Claudia Maggioni

Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform.

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.

Circulating Cell-Free DNA and Circulating Tumor Cells as Prognostic and Predictive Biomarkers in Advanced Non-Small Cell Lung Cancer Patients Treated with First-Line Chemotherapy

Cell-free DNA (cfDNA) and circulating tumor cells (CTCs) are promising prognostic and predictive biomarkers in non-small cell lung cancer (NSCLC). In this study, we examined the prognostic role of cfDNA and CTCs, in separate and joint analyses, in NSCLC patients receiving first line chemotherapy. Seventy-three patients with advanced NSCLC were enrolled in this study. CfDNA and CTC were analyzed at baseline and after two cycles of chemotherapy. Plasma cfDNA quantification was performed by quantitative PCR (qPCR) whereas CTCs were isolated by the ScreenCell Cyto (ScreenCell, Paris, France) device and enumerated according to malignant features. Patients with baseline cfDNA higher than the median value (96.3 hTERT copy number) had a significantly worse overall survival (OS) and double the risk of death (hazard ratio (HR): 2.14; 95% confidence limits (CL) = 1.24-3.68; p-value = 0.006). Conversely, an inverse relationship between CTC median baseline number (6 CTC/3 mL of blood) and OS was observed. In addition, we found that in patients reporting stable disease (SD), the baseline cfDNA and CTCs were able to discriminate patients at high risk of poor survival. cfDNA demonstrated a more reliable biomarker than CTCs in the overall population. In the subgroup of SD patients, both biomarkers identified patients at high risk of poor prognosis who might deserve additional/alternative therapeutic interventions.

Serum proteomic test in advanced non-squamous non-small cell lung cancer treated in first line with standard chemotherapy

Background:VeriStrat is a blood-based proteomic test with predictive and prognostic significance in second-line treatments for non-small cell lung cancer (NSCLC). This trial was designed to investigate the role of VeriStrat in first-line treatment of advanced NSCLC with standard chemotherapy. Here we present the results for 76 non-squamous patients treated with a combination of carboplatin or cisplatin with pemetrexed.Methods:The test-assigned classifications of VeriStrat Good or VeriStrat Poor to samples collected at baseline. The primary end point was progression-free survival (PFS); secondary end points included overall survival (OS) and objective response. Exploratory analyses of end points separately in carboplatin/pemetrexed and cisplatin/pemetrexed subgroups were also conducted.Results:Patients classified as VeriStrat Good had longer PFS and OS than VeriStrat Poor: 6.5 vs 1.6 months and 10.8 vs 3.4 months, respectively; the corresponding hazard ratios (HRs) were 0.36 (P<0.0001) and 0.26 (P<0.0001); they were also more likely to achieve objective response. Prognostic significance of VeriStrat was confirmed in multivariate analysis. Significant differences in OS and PFS between Veristrat classifications were also found when treatment subgroups were analysed separately.Conclusions:The trial demonstrated clinical utility of VeriStrat as a prognostic test for standard first-line chemotherapy of non-squamous advanced NSCLC.

Prognostic and Therapeutic Implications of MicroRNA in Malignant Pleural Mesothelioma

Malignant Pleural Mesothelioma (MPM) is an aggressive disease characterized by a dismal prognosis, mainly due to late diagnosis. To date, there are very few treatment options available and the refractoriness to the majority of therapeutic strategies, leading to consider MPM a relevant problem in public health. Therefore, the identification of novel prognostic markers and alternative therapeutic strategies remain a top priority. Several efforts have been made in this direction and to date a number of studies have investigated the role of microRNA as biomarkers in MPM, identifying the potential prognostic role of miR-29c* and miR-31. Very recently, the first microRNA signature able to discriminate poor or and good prognosis of MPM patients underwent surgery has been published. Very interestingly, several microRNA such as miR-1, miR-16, and miR-34b/c have been identified as potential therapeutic agents. Indeed, the forced expression of these microRNA resulted in anti-tumor effects both in vitro and in vivo. Besides, the introduction of microRNA mimic, some agents such as EphrinA1 and Onconase, seemed to exert anti-tumor effects through specific microRNA. Moreover, microRNA have also been reported to play a role in chemoresistance enhancing the sensitivity to specific drug such as pemetrexed. In this review the most relevant and updated data about the role of microRNA as prognostic markers and therapeutic agents in MPM will be presented, opening new avenues towards improved management of this aggressive disease.

Belagenpumatucel-L for the treatment of non-small cell lung cancer.

Introduction: Immunotherapy has become a promising approach for the treatment of NSCLC. In order to stimulate the host immune system against tumour antigens, several cancer vaccines have been generated and evaluated. Belagenpumatucel-L is a whole tumour cell vaccine expressing the antisense strand of the TGF-β2 gene. Areas covered: The purpose of this article is to review the most relevant findings of clinical trials testing belagenpumatucel-L in advanced NSCLC patients. Expert opinion: Although the Phase III trial investigating belagenpumatucel-L in stage III/IV patients did not meet its primary end point, a survival benefit was observed in several subgroups of patients. Further studies are needed in order to select patients who may benefit from this vaccine.

Afatinib for the treatment of non-small cell lung cancer

Introduction: Tyrosine kinase inhibitors (TKI) are currently the most effective therapy for patients with NSCLC and common activating mutations of EGFR. Afatinib, an irreversible EGFR, HER2, and HER4 inhibitor, has recently been approved at the dose of 40 mg daily as front-line treatment for advanced NSCLC with common EGFR-activating mutations. Areas covered: The review analyzes the pharmacodynamics, pharmacokinetics, safety, and clinical efficacy of afatinb in NSCLC. This irreversible TKI was initially investigated in EGFR-unselected patients who progressed after TKIs, but the subsequent observation of its major activity against exon 19 mutant determined its indication in common EGFR mutations. Several Phase I to Phase III studies explored the role of afatinib in different settings of patients. Expert opinion: Afatinib showed efficacy as a front-line treatment in patients with EGFR common mutations. Currently, no statistically significant differences between afatinib, gefitinib, and erlotinib have been detected, as they all improve progression-free survival, disease control rate, and control of symptoms in NSCLC EGFR mutant patients; the pooled analysis of LUX-Lung 3 and 6 data in patients harboring Del19 is the only demonstration of a statistical improvement in overall survival with afatinib.