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The Biospecimen Pre-analytical Variables (BPV) Program of the Biorepositories and Biospecimen Research Branch (BBRB) at the National Cancer Institute (NCI) is designed to systematically investigate the effects of preanalytical factors on the molecular integrity of biospecimens. Specific parameters related to formalin fixation and paraffin embedding (FFPE) of cancer tissues were examined, including the effects of cold ischemic time (delay to fixation (DTF)) and time in fixative (TIF). Additional preanalytical studies focus on snap freezing method for tissue specimens (dry Ice vs. LN2 vapor), storage temperature (-80°C vs. LN2 vapor), and duration of storage for frozen specimens. Biospecimens for the BPV program were collected at four medical centers, within a tightly regulated infrastructure and strict adherence to SOPs, to enable consistent collection and handling across all sites. Biospecimens were collected from research participants undergoing surgical treatment for renal cell carcinoma; ovarian, fallopian tube, and peritoneal carcinoma; lung adenocarcinoma and squamous cell carcinoma; and colorectal adenocarcinoma. Biospecimens were subjected to specific experimental protocols to systematically vary the preanalytical factors of interest. Extensive annotation was performed with 300+ data elements that include steps in the collection, handling, and processing of the biospecimens, pathological evaluation of the tumor, and clinical information collected from donors. Biospecimen collections concluded in April 2015 with a total of 364 tumor tissue cases collected. The BPV program has conducted multiple, simultaneous molecular analyses to understand the impact of FFPE preanalytical factors on the expression and detection of various molecular analytes. Initial efforts were focused on evaluating the impact of DTF and TIF on the quality of DNA and RNA from FFPE samples using multiple methods and approaches such as NanoDrop 8000 UV-Vis spectrophotometer, Qubit 2.0 Fluorometer, Agilent Bioanalyzer and KAPA Human Genomic DNA Quantification. The QC data from both RIN and Kappa assays showed that FFPE samples have a significant drop in RNA and DNA quality compared with matched frozen samples. 72 hr TIF samples showed a significant drop in both RNA and DNA quality compared to shorter time points (6, 12, or 23 hr TIF), as measured by DV200 and Kappa assays. No significant differences were observed in RNA/DNA quality between the shorter TIF time points or between the DTF time points (1, 2, 3, 12 hr DTF). Further studies are now underway to evaluate additional preanalytical factors. The data from BPV studies will be widely shared with the research community through publication and deposition at a public data repository. The results from these studies will be used to develop evidence-based protocols and best practices for fit-for-purpose collection, processing, and storage of biospecimens. This project is funded by NCI Contract No. HHSN261200800001E. Citation Format: Rachana Agarwal, Ping Guan, Mary Barcus, Jasmin Bavarva, Robin Burges, Philip Branton, Latarsha Carithers, Corinne Camalier, Biswajit Das, Jason Lih, Hana Odeh, Nancy Roche, Dan Rohrer, Michael Sachs, Leslie Sobin, Jewell Scott, Anna Smith, Conrado Soria, Kimberly Valentino, Dana Valley, Mickey Williams, Helen Moore. Impact of biospecimen pre-analytical factors on molecular analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1377.

The Multiple Myeloma Research Foundation (MMRF) CoMMpass®; trial opened in July 2011 and will follow 1000 patients with newly diagnosed multiple myeloma (ND-MM) [http://clinicaltrials.gov/ct2/show/NCT01454297]. The goal of this study is to understand the molecular and genetic changes underpinning the evolution of myeloma in individuals and to facilitate future clinical trials based on those results. Comprehensive clinical annotation will accompany individual molecular profiles at baseline, treatment response and disease recurrence over the course of 5-8 years. The study will yield an extensive molecular and clinical database providing information on the longitudinal aspect of multiple myeloma disease progression in individual patients. Eligible patients include those with symptomatic ND-MM who have not received prior therapy. Initial treatments permitted in the study include those containing either a proteasome inhibitor, an IMiD or both. Centralized bone marrow aspirate and peripheral blood specimen processing building upon the experience of the Multiple Myeloma Research Consortium (MMRC) Tissue Bank is being conducted to support profiling of the patient samples. Here we describe the workflow for biospecimens and characterization including the analysis of bone marrow aspirates by multiplex flow cytometry to identify patient-specific immunophenotypic markers and sequencing of BRAF V600 mutations. CD138-enriched tumor cells derived from the aspirates will enable a breadth of sequencing analysis comprising whole genome and whole exome sequencing, as well as, transcriptome analysis via RNA Seq. Chromosomal alterations and ploidy will be assessed by cytogenetic and FISH analysis. Altogether, these efforts will provide an unprecedented myeloma dataset on copy number variation, mutation and levels of gene expression. Samples collected at suspected complete response will be evaluated by quantitative flow cytometry using multiple markers to follow immunophenotypic changes and to monitor minimal residual disease. The MMRF CoMMpass® study aims to comprehensively catalog genetic alterations in patients treated with standard-of-care 1st line agents and at milestones of relapse. The knowledge obtained in CoMMpass® will provide key insights into mechanisms of disease and drug response in myeloma, reveal targets and biomarkers for drug development, provide insights into refined classification of subtypes of myeloma and be used as a foundation to build personalized medical care initiatives for myeloma patients. 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 4561. doi:1538-7445.AM2012-4561