Circulating tumor DNA and radiological tumor volume identify patients at risk for relapse with resected, early-stage non-small-cell lung cancer.

BACKGROUND: Predicting relapse and overall survival (OS) in early-stage non-small-cell lung cancer (NSCLC) patients remains challenging. Therefore, we hypothesized that detection of circulating tumor DNA (ctDNA) can identify patients with increased risk of relapse and that integrating radiological tumor volume measurement along with ctDNA detectability improves prediction of outcome. PATIENTS AND METHODS: We analyzed 366 serial plasma samples from 85 patients who underwent surgical resections and assessed ctDNA using a next-generation sequencing liquid biopsy assay, and measured tumor volume using a computed tomography-based three-dimensional annotation. RESULTS: Our results showed that patients with detectable ctDNA at baseline or after treatment and patients who did not clear ctDNA after treatment had a significantly worse clinical outcome. Integrating radiological analysis allowed the stratification in risk groups prognostic of clinical outcome as confirmed in an independent cohort of 32 patients. CONCLUSIONS: Our findings suggest ctDNA and radiological monitoring could be valuable tools for guiding follow-up care and treatment decisions for early-stage NSCLC patients.

Distinct tumor-infiltrating lymphocyte landscapes are associated with clinical outcomes in localized non-small-cell lung cancer.

BACKGROUND: Despite the importance of tumor-infiltrating T lymphocytes (TILs) in cancer biology, the relationship between TIL phenotypes and their prognostic relevance for localized non-small-cell lung cancer (NSCLC) has not been well established. PATIENTS AND METHODS: Fresh tumor and normal adjacent tissue was prospectively collected from 150 patients with localized NSCLC. Tissue was comprehensively characterized by high-dimensional flow cytometry of TILs integrated with immunogenomic data from multiplex immunofluorescence, T-cell receptor sequencing, exome sequencing, RNA sequencing, targeted proteomics, and clinicopathologic features. RESULTS: While neither the magnitude of TIL infiltration nor specific TIL subsets were significantly prognostic alone, the integration of high-dimensional flow cytometry data identified two major immunotypes (IM1 and IM2) that were predictive of recurrence-free survival independent of clinical characteristics. IM2 was associated with poor prognosis and characterized by the presence of proliferating TILs expressing cluster of differentiation 103, programmed cell death protein 1, T-cell immunoglobulin and mucin-domain containing protein 3, and inducible T-cell costimulator. Conversely, IM1 was associated with good prognosis and differentiated by an abundance of CD8+ T cells expressing cytolytic enzymes, CD4+ T cells lacking the expression of inhibitory receptors, and increased levels of B-cell infiltrates and tertiary lymphoid structures. While increased B-cell infiltration was associated with good prognosis, the best prognosis was observed in patients with tumors exhibiting high levels of both B cells and T cells. These findings were validated in patient tumors from The Cancer Genome Atlas. CONCLUSIONS: Our study suggests that although the number of infiltrating T cells is not associated with patient survival, the nature of the infiltrating T cells, resolved in distinct TIL immunotypes, is prognostically relevant in NSCLC and may inform therapeutic approaches to clinical care.

Antitumour efficacy of MEK inhibitors in human lung cancer cells and their derivatives with acquired resistance to different tyrosine kinase inhibitors.

BACKGROUND: To study the molecular mechanisms regulating cancer cell resistance to four different tyrosine kinase inhibitors (TKIs): erlotinib, gefitinib, vandetanib and sorafenib. METHODS: An in vitro model of acquired resistance to these TKIs was developed by continuously treating the human lung adenocarcinoma cell line CALU-3 with escalating doses of each drug. Transcriptional profiling was performed with Agilent whole genome microarrays. Western blot analysis, enzyme-linked immunosorbent (ELISA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation, migration, invasion and anchorage-independent colony growth assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in parental (P) and TKI-resistant (R) CALU-3 cell lines. RESULTS: As compared with P-CALU-3 cells, in TKI-R CALU-3 cell lines a significant increase in the expression of activated, phosphorylated MET, IGF-1R, AKT, MEK, MAPK and of survivin was observed. Downregulation of E-cadherin and amphiregulin mRNAs and upregulation of vimentin, VE-cadherin, HIF-1α and vascular endothelial growth factor receptor-1 mRNAs were observed in all four TKI-R CALU-3 cell lines. All four TKI-R CALU-3 cells showed increased invasion, migration and anchorage-independent growth. Together, these data suggest epithelial to mesenchymal transition (EMT) in TKI-R CALU-3 cells. Treatment with several agents that target AKT, MET or IGF-1R did not affect TKI-R CALU-3 cell proliferation. In contrast, treatment with MSC19363669B and selumetinib, two selective MEK inhibitors, caused inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumour growth in vivo of all four TKI-R CALU-3 cell lines. CONCLUSION: These data suggest that resistance to four different TKIs is characterised by EMT, which is MEK-inhibitor sensitive in human CALU-3 lung adenocarcinoma.

Safety and activity of vandetanib in combination with everolimus in patients with advanced solid tumors: a phase I study.

BACKGROUND: Preclinical studies suggest that combining vandetanib (VAN), a multi-tyrosine kinase inhibitor of rearranged during transfection (RET) proto-oncogene, vascular endothelial growth factor receptor (VEGFR), and epidermal growth factor receptor (EGFR), with everolimus (EV), a mammalian target of rapamycin (mTOR) inhibitor, may improve antitumor activity. We determined the safety, maximum tolerated dose (MTD), recommended phase II dose (RP2D), and dose-limiting toxicities (DLTs) of VAN + EV in patients with advanced solid cancers and the effect of combination therapy on cancer cell proliferation and intracellular pathways. PATIENTS AND METHODS: Patients with refractory solid tumors were enrolled in a phase I dose-escalation trial testing VAN (100-300 mg orally daily) + EV (2.5-10 mg orally daily). Objective responses were evaluated using RECIST v1.1. RET mutant cancer cell lines were used in cell-based studies. RESULTS: Among 80 patients enrolled, 72 (90%) patients were evaluable: 7 achieved partial response (PR) (10%) and 37 had stable disease (SD) (51%; duration range: 1-27 cycles). Clinical benefit (SD or PR ≥ 6 months) was observed in 26 evaluable patients [36%, 95% confidence intervals (CI) (25% to 49%)]. In 80 patients, median overall survival (OS) was 10.5 months [95% CI (8.5-16.1)] and median progression-free survival (PFS) 4.1 months [95% CI (3.4-7.3)]. Six patients (7.5%) experienced DLTs and 20 (25%) required dose modifications. VAN + EV was safe, with fatigue, rash, diarrhea, and mucositis being the most common toxicities. In cell-based studies, combination therapy was superior to monotherapy at inhibiting cancer cell proliferation and intracellular signaling. CONCLUSIONS: The MTDs and RP2Ds of VAN + EV are 300 mg and 10 mg, respectively. VAN + EV combination is safe and active in refractory solid tumors. Further investigation is warranted in RET pathway aberrant tumors.

Serum vascular endothelial growth factor (VEGF) levels correlate with tumor VEGF and p53 overexpression in endocrine positive primary breast cancer.

Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, associated with unfavorable clinical characteristics in breast cancer. The aim of this study was to evaluate different angiogenic markers in endocrine-positive breast cancer patients. The authors analyzed serum and tumor samples from 71 patients with endocrine-positive operable primary breast cancer to determine the expression and the possible relationship between circulating serum VEGF levels, tumor VEGF expression, microvessel density (MVD), and other immunohistochemical parameters. Basal VEGF serum levels were significantly higher in breast cancer patients than in healthy controls. A significant correlation was observed between basal VEGF serum concentrations, microvessel density (p = 0.01) and p53 status (p = 0.004). Intratumoral VEGF expression was significantly associated with neoplastic embolization (p = 0.041) and circulating VEGF levels (p = 0.047). The results confirm that in primary endocrine-positive breast cancer serum VEGF levels are elevated and show a positive relationship with tumor VEGF and p53 overexpression.

Interaction between the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF) pathways: a rational approach for multi-target anticancer therapy.

Over the last decade, the concept of targeted biological therapy for the treatment of cancer has emerged. However, a better understanding of these targets and their role in tumor cells and in the surrounding stromal cells is required. Two interesting biological targets are the epidermal growth factor receptor (EGFR) and the vascular endothelia growth factor (VEGF) and its receptors. A number of agents that target these pathways have been described. Many of these are currently in clinical trials and a few have recently been approved by the regulatory authorities in USA and in the European Union. The molecular pathways involved in the proliferation of cancer cells and in tumor-related angiogenesis are very complex and the interference with only a single step of these pathways may often reveal an insufficient therapeutic approach. Moreover, cancer cells have an inherent ability to harness different growth factor signaling pathways for growth advantage and cell survival, a process that may even be facilitated by the use of selective targeted agents. Because of these escape mechanisms, monotherapy with selective targeted agents is unlikely to be a fully effective cancer treatment. For these reasons, targeting different pathways is an attractive and effective therapeutic strategy with a strong rationale for investigating this approach in the clinic. This review focuses on the preclinical rationale of combining targeted agents such as EGFR and VEGF inhibitors in the treatment of cancer and on the clinical trials that have emerged from these studies.

Sequence-dependent antiproliferative effects of cytotoxic drugs and epidermal growth factor receptor inhibitors.

BACKGROUND: Epidermal growth factor receptor (EGFR) inhibitors are in clinical development in cancer treatment. Preclinical studies have shown potential antitumor efficacy of these agents in combination with chemotherapy or with radiotherapy. However, controversial results have been obtained in different clinical trials. MATERIALS AND METHODS: The effects on proliferation, cell cycle distribution and induction of apoptosis of three different anti-EGFR agents (gefitinib, ZD6474, cetuximab) were evaluated in different sequences of combination with either a platinum derivative (cisplatin, carboplatin, oxaliplatin) or a taxane (docetaxel, paclitaxel) in KYSE30 cells, a model of a human cancer cell line with a functional EGFR autocrine pathway. RESULTS: The combination of a cytotoxic drug with an EGFR inhibitor caused different antiproliferative effects on KYSE30 cancer cells depending on the treatment schedule. An antagonistic effect was observed when treatment with each EGFR inhibitor was done before chemotherapy. In contrast, a synergistic antiproliferative activity was obtained when chemotherapy was followed by treatment with EGFR antagonists. This effect was accompanied by potentiation of apoptosis and arrest of the surviving cancer cells in the G(2)/M phases of the cell cycle. CONCLUSIONS: This study provides a rationale for the evaluation of a potentially synergistic sequence of cytotoxic drugs and EGFR inhibitors in a clinical setting.

Multiple receptor tyrosine kinases regulate HIF-1alpha and HIF-2alpha in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors.

Novel treatment approaches are needed for children with advanced neuroblastoma. Studies with neuroblastoma cells have indicated the presence of a hypoxia-driven vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-1 autocrine loop modulating hypoxia-inducible factor-1alpha (HIF-1alpha). Whether other receptor tyrosine kinases (RTKs) are capable of modulating HIF-1alpha levels and whether RTKs can regulate HIF-2alpha as well is largely unknown. We evaluated neuroblastoma cell lines for expression of various RTKs. Although cell lines were heterogeneous in the expression of VEGFR-1, -3, c-Kit and RET, most cells expressed PDGFR-alpha and -beta. Ligand-induced activation of multiple RTKs upregulated HIF-1alpha levels, whereas activation of VEGFR-1 alone upregulated HIF-2alpha. Multitargeted tyrosine kinase inhibitor sunitinib reduced hypoxia-induced rises in HIF-1alpha and HIF-2alpha through mechanisms involving effects on both mRNA levels and protein stability. In addition, sunitinib and sorafenib had direct effects on tumor cell viability in vitro. In a neuroblastoma xenograft model, tumor growth inhibition by sunitinib was associated with inhibition of angiogenesis and reduced HIF-1alpha levels. These findings show that multiple RTKs may regulate the HIF axis in normoxia and hypoxia and suggest that multikinase inhibitors may exert antiangiogenic effects not only by direct effects on endothelial cells, but also by blocking compensatory hypoxia- and ligand-induced changes in HIF-1alpha and HIF-2alpha.

Epidermal growth factor receptor regulates MET levels and invasiveness through hypoxia-inducible factor-1alpha in non-small cell lung cancer cells.

Recent studies have established that amplification of the MET proto-oncogene can cause resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cell lines with EGFR-activating mutations. The role of non-amplified MET in EGFR-dependent signaling before TKI resistance, however, is not well understood. Using NSCLC cell lines and transgenic models, we demonstrate here that EGFR activation by either mutation or ligand binding increases MET gene expression and protein levels. Our analysis of 202 NSCLC patient specimens was consistent with these observations: levels of MET were significantly higher in NSCLC with EGFR mutations than in NSCLC with wild-type EGFR. EGFR regulation of MET levels in cell lines occurred through the hypoxia-inducible factor (HIF)-1alpha pathway in a hypoxia-independent manner. This regulation was lost, however, after MET gene amplification or overexpression of a constitutively active form of HIF-1alpha. EGFR- and hypoxia-induced invasiveness of NSCLC cells, but not cell survival, were found to be MET dependent. These findings establish that, absent MET amplification, EGFR signaling can regulate MET levels through HIF-1alpha and that MET is a key downstream mediator of EGFR-induced invasiveness in EGFR-dependent NSCLC cells.

Small molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer.

Despite recent developments in the diagnosis and conventional treatment of non small cell lung cancer (NSCLC), the prognosis remains unsatisfactory, with 5-year survival rates of approximately 15% for all stages. To date, chemotherapy represents the standard treatment for advanced-non small lung cancer, but efficacy of currently available cytotoxic drugs is modest. Median survival does not exceed 8-10 months. New treatment strategies are needed and considerable hope has been placed in therapies that specifically target the molecular mechanisms of tumour growth. One molecular target of particular relevance to lung cancer pathogenesis is the epidermal growth factor receptor (EGFR), a cell membrane receptor tyrosine kinase. Several inhibitors of EGFR fuctinonal activation have been developed. Amon these, erlotinib (Tarceva) and gefitinib (Iressa) are two orally bioavailable, small molecule EGFR inhibitors of the tyrosine kinase enzymatic activity which prevent EGFR autophosphorylation and activation. In monotherapy, gefitinib and erlotinib have determinated a 10-20% response rate and a 30-50% symptom improvement in previously treated, chemotherapy refractory, advanced NSCLC patients. Furthermore, a randomized, placebo controlled, multicenter phase III study has shown a two months improvement in median survival with erlotinib in the second or third line treatment of metastatic NSCLC patients. We will summarize the clinical evidence on the anticancer activity of small molecule EGFR inhibitors.