Immunohistochemical scoring of LAG-3 in conjunction with CD8 in the tumor microenvironment predicts response to immunotherapy in hepatocellular carcinoma.

Introduction: Immune checkpoint blockade (ICB) is a systemic therapeutic option for advanced hepatocellular carcinoma (HCC). However, low patient response rates necessitate the development of robust predictive biomarkers that identify individuals who will benefit from ICB. A 4-gene inflammatory signature, comprising CD8, PD-L1, LAG-3, and STAT1, was recently shown to be associated with a better overall response to ICB in various cancer types. Here, we examined whether tissue protein expression of CD8, PD-L1, LAG-3, and STAT1 predicts response to ICB in HCC. Methods: HCC samples from 191 Asian patients, comprising resection specimens from 124 patients (ICB-naïve) and pre-treatment specimens from 67 advanced HCC patients treated with ICB (ICB-treated), were analyzed for CD8, PD-L1, LAG-3, and STAT1 tissue expression using multiplex immunohistochemistry followed by statistical and survival analyses. Results: Immunohistochemical and survival analyses of ICB-naïve samples showed that high LAG-3 expression was associated with shorter median progression-free survival (mPFS) and overall survival (mOS). Analysis of ICB-treated samples revealed that high proportions of LAG-3+ and LAG-3+CD8+ cells pre-treatment were most closely associated with longer mPFS and mOS. Using a log-likelihood model, adding the total LAG-3+ cell proportion to the total CD8+ cell proportion significantly increased the predictive values for mPFS and mOS, compared with the total CD8+ cell proportion alone. Moreover, levels of CD8 and STAT1, but not PD-L1, were significantly correlated with better responses to ICB. After analyzing viral-related and non-viral HCC samples separately, only the LAG3+CD8+ cell proportion was significantly associated with responses to ICB regardless of viral status. Conclusion: Immunohistochemical scoring of pre-treatment levels of LAG-3 and CD8 in the tumor microenvironment may help predict ICB benefits in HCC patients. Furthermore, immunohistochemistry-based techniques offer the advantage of being readily translatable in the clinical setting.

PLK1 inhibition selectively induces apoptosis in ARID1A deficient cells through uncoupling of oxygen consumption from ATP production.

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer.

Intratumoral CD39+CD8+ T Cells Predict Response to Programmed Cell Death Protein-1 or Programmed Death Ligand-1 Blockade in Patients With NSCLC.

INTRODUCTION: Programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1) blockade is currently widely used in the treatment of metastatic NSCLC. Despite available biomarker stratification, clinical responses vary. Thus, the search for novel biomarkers with improved response prediction is ongoing. Previously, using mass cytometry or cytometry by time-of-flight (CyTOF), our group demonstrated that CD39+CD8+ immune cells represent tumor antigen-specific, cytotoxic T cells in treatment-naive NSCLC. We hypothesized that accurate quantitation of this T cell subset would predict immunotherapy outcome. METHODS: To translate this to a clinical setting, the present study compared CyTOF data with a range of clinically relevant methods, including conventional immunohistochemistry (IHC), multiplex IHC or immunofluorescence (mIHC), and gene expression assay by NanoString. RESULTS: Quantification using mIHC but not conventional IHC or NanoString correlated with the CyTOF results. The specificity and sensitivity of mIHC were then evaluated in a separate retrospective NSCLC cohort. CD39+CD8+ T cell proportion, as determined by mIHC, successfully stratified responders and nonresponders to PD-1 or PD-L1 inhibitors (objective response rate of 63.6%, compared with 0% for the negative group). This predictive capability was independent from other confounding factors, such as total CD8+ T cell proportion, CD39+ lymphocyte proportion, PD-L1 positivity, EGFR mutation status, and other clinicopathologic parameters. CONCLUSIONS: Our results suggest that the mIHC platform is a clinically relevant method to evaluate CD39+CD8+ T cell proportion and that this marker can serve as a potential biomarker that predicts response to PD-1 or PD-L1 blockade in patients with NSCLC. Further validation in additional NSCLC cohorts is warranted.

Quantitative imaging of RAD51 expression as a marker of platinum resistance in ovarian cancer.

Early relapse after platinum chemotherapy in epithelial ovarian cancer (EOC) portends poor survival. A-priori identification of platinum resistance is therefore crucial to improve on standard first-line carboplatin-paclitaxel treatment. The DNA repair pathway homologous recombination (HR) repairs platinum-induced damage, and the HR recombinase RAD51 is overexpressed in cancer. We therefore designed a REMARK-compliant study of pre-treatment RAD51 expression in EOC, using fluorescent quantitative immunohistochemistry (qIHC) to overcome challenges in quantitation of protein expression in situ. In a discovery cohort (n = 284), RAD51-High tumours had shorter progression-free and overall survival compared to RAD51-Low cases in univariate and multivariate analyses. The association of RAD51 with relapse/survival was validated in a carboplatin monotherapy SCOTROC4 clinical trial cohort (n = 264) and was predominantly noted in HR-proficient cancers (Myriad HRDscore < 42). Interestingly, overexpression of RAD51 modified expression of immune-regulatory pathways in vitro, while RAD51-High tumours showed exclusion of cytotoxic T cells in situ. Our findings highlight RAD51 expression as a determinant of platinum resistance and suggest possible roles for therapy to overcome immune exclusion in RAD51-High EOC. The qIHC approach is generalizable to other proteins with a continuum instead of discrete/bimodal expression.

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.

Establishment and Characterization of Humanized Mouse NPC-PDX Model for Testing Immunotherapy.

Immune checkpoint blockade (ICB) monotherapy shows early promise for the treatment of nasopharyngeal carcinoma (NPC) in patients. Nevertheless, limited representative NPC models hamper preclinical studies to evaluate the efficacy of novel ICB and combination regimens. In the present study, we engrafted NPC biopsies in non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain-null (NSG) mice and established humanized mouse NPC-patient-derived xenograft (NPC-PDX) model successfully. Epstein-Barr virus was detected in the NPC in both NSG and humanized mice as revealed by Epstein-Barr virus-encoded small RNA (EBER) in situ hybridization (ISH) and immunohistochemical (IHC) staining. In the NPC-bearing humanized mice, the percentage of tumor-infiltrating CD8+ cytotoxic T cells was lowered, and the T cells expressed higher levels of various inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) than those in blood. The mice were then treated with nivolumab and ipilimumab, and the anti-tumor efficacy of combination immunotherapy was examined. In line with paired clinical data, the NPC-PDX did not respond to the treatment in terms of tumor burden, whilst an immunomodulatory response was elicited in the humanized mice. From our results, human proinflammatory cytokines, such as interferon-gamma (IFN-γ) and interleukin-6 (IL-6) were significantly upregulated in plasma. After treatment, there was a decrease in CD4/CD8 ratio in the NPC-PDX, which also simulated the modulation of intratumoral CD4/CD8 profile from the corresponding donor. In addition, tumor-infiltrating T cells were re-activated and secreted more IFN-γ towards ex vivo stimulation, suggesting that other factors, including soluble mediators and metabolic milieu in tumor microenvironment may counteract the effect of ICB treatment and contribute to the tumor progression in the mice. Taken together, we have established and characterized a novel humanized mouse NPC-PDX model, which plausibly serves as a robust platform to test for the efficacy of immunotherapy and may predict clinical outcomes in NPC patients.

The Roles of CD38 and CD157 in the Solid Tumor Microenvironment and Cancer Immunotherapy.

The tumor microenvironment (TME) consists of extracellular matrix proteins, immune cells, vascular cells, lymphatics and fibroblasts. Under normal physiological conditions, tissue homeostasis protects against tumor development. However, under pathological conditions, interplay between the tumor and its microenvironment can promote tumor initiation, growth and metastasis. Immune cells within the TME have an important role in the formation, growth and metastasis of tumors, and in the responsiveness of these tumors to immunotherapy. Recent breakthroughs in the field of cancer immunotherapy have further highlighted the potential of targeting TME elements, including these immune cells, to improve the efficacy of cancer prognostics and immunotherapy. CD38 and CD157 are glycoproteins that contribute to the tumorigenic properties of the TME. For example, in the hypoxic TME, the enzymatic functions of CD38 result in an immunosuppressive environment. This leads to increased immune resistance in tumor cells and allows faster growth and proliferation rates. CD157 may also aid the production of an immunosuppressive TME, and confers increased malignancy to tumor cells through the promotion of tumor invasion and metastasis. An improved understanding of CD38 and CD157 in the TME, and how these glycoproteins affect cancer progression, will be useful to develop both cancer prognosis and treatment methods. This review aims to discuss the roles of CD38 and CD157 in the TME and cancer immunotherapy of a range of solid tumor types.