Necela, Brian M.

Abstract Background: Understanding heterogeneity within individual breast tumors is key to the ability to predict therapeutic outcome. Molecular heterogeneity is commonly evaluated based on genomic features, including mRNA abundance, gene copy number events, and somatic mutations. The expression profile and activation state of key proteins is widely recognized as another key element in defining tumor heterogeneity. We have taken advantage of NanoString 3D Biology™ technology (for research use only) and curated nCounter Vantage 3DTM Solid Tumor Assay to interrogate a survey panel of HER2-positive breast tumors with the ultimate goal of determining key relationships between multiple genomic and proteomic profiles in individual tumors. Methods: We analyzed samples from 24 HER2+ breast cancer patients using NanoString technology to quantify the expression profile for over 25 total and phospho signaling proteins, including PI3K/MAPK/EGFR/HER2, 770 mRNA corresponding to 13 canonical cancer pathways, and 104 somatic mutations and small INDELS that are commonly associated with cancer, including 8 known PIK3CA mutations. These analyses were carried out in a matched fresh frozen and FFPE samples on the nCounter paltform. Data were analyzed by nSolver to identify genotype specific expression profiles across the 24 samples. Results: In our proof-of-concept data set, we successfully demonstrate that NanoString’s 3D biology Technology shows concordance across both FFPE and fresh frozen sample types for DNA, RNA, and protein. NanoString analysis also showed high concordance to gold-standard techniques used to assess genotype and RNA expression profiles. The combination of digital DNA, RNA, and protein data from our HER2+ breast cancer samples yielded potentially actionable data based on mapping of mutational status as the driver of key differences in protein expression and mRNA abundance of the signaling targets profiled. This work sheds new light on HER2+ breast cancer biology and the interplay between genomic and proteomic profiles while setting the stage for future studies that further probe the differences observed in this sample set. Conclusions: Simultaneous analysis of mutational status (SNV) and expression at the level of both mRNA and protein promises to enable a more detailed view of the relationship between genotype and the biological and clinical behavior of key tumor types. The NanoString Vantage 3DTM Solid Tumor platform provides a rapid, reliable, and economic means of assessing these analytes simultaneously. The application of these analytes to models that make clinically actionable predictions will require additional analyses of large sample cohorts, but such analysis is quite feasible using a variety of sample types. Acknowledgements: Supported in part by grants from the Breast Cancer Research Foundation and the 26.2 with Donna Foundation. Citation Format: Sarayna Chumsri, Daniel J. Serie, Brian M. Necela, Jennifer M. Kachergus, Bianca C. Axenfeld, Gokhan Demirkan, Gavin Meredith, P. Martin Ross, Anisha Kharkia, Erin Piazza, Afshin Mashadi-Hossein, Sarah Warren, Sarah A. McLaughlin, Joseph Beechem, Gary Geiss, E. Aubrey Thompson. Simultaneous analysis of the mutational landscape and RNA and protein expression profile of HER2-positive breast cancer using 3D BiologyTM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3377. doi:10.1158/1538-7445.AM2017-3377

Doxorubicin and the ERBB2 targeted therapy, trastuzumab, are routinely used in the treatment of HER2+ breast cancer. In mouse models, doxorubicin is known to cause cardiomyopathy and conditional cardiac knock out of Erbb2 results in dilated cardiomyopathy and increased sensitivity to doxorubicin-induced cell death. In humans, these drugs also result in cardiac phenotypes, but severity and reversibility is highly variable. We examined the association of decline in left ventricular ejection fraction (LVEF) at 15,204 single nucleotide polymorphisms (SNPs) spanning 72 cardiomyopathy genes, in 800 breast cancer patients who received doxorubicin and trastuzumab. For 7033 common SNPs (minor allele frequency (MAF) > 0.01) we performed single marker linear regression. For all SNPs, we performed gene-based testing with SNP-set (Sequence) Kernel Association Tests: SKAT, SKAT-O and SKAT-common/rare under rare variant non-burden; rare variant optimized burden and non-burden tests; and a combination of rare and common variants respectively. Single marker analyses identified seven missense variants in OBSCN (p = 0.0045–0.0009, MAF = 0.18–0.50) and two in TTN (both p = 0.04, MAF = 0.22). Gene-based rare variant analyses, SKAT and SKAT-O, performed very similarly (ILK, TCAP, DSC2, VCL, FXN, DSP and KCNQ1, p = 0.042–0.006). Gene-based tests of rare/common variants were significant at the nominal 5% level for OBSCN as well as TCAP, DSC2, VCL, NEXN, KCNJ2 and DMD (p = 0.044–0.008). Our results suggest that rare and common variants in OBSCN, as well as in other genes, could have modifying effects in cardiomyopathy.

AbstractBackgroundThe targeted ERBB2 therapy, trastuzumab, has had a tremendous impact on management of patients with HER2+ breast cancer, leading to development and increased use of further HER2 targeted therapies. The major clinical side effect is cardiotoxicity but the mechanism is largely unknown. On the basis that gene expression is known to be altered in multiple models of heart failure, we examined differential gene expression of iPSC‐derived cardiomyocytes treated at day 11 with the ERBB2 targeted monoclonal antibody, trastuzumab for 48 h and the small molecule tyrosine kinase inhibitor of EGFR and ERBB2.ResultsTranscriptome sequencing was performed on four replicates from each group (48 h untreated, 48 h trastuzumab and 48 h lapatinib) and differential gene expression analyses were performed on each treatment group relative to untreated cardiomyocytes. 517 and 1358 genes were differentially expressed, p < 0.05, respectively in cardiomyocytes treated with trastuzumab and lapatinib. Gene ontology analyses revealed in cardiomyocytes treated with trastuzumab, significant down‐regulation of genes involved in small molecule metabolism (p = 3.22 × 10−9) and cholesterol (p = 0.01) and sterol (p = 0.03) processing. We next measured glucose uptake and lactate production in iPSC‐derived cardiomyocytes 13 days post‐plating, treated with trastuzumab up to 96 h. We observed significantly decreased glucose uptake from the media of iPSC‐derived cardiomyocytes treated with trastuzumab as early as 24 h (p = 0.001) and consistently up to 96 h (p = 0.03).ConclusionsOur study suggests dysregulation of cardiac gene expression and metabolism as key elements of ERBB2 signaling that could potentially be early biomarkers of cardiotoxicity.

Abstract Background: Extranodal follicular dendritic cell sarcoma (FDCS) is an exceedingly rare tumor type that is limited to few cases in the literature. Given the rarity of this tumor, there is a lack of genetic understanding of this disease resulting in a non-individualized plan of care for patients with this disease. Herein, we are the first to describe extensively molecularly the DNA and RNA of this tumor. Methods: Under an IRB approved protocol, the patient's tumor and whole blood was freshly frozen for molecular analysis. We performed Next Generation Sequencing (NGS) genomic profiling including exome capture, mate pair and mRNA sequencing on this tumor. Exome and mRNA data were processed through Mayo's Bioinformatics Core standard pipelines, GENOME GPS 1.2.1 and MAPRSeq v1.2.1. Mutations and indels were prioritized depending on whether or not they belonged to known cancer associated genes. Copy number variations (CNVs) in Exome and Mate Pair data was found through PatternCNV v 1.0.1 and fusion transcripts were found using SNOWSHOES-FTD and Tophat-Fusion 2.0.6. Results: At the genomic level large scale genomic rearrangements were detected, including chr19 and chr20 trisomy, chr15q and chr17q segmental duplications result in two intra-chromosomal fusion genes, and translocations leading to four inter-chromosomal fusion genes between chr15 and chr17. Of particular interest is a stop codon gain in the CLTC1 (chr22) gene, which is involved in the mitotic spindle organization which has been associated with meningiomas and breast tumorigenesis. The new stop codon is before the site required for the recruitment of the proteins to the spindle. The large scale genomic rearrangement described above is consistent with this defective spindle assembly hypothesis. Another intriguing mutation detected in the VEGFR1 (chr13) gene leads to a 45% mis-spliced transcript. VEGFR1 is thought to modulate VEGFR2 which plays an essential role in angiogenesis, by acting as a decoy receptor. Therefore the loss of function of VEGFR1 might lead to hyper-activity of VEGFR2 pathway and a potential therapeutic strategy is to directly inhibit the VEGFR2 pathway. Furthermore missense mutations in the ATM (chr11) and TP53 (chr17) genes were found. PARP inhibitors have been used with success for cancer with defects in the ATM gene. Finally a novel fusion transcript of HDGRFP3 (chr15) and SHC4 (chr15) was identified. Such fusion results in an overexpressed SHC4 gene which has been associated with metastatic melanomas. Conclusions: In summary, genomic profiling of a rare thyroid FDCS identified drug targetable genes as well as changes related to tumor etiology. Future work is necessary to determine the functional significance of these molecular findings and whether they will predict benefit to targeted therapies. Citation Format: Jaime I. Davila, Jason Starr, Steven Attia, Chen Wang, Xue Wang, Brian Necela, Casler John, David Menke, Vivek Sarangi, Gavin Oliver, Richard Joseph, John Copland, Alexander Parker, E. Aubrey Thompson, Robert Smallridge, Yan W. Asmann. Extensive genomic profiling of a rare extranodal-follicular dendritic cell sarcoma: Implications for future individualized therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1894. doi:10.1158/1538-7445.AM2014-1894

Abstract Objectives and Rationale: Structural variants (SV) including large copy number aberrations (CNV), translocations, inversions, and large insertions and deletions (INDEL) play a critical role in tumorigenesis and progression. In fact, we now know that tumors can be categorized according to the size of mutations harbored. In several cancers, including ovarian and breast cancer, the large structural mutations, rather than single site mutations, play a dominate role in tumor etiology. Therefore, it's critical to implement reliable algorithm for the detection of structural variants in DNA sequencing data. Mate-pair sequencing is a protocol specifically implemented for detection of the whole-genome level structural variants. It requires less sequencing depth therefor is cost effective, and enables the detection of CNVs, translocations, and inversions simultaneously. However, so far there has been no reliable bioinformatics pipeline for the analyses of the mate-pair sequencing data. Methods: Our novel algorithm, the SnowShoes-SV, is an exhaustive search algorithm designed specifically for mate-pair DNA sequencing data analyses. It calls the SVs based on disconcordant read pairs. The false SVs are filtered according to the following criteria: (i) the number of the supporting read pairs; (ii) the lack of reads from control data that implicate SV at the same region; (iii) the mappability and uniqueness of the region based on data from the ENCODE project; (iv) consistencies of the mapping orientations of the supporting read pairs; (v) the similar sizes between sequencing library and the two end read clusters. Results: Using a set of samples previously genotyped by aCGH, the SnowShoes-SV successfully detected all known CNVs and other SVs. It also identified copy number neutral translocations and inversions previously not identified by aCGH. In addition, the algorithm nominated novel SVs which are to be validated by PCR. Conclusions: SnowShoes-SV is a highly sensitive and specific algorithm for SV detection from the mate-pair DNA sequencing data. Citation Format: Yan W Asmann, Chen Wang, Brian M Necela, Xianfeng Chen, Jean-Pierre A Kocher, Matthew J Maurer, Thomas M Habermann, Susan L Slager, Andrew L Feldman, Anne J Novak, James R Cerhan, Edith A Perez, E Aubrey Thompson. An exhaustive algorithm for detecting copy number aberrations and large structural variants in whole-genome mate-pair sequencing data [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 P6-07-03.

Context and Objective:Oncocytic thyroid carcinoma, also known as Hürthle cell thyroid carcinoma, accounts for only a small percentage of all thyroid cancers. However, this malignancy often presents at an advanced stage and poses unique challenges to patients and clinicians. Surgical resection of the tumor accompanied in some cases by radioactive iodine treatment, radiation, and chemotherapy are the established modes of therapy. Knowledge of the perturbed oncogenic pathways can provide better understanding of the mechanism of disease and thus opportunities for more effective clinical management.Design and Patients:Initially, two oncocytic thyroid carcinomas and their matched normal tissues were profiled using whole genome sequencing. Subsequently, 72 oncocytic thyroid carcinomas, one cell line, and five Hürthle cell adenomas were examined by targeted sequencing for the presence of mutations in the multiple endocrine neoplasia I (MEN1) gene.Results:Here we report the identification of MEN1 loss-of-function mutations in 4% of patients diagnosed with oncocytic thyroid carcinoma. Whole genome sequence data also revealed large regions of copy number variation encompassing nearly the entire genomes of these tumors.Conclusion:Menin, a ubiquitously expressed nuclear protein, is a well-characterized tumor suppressor whose loss is the cause of MEN1 syndrome. Menin is involved in several major cellular pathways such as regulation of transcription, control of cell cycle, apoptosis, and DNA damage repair pathways. Mutations of this gene in a subset of Hürthle cell tumors point to a potential role for this protein and its associated pathways in thyroid tumorigenesis.

Abstract Desmoplasia and an inflammatory environment are defining features of pancreatic cancer. Unclear is how pancreatic cells that undergo oncogenic transformation can cross-talk with immune cells and how this contributes to the development of pancreatic lesions. Here, we demonstrate that pancreatic acinar cells expressing mutant KRAS can expedite their transformation to a duct-like phenotype by inducing local inflammation. Specifically, we show that KRASG12D induces the expression of intercellular adhesion molecule-1 (ICAM-1), which serves as chemoattractant for macrophages. Infiltrating macrophages amplify the formation of KRASG12D-caused abnormal pancreatic structures by remodeling the extracellular matrix and providing cytokines such as TNF. Depletion of macrophages or treatment with a neutralizing antibody for ICAM-1 in mice expressing oncogenic Kras under an acinar cell–specific promoter resulted in both a decreased formation of abnormal structures and decreased progression of acinar-to-ductal metaplasia to pancreatic intraepithelial neoplastic lesions. Significance: We here show that oncogenic KRAS in pancreatic acinar cells upregulates the expression of ICAM-1 to attract macrophages. Hence, our results reveal a direct cooperative mechanism between oncogenic Kras mutations and the inflammatory environment to drive the initiation of pancreatic cancer. Cancer Discov; 5(1); 52–63. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1

Abstract Context: Hürthle cell cancer (HCC) of the thyroid remains the subject of controversy with respect to natural course, treatment, and follow-up. Objective: The objective of the study was to evaluate the clinical and molecular features associated with outcome in HCC. Design: The study was a review of 173 HCC cases treated at Mayo Clinic over 11 years with a median 5.8-year follow-up. Results: None of the patients with minimally invasive histology had persistent disease, clinical recurrence, or disease-related death. Male gender and TNM stage were independently associated with increased risk of clinical recurrence or death in widely invasive patients. The 5-year cumulative probability of clinical recurrence or death was higher in patients with TNM stage III–IV (females, 74%; males, 91%) compared with patients with TNM stage I–II (females, 0%; males, 17%). Pulmonary metastases were best identified by computed tomography, whereas radioactive iodine scans were positive in only two of 27 cases. Thyroglobulin was detectable in patients with clinical disease, with the notable exception of five patients with distant metastases. The common TERT C228T promoter mutation was detected in both widely invasive and minimally invasive tumors. TERT mRNA was below the limit of detection in all samples. Conclusion: Widely invasive HCC with TNM stage III–IV is aggressive, with low probability of recurrence-free survival. Males have worse outcomes than females. Minimally invasive HCC appears to be considerably less aggressive. Radioactive iodine scan performs poorly in detecting distant disease. Although the TERT gene is mutated in HCC, the role of this mutation remains to be demonstrated.

Abstract Next Generation Sequencing (NGS) technologies provide rapid genomic analyses of single nucleotide variants, RNA expression and DNA copy number. Application of these technologies to material isolated from formalin fixed paraffin embedded (FFPE) tissue and even degraded frozen material could provide powerful replication samples but remains challenging. We tested the nanoString platform to validate deep sequence analysis of gene expression and DNA copy number in degraded and FFPE material. Firstly, RNA from the Universal Human Reference RNA and a breast cancer cell line (MDA-MB-436) was artificially degraded to different degrees (RIN 1.2-6.8). We used the nanoString platform to simultaneously measure RNA expression across 226 genes in each degraded sample and the corresponding undegraded RNA. Secondly we isolated RNA and DNA from matched fresh frozen and FFPE tissues from nine breast cancer patients (3 HER2+/ER+/PR+, 2 HER2+/ER-/PR-, 2 HER2+/ER+/PR-, 2 HER2-/ER+/PR+) using the nanoString platform to compare expression and copy number across 226 and 86 genes respectively. Finally, we correlated expression and copy number data generated by nanoString with Illumina transcriptome and whole genome sequencing (WGS). NanoString log2 expression fold-change between all artificially degraded samples and their undegraded counterpart showed extremely high correlation (r2&gt;0.91). NanoString DNA copy number between matched fresh-frozen and FFPE showed a high degree of correlation (r2=0.71). All gene amplifications with copy number ≥ 5 in DNA from fresh-frozen material (N=9) were successfully identified in DNA from FFPE material. We also observed good correlation of gene expression between whole transcriptome sequencing and the nanoString platform (r2 0.59 - 0.72) in FFPE and artificially degraded material and for DNA copy number between WGS and nanoString in DNA isolated from cancer cell lines (r2=0.96). The nanoString platform provides reliable data from highly degraded and FFPE material and correlates with sequence analysis of both expression and copy number from NGS platforms demonstrating potential for large-scale replication studies in FFPE material. Citation Format: Nadine Norton, Edith A. Perez, Yan W. Asmann, Jennifer M. Carr, Brian M. Necela, Jennifer M. Kachergus, Jin Jen, Bruce W. Eckloff, E Aubrey Thompson. Analysis of gene expression and copy number variation in breast tumors using both sequencing and hybridization-based platforms. [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 2005. doi:10.1158/1538-7445.AM2013-2005

Context: The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression. Objective: RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status. Design: BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2–3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes. Patients: BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes. Results: RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR. Conclusion: BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.