Junko Stevens

Abstract 4218: Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies.

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

Abstract 2100: Cancer biomarker research using castPCR technology

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Cancer Biomarkers have applications in the diagnosis, staging, prognosis and monitoring of disease progression, as well as in the predication and monitoring of drug response. Profiling and validation research tools are needed that exhibit the combined features of high sensitivity and high specificity for cancers. However, the sensitivity of molecular methods such as DNA sequencing and conventional genotyping in tumor samples is limited, typically ranging from 5-20%. We have recently developed TaqMan® Mutation Detection Assays using our competitive allele specific TaqMan® PCR (castPCR) technology for cancer biomarker research. castPCR assays were tested with >300 tumor research samples (either fresh/frozen or formalin-fixed, paraffin-embedded samples) and cell lines to assess mutation status. The results showed that castPCR technology can robustly detect mutations as low as 0.1% and has >99% concordance to other technologies including PCR-based technology and sequencing. In this study, a large panel of castPCR assays for AKT1, APC, BRAF, CTNNB1, HRAS, KRAS, NRAS, PIK3CA, PTEN and TP53 genes were used for investigating somatic mutations in breast tumor research samples. Initially, 4 model FFPE cell lines were used to validate the assays. Mutant DNAs were titrated in the wild type DNAs from 50% to 0.1%. Mutations were identified down to 0.1% titration with high reproducibility. No false positives were found in non-tumor samples. The results obtained by castPCR assays for 20 breast tumor samples were concordant to those reported by other methods. Our data showed that castPCR technology provides an excellent tool for identifying cancer biomarkers or confirming potential cancer markers such as those obtained by next-generation sequencing and other technologies. 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 2100. doi:1538-7445.AM2012-2100

Abstract 5001: Epigenetic networks in breast cancer

Epigenetic mechanisms are essential for normal development and maintenance of normal gene expression patterns in humans. Recent studies suggest that epigenetic alterations may be the key initiating events in some forms of cancers and global changes in the epigenome are a hallmark of cancers. Phospholipase D (PLD) catalyzes the hydrolysis of phophatidylcholine to generate the lipid second messenger phosphatidic acid (PA) and choline and regulates multiple cellular pathways. Elevated expression of PLD1 promoted cell proliferation and has been associated with the progression and metastasis of multiple cancers. In this work, we showcased the applications of our new epigenetic tools by elucidating the changes in chromatin organization (3C), DNA methylation and microRNA expression that underlies the aberrant expression level of PLD1 in breast cancers. Differences in PLD1 expression and the epigenetic network between tamoxifen responsive (MCF-7) and non-responsive (MDA-MB-231) breast cancer cell lines were compared to associate epigenetic changes with their cancer phenotypes. Effects of DNA demethylation and histone acetylation induced by methyltransferase inhibitor (5-aza-2′-deoxycytidine) and histone deacetylase inhibitor (Trichostatin A) were also investigated to provide insights into the molecular mechanisms of epigenetic drugs. 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 5001. doi:1538-7445.AM2012-5001

Abstract 3070: Simultaneous amplification of multiple genetic/epigenetic tumor markers in a single reaction for high throughput genotyping and aberrant methylation profiling

Matching appropriate therapeutic regimes with tumor genotypes and epigenotypes is of great medical and economical importance. A prerequisite for this approach is thorough and accurate genotype and epigenotype profiling of tumor specimens, which should be performed in precisely defined homogeneous tumor cell populations with minimum non-tumor cell contaminations. However, this can be very challenging due to the genetic and epigenetic heterogeneities and the scarcity of homogeneous tumor cells within a tumor specimen. Multiplex PCR allows high throughput profiling by maximizing the number of targets/reaction in each sample and can reduce the limitations of such sample shortage. Although several multiplex PCR kits have been commercially available for this purpose for some time, their usefulness is restricted by small amplicon size range, less satisfactory amplification uniformity of different amplicons, and occasional failure in amplifying some amplicons, or false positive amplifications. To solve these problems, we have devised a highly specific, optimization-free multiplex PCR formulation (Applied Biosystems Platinum® Multiplex PCR Master Mix) with novel reaction chemistry. We show here that this new formulation is excellent for simultaneous amplification and high throughput profiling of SNPs, deletions, insertions and their wild types of many targets in a single reaction in terms of robustness, uniformity, amplicon size range, compatibility with amplicon GC contents, and the reduction of non-specific primer dimers and false positive/negative products in multiplex PCR. In combination with a primer system of varied lengths and compositions of 5’ stem-loop tag sequences, we demonstrate that even mixtures of multiple adjacent or overlapping alleles in the same locus of EGFR/KRAS/BRAF mutations can be specifically amplified in the same reaction and distinguished by amplicon size differentiation in a single lane of an agarose gel. Moreover, using primer sets specific for methylated and converted DNA, and those specific for unmethylated and converted DNA, we demonstrate that EGFR/KRAS/BRAF mutations and their associated promoter methylation status can be amplified from bisulfite-treated genomic DNA of colon cancer cell lines in a single reaction. In conclusion, the Applied Biosystems Platinum® Multiplex PCR Master Mix and the stem-loop primer system are a “best in class” solution to simultaneous amplification of various types of genetic and epigenetic tumor markers of multiple targets for high throughput genotyping and aberrant methylation profiling. 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 3070. doi:10.1158/1538-7445.AM2011-3070

Abstract 182: Direct bisulfite conversion from archived tumor samples for methylation detection

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC DNA methylation is an important epigenetic mechanism of transcriptional control. DNA methylation plays an essential role in maintaining cellular function, and changes in methylation patterns may contribute to the development of cancer. Aberrant methylation of DNA is frequently found in tumor cells. Formalin-fixed and paraffin-embedded (FFPE) tissue sections are used for routine histopathological diagnostics, but they have increasingly become material for molecular studies of genome using molecular biological techniques such as PCR, qPCR, sequencing, genotyping etc. A major limitation of FFPE samples is the significant degree of degradation of the nucleic acids recovered from fixed tissues especially for the aged, long term archived samples. Since FFPE samples provide such huge collections of clinical tissue bank, more and more efforts have been spent in recovering the high quality of DNA from these specimens. Although the number of epigenetic cancer studies continues to grow, the wealth of FFPE samples available remains largely untapped. The value of FFPE samples and the potential they have to contribute to studies of the epigenome and the role of DNA methylation in numerous biological processes and disease, including cancer has been recognized more and more. Couple methods have been wildly used for methylation detection nowadays. With all the methods available, The bisulfite conversion method remains to be the most commonly used technique today as it can provide single CpG site methylation detection. Currently available methods for genome study or epigenetic study require isolation of gDNA from FFPE samples. With that, all the protocols require deparaffinization of the specimen prior to DNA extraction. Deparaffinization is usually accomplished with 2 to 3 incubations in xylene and can be a nuisance, particularly if there are more than just a few specimens to deal with. We, hereby, develop a new method to not only eliminate the deparaffinization but also eliminate the genomic DNA purification procedure. In our procedure, FFPE samples are directly threw in the denaturation/lysis reagent followed by bisulfite conversion and desulfonation procedure. The whole process can be completed within 2 hours. Even for long term archived samples, fair length of DNA fragments can be captured after the conversion. High quality of converted gDNA fragmented are produced which can be used for methylation detection at single CpG site level such as methylation profiling, deep sequencing etc. Clinical archived breast cancer and adjacent normal tissues as well as paired lung cancer samples were directly bisulfite converted and both breast cancer and lung cancer related promoter regions such as APC, BCL2, PTEN, and SCGB3A1 were compared for the methylation pattern changes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 182.

Abstract 2187: Optimization free oncogene mutation screening work flow

BRCA1 and BRCA2 are the two primary breast cancer susceptibility genes in which identifying mutations is important to access cancer risk and decision for treatment. Prescreening of exons by High Resolution Melt (HRM) prior to sequencing can reduce effort in mutation screening in large transcripts such as the BRCA genes. Published methods for BRCA1 and 2 HRM screening have relied on complicated touchdown cycling protocols and custom reagent mixes that can lead to variable result. We have validated a streamlined workflow for adaptability in a clinical diagnostic environment. First, genomic DNA template was extracted on an automated sample processor using magnetic bead technology to purify genomic DNA directly from blood sample. Then, the PCR was performed using a PCR master mix optimized for HRM coupled with universal cycling conditions. We have successfully amplified all 120 amplicons covering exons of both BRCA genes using universal PCR condition. Potential variations identified by HRM screening were confirmed by direct sequencing of HRM PCR amplicons with M13 universal tags. By using unified protocol for PCR and universal sequencing for all amplicons the risk of incorrect procedure is greatly reduced. This simplified workflow for oncogene mutation screening using automated sample extraction and universal protocol for PCR and sequencing provides significant advantages for application in diagnostic settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2187.