Leslie Rozeboom

Lymphocyte-activation gene-3, an important immune checkpoint in cancer.

Immunotherapy has recently become widely used in lung cancer. Many oncologists are focused on cytotoxic T lymphocyte antigen‐4 (CTLA‐4), programmed cell death ligand‐1 (PD‐L1) and programmed cell death‐1 (PD‐1). Immunotherapy targeting the PD‐1/PD‐L1 checkpoints has shown promising efficacy in non‐small cell lung cancer (NSCLC), but questions remain to be answered. Among them is whether the simultaneous inhibition of other checkpoints could improve outcomes. Lymphocyte‐activation gene‐3 (LAG‐3) is another vital checkpoint that may have a synergistic interaction with PD‐1/PD‐L1. Here we review the LAG‐3 function in cancer, clinical trials with agents targeting LAG‐3 and the correlation of LAG‐3 with other checkpoints.

Endobronchial miRNAs as Biomarkers in Lung Cancer Chemoprevention

Lung cancers express lower levels of prostacyclin than normal lung tissues. Prostacyclin prevents lung cancer in a variety of mouse models. A randomized phase II trial comparing oral iloprost (a prostacyclin analog) with placebo in high-risk subjects showed improvement in bronchial histology in former, but not current, smokers. This placebo-controlled study offered the opportunity for investigation of other potential intermediate endpoint and predictive biomarkers to incorporate into chemoprevention trials. Matched bronchial biopsies were obtained at baseline and at 6-month follow-up from 125 high-risk individuals who completed the trial: 31/29 and 37/28 current/former smokers in the iloprost and placebo arm, respectively. We analyzed the expression of 14 selected miRNAs by Real Time PCR in 496 biopsies. The expression of seven miRNAs was significantly correlated with histology at baseline. The expression of miR-34c was inversely correlated with histology at baseline (P < 0.0001) and with change in histology at follow-up (P = 0.0003), independent of treatment or smoking status. Several miRNAs were also found to be differentially expressed in current smokers as compared with former smokers. In current smokers, miR-375 was upregulated at baseline (P < 0.0001) and downregulated after treatment with iloprost (P = 0.0023). No miRNA at baseline reliably predicted a response to iloprost. No biomarker predictive of response to iloprost was found. MiR-34c was inversely correlated with baseline histology and with histology changes. Mir-34c changes at follow-up could be used as a quantitative biomarker that parallels histologic response in formalin-fixed bronchial biopsies in future lung cancer chemoprevention studies. Cancer Prev Res; 6(2); 100–8. ©2012 AACR.

LAG-3 Protein Expression in Non–Small Cell Lung Cancer and Its Relationship with PD-1/PD-L1 and Tumor-Infiltrating Lymphocytes

Introduction: Immunotherapy targeting the programmed death 1 (PD‐1)/programmed death ligand 1 (PD‐L1) checkpoint has shown promising efficacy in patients with NSCLC. Lymphocyte activating 3 gene (LAG‐3) is another important checkpoint, and its role in NSCLC is still not clear. In this study we investigated lymphocyte activing 3 (LAG‐3) protein expression; its correlation with PD‐1, PD‐L1, and tumor‐infiltrating lymphocytes (TILs); and its association with survival in NSCLC. Methods: The expression of LAG‐3 (EPR4392 [Abcam, Cambridge, MA]) protein was assessed in 55 NSCLC cell lines by immunohistochemistry. LAG‐3, PD‐1 (NAT 105 [Cell Marque, Rocklin, CA]), and PD‐L1 (22C3 [Dako, Carpenteria, CA]) protein expression was evaluated by immunohistochemistry, and TIL abundance was scored in 139 surgically resected specimens from patients with NSCLC. We also verified results in samples from 62 patients with untreated NSCLC and detected a correlation between LAG‐3 expression and EGFR and KRAS mutation and echinoderm microtubule associated protein like 4 gene (EML4)–anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement. Results: LAG‐3 was not expressed on any of the 55 NSCLC cell lines. However, LAG‐3 was expressed on the TILs in 36 patients with NSCLC (25.9%). Sixty patient samples (43.2%) were positive for PD‐1 on the TILs, and 25 (18.0%) were positive for PD‐L1 on tumor cells. Neither LAG‐3 nor PD‐1 was expressed on the tumor cells. LAG‐3 was overexpressed on the TILs in nonadenocarcinoma compared with in adenocarcinoma (p = 0.031). LAG‐3 expression on TILs was significantly correlated with that of PD‐1 on TILs (p < 0.001) and PD‐L1 on tumor cells (p = 0.041) but not with TIL percentage (p = 0.244). With the logistic regression model, the ORs for LAG‐3 were 0.320 (95% confidence interval [CI]: 0.110–0.929) and 4.364 (95% CI: 1.898–10.031) when nonadenocarcinoma was compared with adenocarcinoma and TILs that were negative for PD‐1 were compared with those positive for PD‐1. Recurrence‐free survival was significantly different in patients whose TILs were LAG‐3–negative as opposed to LAG‐3–positive (1.91 years [95% CI: 0.76–3.06] versus 0.87 years [95% CI: 0.27–1.47] [p = 0.025]). Likewise, LAG‐3 status of TILs (negative versus positive) did significantly affect overall survival (OS) (3.04 years [95% CI: 2.76–3.32] versus 1.08 years [95% CI: 0.42–1.74] [p = 0.039]). Using Kaplan‐Meier analysis, we found that patients with both PD‐L1–negative tumor cells and LAG‐3–negative TILs have longer recurrence‐free survival than patients who are either PD‐L1– or LAG‐3–positive or both PD‐L1– and LAG‐3–positive (2.09 years [95% CI: 0.90–3.28] versus 1.42 years [95% CI: 0.46–2.34] versus 0.67 years [95% CI: 0.00–1.45] [p = 0.007]). In the verification stage, high expression of LAG‐3 was also significantly correlated with higher expression of PD‐1 on TILs (p = 0.016) and PD‐L1 on tumor cells (p = 0.014). There was no correlation between LAG‐3 expression and EGFR (p = 0.325) and KRAS mutation (p = 1.000) and ALK fusion (p = 0.562). Conclusions: LAG‐3 is expressed on TILs in tumor tissues of some patients with NSCLC. Its expression was higher in nonadenocarcinoma and correlated with PD‐1/PD‐L1 expression. LAG‐3 positivity or both LAG‐3 and PD‐L1 positivity was correlated with early postoperative recurrence. LAG‐3 was related to poor prognosis.

Independent validation test of the vote-counting strategy used to rank biomarkers from published studies.

AIM Vote counting is frequently used in meta-analyses to rank biomarker candidates, but to our knowledge, there have been no independent assessments of its validity. Here, we used predictions from a recent meta-analysis to determine how well number of supporting studies, combined sample size and mean fold change performed as vote-counting strategy criteria. MATERIALS & METHODS Fifty miRNAs previously ranked for their ability to distinguish lung cancer tissue from normal were assayed by RT-qPCR using 45 paired tumor-normal samples. RESULTS Number of supporting studies predicted biomarker performance (p = 0.0006; r = 0.44), but sample size and fold change did not (p > 0.2). CONCLUSION Despite limitations, counting the number supporting studies appears to be an effective criterion for ranking biomarkers. Predictions based on sample size and fold change provided little added value. External validation studies should be conducted to establish the performance characteristics of strategies used to rank biomarkers.

PD-1, PD-L1 Protein Expression in Non-Small Cell Lung Cancer and Their Relationship with Tumor-Infiltrating Lymphocytes

Background Immunotherapy targeting the programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint has shown the good outcomes in non-small cell lung cancer (NSCLC). We investigated PD-1 and PD-L1 protein expression and their correlation with tumor-infiltrating lymphocytes (TILs), and association with survival in NSCLC. Material/Methods The expression of PD-1 (NAT105, Cell Marque) and PD-L1 (28-8, Dako) protein was assessed in 55 NSCLC cell lines by immunohistochemistry (IHC). PD-1 (NAT105, Cell Marque) and PD-L1 (22C3, Dako) protein expression was evaluated by IHC, and TIL percentage was scored, in 139 surgically resected specimens from patients with NSCLC. Results PD-1 was not expressed on NSCLC cell lines. PD-L1 was expressed on 20 NSCLC cell lines (36.4%). A total of 60 patient samples (43.2%) were positive for PD-1 on the TILs, and 25 (18.0%) were positive for PD-L1 on tumor cells. High expression of PD-1 on tumor cells was significantly correlated with higher expression of PD-L1 (P=0.026) and a higher percentage of TILs (P<0.001). In the Cox regression model, the odds ratio for PD-1 was 2.828 (95% CI: 1.325–11.165; P=0.013) and 8.579 (95% CI: 4.148–22.676; P<0.001) when PD-L1 and TILs were positive. Patients whose tumor cells were PD-L1 negative had a tendency for longer relapse-free survival (RFS) than patients who were PD-L1 positive (1.85 years, 95% CI: 0.77–2.93 vs. 0.97 years, 95% CI: 0.71–1.23; P=0.054). Conclusions PD-1 was expressed on TILs in tumor tissues in NSCLC patients. PD-L1 was expressed on both TILs and tumor tissues. PD-1 expression was correlated with PD-L1 on tumor cells and TILs. Patients who were PD-L1 positive tended to experience progression after surgery.

Therapy-induced E-cadherin downregulation alters expression of programmed death ligand-1 in lung cancer cells

OBJECTIVES Immunotherapy that targets the programmed death-1/programmed death-ligand 1 (PD-L1) axis has been approved for treatment of non-small cell lung cancer (NSCLC) patients in many countries. However, our current understanding of the role of immunotherapies on NSCLC patients with epidermal growth factor receptor (EGFR) mutation, following acquisition of resistance to EGFR tyrosine kinase inhibitors (TKIs), is so far unclear. Especially, there is little data on if each acquired resistance mechanism to EGFR-TKIs alters PD-L1 expression status which is employed as an important predictive biomarker for PD-1/PD-L1 targeting agents. MATERIALS AND METHODS Lung cancer cell lines (HCC827, HCC4006, PC9, H1975, H358, SW900, and H647) and their daughter cells that acquired resistance to EGFR-TKIs or cytotoxic drugs (cisplatin or vinorelbine) were examined. PD-L1 expression was analyzed by immunohistochemistry, immunoblotting, and/or fluorescent imaging. Published microarray data were also employed to evaluate our findings. RESULTS AND CONCLUSION We found correlations between therapy-induced E-cadherin downregulation and decreased PD-L1 expression using our cell lines and published microarray data. ShRNA mediated E-cadherin knockdown decreased PD-L1 expression in parental cells, and dual immunofluorescent staining of E-cadherin and PD-L1 suggests co-localization of both molecules. We also observed marked downregulation of PD-L1 in cells with E-cadherin downregulation after chronic treatment with vinorelbine. These results indicate a correlation between therapy-induced E-cadherin downregulation and decreased PD-L1 expression, highlighting the importance of re-biopsy after acquisition of resistance to EGFR-TKIs, not only for the evaluation of resistance mechanisms but also for the determination of PD-L1 expression status.

Seven-microRNA panel for lung adenocarcinoma early diagnosis in patients presenting with ground-glass nodules

Background MicroRNA (miRNA) expression is correlated with tumor histology, differentiation, invasiveness and treatment outcome. We aimed to identify miRNAs whose differential expression might enable early diagnosis of lung adenocarcinoma in patients presenting with ground-glass nodules (GGNs). Methods To identify potential miRNAs of interest, we analyzed the miRNA expression profile of tumor and adjacent non-para-tumor tissue in three participants by next-generation sequencing (NGS). We then assessed the expression levels of the miRNAs of interest in 73 lung adenocarcinomas presenting with GGNs with matched adjacent non-tumor tissue by quantitative real-time polymerase chain reaction (qRT-PCR). We also detected the miRNA panel in 66 lung benign diseases and 66 lung adenocarcinomas presenting with GGN lesion tissues by qRT-PCR. Target genes of our selected miRNA panel were predicted using Miranda with default parameters. Results Twenty-three miRNAs showed differential expression between tumor and adjacent non-tumor tissue by NGS. Five miRNAs exhibited higher expression in tumor tissue compared to adjacent non-tumor tissue (P<0.05); 18 miRNAs demonstrated lower expression in tumor tissue versus adjacent non-tumor tissue (P<0.05). When qRT-PCR was performed for the 23 miRNAs identified by NGS in the pilot stage, seven were found to have statistically significant expression in tumor versus adjacent non-tumor tissue (P<0.05). The sensitivity and specificity of seven-miRNA panel were 86.4% and 60.6%, respectively. Conclusion The predicted targets of our miRNAs of interest are frequently associated with cancer signaling pathways. We developed a miRNA panel that could potentially predict the presence of lung adenocarcinoma in patients presenting with GGNs.

CD44 facilitates epithelial to mesenchymal transition phenotypic change at acquisition of resistance to EGFR kinase inhibitors in lung cancer

Epithelial-to-mesenchymal transition (EMT) is one of the acquired resistance mechanisms to EGFR tyrosine kinase inhibitors (TKI) in lung cancers. Because EMT is related to tumor invasion, metastases, and resistance to various treatments, it is important to prevent the emergence of EMT. However, molecular mechanism(s) underlying EMT phenotypic changes, as well as biomarker(s) that predict the emergence of EMT in EGFR-mutated lung cancers, are unclear to date. Through the comparison of expression data between isogenic lung cancer cell lines that acquired resistance to EGFR-TKI(s), we identified that high CD44 expression is related to a mesenchymal phenotype and that shRNA-mediated knockdown of CD44 reversed the EMT change. High membranous CD44 expression was identified in lesions with mesenchymal phenotype that were obtained from lung cancer patients who developed acquired resistance to gefitinib or afatinib, whereas isogenic lesions without EMT change showed negative/weak staining for CD44. Immunohistochemistry for treatment-naïve lung cancer cell lines with EGFR mutations found those that acquire resistance to EGFR-TKIs via EMT (HCC4006 and H1975 cells) had strong membranous CD44 expression compared with non–EMT-transforming lines which demonstrated negative or weak staining (Fisher exact test P value = 0.036). shRNA-mediated CD44 knockdown in HCC4006 cells prevented the emergence of EMT after chronic exposure to osimertinib. These results suggest that upregulation of CD44 facilitates EMT-phenotypic change in lung cancers with EGFR mutations when treated with EGFR-TKIs. In addition, our results suggest that CD44 can be a useful biomarker to predict the emergence of EMT upon EGFR-TKI monotherapy. Mol Cancer Ther; 17(10); 2257–65. ©2018 AACR.

The immune checkpoint, HVEM may contribute to immune escape in non-small cell lung cancer lacking PD-L1 expression

BACKGROUND Herpes Virus Entry Mediator (HVEM) is an important immune checkpoint in cancer recognition. HVEM expressed on tumor cell membranes activates immune cell signaling pathways leading to either inhibition of activity (B- and T-lymphocyte attenuator, BTLA) or activation of immune activity (LIGHT). The aim of this study is to investigate the prevalence of HVEM expression and its association with PDL1 expression in NSCLC. METHODS A TMA of 527 resected NSCLC samples and 56 NSCLC cell lines were evaluated for HVEM and PD-L1 expression. The IHC assay for HVEM was optimized on the Dako Link48 autostainer using a polyclonal antibody from R&D Systems(AF356). PD-L1 IHC was performed on the Dako Link48 autostainer using the PD-L1 28-8 pharmDx kit. Scoring HVEM employed the H-score system while for PD-L1 the tumor proportion score (TPS) was used. RESULTS HVEM expression in the NSCLC resected samples and cell lines revealed a positive H-score more than 1 was 18.6% (77/415) and 48.2% (27/56) respectively. HVEM expression was significantly higher in patients with lymph node N2 metastasis (25.5% vs 7.9% vs 17.5%, p = 0.046) when comparing with N1 or no lymph node metastasis, and was marginally significantly higher in patients with stage III/IV disease (24.5% vs 16.4%, p = 0.059). Subgroup analysis showed that HVEM (median 45 vs 36 months, p = 0.706) and PD-L1 expression (median 45 vs 48 months, p = 0.178) status was not predictive of overall survival. HVEM was found to have a significant negative correlation with PD-L1 expression (r=-0.274, p < 0.001) in patients with NSCLC and also a weak negative correlation in NSCLC cell lines (r=-0.162, p = 0.352). CONCLUSION HVEM was found to be overexpressed in NSCLC patients of N2 lymph node metastasis or later stage and has a negative co-relationship with PD-L1 expression. HVEM was not prognostic for NSCLC patients.

EGFR-directed monoclonal antibodies in combination with chemotherapy for treatment of non-small-cell lung cancer: an updated review of clinical trials and new perspectives in biomarkers analysis

Lung cancer still represents one of the most common and fatal neoplasm, accounting for nearly 30% of all cancer-related deaths. Targeted therapies based on molecular tumor features and programmed death-1 (PD-1)/programmed death ligand-1 (PDL-1) blockade immunotherapy have offered new therapeutic options for patients with advanced non-small-cell lung cancer (NSCLC). Activation of the epidermal growth factor receptor (EGFR)-pathway promotes tumor growth and progression, including angiogenesis, invasion, metastasis and inhibition of apoptosis, providing a strong rationale for targeting this pathway. EGFR expression is detected in up to 85% of NSCLC and has been demonstrated to be associated with poor prognosis. Two approaches for blocking EGFR signaling are available: prevention of ligand binding to the extracellular domain with monoclonal antibodies (mAbs) and inhibition of the intracellular tyrosine kinase activity with small molecules. There is a strong rationale to consider the tumors level of EGFR expression as one of the most significant predictive biomarkers in this setting. In this paper we provide an update focusing on the current status of EGFR-directed mAbs use for the treatment of patients with advanced NSCLC, through a review of all clinical trials involving anti-EGFR mAbs in combination with chemotherapy (CT) for advanced disease and with chemo-radiotherapy for stage III disease. Here we also discuss the current status of predictive biomarkers for anti-EGFR mAbs when added to first-line CT in patients with advanced NSCLC. Finally, we focused on the relevance of EGFR fluorescence in situ hybridization (FISH)+ and immunohistochemistry (IHC)-Score ≥ 200 as predictive biomarkers for the selection of patients who would be most likely to derive a clinical benefit from treatment with CT in combination with anti-EGFR mAbs, with particular reference also to histology.