Robust detection of immune transcripts in FFPE samples using targeted RNA sequencing.
Author(s): Paluch BE, Glenn ST, Conroy JM, Papanicolau-Sengos A, Bshara W, Omilian AR, Brese E, Nesline M, Burgher B, Andreas J, Odunsi K, Eng K, He J, Qin M, Gardner M, Galluzzi L, Morrison CD
Publication: Oncotarget, 2017, Vol. 8, Page 3197-3205
PubMed ID: 27911273 PubMed Review Paper? No
Purpose of Paper
This paper compared next-generation sequencing results in paired formalin-fixed paraffin-embedded (FFPE) and frozen ovarian tumors and investigated the relationship between normalized reads per million (nRPM) and levels of CD8, New York esophageal squamous cell carcinoma-1 (NY-ESO-1, CTAG), and programmed death-ligand 1 (PD-L1, CD274) as determined by real-time PCR and immunohistochemistry (IHC).
Conclusion of Paper
One of the fourteen FFPE specimens failed quality control and produced far fewer reads and lower percentage on-target mapped reads and thus the pair was excluded from further analysis. nRPM were strongly correlated for the other 13 pairs. nRPM values were very strongly correlated with the GAPDH-normalized real-time PCR CT values. Samples considered positive based on NY-ESO-1 IHC had higher nRPMs and inversed normalized CT values than those considered and specimens with higher nRPM generally had higher IHC levels.
Studies
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Study Purpose
This study compared next-generation sequencing results in paired FFPE and frozen ovarian tumors and investigated the relationship between nRPM and levels of CD8, NY-ESO-1, and PD-L1 as determined by real-time PCR and immunohistochemistry. Fourteen ovarian cancer specimens were split into two pieces. The first piece was frozen in OCT and the other half was processed for FFPE. Details of specimen procurement and processing were not provided. RNA was extracted from FFPE specimens using truXTRAC FFPE RNA kit and from frozen specimens using the AllPrep DNA/RNA kit. Oncomine Immune Response Research Assay libraries were sequenced using the Ion Proton PIv3. NY-ESO, PD-L1, and CD8 expression were also evaluated by IHC. NY-ESO positivity was defined as moderate to strong cytoplasmic staining with distinct membrane accent in at least 5% of tumor cells. PD-L1 positivity was defined as partial or complete staining in >50% of tumor cells. CD8 staining was assessed by Aperio Scanscope. CTAG1B/1A (NY-ESO-1) and CD274 (PD-L1) were quantified by real-time PCR and normalized by GAPDH.
Summary of Findings:
The FFPE specimen of one of the fourteen cases failed quality control and produced fewer reads (2,354 versus 2,554,065-6,866,821) and lower percentage on-target mapped reads (38% versus 79.96-94.14%) and thus the pair was excluded from further analysis. nRPM were strongly correlated for the other 13 pairs (mean r =0.920, range 0.837-0.969) and with the GAPDH-normalized real-time PCR CT values for NY-ESO-1 (ρ=0.9402, P<0.0001), CD8 (ρ=0.9063, P<0.0001), and PDL1 (ρ=0.9132, P<0.0001). Samples considered positive based on NY-ESO-1 IHC had higher nRPMs than those considered negative and normalized CT values were inversed. Similarly, the number of log-transformed CD8-positive T-cells per mm3 was significantly correlated with log-transformed nRPM (R=0.8323, P<0.0001) and the inverse of the normalized CT values (R=0.9063, P<0.0001). The four specimens with some PD-L1 staining also had higher nRPM values but the specimen with the highest nRPM values was considered PD-L1 negative as it only had staining in 35% of cells.
Biospecimens
Preservative Types
- Formalin
- OCT
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Next generation sequencing Protein Immunohistochemistry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
OCT
Immunohistochemistry Specific Targeted peptide/protein CD8
PD-L1
NY-ESO-1
Real-time qRT-PCR Specific Targeted nucleic acid CD8
PD-L1
NY-ESO-1
GAPDH
Next generation sequencing Specific Technology platform Real-time PCR
IHC