Immunohistochemical scoring of CD38 in the tumor microenvironment predicts responsiveness to anti-PD-1/PD-L1 immunotherapy in hepatocellular carcinoma

Introduction Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated mortality globally. Immune-checkpoint blockade (ICB) is one of the systemic therapy options for HCC. However, response rates remain low, necessitating robust predictive biomarkers. In the present study, we examined the expression of CD38, a molecule involved in the immunosuppressive adenosinergic pathway, on immune cells present in the tumor microenvironment. We then investigated the association between CD38 and ICB treatment outcomes in advanced HCC. Methods Clinically annotated samples from 49 patients with advanced HCC treated with ICB were analyzed for CD38 expression using immunohistochemistry (IHC), multiplex immunohistochemistry/immunofluorescence (mIHC/IF) and multiplex cytokine analysis. Results IHC and mIHC/IF analyses revealed that higher intratumoral CD38+ cell proportion was strongly associated with improved response to ICB. The overall response rates to ICB was significantly higher among patients with high proportion of total CD38+cells compared with patients with low proportion (43.5% vs 3.9%, p=0.019). Higher responses seen among patients with a high intratumoral CD38+cell proportion translated to a longer median progression-free survival (mPFS, 8.21 months vs 1.64 months, p=0.0065) and median overall survival (mOS, 19.06 months vs 9.59 months, p=0.0295). Patients with high CD38+CD68+macrophage density had a better mOS of 34.43 months compared with 9.66 months in patients with low CD38+CD68+ macrophage density. CD38hi macrophages produce more interferon γ (IFN-γ) and related cytokines, which may explain its predictive value when treated with ICB. Conclusions A high proportion of CD38+ cells, determined by IHC, predicts response to ICB and is associated with superior mPFS and OS in advanced HCC.

[1]  S. Lim,et al.  Immune gene signatures for predicting durable clinical benefit of anti-PD-1 immunotherapy in patients with non-small cell lung cancer , 2020, Scientific Reports.

[2]  K. Horimoto,et al.  CD4+ T-cell Immunity in the Peripheral Blood Correlates with Response to Anti-PD-1 Therapy , 2019, Cancer Immunology Research.

[3]  Sherlly Lim,et al.  The Roles of CD38 and CD157 in the Solid Tumor Microenvironment and Cancer Immunotherapy , 2019, Cells.

[4]  G. Yegutkin,et al.  Adenosine Metabolism: Emerging Concepts for Cancer Therapy. , 2019, Cancer cell.

[5]  I. Jacobs,et al.  Peripheral immune-based biomarkers in cancer immunotherapy: can we realize their predictive potential? , 2019, Journal of Immunotherapy for Cancer.

[6]  M. Kudo,et al.  IMbrave150: Efficacy and safety results from a ph III study evaluating atezolizumab (atezo) + bevacizumab (bev) vs sorafenib (Sor) as first treatment (tx) for patients (pts) with unresectable hepatocellular carcinoma (HCC) , 2019, Annals of Oncology.

[7]  M. Kudo,et al.  CheckMate 459: A randomized, multi-center phase III study of nivolumab (NIVO) vs sorafenib (SOR) as first-line (1L) treatment in patients (pts) with advanced hepatocellular carcinoma (aHCC) , 2019, Annals of Oncology.

[8]  Jonathan M. Hernandez,et al.  Tumor Cell Biodiversity Drives Microenvironmental Reprogramming in Liver Cancer. , 2019, Cancer cell.

[9]  S. Choo,et al.  Expression of CD38 on Macrophages Predicts Improved Prognosis in Hepatocellular Carcinoma , 2019, Front. Immunol..

[10]  P. Tan,et al.  Evaluation of phospho-histone H3 in Asian triple-negative breast cancer using multiplex immunofluorescence , 2019, Breast Cancer Research and Treatment.

[11]  P. Tan,et al.  The role of Ki-67 in Asian triple negative breast cancers: a novel combinatory panel approach , 2019, Virchows Archiv.

[12]  N. Hacohen,et al.  PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance , 2019, Nature Immunology.

[13]  J. Furuse,et al.  Abstract 2675: Assessment of inflammation biomarkers in relation to clinical outcomes in nivolumab-treated patients with advanced hepatocellular carcinoma in CheckMate 040 , 2019, Clinical research.

[14]  D. Schadendorf,et al.  Abstract CT037: Genomic analyses and immunotherapy in advanced melanoma , 2019, Clinical Trials.

[15]  P. Sharma,et al.  Abstract 2673: Association of PD-L1 combined positive score and immune gene signatures with efficacy of nivolumab (NIVO) ± ipilimumab (IPI) in patients with metastatic gastroesophageal cancer (mGEC) , 2019, Clinical Research (Excluding Clinical Trials).

[16]  S. Millis,et al.  Prevalence of established and emerging biomarkers of immune checkpoint inhibitor response in advanced hepatocellular carcinoma , 2019, Oncotarget.

[17]  P. Chow,et al.  CD38 is a good predictor of anti-PD-1 immunotherapy responsiveness in hepatocellular carcinoma , 2019, bioRxiv.

[18]  Duo-qing Wu,et al.  Comparison Between UMAP and t-SNE for Multiplex-Immunofluorescence Derived Single-Cell Data from Tissue Sections , 2019, bioRxiv.

[19]  P. Tan,et al.  Prognostic value of CD8 + PD-1+ immune infiltrates and PDCD1 gene expression in triple negative breast cancer , 2019, Journal of Immunotherapy for Cancer.

[20]  Sol Efroni,et al.  The Immune System Computes the State of the Body: Crowd Wisdom, Machine Learning, and Immune Cell Reference Repertoires Help Manage Inflammation , 2019, Front. Immunol..

[21]  N. Schultz,et al.  Prospective Genotyping of Hepatocellular Carcinoma: Clinical Implications of Next-Generation Sequencing for Matching Patients to Targeted and Immune Therapies , 2018, Clinical Cancer Research.

[22]  Gary D Bader,et al.  Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations , 2018, Nature Communications.

[23]  J. Lunceford,et al.  Pan-tumor genomic biomarkers for PD-1 checkpoint blockade–based immunotherapy , 2018, Science.

[24]  E. Newell,et al.  An integrated automated multispectral imaging technique that simultaneously detects and quantitates viral RNA and immune cell protein markers in fixed sections from Epstein-Barr virus-related tumours. , 2018, Annals of diagnostic pathology.

[25]  E. Dmitrovsky,et al.  CD38-Mediated Immunosuppression as a Mechanism of Tumor Cell Escape from PD-1/PD-L1 Blockade. , 2018, Cancer discovery.

[26]  T. Papenfuss,et al.  CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions , 2018, Front. Immunol..

[27]  P. Chow,et al.  Multidimensional analyses reveal distinct immune microenvironment in hepatitis B virus-related hepatocellular carcinoma , 2018, Gut.

[28]  S. Benz,et al.  Abstract 5725: Systematic identification of tumour-specific neoantigens(by whole-genome sequencing) and correlation between tumour neoantigen burden, PD-L1 expression and immune infiltrates in 158Asian colorectal cancers , 2018, Immunology.

[29]  M. Kudo,et al.  Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial. , 2018, The Lancet. Oncology.

[30]  P. Tan,et al.  High Densities of Tumor-Associated Plasma Cells Predict Improved Prognosis in Triple Negative Breast Cancer , 2018, Front. Immunol..

[31]  Charles J. Vaske,et al.  RNA-Seq analyses of immune cell-type enrichments in 158 Asian colorectal cancers (CRCs). , 2018 .

[32]  M. Fehlings,et al.  Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates , 2018, Nature.

[33]  N. Reguart,et al.  PD-L1 expression testing in non-small cell lung cancer , 2018, Therapeutic advances in medical oncology.

[34]  Puay Hoon Tan,et al.  An automated staining protocol for seven-colour immunofluorescence of human tissue sections for diagnostic and prognostic use. , 2018, Pathology.

[35]  M. Kudo,et al.  Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial , 2018, The Lancet.

[36]  Eric Faulkner,et al.  PD-L1 diagnostic tests: a systematic literature review of scoring algorithms and test-validation metrics , 2018, Diagnostic Pathology.

[37]  Reinhard Dummer,et al.  High-dimensional single-cell analysis predicts response to anti-PD-1 immunotherapy , 2018, Nature Network Boston.

[38]  P. Richardson,et al.  CD38 antibodies in multiple myeloma: back to the future. , 2018, Blood.

[39]  T. Chan,et al.  Tumor and Microenvironment Evolution during Immunotherapy with Nivolumab , 2017, Cell.

[40]  F. Aversa,et al.  Low PD-1 Expression in Cytotoxic CD8+ Tumor-Infiltrating Lymphocytes Confers an Immune-Privileged Tissue Microenvironment in NSCLC with a Prognostic and Predictive Value , 2017, Clinical Cancer Research.

[41]  M. Smyth,et al.  Targeting immunosuppressive adenosine in cancer , 2017, Nature Reviews Cancer.

[42]  Alan D. Lopez,et al.  The Burden of Primary Liver Cancer and Underlying Etiologies From 1990 to 2015 at the Global, Regional, and National Level , 2017, JAMA oncology.

[43]  W. Lau,et al.  Comment on "Prediction of Hepatocellular Carcinoma Recurrence Beyond Milan Criteria After Resection: Validation of a Clinical Risk Score in Aninternational Cohort". , 2017, Annals of surgery.

[44]  K. Goldberg,et al.  FDA Approval Summary: Pembrolizumab for Treatment of Metastatic Non‐Small Cell Lung Cancer: First‐Line Therapy and Beyond , 2017, The oncologist.

[45]  J. Lunceford,et al.  IFN-&ggr;–related mRNA profile predicts clinical response to PD-1 blockade , 2017, The Journal of clinical investigation.

[46]  P. Tan,et al.  Caveolin-1 expression as a prognostic marker in triple negative breast cancers of Asian women , 2017, Journal of Clinical Pathology.

[47]  Tuan Bui,et al.  Multiparametric immune profiling in HPV- oral squamous cell cancer. , 2017, JCI insight.

[48]  Toby C. Cornish,et al.  Association of HIV Status With Local Immune Response to Anal Squamous Cell Carcinoma: Implications for Immunotherapy , 2017, JAMA oncology.

[49]  M. Kudo,et al.  Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial , 2017, The Lancet.

[50]  Steven J. M. Jones,et al.  Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma , 2017, Cell.

[51]  A. Kulkarni,et al.  Blockade of adenosine A2A receptor enhances CD8+ T cells response and decreases regulatory T cells in head and neck squamous cell carcinoma , 2017, Molecular Cancer.

[52]  A. Ravaud,et al.  Avelumab, an Anti–Programmed Death-Ligand 1 Antibody, In Patients With Refractory Metastatic Urothelial Carcinoma: Results From a Multicenter, Phase Ib Study , 2017, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[53]  P. Tan,et al.  Higher densities of Foxp3+ regulatory T cells are associated with better prognosis in triple-negative breast cancer , 2017, Breast Cancer Research and Treatment.

[54]  J. Sosman,et al.  Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma , 2017, Cell.

[55]  C. Rao,et al.  Molecular Pathways: Mucins and Drug Delivery in Cancer , 2016, Clinical Cancer Research.

[56]  R. Berger,et al.  Antitumor Activity of Pembrolizumab in Biomarker-Unselected Patients With Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma: Results From the Phase Ib KEYNOTE-012 Expansion Cohort , 2016, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[57]  Dong-hao Wu,et al.  Nomogram for preoperative estimation of long-term survival of patients who underwent curative resection with hepatocellular carcinoma beyond Barcelona clinic liver cancer stage A1 , 2016, Oncotarget.

[58]  N. Rizvi,et al.  PD-L1 biomarker testing for non-small cell lung cancer: truth or fiction? , 2016, Journal of Immunotherapy for Cancer.

[59]  Michael Neuberger,et al.  Multispectral Imaging of T and B Cells in Murine Spleen and Tumor , 2016, The Journal of Immunology.

[60]  Drew M. Pardoll,et al.  PD-1 Blockade with Pembrolizumab in Advanced Merkel-Cell Carcinoma. , 2016, The New England journal of medicine.

[61]  A. Ohta.  A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment , 2016, Front. Immunol..

[62]  P. Keegan,et al.  FDA Approval Summary: Pembrolizumab for the Treatment of Patients With Metastatic Non-Small Cell Lung Cancer Whose Tumors Express Programmed Death-Ligand 1 , 2016, The oncologist.

[63]  D. Inman,et al.  COX-2 modulates mammary tumor progression in response to collagen density , 2016, Breast Cancer Research.

[64]  P. Tan,et al.  Increased CD4 and CD8-positive T cell infiltrate signifies good prognosis in a subset of triple-negative breast cancer , 2016, Breast Cancer Research and Treatment.

[65]  Michael R. Speicher,et al.  The tumor microenvironment and Immunoscore are critical determinants of dissemination to distant metastasis , 2016, Science Translational Medicine.

[66]  Y. Maehara,et al.  PD‐L1 Is Upregulated by Simultaneous Amplification of the PD‐L1 and JAK2 Genes in Non–Small Cell Lung Cancer , 2016, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[67]  M. Garnelo,et al.  Interaction between tumour-infiltrating B cells and T cells controls the progression of hepatocellular carcinoma , 2015, Gut.

[68]  M. Valsecchi.  Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. , 2015, The New England journal of medicine.

[69]  F. Malavasi,et al.  NAD+-Metabolizing Ectoenzymes in Remodeling Tumor–Host Interactions: The Human Myeloma Model , 2015, Cells.

[70]  X. Liu,et al.  Advanced Lung Cancer Is Associated with Decreased Expression of Perforin, CD95, CD38 by Circulating CD3+CD8+ T Lymphocytes. , 2015, Annals of clinical and laboratory science.

[71]  R. Kalluri,et al.  Epithelial to Mesenchymal Transition induces cell cycle arrest and parenchymal damage in renal fibrosis , 2015, Nature Medicine.

[72]  Dirk Schadendorf,et al.  Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. , 2015, The New England journal of medicine.

[73]  Yujin Hoshida,et al.  Hepatitis C virus-induced hepatocellular carcinoma , 2015, Clinical and molecular hepatology.

[74]  Akio Ohta,et al.  Immunological mechanisms of the antitumor effects of supplemental oxygenation , 2015, Science Translational Medicine.

[75]  R. Cone,et al.  G-protein independent coupling of the MC4R to Kir 7.1 in hypothalamic neurons , 2014, Nature.

[76]  Chichung Wang,et al.  Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. , 2014, Methods.

[77]  Jonathan C. Cohen,et al.  ANGPTL8/Betatrophin Does Not Control Pancreatic Beta Cell Expansion , 2014, Cell.

[78]  R. Emerson,et al.  PD-1 blockade induces responses by inhibiting adaptive immune resistance , 2014, Nature.

[79]  M. Smyth,et al.  Antimetastatic effects of blocking PD-1 and the adenosine A2A receptor. , 2014, Cancer research.

[80]  Wei Huang,et al.  Analysis and validation of tissue biomarkers for renal cell carcinoma using automated high-throughput evaluation of protein expression. , 2014, Human pathology.

[81]  Lin Li,et al.  Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma , 2013, Genome research.

[82]  H. Mittrücker,et al.  CD38 Controls the Innate Immune Response against Listeria monocytogenes , 2013, Infection and Immunity.

[83]  M. Smyth,et al.  Blockade of A2A receptors potently suppresses the metastasis of CD73+ tumors , 2013, Proceedings of the National Academy of Sciences.

[84]  Hon Cheung Lee,et al.  Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) as Messengers for Calcium Mobilization* , 2012, The Journal of Biological Chemistry.

[85]  M. Horton,et al.  Enhancement of tumor immunotherapy by deletion of the A2A adenosine receptor , 2012, Cancer Immunology, Immunotherapy.

[86]  Katja Fall,et al.  Utility of multispectral imaging in automated quantitative scoring of immunohistochemistry , 2012, Journal of Clinical Pathology.

[87]  Kwang-Hyun Park,et al.  The Role of CD38 in Fcγ Receptor (FcγR)-mediated Phagocytosis in Murine Macrophages* , 2012, The Journal of Biological Chemistry.

[88]  I. Ng,et al.  Chemokine-driven lymphocyte infiltration: an early intratumoural event determining long-term survival in resectable hepatocellular carcinoma , 2011, Gut.

[89]  S. Deaglio,et al.  NAD+‐metabolizing ecto‐enzymes shape tumor–host interactions: The chronic lymphocytic leukemia model , 2011, FEBS letters.

[90]  M. Smyth,et al.  Extracellular adenosine triphosphate and adenosine in cancer , 2010, Oncogene.

[91]  Dieter Häussinger,et al.  Sorafenib in advanced hepatocellular carcinoma. , 2008, The New England journal of medicine.

[92]  S. Deaglio,et al.  Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology. , 2008, Physiological reviews.

[93]  X-p Chen,et al.  Long‐term outcome of resection of large hepatocellular carcinoma , 2006, The British journal of surgery.

[94]  N. Solvason,et al.  CD38: a new paradigm in lymphocyte activation and signal transduction , 1998, Immunological reviews.

[95]  H. Edmondson,et al.  Primary carcinoma of the liver. A study of 100 cases among 48,900 necropsies , 1954, Cancer.

[96]  S. Bürgler.  Role of CD38 Expression in Diagnosis and Pathogenesis of Chronic Lymphocytic Leukemia and Its Potential as Therapeutic Target. , 2015 .

[97]  Grace S. Shieh,et al.  Identification of the common regulators for hepatocellular carcinoma induced by hepatitis B virus X antigen in a mouse model. , 2012, Carcinogenesis.

[98]  L. Schwartz,et al.  New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). , 2009, European journal of cancer.

[99]  B. Hillner,et al.  Should women with node-negative breast cancer receive adjuvant chemotherapy? — Insights from a decision analysis model , 2005, Breast Cancer Research and Treatment.

[100]  J. Dürig,et al.  CD38 expression is an important prognostic marker in chronic lymphocytic leukaemia , 2002, Leukemia.

[101]  J. Bruix,et al.  Prognosis of Hepatocellular Carcinoma: The BCLC Staging Classification , 1999, Seminars in liver disease.