NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Pre-analytical effects on whole transcriptome and targeted RNA sequencing analysis in cytology: The effects of prolonged time in storage of effusion specimens prior to preservation.

Author(s): Sura GH, Tran K, Fu C, Du L, Marczyk M, Gould RE, Chen E, Tasto AM, Tinnirello AA, Symmans WF

Publication: Cytopathology, 2023, Vol. , Page

PubMed ID: 37712171 PubMed Review Paper? No

Purpose of Paper

This paper compared targeted and whole transcriptome RNA sequencing (wtRNASeq) of transcriptomes in cytology specimens stored at 4°C for up to one week that were either preserved in RNAlater and frozen or prepared as either Carnoy’s- or ethanol-fixed cytospins before analysis.

Conclusion of Paper

Generally, SETER/PR and PI3Kges gene expression, which were analyzed by targeted RNASeq and wtRNASeq, were unchanged or decreased slightly after refrigerated storage of effusion specimens for up to one week.  Concordance and bias coefficients were higher for RNAlater-preserved cytology specimens when the cytospin specimen was fixed in Carnoy’s rather than ethanol. Principal component analysis showed that most of the observed variability was due to patient source, followed by storage, and then the method of cytological preservation. Mixed effects modeling identified significantly lower expression of the PI3Kges signature in ethanol-fixed cytospin specimens than in RNAlater-fixed specimens. Similarly, expression of the SETER/PR signature decreased with storage and had a higher variance in cytospin samples fixed in 95% ethanol.  The mixed effects model showed that overall gene expression was comparable between RNAlater- and cytospin-preserved specimens and storage timepoints.  Importantly, all mutations detected in specimens stored for 0 days were found at all subsequent storage timepoints and, generally, the allele frequency remained the same with storage.    

Studies

  1. Study Purpose

    This study compared targeted and wtRNASeq of transcriptomes in cytology specimens stored at 4°C for one week and preserved in RNAlater and frozen or prepared as either Carnoy’s- or ethanol-fixed cytospins. Ten leftover effusion specimens containing metastic breast cancer cells were stored at 4°C for 0, 1, 4, and 7 days before processing. After storage, aliquots were filtered through gauze and then centrifuged at 504 g for 10 min. For specimens that were blood-tinged, blood was removed using Ficoll-Hypaque gradient separation. Matched specimens were placed in RNAlater (3 drops in 0.8 mL and stored at -80°C), or placed in a cytospin device (cytocentrifugation at 64 g, smeared, fixed in Carnoys fixative or 95% ethanol, stained with Papanicolaou, and stored at room temperature). A cytopathologist reviewed all stained slides and estimatedtumor cellularity to be >10% for all specimens. The RNAlater specimen was thawed at room temperature, and an aliquot was mixed with PBS, centrifuged at 5000 g for 10 min, resuspended in Qiazol, and phase separated with chloroform before RNA extraction using the RNeasy Micro Kit.  Cytospin slides were soaked in xylene, scraped into tubes, washed in ethanol, and air-dried before RNA extraction with a PicoPure RNA isolation Kit. When the ratio of absorbance at 260 nm to 230 nm was less than 1, RNA was reprecipitated with ethanol. RNA was quantified by Nanodrop spectrophotometry. Targeted RNA sequencing was performed using the SET4 assay and sequenced on an Illumina MiSeq instrument. wtRNASeq libraries were prepared using a RNA Hyper-Prep Kit with RiboErase and pair-end sequenced on a NovaSeq 6000 instrument using the S4 Reagent Kit. The SET4 assay includes SETER/PR (ratio of transcripts with ESR1 and PGR activating mutations to reference) and PI3Kges (ratio of transcripts with activating mutations in PIK3CA mutations in exon 9 and 20 and a 9 gene transcriptional signature).

    Summary of Findings:

    Generally, SETER/PR and PI3Kges gene expression, assessed by targeted RNASeq and wtRNASeq, were unchanged or decreased slightly after refrigerated storage of effusion specimens.  Concordance was higher with the RNAlater specimen when the cytospin specimen was fixed in Carnoy’s rather than in ethanol (ρ=0.98-0.99 versus ρ=0.84-0.98 for targeted RNASeq and ρ=0.93-0.99 versus ρ=0.71-0.99 for wtRNASeq). Similarly, the bias coefficient was lower when the cytospin sample fixed in Carnoy’s (as opposed to  ethanol) was used for comparison (0.97-1.0 versus 0.87-0.99).  Principal component analysis showed that most of the observed variability was due to patient source, followed by storage, and then cytological preservation method.  Gene signatures using either sequencing method were highly reproducible between replicates at all timepoints, with the exception of SETER/PR in ethanol-fixed cytospin specimens on day 0. The mixed effects model identified significantly lower expression of the PI3Kges signature in ethanol-fixed cytospin specimens than RNAlater-fixed specimens (P=1.3E-07), but no effect of storage was observed. Similarly, expression of the SETER/PR signature decreased with storage (P=9.3E-07) and had a higher variance in cytospin specimens that were fixed in 95% ethanol (P=5.0E-11).  The mixed effects model showed that overall gene expression was comparable between RNAlater and cytospin specimens and storage timepoints.  Importantly, all mutations detected in specimens stored for 0 days were also found at all subsequent timepoints and, generally, the allele frequency remained the same with storage.    

    Biospecimens
    Preservative Types
    • Ethanol
    • Other Preservative
    • RNAlater
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    RNA Spectrophotometry
    RNA Next generation sequencing
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 0 days
    1 day
    4 days
    7 days
    Biospecimen Preservation Type of fixation/preservation Carnoy's solution
    Ethanol
    RNAlater

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