ctDNA analysis reveals different molecular patterns upon disease progression in patients treated with osimertinib.

BACKGROUND: Several clinical trials have demonstrated the efficacy and safety of osimertinib in advanced non-small-cell lung cancer (NSCLC). However, there is significant unexplained variability in treatment outcome. METHODS: Observational prospective cohort of 22 pre-treated patients with stage IV NSCLC harboring the epidermal growth factor receptor (EGFR) p.T790M resistance mutation and who were treated with osimertinib. Three hundred and twenty-six serial plasma samples were collected and analyzed by digital PCR (dPCR) and next-generation sequencing (NGS). RESULTS: The median progression-free survival (PFS), since the start of osimertinib, was 8.9 [interquartile range (IQR): 4.6-18.0] months. The median treatment durations of sequential gefitinib + osimertinib, afatinib + osimertinib and erlotinib + osimertinib treatments were 30.1, 24.6 and 21.1 months, respectively. The p.T790M mutation was detected in 19 (86%) pre-treatment blood samples. Undetectable levels of the original EGFR-sensitizing mutation after 3 months of treatment were associated with superior PFS (HR: 0.2, 95% CI: 0.05-0.7). Likewise, re-emergence of the original EGFR mutation, alone or together with the p.T790M mutation was significantly associated with shorter PFS (HR: 8.8, 95% CI: 1.1-70.7 and HR: 5.9, 95% CI: 1.2-27.9, respectively). Blood-based monitoring revealed three molecular patterns upon progression to osimertinib: sensitizing+/T790M+/C797S+, sensitizing+/T790M+/C797S-, and sensitizing+/T790M-/C797S-. Median time to progression in patients showing the triplet pattern (sensitizing+/T790M+/C797S+) was 12.27 months compared with 4.87 months in patients in whom only the original EGFR sensitizing was detected, and 2.17 months in patients showing the duplet pattern (sensitizing+/T790M+). Finally, we found that mutations in exon 545 of the PIK3CA gene were the most frequent alteration detected upon disease progression in patients without acquired EGFR-resistance mutations. CONCLUSIONS: Different molecular patterns identified by plasma genotyping may be of prognostic significance, suggesting that the use of liquid biopsy is a valuable approach for tumor monitoring.

Cisplatin resistance involves a metabolic reprogramming through ROS and PGC-1α in NSCLC which can be overcome by OXPHOS inhibition.

BACKGROUND: Platinum-based chemotherapy remains the standard of care for most lung cancer cases. However chemoresistance is often developed during the treatment, limiting clinical utility of this drug. Recently, the ability of tumor cells to adapt their metabolism has been associated to resistance to therapies. In this study, we first described the metabolic reprogramming of Non-Small Cell Lung Cancer (NSCLC) in response to cisplatin treatment. METHODS: Cisplatin-resistant versions of the A549, H1299, and H460 cell lines were generated by continuous drug exposure. The long-term metabolic changes, as well as, the early response to cisplatin treatment were analyzed in both, parental and cisplatin-resistant cell lines. In addition, four Patient-derived xenograft models treated with cisplatin along with paired pre- and post-treatment biopsies from patients were studied. Furthermore, metabolic targeting of these changes in cell lines was performed downregulating PGC-1α expression through siRNA or using OXPHOS inhibitors (metformin and rotenone). RESULTS: Two out of three cisplatin-resistant cell lines showed a stable increase in mitochondrial function, PGC1-α and mitochondrial mass with reduced glycolisis, that did not affect the cell cycle. This phenomenon was confirmed in vivo. Post-treatment NSCLC tumors showed an increase in mitochondrial mass, PGC-1α, and a decrease in the GAPDH/MT-CO1 ratio. In addition, we demonstrated how a ROS-mediated metabolism reprogramming, involving PGC-1α and increased mitochondrial mass, is induced during short-time cisplatin exposure. Moreover, we tested how cells with increased PGC-1a induced by ZLN005 treatment, showed reduced cisplatin-driven apoptosis. Remarkably, the long-term metabolic changes, as well as the metabolic reprogramming during short-time cisplatin exposure can be exploited as an Achilles' heel of NSCLC cells, as demonstrated by the increased sensitivity to PGC-1α interference or OXPHOS inhibition using metformin or rotenone. CONCLUSION: These results describe a new cisplatin resistance mechanism in NSCLC based on a metabolic reprogramming that is therapeutically exploitable through PGC-1α downregulation or OXPHOS inhibitors.

Cancer-associated fibroblasts modify lung cancer metabolism involving ROS and TGF-ß signaling.

Lung cancer is a major public health problem due to its high incidence and mortality rate. The altered metabolism in lung cancer is key for the diagnosis and has implications on both, the prognosis and the response to treatments. Although Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor microenvironment, little is known about their role in lung cancer metabolism. We studied tumor biopsies from a cohort of 12 stage IIIA lung adenocarcinoma patients and saw a positive correlation between the grade of fibrosis and the glycolysis phenotype (Low PGC-1α and High GAPDH/MT-CO1 ratio mRNA levels). These results were confirmed and extended to other metabolism-related genes through the in silico data analysis from 73 stage IIIA lung adenocarcinoma patients available in TCGA. Interestingly, these relationships are not observed with the CAFs marker α-SMA in both cohorts. To characterize the mechanism, in vitro co-culture studies were carried out using two NSCLC cell lines (A549 and H1299 cells) and two different fibroblast cell lines. Our results confirm that a metabolic reprogramming involving ROS and TGF-ß signaling occurs in lung cancer cells and fibroblasts independently of α-SMA induction. Under co-culture conditions, Cancer-Associated fibroblasts increase their glycolytic ability. On the other hand, tumor cells increase their mitochondrial function. Moreover, the differential capability among tumor cells to induce this metabolic shift and also the role of the basal fibroblasts Oxphos Phosphorylation (OXPHOS) function modifying this phenomenon could have implications on both, the diagnosis and prognosis of patients. Further knowledge in the mechanism involved may allow the development of new therapies.

Screening for metabolites from Penicillium novae-zeelandiae displaying radical-scavenging activity and oxidative mutagenicity: isolation of gentisyl alcohol.

In the search for new natural products with anti-oxidant activity, we have combined the cell-free assay based on the scavenging of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), with a bioassay that detects oxidative mutagens. This bioassay uses a new Escherichia coli tester strain, IC203, specifically sensitive to oxidative stress due to a deficiency in the OxyR function. OxyR is a redox-sensitive transcriptional activator of genes encoding anti-oxidant enzymes such as catalase and peroxiredoxin alkyl hydroperoxide reductase. The positive response observed in E. coli IC203 with several known anti-oxidants, including cysteine, catechol and ascorbic acid, suggested to us the usefulness of the mutagenicity assay for a rapid screening of anti-oxidant compounds. The extract from Penicillium novae-zeelandiae was found to scavenge the DPPH radical. Subsequently, guided by the DPPH-scavenging assay and the oxidative mutagenesis assay, we isolated and identified three compounds in fractions from that active extract: patulin (1). 3-hydroxybenzyl alcohol (2). and gentisyl alcohol (2,5-dihydroxybenzyl alcohol) (3). Of these, gentisyl alcohol showed both DPPH-scavenging activity and oxidative mutagenicity. This compound also gave rise to intracellular formation of superoxide, evaluated by monitoring the oxidation of dihydroethidium, and was able to inhibit mutagenesis induced by the model oxidant t-butyl hydroperoxide (t-BuOOH).