Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Next-generation biobanking of metastases to enable multidimensional molecular profiling in personalized medicine.

Author(s): Diaz Z, Aguilar-Mahecha A, Paquet ER, Basik M, Orain M, Camlioglu E, Constantin A, Benlimame N, Bachvarov D, Jannot G, Simard MJ, Chabot B, Gologan A, Klinck R, Gagnon-Kugler T, Lespérance B, Samson B, Kavan P, Alcindor T, Dalfen R, Lan C, Chabot C, Buchanan M, Przybytkowski E, Qureshi S, Rousseau C, Spatz A, Têtu B, Batist G

Publication: Mod Pathol, 2013, Vol. 26, Page 1413-24

PubMed ID: 23743930 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the morphology of formalin-fixed, paraffin-embedded (FFPE) specimens; OCT-embedded, snap-frozen specimens; and OCT-embedded, RNAlater-preserved specimens stored at 4˚C or -150˚C with or without washing in PBS. DNA and RNA purity and integrity of OCT-embedded snap-frozen and RNAlater-preserved specimens was also compared. The analytical suitability of DNA and RNA extracted from specimens that were RNAlater-preserved, OCT-embedded was also discussed. Multiple biopsies were obtained using a 16-gauge BioPince full-core, end-cutting, tri-axial automated biopsy device from hepatectomy specimens of patients diagnosed with colorectal cancer. Specimens were: (A) fixed in formalin for 48 h at room temperature and embedded in paraffin, (B) snap frozen in liquid nitrogen and stored at -150˚C for 72 h and embedded in OCT, (C) stored in RNA later at 4˚C for 72 h and OCT-embedded, (D) stored in RNA later at -150˚C for 72 h, washed in PBS on dry ice, and OCT-embedded, or (E) stored in RNAlater at 4˚C for 72 h, washed in PBS on dry ice, and OCT embedded. All OCT-embedded specimens were stored at -80˚C until sectioning at -20˚C. All specimens were sectioned at 5 µM.  DNA and RNA were isolated from snap-frozen specimens and specimens stored in RNAlater at 4˚C for 72 h and washed in PBS on dry ice using the Qiagen AllPrep DNA/RNA Mini Kit. To investigate the suitability of protocol E for molecular analysis, biopsies containing >60% tumor and <20% necrotic cells were obtained from each of 3 patients and neoplastic cells were removed by macrodissection using an H&E stained reference slide.

   Summary of Findings:

   Optimal morphological preservation was observed with the FFPE specimen, but suitable preservation was observed among OCT-embedded, snap-frozen specimens and specimens preserved in RNAlater at 4˚C, washed in PBS, then OCT-embedded (protocol E). RNAlater-preserved specimens that were OCT-embedded but not washed (protocols C and D) did not stain consistently and when frozen, did not adhere to slides. Despite containing 100% tumor, the authors contend some specimens contained so many necrotic and stromal cells that there were not enough neoplastic cells for molecular analysis.  DNA and RNA yield and purity as determined by spectrophotometer and RNA integrity numbers (RIN) were comparable between the OCT-embedded, snap-frozen specimens and RNAlater-preserved specimens (4˚C) that were washed prior to OCT-embedding, but DNA yield was more variable among  RNAlater specimens, which the authors attribute to increased difficulty in homogenization. High molecular weight DNA was obtained from the OCT-embedded, snap-frozen specimens and RNAlater-preserved (4˚C) specimens that were washed prior to OCT-embedding.  DNA from RNAlater-preserved (4˚C) specimens that were washed prior to OCT-embedding was sufficient to detect the expected chromosomal gains (20q, 13 and 8q) and losses (18, 20 and 8p) by array cGH, as well as to detect global hypomethylation and hypomethylation of the CpG  islands in Jun proto-oncogene (JUN), FBJ murine osteosarcoma viral oncogene homolog (FOS), and avian myelocytomatosis viral oncogene homolog (MYC). Using RNA isolated from the three RNAlater-preserved, OCT-embedded specimens it was possible to detect all 138 microRNAs (miRNA) on the low density array, 79 of 96 alternative splice events analyzed using a high-throughput RT-PCR method, and 12,999 expressed genes on the microarray. Importantly, 12 of 25 miRNA previously implicated in colorectal cancer were upregulated in all three specimens and mRNA levels were strongly correlated among the specimens (r=0.88-0.91). Finally, when the data was combined, RNA levels, methylation status, and copy number results all agreed with one another.

   Biospecimens

   • Tissue - Liver

   Preservative Types

   • Formalin
   • OCT
   • RNAlater

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   Morphology	H-and-E microscopy
   RNA	Low density array
   RNA	Automated electrophoresis/Bioanalyzer
   DNA	Electrophoresis
   RNA	DNA microarray
   RNA	Spectrophotometry
   DNA	Array CGH
   DNA	Bisulfite conversion assay
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	RNAlater Snap frozen Formalin (buffered)
   DNA microarray Specific	Technology platform	aCGH Methylation array
   Bisulfite conversion assay Specific	Targeted nucleic acid	JUN FOS MYC
   Biospecimen Preservation	Temperature of fixation/preservation	4˚C -150˚C
   Analyte Extraction and Purification	Washing	Washed in PBS Not washed

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