Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Biobanking of Fresh-Frozen Human Adenocarcinomatous and Normal Colon Tissues: Which Parameters Influence RNA Quality?

Author(s): Galissier T, Schneider C, Nasri S, Kanagaratnam L, Fichel C, Coquelet C, Diebold MD, Kianmanesh R, Bellon G, Dedieu S, Marchal Bressenot A, Boulagnon-Rombi C

Publication: PLoS One, 2016, Vol. 11, Page e0154326

PubMed ID: 27124490 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of patient age and sex, location of the tumor within the colon, kind of surgery (right hemicolectomy; transverse, left or total colectomy; sigmoidectomy; or high anterior resection), surgical approach (laparoscopic vs. open colectomy), cold ischemia time, specimen weight, cellular composition,  storage duration,  IHC staining of apoptotic (caspase 3) or ischemia markers (HIF-1α), and mutational status on the percentage of normal and adenocarcinoma colon specimens with an RNA quality index (RQI) of greater than 5 The study also evaluated if RNA integrity of select specimen by real-time RT-PCR amplification of products from the 3’ and 5’ ends. Specimens (241 adenocarcinoma tumor and 115 normal colon tissues) collected from October 2006 to December 2012 were split in half and snap-frozen in LN followed by -80°C storage or formalin-fixed and paraffin-embedded (FFPE) for histopathological analysis. FFPE sections were immunohistochemically stained or hematoxylin-phloxine-saffron (HPS) stained. HPS-stained sections were evaluated by two pathologists for the ratio of malignant to stromal cells (percentages of stromal inflammatory cells, endothelial cells, and fibroblasts were assessed separately), and cellularity density score (0 = low, 1 = moderate, and 2: high) among other parameters. RNA was extracted from the frozen specimens using the Promega Maxwell®16 LEV simply RNA tissue kit, measured spectrophotometrically, and RNA quality index (RQI) was determined using the Bio-Rad Experion™ automated electrophoresis system. Twelve specimens with RQI between 2.4 and 9.3 were analyzed by real-time qRT-PCR amplification of both the 3’ and 5’ ends of TATA-Binding Protein (TBP) and Beta-2-microglobulin (B2M). BRAFV600E mutation detection was performed using TaqMan, KRAS gene mutations were searched by pyrosequencing analysis using the Qiagen Pyromark Q96 kit, and MSI status was determined by multiplex PCR using the Promega MSI Analysis System kit, Version 1.2.

   Summary of Findings:

   Mean RQI was 1.7 times higher for tumor specimens than matched normal specimens (P<0.0001). Although patient age and sex, tumor location (left versus right colon), surgical approach and type, cold ischemia time, specimen weight, and storage duration were not significantly different among normal or tumor specimens with an RQI < 5 and those with an RQI ≥ 5 (P>0.05 for all), an RQI ≥ 5 was found for significantly more left-sided colon adenocarcinomas than right-sided adenocarcinomas (115/137, 83.9% vs. 73/104, 70.2%; P=0.01) and for more tumors obtained by open colectomies than laproscopic colectomies (97.7 versus 2.3%, P=0.01).  Further, lower RNA quality in tumor specimens was found to be associated with occurrence of anastomotic leakage (OR=0.03).  MSI, BRAF, or KRAS mutational status; and caspase 3 and HIF-1α IHC staining were not significantly different among specimens with an RQI < 5 and those with an RQI ≥ 5. While RQI was not affected by percentage of stromal inflammatory cells, endothelial cells, or fibroblasts (P = 0.95, P = 0.41, and P = 0.86; respectively); specimens with RQI ≥ 5 had a higher stromal cell to malignant cell ratio (mean of +8.7%, P <0.05). As expected, specimens with an RQI > 5 had lower ratios of 3’ to 5’ PCR products than those with an RQI < 5 for B2M (2.89 versus 5.01, respectively) and TBP (1.36 and 3.18, respectively). Only 66 of the 241 specimens (27.3%) contained >60% malignant cells and in seven of these necrosis affected >20% of the specimen surface. Thus only 59 of 241 cases met the requirements for molecular analysis according to the Cancer Genome Atlas (TGCA).  

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   Morphology	Light microscopy
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Preaquisition	Patient age	43–90 Years
   Preaquisition	Surgical procedure type	Right hemicolectomy Transverse colectomy Left colectomy Total colectomy Sigmoidectomy High anterior resection
   Preaquisition	Diagnosis/ patient condition	Healthy Adenocarcinoma
   Storage	Storage duration	6.2 ±1.9 years 6. ±1.8 years
   Real-time qRT-PCR Specific	Targeted nucleic acid	TBM B2M KRAS BRAF MSI MSI
   Biospecimen Aliquots and Components	Aliquot size/volume	0.18 ± 0.12 0.19 ± 0.07
   Biospecimen Acquisition	Cold ischemia time	70.2 ± 50.9 minutes
   Immunohistochemistry Specific	Targeted peptide/protein	Caspase 3 HIF-1α
   Biospecimen Acquisition	Biospecimen location	Right colon Left colon
   Biospecimen Acquisition	Method of tissue acquisition	Laproscopic surgery Open surgery

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