CEA, CYFRA21-1 and NSE are tumor markers used for monitoring the response to chemotherapy in advanced adenocarcinoma, squamous cell carcinoma and small-cell lung cancer, respectively. Their role in cancer immunotherapy needs to be elucidated. Patients with advanced non-small cell lung cancer (NSCLC) were treated with nivolumab 3 mg/kg every 2 weeks within the Italian Nivolumab Expanded Access Program. Blood samples were collected at baseline, at each cycle up to cycle 5 and then every two cycles until patient’s withdrawn from the study. All patients underwent a CT-scan after every 4 cycles of treatment and responses were classified according to RECIST 1.1. The biomarkers serum levels were measured with a chemiluminescent microparticle immunoassay for CEA and with an immuno radiometric assay for CYFRA21-1 and NSE. The markers values at baseline and after 4 cycles were used to analyze the relationship between their variation over baseline and the tumor response, evaluated as disease control rate (DCR: CR + PR + SD), and survival (PFS and OS). A total of 70 patients were evaluable for the analysis. Overall, a disease control was obtained in 24 patients (35.8%, 4 PR + 20 SD). After 4 cycles of nivolumab a CEA or CYFRA21-1 reduction ≥ 20% over the baseline was significantly associated with DCR (CEA, p = 0.021; CYFRA21-1, p < 0.001), PFS (CEA, p = 0.028; CYFRA21-1, p < 0.001) and OS (CEA, p = 0.026; CYFRA21-1, p = 0.019). Multivariate analysis confirmed the ability of CYFRA21-1 reduction ≥ 20% to predict DCR (p = 0.002) and PFS (p < 0.001). The reduction in serum level of CYFRA21-1 or CEA might be a reliable biomarker to predict immunotherapy efficacy in NSCLC patients. NSE was not significant for monitoring the efficacy of nivolumab.

Non-small cell lung cancer is one leading cause of death worldwide, and patients would greatly benefit from an early diagnosis. Since targeted and immunotherapies have emerged as novel approaches for more tailored treatments, repeated assessments of the tumor biology have become pivotal to drive clinical decisions. Currently, tumor tissue biopsy is the gold standard to investigate potentially actionable biomarkers, but this procedure is invasive and may prove inadequate to represent the whole malignancy. In this regard, liquid biopsy represents a minimally invasive and more comprehensive option for early detection and investigation of this tumor. Today, cell-free DNA is the only approved circulating marker to select patients for a targeted therapy. Conversely, the other tumor-derived markers (i.e., circulating tumor cells, miRNAs, exosomes, and tumor educated platelets) are still at a pre-clinical phase, although they show promising results for their application in screening programs or as prognostic/predictive biomarkers. The main challenges for their clinical translation are the lack of reliable cutoffs and, especially for miRNAs, the great variability among the studies. Moreover, no established tool has been approved for circulating tumor cells and exosome isolation. Finally, large prospective clinical trials are mandatory to provide evidence of their clinical utility.

Glycolysis in presence of oxygen with high glucose consumption is known to be the metabolism of choice in many tumors. In lung cancer this phenomenon is routinely exploited in diagnostic PET imaging of fluorodeoxyglucose uptake, but not much is known about the prognostic capabilities of glycolysis level assessment in resected lung tumor samples.

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.

Reliable predictors of benefit from immune checkpoint inhibitors in non-small-cell lung cancer (NSCLC) are still limited. We aimed to evaluate the association between the expression of selected molecules involved in immune response and clinical outcomes in NSCLC patients receiving nivolumab. In our study, the outcomes of 46 NSCLC patients treated with nivolumab in second or subsequent lines (Nivolumab Cohort) were compared with the expression of PD-L1, PD-L2, PD-1, B7-H3, and B7-H4 assessed by immunohistochemistry (IHC). Samples from 17 patients (37.0%) in the Nivolumab Cohort were positive for B7-H4 expression. At univariate analyses, only B7-H4 expression was associated with significantly decreased progression-free survival (PFS; 1.7 vs. 2.0 months; p = 0.026) and with a disadvantage in terms of overall survival (OS) close to statistical significance (4.4 vs. 9.8 months; p = 0.064). At multivariate analyses, B7-H4 expression was significantly associated with decreased PFS (hazard ratio (HR) = 2.28; p = 0.021) and OS (HR = 2.38; p = 0.022). Subsequently, B7-H4 expression was compared with clinical outcomes of 27 NSCLC patients receiving platinum-based chemotherapy (Chemotherapy Cohort), but no significant association was observed. Our results suggest a negative predictive role of B7-H4 in a population of NSCLC treated with immune checkpoint inhibitors, which deserves further research.

Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v.3.0 (Roche NimbleGen) and SureSelect XT Human All Exon v.5 (Agilent Technologies). Sequencing metrics on Illumina HiSeq were optimal for both exome systems and comparable among FFPE and FF samples, with a slight increase of PCR duplicates in FFPE, mainly in Roche NimbleGen libraries. Comparison of single nucleotide variants (SNVs) between FFPE-FF pairs reached overlapping values >90 % in both systems. Both WES showed high concordance with target re-sequencing data by Ion PGM™ in 22 lung-cancer genes, regardless the source of samples. Exon coverage of 623 cancer-related genes revealed high coverage efficiency of both kits, proposing WES as a valid alternative to target re-sequencing. High-quality and reliable data can be successfully obtained from WES of FFPE samples starting from a relatively low amount of input gDNA, suggesting the inclusion of NGS-based tests into clinical contest. In conclusion, our analysis suggests that the WES approach could be extended to a translational research context as well as to the clinic (e.g. to study rare malignancies), where the simultaneous analysis of the whole coding region of the genome may help in the detection of cancer-linked variants.