Mehmet Altan

Differential Expression and Significance of PD-L1, IDO-1, and B7-H4 in Human Lung Cancer

Purpose: To determine the expression level, associations, and biological role of PD-L1, IDO-1, and B7-H4 in non–small cell lung cancer (NSCLC). Experimental Design: Using multiplexed quantitative immunofluorescence (QIF), we measured the levels of PD-L1, IDO-1, B7-H4, and different tumor-infiltrating lymphoycte (TIL) subsets in 552 stages I–IV lung carcinomas from two independent populations. Associations between the marker levels, TILs, and major clinicopathologic variables were determined. Validation of findings was performed using mRNA expression data from The Cancer Genome Atlas (TCGA) and in vitro stimulation of lung adenocarcinoma A549 cells with IFNγ and IL10. Results: PD-L1 was detected in 16.9% and 21.8% of cases in each population. IDO-1 was expressed in 42.6% and 49.8%; and B7-H4 in 12.8% and 22.6% of cases, respectively. Elevated PD-L1 and IDO-1 were consistently associated with prominent B- and T-cell infiltrates, but B7-H4 was not. Coexpression of the three protein markers was infrequent, and comparable results were seen in the lung cancer TCGA dataset. Levels of PD-L1 and IDO-1 (but not B7-H4) were increased by IFNγ stimulation in A549 cells. Treatment with IL10 upregulated B7-H4 but did not affect PD-L1 and IDO-1 levels. Conclusions: PD-L1, IDO-1, and B7-H4 are differentially expressed in human lung carcinomas and show limited co-expression. While PD-L1 and IDO-1 are associated with increased TILs and IFNγ stimulation, B7-H4 is not. The preferential expression of discrete immune evasion pathways in lung cancer could participate in therapeutic resistance and support design of optimal clinical trials. Clin Cancer Res; 23(2); 370–8. ©2016 AACR.

B7-H3 expression in NSCLC and its association with B7-H4, PD-L1 and Tumor Infiltrating Lymphocytes

Purpose: The immune checkpoint PD-1 and its receptor B7-H1 (PD-L1) are successful therapeutic targets in cancer but less is known about other B7 family members. Here, we determined the expression level of B7-H3 protein in non–small cell lung cancer (NSCLC) and evaluated its association with tumor-infiltrating lymphocytes (TIL), PD-L1, B7-H4, and major clinicopathologic characteristics is in 3 NSCLC cohorts. Experimental design: We used multiplexed automated quantitative immunofluorescence (QIF) to assess the levels of B7-H3, PD-L1, B7-H4, and TILs in 634 NSCLC cases with validated antibodies. Associations between the marker levels, major clinicopathologic variables and survival were analyzed. Results: Expression of B7-H3 protein was found in 80.4% (510/634) of the cases. High B7-H3 protein level (top 10 percentile) was associated with poor overall survival (P < 0.05). Elevated B7-H3 was consistently associated with smoking history across the 3 cohorts, but not with sex, age, clinical stage, and histology. Coexpression of B7-H3 and PD-L1 was found in 17.6% of the cases (112/634) and with B7-H4 in 10% (63/634). B7-H4 and PD-L1 were simultaneously detected only in 1.8% of NSCLCs (12/634). The expression of B7-H3 was not associated with the levels of CD3-, CD8-, and CD20-positive TILs. Conclusions: B7-H3 protein is expressed in the majority of NSCLCs and is associated with smoking history. High levels of B7-H3 protein have a negative prognostic impact in lung carcinomas. Coexpression of B7-H3 with PD-L1 and B7-H4 is relatively low, suggesting a nonredundant biological role of these targets. Clin Cancer Res; 23(17); 5202–9. ©2017 AACR.

Erratum to: Effect of neoadjuvant chemotherapy on tumor-infiltrating lymphocytes and PD-L1 expression in breast cancer and its clinical significance

BackgroundThe effects of neoadjuvant chemotherapy on immune markers remain largely unknown. The specific aim of this study was to assess stromal tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) protein expression in a cohort of breast cancer patients treated with neoadjuvant chemotherapy.MethodsUsing quantitative immunofluorescence, we investigated stromal TILs and PD-L1 protein expression in pre-treatment and residual breast cancer tissue from a Yale Cancer Center patient cohort of 58 patients diagnosed with breast cancer from 2003 to 2009 and treated with neoadjuvant chemotherapy. We compared the TIL count and PD-L1 status in paired pre-treatment and residual cancer tissues and correlated changes and baseline levels with survival.ResultsOf the 58 patients, 46 (79.3%) had hormone-positive and 34 (58.6%) had node-positive breast cancer. Eighty-six percent of residual cancer tissues had TIL infiltration and 17% had PD-L1 expression. There was a trend for higher TIL counts in postchemotherapy compared to prechemotherapy samples (p = 0.09). Increase in TIL count was associated with longer 5-year recurrence-free survival (p = 0.02, HR = 3.9, 95% CI = 1.179–15.39). PD-L1 expression (both stromal and tumor cells) was significantly lower in post-treatment samples (p = 0.001). Change in PD-L1 expression after therapy or TILs and PD-L1 expression in the posttreatment samples did not correlate with survival.ConclusionsIncrease in stromal TILs in residual cancer compared to pretreatment tissue is associated with improved recurrence-free survival. Despite a trend for increasing TIL counts, PD-L1 expression decreased in residual disease compared to pretreatment samples.

Gene Signature–Guided Dasatinib Therapy in Metastatic Breast Cancer

Purpose: Dasatinib has limited single-agent activity in unselected patients with metastatic breast cancer. Several gene signatures predictive of dasatinib response in vitro have been reported. The purpose of this three-arm, phase II study was to prospectively assess the utility of three previously published gene signatures to select patients with clinical benefit from dasatinib. Experimental Design: Patients with metastatic breast cancer underwent biopsy for gene expression profiling in an academic CLIA laboratory; those who were positive for any one of three predictive gene signatures (dasatinib sensitivity signature, SRC pathway activity signature, and dasatinib target index) received dasatinib 100 mg orally daily. The three marker-defined cohorts were analyzed separately, using early stopping rules for futility. Results: Ninety-seven patients were enrolled, 93 underwent biopsy, and 80% of the biopsies were sufficient for molecular testing. Thirty patients were positive for at least one signature and received treatment. Only 1 patient experienced clinical benefit and had stable disease over 300 days. All three arms were closed early for futility. There was one serious biopsy-related adverse event (hematoma and pain following a liver biopsy). There were no unexpected toxicities from dasatinib. Conclusion: None of the three predictive gene signatures, although supported by preclinical evidence, defined tumors clinically sensitive to dasatinib as a single agent. Clin Cancer Res; 20(20); 5265–71. ©2014 AACR.

Mechanisms and clinical activity of an EGFR and HER2 exon 20–selective kinase inhibitor in non–small cell lung cancer

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non–small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations.Poziotinib is a candidate inhibitor for a subset of EGFR or HER2 mutant non–small cell lung cancers that lack effective therapy.

Safety of Programmed Death–1 Pathway Inhibitors Among Patients With Non–Small-Cell Lung Cancer and Preexisting Autoimmune Disorders

Purpose Although programmed death (PD)-1 pathway inhibitors are now used in nearly all patients with advanced non-small-cell lung cancer (NSCLC), the large number of patients with NSCLC and concurrent autoimmune disease (AID) have been universally excluded from immunotherapy clinical trials. Therefore, the safety of PD-1 and PD-ligand 1 (PD-L1) inhibitors in patients with NSCLC and underlying AID is currently unknown. Methods As part of a multi-institutional effort, we retrospectively collected clinicopathologic data from patients with NSCLC and a history of AID who received monotherapy with either a PD-1 or a PD-L1 (herein referred to as PD-[L]1) inhibitor. Qualifying AIDs included but were not limited to: rheumatologic, neurologic, endocrine, GI, and dermatologic conditions. Results We identified 56 patients with NSCLC and an AID who received a PD-(L)1 inhibitor. At the time of treatment initiation, 18% of patients had active AID symptoms and 20% were receiving immunomodulatory agents for their AID. A total of 55% of patients developed an AID flare and/or an immune-related adverse event (irAE). Exacerbation of the AID occurred in 13 patients (23% of the whole cohort), four of whom required systemic corticosteroids. Immune-related adverse events occurred in 21 patients (38%). Among irAEs, 74% were grade 1 or 2 and 26% were grade 3 or 4; eight patients required corticosteroids for irAE management. PD-(L)1 therapy was permanently discontinued in eight patients (14%) because of irAEs. The overall response rate to immunotherapy in this population was 22%. Conclusion In patients with NSCLC with AID treated with a PD-(L)1 inhibitor, exacerbation of AID occurred in a minority of patients. The incidence of irAEs was similar to reported rates in clinical trials where patients with AID were excluded. Adverse events were generally manageable and infrequently led to permanent discontinuation of immunotherapy.

EGFR-directed antibodies increase the risk of severe infection in cancer patients

Monoclonal antibodies directed to the epidermal growth factor receptor (EGFR) have a role in the management of several solid tumors, alone or in combination with chemotherapy or radiation therapy. Recognized toxicities have included hypersensitivity reactions, rash, hypomagnesemia, and constitutional symptoms, but the possibility that the agents lead to immunosuppression or increase the risk of infection has only recently been recognized. Two latest meta-analyses, including the recently published article by Qi et al., highlight the increased risk of severe infections with EGFR-directed monoclonal antibodies. Further studies are needed to better identify the association between EGFR-directed monoclonal antibody treatment and infection, as well as to elucidate the mechanism of this toxicity and to develop tools to identify patients at increased risk for these complications. In the meantime, awareness of the role of EGFR-directed antibodies in increased infection risk may have implications for dose modification strategies in both clinical trial design and the practice of oncology.Please see related article: http://www.biomedcentral.com/1741-7015/12/203.

EGFR-GRB2 Protein Colocalization Is a Prognostic Factor Unrelated to Overall EGFR Expression or EGFR Mutation in Lung Adenocarcinoma

Introduction EGFR is a therapeutic target in NSCLC for EGFR‐mutant patients. Proximity ligation assay (PLA) is a method to detect functional signaling associated protein complexes. Growth factor receptor bound protein 2 (GRB2) is an adaptor protein that binds to the phosphorylated residues of active EGFR. Interaction of EGFR and GRB2 correlates with active EGFR signaling and leads to activation of the MAPK/ERK pathway. Methods A PLA developed to detect EGFR‐GRB2 interaction was measured by quantitative immunofluorescence using Automated Quantitative Analysis technology. EGFR pathway activation was assessed in patients with NSCLC with different mutation status along with overall EGFR expression. Additionally, the PLA to detect EGFR‐GRB2 interaction was evaluated as a prognostic marker in two cohorts of patients with lung adenocarcinoma. Results The PLA to detect EGFR‐GRB2 interaction was unrelated to overall EGFR expression or mutation in a series of patients with NSCLC with known mutation status. EGFR‐mutant (p = 0.04) and EGFR/KRAS wild‐type tumors (p = 0.0049) had significantly higher EGFR pathway activation compared with KRAS‐mutant cases, with no significant difference shown between mutation sites. In two series of patients with lung adenocarcinoma, the PLA to detect EGFR‐GRB2 interaction was independently associated with longer survival (hazard ratio = 0.46, 95% confidence interval: 0.2–0.78, p = 0.0085 and hazard ratio = 0.48, 95% confidence interval: 0.2–0.85, p = 0.017). Total EGFR protein expression alone was not correlated with outcome. Conclusions EGFR colocalization with GRB2 as assessed by PLA is not correlated with EGFR expression levels or mutation status, defining a patient group that may show EGFR pathway activation, as illustrated by its prognostic value. Future studies may determine whether this group is more likely to respond to EGFR‐targeted therapies.

An assessment of neuronal calcium sensor-1 and response to neoadjuvant chemotherapy in breast cancer patients

Neuronal calcium sensor-1 (NCS-1) has been identified as a binding partner of the taxane, paclitaxel. Our previous study showed that overexpression of NCS-1 increased the efficacy of paclitaxel in vitro, but was associated with poor clinical outcome. Here, we determine if NCS-1 expression is associated with pathological complete response (pCR) to taxane-based neoadjuvant chemotherapy in 105 pre-treatment breast cancer biopsies. Elevated expression of NCS-1 was found to be positively associated with pCR. These results suggest that NCS-1 may be a predictive biomarker for response to taxane-based neoadjuvant chemotherapy in breast cancer.

Immune marker profiling and PD-L1 expression across Non-Small Cell Lung Cancer mutations.

Introduction: Programmed death 1/programmed death ligand 1 (PD‐L1) axis inhibitors have been proven effective, especially in patients with tumors expressing PD‐L1. Their clinical efficacy in patients with EGFR‐activating mutations is still unclear, whereas KRAS mutations seem to be associated with good response. Methods: We used multiplexed quantitative immunofluorescence to investigate PD‐L1 expression and to characterize tumor infiltrating lymphocyte (TIL) populations and their activation status in more than 150 NSCLC patients with known mutation status. Results: PD‐L1 expression was significantly lower in EGFR‐mutant compared to KRAS‐mutant, and EGFR/KRAS wild‐type (WT) tumors. KRAS mutant tumors were more inflamed with higher CD4+, CD8+ and CD20+ TILs. Subgroup analysis by TIL activation status revealed that EGFR mutants had a high frequency of inactive TILs even though lymphocytes were present in the tumor microenvironment. In contrast, in KRAS mutants, when TILs were present they were almost always active. Additionally, we found differences between EGFR mutation sites in CD8+ expression and the TIL activation profile. Finally, activated EGFR correlated with increased PD‐L1 expression in EGFR mutants but not in EGFR WT, whereas TIL activation was associated with higher PD‐L1 only in EGFR/KRAS WT. Conclusions: Our findings show the unique immune profile of EGFR‐mutant tumors. The high frequency of inactive TILs could explain the low immunotherapy response rates in these patients, whereas PD‐L1 as a predictive biomarker may reflect the constitutive oncogenic signaling rather than immune signaling, which would be associated with high PD‐L1 levels and TILs activation.