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Supplementary Figures S1-2, Tables S1-3. Figure S1. Comparison of the number of high confidence, non-synonymous somatic point mutations/indels and high level copy number alterations (CNAs) by tumor grade. Figure S2. Copy number plots of representative benign and borderline phyllodes tumors. Table S2: Somatic non-synonymous mutations identified in FFPE phyllodes tumors subjected to targeted next generation sequencing. Table S3: Detailed information about identified somatic non-synonymous mutations.

Supplementary Figures S1-S5; Supplementary Methods; Supplementary Tables S1-S2.

Purpose: Chromosomal gain at 7q21 is a frequent event in esophageal adenocarcinoma (EAC). However, this event has not been mapped with fine resolution in a large EAC cohort, and its association with clinical endpoints and functional relevance are unclear.Experimental Design: We used a cohort of 116 patients to fine map the 7q21 amplification using SNP microarrays. Prognostic significance and functional role of 7q21 amplification and its gene expression were explored.Results: Amplification of the 7q21 region was observed in 35% of tumors with a focal, minimal amplicon containing six genes. 7q21 amplification was associated with poor survival and analysis of gene expression identified cyclin-dependent kinase 6 (CDK6) as the only gene in the minimal amplicon whose expression was also associated with poor survival. A low-level amplification (10%) was observed at the 12q13 region containing the CDK6 homologue cyclin-dependent kinase 4 (CDK4). Both amplification and expression of CDK4 correlated with poor survival. A combined model of both CDK6 and CDK4 expressions is a superior predictor of survival than either alone. Specific knockdown of CDK4 and/or CDK6 by siRNAs shows that they are required for proliferation of EAC cells and that their function is additive. PD-0332991 targets the kinase activity of both molecules and suppresses proliferation and anchorage independence of EAC cells through activation of the pRB pathway.Conclusions: We suggest that CDK6 is the driver of 7q21 amplification and that both CDK4 and CDK6 are prognostic markers and bona fide oncogenes in EAC. Targeting these molecules may constitute a viable new therapy for this disease. Clin Cancer Res; 17(13); 4513–22. ©2011 AACR.

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PDF file - 50K, Description of Combining SNP6.0 and 250K Platforms

PDF file - 64K, Analysis of SNP6.0 Data Only

A more detailed understanding of the somatic genetic events that drive gastrointestinal adenocarcinomas is necessary to improve diagnosis and therapy. Using data from high-density genomic profiling arrays, we conducted an analysis of somatic copy-number aberrations in 486 gastrointestinal adenocarcinomas including 296 esophageal and gastric cancers. Focal amplifications were substantially more prevalent in gastric/esophageal adenocarcinomas than colorectal tumors. We identified 64 regions of significant recurrent amplification and deletion, some shared and others unique to the adenocarcinoma types examined. Amplified genes were noted in 37% of gastric/esophageal tumors, including in therapeutically targetable kinases such as ERBB2, FGFR1, FGFR2, EGFR, and MET, suggesting the potential use of genomic amplifications as biomarkers to guide therapy of gastric and esophageal cancers where targeted therapeutics have been less developed compared with colorectal cancers. Amplified loci implicated genes with known involvement in carcinogenesis but also pointed to regions harboring potentially novel cancer genes, including a recurrent deletion found in 15% of esophageal tumors where the Runt transcription factor subunit RUNX1 was implicated, including by functional experiments in tissue culture. Together, our results defined genomic features that were common and distinct to various gut-derived adenocarcinomas, potentially informing novel opportunities for targeted therapeutic interventions. Cancer Res; 72(17); 4383–93. ©2012 AACR.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Penile squamous cell carcinoma (PeSCCA) is a rare malignancy for which there are limited treatment options due to a poor understanding of the molecular alterations underlying disease development and progression. Therefore, we performed comprehensive, targeted next-generation sequencing to identify relevant somatic genomic alterations in a retrospective cohort of 60 fixed tumor samples from 43 PeSCCA cases (including 14 matched primary/metastasis pairs). We identified a median of two relevant somatic mutations and one high-level copy-number alteration per sample (range, 0–5 and 0–6, respectively). Expression of HPV and p16 was detectable in 12% and 28% of patients, respectively. Furthermore, advanced clinical stage, lack of p16 expression, and MYC and CCND1 amplifications were significantly associated with shorter time to progression or PeSCCA-specific survival. Notably, four cases harbored EGFR amplifications and one demonstrated CDK4 amplification, genes for which approved and investigational targeted therapies are available. Importantly, although paired primary tumors and lymph node metastases were largely homogeneous for relevant somatic mutations, we identified heterogeneous EGFR amplification in primary tumor/lymph node metastases in 4 of 14 cases, despite uniform EGFR protein overexpression. Likewise, activating HRAS mutations occurred in 8 of 43 cases. Taken together, we provide the first comprehensive molecular PeSCCA analysis, which offers new insight into potential precision medicine approaches for this disease, including strategies targeting EGFR. Cancer Res; 75(24); 5219–27. ©2015 AACR.

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Sanger sequencing validation of next generation sequencing (NGS) variant calls ; Alternative view of integrative molecular profiling heatmap ; p16 expression via immunohistochemistry ; Kaplan-Meier analysis of histologic subtypes of PeSCCA ; Comparison of prioritized molecular alterations in penile (PN) squamous cell carcinomas (SCCA) compared to other SCCAs .

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Clinicopathologic data for all profiled penile squamous cell carcinoma (PeSCCA) samples ; Sequencing data for all profiled penile squamous cell carcinoma (PeSCCA) samples ; Detailed somatic variant information from PeSCCA profiled by next generation sequenicng ; PeSCCA HPV genotyping and p16 immunohistochemistry (IHC) results .

PDF file - 1MB, Supplemental Figures 1-13 - Figure S1. Average levels of focal amplification and deletion along the chromosome arm; Figure S2. Patterns of chromosomal instability differ across adenocarcinomas of the gastrointestinal tract after removing samples without arm-level alterations; Figure S3. Numbers of high-level amplifications and deletions using arm-level based thresholds; Figure S4. Identification of arm-level alterations in esophageal, gastric, and colorectal adenocarcinomas; Figure S5. Comparison of SNP6.0 and 250K adenocarcinoma data to Progenetix database; Figure S6. Significance of focal amplifications in esophageal, gastric, and colorectal adenocarcinomas; Figure S7. Variable levels of focal alterations across gut adenocarcinomas; Figure S8. KRAS alterations differ across gut adenocarcinomas; Figure S9. Quantitative real-time PCR confirms FGFR2 amplification in EA tumors; Figure S10. Focal alterations in gut adenocarcinomas shared with all cancers; Figure S11. Significance of focal deletions in esophageal, gastric, and colorectal adenocarcinomas; Figure S12. Length distribution of SCNAs in gut adenocarcinomas; Figure S13. Observed distribution of copy number event amplitudes in the data set.

Introduction: We present the comprehensive genomic profiling performance of the Ion Torrent Genexus system using the Oncomine Comprehensive Assay Plus (OCA Plus), a 500+ gene targeted AmpliSeq-based oncology research panel that evaluates DNA variants (including copy number alterations), RNA fusions, and key oncology research endpoints including tumor mutational burden (TMB), microsatellite instability (MSI), and homologous recombination repair deficiency (HRD) via characterization of genomic instability by the newly introduced Genomic Instability Metric (GIM). Methods: The Ion Torrent Genexus System provides comprehensive genomic profiling via automated sample-to-report workflow with next day results. The Genexus System supports oncology research panels such as OCA Plus, which is comprised of over 13,000 amplicons, and enables low input requirements of just 20ng of FFPE DNA and RNA. This study utilized cell lines, reference controls, and orthogonally tested FFPE research samples to evaluate detection of DNA variants, copy number alterations, RNA fusions, and key research endpoints, including MSI, TMB, and HRD. The OCA Plus panel was also evaluated for the ability to detect arm-level copy number changes in orthogonally validated FFPE samples. Results: Commercial reference controls and FFPE research samples were sequenced using OCA Plus on the Genexus System to an average depth of ≥24 million reads per sample, with four DNA and RNA samples supported per run. SNV and MNV calling performance was assessed using the AcroMetrix Oncology Hotspot Control which has 377 variants covered by OCA Plus and delivered SNV sensitivity and PPV >99% and MNV sensitivity of >99% and PPV >95%. MSI status was assessed using orthogonally tested FFPE samples from various tumor tissues (stomach, endometrial, colorectal) and returned status concordance of 99.4% with sensitivity and PPV >99%. The TMB endpoint was tested using commercial controls and FFPE samples with a correlation of r2 > 0.90 to orthogonal measurements. RNA Fusion reference controls showed 100% positive correlation. Copy number gain detection shows sensitivity of 99% and PPV >95%, while homologous copy loss gives 100% PPV and sensitivity of >90%. We also demonstrate high concordance to orthogonal methods in detection of HER2 amplifications, and the ability to detect arm-level copy number alterations such as 1p/19q co-deletions in IDH1 positive glioma samples. Conclusion: The Genexus System combines minimal touch points and a rapid turnaround time to enable comprehensive genomic profiling for research assays such as OCA Plus for detection of rare variants and low-level fusion transcripts. Further, by providing accurate characterization of key oncology research endpoints, the Genexus System can accelerate research in oncology. For research use only. Not for use in diagnostic procedures. Citation Format: Geoffrey Marc Lowman, Dinesh Cyanam, Emily Norris, Michelle Toro, Coleen Nemes, Tanaya Puranik, Yan Zhu, Alex Phan, Derek Wong, Portia Bernado, Anelia Kralcheva, Srinivas Nallandhighal, Loni Pickle, April Bigley, Mohit Gupta, Ying Jin, Sameh El-Difrawy, Amir Marcovitz, Fatima Zare, Charles Scafe, Yu-Ting Tseng, Jianjun Guo, Vinay Mittal, Scott Myrand, Santhoshi Bandla, Paul Williams, Eugene Ingerman, Elaine Wong-Ho, Seth Sadis, Mark Andersen, Rob Bennett. Fully automated comprehensive genomic profiling for detection of cancer variants, gene fusions, and complex oncology endpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 232.

Comprehensive genomic profiling (CGP) of tumor samples by next-generation sequencing is used to support clinical and translational research into the genetic variants that serve as biomarkers for diagnosis, prognosis and potential therapeutic response. However, as these assays grow in size to meet the expanding demands of users, it is challenging to maintain performance in the face of limited sample input necessitated by small sample volumes and to provide a simple and fast sample to report workflow with limited hands-on time. We, therefore, developed OCA Plus to meet user needs for a large CGP assay with excellent performance. Gene content was prioritized based on potential clinical relevance and variant prevalence in solid tumors. Over 500 genes were selected including genes in the indication statements of approved drug labels, clinical guidelines, and in the enrollment criteria of clinical trials. In addition, driver genes were selected in key pathways including DNA repair and immune checkpoint response. Amplicon design strategies were optimized accordingly for key hotspots, full coding sequences or copy number variation (CNV). The assay used Ion AmpliSeq™ technology with manual library preparation or automated templating on the Ion Chef™ System and sequencing on the Ion GeneStudio™ S5 platform. Twenty ng of purified DNA was routinely used as input. An automated tumor-only workflow for variant calling and sample quality reporting was provided within Ion Reporter™ Software. Streamlined access to reporting of variant relevance was enabled by Oncomine™ Reporter. In development studies of cancer cell line and formaldehyde-fixed, paraffin-embedded (FFPE) tumor samples, the assay displayed excellent uniformity (98% and 94%, respectively). Detection of single nucleotide variants and indels in cell lines and FFPE samples showed >95% sensitivity and PPV. Detection of CNV gain and loss in cell lines and FFPE samples showed >95% sensitivity and PPV. Assessment of tumor mutational burden (TMB) using publicly available whole-exome cancer sequencing data as well as test cell lines and FFPE samples showed high concordance with whole exome sequencing (R2 > 0.90). MSI sensitivity and specificity was >95% as tested using a diverse set of tumor samples. Targeted fusions were reported with 100% sensitivity and specificity when tested with commercially available controls. Total time from purified DNA to end of sequencing was < 2 days with < 3 hours of hands-on time and the time from post-sequencing to report generation was < 2 hours. Oncomine Comprehensive Assay (OCA Plus) was developed to support CGP and routine clinical research in oncology. The assay design and informatics workflow were optimized to support low input and rapid sample-to-report turn-around time, which will accelerate clinical and translational research. Citation Format: Vinay Mittal, Jennifer Kilzer, Dinesh Cyanam, Janice Au-Young, Santhoshi Bandla, Gary Bee, Sameh El-Difrawy, Aren Ewing, Rajesh Gottimukkala, Mohit Gupta, Nickolay Khazanov, Anelia Kraltcheva, Amir Marcovitz, Scott Myrand, Rose Putler, Yu-Ting Tseng, Warren Tom, Cristina Van Loy, James Veitch, Paul Williams, Elaine Wong-Ho, Huimin Xie, Chenchen Yang, Zheng Zang, Seth Sadis. Comprehensive genomic profiling of solid tumors for key targeted and immuno-oncology biomarkers using Ion Torrent NGS technology on the Oncomine Comprehensive Assay Plus (OCA Plus) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 179.

Introduction: Lymphomas, including diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, and other lymphomas, are clinically heterogeneous: some respond well to therapy, but many fail to respond. Much of this variability in response is reported to reflect molecular heterogeneity of the tumor. Identifying somatic variants including SNVs, insertions, deletions, and copy number variations (CNVs) is important in characterizing these samples. Robust detection of variants in multiple genes using fine needle aspirate (FNA) samples, low abundance DNA, and FFPE samples is needed. Methods: We describe a next-generation sequencing assay with 25 genes, the Oncomine Lymphoma Panel, including ARID1A, ATM, B2M, BCL2, BCL6, BRAF, BTK, CARD11, CD79B, CDKN2A, CREBBP, EZH2, GNA13, HIST1H1E, KMT2D, MTOR, MYC, MYD88, PIM1, SF3B1, SOCS1, TNFAIP3, TNFRSF14, TP53, and XPO1. This panel comprises 976 amplicons in total. The assays for these genes have been optimized, and performance has been tested on control samples and on representative clinical research samples. Another 300 genes, with optimized and verified performance, can be added to customize the panel. This panel is designed to work with 20 ng input DNA from FFPE samples and can also be used with samples having non-degraded DNA. A comprehensive bioinformatics analysis solution was developed to detect SNPs, indels, and CNVs; to perform filtering for the most relevant variants; to annotate these variants with a wide variety of bioinformatics databases; and to report on the interpretation of the selected variants. Results: We tested this panel on various types of input material, including control samples and FFPE samples. Uniformity, coverage, on target mapping, reproducibility, and sensitivity to detect variants were high in all cases and above established quality criteria (>90% or > 95%). Finally, a coordinated analysis solution uses information about the panel and provides an integrated analysis pipeline with a simple and powerful visual interface, including variant calling, CNV detection, functional annotation, population MAF, predicted protein effect, and annotations including ClinVar, COSMIC, etc. Filtering tools utilizing this information facilitate variant prioritization. Conclusions: An NGS lymphoma assay with a comprehensive data analysis approach was developed and analytically validated. The system is capable of detecting both small mutations and CNVs simultaneously with high sensitivity in FFPE samples for lymphoma translational research. For research use only. Not for use in diagnostic procedures. Citation Format: Fiona Hyland, Charles Scafe, Yun Zhu, Chenchen Yang, Yu-Ting Tseng, Brooke McKnight, Fernando Farfan, Santhoshi Bandla, Seth Sadis, Steve Roman. Development and analytical validation of a novel next-generation DNA sequencing assay, the oncomine lymphoma panel, to detect SNV, insertion, deletion and copy number variants in 25 Lymphoma genes in FFPE samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 170.

Penile squamous cell carcinoma (PeSCCA) is a rare malignancy for which there are limited treatment options due to a poor understanding of the molecular alterations underlying disease development and progression. Therefore, we performed comprehensive, targeted next-generation sequencing to identify relevant somatic genomic alterations in a retrospective cohort of 60 fixed tumor samples from 43 PeSCCA cases (including 14 matched primary/metastasis pairs). We identified a median of two relevant somatic mutations and one high-level copy-number alteration per sample (range, 0–5 and 0–6, respectively). Expression of HPV and p16 was detectable in 12% and 28% of patients, respectively. Furthermore, advanced clinical stage, lack of p16 expression, and MYC and CCND1 amplifications were significantly associated with shorter time to progression or PeSCCA-specific survival. Notably, four cases harbored EGFR amplifications and one demonstrated CDK4 amplification, genes for which approved and investigational targeted therapies are available. Importantly, although paired primary tumors and lymph node metastases were largely homogeneous for relevant somatic mutations, we identified heterogeneous EGFR amplification in primary tumor/lymph node metastases in 4 of 14 cases, despite uniform EGFR protein overexpression. Likewise, activating HRAS mutations occurred in 8 of 43 cases. Taken together, we provide the first comprehensive molecular PeSCCA analysis, which offers new insight into potential precision medicine approaches for this disease, including strategies targeting EGFR. Cancer Res; 75(24); 5219–27. ©2015 AACR.

To determine and compare the frequency of cancer-associated genetic abnormalities in esophageal metaplasia biopsies with and without goblet cells.Barrett's esophagus is associated with increased risk of esophageal adenocarcinoma (EAC), but the appropriate histologic definition of Barrett's esophagus is debated. Intestinal metaplasia (IM) is defined by the presence of goblet cells whereas nongoblet cell metaplasia (NGM) lacks goblet cells. Both have been implicated in EAC risk but this is controversial. Although IM is known to harbor genetic changes associated with EAC, little is known about NGM. We hypothesized that if NGM and IM infer similar EAC risk, then they would harbor similar genetic aberrations in genes associated with EAC.Ninety frozen NGM, IM, and normal tissues from 45 subjects were studied. DNA copy number abnormalities were identified using microarrays and fluorescence in situ hybridization. Targeted sequencing of all exons from 20 EAC-associated genes was performed on metaplasia biopsies using Ion AmpliSeq DNA sequencing.Frequent copy number abnormalities targeting cancer-associated genes were found in IM whereas no such changes were observed in NGM. In 1 subject, fluorescence in situ hybridization confirmed loss of CDKN2A and amplification of chromosome 8 in IM but not in a nearby NGM biopsy. Targeted sequencing revealed 11 nonsynonymous mutations in 16 IM samples and 2 mutations in 19 NGM samples.This study reports the largest and most comprehensive comparison of DNA aberrations in IM and NGM genomes. Our results show that IM has a much higher frequency of cancer-associated mutations than NGM.

The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a 5-year survival rate of ~15%, the identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole-exome sequencing of 149 EAC tumor-normal pairs, 15 of which have also been subjected to whole-genome sequencing. We identify a mutational signature defined by a high prevalence of A>C transversions at AA dinucleotides. Statistical analysis of exome data identified 26 significantly mutated genes. Of these genes, five (TP53, CDKN2A, SMAD4, ARID1A and PIK3CA) have previously been implicated in EAC. The new significantly mutated genes include chromatin-modifying factors and candidate contributors SPG20, TLR4, ELMO1 and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 identifies increased cellular invasion. Therefore, we suggest the potential activation of the RAC1 pathway as a contributor to EAC tumorigenesis., National Human Genome Research Institute (U.S.) (Large Scale Sequencing Program Grant U54 HG003067)

Objective This study aimed to identify pathways and cellular processes that are modulated by exposure of normal esophageal cells to bile and acid. Background Barrett's esophagus most likely develops as a response of esophageal stem cells to the abnormal reflux environment. Although insights into the underlying molecular mechanisms are slowly emerging, much of the metaplastic process remains unknown. Methods We performed a global analysis of gene expression in normal squamous esophageal cells in response to bile or acid exposure. Differentially expressed genes were classified into major biological functions using pathway analysis and interaction network software. Array data were verified by quantitative PCR and western blot both in vitro and in human esophageal biopsies. Results Bile modulated expression of 202 genes, and acid modulated expression of 103 genes. Genes involved in squamous differentiation formed the largest functional group (n = 45) all of which were downregulated by bile exposure. This included genes such as involucrin (IVL), keratinocyte differentiation-associated protein (KRTDAP), grainyhead-like 1 (GRHL1), and desmoglein1 (DSG1) the downregulation of which was confirmed by quantitative PCR and western blot. Bile also caused expression changes in genes involved in cell adhesion, DNA repair, oxidative stress, cell cycle, Wnt signaling, and lipid metabolism. Analysis of human esophageal biopsies demonstrated greatly reduced expression of IVL, KRTDAP, DSG1, and GRHL1 in metaplastic compared to squamous epithelia. Conclusions We report for the first time that bile inhibits the squamous differentiation program of esophageal epithelial cells. This, coordinated with induction of genes driving intestinal differentiation, may be required for the development of Barrett's esophagus.

Background Esophageal cancer consists of two major histologic types: esophageal squamous cell carcinoma (ESCC), predominant globally, and esophageal adenocarcinoma (EAC), which has a higher incidence in westernized countries. Five-year overall survival is 15%. Clinical trials frequently combine histologic types although they are different diseases with distinct origins. In the evolving era of personalized medicine and targeted therapies, we hypothesized that ESCC and EAC have genomic differences important for developing new therapeutic strategies for esophageal cancer. Methods We explored DNA copy number abnormalities in 70 ESCCs with publicly available array data and 189 EACs from our group. All data was from single nucleotide polymorphism arrays. Analysis was performed using a segmentation algorithm. Log ratio thresholds for copy number gain and loss were set at ±0.2 (approximately 2.3 and 1.7 copies, respectively). Results The ESCC and EAC genomes showed some copy number abnormalities with similar frequencies (eg, CDKN2A, EGFR, KRAS, MYC, CDK6, MET) but also many copy number abnormalities with different frequencies between histologic types, most of which were amplification events. Some of these regions harbor genes for which targeted therapies are currently available (VEGFA, ERBB2) or for which agents are in clinical trials (PIK3CA, FGFR1). Other regions contain putative oncogenes that may be targeted in the future. Conclusions Using single nucleotide polymorphism arrays we compared genomic abnormalities in a large cohort of EACs and ESCCs. We report here the similar and different frequencies of copy number abnormalities in ESCC and EAC. These results may allow development of histology-specific therapeutic agents for esophageal cancer.

Purpose: Chromosomal gain at 7q21 is a frequent event in esophageal adenocarcinoma (EAC). However, this event has not been mapped with fine resolution in a large EAC cohort, and its association with clinical endpoints and functional relevance are unclear. Experimental Design: We used a cohort of 116 patients to fine map the 7q21 amplification using SNP microarrays. Prognostic significance and functional role of 7q21 amplification and its gene expression were explored. Results: Amplification of the 7q21 region was observed in 35% of tumors with a focal, minimal amplicon containing six genes. 7q21 amplification was associated with poor survival and analysis of gene expression identified cyclin-dependent kinase 6 (CDK6) as the only gene in the minimal amplicon whose expression was also associated with poor survival. A low-level amplification (10%) was observed at the 12q13 region containing the CDK6 homologue cyclin-dependent kinase 4 (CDK4). Both amplification and expression of CDK4 correlated with poor survival. A combined model of both CDK6 and CDK4 expressions is a superior predictor of survival than either alone. Specific knockdown of CDK4 and/or CDK6 by siRNAs shows that they are required for proliferation of EAC cells and that their function is additive. PD-0332991 targets the kinase activity of both molecules and suppresses proliferation and anchorage independence of EAC cells through activation of the pRB pathway. Conclusions: We suggest that CDK6 is the driver of 7q21 amplification and that both CDK4 and CDK6 are prognostic markers and bona fide oncogenes in EAC. Targeting these molecules may constitute a viable new therapy for this disease. Clin Cancer Res; 17(13); 4513–22. ©2011 AACR.

Background The incidence of esophageal adenocarcinoma is rapidly increasing and is now one of the leading causes of cancer death in the western world. MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of protein-encoding genes and are involved in the development, progression and prognosis of other malignancies. We hypothesized that global miRNA expression would predict survival and lymph node involvement in a cohort of surgically resected esophagus cancer patients. Methods The miRNA analysis was performed using a custom Affymetrix microarray with probes for 462 known human, 2,102 predicted human, 357 mouse, and 238 rat miRNAs. Expression of miRNA was evaluated in 45 primary tumors, and the association of miRNA expression with patient survival and lymph node metastasis was assessed. The prognostic impact of identified unique miRNAs was verified with quantitative reverse transcriptase polymerase chain reaction. Results Our data indicate that the expression of individual human miRNA species is significantly associated with postresection patient survival. Using data from five unique miRNAs, we were further able to generate a combined miRNA expression signature that is associated with patient survival ( p = 0.005; hazard ratio 3.6) independent of node involvement and overall stage. The expression of three miRNAs (miR-99b and miR-199a_3p and _5p) was also associated with the presence of lymph node metastasis. Conclusions These results suggest miRNA expression profiling could provide prognostic utility in staging esophagus cancer patients and treatment planning with endoscopic and neoadjuvant therapies. The alterations of specific miRNAs may further elucidate steps in the metastatic pathway and allow for development of targeted therapy.

Title: Analytical performance of a novel next generation sequencing assay for myeloid cancers Nick Khazanov, Wally Zhang, Dinesh Cyanam, Scott P. Myrand, Denis Kaznadzey, Paul Williams, Vinay Mittal, Dan Mazur, Sihong Chen, Jason Wustman, Efren Ballesteros-Villagrana, Goutam Nistala, Jon Sherlock, Michael Hogan, Jim Veitch, John Bishop, Seth Sadis Introduction: Myeloid malignancies contain a diverse and heterogeneous set of genomic alterations that include recurrent somatic mutations in key driver genes as well as frequent and diagnostic chromosomal rearrangements that generate a wide array of gene fusion products. To support clinical and translational research into precision oncology strategies for myeloid cancers, a next-generation sequencing (NGS) assay was generated to detect common and relevant somatic alterations. Methods: To define gene targets that were recurrently altered in myeloid cancers and relevant for clinical and translational research, an extensive survey of investigators at hematology oncology research labs was performed. The gene targets identified by researchers were complemented by a comprehensive survey of literature and genomic databases. Clinical guidelines for myeloid diseases in the US and in Europe were reviewed to ensure representation of relevant alterations. A targeted Ion AmpliSeq panel was generated to support the detection of recurrent single-nucleotide variants, insertions/deletions, and gene fusions from blood or bone marrow samples. The panel was developed for manual or automated library preparation and sequencing on the Ion Torrent PGM or Ion S5 instruments. Results: The Oncomine™ Myeloid Research Assay gene panel included 58 genes and generated an average read depth of >2,000 reads per targeted amplicon with an average uniformity of >95%. Important GC-rich targets such as CEBPA generated sufficient balanced read depth to support variant detection. A cohort of samples positive for FLT3 alterations was analyzed and successful detection of FLT3-internal tandem repeat variants was demonstrated. Several gene fusion transcripts common to myeloid cancers were detected. Comparable results were observed on Ion Torrent PGM and Ion S5 instruments. Conclusions: A novel myeloid specific NGS assay capable of detecting relevant DNA and RNA alterations from the same sample was developed. The assay is useful for characterizing relevant alterations in a range of myeloid diseases including acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelogenous leukemia, chronic myelomonocytic leukemia, and juvenile myelomonocytic leukemia. A review of the analytical studies will be presented. Citation Format: Nickolay Khazanov, Wally Zhang, Dinesh Cyanam, Scott P. Myrand, Denis Kaznadzey, Paul D. Williams, Vinay Mittal, Daniel J. Mazur, Sihong Chen, Jason Wustman, Efren Ballesteros-Villagrana, Goutam Nistala, Santhoshi Bandla, Jim Veitch, Jon Sherlock, John Bishop, Seth Sadis. Analytical performance of a novel next generation sequencing assay for myeloid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3863.

Phyllodes tumors are rare fibroepithelial tumors with variable clinical behavior accounting for a small subset of all breast neoplasms, yet little is known about the genetic alterations that drive tumor initiation and/or progression. Here, targeted next-generation sequencing (NGS) was used to identify somatic alterations in formalin-fixed paraffin-embedded (FFPE) patient specimens from malignant, borderline, and benign cases. NGS revealed mutations in mediator complex subunit 12 (MED12) affecting the G44 hotspot residue in the majority (67%) of cases spanning all three histologic grades. In addition, loss-of-function mutations in p53 (TP53) as well as deleterious mutations in the tumor suppressors retinoblastoma (RB1) and neurofibromin 1 (NF1) were identified exclusively in malignant tumors. High-level copy-number alterations (CNA) were nearly exclusively confined to malignant tumors, including potentially clinically actionable gene amplifications in IGF1R and EGFR. Taken together, this study defines the genomic landscape underlying phyllodes tumor development, suggests potential molecular correlates to histologic grade, expands the spectrum of human tumors with frequent recurrent MED12 mutations, and identifies IGF1R and EGFR as potential therapeutic targets in malignant cases. Implications: Integrated genomic sequencing and mutational profiling provides insight into the molecular origin of phyllodes tumors and indicates potential druggable targets in malignant disease. Visual Overview: http://mcr.aacrjournals.org/content/early/2015/04/02/1541-7786.MCR-14-0578/F1.large.jpg. Mol Cancer Res; 13(4); 613–9. ©2015 AACR.

Background Cyclin E is a cell cycle regulator which is critical for driving G1/S transition. Abnormal levels of cyclin E have been found in many cancers. However, the level changes of cyclin E in esophageal adenocarcinoma and its precancerous lesion have not been well studied. Here, we focus on the gene amplification and expression of cyclin E in these lesions, and aim to ascertain the relationship with clinicopathological characteristics. Methods Genomic DNA was analyzed from 116 esophageal adenocarcinoma and 26 precancerous lesion patients using Affymetrix SNP 6.0 arrays. The protein overexpression of cyclin E was also detected using immunohistochemistry from tissue microarrays containing esophageal adenocarcinoma and precancerous lesions. Patient survival and other clinical data were collected and analyzed. The intensity and percentage of the cyclin E expressing cells in tissue microarrays were scored by two pathologists. Fisher exact tests and Kaplan-Meier methods were used to analyze data. Results By genomic analysis, cyclin E was amplified in 19.0% of the EAC samples. By immunohistochemistry, high expression of cyclin E was observed in 2.3% of squamous mucosa tissues, 3.7% in columnar cell metaplasia, 5.8% in Barrett’s esophagus, 19.0% in low grade dysplasia, 35.7% in high grade dysplasia, and 16.7% in esophageal adenocarcinoma. The differences in cyclin E high expression between neoplastic groups and non-dysplasia groups are statistically significant (p

Bile acid reflux into the esophagus is important in the development of esophageal adenocarcinoma (EAC). Recently, vitamin D receptor (VDR) was recognized as a bile acid receptor as well as a vitamin receptor. Expression of VDR is reported to influence the development of various types of cancer, such as those of the breast, liver, and colon. However, little is known about the role of VDR in esophageal neoplasms. We investigated the clinicopathological role of VDR in esophageal tumors. We analyzed genomic DNA from 116 EACs for copy number aberrations. The VDR locus was amplified in 7% of EACs. Expression of the VDR protein was also detected by immunohistochemistry from tissue microarrays created from tissues of Barrett esophagus (BE), low-grade (LGD) and high-grade dysplasia (HGD), columnar cell metaplasia (CCM), squamous epithelium (SE), EAC, and esophageal squamous cell carcinoma (ESCC). The protein was highly expressed in 88% of CCM (58/66), 95% of BE (35/37), 100% of the 19 LGD, 94% of HGD (15/16), and 79% of EAC (86/109), but expression in SE and ESCC was rare. Female patients with EAC and CCM were significantly less likely to have high VDR expression than male patients. The overall survival rate was significantly different for patients with tumors exhibiting VDR amplification versus nonamplification. Our findings suggest that VDR plays a role in the early development of EAC through a bile acid ligand. The sex difference in VDR expression may help to explain why men have a high incidence of EAC.

e20667Background: Next-generation sequencing (NGS) is an emerging technology for molecular diagnostics enabling the identification of multiple variants from a small tissue sample, thus displacing s...

e23224Background: Development of scalable and cost-effective NGS solutions to accurately assess relevant genomic alterations is necessary to support translational research and a future precision on...

A more detailed understanding of the somatic genetic events that drive gastrointestinal adenocarcinomas is necessary to improve diagnosis and therapy. Using data from high-density genomic profiling arrays, we conducted an analysis of somatic copy-number aberrations in 486 gastrointestinal adenocarcinomas including 296 esophageal and gastric cancers. Focal amplifications were substantially more prevalent in gastric/esophageal adenocarcinomas than colorectal tumors. We identified 64 regions of significant recurrent amplification and deletion, some shared and others unique to the adenocarcinoma types examined. Amplified genes were noted in 37% of gastric/esophageal tumors, including in therapeutically targetable kinases such as ERBB2, FGFR1, FGFR2, EGFR, and MET, suggesting the potential use of genomic amplifications as biomarkers to guide therapy of gastric and esophageal cancers where targeted therapeutics have been less developed compared with colorectal cancers. Amplified loci implicated genes with known involvement in carcinogenesis but also pointed to regions harboring potentially novel cancer genes, including a recurrent deletion found in 15% of esophageal tumors where the Runt transcription factor subunit RUNX1 was implicated, including by functional experiments in tissue culture. Together, our results defined genomic features that were common and distinct to various gut-derived adenocarcinomas, potentially informing novel opportunities for targeted therapeutic interventions. Cancer Res; 72(17); 4383–93. ©2012 AACR.

Chromosomal gain at 7q21 is a frequent event in esophageal adenocarcinoma (EAC). However, this event has not been mapped with fine resolution in a large EAC cohort, and its association with clinical endpoints and functional relevance are unclear.We used a cohort of 116 patients to fine map the 7q21 amplification using SNP microarrays. Prognostic significance and functional role of 7q21 amplification and its gene expression were explored.Amplification of the 7q21 region was observed in 35% of tumors with a focal, minimal amplicon containing six genes. 7q21 amplification was associated with poor survival and analysis of gene expression identified cyclin-dependent kinase 6 (CDK6) as the only gene in the minimal amplicon whose expression was also associated with poor survival. A low-level amplification (10%) was observed at the 12q13 region containing the CDK6 homologue cyclin-dependent kinase 4 (CDK4). Both amplification and expression of CDK4 correlated with poor survival. A combined model of both CDK6 and CDK4 expressions is a superior predictor of survival than either alone. Specific knockdown of CDK4 and/or CDK6 by siRNAs shows that they are required for proliferation of EAC cells and that their function is additive. PD-0332991 targets the kinase activity of both molecules and suppresses proliferation and anchorage independence of EAC cells through activation of the pRB pathway.We suggest that CDK6 is the driver of 7q21 amplification and that both CDK4 and CDK6 are prognostic markers and bona fide oncogenes in EAC. Targeting these molecules may constitute a viable new therapy for this disease.

The HER2 oncogene was recently reported to be amplified and overexpressed in esophageal adenocarcinoma. However, the relationship of HER2 amplification in esophageal adenocarcinoma with prognosis has not been well defined. The scoring systems for clinically evaluating HER2 in esophageal adenocarcinoma are not established. The aims of the study were to establish a HER2 scoring system and comprehensively investigate HER2 amplification and overexpression in esophageal adenocarcinoma and its precursor lesion. Using a tissue microarray, containing 116 cases of esophageal adenocarcinoma, 34 cases of Barrett's esophagus, 18 cases of low-grade dysplasia and 15 cases of high-grade dysplasia, HER2 amplification and overexpression were analyzed by HercepTest and chromogenic in situ hybridization methods. The amplification frequency in an independent series of 116 esophageal adenocarcinoma samples was also analyzed using Affymetrix SNP 6.0 microarrays. In our studies, we have found that HER2 amplification does not associate with poor prognosis in total 232 esophageal adenocarcinoma patients by chromogenic in situ hybridization and high-density microarrays. We further confirm the similar frequency of HER2 amplification by chromogenic in situ hybridization (18%; 21 out of 116) and SNP 6.0 microarrays (16%, 19 out of 116) in esophageal adenocarcinoma. HER2 protein overexpression was observed in 12% (14 out of 116) of esophageal adenocarcinoma and 7% (1 out of 15) of high-grade dysplasia. No HER2 amplification or overexpression was identified in Barrett's esophagus or low-grade dysplasia. All HER2 protein overexpression cases showed HER2 gene amplification. Gene amplification was found to be more frequent by chromogenic in situ hybridization than protein overexpression in esophageal adenocarcinoma (18 vs 12%). A modified two-step model for esophageal adenocarcinoma HER2 testing is recommended for clinical esophageal adenocarcinoma HER2 trial.

e22164 Background: Next generation sequencing (NGS) has enabled genomewide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required for action...

Introduction: Treating cancer effectively requires an understanding of the molecular alterations driving each patient's tumor. Prevalent and recurrent cancer-associated somatic alterations have been defined using whole genome, exome, and transcriptome sequencing across thousands of cases. Targeted sequencing efforts that characterize prevalent somatic alterations and require limited sample input may provide an effective diagnostic approach in the future. Materials and Methods: Herein, we describe the design and initial characterization of the Oncomine Cancer Research Panel (OCP) that includes recurrent somatic alterations in solid tumors derived from the Oncomine™ cancer database. Using Ion AmpliSeq™, we designed a DNA panel that includes assays for 73 oncogenes with 1, 826 recurrent hotspot mutations, 26 tumor suppressor genes enriched for deleterious mutations, as well as 75 genes subject to recurrent focal copy gain or loss. A complementary RNA panel includes 183 assays for relevant gene fusions involving 22 fusion driver genes. Recommended sample inputs were 20 ng of DNA and 10 ng of RNA. Sequencing libraries were analyzed on an Ion Torrent Personal Genome Machine. Results: Initial testing revealed an average read depth of > 1, 500X with > 95% uniformity and on target frequency. The panel was shown to reliably detect known hotspots, insertions/deletions, gene copy changes, and gene fusions in molecular standards, cell lines and formalin-fixed paraffin embedded clinical specimens. Retrospective analysis of large sample cohorts has been completed and the results of analysis of 100 lung cancer and 100 prostate cancer samples will be summarized. In addition, a cohort of 100 tumor samples from the University of Michigan Molecular Diagnostics laboratory, which were submitted for molecular testing (KRAS, BRAF, EGFR, ALK), were also profiled with OCP. Overall, we achieved >95% sensitivity and specificity for detection of KRAS, BRAF, and EGFR mutations, as well as ALK gene fusions. Conclusions: We designed OCP to include prevalent and relevant somatic alterations to aid in cancer research exploring whether targeted NGS panels may have utility in future diagnostic applications.

The MAPK and PI3K pathways are frequently altered in human cancer and are targeted by dozens of agents in clinical trials. The successful application of these therapies, alone or in combination, may depend on the activation status of both pathways. Next-generation sequencing of cancer exomes provides a unique opportunity to systematically survey pathway alterations in cancer. Using somatic mutation data obtained from The Cancer Genome Atlas, we sought to catalog the members of the MAPK and PI3K pathways with driver mutations, the frequency of occurrence in common cancers and the frequency of co-occurrence. Furthermore, we sought to characterize the association of pathway mutation status with drug response in pre-clinical models. While the MAPK and PI3K pathways were frequently altered, the frequency of single and dual pathway alteration and the altered genes varied substantially across cancer types. The MAPK pathway was most frequently altered in rectal (62%), colon (59%), uterine (31%) and lung adenocarcinoma (45%) but infrequently altered in and breast cancer (4%). KRAS, BRAF and NRAS hotspot mutations were the most common pathway drivers, along with NF1 deleterious mutations in certain cancer types. The PI3K pathway was most frequently altered in uterine (84%), breast (40%) and glioblastoma (41%) but was rarely altered in lung adenocarcinoma (9%). Hotspot mutations in PIK3CA and hotspot and deleterious mutations in PTEN were the most common pathway alterations. In addition, predicted driver mutations occurred in PIK3R1, PIK3R3, MTOR, AKT1 and AKT3. Notably, MAPK and PI3K pathway alterations co-occurred in uterine (30%), colon (17%) and gastric (12%) cancers more so than would be expected by chance (p < 0.02). In contrast, other cancer types favored one pathway almost exclusively and thus had little co-occurrence. For example, breast cancer significantly favored PI3K pathway whereas lung adenocarcinoma favored MAPK pathway. To assess the effect of pathway mutation status on treatment response, we integrated hybrid-capture sequencing data from the Cancer Cell Line Encyclopedia with pharmacological data from over 150 compounds. We found that MAPK and PI3K pathway mutations most significantly associated with sensitivity to MEK and PI3K/AKT/mTOR inhibitors, respectively. Notably though, cell lines with co-occurring MAPK pathway and PIK3CA mutations were insensitive to MEK inhibitors and cell lines with co-occurring PI3K pathway and KRAS mutations were insensitive to PI3K inhibitors. Also, not all pathway mutations conferred equal sensitivity. For example, BRAF mutants were generally sensitive, KRAS mutants were mixed and NF1 mutants were generally insensitive to MEK inhibitors. Taken together, our work highlights the need to consider pathways and co-occurrence in the development of targeted therapies. Citation Format: Mark Tomilo, Paul D. Williams, Emma T. Bowden, Supra R. Gajjala, Santhoshi Bandla, Sean F. Eddy, Seth E. Sadis, Peter J. Wyngaard, Nickolay A. Khazanov, Daniel R. Rhodes. Discovery and characterization of driver MAPK and PI3K pathway mutations in tumors and association with drug response in cell lines. [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 2897. doi:10.1158/1538-7445.AM2013-2897

Introduction: Esophageal cancer occurs with two distinct histologies_ squamous cell carcinoma (SCC) and esophageal adenocarcinoma (EA). The incidence of EA has risen over the past few decades and is now the most common esophageal malignancy in the United States and Western Europe. The genome of esophageal adenocarcinoma is highly unstable due to the large number of amplification and deletion events. Presumably, such genomic events drive tumor formation and disease progression by altering the expression of genes within these regions. Several studies have reported large regions of genomic instability especially spanning chromosomal arms or in gene dense regions. Using a large patient cohort and high-resolution data for DNA copy number and expression, this study aims to identify potential driver genes and prognostic markers especially in whole chromosomal aberrations and gene dense regions. Materials & Methods: DNA copy number aberrations were explored in 116 esophageal adenocarcinomas (EA) using Affymetrix SNP 6.0 microarrays. Additionally, gene expression data from a subset of 110 of these tumors using Affymetrix U133 plus 2.0 microarrays were analyzed. DNA copy number aberrations were analyzed using Nexus 5.0 and GISTIC software while the gene expression data was processed in Partek Genomic Suite. Copy number and expression correlations for each gene in the regions of copy number aberrations were calculated using t-test to compare samples with the changes versus samples without the genomic aberrations. Results: We identified at least 50 regions of gains and losses of which 12 regions are either whole chromosomal arm aberrations or aberrations in gene dense regions. Some regions with large number genes include gains at 1q (460 genes), 7q21-22 (80 genes), 10q21-22 (100 genes), and losses at 4q (98 genes), 5q (283 genes), 21q (82 genes). Copy number and expression correlations for each gene in these regions considerably reduced the number of genes that could be considered as candidate driver genes. Furthermore, scouring the literature helped identify potential candidate driver genes such as ANP32E (1q), VCL (10q), CDKN2AIP (4q), and ING2 (4q). Additionally, we report identifying copy number and expression correlations of potential prognostic markers such as MCM4 (8q), CA9 (9p), KLF5 (13q) that lie in focal/smaller regions of genomic instability but at high frequencies. Conclusion: Analysis of the DNA copy number changes identified several regions of frequent gains and losses. At least 12 regions span the chromosomal arm or are in the gene dense regions. Using an integrated genomics approach with high-resolution DNA copy number and expression data, we are able to identify potential driver genes and prognostic markers that lie in these large amplification/deletion regions in EA. 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 5073. doi:1538-7445.AM2012-5073

Background: The incidence of adenocarcinoma of the esophagus and gastroesophageal junction (EAC) has grown faster than any tumor type in the United States and westernized countries, outpacing the next closest cancer by almost 3 times. This rising incidence of EAC is associated with the increasing prevalence of obesity and gastroesophageal reflux disease (GERD). Due to the limited effect of current chemotherapy for this cancer, the search for new therapeutic targets is disparately needed. Chromosomal amplification is one mechanism by which cancer cells acquire and maintain neoplastic phenotype and genes that are amplified and overexpressed may represent potential therapeutic targets. Methods: DNA and RNA from a cohort of 116 patients were analyzed using SNP and mRNA expression microarrays respectively. Gene amplification was explored for correlation with expression and patient survival. Results: 25% of tumors harbor amplification at chromosome 1q which is significantly associated with poor survival. Although the amplicon is non-focal spanning almost the entire arm of chromosome 1q, only a subset of amplified genes (including The CDC28 protein kinase subunit 1B, CKS1B) are significantly overexpressed. CKS1B is an auxiliary protein required for ubiquitination and cytosomal degradation of the CDK inhibitors p27Kip1 and p21Cip1/Waf1 by the SCFSKP2 E3 ubiquitin ligase complex. Both p27Kip1 and p21Cip1/Waf1 inhibits CDK2/Cyclin E complex during the G1/S cell cycle transition. The endogenous protein expression of p27Kip1 is low among EAC cell lines. However the expression of p21Cip1/Waf1 is differential being very high in a subset of cells and low or undetectable in others. Knocking down CKS1B by siRNA in six EAC cell lines results in accumulation of p27 Kip1 with little or no effect on p21Cip1/Waf1 expression. CKS1B knockdown inhibits monolayer cell proliferation only in cells with high endogenous expression of p21Cip1/Waf1. Conclusion: We suggest that the combined action of accumulation of p27 Kip1 and the endogenously highly expressed p21Cip1/Waf1 triggers the response to CKS1B knockdown in EAC cell lines. We thus propose the potential use of CKS1B as a novel therapeutic target in esophageal adenocarcinomas with high p21Cip1/Waf1 expression. 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 3051. doi:1538-7445.AM2012-3051

Introduction: Whole genome and whole exome sequencing by large collaborative research groups is providing us with an unprecedented view of the mutations that occur in cancer. Identifying clinically-relevant and actionable mutations however remains a challenge that will most likely be met by individual groups focused on specific clinical questions and scenarios. Such studies will rely on targeted resequencing of selected genes in large numbers of well-annotated clinical specimens. These specimens are most often formalin-fixed and paraffin-embedded (FFPE) and some of the most valuable samples are small biopsies or fine needle aspirates (FNA's). Translational and clinical researchers require rapid and flexible resequencing approaches that are compatible with the small amounts of highly degraded nucleic acids derived from these challenging samples. Materials & Methods: Using Illumina whole exome sequence data from >60 esophageal adenocarcinoma (EAC) specimens, we identified a set of 20 genes that are frequently mutated in this tumor. PCR amplicons no larger than 140bp were designed to cover all exons from these genes, using the Ion Torrent AmpliSeqTM Designer pipeline. Primers were pooled for a 1285-plex multiplex PCR preamplification step starting with 1-20ng of input DNA from both FFPE and frozen tissue and the resulting amplicon products were used to generate sequencing libraries. Semiconductor sequencing was performed on the Ion Personal Genome MachineTM (PGMTM) sequencer. Metrics evaluated included reads on target, base accuracy, uniform coverage of bases and sequencing base read depth. Mutations detected in EAC samples were compared between the two sequencing platforms. Data from dysplastic and metaplastic esophageal biopsies are now being generated to identify mutations that occur in the development of EAC. Results: 1285 PCR primer pairs were designed that covered 66103bp of the coding region of the 20 target genes. Sequencing data covered >99% with at least 100 reads indicating minimal drop-out during preamplification. Average base read depth was >1900X and no significant differences were observed between FFPE and frozen tissue derived DNA. Initial studies demonstrate 100% concordance of mutations (n=42) detected in the 20 target genes and mutant allele frequencies are highly correlated between the two sequencing approaches (slope =0.96; R2 =0.85; range 4-78%). Conclusion: We have used the Ion Torrent AmpliSeqTM technology to detect mutations in EAC specimens and precursor lesions with high sensitivity and accuracy. The flexible primer design pipeline combined with AmpliSeqTM technology provides a fast and easy approach to targeted resequencing in particularly challenging clinical specimens including FFPE biopsies. 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 LB-410. doi:1538-7445.AM2012-LB-410