Abstract 3492: A next generation sequencing biopsy protocol
Authors: Kossai Myriam , Schiffman Marc , Elemento Olivier , Sboner Andrea , Fontugne Jacqueline , Kim Rob , Mosquera Juan Miguel , Beltran Himisha , Rubin Mark A. 
Published: May 2015

Abstract
Background
Sampling metastatic tumor sites, especially bone, can be challenging, at times yielding suboptimal tissue even for diagnosis. With introduction of next generation sequencing (NGS) in cancer care, high quality genomic material is required. The following is our multi-disciplinary approach to validate an effective biopsy protocol and tissue processing that generates superior samples for clinical and research application.
Design
After informed consent as part of an IRB approved protocol, image-guided biopsies of metastatic sites were performed on 42 consecutive cancer patients. Novel interventional radiology tools were implemented on a case-by case basis (i.e. OnControl drill for sclerotic bone metastases). Fresh tissue was transported on wet ice to Pathology. After gross examination, a photograph was taken and tissue was placed on Tissue Tek™ OCT, frozen in isopentane/dry ice combination to avoid freeze artifact. An H&E stained section was reviewed for diagnosis confirmation and evaluation of tumor content. If there were multiple biopsies, each core was embedded separately. After evaluation of all slides, a selected sample was converted to formalin-fixed paraffin embedded tissue for further assays (i.e. IHC, FISH). Slides were annotated to highlight areas for DNA/RNA extraction, then scanned. Real-time updates were provided until pathologic evaluation is finalized. A minimum of 2µg DNA and RNA (RIN&gt; 6) was extracted for sequencing. Corresponding germline DNA from blood is extracted and used to prepare sequencing libraries to cover the whole exome. Sequencing was performed using Illumina HiSeq2500 under rapid run mode (average duration 1.5 days). When amount of tissue was enough, samples were utilized for organoid and xenograft model development.
Results
We achieved &gt;95% success rate in obtaining high-quality material for NGS using the OnControl drill to biopsy bone sites, 100% success rate for all other sites. The amount of extracted DNA ranged between 200 and 9339.14ng (median 2221.2ng). On average, 99.5% (&gt;170M) and 95.8% (&gt;70M) of paired-end reads generated have been mappable, confirming the high quality of extracted DNA and RNA, respectively.
Conclusion
We have developed a multi-disciplinary approach to validate an effective biopsy protocol that increases the efficiency and quality of metastatic tumor samples for NGS sequencing. The essential role of the pathologist is highlighted. This protocol has been implemented for Precision Care and Clinical Trials at our Institution and has now recently been successfully implemented by various other sites as part of an ongoing Stand Up To Cancer -PCF Dream Team grant, thus demonstrating wide applicability.
Citation Format: Myriam Kossai, Marc Schiffman, Olivier Elemento, Andrea Sboner, Jacqueline Fontugne, Rob Kim, Juan Miguel Mosquera, Himisha Beltran, Mark A. Rubin. A next generation sequencing biopsy protocol. [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 3492. doi:10.1158/1538-7445.AM2014-3492