Next-generation prostate cancer biobanking: toward a processing protocol amenable for the International Cancer Genome Consortium

Abstract

Next-generation DNA and RNA sequencing requires intact nucleic acids from high-quality human tissue samples to better elucidate the molecular basis of cancer. We have developed a prostate biobanking protocol to acquire suitable samples for sequencing without compromising the accuracy of clinical diagnosis. To assess the clinical implications of implementing this protocol, we evaluated 105 consecutive radical prostatectomy specimens from November 2008 to February 2009. Alternating levels of prostate samples were submitted to Surgical Pathology as formalin-fixed, paraffin-embedded blocks and to the institutional biobank as frozen blocks. Differences in reported pathologic characteristics between clinical and procured specimens were compared. Clinical staging and grading were not affected by the biobank protocol. Tumor foci on frozen hematoxylin and eosin slides were identified and high-density tumor foci were scored and processed for DNA and RNA extractions for sequencing. Both DNA and RNA were extracted from 22 cases of 44 with high-density tumor foci. Eighty-two percent (18/22) of the samples passed rigorous quality control steps for DNA and RNA sequencing. To date, DNA extracted from 7 cases has undergone whole-genome sequencing, and RNA from 18 cases has been RNA sequenced. This protocol provides prostate tissue for high-throughput biomedical research and confirms the feasibility of actively integrating prostate cancer into The Cancer Genome Atlas Program, a member of the International Cancer Genome Consortium.

Figure 1

Biobank Workflow: Illustrates the Standard Operating Procedures of the Weill Cornell Medical College Biobank for patient consent, specimen collection, specimen processing, pathology examination, data collection, and storing samples and slides. The workflow is designed to provide the best clinical care with concurrent collection of well-annotated biospecimens for future research.

Figure 2

Illustrates the grossing protocol for prostate gland from radical prostatectomy. The seminal vesicles and vas deferens are removed at the prostate base. The base and apical margins are resected from the prostate gland and cut into perpendicular sections. The remaining prostate is sectioned perpendicular to urethral axis and each level is quartered retaining glandular orientation.

Figure 3

Digital images by Ariol Platform (Genetix, San Jose, CA) demonstrate how blue and red framings are marked on frozen H&E slides. Corresponding 20X photomicrographs show tumor foci with >90% tumor area and <90% tumor area, respectively. DNA samples from STID 3027 and 3043 were sequenced at the Broad Institute (Cambridge, MA).

Figure 4

A To assess the integrity of the genomic DNA, gel electrophoresis is performed with 50ng of DNA. Only the samples with DNA of high molecular weight as the large majority were selected as candidates for SNP arrays and whole genome sequencing assays. B The quality of the RNA is determined using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with Agilent RNA 6000 Nano Kit. RIN above 7 is required to be considered for gene expression studies.