Abstract 64: Quality and feasibility of a protocol for simultaneous isolation of RNA, DNA and tissue for IHC from needle core lymph node biopsies in DLBCL adapted for multi-center trials.
Authors: Nielsen Torsten H. , Diaz Zuanel , Christodoulopoulos Rosa , Yao Lu , Qureshi Samia , Benlimame Naciba , Camlioglu Errol , Crump Michael , Morin Ryan D. , Johnson Nathalie , Haliotis Tina P. , Miller Wilson H. , Assouline Sarit , Mann Koren K. 
Published: August 2014

Abstract
Introduction: Personalized medicine strategies targeting specific aberrations unique to an individual tumor, are promising to become a new paradigm in the fight against cancer. Such tailored approaches rely on the thorough profiling of malignant tissues by high-throughput technologies followed by selection of appropriate treatment targeting tumor-specific aberrations. One fundamental challenge, whose resolution is necessary for any successful personalized medicine endeavor, is isolation of useful amounts of good quality molecular material for reliable tumor characterization in downstream assays. Here, we describe our standardized protocol for biopsy collection and results from a multi-center phase II clinical trial investigating the use of panobinostat with or without rituximab in relapsed diffuse large B-cell lymphoma (NCT01238692).
Results: Biopsies are performed with patient consent, before treatment initiation and after 15 days of treatment, in patients with safely accessible lesions. Four needle core biopsies are collected at each time point using standard operating procedures to limit pre-analytical variability. Of these, one is preserved in formalin for immunohistochemical (IHC) analyses, while the remaining three are pooled together in RPMI 1640 media. These samples are sent to a central laboratory for further processing. The needle cores in RPMI 1640 undergo B-cell purification using a negative selection kit (StemCell Technologies). Isolation of tumor RNA and DNA is performed using a commercially available kit (Qiagen).
Of the 23 patients currently enrolled, 16 (70%) have had a pre-treatment biopsy performed and of these, 7 (44% of those biopsied, 30% of all patients) have also had a post-treatment biopsy, demonstrating the feasibility of tissue collection at participating sites. We assess the effect of transport on the quality and yield of RNA and DNA as well as differences in quality and yield between pre-treatment biopsy vs. biopsies at day 15. The material isolated from these biopsies is evaluated for suitability in downstream applications, namely IHC, gene expression microarray and DNA exome sequencing.
Conclusions: The development of standard operating procedures for the collection and processing of biospecimens is essential to control for pre-analytical variability inherent to multicenter trials. Our experience shows that this is both feasible and crucial to understanding of tumor molecular profile changes associated with treatment. Our protocol allows extraction of RNA and DNA of sufficient quality and quantity to permit downstream multi-dimensional analysis. Patient acceptance of research biopsies has been high although the rate of pre-treatment biopsies is greater than that of post-treatment biopsies. Finally, our work demonstrates that the timing of post-treatment biopsies is critical.
Citation Format: Torsten H. Nielsen, Zuanel Diaz, Rosa Christodoulopoulos, Lu Yao, Samia Qureshi, Naciba Benlimame, Errol Camlioglu, Michael Crump, Ryan D. Morin, Nathalie Johnson, Tina P. Haliotis, Wilson H. Miller, Sarit Assouline, Koren K. Mann. Quality and feasibility of a protocol for simultaneous isolation of RNA, DNA and tissue for IHC from needle core lymph node biopsies in DLBCL adapted for multi-center trials. [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 64. doi:10.1158/1538-7445.AM2013-64