Etienne Giroux Leprieur

Outcome of EGFR-mutated NSCLC patients with MET-driven resistance to EGFR tyrosine kinase inhibitors

Background Several mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC have been described including the T790M mutation and MET amplification. Whereas T790M mutation confers prolonged survival and sensitivity to 3rd generation TKIs, data are lacking on clinical features and outcome of MET-driven resistant EGFR-mutated NSCLC patients. Methods Patients with metastatic EGFR-mutated NSCLC displaying high MET overexpression or MET amplification, detected on a biopsy performed after progression on EGFR TKI, were identified in 15 centers. Clinical and molecular data were retrospectively collected. Results Forty two patients were included. The median overall survival (OS), and the median post EGFR TKI progression overall survival (PPOS) were 36.2 months [95%CI 27.3-66.5] and 18.5 months [95%CI 10.6-27.4] respectively. Nineteen out of 36 tumors tested for MET FISH had MET amplification. A T790M mutation was found in 11/41 (26.8%) patients. T790M-positive patients had a better OS than T790M-negative patients (p=0.0224). Nineteen patients received a MET TKI. Objective response was reported in 1 out of 12 evaluable patients treated with a MET inhibitor as a single agent and in 1 of 2 patients treated with a combination of MET and EGFR TKIs. Conclusion MET-driven resistance to EGFR TKI defines a specific pattern of resistance characterized by low objective response rate to MET inhibitors given alone and overlapping with T790M mutations. Further studies are warranted to define adequate therapeutic strategies for MET-driven resistance to EGFR TKI.

Modalités d’utilisation du ceritinib (Zykadia™), inhibiteur de ALK de 2e génération, dans le cancer bronchique non à petites cellules de stade avancé

Around 4% of advanced non-small cell lung cancers (NSCLC) harbor a ALK rearrangement, with high sensitivity to ALK inhibitor as crizotinib. However, the vast majority of these tumors end with a tumor progression after several months of treatment with crizotinib. Ceritinib is a 2nd generation ALK inhibitor, which showed high efficiency in NSCLC with ALK rearrangement. Results from phase I trial showed a response rate at 58% in these tumors, with a similar rate for previously crizotinib-treated patients or crizotinib-naïve patients. Moreover, cerebral responses were observed with ceritinib. Preliminary date from a phase 2 trial confirmed these results. These promising results allowed a European marketing authorization (autorisation de mise sur le marché [AMM]) since May 2015 for the treatment of advanced NSCLC with ALK rearrangement and resistance or intolerance to crizotinib.

Spotlight on crizotinib in the first-line treatment of ALK-positive advanced non-small-cell lung cancer: patients selection and perspectives

Around 4% of advanced non-small-cell lung cancers (NSCLCs) have an ALK rearrangement at the time of diagnosis. This molecular feature is more frequent in young patients, with no/light smoking habit and with adenocarcinoma pathological subtype. Crizotinib is a tyrosine kinase inhibitor, targeting ALK, ROS1, RON, and MET. The preclinical efficacy results led to a fast-track clinical development. The US Food and Drug Administration (FDA) approval was achieved after the Phase I clinical trial in 2011 in ALK-rearranged advanced NSCLC progressing after a first-line treatment. In 2013, the randomized Phase III trial PROFILE-1007 confirmed the efficacy of crizotinib in ALK-rearranged NSCLC, compared to cytotoxic chemotherapy, in second-line setting or more. In 2014, the PROFILE-1014 trial showed the superiority of crizotinib in the first-line setting compared to the pemetrexed platinum doublet chemotherapy. The response rate was 74%, and the progression-free survival was 10.9 months with crizotinib. Based on these results, crizotinib received approval from the FDA and European Medicines Agency for first-line treatment of ALK-rearranged NSCLC. The various molecular mechanisms at the time of the progression (ALK mutations or amplification, ALK-independent mechanisms) encourage performing re-biopsy at the time of progression under crizotinib. The best treatment strategy at the progression (crizotinib continuation beyond progression, switch to second-generation tyrosine kinase inhibitors, or cytotoxic chemotherapy) depends on the phenotype of the progression, the molecular status, and the physical condition of the patient.

The homeobox gene EMX2 is a prognostic and predictive marker in malignant pleural mesothelioma.

OBJECTIVES Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm with a poor prognosis and limited treatment options. EMX2 is a homeobox transcription factor that may regulate key developmental pathways known to promote tumorigenesis. In this study, we evaluated the prognostic and predictive significance of EMX2 expression in MPM. MATERIALS AND METHODS Fifty surgically resected MPM specimens were studied. Quantitative real-time RT-PCR was used to analyze EMX2 mRNA expression. Association of EMX2 levels with clinical outcomes was evaluated with using the Kaplan-Meier method and a multivariate Cox proportional hazards regression model. RESULTS EMX2 expression was significantly associated with IMIG stage (p<0.001) and smoking history (p=0.006). Cox hazard regression modeling identified low-EMX2 expression as a negative prognostic factor in progression-free survival by both univariate (p=0.002) and multivariate analysis (p=0.002). Kaplan-Meier analysis revealed significant differences in progression-free survival between low- and high-EMX expressing groups in all patients (p=0.001), and also when grouped by early (I/II) stage disease (p<0.001), patients undergoing pleurectomy (p<0.001) and patients with an epitheliod subtype (p<0.004). Furthermore, EMX2 expression predicted response to neoadjuvant chemotherapy. High-EMX2 expression was associated with decreased progression-free survival after neoadjuvant therapy, suggesting that induction therapy should be avoided in these patients. CONCLUSIONS EMX2 expression is downregulated in advanced cases of malignant pleural mesothelioma and may serve as an important prognostic and predictive molecular biomarker of progression-free survival.

Preclinical characterization of therapeutic antibodies targeted at the carboxy-terminus of Sonic hedgehog

The Sonic Hedgehog (Shh) signaling pathway has been implicated in the development and tumor progression of a number of human cancers. Using synthetic peptide mimics to mount an immune response, we generated a mouse mAb to the carboxy (C)-terminus of the Shh protein and characterized its preclinical antitumor effects. In vitro screening guided selection of the best candidate for mAb scale-up production and therapeutic development. C-term anti-Shh, Ab 1C11-2G4 was selected based on ELISA screens, Western blotting, and flow cytometric analyses. Purified Ab 1C11-2G4 was shown to recognize and bind both Shh peptide mimics and cell surface Shh. Administration of Ab 1C11-2G4 not only reduced cell viability in 7 cancer cell lines but also significantly inhibitted tumor growth in a xenograft model of A549 lung cancer cells. Ex vivo analyses of xenograft tumors revealed a reduction in Shh signal transduction and apoptosis in 2G4-treated mice. Collectively, our results provide early demonstration of the antitumor utility of antibodies specific for the C-terminal region of Shh, and support continued development to evaluate their potential efficacy in cancers in which Shh activity is elevated.

Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer

The mechanism of Sonic Hedgehog (Shh) pathway activation in non-small cell lung cancer (NSCLC) is poorly described. Using an antibody against the Shh C-terminal domain, we found a small population of Shh-positive (Shh+) cells in NSCLC cells. The objective of this study was to characterize these Shh+ cells. Shh+ and Shh- cells were sorted by using Fluorescence Activated Cell Sorting (FACS) on 12 commercial NSCLC cell lines. Functional analyses on sorted cells were performed with gene expression assays (qRT-PCR and microarray) and cells were treated with cytotoxic chemotherapy and a targeted inhibitor of Shh signaling (GDC0449). We used in vivo models of nude mice inoculated with Shh+ and Shh- sorted cells and drug-treated cells. Finally, we confirmed our results in fresh human NSCLC samples (n=48) paired with normal lung tissue. We found that Shh+ cells produced an uncleaved, full-length Shh protein detected on the membranes of these cells. Shh+ cells exerted a paracrine effect on Shh- cells, inducing their proliferation and migration. Shh+ cells were chemo-resistant and showed features of cancer stem cells (CSCs) in vitro and in vivo. Pharmacological inhibition of the Shh pathway suppressed their CSC features. A high percentage of Shh+ cells was associated with poor prognosis in early-stage NSCLC patients. In conclusion, we describe for the first time the presence of an abnormal membrane-bound full-length Shh protein in human cancer cells that allows the identification of CSCs in vitro and in vivo.

Old Sonic Hedgehog, new tricks: a new paradigm in thoracic malignancies

The Sonic Hedgehog (Shh) pathway is physiologically involved during embryogenesis, but is also activated in several diseases, including solid cancers. Previous studies have demonstrated that the Shh pathway is involved in oncogenesis, tumor progression and chemoresistance in lung cancer and mesothelioma. The Shh pathway is also closely associated with epithelial-mesenchymal transition and cancer stem cells. Recent findings have revealed that a small proportion of lung cancer cells expressed an abnormal full-length Shh protein, associated with cancer stem cell features. In this paper, we review the role of the Shh pathway in thoracic cancers (small cell lung cancer, non-small cell lung cancer, and mesothelioma) and discuss the new perspectives of cancer research highlighted by the recent data of the literature.

Clinical factors associated with early progression and grade 3–4 toxicity in patients with advanced non-small-cell lung cancers treated with nivolumab

Introduction The prognosis of advanced non-small-cell lung cancer (NSCLC) has been improved by development of immune checkpoint inhibitors (ICIs) such as nivolumab for second-line treatment. As phase III trials include only selected patients, we here investigated the clinical factors associated with efficacy and safety of nivolumab in ‘real life’ patients with advanced NSCLC. Methods Clinical and histological characteristics, therapies and survival data of all consecutive patients with advanced NSCLC included prospectively and treated by nivolumab in two French academic hospitals between February 2015 and December 2016 were examined. Results Sixty-seven patients were included, mostly male (69%), current or former smokers (87%) with PS <2 (73%). Median age was 68.5 years and 42% were aged ≥70 years. According to uni- and multi-variate analyses, only PS 2 (OR = 0.17, 95% CI 0.03–0.99, p = 0.049) and number of previous treatment lines (OR = 0.33, 95% CI 0.13–0.85, p = 0.022) were significantly negatively associated with tumor control. Worse progression-free survival (PFS) was significantly associated with PS 2 (HR = 5.17, 95% CI 1.99–13.43, p = 0.001) and use of steroids (HR = 3.27, 95% CI 1.39–7.69, p = 0.006). Worse overall survival was associated with symptomatic brain metastasis (HR = 3.15, 95% CI 1.23–8.85, p = 0.029). Treatment-related adverse events occurred in 47 patients (70%), symptomatic brain metastasis being significantly associated with Grade ≥3 toxicity (OR = 8.13, 95% CI 1.21–55.56, p = 0.031). Age and nutritional status were not associated with response, PFS, OS or toxicity. Conclusion Our results suggest that nivolumab is not beneficial or safe for patients with PS 2 and symptomatic brain metastases.

Brigatinib, a new treatment option in ALK-rearranged advanced non-small cell lung carcinoma

Anaplastic lymphoma kinase (ALK) rearrangement is a rare feature in advanced non-small cell lung cancer (NSCLC), occurring in around 5% of the patients (1). This oncogenic addiction is more frequent in no/light-smokers, in thyroid transcription factor-1 (TTF1)-positive adenocarcinoma, with cribriform architecture and ring cells (2). ALK rearrangement—most of the time a translocation with a partner gene—induces the formation of a fusion protein with oncogenic properties. The first targeted therapy developed for ALK -rearranged NSCLC was crizotinib, an ALK tyrosine kinase inhibitor (TKI). Crizotinib was proved to be superior to chemotherapy in first-line setting in ALK -rearranged NSCLC, with an overall response rate (ORR) of 74% and a progression-free survival (PFS) of 10.9 months (3). Crizotinib in first-line treatment is now widely used. Other ALK TKIs have been developed, as brigatinib. Results of the phase I-II trial published in 2016 showed in 79 advanced NSCLC patients treated with brigatinib an ORR at 75% [72% in previously crizotinib treated patients (n=71)] and a PFS (median) of 13.2 months (4). Safety profile showed specific lung toxicity, occurring at the beginning of the treatment (median 2 days) in 8% of the patients, with incidence increasing with the dose of brigatinib. The ALTA trial, published in May 2017 in Journal of Clinical Oncology by Kim et al . was a phase II trial that randomized patients with advanced ALK -rearranged NSCLC between 2 doses of brigatinib (90 mg daily and 90 mg daily for 7 days then 180 mg daily) (5). The primary endpoint was ORR, and no comparison between the 2 arms was pre-planned. Assuming an ORR ≥35% with a power of 90% and a bilateral alpha risk at 0.025, the study had to include 109 patients per arm. Patients should have an advanced ALK -rearranged NSCLC with progression with crizotinib and a performance status (PS) between 0 and 2. Brain metastases were allowed only if asymptomatic and with stable doses of steroid treatment. Two-hundred and twenty-two patients were randomized [112 patients arm A (90 mg) and 110 patients arm B (90 mg then 180 mg)]. Both arms were well-balanced, with a median age at 54 years, 57% of women, 31% of Asian, 60% of non-smokers, 97% of adenocarcinoma, 69% of brain metastases. ORR (investigator-assessed) was 45% in arm A and 54% in arm B, and disease-control rate was 82% (arm A) and 86% (arm B). Median time to response was 1.8 months (arm A) and 1.9 months (arm B), and duration of response was 13.8 months (arm A) and 11.8 months (arm B). Median PFS was 9.2 months (arm A) and 12.9 months (arm B). Post-hoc comparison between the 2 arms for PFS showed a clear benefit for arm B, with a hazard ratio (HR) at 0.55 [95% confidence interval (CI), 0.36–0.86]. One-year overall survival (OS) probability was 71% (arm A) and 80% (arm B). As shown in the phase I-II trial, brigatinib had intracranial efficacy. ORR for measurable brain metastases was 42% (arm A, n=26) and 67% (arm B, n=18). For active brain metastases (i.e., metastases without previous brain radiotherapy, or progression after brain radiotherapy), intracranial ORR was 42% (arm A, n=19) and 73% (arm B, n=15). Intracranial PFS (median) was 15.6 months (arm A) and 12.8 months (arm B), with a duration of intracranial response (median) not reached in both arms. In term of safety, no new signal was detected. In both arms, main toxicities (≥10% patients) were nausea (33%/40%), diarrhea (19%/38%), vomiting (24%/23%), headache (28%/27%), decrease appetite (22%/15%) and hypertension (11%/21%). Grade 3–4 toxicities were rare: hypertension (6%/6%), increased creatine phosphokinase (3%/9%) and rash (1%/3%), mainly. Pulmonary early toxicity occurred in 6% of patients (n=14), during the first 2 days of treatment (always at 90mg daily of brigatinib). Grade 3–5 pulmonary toxicities concerned 3% of patients (n=7). Half of the patients (n=7) had successfully continued brigatinib after suspension, whereas 7 patients stopped brigatinib definitely. One patient died from acute respiratory failure (<1%). Multivariate analysis showed that advanced age [odds ratio (OR) =2.10; 95% CI, 1.21–3.65] and time from last crizotinib dose to first brigatinib dose less than 7 days (OR =3.88; 95% CI, 1.10–13.68) were associated with early pulmonary toxicity. Seven percent (arm A) and 20% (arm B) patients had dose reduction, due to increase of creatine phosphokinase, pneumonitis and rash.

Molecular characterization of circulating tumor cells in lung cancer: moving beyond enumeration

Molecular characterization of tumor cells is a key step in the diagnosis and optimal treatment of lung cancer. However, analysis of tumor samples, often corresponding to small biopsies, can be difficult and does not accurately reflect tumor heterogeneity. Recent studies have shown that isolation of circulating tumor cells (CTCs) is feasible in non-small cell lung cancer patients, even at early disease stages. The amount of CTCs corresponds to the metastatic potential of the tumor and to patient prognosis. Moreover, molecular analyses, even at the single-cell level, can be performed on CTCs. This review describes the technologies currently available for detecting and capturing CTCs, the potential for downstream molecular diagnostics, and the clinical applications of CTCs isolated from lung cancer patients as screening, prognostic, and predictive tools. Main limitations of CTCs are also discussed.