DNA copy number (CN) studies hold great promise for the discovery of clinical biomarkers to predict disease course, recurrence risk, and response to therapy. The molecular characterization of a tumor genome across many samples helps to classify cancers in a biologically and clinically relevant manner. While exciting results have been found with genes already known to be involved in key pathways, confirming early results and genome-wide testing requires large numbers of well-characterized clinical samples. A vast collection of hundreds of millions of stored FFPE samples already exists. Unfortunately, many genomic assays fail to produce high-quality CN and genotype data from FFPE samples, given the degradation and chemical changes to the DNA and the small quantity obtained from tiny tissue samples. The higher rate of sequencing artifacts and genotyping failures in FFPE samples restricts the application of these promising whole-genome scanning technologies to the limited number of fresh-frozen samples. OncoScan™ FFPE Assay Kit is designed to interrogate the whole genome for copy number aberrations (CNAs), loss of heterozygosity (LOH), and selected somatic mutations (SMs) and captures the alleles of over 220,000 SNPs at carefully selected genomic locations. These SNPs are evenly distributed across the genome and with increased density within ∼900 cancer-related genes. Based on Molecular Inversion Probe (MIP) technology, several OncoScan Assay Kit features make it highly suitable for the analysis of FFPE tissue-derived DNA. The assay uses only 80 ng input DNA with no requirement for amplification of the genomic DNA; rather, the probe itself is amplified, leading to a high signal-to-noise ratio. The entire assay can be run such that results can be obtained within 48 hours of gDNA extraction. Cancer sample data are complex because each sample is inherently mosaic, comprised of a mixture of normal cells along with one or multiple tumor clones. OncoScan FFPE Assay Kit includes a component algorithm that specifically addresses the issue of normal contamination in the tumor sample. In this study, we will describe the characterization of genomic variations in a set of 67 lung tumor samples collected in collaboration with Cancer Research UK (CRUK). From each sample, 80 ng FFPE gDNA has been extracted and purified as input to OncoScan Assay Kit. Collectively, the tumor genomes showed frequent CN gains on chromosomes 1q, 5p, 6p, 7, 8q, and 17q and frequent CN losses on chromosomes 6q and 17p. The number of structural aberrations per sample ranged from 3 to 340, with an average of 94. The assay simultaneously interrogates 74 SMs of interest using a panel of 64 MIPs. Forty-nine samples had at least 1 SM call (1 sample having a maximum of 4). Citation Format: Ron Sapolsky, Anju Shukla, Sumathi Venkatapathy, Chuan Chen, Carsten Bruckner, Vicky Huynh, Liansen Liu, Xuan Shen, Kent Suyenaga, Patrick Weaver, Wai Wu, Bitao Liu, Matt Ghent, Benjamin Bolstad, Farooq Siddiqui, Diana Abdueva, Mirjana Alvi, Eric Fung, Jeanette Schmidt, Lawrence Greenfield. Molecular Inversion Probe analysis using OncoScan™ FFPE Assay Kit to detect copy number aberrations and somatic mutations in lung tumor DNA samples from formalin-fixed paraffin-embedded (FFPE) tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4655. doi:10.1158/1538-7445.AM2014-4655

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