Sequential liquid biopsies reveal dynamic alterations of EGFR driver mutations and indicate EGFR amplification as a new mechanism of resistance to osimertinib in NSCLC.

Osimertinib is an EGFR-T790M-specific TKI, which has demonstrated impressive response rates in NSCLC, after failure to first-line anti-EGFR TKIs. However, acquired resistance to osimertinib is also observed and the molecular mechanisms of resistance are not yet fully understood. Monitoring and managing NSCLC patients who progressed on osimertinib is, therefore, emerging as an important clinical challenge. Sequential liquid biopsies were used to monitor a patient with EGFR-exon19del positive NSCLC, who received erlotinib and progressed through the acquisition of the EGFR-T790M mutation. Erlotinib was discontinued and osimertinib was initiated. Blood samples were collected at erlotinib progression and during osimertinib treatment for the detection of the activating (EGFR-exon19del) and resistance mutations (EGFR-T790M, EGFR-C797S, BRAF-V600E, METamp and ERBB2amp) in the plasma DNA using digital droplet PCR. Plasma levels of the activating EGFR-exon19del accurately paralleled the clinical and radiological progression of disease and allowed early detection of AR to osimertinib. Resistance to osimertinib coincided with the emergence of a small tumor cell subpopulation carrying the known EGFR-C797S resistance mutation and an additional subpopulation carrying amplified copies of EGFR-exon19del. Given the existence of multiple AR mechanisms, quantification of the original EGFR activation mutation, instead of the resistance mutations, can be efficiently used to monitor response to osimertinib, allowing early detection of AR. Absolute quantification of both activation and resistance mutations can provide important information on tumor clonal evolution upon progression to osimertinib. Selective amplification of the EGFR-exon19del allele may represent a novel resistance mechanism to osimertinib.

Dual function of the long pentraxin PTX3 in resistance against pulmonary infection with Klebsiella pneumoniae in transgenic mice.

The long pentraxin PTX3 is expressed during acute inflammation and appears to control nitric oxide (NO) and tumor necrosis factor (TNF)-alpha production. In the present study, the physiological function of PTX3 was investigated in a model of pulmonary infection caused by the Gram-negative bacterium Klebsiella pneumoniae. Transgenic mice expressing multiple copies of PTX3 under the control of its own promoter were used to assess lethality rates, bacterial counts and inflammatory indices following pulmonary infection of mice. Expression of PTX3 is enhanced during pulmonary infection in wild-type mice. In transgenic mice given a high inoculum, overt PTX3 expression was associated with faster lethality. Faster lethality correlated with enhanced nitrate in plasma, an inability of neutrophils to migrate to lung tissue and greater dissemination of bacteria to blood at 20h after infection. In contrast, transgenic PTX3 expression conferred protection to mice given lower pulmonary inocula. In the latter experiments, there was enhanced TNF-alpha production, greater neutrophil influx and phagocytosis of bacteria by migrated neutrophils. By controlling the production of TNF-alpha and NO, and depending on the intensity of the inflammatory response induced by a given inoculum, the expression of PTX3 may favor or disfavor the influx of neutrophils and the ability of the murine host to deal with pulmonary infection with K. pneumoniae. These experiments highlight the delicate balance that exists among the various mediators that control the inflammatory response and suggest that PTX3 is an essential part of the ability of a host to deal with bacterial infection.

Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy.

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of "general" immune checkpoint drugs in this subset of patients.