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

// Benjamin E. Paluch 1 , Sean T. Glenn 1, 2 , Jeffrey M. Conroy 1, 3 , Antonios Papanicolau-Sengos 1 , Wiam Bshara 4 , Angela R. Omilian 4 , Elizabeth Brese 4 , Mary Nesline 1 , Blake Burgher 1 , Jonathan Andreas 1 , Kunle Odunsi 5 , Kevin Eng 6 , Ji He 1 , Maochun Qin 1 , Mark Gardner 1 , Lorenzo Galluzzi 7, 8, 9, 10, 11, 12 , Carl D. Morrison 1, 3 1 Omniseq LLC, Buffalo, NY 14203, USA 2 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 3 Center for Personalized Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 4 Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 5 Department of Gynecologic Oncology, Center for Immunotherapy, Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 6 Department of Biostatistics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 7 Department of Radiation Oncology, Weill Cornell Medical College, New York, NY 10065, USA 8 Equipe 11 labellisée Ligue contre le Cancer, Centre de Recherche des Cordeliers, 75006 Paris, France 9 INSERM, U1138, 75006 Paris, France 10 Université Paris Descartes/Paris V, Sorbonne Paris Cité, 75006 Paris, France 11 Université Pierre et Marie Curie/Paris VI, 75006 Paris, France 12 Gustave Roussy Cancer Campus, 94805 Villejuif, France Correspondence to: Carl D. Morrison, email: carl.morrison@omniseq.com Keywords: cancer immunotherapy, CD8+ cytotoxic T lymphocytes, nivolumab, NY-ESO-1, PD-L1 Received: October 21, 2016 Accepted: November 21, 2016 Published: November 29, 2016 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.