Efficacy of Immunotherapy in Second-Line Treatment of KRAS-Mutated Patients with Non-Small-Cell Lung Cancer-Data from Daily Practice.

Background The implementation of next-generation sequencing (NGS) into daily practice allows for the identification of a greater number of molecular abnormalities. We aimed to confirm the benefits of immunotherapy in the group of patients with KRAS aberrations treated within clinical practice. Methods This study was a retrospective analysis of the patients (pts) treated in routine practice within the National Drug Programme in Poland. The NGS was performed using a FusionPlex Comprehensive Thyroid and Lung (CTL) kit (ArcherDx) and sequenced using a MiniSeq (Illumina). The analyses were performed with the R language environment, version 4.1.3. Results A total of 96 pts with chemotherapy-pre-treated advanced NSCLC (CS III−IV) were qualified for nivolumab or atezolizumab treatment following a molecular diagnosis by the NGS and the exclusion of EGFR and ALK gene abnormalities. A mutation in the KRAS gene was found in 26 patients (27%); among them, the variant p.Gly12Cyc (G12C) was the most common (42%). The median PFS and OS for the overall population were 2 months (95% CI: 1.8−2.75) and 10 months (95% CI: 6.9−16.2), respectively. No differences were observed in terms of the mPFS between the KRAS-mutated and KRAS wild-type (WT) patients. A trend toward a longer OS was observed in the group of patients with the KRAS mutation, but the difference was not statistically significant (p = 0.43). In the multivariate analysis, the presence of mutations in the KRAS gene had no prognostic significance, while the occurence of grade 3 toxicity and the neutrophil-to-lymphocyte ratio (NLR) > 3.5 were found as statistically significant factors. Conclusions Immunotherapy in the second-line treatment of advanced NSCLC allows for a benefit regardless of the KRAS gene mutation status. The treatment sequence, including molecularly targeted drugs such as sotorasib and adagrasib, is still discussed. The NGS is a valuable method to identify a variety of molecular abnormalities in patients with NSCLC in daily practice.

Changes in the activity of acetylcholinesterase and Na,K-ATPase in human erythrocytes irradiated with X-rays.

The response of human erythrocytes to X-rays in the dose range from 40 Gy to 600 Gy was determined on the basis of changes in the activities of AChE and ATPase. The Na,K-ATPase activity increased above the control value at doses below 200 Gy, while at the doses higher than 200 Gy, it decreased, reaching 96% of the control value at a dose of 600 Gy. In the range of doses up to 200 Gy, the AChE activity, expressed as Vmax, did not change. At higher doses, it fell drastically, reaching 33% of the control value at a dose of 600 Gy. Simultaneously, the enzyme substrate affinity decreased at 200 Gy, and then started to increase at lower values of Vmax. The obtained results suggest that under appropriate conditions, low doses of radiation may have the opposite effects to high doses.

The influence of radiation quality on radiation-induced hemolysis and hemoglobin oxidation of human erythrocytes.

Human erythrocytes were exposed to gamma-rays and alpha-particles to assess radiation-induced membrane damage and hemoglobin oxidation and denaturation. With all parameters measured, the alpha-particles proved to be less efficient than the gamma-rays. The time-dependence of hemolysis showed also clear differences: with the gamma-rays the process was faster, reaching saturation after 40-90 min (depending on dose), but with the alpha-particles the final level was attained only after about 3-7 h. Hemoglobin oxidation and denaturation could be measured only after gamma-exposure, but they were negligible with the alpha-particles when comparable doses were applied. These results are interpreted by proposing that OH-radicals, whose yields are smaller with densely ionizing radiation, play a crucial role in the induction of the processes for radiation-induced erythrocyte damage.

The influence of split doses of gamma-radiation on human erythrocytes.

Human erythrocyte suspensions in an isotonic Na-phosphate buffer, pH 7.4, of hematocrit of 2% were exposed under air to gamma radiation at a dose rate of 2.2 kGy. Erythrocytes were irradiated with single doses, and identical doses split into two fractions with an interval time of 3.5 h between following exposures. The obtained results indicated that the irradiation of enucleated human erythrocytes with split doses caused a reduction of hemolysis (2.4 times), a decrease in the level of damage to membrane lipids and the contents of MetHb, compared with identical single doses. However, the splitting of radiation doses did not change the level of damage to the membrane proteins, as was estimated with a maleimide spin label. The obtained results suggest that a decrease in the level of damage to lipids was related to a decrease in hemolysis.

The influence of X-rays on human erythrocytes. Primary radicals.

The effects on human erythrocytes of water-derived radicals generated by X-rays were studied under anaerobic conditions and in the presence of oxygen. Erythrocyte damage was estimated on the basis of the reduced GSH and MetHb content in the erythrocytes, the -SH group content in the membrane proteins and the amount of K(+)released from the erythrocytes. The results obtained show that the level of reduced GSH was the most sensitive indicator of erythrocyte damage by X-rays followed by the efflux of K(+). The processes of GSH oxidation took place most rapidly under air. At a dose of 100 Gy, the level of GSH fell to about 50%, whereas under argon and N(2)O to about 75% and 65%, respectively. A slight increase in the efflux of K(+)was observed in preparations irradiated under air. However, when erythrocytes were irradiated under argon and N(2)O, the loss of K(+)occurred at a dose 8-times higher. Changes in the remaining parameters occurred at considerably higher doses. On the basis of the results obtained one can say that oxygen is a factor increasing the toxicity of(.)OH radicals towards erythrocytes; however, e(-)(aq)present in the system can cause a decrease in damage to certain cellular components.

Damage to human erythrocytes by radiation-generated HO* radicals: molecular changes in erythrocyte membranes.

The effectiveness of radiation-generated HO* radicals in initiating erythrocyte hemolysis in the presence of oxygen and under anaerobic conditions and prehemolytic structural changes in the plasma-erythrocyte membrane were studied. Under anaerobic conditions the efficacy of HO* radicals in induction of hemolysis was 16-fold lower than under air. In both conditions, hemolysis was the final consequence of changes of the erythrocyte membrane. Preceding hemolysis, the dominating process under anaerobic conditions was the aggregation of membrane proteins. The aggregates were principally formed by -S-S- bridges. A decrease in spectrin and protein of band 3 content suggests their participation in the formation of the aggregates. These processes were accompanied by changes in protein conformation determined by means of 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (MSL) spin label attached to membrane proteins. Under anaerobic conditions, in the range of prehemolytical doses, the reaction of HO* with lipids caused a slight (10-16%) increase in fluidity of the lipid bilayer in its hydrophobic region with a lack of lipid peroxidation. However, in the presence of oxygen, hemolysis was preceded by intense lipid peroxidation and by profound changes in the conformation of membrane proteins. At the radiation dose that normally initiates hemolysis a slight aggregation of proteins was observed. Changes were not observed in particular protein fractions. It can be suggested the cross-linking induced by HO* radicals under anaerobic conditions and a lack of lipid peroxidation are the cause of a decrease in erythrocyte sensitivity to hemolysis. Contrary, under aerobic conditions, molecular oxygen suppresses cross-linking, catalysing further steps of protein and lipid oxidation, which accelerate hemolysis.