Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Rapid Loss of RNA Detection by In Situ Hybridization in Stored Tissue Blocks and Preservation by Cold Storage of Unstained Slides.

Author(s): Baena-Del Valle JA, Zheng Q, Hicks JL, Fedor H, Trock BJ, Morrissey C, Corey E, Cornish TC, Sfanos KS, De Marzo AM

Publication: Am J Clin Pathol, 2017, Vol. 148, Page 398-415

PubMed ID: 29106457 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated how Z-pair/branched in situ hybridization (RNAscope) results in prostate adenocarcinoma, normal adjacent, and benign specimens were affected by formalin-fixed, paraffin-embedded (FFPE) block storage duration.  Effects of FFPE block storage was evaluated in 25 specimens collected during a radical retropubic prostatectomy that were selected to represent different storage periods (10 months up to 11 y).  Cores (3-5 per case) from another 30 prostatectomy cases that had been stored as FFPE blocks for 6 months to 13 y were freshly assembled in a tissue microarray (TMA) and used to validate initial findings, as were stored FFPE blocks containing patient-derived xenograft (PDX) specimens. A snap-frozen radical retropubic prostatectomy sample that was stored for 10 y was also used for comparison.  Although details were not provided, the authors note that all specimens were fixed and processed in the same lab using extremely similar fixation and processing protocols. RNA in situ hybridization was performed on FFPE sections with the RNAscope 2.0 FFPE Brown Reagent Kit and four probe sets to identify benign and normal adjacent (TP63, PTEN) and carcinoma (MVC, PCA3) specimens; an additional PPIB probe set was also assessed in TMA sections.  RNAscope probe signals were manually scored by a pathologist who was blind to FFPE block age using a semi-quantitative scale based on staining extent (percentage of stained cells) and intensity (intensity 0-4), as well by quantitative image analysis with FrIDA.

   Summary of Findings:

   Slides from standard FFPE blocks that were stored for ≥5 y displayed lower RNAscope signal scores for all four probe sets compared to blocks that were stored for ≤1 y. The differences in manual semi-quantitative signal scores (intensity x extent staining) were significant when FFPE blocks were stratified into three different groups based on the duration of FFPE storage (<1 y, 5-7 y, 8-11 y; P=0.0001 for TP63, P=0.0005 for PTEN, P=0.0018 for MYC, P=0.0064 for PCA3).  Similarly, TMA RNAscope signals for MYC and PPIB, which were quantitatively scored, were significantly lower among cores that were stored as FFPE blocks for 3 y or longer for both benign and carcinoma tissue specimens. The median area score for PPIB was 0.063 for the cores of specimens stored <1 y, 0.01 for specimens stored for 3-7 y, and 0.001 for specimens stored for 8-13 y. Lower signals were also observed upon visual inspection of RNAscope data of PDX-derived FFPE blocks stored for 5 y compared to those stored for <1 y. Three snap-frozen RRP specimens that were stored for 10 y displayed much more robust RNAscope signals for the 4 probes set evaluated (PPIB, TP63, PTEN, 5,8S rRNA) than FFPE blocks stored for a comparable duration at room temperature; however, stored snap-frozen specimens and freshly processed FFPE specimens displayed similar signal scores.

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Normal Adjacent
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   RNA	Tissue microarray
   RNA	In situ hybridization

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	<1 y 5-7 y 8-11 y
   In situ hybridization Specific	Targeted nucleic acid	TP63 PTEN MVC PCA3 PPIB 5.8 rRNA
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen

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2. Study Purpose

   This study investigated how Z-pair/branched in situ hybridization (RNAscope) results in prostate adenocarcinoma, normal adjacent, and benign specimens were affected by storage of slide-mounted FFPE sections at different temperatures (ambient, -20°C), under different conditions (with and without desiccant), and for different durations (1 month – 2 y).  Effects of FFPE slide-mounted section storage temperature was evaluated in 4 adjacent slides from three different FFPE tissue blocks that were stored at -20°C or at ambient temperature for 5 years. Potential effects of FFPE slide-mounted sections after storage at different temperatures and durations with and without desiccant were also examined after deep sectioning of a TMA FFPE block (stored for no longer than 3 months) in 4 adjacent slides that were (1) stored at ambient temperature, (2) stored at ambient temperature with desiccant, (3) stored at -20°C, and (4) stored at -20°C with desiccant. Although details were not provided, the authors note that all specimens were fixed and processed in the same lab using extremely similar fixation and processing protocols. RNA in situ hybridization was performed on FFPE sections with the RNAscope 2.0 FFPE Brown Reagent Kit and four probe sets to identify benign and normal adjacent (TP63, PTEN) and carcinoma (MVC, PCA3) specimens; an additional PPIB probe set was also assessed in TMA sections.  RNAscope probe signals were scored by quantitative image analysis with FrIDA.

   Summary of Findings:

   RNAscope probe signal was superiorly preserved when unstained slide-mounted FFPE tissue sections were stored at -20°C compared to those stored at ambient temperature; this was true for 3 of the 4 probe sets evaluated after 6 months of slide storage .  The presence of desiccant did not impact probe signal during FFPE slide storage at -20°C, although some benefit was observable during slide storage at ambient temperature.  The authors note that 5.8S rRNA signals were not as strongly affected by the temperature of FFPE slide storage.

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Benign
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	In situ hybridization

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	1 month 6 months 1 y 2 y
   Storage	Storage temperature	Ambient temperature -20°C
   Storage	Storage conditions	With desiccant Without desiccant

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