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Treatment with the tyrosine kinase inhibitor (TKI) osimertinib is the standard of care for non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR). Osimertinib is also used in T790M-positive NSCLC that may occur de novo or be acquired following first-line treatment with other EGFR TKIs (i.e., gefitinib, erlotinib, afatinib, or dacomitinib). However, patients treated with osimertinib have a high risk of developing resistance to the treatment. A substantial fraction of the mechanisms for resistance is unknown and may involve RNA and/or protein alterations. In this study, we investigated the full transcriptome of parental and osimertinib-resistant cell lines, revealing 131 differentially expressed genes. Knockdown screening of the genes upregulated in resistant cell lines uncovered eight genes to partly confer resistance to osimertinib. Among them, we detected the expression of Ras-related protein Rab-32 (RAB32) and thrombospondin 1 (THBS1) in plasmas sampled at baseline and at disease progression from EGFR-positive NSCLC patients treated with osimertinib. Both genes were upregulated in progression samples. Moreover, we found that knockdown of RAB32 and THBS1 reduced the expression of phosphorylated focal adhesion kinase (FAK). Combination of osimertinib with a FAK inhibitor resulted in synergistic toxicity in osimertinib-resistant cells, suggesting a potential therapeutic drug combination for overcoming resistance to osimertinib in NSCLC patients.

Nuclear factor erythroid 2-related factor 2 (NRF2) protein expression promotes cancer progression in non-small cell lung cancer (NSCLC). However, its role in the clinical setting has not been established. We retrospectively analyzed data from 304 patients with surgically removed NSCLC. Multiplex antibody staining of NRF2 and thioredoxin reductase 1 (TrxR1) was conducted and scored in cytokeratin-positive (CK+) cells within the whole-tissue core as well as the tumor and stromal compartments of each tissue microarray (TMA) core. A high density of NRF2+/CK+ cells in the whole-tissue core compartment was correlated with a higher risk of central nervous system (CNS) relapse OR = 7.36 (95% CI: 1.64-33.06). The multivariate analysis showed an OR = 8.00 (95% CI: 1.70-37.60) for CNS relapse in NRF2+/CK+ high-density cases. The density of TrxR1+/CK+ cells failed to show any statistically significant risk of relapse. The OS analyses for NRF2+/CK+ and TrxR1+/CK+ cell density failed to show any statistical significance. This is the first study to report a correlation between NRF2+/CK+ cell density and the risk of CNS relapse in early-stage NSCLC. The results of our study may impact the follow-up strategy for early-stage NSCLC patients and eventually improve their prognosis.

There is a paucity of biomarkers for the prediction of intracranial (IC) outcome in immune checkpoint inhibitor (ICI)-treated non-small cell lung cancer (NSCLC) patients (pts) with brain metastases (BM). We identified 280 NSCLC pts treated with ICIs at Karolinska University Hospital, Sweden, and University Hospital of Heraklion, Greece. The inclusion criteria for response assessment were brain metastases (BM) prior to ICI administration, radiological evaluation with CT or MRI for IC response assessment, PD-1/PD-L1 inhibitors as monotherapy, and no local central nervous system (CNS) treatment modalities for ≥3 months before ICI initiation. In the IC response analysis, 33 pts were included. Non-primary (BM not present at diagnosis) BM, odds ratio (OR): 13.33 (95% CI: 1.424-124.880, p = 0.023); no previous brain radiation therapy (RT), OR: 5.49 (95% CI: 1.210-25.000, p = 0.027); and age ≥70 years, OR: 6.19 (95% CI: 1.27-30.170, p = 0.024) were associated with increased probability of IC disease progression. Two prognostic groups (immunotherapy (I-O) CNS score) were created based on the abovementioned parameters. The I-O CNS poor prognostic group B exhibited a higher probability for IC disease progression, OR: 27.50 (95% CI: 2.88-262.34, p = 0.004). Age, CNS radiotherapy before the start of ICI treatment, and primary brain metastatic disease can potentially affect the IC outcome of NSCLC pts with BM.

Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an EGFR -driven NSCLC adenocarcinoma than in sEVs from PE samples of non- EGFR driven adenocarcinoma patients or in samples from patients with benign lung disease. In summary, we have developed a diagnostic method for sEVs in liquid biopsies of cancer patients which may be used for longitudinal treatment monitoring to detect emerging bypassing resistance mechanisms in a noninvasive way.

There is lack of data addressing the intracranial (IC) efficacy of immune checkpoint inhibitors (ICIs) on brain metastases (BM) in non-small cell lung cancer (NSCLC). This patient category is underrepresented in randomized clinical trials. We retrospectively collected clinical data on patients with non-oncogenic driven NSCLC with BM who were treated with ICIs at two medical oncology institutes in Sweden and Greece from 2016 to 2019. IC efficacy was assessed in patients who had not received local treatment for BM less than three months prior to the initiation of ICIs and had adequate radiological evaluation. We screened 280 patients, of which 51 had BM. BM was an independent predictor for inferior PFS (HR = 2.27; 95% CI, 1.53-3.36) but not OS (HR = 1.58; 95% CI, 0.97-2.60) for the whole patient population. IC response assessment was done on 33 patients. IC objective response rate (ORR) was 24.2%. The presence of neurological symptoms related to BM did not affect IC ORR ( p = 0.48). High PD-L1 levels from extracranial biopsies were not a predictive factor for IC ORR ( p = 0.13). ICIs are active in NSCLC patients with BM regardless of the presence of neurological symptoms and can achieve durable IC disease stabilization in a subgroup of patients.

Background: Disease-specific Graded Prognostic Assessment (DS-GPA) is the most validated prognostic tool for patients with brain metastasized lung cancer. The Lung-molGPA scoring system was recently introduced for oncogenic-driven brain metastasized lung cancer, but has not yet been validated in cohorts including only ALK-translocated tumors., Methods: We designed a retrospective cohort study consisting of 44 patients with brain metastasized ALK-positive, non-small cell lung cancer (NSCLC) who were treated between January 2009 and November 2019 at Karolinska University Hospital in Stockholm, Sweden. Information about demographics and clinicopathological parameters were collected. Predictors of overall survival (OS) were identified by Cox regression analyses. A bootstrap validation with 1000 samples was performed in order to compare the different prognostic scores., Results: The variables found to independently influence OS in the multivariate analysis, i.e., PS, sex and brain metastases at diagnosis, were used as prognostic variables in our new prognostic index (ALK-BPI). Patients were divided into two prognostic groups. The median OS was 65.7 months for the good prognostic group and 22.7 months for the poor prognostic group ( p = 0.0068). In the univariate analysis of the different prognostic scores, ALK-BPI performed better than the others (HR = 3.6; 95% CI: 1.3-9.9). The mean C-statistics of the different prognostic scores were compared to each other, and no significant difference was observed., Conclusion: We propose the ALK-BPI score as a new prognostic tool that can easily be applied for ALK-positive lung cancer patients with brain metastases in daily clinical practice, as it has at least the same prognostic value as Lung-molGPA.