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.
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References
- Sharma P, Allison JP. The future of immune checkpoint therapy. Science. 2015;348:56–61.
- Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R, Robert C, Schadendorf D, Hassel JC, Akerley W, van den Eertwegh AJ, Lutzky J, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711–723.
- Robert C, Thomas L, Bondarenko I, O'Day S, Weber J, Garbe C, Lebbe C, Baurain JF, Testori A, Grob JJ, Davidson N, Richards J, Maio M, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med. 2011;364:2517–2526.
- Wolchok JD, Neyns B, Linette G, Negrier S, Lutzky J, Thomas L, Waterfield W, Schadendorf D, Smylie M, Guthrie T, Jr, Grob JJ, Chesney J, Chin K, et al. Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind, multicentre, phase 2, dose-ranging study. Lancet Oncol. 2010;11:155–164.
- Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, Chow LQ, Vokes EE, Felip E, Holgado E, Barlesi F, Kohlhaufl M, Arrieta O, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med. 2015;373:1627–1639.
- Brahmer J, Reckamp KL, Baas P, Crino L, Eberhardt WE, Poddubskaya E, Antonia S, Pluzanski A, Vokes EE, Holgado E, Waterhouse D, Ready N, Gainor J, et al. Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer. N Engl J Med. 2015;373:123–135.
- Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, Tykodi SS, Sosman JA, Procopio G, Plimack ER, Castellano D, Choueiri TK, Gurney H, et al. Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med. 2015;373:1803–1813.
- Postow MA, Chesney J, Pavlick AC, Robert C, Grossmann K, McDermott D, Linette GP, Meyer N, Giguere JK, Agarwala SS, Shaheen M, Ernstoff MS, Minor D, et al. Nivolumab and ipilimumab versus ipilimumab in untreated melanoma. N Engl J Med. 2015;372:2006–2017.
- Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L, Hassel JC, Rutkowski P, McNeil C, Kalinka-Warzocha E, Savage KJ, Hernberg MM, Lebbe C, et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med. 2015;372:320–330.
- Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, Segal NH, Ariyan CE, Gordon RA, Reed K, Burke MM, Caldwell A, Kronenberg SA, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013;369:122–133.
- Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, Patnaik A, Aggarwal C, Gubens M, Horn L, Carcereny E, Ahn MJ, Felip E, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372:2018–2028.
- Ribas A, Puzanov I, Dummer R, Schadendorf D, Hamid O, Robert C, Hodi FS, Schachter J, Pavlick AC, Lewis KD, Cranmer LD, Blank CU, O'Day SJ, et al. Pembrolizumab versus investigator-choice chemotherapy for ipilimumab-refractory melanoma (KEYNOTE-002): a randomised, controlled, phase 2 trial. Lancet Oncol. 2015;16:908–918.
- Robert C, Ribas A, Wolchok JD, Hodi FS, Hamid O, Kefford R, Weber JS, Joshua AM, Hwu WJ, Gangadhar TC, Patnaik A, Dronca R, Zarour H, et al. Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial. Lancet. 2014;384:1109–1117.
- Robert C, Schachter J, Long GV, Arance A, Grob JJ, Mortier L, Daud A, Carlino MS, McNeil C, Lotem M, Larkin J, Lorigan P, Neyns B, et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. N Engl J Med. 2015;372:2521–2532.
- McDermott DF, Sosman JA, Sznol M, Massard C, Gordon MS, Hamid O, Powderly JD, Infante JR, Fasso M, Wang YV, Zou W, Hegde PS, Fine GD, et al. Atezolizumab, an Anti-Programmed Death-Ligand 1 Antibody, in Metastatic Renal Cell Carcinoma: Long-Term Safety, Clinical Activity, and Immune Correlates From a Phase Ia Study. J Clin Oncol. 2016;34:833–842.
- Rosenberg JE, Hoffman-Censits J, Powles T, van der Heijden MS, Balar AV, Necchi A, Dawson N, O'Donnell PH, Balmanoukian A, Loriot Y, Srinivas S, Retz MM, Grivas P, et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Lancet. 2016;387:1909–1920.
- Topalian SL, Taube JM, Anders RA, Pardoll DM. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer. 2016;16:275–287.
- Webster RM. The immune checkpoint inhibitors: where are we now? Nat Rev Drug Discov. 2014;13:883–884.
- Patel SP, Kurzrock R. PD-L1 Expression as a Predictive Biomarker in Cancer Immunotherapy. Mol Cancer Ther. 2015;14:847–856.
- Roach C, Zhang N, Corigliano E, Jansson M, Toland G, Ponto G, Dolled-Filhart M, Emancipator K, Stanforth D, Kulangara K. Development of a Companion Diagnostic PD-L1 Immunohistochemistry Assay for Pembrolizumab Therapy in Non-Small-cell Lung Cancer. Appl Immunohistochem Mol Morphol. 2016;24:392–397.
- Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J, Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature. 2014;515:577–581.
- Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, Lee W, Yuan J, Wong P, Ho TS, Miller ML, Rekhtman N, Moreira AL, et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science. 2015;348:124–128.
- Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A, Walsh LA, Postow MA, Wong P, Ho TS, Hollmann TJ, Bruggeman C, Kannan K, et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med. 2014;371:2189–2199.
- Hamid O, Schmidt H, Nissan A, Ridolfi L, Aamdal S, Hansson J, Guida M, Hyams DM, Gomez H, Bastholt L, Chasalow SD, Berman D. A prospective phase II trial exploring the association between tumor microenvironment biomarkers and clinical activity of ipilimumab in advanced melanoma. J Transl Med. 2011;9:204.
- Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon MS, Sosman JA, McDermott DF, Powderly JD, Gettinger SN, Kohrt HE, Horn L, Lawrence DP, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 2014;515:563–567.
- Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, West AN, Carmona M, Kivork C, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515:568–571.
- Medeiros F, Rigl CT, Anderson GG, Becker SH, Halling KC. Tissue handling for genome-wide expression analysis: a review of the issues, evidence, and opportunities. Arch Pathol Lab Med. 2007;131:1805–1816.
- Penland SK, Keku TO, Torrice C, He X, Krishnamurthy J, Hoadley KA, Woosley JT, Thomas NE, Perou CM, Sandler RS, Sharpless NE. RNA expression analysis of formalin-fixed paraffin-embedded tumors. Lab Invest. 2007;87:383–391.
- Odunsi K, Jungbluth AA, Stockert E, Qian F, Gnjatic S, Tammela J, Intengan M, Beck A, Keitz B, Santiago D, Williamson B, Scanlan MJ, Ritter G, et al. NY-ESO-1 and LAGE-1 cancer-testis antigens are potential targets for immunotherapy in epithelial ovarian cancer. Cancer Res. 2003;63:6076–6083.
- Senovilla L, Vacchelli E, Galon J, Adjemian S, Eggermont A, Fridman WH, Sautes-Fridman C, Ma Y, Tartour E, Zitvogel L, Kroemer G, Galluzzi L. Trial watch: Prognostic and predictive value of the immune infiltrate in cancer. Oncoimmunology. 2012;1:1323–1343.
- Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12:298–306.
- Zou W, Chen L. Inhibitory B7-family molecules in the tumour microenvironment. Nat Rev Immunol. 2008;8:467–477.
- Conesa A, Madrigal P, Tarazona S, Gomez-Cabrero D, Cervera A, McPherson A, Szczesniak MW, Gaffney DJ, Elo LL, Zhang X, Mortazavi A. A survey of best practices for RNA-seq data analysis. Genome Biol. 2016;17:13.
- Auerbach SS, Phadke DP, Mav D, Holmgren S, Gao Y, Xie B, Shin JH, Shah RR, Merrick BA, Tice RR. RNA-Seq-based toxicogenomic assessment of fresh frozen and formalin-fixed tissues yields similar mechanistic insights. J Appl Toxicol. 2015;35:766–780.
- Hedegaard J, Thorsen K, Lund MK, Hein AM, Hamilton-Dutoit SJ, Vang S, Nordentoft I, Birkenkamp-Demtroder K, Kruhoffer M, Hager H, Knudsen B, Andersen CL, Sorensen KD, et al. Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue. PLoS One. 2014;9:e98187.
- Li P, Conley A, Zhang H, Kim HL. Whole-Transcriptome profiling of formalin-fixed, paraffin-embedded renal cell carcinoma by RNA-seq. BMC Genomics. 2014;15:1087.
- Norton N, Sun Z, Asmann YW, Serie DJ, Necela BM, Bhagwate A, Jen J, Eckloff BW, Kalari KR, Thompson KJ, Carr JM, Kachergus JM, Geiger XJ, et al. Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors. PLoS One. 2013;8:e81925.
- Webster AF, Zumbo P, Fostel J, Gandara J, Hester SD, Recio L, Williams A, Wood CE, Yauk CL, Mason CE. Mining the Archives: A Cross-Platform Analysis of Gene Expression Profiles in Archival Formalin-Fixed Paraffin-Embedded Tissues. Toxicol Sci. 2015;148:460–472.
- Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoue F, Bruneval P, Cugnenc PH, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006;313:1960–1964.
- Mlecnik B, Bindea G, Kirilovsky A, Angell HK, Obenauf AC, Tosolini M, Church SE, Maby P, Vasaturo A, Angelova M, Fredriksen T, Mauger S, Waldner M, et al. The tumor microenvironment and Immunoscore are critical determinants of dissemination to distant metastasis. Sci Transl Med. 2016;8:327ra326.
- Mlecnik B, Tosolini M, Kirilovsky A, Berger A, Bindea G, Meatchi T, Bruneval P, Trajanoski Z, Fridman WH, Pages F, Galon J. Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction. J Clin Oncol. 2011;29:610–618.
- Galon J, Pages F, Marincola FM, Angell HK, Thurin M, Lugli A, Zlobec I, Berger A, Bifulco C, Botti G, Tatangelo F, Britten CM, Kreiter S, et al. Cancer classification using the Immunoscore: a worldwide task force. J Transl Med. 2012;10:205.
- Taube JM, Klein A, Brahmer JR, Xu H, Pan X, Kim JH, Chen L, Pardoll DM, Topalian SL, Anders RA. Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy. Clin Cancer Res. 2014;20:5064–5074.
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