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Accurate and early detection of oncogenic markers may one day be the key to fighting cancer. However, with complex workflows, long turnaround times, and numerous user touch points of most sequencing platforms does not make the process attainable. In contrast, the fully automated Genexus࣪ system provides a specimen-to-report workflow for cancer research with minimal user touchpoints and single day turnaround time. FFPE (formalin-fixed paraffin embedded) tissues and liquid biopsy are two of the main sample types used in oncology research. Here we report the use of the Oncomine࣪ Precision Assay (OPA) with the Genexus࣪ System which provides a comprehensive genetic profile across 50 key genes using DNA and RNA from FFPE tissues or cfTNA (cell free total nucleic acid) from liquid biopsy samples. To evaluate the nucleic acid (NA) extraction and sequencing performance of the Genexus࣪ system, contrived control samples with known variants and clinical research samples were used in the OPA FFPE and liquid biopsy workflows (n = 30). NA was quantified using the Genexus࣪ purification instrument’s onboard Qubit࣪ quantitation feature after purification. Output plates with extracted NA were transferred to the Genexus࣪ Integrated Sequencer for library preparation and sequencing using the OPA assay. Data were analyzed using the Ion torrent Genexus࣪ software to evaluate assay performance and variant calling.The OPA assay only requires 10ng of DNA and RNA from FFPE samples and 20ng of cfTNA from liquid biopsy samples. Genexus࣪ purification instrument onboard quantitation data showed successful extraction of NA exceeding the required yields for library preparation. Excess NA was automatically aliquoted into an archive plate and stored for future use. Sequencing results for four samples of FFPE or liquid biopsy were reported within 24 hours. Both Control and clinical research samples showed expected assay metrices including read coverage, molecular coverage, and uniformity. The results reported all expected variants at correct allele frequencies, including BRAF V600E, KRAS G12C, PIK3CA N345K, AKT1 etc. Overall, this study demonstrates that the Genexus࣪ system provides a user-friendly workflow with automated NA purification, quantitation, sample dilution, library preparation, sequencing, and data analysis with minimal hands-on time that can be performed with limited expertise to obtain results within 24 hours. Reliable identification of variants from control and clinical research samples of FFPE and liquid biopsy origin with optimal assay metrics demonstrates the successful use of the OPA assay and Genexus࣪ system that can be confidently used in clinical oncology research.(For Research Use Only. Not for use in diagnostic procedures.) Citation Format: Thilanka Jayaweera, Nicholas Siepert, Brian Hradecky, Emilia Ostrowska, Iris Casuga, kristy Lea, Priyanka Kshatriya, Jian Gu, Jeff Schageman, Ru Cao, Angie Cheng, Kelli Bramlett. Automation meets reliability: Use of Oncomine࣪precision assay on the Genexus࣪system for accurate identification of cancer biomarkers in FFPE and liquid biopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2939.

Next-Generation Sequencing technology has enhanced oncology research by enabling the detection of all cancer related variants into one assay for research and drug discovery programs. The Oncomine࣪ Comprehensive Assay v3, a pan-cancer panel, used with the Ion Torrent࣪ Genexus࣪ System allows for formalin-fixed paraffin embedded (FFPE) samples to be examined across 161 unique genes in an automated sample to result workflow in 30hrs. This study demonstrates ≥ 95% Sensitivity and PPV for detecting SNV, Indel, and Copy Number variants of clinical FFPE samples along with fusions. When tested using analytical controls, the Genexus System achieves ≥ 98% Sensitivity for hotspot variants, ≥ 95% for de novo variants, and 100% Sensitivity for fusion variants. The Ion Torrent࣪ Genexus࣪ System is fully automated and consists of two software linked instruments, the Ion Torrent࣪ Genexus࣪ Purification System and the Ion Torrent࣪ Genexus࣪ Integrated Sequencer. For this study, the purification system was used to sequentially extract and quantify DNA & RNA samples from human colon and lung FFPE tumor tissue. The purification instrument provides minimal hands-on-time, ease of use and compatibility with the integrated sequencer. It extracts and quantifies the nucleic acids, records the quantitation values and transfers the nucleic acids to an output plate. The plate is transferred from the purification instrument directly to the sequencer for sample dilution, library preparation, and sequencing using the Oncomine࣪ Comprehensive Assay v3 panel. Variant calling analysis of the DNA & RNA pairs is completed immediately following sequencing. Results show that the variants are detected reproducibly with ≥ 95% Sensitivity and PPV when compared to the Ion GeneStudio࣪ S5 System as an orthogonal method. All variants in the report were correct with p-values ≤ 10-5. Additionally, a subset of the extracted samples was evaluated using Sanger Sequencing to verify hotspot mutations found in samples sequenced on both the Genexus࣪ System and GeneStudio࣪ S5 System. Results show that all expected hotspot variants were detected. In summary, the Ion Torrent࣪ Genexus࣪ System is a reliable and fast turnaround solution for sample-to-variant calling results. When used with the Oncomine࣪ Comprehensive Assay v3 panel, the system provides accurate identification of tumor markers for oncology research. Citation Format: Iris Casuga, Frances Chan, Milton Huynh, Gregory R. Govoni, Kayla Zochowski, Thilanka Jayaweera, Janice Au-Young. Rapid and accurate variant calling of FFPE samples with the Genexus System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2944.

Introduction: Myeloid malignancies are associated with a broad and diverse set of genomic alterations, including SNVs, insertions, deletions and gene fusions. Comprehensive characterization of genetic mutations in hematological disorders currently requires a variety of diagnostic tests and takes multiple days to complete. We developed a fully automated NGS Myeloid Assay that offers an easy to use sample-to-report workflow and the capability for processing up to 8 samples per day. Methods: The Genexus System is comprised of two software linked instruments, the Genexus Purification System and the Genexus Integrated Sequencer. The Genexus Purification System was used to isolate the DNA and RNA from blood or bone marrow samples from precharacterized myeloid samples representative of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) and from the blood of healthy donors. The Genexus Integrated Sequencer was used to dilute the nucleic acids to optimal concentration and to sequence the samples in replicates with Oncomine Myeloid Genexus v2 Assay. Six DNA and RNA samples were sequenced per run per day along with commercially available analytical controls and a No Template Control. The report was generated by the Genexus Software analysis pipeline optimized to detect different variant types with high sensitivity and specificity. Results: The purification workflows were tested with blood input of 50-400uL for DNA and 50-150uL for RNA. Genexus Purification System extracted and quantified nucleic acids showed input dependent yields. DNA and RNA yields obtained with 50uL sample inputs consistently met the 27.5ng DNA and 15ng RNA minimum requirements for Genexus Integrated Sequencer workflows. DNA libraries had >97% Uniformity of Amplicon Coverage and >95% Target Base Coverage at 350x. The percentage ratio of Mapped Reads for DNA and RNA libraries was approximately 80:20. Detected genetic variations included key hotspots in CEBPA, FLT3, IDH1/2, NPM1, NRAS, RUNX1, and U2AF1 genes that are prevalent in AML and MDS. Genexus Variant Calling results showed high reproducibility and high concordance to the Ion GeneStudio S5 sequencing platform (>95%). The analytical controls, AcroMetrix Oncology Hotspot Control, Seraseq Myeloid Mutation DNA Mix and Seraseq Myeloid Fusion RNA Mix, were sequenced with Sensitivity and PPV >95%. Conclusion: The Genexus System offers an automated sample-to-report workflow with minimal hands-on-time and run results in 30 hours which allows an easy to use solution for next day turnaround time. When used with the Oncomine Myeloid GX v2 Assay, it provides accurate and comprehensive information on diverse mutations including fusions that are relevant to the study of myeloid cancers. For research use only. Not for use in diagnostic procedures. Citation Format: Marina Sedova, Alexy Ongpin, Jennifer Burke, Collyn Seeger, Sarah Brozio, Janice Au-Young, Jiajie Huang, Thilanka Jayaweera, Iris Casuga, Milton Huynh, Fiona Hyland. Fully automated sample-to-report NGS workflow for comprehensive genomic profiling for myeloid neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 764.

Introduction: The Ion Torrent Genexus System has redefined the genomic profiling paradigm as the first fully-integrated, next-generation sequencing (NGS) research platform to provide an automated sample-to-report workflow with results in a single day. With a purification instrument, an enhanced chip architecture, and downstream reporting, the Genexus System provides a convenient solution to enable oncology research. Here we highlight the oncology research applications and high-throughput NGS capabilities of the Genexus System with Oncomine Comprehensive Assay Plus (OCA Plus), an oncology research panel that can detect variants, fusions, and evaluate key oncology research endpoints. We demonstrate the ability of OCA Plus on Genexus to evaluate tumor mutational burden (TMB), microsatellite instability (MSI), loss of heterozygosity (LOH), and homologous recombination repair deficiency (HRD). Methods: The high-throughput capabilities of the Genexus System enable it to support large oncology research panels such as OCA Plus, which comprises over 13,000 amplicons. The extensive per sample coverage allows for comprehensive DNA and RNA genomic profiling of relevant cancer biomarkers in over 500 genes including detection of over 1,300 fusion isoforms. We utilized high-molecular weight and FFPE samples, reference controls, and orthogonally tested research samples to evaluate DNA variant calling, RNA fusion calling, and key oncology research endpoints, including MSI, LOH, TMB, and HRD. Results: Commercially sourced reference control and research samples were sequenced using OCA Plus on the Genexus System. Sequencing data metrics showed ≥24 million reads per sample, with four samples supported per run. The high-throughput capacity of the Genexus chip architecture results in >95% of amplicons achieving a minimum of 500X coverage with an average coverage uniformity of ≥95% when evaluated across all >13,000 amplicons. Variant calling was assessed using the AcroMetrix Oncology Hotspot Control which has 328 hotspot variants covered by OCA Plus. Variant calling performance showed a hotspot and de novo variant sensitivity and PPV >95%. MSI score and status were assessed using known MSI-High and microsatellite stable (MSS) research samples and FFPE samples of interest. Results show high concordance with data from OCA Plus on GeneStudio. Conclusions: The increased throughput of the Genexus System combined with minimal touchpoints and a rapid turnaround time enables comprehensive genomic profiling for research assays such as OCA Plus where an increased number of sequencing reads leads to greater sensitivity for detecting rare variants and low-level fusion transcripts. Further, accurate characterization of key oncology research endpoints, such as TMB, MSI, LOH, and HRD, allow the Genexus System to accelerate research in the field of oncology. Citation Format: Kayla Zochowski, Dinesh Cyanam, Geoffrey Lowman, Jennifer Kilzer, Sameh El-Difrawy, Yan Zhu, Tanaya Puranik, Alex Phan, Derek Wong, Edith Kwong, Coleen Nemes, Iris Casuga, Frances Chan, Eun Ji Kim, Jianjun Guo, Vinay Mittal, Emily Norris, Shrutii Sarda, Mohit Gupta, Fan Shen, Steven Roman, Gabriel Vargas, Ying Jin, Annie Kraltcheva, Paul Williams, Amneet Gulati, Justin Rigby, Christopher Hoff, Richard Meade, Elaine Wong-Ho, Andrew Wong, Jamsheed Ghadiri, David Garcia-Viramontes, Scott Myrand, Seth Sadis. High-throughput next-generation sequencing research solutions for detection of oncology variants, gene fusion events, and key oncology endpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 42.

Next Generation Sequencing technology has enhanced oncology research by enabling the detection of relevant variants for clinical and drug discovery programs. The Oncomine Comprehensive Assay v3, a pan-cancer panel, used with the Ion Torrent Genexus System allows for formalin-fixed paraffin embedded (FFPE) samples to be examined across 161 unique genes in an automated workflow from sample to result in 30hrs. This study demonstrates ≥ 90% accuracy for detecting SNV/Indel/CNV and Fusion variants. The Genexus System consists of two software linked instruments, the Genexus Purification System and the Genexus Integrated Sequencer. The purification system is used first to extract and quantify 6 FFPE sections from human colon and lung tissue for matching DNA & RNA pairs. The nucleic acid output plate from the purification instrument is directly transferred to the sequencer for library preparation and sequencing using the Oncomine Comprehensive Assay v3. Variant calling analysis of 6 DNA & RNA pairs are completed the following day. Results show that the variants were detected with ≥ 90% Sensitivity when compared to the Ion Torrent GeneStudio S5 System as an orthogonal method. All variants report p-values ≤ 10^-5 signifying more confidence that the variant call is correct. The Genexus System is a seamless solution for sample to variant calling results. Citation Format: Iris Casuga, Jason Gioia, Janice Au-Young, Frances Chan, Milton Huynh, Michael Crowe. Rapid and accurate variant calling of FFPE samples with the genexus system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2282.

The purpose of this study is to test the speed, scalability, and flexibility of variant detection with the Genexus Integrated Sequencer. This research use only instrument uses nucleic acid input and executes the steps of library preparation, sequencing, analysis, and variant calling. Taking advantage of the instrument's flexibility in sample plexy, the authors used the Oncomine™ Comprehensive Assay v3 and commercially available control samples to detect SNPs, InDels, and fusions reliably at up to 6-plex paired DNA and RNA samples. The instrument can accommodate 1, 2, 4, or 6 paired DNA and RNA samples when using 1, 2, 3, or 4 lanes of the GX5 chip. Regardless of the number of samples analyzed, the system achieved at least 98% sensitivity, 98% positive predictive value, 95% agreement to known truth for de novo variants, and 98% agreement to known truth for hotspot variants in the Acrometrix™ Oncomine Hotspot Control. Also regardless of sample plexy, the system achieved at least 97% agreement to known truth for Fusions within the SeraSeq® Fusion RNA Mix v3. The flexible workflows allow for fast turn-around times of as little as 18 hours for a single sample pair and less than 30 hours for 6 paired DNA and RNA samples. Stability of system initialization is an important feature for flexible use of an NGS system, and read length is a limiting performance metric for evaluating stability. In order to test the Genexus system's read length and stability features the Oncomine™ TCR Beta-LR Assay GX was used. Instruments were initialized with sequencing consumables and tested for performance on day 1 and day 15. There was no statistically significant difference in total reads (3.5 ± 1.0 M at day 1 vs. 3.1 ± 1.0 M at day 15), the fraction of reads that are low quality reads (13% ± 2% vs. 14% ± 2%), or mean read length (301 ± 6 bases vs. 296 ± 6 bases) between the two time points. In conclusion, the Genexus integrated sequencer is a robust NGS system that facilitates rapid turn-around time, flexibility in sample plexy, and stability in system consumables use. Citation Format: Jason Gioia, Iris R. Casuga, Frances Chan, James Commins, David Conners, Marla Kratzer, Collyn Seeger, David Yaccarino. Scalable single-day workflows for variant calling with the Genexus Integrated Sequencer [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 1330.

We report a recurrent microdeletion syndrome causing mental retardation, epilepsy and variable facial and digital dysmorphisms. We describe nine affected individuals, including six probands: two with de novo deletions, two who inherited the deletion from an affected parent and two with unknown inheritance. The proximal breakpoint of the largest deletion is contiguous with breakpoint 3 (BP3) of the Prader-Willi and Angelman syndrome region, extending 3.95 Mb distally to BP5. A smaller 1.5-Mb deletion has a proximal breakpoint within the larger deletion (BP4) and shares the same distal BP5. This recurrent 1.5-Mb deletion contains six genes, including a candidate gene for epilepsy (CHRNA7) that is probably responsible for the observed seizure phenotype. The BP4-BP5 region undergoes frequent inversion, suggesting a possible link between this inversion polymorphism and recurrent deletion. The frequency of these microdeletions in mental retardation cases is approximately 0.3% (6/2,082 tested), a prevalence comparable to that of Williams, Angelman and Prader-Willi syndromes.

PUBLISHED, Background Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. Methods We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. Results We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10?7). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. Conclusions We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype., Supported in part by grants from the National Institutes of Health (NIH) (HD043569, to Dr. Eichler), the South Carolina Department of Disabilities and Special Needs (to Drs. Skinner, Stevenson, and Schwartz), the Wellcome Trust (061183, to Dr. Tassabehji), the Andre & Cyprien Foundation and the University Hospitals of Geneva (to Drs. Antonarakis, Bena, and Gallati), and the European Union (EU) (project 219250, to Dr. Sharp; AnEUploidy project 037627, to Drs. Leeuw, Armengol, Antonarakis, Estivill, Veltman, and de Vries). The Irish Autism Study was funded by the Wellcome Trust and the Health Research Board (a grant to Drs. Gallagher and Gill). Dr. Poot was supported by a grant from the Dutch Foundation for Brain Research (Hersenstichting grant 2008(1) 34); Drs. Regan and Knight, by the Oxford Partnership Comprehensive Biomedical Research Centre; Dr. Willatt, by the Cambridge Biomedical Research Centre, with funding from the United Kingdom Department of Health's National Institute for Health Research Biomedical Research Centres funding scheme; Drs. Huang and Maloney, as part of the National Genetics Reference Laboratory (Wessex) by the United Kingdom Department of Health; Ms. Buysse, as a research assistant of the Research Foundation?Flanders (FWO?Vlaanderen); and Dr. Eichler, as an investigator of the Howard Hughes Medical Institute.

Somatic mutation has been implicated in many aspects of cancer such as susceptibility, diagnosis, prognosis, drug response and tumor progress. Detection of somatic mutation is of wide interest in cancer research. The rapid advances in next generation sequencing (NGS) technologies have transformed cancer research. For example, the Ion AmpliSeq™ technology enables the selective amplification of 10s to 1000s of target sequences in a single multiplexed PCR and meshes seamlessly with the Ion semiconductor sequencing platform. The Ion Ampliseq™ Comprehensive Cancer Panel (CCP) provides ready-access to hundreds of genes, making it ideal for broad targeted re-sequencing studies. Alternative technologies are in demand to validate the NGS data orthogonally and screen hundreds or more cancer samples to evaluate mutation patterns, prevalence and frequencies in population. Here, we have demonstrated the utility of TaqMan® Mutation Detection Assays using our competitive allele specific TaqMan® PCR (castPCR™) technology in validation of Ion Torrent sequencing data. In this study, we applied the Ion Ampliseq™ Comprehensive Cancer Panel (CCP) panel to NCI-60 cell lines (MCF-7, MDA-MB-231, DU-145, PC-3, SK-MEL- 28) derived from breast, prostate, and skin cancers on Ion Torrent PGM sequencer. We confirmed previously reported mutations in these cell lines and identified the mutations that were not reported before, including missense and non-coding mutations. We then selected a subset of mutation targets from the cancer panel for castPCR validation including the genes of KRAS. EGFR, BRAF, NRAS, PIK3CA, PTEN, KIT, TP53, and more. Since limited sample quantity has been a challenging issue for most cancer researchers, especially for those who are interested in testing multi-targets by qPCR, we developed a preamplification method to enrich the targets of interest prior to running castPCR assays. We compared the Cq values of on-targets and off-targets from preamplified samples and non-preamplified samples, and the data indicates that our preamplification strategy not only provides roughly100 fold target enrichment but also maintains specificity during the preamplification process. For mutation detection, we showed that the mutation data from castPCR™ and from Ion Torrent PGM sequencer share high concordance for any given mutations. The detailed comparison and analysis will be presented and discussed. Our results demonstrate that castPCR™ technology is a valuable validation tool for next generation sequencing. The combination of Ion Torrent sequencing and castPCR validation empowers cancer researchers to understand the roles that somatic mutation plays in cancer. Citation Format: Kelly Li, Cora Woo, Mokang Mouanoutoua, Bonnie Moy, Emanuel Langit, Pius Brzoska, Xiaoqing You, Sejal Desai, David Keys, Junko Stevens, Benjamin Kong, Mark Shannon, Shiaw-Min Chen, David Ruff, Chieh-Yuan Li, David Joun, Iris Casuga, Robert Bennett. Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies. [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 4218. doi:10.1158/1538-7445.AM2013-4218

The discovery of pivotal genetic alterations and the understanding of their role in cancer is leading to remarkable successes in therapeutics and patient care. Molecular diagnosis methods such as DNA sequencing and conventional genotyping of tumor biopsies have advanced research in this field, but are limited in sensitivity due to stromal contamination and by genetic heterogeneity in cancer. We have recently developed competitive allele specific TaqMan® PCR (castPCR) assays assays for detecting cancer-associated sequence variations. CastPCR not only maintains the wide dynamic range, high sensitivity and reproducibility of TaqMan® assays but also greatly improves the specificity. The technology enables detection, of as little as 1 mutant allele molecule in 10,000,000 wild type molecules. We report here sensitive and accurate detection of cancer-associated KRAS mutations within formalin-fixed paraffin-embedded (FFPE) heterogeneous cancer specimens. Eight FFPE model cell lines were initially used to validate the assays (NCI-H2009:p.G12A; SW1463:p.G12C; PANC-1:p.G12D; PSN-1: p.G12R; A549:p.G12S; SW480:p.G12V; DLD-1:p.G13D; Jurkat:Wild Type). Mutant FFPE cell line DNAs were titrated in the FFPE wild type cell line DNAs from 100% to 0.1%. Mutations were easily identified at the level of 0.1% with high reproducibility. 24 anonymous tumor tissues and 12 non-tumor tissues from FFPE specimens were also examined. No positive samples were found in non-tumor tissues. The results obtained by castPCR for the 24 tumor tissues were concordant to those previously reported by three different methods (Taqman® PCR, Taqman® PCR + PNA and Sequencing). Our results demonstrate that castPCR, as a new rare mutation detection technology, has greater sensitivity, specificity and can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies. 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 3071. doi:10.1158/1538-7445.AM2011-3071