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Feasibility of Prostate PAXgene Fixation for Molecular Research and Diagnostic Surgical Pathology: Comparison of Matched Fresh Frozen, FFPE, and PFPE Tissues.

Author(s): Högnäs G, Kivinummi K, Kallio HML, Hieta R, Ruusuvuori P, Koskenalho A, Kesseli J, Tammela TLJ, Riikonen J, Ilvesaro J, Kares S, Hirvikoski PP, Laurila M, Mirtti T, Nykter M, Kujala PM, Visakorpi T, Tolonen T, Bova GS

Publication: Am J Surg Pathol, 2018, Vol. 42, Page 103-115

PubMed ID: 28984675 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared H&E staining and immunostaining in frozen OCT-embedded, PAXgene-fixed paraffin-embedded, and FFPE prostate specimens. Thirty-six robot-assisted laparoscopic prostatectomy (RALP) specimens were delivered by pneumatic tube to the laboratory and 4˚C saline was added (14-15 min after excision). The bag containing tissue was placed on ice and cooled until it reached <15˚C. Four 8 mm core punches were obtained from 20 of the RALP specimens, three 6 mm cores from 16 of the specimens, and any remaining specimen was fixed in 10% buffered formalin an average of 52 min (8 mm cores) or 40 min (6 mm cores) after excision. For the twenty specimens with four cores, one core was embedded in OCT, snap-frozen in isopentane, and stored at -80˚C; one core was fixed in 10% buffered formalin for 24 h and embedded in Histowax paraffin using the Pathos Delta standard program (23 h); and two cores were fixed in PAXgene for 4 h and then stored in PAXgene stabilizer at 4˚C for <2 weeks followed by embedding in Histowax paraffin using either the Citadel 2000 program for 8 h 30 min (one core from each specimen)or the Pathos Delta standard program for 8 h 30 min (one core from each specimen). For the sixteen specimens with three cores, the first core from each specimen was embedded in OCT and snap-frozen, the second core was fixed in formalin for 24 h and embedded in Histowax as described above, and the remaining one was fixed in PAXgene for 4 h, stored in PAXgene stabilizer at 4˚C for <2 weeks and embedded in ParaPlast Xtra paraffin using a Leica TP1020 program (11 h 5 min).The first 6 µm section of OCT specimens was used for H&E staining and the remaining specimen was sectioned at 10 µm for nucleic acid extraction. For the paraffin embedded specimens, the first 4 µm section was H&E stained; the next six 4 µm sections were used for immunohistochemistry staining of PSA, ERG, vimentin, and a combination of ck5/6, p63, and AMACR by 2IHC; then the excess paraffin was trimmed. For DNA and RNA extraction 12 10 µm sections of 8mm cores (6.0 mm³) or 22 10 µm sections of 6 mm cores (6.2 mm³) were placed in flip-top tubes. DNA and RNA were extracted from frozen specimens using the AllPrep DNA/RNA/miRNA Universal kit, from FFPE sections using the Allprep DNA/RNA FFPE Kit, and from PAXgene sections using the PAXgene Tissue Allprep DNA/RNA/miRNA method.  Deparaffinization of FFPE and PAXgene specimens was in heptane (the 8 mm cores) or xylene (the 6 mm cores). DNA was stored at -20˚C and concentration was determined by fluorometry, integrity investigated by electrophoresis and a fragment analyzer, and, in the case of the 6 mm cores, by the real-time PCR based FFPE QC kit. RNA was stored at -80˚C and analyzed using a fragment analyzer. Whole slide images of H&E and IHC staining were obtained using a 40X objective and an automated scanner and then analyzed by five pathologists. Androgen receptor and 35 other genes and Androgen receptor (AR) and 21 other transcripts were sequenced after rRNA depletion using the SureSelect Target Enrichment Systems on the Illumina MiSeq platform.

   Summary of Findings:

   Staining of PSA, ERG, vimentin, and 2IHC was judged by the pathologists to be adequate for routine use in 100% of FFPE and 96% of PAXgene specimens, regardless of fixative, and no differences in quality were reported. By examining the H&E and 2IHC staining, four of the pathologists were unable to correctly identify the fixative, but the remaining pathologist who had the most experience comparing these fixatives was able to properly identify 13 of 14 FFPE and 14 of 15 PAXgene specimens based on red blood cell morphology and excess hematoxylin staining in the PAXgene specimen. As expected, fixative did not affect the cancer cell fraction and nuclear counts were similar for all three preservation methods (frozen mean: 79839, formalin mean: 98609, and PAX mean: 86892).

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Formalin
   • PAXgene
   • OCT

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Morphology	H-and-E microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Immunohistochemistry Specific	Targeted peptide/protein	PSA ERG Vimentin 2IHC
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) OCT PAXgene

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2. Study Purpose

   This study compared RNA and DNA quantity and quality in frozen OCT-embedded, PAXgene-fixed paraffin-embedded, and FFPE prostate specimens. The effects of PAXgene processing method and storage time in PAXgene stabilizer on RNA quality were also examined. Thirty-six RALP specimens were delivered by pneumatic tube to the lab and 4˚C saline was added (14-15 min after excision). The bag containing tissue was placed on ice and cooled until it reached <15˚C. Four 8 mm core punches were obtained from 20 of the RALP specimens, three 6 mm cores from the 16 other specimens, and any remaining specimen was fixed in 10% buffered formalin an average of 52 min (8 mm cores) or 40 min (6 mm cores) after excision.  For the twenty specimens with four cores, one core was embedded in OCT, snap-frozen in isopentane, and stored at -80˚C; one core was fixed in 10% buffered formalin for 24 h and embedded in Histowax paraffin using the Pathos Delta standard program (23 h); and two cores were fixed in PAXgene for 4 h and then stored in PAXgene stabilizer at 4˚C for <2 weeks followed by embedding in Histowax paraffin using either the Citadel 2000 program for 8 h 30 min (one core from each specimen) or the Pathos Delta standard program for 8 h 30 min (one core from each specimen). For the sixteen specimens with three cores, the first core from each specimen was embedded in OCT and snap-frozen, the second core was fixed in formalin for 24 h and embedded in Histowax as described above, and the remaining core was fixed in PAXgene for 4 h, stored in PAXgene stabilizer at 4˚C for <2 weeks and embedded in ParaPlast Xtra paraffin using a Leica TP1020 program (11 h 5 min).For DNA and RNA extraction, 12 sections of 8 mm cores (6.0 mm³) or 22 sections of 6 mm cores (6.2 mm³) were placed in flip-top tubes. DNA and RNA were extracted from frozen specimens using the AllPrep DNA/RNA/miRNA Universal kit, from FFPE sections using the Allprep DNA/RNA FFPE Kit, and from PAXgene sections using the PAXgene Tissue Allprep DNA/RNA/miRNA method.  Deparaffinization of FFPE and PAXgene specimens was in heptane (the 8 mm cores) or xylene (the 6 mm cores). DNA was stored at -20˚C, concentration was determined by fluorometry, integrity investigated by electrophoresis and a fragment analyzer, and, in the case of the 6 mm cores, by the real-time PCR based FFPE QC kit. RNA was stored at -80˚C, and analyzed using a fragment analyzer. Androgen receptor and 35 other genes and AR and 21 other transcripts were sequenced after rRNA depletion using the SureSelect Target Enrichment Systems on the Illumina MiSeq platform.

   Summary of Findings:

   DNA genomic quality numbers were significantly higher in frozen specimens than in formalin or PAXgene –fixed specimens (7.9 versus 3.2 and 6.2, P<0.0001) and in PAXgene than formalin-fixed specimens (6.2 versus 3.2, P<0.0001). Similarly, DNA fragments were largest in frozen specimens followed by PAXgene-fixed and formalin specimens (59 kb, 41 kb, and 20 kb, respectively) and the fragment analyzer showed a bell-shaped curve for frozen and PAXgene specimens with peaks greater than 48,500 bp, but formalin-fixed specimens had a small peak at 300 bp and another at 20 kb. The differences in the CT values between reference DNA and the specimens in the QC assay were negative for DNA from frozen and PAXgene specimens (-0.7 and -0.6, respectively), indicating intact DNA, but, as expected based on degradation, were significantly higher for FFPE specimens (1.9, P<0.0001, both). Two of the four FFPE specimens had an increased level of C>A changes but all other artifacts occurred at similar frequencies and the other two FFPE specimens had comparable numbers of C>A changes to PAXgene and formalin-fixed specimens. The DNA yield was significantly lower from frozen specimens (1.3 µg) than formalin (2.8 µg, P<0.001) or PAXgene-fixed specimens (2.7 µg, P<0.01) and the differences remained significant when normalized to the total nuclear count (P<0.01, both).

   RNA quality numbers (RQN) were significantly higher from frozen specimens than PAXgene or formalin-fixed specimens (P<0.001, both) and from PAXgene-fixed specimens than formalin-fixed specimens (P<0.001), but no correlations with GQN were observed. The RQN was significantly higher when PAXgene specimens were embedded in ParaPlast Xtra paraffin using a Leica TP1020 program than when embedded in Histowax paraffin using the Citadel 2000 program or the Pathos Delta standard program (P<0.001 and P<0.01, respectively). Interestingly, the RQN for PAXgene specimens was inversely correlated with storage time in the stabilizer (R=-0.75, P<0.001). The percentage of fragments greater than 200 nt (DV200) was highest for frozen specimens followed by PAXgene and formalin-fixed specimens, respectively (81%, 78%, and 50%; respectively). The electropherograms showed clear peaks for the rRNA at 2 kb and 5 kb in the frozen specimens and, to a lesser extent, in the PAXgene specimens, but these peaks were not observed in the electropherograms of formalin-fixed specimens. While there were no differences in mean number of reads between fixatives, FFPE specimens had lower coverage than either frozen or PAXgene-preserved specimens (P<0.01 and P<0.05, respectively) and PAXgene specimens had lower coverage than frozen specimens (P<0.05). RQN values were significantly correlated with coverage (R=0.87, P<0.001). RNA yields from FFPE and frozen specimens were comparable, but yields from PAXgene specimens were higher (P=0.0002 and P=0.0005, respectively).

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Frozen
   • PAXgene
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Electrophoresis
   DNA	Automated electrophoresis/Bioanalyzer
   RNA	Automated electrophoresis/Bioanalyzer
   DNA	Fluorometry
   RNA	Next generation sequencing
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) OCT PAXgene
   Biospecimen Preservation	Duration of tissue processing	Citadel 2000 program (8 h 30 min) Pathos Delta standard program (8 h 30 min) Pathos Delta standard program (23 h)
   Storage	Storage duration	16 h-20 days

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