Gene expression microarray analysis is performed at John Hopkins University using Affymetrix U133 Plus 2.0 gene arrays. For CGH analysis flow sorted nuclei of diploid and aneuploid tumor cell populations are processed and hybridized to 400,000 feature CGH arrays. We have identified distinct high level focal amplicons targeting AKT2, LYN, RET, CDK6, K-RAS, MYCBP, BCL11A, RASA1 and WNT6, and gene specific homozygous deletions including CDKN2A, PTEN, MAP2K4, RASA1 and PARK2 in the sorted aneuploid tumor nuclei. In an effort to elucidate new targets and potential contexts of vulnerability based on the results of this trial, we have extracted combined gene level data for each patient and present these mapped to 32 pancreatic specific pathways and processes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3697. doi:1538-7445.AM2012-3697

Breast cancer is known to be heterogeneous and choices of targeted therapy are still limited for refractory estrogen receptor positive (ER+), refractory HER2+, and triple negative (TNBC) breast cancer. In order to identify novel drug targets that are either focally amplified or involved in fusion or translocation events in such disease segments, we applied high resolution DNA copy number profiling and whole genome sequencing to a set of DNA content sorted breast cancer samples. We first applied DNA-content based flow cytometry to purify diploid and aneuploid cells in each of 54 samples. Purified populations of both aneuploid and diploid cells were then subjected to high resolution aCGH profiling. Our initial analysis of high resolution copy number profiling data identified a list of genes focally amplified and harboring break points. We then selected 8 of these samples (four ER+ and four triple negative) for whole genome sequencing to identify their fusion partners. Paired-end sequencing data from HiSeq for each flow sorted sample was aligned against the human genome. A custom analysis workflow based on published software tools and custom developed scripts for computationally predicting structural variation events was implemented. Paired ends that mapped to discontinuous regions in the genome were merged with our aCGH data to identify candidate gene fusions, inversions, and translocations in each sample. Automatic scan and custom examination for pair-ends mapped to discontinuous genomic regions identified a list of novel gene fusion and translocation candidates. A subset of such fusion candidates showed precise break point position concordance across the NGS and aCGH data and equal copy number amplification of two fusion partners, further strengthening our confidence that such genes are indeed involved in fusion and translocation events. These candidates include transcription factors (e.g. ZNF492), kinases (e.g. DYRK1A), and phosphatases (e.g. PTPRG). Some of these fusion genes are known to be involved in fusion in other diseases or tumor types but have not been reported in breast cancer, such as AUTS2, which is reported as translocation partner in autism and mental retardation patients and B-cell ALL, and MECOM (aka EVI1/MDS1), which is fused with AML1 in AML patients. Others are completely novel such as BICD1-ZNF492. In conclusion, we have successfully identified a list of fusions genes in ER+ refractory and triple negative breast cancer patients by combining high resolution aCGH profiling of DNA copy number analysis and whole genome sequencing. The next step is to validate the expression of these fusion proteins and their functional relevance to breast cancer. The driver fusion genes identified can provide potential therapeutic targets for ER+ refractory and TN breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 969. doi:1538-7445.AM2012-969

One of the challenges for the application of emerging genome technologies to the study of human cancer in patients in vivo is the presence of admixtures of normal cells and stroma in patient samples. Furthermore, tumor samples frequently contain multiple neoplastic populations even in small clinical biopsies. Consequently, it is difficult to accurately detect the multiple genomic aberrations typically present in a biopsy of interest and to distinguish whether they arise concurrently or are present in distinct cell populations in the same sample. Recent advances in flow cytometry technology provide high throughput flow rates and the detection of relatively rare events in dilute samples. Coupling this technology with robust methods to label cells and advanced data acquisition and analyses methods enables the application of flow cytometry to high definition cancer genomics in patients. In our current studies of pancreatic ductal adenocarcinoma (PDA) we developed methods that enable whole genome analyses of clonal populations of sorted aneuploid and diploid nuclei from patient biopsies with as little as 1–5% tumor cell content. Our aCGH studies using Agilent 400k oligonucleotide arrays and flow sorted samples identified 19q13.2 as a region that is recurrently targeted by gene amplification in PDA genomes. Significantly, we identified this amplicon in two distinct aneuploid populations representing 19.8% and 17.4% of the total cellular fraction of a PDA biopsy. We mapped the amplicon to an interval spanning from 43,526,781-cen to 46,171,091-tel, a region targeting 2,644,310 bases in each of the aneuploid populations. This region of interest is relatively gene rich and contains a series of cancer associated genes of interest (e.g. PAK4, AKT2, HIPK4, MAP3K10, RAB4B) to PDA. In addition, our data suggests this region contains variable internal regions of copy number changes suggesting structural rearrangements within the amplicon and its immediate boundaries. To further study this clonally selected region we used the Agilent Sure Select Target Enrichment System to design capture oligonucleotides to sample the 19q13.2 amplicon and its boundaries in the aneuploid PDA genome. These sequences were then interrogated with the PacBio RS system. The integration of these data provides a high resolution clonal analysis of genomic aberrations including point mutations, inversions and insertions that are present in the 19q13.2 amplicon, its immediate boundaries, and the genomes in which it arose. These data provide a highly unique and valuable proof of concept for the application of our integrated clonal genomics methods to the advancement of personalized medicine for patients with PDA and other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-266. doi:10.1158/1538-7445.AM2011-LB-266