Clinical and molecular analysis of lung cancers associated with fibrosing interstitial lung disease.

BACKGROUND: Interstitial lung disease is a heterogeneous group of diseases, some of which are known to present an independent risk factor for lung cancer. Its pathophysiological mechanism has not been fully elucidated and therapeutic management is also complex. We aim to both describe a cohort of patients with lung cancer associated with pre-existing fibrosing interstitial lung disease and to characterize their molecular profile. METHODS: We conducted a retrospective, single centre cohort study, at Toulouse University Hospital. Immuno-histochemical (PD-L1, CD8) and molecular analysis was performed on archived tumour sample. Molecular signalling pathways involved were analysed with the Reactome Pathway Database. RESULTS: Forty-nine patients were analysed. Most common histology was adenocarcinoma (65,3%), followed by squamous cell carcinoma (30.6%). Idiopathic pulmonary fibrosis (30,6%) and interstitial lung disease associated with connective tissue disease (22,4%) were mostly diagnosed. Usual interstitial pneumonia dominated the scans patterns. A high proportion of early tumour stages was observed and overall survival was 34,5 months. In metastatic stages response rate to first line chemotherapy was 38% and overall survival was 11,2 months. Main cause of death was complex cancer progression. PD-L1 expression (n=23) was low (0%) to intermediate (1-49%). Tumour mutational burden was low in 69,2% of analysed cases (n=12) and microsatellite status was stable in all cases (n=13). Sample genotyping (n=14) showed frequent involvement of the TP53 gene and the implication of signalling pathways common to fibrotic processes such as TGFß and PI3K/AKT. CONCLUSIONS: We suggest a particular phenotype of lung cancer associated with fibrosing interstitial lung disease that could provide the basis for specific therapeutic strategies.

Antilisterial activity of dromedary lactoferrin peptic hydrolysates.

The aim of this study was to explore the antibacterial peptides derived from dromedary lactoferrin (LFc). The LFc was purified from colostrum using a batch procedure with a cation exchange chromatography support and was hydrolyzed with pepsin to generate peptic digest. This peptic digest was fractionated by cation exchange chromatography, and the antilisterial activity of LFc, peptic digest, and obtained fractions was investigated using the bioscreen method. The growth of Listeria innocua ATCC 33090 and LRGIA 01 strains was not inhibited by LFc and its hydrolysates. Two fractions of dromedary lactoferrin peptic hydrolysate were active against both strains. A tandem mass spectroscopy analysis revealed that the 2 active fractions comprised at least 227 different peptides. Among these peptides, 9 found in the first fraction had at least 50% similarity with 10 known antimicrobial peptides (following sequence alignments with the antimicrobial peptide database from the University of Nebraska Medical Center, Omaha). Whereas 9 of these peptides presented homology with honeybee, frog, or amphibian peptides, the 10th peptide, F152SASCVPCVDGKEYPNLCQLCAGTGENKCACSSQEPYFGY192 (specifically found in 1 separated fraction), exibited 54% homology with a synthetic antibacterial peptide (AP00481) derived from human lactoferrin named kaliocin-1. Similarly, the second fraction contained 1 peptide similar to lactoferrampin B, an antibacterial peptide derived from bovine milk. This result suggests that peptic hydrolysis of LFc releases more active antimicrobial peptides than their protein source and thus provides an opportunity for their potential use to improve food safety by inhibiting undesirable and spoilage bacteria.

PD-L1 expression in circulating tumor cells of advanced non-small cell lung cancer patients treated with nivolumab.

BACKGROUND: Inhibitors of the PD-1/PD-L1 immune checkpoint have become a standard of care in non-small cell lung cancer (NSCLC). Patient selection, currently based on PD-L1 expression on tumor tissue, is limited by its temporal and spatial heterogeneity. We hypothesized that liquid biopsy with PD-L1 analysis on circulating tumor cells (CTCs) might overcome this limitation. METHODS: Blood samples were prospectively collected from patients with advanced NSCLC before nivolumab treatment and at the time of progression. CTCs were isolated using a cell size-based technology. PD-L1 expression was assessed by immunofluorescence on CTCs and immunohistochemistry on tissue biopsies. RESULTS: 113 specimens from 96 patients were collected. Baseline PD-L1 expression could be assessed on 72% and 93% of tissue and CTC, respectively. CTCs were more frequently found to be PD-L1 positive than tissue (83% vs. 41%) and no correlation was observed between tissue and CTC PD-L1 expression (r = 0.04, p = 0.77). Pre-treatment high CTC number was associated with increased risk of death and progression (HR1.06, p = 0.03 for OS; HR1.05, p = 0.02 for PFS). The presence of pre-treatment PD-L1+CTC was not significantly correlated with outcomes but a higher baseline PD-L1+ CTC number (≥1%) was observed in the "non-responders" group (PFS <6 months) (p = 0.04) and PD-L1+CTC were seen in all patients at progression. CONCLUSION: Assessment of PD-L1 expression in CTCs is feasible and CTCs are more often positive than in tissue. Pre-treatment PD-L1+CTCs are associated with bad prognosis in patients treated with PD-1 inhibitors.

Monitoring KRAS mutations in circulating DNA and tumor cells using digital droplet PCR during treatment of KRAS-mutated lung adenocarcinoma.

Liquid biopsies are a new non-invasive strategy to detect and monitor the biology of non-small-cell lung cancer (NSCLC) in the era of personalized medicine. KRAS is an oncogenic driver that is mutated in 30% of NSCLCs and is associated with a poor prognosis. 62 samples from 32 patients, treated for metastatic KRAS-mutated lung adenocarcinoma, had DNA extracted from plasma and circulating tumor cells (CTCs) prospectively tested for the presence of KRAS mutations using droplet digital PCR. A KRAS mutation was detected in 82% of patients. Sensitivity was 78% for circulating free DNA (cfDNA) and 34% for CTCs. The presence of a KRAS mutation in cfDNA was correlated with a poor response to chemotherapy or targeted therapy. When a KRAS-mutated-DNA was detected and then monitored in cfDNA, its variation during targeted or conventional therapy was correlated with response, according to RECIST criteria, in 87.5% of cases (n=14/16), whereas this correlation was observed in 37.5% of cases for CTCs (n=3/8). We report the usefulness of using digital droplet PCR on liquid biopsies to predict and monitor responses to treatment of KRAS-mutated lung adenocarcinoma. ctDNA was much more sensitive than CTCs in this context.

Alternative processing of the U2 small nuclear RNA produces a 19-22nt fragment with relevance for the detection of non-small cell lung cancer in human serum.

RNU2 exists in two functional forms (RNU2-1 and RNU2-2) distinguishable by the presence of a unique 4-bases motif. Detailed investigation of datasets obtained from deep sequencing of five human lung primary tumors revealed that both forms express at a high rate a 19-22nt fragment (miR-U2-1 and -2) from its 3' region and contains the 4-bases motif. Deep sequencing of independent pools of serum samples from healthy donors and lung cancer patients revealed that miR-U2-1 and -2 are pervasively processed in lung tissue by means of endonucleolytic cleavages and stably exported to the blood. Then, microarrays hybridization experiments of matched normal/tumor samples revealed a significant over-expression of miR-U2-1 in 14 of 18 lung primary tumors. Subsequently, qRT-PCR of miR-U2-1 using serum from 62 lung cancer patients and 96 various controls demonstrated that its expression levels identify lung cancer patients with 79% sensitivity and 80% specificity. miR-U2-1 expression correlated with the presence or absence of lung cancer in patients with chronic obstructive pulmonary disease (COPD), other diseases of the lung - not cancer, and in healthy controls. These data suggest that RNU2-1 is a new bi-functional ncRNA that produces a 19-22nt fragment which may be useful in detecting lung cancer non-invasively in high risk patients.