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

Molecular testing opportunities on cytology effusion specimens: the pre-analytic effects of various body fluid cytology preparation methods on RNA extraction quality and targeted sequencing.

Author(s): Sura GH, Tran K, Fu C, Du L, Marczyk M, Martinez Y, Tinnirello AA, Gould RE, Lau R, Symmans WF

Publication: J Am Soc Cytopathol, 2023, Vol. 12, Page 10-19

PubMed ID: 36270909 PubMed Review Paper? No

Purpose of Paper

This paper compared the yield and integrity of RNA isolated from matched breast tumor specimens preserved as frozen RNAlater specimens; formalin-fixed paraffin-embedded (FFPE) cell blocks; ThinPrep specimens; SurePath specimens; and cytospins fixed in Carnoy’s fixative or 95% EtOH and Papanicolaou stained or air-dried; methanol-fixed; and Diff-Quik stained. RNA was extracted with Norgen RNA Purification Kit and the PicoPure RNA Isolation Kit. The concordance of two different breast cancer signatures and mutation variant allele frequency (VAF) were also compared between frozen specimens and the other specimen types.

Conclusion of Paper

RNA quality, as measured by the percentage of fragments that were greater than 200 (DV200), 500 (DV500) and 1000 (DV1000) bp, was higher when RNA was extracted from Carnoy’s fixed, 95% EtOH fixed, air-dried Diff-Quik, or ThinPrep specimens that were extracted with the PicoPure RNA Isolation Kit (rather than the Norgen RNA Purification Kit). On average, the DV200 was 24% higher when extraction was with PicoPure than Norgen. Relative to matched frozen specimens, significant differences in the average DV200 were limited to DiffQuik cytospins (-14%) and SurePath preparations (-42%) when RNA was extracted with the PicoPure RNA Isolation Kit and the FFPE cell block specimen (-26%) when extraction was with the Norgen Purification Kit. The quality of the RNA isolated from SurePath specimens was inadequate for sequencing. For the SETER/PR index, the highest concordance with matched frozen specimens was observed for air-dried Diff-Quik cytospins (CCC =0.843), followed by Carnoy’s fixed cytospins (CCC = 0.783), 95% ethanol-fixed cytospins (concordance correlation coefficient, CCC=0.769), ThinPrep specimens (CCC=0.755), and FFPE cell blocks (CCC=0.562). For the PIK3CA index, the highest concordance with matched frozen specimens was observed for Carnoy’s fixed cytospins (CCC = 0.874), followed by 95% ethanol-fixed cytospins (CCC=0.854), ThinPrep specimens (CCC=0.717), air-dried Diff-Quik cytospins (CCC =0.676), and FFPE cell blocks (CCC=0.565).  Relative to frozen specimens, FFPE cell block specimens had the lowest concordance for breast cancer signatures and greater variance and bias in the signatures than other specimen types. Finally, VAFs were similar between frozen specimens and the other specimen types examined.

 

Studies

  1. Study Purpose

    This study compared the yield and integrity of RNA isolated from matched breast tumor specimens preserved as frozen RNAlater specimens; FFPE cell blocks; ThinPrep specimens; SurePath specimens; and cytospins fixed in Carnoy’s or 95% EtOH and Papanicolaou stained, or air-dried, methanol-fixed and Diff-Quik stained. RNA was extracted with either the Norgen RNA Purification Kit or the PicoPure RNA Isolation Kit. The concordance of two different breast cancer signatures and mutation variant allele frequency (VAF) were also compared between frozen specimens and the other specimen types. Effusion specimens from thirteen patients with metastatic breast cancer were stored at 20°C for an average of 24 h before processing. Cell blocks were prepared by filtering the effusions through gauze to remove fibrin clots, centrifugation at 1500 rpm for 10 min, fixation of cells in 1:1 10% formalin and 95% ethanol for 8-48 h, and pelleting cells and processing as a cell block (details not provided).  For blood-tinged effusions, blood contents were removed by Ficoll-Hypaque gradient separation prior to cytospin preparation. The cell pellet was resuspended in RPMI and were used to prepare 18 cytospin specimens as well as ThinPrep and SurePath specimens (3 drops per container), and a frozen control (3 drops mixed with 0.8 mL RNAlater stored at -80°C). Before drying, the cytospin specimens were placed in Carnoy’s solution and Papanicolaou stained; fixed in 95% ethanol and Papanicolaou stained; or air-dried, fixed in , and Diff-Quik stained. Frozen specimens were thawed at room temperature and mixed with phosphate-buffered saline (pH 7.4) and centrifuged at 5000 g for 10 minutes, then suspended in QIAzol, phase separated with chloroform, ethanol precipitated; RNA was extracted using the RNeasy Micro Kit. RNA was extracted from xylene deparaffinized 10 µm sections of the FFPE cell block using the Norgen RNA Purification Kit. Prior to RNA extraction, coverslips were removed from cytospins, ThinPrep, and SurePath specimens using xylene, and RNA was extracted using the PicoPure RNA Isolation Kit and the Norgen RNA Purification Kit. RNA was quantified by Nanodrop, and RNA integrity was assessed by bioanalyzer. Targeted RNA sequencing libraries were prepared and sequenced on a MiSeq machine.

     

    Summary of Findings:

    RNA quality, as measured by the percentage of fragments greater than 200 (DV200), 500 (DV500), and 1000 (DV1000) bp, was higher when RNA was extracted from Carnoy’s fixed, 95% EtOH fixed, air-dried Diff-Quik, or ThinPrep specimens with the PicoPure RNA Isolation Kit rather than the Norgen RNA Purification Kit (P<0.05, all). On average, the DV200 was 24% higher when RNA extraction was with PicoPure RNA Isolation Kit rather than the Norgen Purification Kit, with the largest increases observed for Diff-Quik specimens (40.23%). Relative to matched frozen specimens, significant differences in the average DV200 were limited to DiffQuik cytospins (-14%) and SurePath preparations (-42%) when RNA was extracted with the PicoPure RNA Isolation Kit and the FFPE cell block specimen (-26%) when extraction was with the Norgen Purification Kit. The quality of RNA extracted from SurePath specimens was inadequate for sequencing. For the SETER/PR index, the highest concordance with matched frozen specimens was observed for air-dried Diff-Quik cytospins (CCC =0.843), followed by Carnoy’s fixed cytospins (CCC = 0.783), 95% ethanol-fixed cytospins (concordance correlation coefficient, CCC=0.769), ThinPrep specimens (CCC=0.755), and FFPE cell blocks (CCC=0.562). For the PIK3CA index, the highest concordance with matched frozen specimens was observed for Carnoy’s fixed cytospins (CCC = 0.874), followed by 95% ethanol-fixed cytospins (CCC=0.854), ThinPrep specimens (CCC=0.717), air-dried Diff-Quik cytospins (CCC =0.676), and FFPE cell blocks (CCC=0.565).  Relative to frozen specimens, FFPE cell block specimens had the lowest concordance in the breast cancer signatures and greater variance and bias in the signatures than the other specimen types examined. Finally, VAFs were similar between frozen specimens and the other specimen types examined.

    Biospecimens
    Preservative Types
    • Formalin
    • Ethanol
    • RNAlater
    • Other Preservative
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    RNA Next generation sequencing
    RNA Spectrophotometry
    RNA Automated electrophoresis/Bioanalyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method Norgen RNA purification Kit
    PicoPure RNA isolation kit
    Next generation sequencing Specific Data handling SETER/PR index
    PIK3CA index
    Biospecimen Preservation Type of fixation/preservation Ethanol
    Formalin (buffered)
    Methanol
    RNAlater
    Frozen
    SurePath
    ThinPrep

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