Accepted Manuscript. Next Steps for Immune Checkpoints in Hepatocellular Carcinoma. Patricia M. Santos, Lisa H. Butterfield

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1 Accepted Manuscript Next Steps for Immune Checkpoints in Hepatocellular Carcinoma Patricia M. Santos, Lisa H. Butterfield PII: S (18) DOI: Reference: YGAST To appear in: Gastroenterology Please cite this article as: Santos PM, Butterfield LH, Next Steps for Immune Checkpoints in Hepatocellular Carcinoma, Gastroenterology (2018), doi: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 <DOC>Editorials <AT>Next Steps for Immune Checkpoints in Hepatocellular Carcinoma <AU>Patricia M. Santos <AFN>Department of Medicine University of Pittsburgh Pittsburgh, Pennsylvania <AU>Lisa H. Butterfield <AFN>Department of Medicine and Department of Immunology and Department of Surgery and Department of Clinical and Translational Science University of Pittsburgh Pittsburgh, Pennsylvania <COR>Reprint requests Address requests for reprints to: Lisa H. Butterfield, PhD, University of Pittsburgh, UPMC Hillman Cancer Center, 5117 Centre Avenue, Suite 1.27, Pittsburgh, Pennsylavania <ARTFN>Conflicts of Interest The author discloses no conflicts. <ARTFN>Funding This work was supported by the UPMC Hillman Cancer Center, supported in part by award P30 CA <TXBX>See Association between expression level of PD1 by tumor-infiltrating CD8+ T cells and features of hepatocellular carcinoma, by Kim H-D, Song GW, Park S, et al, on page 000.

3 <BEGIN ARTICLE>The field of cancer research has experienced a revolution over the last 10 years. Several decades of dogged investigation into immune-based therapies for different cancers, particularly for melanoma, has paid off, most prominently with the clinical success of checkpoint therapy. The clinical impact of antibodies which target the immune checkpoint molecules CTLA-4 and PD-1/PD-L1 in melanoma (and subsequently many other cancers) has been both potent and durable. September 2017 saw the first US Food and Drug Administration approval in hepatocellular carcinoma (HCC) for PD-1 blockade as a second-line treatment after progression on sorafenib (Checkmate-040). 1 This critical breakthrough in HCC demonstrates the importance of the PD-1/PD-L1 pathway in this disease, and likely other gastrointestinal malignancies. Triggering of PD-1 on T cells by PD-L1 on tumor cells has been shown to be an important escape mechanism. In this issue of Gastroenterology, Kim et al 2 present important new mechanistic data investigating the expression of PD-1 by tumor-infiltrating lymphocytes (TIL) and its association with different aspects of HCC. PD-1, expressed by activated T cells, binds to PD-L1 and PD-L2, which can be expressed by various immune cells and tumor cells in response to inflammatory cytokines such as interferon-γ/ 3 Recently, Hui et al 4 have shown that, upon PD-1/PD-L1 binding, the cytoplasmic tail of PD-1 becomes phosphorylated, allowing for the recruitment and binding of Shp2. The PD- 1/Shp2 complex dephosphorylates CD28, resulting in the inhibition of CD28 costimulatory signaling. 4 For cancer immunotherapy, PD-1 blockade involves the use of monoclonal antibody that binds to PD-1, disrupting PD-1/PD-L1 interactions. Therefore, the inhibitory signaling cascade that normally occurs upon PD-1/PD-L1 binding in T cells is blocked, allowing for restoration of antigen-specific T-cell activation and expansion and could also promote neoantigen-specific T-cell response, 5 all collectively resulting in a functional antitumor response.

4 T-cell activation results from antigen-specific activation of the T-cell receptor (signal 1) in addition to antigen-independent signaling by costimulatory receptors (signal 2). Coinhibitory receptors such as PD-1 and CTLA-4 provide a counterbalance to the positive signals of costimulatory receptors, thus maintaining immune homeostasis. Even though only checkpoint blockade against CTLA-4 and PD-1/PD-L1 are currently approved for immunotherapy, other inhibitory molecules such as LAG3, TIM3, TIGIT and VISTA can also be expressed by T cells. 5,6 Pioneering studies of inhibitory receptors in chronic viral infection models show that the number and pattern of inhibitory receptors expressed by T cells is indicative of T-cell exhaustion. 7,8 Similar findings have now been observed in TILs in various cancers, including HCC. 2,9-12 Thus, it is unsurprising that there is tremendous effort in investigating checkpoint blockade of other inhibitory molecules whether by itself or in combination with PD-1 and/or CTLA-4. The search for actionable biomarkers predictive of clinical response to checkpoint blockade in general, and PD-1/PD-L1 blockade specifically, is intensive. Simplistically, the expression of the PD-1 ligand PD-L1 on tumor cells should serve as a predictive biomarker, but those data have varied across tumor types and clinical settings and are at most prognostic. The existence of multiple PD-L1 antibodies and immunohistochemistry assays has complicated the situation. The cross-society sponsored Blueprint study 13 identified assay similarities and differences as well as needs in the field. Now that PD-1 blockade has also been approved by the US Food and Drug Administration for microsatellite-unstable tumors across histologies, the similar necessity for greater harmonization and interoperability in that assay is highlighted. The report from Kim et al 2 is not the first to examine PD-1 and other checkpoint molecules on T cells in the setting of HCC. In 2017, Zhou et al 11 published a study in 59 patient

5 specimens, examining CD4 + and CD8 + TIL and peripheral blood mononuclear cells for expression of PD-1, LAG-3, TIM-3, and CTLA-4. They found higher levels of these checkpoints on TIL compared with blood or normal liver (as shown in other tumor histologies). The more checkpoint molecules expressed, the more functionally suppressed the T cells. Tumor antigenspecific T cells were also tested (glypican-3, MAGE-C2) for phenotypic activation, TIL proliferation, and cytokine production. Furthermore, blockade of more than 1 checkpoint molecule by antibodies improved lymphocyte function compared with blocking only one. In this issue, Kim et al2 have been able to confirm findings of Zhou et al, this time examining 90 patients who had more PD-1, LAG-3, and TIM-3 expression in TIL compared with peripheral blood mononuclear cells. Tumor antigen-specific T cells (specific for NY-ESO-1 and an unpublished epitope for alfa-fetoprotein) were tested for proliferation and cytokine production, and again, blockade of more than 1 checkpoint improves lymphocyte function more than blockade of any one. To better study the biology of these cells, the authors purified them into 3 groups, based on low, intermediate. and high expression of PD-1. This factor is important in part because PD-1 expression is a marker of activation 5,6 and only indicative of exhaustion in the context of other signals, like the coexpression of other checkpoint molecules. Central to the data presented by Kim et al is the RNAseq analysis on cells sorted based on PD-1 high, intermediate, and low expression levels. These data identified important pathways that were differentially regulated, and that correlated to different levels of functionality between those 3 groups of T cells. Among the key findings were the similarities between gene profiles in PD-1- high TIL from the HCC tumors and the Mel75 gene set from exhausted TIL in melanoma. The authors also found PD-1 blockade clinical response correlations with several candidate prognostic biomarkers previously reported in other diseases, including the 18 gene T-cell

6 inflamed gene signature, as well as circulating CD14 + /CD16 - /HLA-DR-high monocytes. Last, patients with PD-1-high circulating T cells expressed more PD-L1 on their tumor cells. It is important to the field of cancer immunotherapy to identify biological themes across tumor types that will help the field to more quickly bring promising therapies to patients with cancer across histologies. Mechanistically, the RNAseq dataset from Kim et al will be an important resource for mining additional key genes and pathways in PD-1 signaling and immune response. Trials testing PD-1/PD-L1 blockade as a first-line treatment for HCC and myriad combination trials are currently underway, and the clinical data will likely identify more therapeutic options for patients. Mechanistic studies, like those of Kim et al in this issue of Gastroenterology 2 are also important for identification of patients most likely to benefit from checkpoint blockade. Future studies in HCC will be needed to identify validated, actionable biomarkers of PD-1 response, as well as identify therapeutic options for those whose tumorinduced immune dysfunction cannot be reversed by PD-1 pathway blockade. References 1. El-Khoueiry AB, Sangro B, Yau T, et al. Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet 2017;389: Kim H-D, Song GW, Park S, et al. Association between expression level of PD1 by tumor-infiltrating CD8+ T cells and features of hepatocellular carcinoma. Gastroenterology 2018;000: Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the Pd-1 Immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 2000;192:1027.

7 4. Hui E, Cheung J, Zhu J, et al. T cell costimulatory receptor CD28 is a primary target for PD-1 mediated inhibition. Science 2017;355: Wei SC, Duffy CR, Allison JP. Fundamental mechanisms of immune checkpoint blockade therapy. Cancer Discov 2018;8: Zarour HM. Reversing T-cell dysfunction and exhaustion in cancer. Clin Cancer Res 2016;22: Wherry EJ, Ha SJ, Kaech SM, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 2007;27: Blackburn SD, Shin H, Haining WN, et al. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol 2009;10: Fourcade J, Sun Z, Benallaoua M, et al. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen specific CD8 + ; T cell dysfunction in melanoma patients. J Exp Med 2010;207: Baitsch L, Baumgaertner P, Devevre E, et al. Exhaustion of tumor-specific CD8(+) T cells in metastases from melanoma patients. J Clin Invest 2011;121: Zhou G, Sprengers D, Boor PPC, et al. Antibodies against immune checkpoint molecules restore functions of tumor-infiltrating T cells in hepatocellular carcinomas. Gastroenterology 2017;153: e Matsuzaki J, Gnjatic S, Mhawech-Fauceglia P, et al. Tumor-infiltrating NY-ESO-1 specific CD8 T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer. Proc Natl Acad Sci U S A 2010;107: Tsao M, Kerr K, Yatabe Y, et al. PL Blueprint 2: PD-L1 Immunohistochemistry comparability study in real-life, clinical samples. J Thorac Oncol 2017;12:S1606.

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