Robust detection of immune transcripts in FFPE samples using targeted RNA sequencing

Benjamin E Paluch, Sean T Glenn, Jeffrey M Conroy, Antonios Papanicolau-Sengos, Wiam Bshara, Angela R Omilian, Elizabeth Brese, Mary Nesline, Blake Burgher, Jonathan Andreas, Kunle Odunsi, Kevin Eng, Ji He, Maochun Qin, Mark Gardner, Lorenzo Galluzzi, Carl D Morrison, Benjamin E Paluch, Sean T Glenn, Jeffrey M Conroy, Antonios Papanicolau-Sengos, Wiam Bshara, Angela R Omilian, Elizabeth Brese, Mary Nesline, Blake Burgher, Jonathan Andreas, Kunle Odunsi, Kevin Eng, Ji He, Maochun Qin, Mark Gardner, Lorenzo Galluzzi, Carl D Morrison

Abstract

Current criteria for identifying cancer patients suitable for immunotherapy with immune checkpoint blockers (ICBs) are subjective and prone to misinterpretation, as they mainly rely on the visual assessment of CD274 (best known as PD-L1) expression levels by immunohistochemistry (IHC). To address this issue, we developed a RNA sequencing (RNAseq)-based approach that specifically measures the abundance of immune transcripts in formalin-fixed paraffin embedded (FFPE) specimens. Besides exhibiting superior sensitivity as compared to whole transcriptome RNAseq, our assay requires little starting material, implying that it is compatible with RNA degradation normally caused by formalin. Here, we demonstrate that a targeted RNAseq panel reliably profiles mRNA expression levels in FFPE samples from a cohort of ovarian carcinoma patients. The expression profile of immune transcripts as measured by targeted RNAseq in FFPE versus freshly frozen (FF) samples from the same tumor was highly concordant, in spite of the RNA quality issues associated with formalin fixation. Moreover, the results of targeted RNAseq on FFPE specimens exhibited a robust correlation with mRNA expression levels as measured on the same samples by quantitative RT-PCR, as well as with protein abundance as determined by IHC. These findings demonstrate that RNAseq profiling on archival FFPE tissues can be used reliably in studies assessing the efficacy of cancer immunotherapy.

Keywords: CD8+ cytotoxic T lymphocytes; NY-ESO-1; PD-L1; cancer immunotherapy; nivolumab.

Conflict of interest statement

CONFLICTS OF INTEREST

CM, JC and MN hold minor interest in OmniSeq LLC.

Figures

Figure 1. Immune Advance assay performance on…
Figure 1. Immune Advance assay performance on FFPE versus FF samples
A. Samples from 14 ovarian cancer patients were halved to generate a series of matched fresh frozen (FF) and formalin-fixed paraffin embedded (FFPE) specimens, which were serially sectioned, and processed for further analysis. B. Targeted RNAseq on a panel of immunological transcripts was performed on 13 samples pairs that passed quality control upon RNA extraction, as well as on control sample NA12878 in triplicate runs. Each FFPE/FF sample pair demonstrated unique correlation distinct from all other specimens. The matrix depict inter-sample correlation based on Pearson correlation coefficient (R). R2 are indicated for each sample pair in parentheses.
Figure 2. Validation of the Immune Advance…
Figure 2. Validation of the Immune Advance assay on NY-ESO-1
A-C. Formalin-fixed paraffin embedded (FFPE) samples from 13 ovarian cancer patients were sectioned and processed for immunohistochemical assessment of NY-ESO-1 expression, RNA extraction followed by targeted RNAseq on a panel of immunological transcripts or qRT-PCR-assisted quantification of CTAG1B (NY-ESO-1-coding) mRNA levels (GAPDH expression was monitored as internal reference). A. Representative images of NY-ESO-1 expression levels as assessed by immunohistochemistry (IHC) on samples #5 and #7. Scale bars = 100 μm. B. Summary of results from RNAseq, qRT-PCR and IHC. C. Correlation of RNAseq (log2-transformed normalized reads per million, nRPM) and qRT-PCR (1/ΔCt) results. Samples #5 and #7 are indicated; circles delineate samples with negative (0%) or positive (≥5%) NY-ESO-1 staining by IHC. Pearson correlation coefficient (R) and p value are reported.
Figure 3. Validation of the Immune Advance…
Figure 3. Validation of the Immune Advance assay on CD8
A-D. Formalin-fixed paraffin embedded (FFPE) samples from 13 ovarian cancer patients were sectioned and processed for immunohistochemical assessment of CD8 expression, RNA extraction followed by targeted RNAseq on a panel of immunological transcripts or qRT-PCR-assisted quantification of CD8 mRNA levels (GAPDH expression was monitored as internal reference). A. Representative images of CD8+ T-cell infiltration as assessed by immunohistochemistry (IHC) on samples #5 and #6. Scale bars = 100 μm. B. Summary of results from RNAseq, qRT-PCR and IHC. C. Correlation of RNAseq (log2-transformed normalized reads per million, nRPM) and qRT-PCR (1/ΔCt) results. Samples #5 and #6 are indicated. Linear regression trend, Pearson correlation coefficient (R) and p value are reported. D. Correlation of RNAseq (log2-transformed normalized reads per million, nRPM) and IHC (CD8+ T cells/mm2). Samples #5 and #6 are indicated; Pearson correlation coefficient (R) and p value are reported. See also Supplementary Figure S2.
Figure 4. Validation of the Immune Advance…
Figure 4. Validation of the Immune Advance assay on PD-L1
A-C. Formalin-fixed paraffin embedded (FFPE) samples from 13 ovarian cancer patients were sectioned and processed for immunohistochemical assessment of PD-L1 expression, RNA extraction followed by targeted RNAseq on a panel of immunological transcripts or qRT-PCR-assisted quantification of CD274 (PD-L1-coding) mRNA levels (GAPDH expression was monitored as internal reference). A. Representative images of PD-L1 expression as assessed by immunohistochemistry (IHC) on samples #10 and #12. Scale bars = 100 μm. B. Summary of results from RNAseq, qRT-PCR and IHC. IHC scoring as per Dako HC223 pharmDx guidelines is indicated. C. Correlation of RNAseq (log2-transformed normalized reads per million, nRPM) and qRT-PCR (1/ΔCt) results. Samples #10 and #12 are indicated; circles delineate samples with negative (0%) or positive (≥5%) PD-L1 staining by IHC. Linear regression trend, Pearson correlation coefficient (R) and p value are reported.

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