F. Papaccio

SMO Gene Amplification and Activation of the Hedgehog Pathway as Novel Mechanisms of Resistance to Anti-Epidermal Growth Factor Receptor Drugs in Human Lung Cancer

Purpose: Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype. Experimental Design: Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non–small cell lung cancer (NSCLC) cell lines. Results: Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity. In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy. Conclusions:This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer. Clin Cancer Res; 21(20); 4686–97. ©2015 AACR.

Pulmonary Large-Cell Neuroendocrine Carcinoma: From Epidemiology to Therapy

Lung neuroendocrine tumors are a heterogeneous subtype of pulmonary cancers representing approximately 20% of all lung cancers, including small-cell lung cancer (SCLC) and large-cell neuroendocrine carcinoma (LCNEC). The frequency appears to be approximately 3% for LCNEC. Diagnosis of LCNEC requires attention to neuroendocrine features by light microscopy and confirmation by immunohistochemical staining for neuroendocrine markers. Both SCLC and pulmonary LCNEC are high-grade and poor-prognosis tumors, with higher incidence in males and smokers and peripheral localization. LCNEC is very rare, and the precise diagnosis on small specimens is very difficult, so we have still too few data to define a standard of treatment for pulmonary LCNECs. Data of literature, most based on retrospective analysis, indicated a poor 5-year overall survival, with a high incidence of recurrence after surgery, even in stage I disease. Primary surgery should be the first option in all operable patients because there is no validate therapeutic approach for LCNEC due to lack of clinical trials in this setting. Neoadjuvant platinum-based regimens remain only an option for potentially resectable tumors. In advanced stages, SCLC-like chemotherapy seems the best option of treatment, with a good response rate but a poor overall survival (from 8 to 16 months in different case series). New agents are under clinical investigation to improve LCNEC patients’ outcome. We reviewed all data on treatment options feasible for pulmonary LCNEC, both for localized and extensive disease.

A Multicenter, Open-Label Phase II Study of Metformin With Erlotinib in Second-Line Therapy of Stage IV Non–Small-Cell Lung Cancer Patients: Treatment Rationale and Protocol Dynamics of the METAL Trial

We present the rationale and study design of the METAL (METformin in Advanced Lung cancer) trial (EudraCT number: 2014-000349-59), a multicenter, open label phase II study, designed to evaluate the safety and activity of metformin combined with erlotinib as second-line therapy in patients with stage IV non-small-cell lung cancer. This is a 2-part trial, consisting of a safety run-in part followed by a phase II part. The primary end point for the first part is the maximum tolerated dose and the identification of the recommended phase II dose of metformin in combination with erlotinib. Secondary end points are the study of pharmacokinetics and the antitumor activity evaluation of the experimental combination. The primary end point of part II is the time to disease progression with the combination, and antitumor activity as a secondary end point. Based on the statistical design, we plan to enroll approximately 60 patients.

Results of the safety run-in part of the METAL (METformin in Advanced Lung cancer) study: a multicentre, open-label phase I–II study of metformin with erlotinib in second-line therapy of patients with stage IV non-small-cell lung cancer

Purpose Our previous works demonstrated the ability of metformin to revert resistance to gefitinib, a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, in non-small-cell lung cancer (NSCLC) EGFR/LKB1 wild-type (WT) cell lines. However, the optimal dose of metformin to be used in non-diabetic patients still remains to be defined. The phase I–II trial METformin in Advanced Lung cancer (METAL) was designed to identify the maximum tolerated dose and to evaluate safety and activity of metformin combined with erlotinib in second-line treatment of patients with stage IV NSCLC, whose tumours harbour the WT EGFR gene. Patients and methods We report results from the safety run-in part designed to detect acute toxicities, to study pharmacokinetics and to identify the recommended phase II dose (RPD) to be used for the following phase of the study. In the run-in phase, metformin treatment was administered according to a dose escalation scheme and, subsequently, combined with erlotinib. Results Twelve patients were enrolled. Common adverse events were diarrhoea, decreased appetite, abdominal pain, vomiting and skin toxicity, mostly reversible with symptomatic medical treatment. Dose-limiting toxicities were vomiting and diarrhoea registered in the initial cohort receiving metformin 2000 mg plus erlotinib at 150 mg die, which was declared the maximum administered dose. Only one of nine patients treated at the next lower dose of 1500 mg of metformin plus erlotinib at 150 mg experienced G3 gastrointestinal toxicity. Metformin plasma-concentration profile confirmed the trend already observed in non-diabetic population. Glycemic profiles showed stability of the blood glucose level within the physiological range for non-diabetic subjects. At a follow-up of 30 weeks, six (50%) patients experienced a disease control (5 SD and 1 partial response). Conclusions The RP2D of metformin dose was defined at 1500 mg/day to be combined with erlotinib 150 mg. Trial registration number EudraCT number: 2014-000349-59.

Role of HGF–MET Signaling in Primary and Acquired Resistance to Targeted Therapies in Cancer

The Hepatocyte growth factor (HGF)—mesenchymal-epithelial transition (MET) pathway is deregulated in several cancers and is associated with aggressive phenotype and worse prognosis. MET, a tyrosine kinase receptor activated by HGF, plays a physiological role in embryogenesis, promoting cell growth, survival and motility. HGF–MET aberrant activation in tumorigenesis acts through various mechanisms: paracrine/autocrine HGF production, MET overexpression, MET germ-line and sporadic mutations and cross-talk with other growth factor receptors. In addition, MET activation could represent a mechanism of escape from other targeted therapies, through receptor amplification or over-stimulation by the ligand, as demonstrated in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) models with acquired resistance to epidermal growth factor receptor (EGFR) inhibitors and also in models of melanoma resistant to the BRAF inhibitor vemurafenib. As a consequence, a lot of molecules targeting MET signaling are under clinical investigation as single agent or in combination with other targeted drugs. Patient selection, based on MET expression on tumor samples (eventually, by re-biopsy of new metastatic sites), and pharmacokinetic/pharmacodynamic markers are needed. Authors review the latest data on the role of MET and the molecular mechanism underlying primary or acquired resistance to biological agents, focusing on NSCLC, CRC and melanoma.

51PD Effect of MEK inhibition on PDL1 and and on cytokinesproduction profilein NSCLC cell lines and in human lymphocites

Background: Preclinical models suggest that MAPK pathway is implicated in the immune-resistance of tumors and MEK-inhibition can increase the CD8+ T-cell infiltration and the efficacy of PD-1/PD-L1 blockade. Methods: First, we evaluated PD-L1 mRNA expression by Real Time qPCR and its protein production, togheter with MAPK proteins in a panel of non-small cell lung cancer (NSCLC) cell lines. Then, we studied the changes in PD-L1 and major histocompatibility complex class-I (MHC-I) expression and cytokines’ production, after inhibition with selumetinib or stimulation of MAPK signalling by phorbol 12-myristate 13-acetate (PMA). In addition, we explored the effect of MEK inhibition on T-cell function by using Peripheral blood mononuclear cells (PBMC) from healthy volunteers. Results: A consistent correlation between PD-L1 mRNA and protein expression across cell lines suggested that expression mainly depends on trascriptional regulation, and it is regulated by MAPK signal, through the bindng of p65 to the PD-L1 promoter. Moreover, MEK inhibition resulted in an increased expression of MHC-I on cancer cells and increased mRNA expression levels of IFN gamma, IL-6, IL-1B, and TNFalpha, all molecules involved in the activation and differentiation of TCD8+ cytotoxic lymphocytes (CTL) subset. In this scenario, we also tested the effect of MEK inhibitor on activated T-lymphocytes from PBMC of healthy volunteers. After five days of treatment, RT-qPCR analysis revealed a significant increase of mRNA expression of some typical CD8+ T cell pro-inflammatory cytokines, like IL-12, TNFalpha and IFNgamma. Conclusions: These results further support the idea that MEK inhibitor reduces PD-L1 expression and this allows the establishment of a pro inflammatory microenvironment. On the other side, pheripheral T cells, treated with selumetinib, produce pro inflammatory cytokines typical of CTL subset, that seems more involved in immune response against cancer. In this context, MEK inhibition may represent a potential mechanism to convert otherwise resistant cancers and suggest new potential treatment combination strategies of MEK-inhibitors with anti-PD-L1 antibodies in NSCLC. Legal entity responsible for the study: University of Campania “L. Vanvitelli” Funding: Has not received any funding Disclosure: All authors have declared no conflicts of interest.

Stemness markers of osteosarcoma

Osteosarcomas (OSs) are a heterogeneous group of rare tumors that affect children and adolescents between 10 and 25 years, and have a second peak of incidence in the elderly, where they are associated with defective bone remodeling. Most OSs are of high grade and frequently develop pulmonary metastases. OSs derived from progenitor cells that have accumulated mutations leading to high proliferation and defects in their ability to differentiate in osteoblasts. A growing body of evidence supports the hypothesis that only a small subset of cells within a tumor, called cancer stem cells (CSCs), is capable to initiate a new tumor and sustain its growth. CSCs share many features with normal stem cells, such as self-renewal, differentiation, proliferation potential, drug resistance and migration capacity. In this chapter, we present current knowledge about the biology of osteosarcoma stem cells, highlighting on markers that are implicated in their detection and selection. Improved understanding into the cell origins of osteosarcomas and further refinements in the molecular characterization of OSs will be essential in the discrimination of putative CSCs. In addition, more defined criteria for the definition of putative stem cells identified via the various assays will be paramount to any progress achieved in the study of stem cell biology. In the future, hopefully, the unequivocal identification of osteosarcoma CSCs coupled with the development of drugs targeting CSCs may lead to new view of cancer and above all to eradicate itself.