Phase 1/2 study of rilotumumab (AMG 102), a hepatocyte growth factor inhibitor, and erlotinib in patients with advanced non-small cell lung cancer.

BACKGROUND: Activation of the mesenchymal-epidermal transition factor (MET) tyrosine kinase and its ligand, hepatocyte growth factor (HGF), is implicated in resistance to epidermal growth factor receptor (EGFR) inhibitors. In this phase 1/2 trial, rilotumumab (an anti-HGF antibody) combined with erlotinib was evaluated in patients with metastatic, previously treated non-small cell lung cancer. METHODS: In phase 1, a dose de-escalation design was adopted with rilotumumab starting at 15 mg/kg intravenously every 3 weeks and oral erlotinib 150 mg daily. In phase 2, the disease control rate (DCR) (according to Response Evaluation Criteria in Solid Tumors) of the combination was evaluated using a Simon 2-stage design. The biomarkers examined included 10 plasma-circulating molecules associated with the EGFR and MET pathways. RESULTS: Without indications for de-escalation, the recommended phase 2 dose was dose level 0. Overall, 45 response-evaluable patients were enrolled (13 with squamous carcinoma, 32 with adenocarcinoma; 2 had confirmed EGFR mutations, 33 had confirmed wild-type [WT] EGFR, and 7 had KRAS mutations). The DCR for all patients was 60% (90% confidence interval [CI], 47.1%-71.3%). Median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 6.6 months (90% CI, 5.6-8.9 months). Among patients with WT EGFR, the DCR was 60.6% (90% CI, 46.3%-73.3%), median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 7.0 months (90% CI, 5.6-13.4 months). Elevated baseline levels of neuregulin 1 were associated with longer progression-free survival (hazard ratio, 0.41; 95% CI, 0.19-0.87), whereas elevated amphiregulin levels were associated with more rapid progression (hazard ratio, 2.14; 95% CI, 1.48-3.08). CONCLUSIONS: Combined rilotumumab and erlotinib had an acceptable safety profile, and the DCR met the prespecified criteria for success. In the EGFR WT group, the DCR exceeded published reports for erlotinib alone. High circulating levels of neuregulin 1 may indicate sensitivity to this combination. Cancer 2017;123:2936-44. © 2017 American Cancer Society.

KRAS and TP53 mutations in bronchoscopy samples from former lung cancer patients.

Mutations in the KRAS and TP53 genes have been found frequently in lung tumors and specimens from individuals at high risk for lung cancer and have been suggested as predictive markers for lung cancer. In order to assess the prognostic value of these two genes' mutations in lung cancer recurrence, we analyzed mutations in codon 12 of the KRAS gene and in hotspot codons of the TP53 gene in 176 bronchial biopsies obtained from 77 former lung cancer patients. Forty-seven patients (61.0%) showed mutations, including 35/77 (45.5%) in the KRAS gene and 25/77 (32.5%) in the TP53 gene, among them 13/77 (16.9%) had mutations in both genes. When grouped according to past or current smoking status, a higher proportion of current smokers showed mutations, in particular those in the TP53 gene (P = 0.07), compared with ex-smokers. These mutations were found in both abnormal lesions (8/20 or 40%) and histologically normal tissues (70/156 or 44.9%) (P = 0.812). They consisted primarily of G to A transition and G to T transversion in both the KRAS (41/56 or 73.2%) and TP53 (24/34 or 70.6%) genes, consistent with mutations found in lung tumors of smoking lung cancer patients. Overall, recurrence-free survival (RFS) among all subjects could be explained by age at diagnosis, tumor stage, tumor subtype, and smoking (P < 0.05, Cox proportional hazard). Therefore, KRAS and TP53 mutations were frequently detected in bronchial tissues of former lung cancer patients. However, the presence of mutation of bronchial biopsies was not significantly associated with a shorter RFS time. © 2016 Wiley Periodicals, Inc.

Gastrin-releasing peptide receptor expression in non-cancerous bronchial epithelia is associated with lung cancer: a case-control study.

BACKGROUND: Normal bronchial tissue expression of GRPR, which encodes the gastrin-releasing peptide receptor, has been previously reported by us to be associated with lung cancer risk in 78 subjects, especially in females. We sought to define the contribution of GRPR expression in bronchial epithelia to lung cancer risk in a larger case-control study where adjustments could be made for tobacco exposure and sex. METHODS: We evaluated GRPR mRNA levels in histologically normal bronchial epithelial cells from 224 lung cancer patients and 107 surgical cancer-free controls. Associations with lung cancer were tested using logistic regression models. RESULTS: Bronchial GRPR expression was significantly associated with lung cancer (OR = 4.76; 95% CI = 2.32-9.77) in a multivariable logistic regression (MLR) model adjusted for age, sex, smoking status and pulmonary function. MLR analysis stratified by smoking status indicated that ORs were higher in never and former smokers (OR = 7.74; 95% CI = 2.96-20.25) compared to active smokers (OR = 1.69; 95% CI = 0.46-6.33). GRPR expression did not differ by subject sex, and lung cancer risk associated with GRPR expression was not modified by sex. CONCLUSIONS: GRPR expression in non-cancerous bronchial epithelium was significantly associated with the presence of lung cancer in never and former smokers. The association in never and former smokers was found in males and females. Association with lung cancer did not differ by sex in any smoking group.

Syngeneic tobacco carcinogen-induced mouse lung adenocarcinoma model exhibits PD-L1 expression and high tumor mutational burden.

Human lung adenocarcinoma (LUAD) in current or former smokers exhibits a high tumor mutational burden (TMB) and distinct mutational signatures. Syngeneic mouse models of clinically relevant smoking-related LUAD are lacking. We established and characterized a tobacco-associated, transplantable murine LUAD cell line, designated FVBW-17, from a LUAD induced by the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone in the FVB/N mouse strain. Whole-exome sequencing of FVBW-17 cells identified tobacco-associated KrasG12D and Trp53 mutations and a similar mutation profile to that of classic alkylating agents with a TMB greater than 500. FVBW-17 cells transplanted subcutaneously, via tail vein, and orthotopically generated tumors that were histologically similar to human LUAD in FVB/N mice. FVBW-17 tumors expressed programmed death ligand 1 (PD-L1), were infiltrated with CD8+ T cells, and were responsive to anti-PD-L1 therapy. FVBW-17 cells were also engineered to express green fluorescent protein and luciferase to facilitate detection and quantification of tumor growth. Distant metastases to lung, spleen, liver, and kidney were observed from subcutaneously transplanted tumors. This potentially novel cell line is a robust representation of human smoking-related LUAD biology and provides a much needed preclinical model in which to test promising new agents and combinations, including immune-based therapies.

Phase I Study of Ficlatuzumab and Cetuximab in Cetuximab-Resistant, Recurrent/Metastatic Head and Neck Cancer.

Cetuximab, an anti-EGFR monoclonal antibody (mAb), is approved for advanced head and neck squamous cell carcinoma (HNSCC) but benefits a minority. An established tumor-intrinsic resistance mechanism is cross-talk between the EGFR and hepatocyte growth factor (HGF)/cMet pathways. Dual pathway inhibition may overcome cetuximab resistance. This Phase I study evaluated the combination of cetuximab and ficlatuzumab, an anti-HGF mAb, in patients with recurrent/metastatic HNSCC. The primary objective was to establish the recommended Phase II dose (RP2D). Secondary objectives included overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Mechanistic tumor-intrinsic and immune biomarkers were explored. Thirteen patients enrolled with no dose-limiting toxicities observed at any dose tier. Three evaluable patients were treated at Tier 1 and nine at Tier 2, which was determined to be the RP2D (cetuximab 500 mg/m2 and ficlatuzumab 20 mg/kg every 2 weeks). Median PFS and OS were 5.4 (90% CI = 1.9-11.4) and 8.9 (90% CI = 2.7-15.2) months, respectively, with a confirmed ORR of 2 of 12 (17%; 90% CI = 6-40%). High circulating soluble cMet levels correlated with poor survival. An increase in peripheral T cells, particularly the CD8+ subset, was associated with treatment response whereas progression was associated with expansion of a distinct myeloid population. This well-tolerated combination demonstrated promising activity in cetuximab-resistant, advanced HNSCC.

A preliminary analysis of interleukin-1 ligands as potential predictive biomarkers of response to cetuximab.

BACKGROUND: The epidermal growth factor receptor (EGFR) monoclonal IgG1 antibody cetuximab is approved for first-line treatment of recurrent and metastatic (R/M) HNSCC as a part of the standard of care EXTREME regimen (platinum/5-fluorouracil/cetuximab). This regimen has relatively high response and disease control rates but is generally not curative and many patients will experience recurrent disease and/or metastasis. Therefore, there is a great need to identify predictive biomarkers for recurrence and disease progression in cetuximab-treated HNSCC patients to facilitate patient management and allow for treatment modification. The goal of this work is to assess the potential of activating interleukin-1 (IL-1) ligands (IL-1 alpha [IL-1α], IL-1 beta [IL-1ß]) as predictive biomarkers of survival outcomes in HNSCC patients treated with cetuximab-based chemotherapy. METHODS: Baseline gene, serum and tumor expression of interleukin-1 (IL-1) ligands were analyzed from The Cancer Genome Atlas (TCGA) database or clinical trials of cetuximab-based therapies and interrogated for associations with clinical outcome data. RESULTS: High tumor gene expression of IL-1ß was associated with a more favorable overall survival in cetuximab-treated HNSCC patients but not in non-cetuximab-treated patients. In HNSCC patients treated with cetuximab-based chemotherapy, higher gene and circulating levels of IL-1α and IL-1ß were correlated with a more favorable progression free survival compared to patients with low or undetectable levels of IL-1 ligands. CONCLUSIONS: These findings suggest that IL-1 ligands may function as predictive biomarkers for tumor response to cetuximab-based chemotherapy in HNSCC patients and warrants further investigation and validation in larger clinical studies.

ADAM10 Sheddase Activity is a Potential Lung-Cancer Biomarker.

Background: Increases in expression of ADAM10 and ADAM17 genes and proteins are inconsistently found in cancer lesions, and are not validated as clinically useful biomarkers. The enzyme-specific proteolytic activities, which are solely mediated by the active mature enzymes, directly reflect enzyme cellular functions and might be superior biomarkers than the enzyme gene or protein expressions, which comprise the inactive proenzymes and active and inactivated mature enzymes. Methods: Using a recent modification of the proteolytic activity matrix analysis (PrAMA) measuring specific enzyme activities in cell and tissue lysates, we examined the specific sheddase activities of ADAM10 (ADAM10sa) and ADAM17 (ADAM17sa) in human non-small cell lung-carcinoma (NSCLC) cell lines, patient primary tumors and blood exosomes, and the noncancerous counterparts. Results: NSCLC cell lines and patient tumors and exosomes consistently showed significant increases of ADAM10sa relative to their normal, inflammatory and/or benign-tumor controls. Additionally, stage IA-IIB NSCLC primary tumors of patients who died of the disease exhibited greater increases of ADAM10sa than those of patients who survived 5 years following diagnosis and surgery. In contrast, NSCLC cell lines and patient tumors and exosomes did not display increases of ADAM17sa. Conclusions: This study is the first to investigate enzyme-specific proteolytic activities as potential cancer biomarkers. It provides a proof-of-concept that ADAM10sa could be a biomarker for NSCLC early detection and outcome prediction. To ascertain that ADAM10sa is a useful cancer biomarker, further robust clinical validation studies are needed.

Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates.

Increases in expression of ADAM10 and ADAM17 genes and proteins have been evaluated, but not validated as cancer biomarkers. Specific enzyme activities better reflect enzyme cellular functions, and might be better biomarkers than enzyme genes or proteins. However, no high throughput assay is available to test this possibility. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10-/- and ADAM17-/- mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10-/- MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms.

15q12 variants, sputum gene promoter hypermethylation, and lung cancer risk: a GWAS in smokers.

BACKGROUND: Lung cancer is the leading cause of cancer-related mortality worldwide. Detection of promoter hypermethylation of tumor suppressor genes in exfoliated cells from the lung provides an assessment of field cancerization that in turn predicts lung cancer. The identification of genetic determinants for this validated cancer biomarker should provide novel insights into mechanisms underlying epigenetic reprogramming during lung carcinogenesis. METHODS: A genome-wide association study using generalized estimating equations and logistic regression models was conducted in two geographically independent smoker cohorts to identify loci affecting the propensity for cancer-related gene methylation that was assessed by a 12-gene panel interrogated in sputum. All statistical tests were two-sided. RESULTS: Two single nucleotide polymorphisms (SNPs) at 15q12 (rs73371737 and rs7179575) that drove gene methylation were discovered and replicated with rs73371737 reaching genome-wide significance (P = 3.3×10(-8)). A haplotype carrying risk alleles from the two 15q12 SNPs conferred 57% increased risk for gene methylation (P = 2.5×10(-9)). Rs73371737 reduced GABRB3 expression in lung cells and increased risk for smoking-induced chronic mucous hypersecretion. Furthermore, subjects with variant homozygote of rs73371737 had a two-fold increase in risk for lung cancer (P = .0043). Pathway analysis identified DNA double-strand break repair by homologous recombination (DSBR-HR) as a major pathway affecting susceptibility for gene methylation that was validated by measuring chromatid breaks in lymphocytes challenged by bleomycin. CONCLUSIONS: A functional 15q12 variant was identified as a risk factor for gene methylation and lung cancer. The associations could be mediated by GABAergic signaling that drives the smoking-induced mucous cell metaplasia. Our findings also substantiate DSBR-HR as a critical pathway driving epigenetic gene silencing.

Nicotine signals through muscle-type and neuronal nicotinic acetylcholine receptors in both human bronchial epithelial cells and airway fibroblasts.

BACKGROUND: Non-neuronal cells, including those derived from lung, are reported to express nicotinic acetylcholine receptors (nAChR). We examined nAChR subunit expression in short-term cultures of human airway cells derived from a series of never smokers, ex-smokers, and active smokers. METHODS AND RESULTS: At the mRNA level, human bronchial epithelial (HBE) cells and airway fibroblasts expressed a range of nAChR subunits. In multiple cultures of both cell types, mRNA was detected for subunits that constitute functional muscle-type and neuronal-type pentomeric receptors. Two immortalized cell lines derived from HBE cells also expressed muscle-type and neuronal-type nAChR subunits. Airway fibroblasts expressed mRNA for three muscle-type subunits (alpha1, delta, and epsilon) significantly more often than HBE cells. Immunoblotting of HBE cell and airway fibroblast extracts confirmed that mRNA for many nAChR subunits is translated into detectable levels of protein, and evidence of glycosylation of nAChRs was observed. Some minor differences in nAChR expression were found based on smoking status in fibroblasts or HBE cells. Nicotine triggered calcium influx in the immortalized HBE cell line BEAS2B, which was blocked by alpha-bungarotoxin and to a lesser extent by hexamethonium. Activation of PKC and MAPK p38, but not MAPK p42/44, was observed in BEAS2B cells exposed to nicotine. In contrast, nicotine could activate p42/44 in airway fibroblasts within five minutes of exposure. CONCLUSIONS: These results suggest that muscle-type and neuronal-type nAChRs are functional in airway fibroblasts and HBE cells, that prior tobacco exposure does not appear to be an important variable in nAChR expression, and that distinct signaling pathways are observed in response to nicotine.

Targeting of Both the c-Met and EGFR Pathways Results in Additive Inhibition of Lung Tumorigenesis in Transgenic Mice.

EGFR and c-Met are both overexpressed in lung cancer and initiate similar downstream signaling, which may be redundant. To determine how frequently ligands that initiate signaling of both pathways are found in lung cancer, we analyzed serum for hepatocyte growth factor (HGF), transforming growth factor-alpha, and amphiregulin (AREG) in lung cancer cases and tobacco-exposed controls. HGF and AREG were both significantly elevated in cases compared to controls, suggesting that both HGF/c-Met and AREG/EGFR pathways are frequently active. When both HGF and AREG are present in vitro, downstream signaling to MAPK and Akt in non-small cell lung cancer (NSCLC) cells can only be completely inhibited by targeting both pathways. To test if dual blockade of the pathways could better suppress lung tumorigenesis in an animal model than single blockade, mice transgenic for airway expression of human HGF were treated with inhibitors of both pathways alone and in combination after exposure to a tobacco carcinogen. Mean tumor number in the group using both the HGF neutralizing antibody L2G7 and the EGFR inhibitor gefitinib was significantly lower than with single agents. A higher tumor K-ras mutation rate was observed with L2G7 alone compared to controls, suggesting that agents targeting HGF may be less effective against mutated K-ras lung tumors. This was not observed with combination treatment. A small molecule c-Met inhibitor decreased formation of both K-ras wild-type and mutant tumors and showed additive anti-tumor effects when combined with gefitinib. Dual targeting of c-Met/EGFR may have clinical benefit for lung cancer.

Molecular analysis of plasma DNA for the early detection of lung cancer by quantitative methylation-specific PCR.

PURPOSE: Aberrant promoter hypermethylation of tumor suppressor genes is a promising marker for lung cancer detection. We investigated the likelihood of detecting aberrant DNA methylation of tumor suppressor genes in plasma samples of patients with abnormalities of the lung detected upon computed tomography (CT) scan. EXPERIMENTAL DESIGN: In a small evaluation cohort, four gene promoters (DCC, Kif1a, NISCH, and Rarb) were found to be methylated with increased frequency in samples from cancer patients specifically. We then examined DNA from 93 plasma samples from patients with abnormal findings in the lung detected upon CT scan for aberrant methylation of these four gene promoters by quantitative fluorogenic real-time PCR. The patients were divided into two groups, ground glass opacity (n = 23) and cancerous tumors (n = 70). Plasma DNA from age-matched nodule-free individuals were used as controls (n = 80). RESULTS: In plasma, 73% of patients with cancerous tumors showed methylation of at least one gene with a specificity of 71% (P = 0.0001). Only 22% patients with ground glass opacity exhibited methylation of at least one gene. When smoking history was taken into account, 72% of cancer patients with no smoking history or those who smoked <20 pack-years showed methylation of at least one gene with 100% specificity (P = 0.05) when compared with matched controls. Among heavy smokers with 20+ pack-years of smoking history, 30% of the control group and 73% of the patients with cancerous tumors showed methylation (P = 0.0001). CONCLUSIONS: These biomarkers can distinguish between cancerous and noncancerous abnormal CT findings.

Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib.

Gastrin-releasing peptide (GRP) is a mitogen for lung epithelial cells and initiates signaling through a G-protein-coupled receptor, gastrin-releasing peptide receptor (GRPR). Because GRPR transactivates the epidermal growth factor receptor (EGFR), we investigated induction by GRP of Akt, an EGFR-activated signaling pathway, and examined effects of GRP on viability of non-small cell lung carcinoma (NSCLC) cells exposed to the EGFR tyrosine kinase inhibitor gefitinib. GRP induced Akt activation primarily through c-Src-mediated transactivation of EGFR. Transfection of dominant-negative c-Src abolished GRP-induced EGFR and Akt activation. GRP induced release of amphiregulin, and pre-incubation with human amphiregulin neutralizing antibody eliminated GRP-induced Akt phosphorylation. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely blocked GRP-initiated Akt phosphorylation. These results suggest that GRP stimulates Akt activation primarily via c-Src activation, followed by extracellular release of the EGFR ligand amphiregulin, leading to the activation of EGFR and PI3K. Pretreatment of NSCLC cells with GRP resulted in an increase in the IC(50) of gefitinib of up to 9-fold; this protective effect was mimicked by the pretreatment of cells with amphiregulin and reversed by Akt or PI3K inhibition. GRP appears to rescue NSCLC cells exposed to gefitinib through release of amphiregulin and activation of the Akt pathway, suggesting GRPR and/or EGFR autocrine pathways in NSCLC cells may modulate therapeutic response to EGFR inhibitors.