Characterisation of the Phosphatidylinositol 3-Kinase Pathway in Non-Small Cell Lung Cancer Cells Isolated from Pleural Effusions

Abstract

Objectives: We hypothesised that it was possible to quantify phosphorylation of important nodes in the phosphatidylinositol 3-kinase (PI3K) pathway in cancer cells isolated from pleural effusions of patients with non-small cell lung cancer (NSCLC) and study their correlation to somatic mutations and clinical outcomes. Materials and Methods: Cells were immunomagnetically separated from samples of pleural effusion in patients with NSCLC. p-AKT, p-S6K and p-GSK3β levels were quantified by ELISA; targeted next-generation sequencing was used to characterise mutations in 26 genes. Results: It was possible to quantify phosphoproteins in cells isolated from 38/43 pleural effusions. There was a significant correlation between p-AKT and p-S6K levels [r = 0.85 (95% confidence interval 0.73-0.92), p < 0.0001], but not p-AKT and p-GSK3β levels [r = 0.19 (95% confidence interval -0.16 to 0.5), p = 0.3]. A wide range of mutations was described and p-S6K was higher in samples that harboured at least one mutation compared to those that did not (p = 0.03). On multivariate analysis, p-S6K levels were significantly associated with poor survival (p < 0.01). Conclusion: Our study has shown a correlation between p-AKT levels and p-S6K, but not GSK3β, suggesting differences in regulation of the distal PI3K pathway by AKT. Higher p-S6K levels were associated with adverse survival, making it a critically important target in NSCLC.

A study of PD-L1 expression in KRAS mutant non-small cell lung cancer cell lines exposed to relevant targeted treatments

“…Immunohistochemistry is the most common method of measuring PD-L1 in the clinical context with well documented issues with standardization across platforms [ 24 ] and we propose that the jurkat co-culture system be investigated in the clinical context. This could involve the isolation of cancer cells from circulating tumour cell platform or serous effusions [ 25 ] [ 26 ] and is worthy of further investigation. We recognize the experiments in immunocompetent animal models will offer the most robust validation of the functional relevance of PDL-1 expression as a mechanism of resistance.…”

A transcriptional biosensor to monitor single cancer cell therapeutic responses by bioluminescence microscopy

“…We believe that such a reconstructive approach from single molecular alterations to whole tumor biology has great potential because it has been previously shown that freshly dissociated cells from the tumor mirror the genotypic characteristics of the tumor [24,25]. This method is also versatile because various sources of samples such as biopsies, blood, peritoneal (ascites), urine, pleural or cerebrospinal fluids can be exploited to harvest cancer cells [25,[47][48][49][50]. Moreover, in prostate cancer, the anatomical position of the prostate makes prostate biopsies and urine important sources of cancer cells, which could facilitate the translation of the technology into the clinics.…”

Molecular testing on serous effusions

“…p‐S6K levels were significantly higher in tumors carrying at least one mutation compared with those that did not carry any. Higher levels of p‐AKT and p‐S6K were associated with shorter survival in univariate analysis, and the latter was retained in multivariate analysis 36 …”