Integrative Copy Number and Mutational Analysis Improves Glioma Diagnostics

Abstracts We report on the molecular cytogenetic findings of a 10-yearold male with a history of mild mental retardation (IQ 66), mood disorder, anxiety, disruptive behavior and lazy eye and recent onset of seizures. DNA isolated from peripheral blood was analyzed by chromosome microarray and revealed a mosaic non-integer copy number gain of 29.6 Mb on the short arm of chromosome 5 between bands 5p13.3 and 5p15.3 (copy number state of w2.3) suggesting 30% of cells have a 5p15.3p13.3 duplication. No other relevant copy number variants were detected. The clinical manifestations of patients with partial trisomy of 5p are variable and seem to depend on the position and size of the duplication. More proximal duplications or those involving the entire p-arm usually result in a more severe phenotype in comparison to duplications distal to 5p13. Copy number gains of 5p are uncommon and often the result of an unbalanced translocation. With the exception of a small number of cri-du-chat syndrome patients mosaic for both 5p deletions and 5p duplications, mosaic duplications of this region have not been reported. Additional cytogenetic testing is in progress to further characterize the chromosomal abnormality and to examine the level of mosaicism in other tissues.

Integrative Copy Number and Mutational Analysis Improves Glioma Diagnostics Shakti H. Ramkissoon a,c,f, Wenya L. Bi b,d,e, Steven E. Schumacher a,d,f, Lori A. Ramkissoon c, Sam Haidar c, Adrian M. Dubuc a, Loreal Brown d, Margot Burns d, Jane Cryan a, David A. Reardon d, Eudocia Q. Lee d, Mikael L. Rinne d, Andrew D. Norden d, Lakshmi Nayak d, Sandra Ruland d, Lisa M. Doherty d, Debra C. LaFrankie d, Andrea Russo e, Nils D. Arvold e, Elizabeth B. Claus b, Ossama Al-Mefty b, Mark D. Johnson b, Alexandra Golby b, Ian F. Dunn b, E. Antonio Chiocca b, Sandro Santagata b,f, Rebecca D. Folkerth b,f, Philip Kantoff d, Barrett J. Rollins d, Neal I. Lindeman a, Patrick Y. Wen d, Rameen Beroukhim d, Azra H. Ligon a,f, Brian M. Alexander d, Keith L. Ligon a,f a Department of Pathology, Brigham and Women’s Hospital, Boston, MA; b Department of Neurosurgery, Brigham and Women’s Hospital, Boston, MA; c Center for Molecular Oncologic Pathology, DanaFarber Cancer Institute, Boston, MA; d Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA; e Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA; f Harvard Medical School, Harvard Medical School, Boston, MA

Genomic (copy number, mutational) profiling of formalin-fixed paraffin-embedded (FFPE) samples has potential to improve cancer diagnostics, identifying characteristic aberrations relating to grade and/or histology; however, the use of this information in a CLIA setting is not yet routine. We sought to report our experience with the implementation of comparative hybridization genomic (CGH) and mutation-testing program across a series of > 600 primary brain tumors. For glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor, comparative genomic hybridization (nZ236) and targeted mass spectrometrybased mutation genotyping (nZ94) clinical testing profiles were collected and analyzed. These results were compared to large genomic datasets generated from fresh frozen primary tumors profiled through the TCGA. Across our cohort clinically relevant loci were detected, including: 1p/19q co-deletion, EGFR, EGFRvIII, PDGFRA, CDKN2A and PTEN, as well as recurrent mutations in IDH1, TP53 and PTEN. Patients >40 years old showed a

287 classic copy number profile for GBM, including polysomy 7, EGFR amplification, CDKN2A loss or PTEN deletion.

Detection of Copy Number Variations in Breast Cancer Samples Using Single-nucleotide Polymorphismtargeted Massively Multiplexed PCR Joshua E. Babiarz, Bernhard G. Zimmermann, Tudor Constantin, Ryan Swenerton, Eser Kirkizlar, Nicholas Wayham, Matthew Rabinowitz, Matthew Hill Natera, Inc., San Carlos, CA

Evaluation of copy number variation (CNV) in tumor tissues typically involves SNP microarray or aCGH. These methods have high whole-genome resolution, but require large amounts of input material, have high fixed costs, and falter on FFPE samples. Here, we employ a 28,000-plex SNP-targeted PCR with NGS, targeting chromosomes 1, 2, 13, 18, 21 and X, and regions 4p16, 5p15, 7q11, 15q, 17p, 22q11, and 22q13, to detect CNVs from fresh and FFPE samples. An accuracy of 100% was demonstrated on 96 samples: 25 unaffected and 71 affected (minimum 3 samples per region). Single-molecule sensitivity was established by analyzing single cells. Among five matched tumor and normal cell lines all five tumor samples had multiple amplifications and deletions (median: 13), whereas CNVs were absent in the matched controls. Of 15 fresh frozen and 2 FFPE breast tumors samples, 14 fresh and both FFPEs showed full or partial CNVs in at least one and up to 15 regions (median: 7); all had a CNV of either 1q or 17p, the two most common breast cancer CNVs represented on this panel. Evidence of tumor heterogeneity was also observed. Although not investigated here, the very low input requirements will allow this assay to analyze tumor heterogeneity. A similar assay targeting all chromosome arms would result in a sequencing cost of approximately $5 per sample. Thus, this method offers a powerful, efficient, and scalable approach for investigating large numbers of fresh frozen or FFPE samples.

Non-invasive Cell-free Tumor DNA-based Detection of Breast Cancer-related Copy Number Variations Bernhard G. Zimmermann 1, Eser Kirkizlar 1, Matthew Hill, Tudor Constantin, Styrmir Sigurjonsson, Bin Hoanga, Nikhil Chopra, Matthew Rabinowitz Natera, Inc., San Carlos, CA

Breast cancer screening involves mammography, which has high false positive rates and misses some cancers1-4. Analysis of tumor-derived circulating cell-free DNA (ctDNA) for cancerassociated copy number variations (CNVs) may allow for earlier, safer, and more accurate screening. Here, we employed a singlenucleotide polymorphism (SNP)-based massively multiplex PCR (mmPCR) approach to screen for CNVs in ctDNA isolated from the plasma of breast cancer patients. The mmPCR assay targeted 3,168 SNPs on chromosomes 1, 2, and 22q, which often have CNVs in cancer (e.g., 49% of breast cancer samples have a 22q deletion5). Here, we present case studies of six breast

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These authors contributed equally.