Integrated circulating tumour DNA and cytokine analysis for therapy monitoring of ALK-rearranged lung adenocarcinoma.

BACKGROUND: Detection of circulating tumour DNA (ctDNA) in biological fluids is a minimally invasive alternative to tissue biopsy for therapy monitoring. Cytokines are released in the tumour microenvironment to influence inflammation and tumorigenic mechanisms. Here, we investigated the potential biomarker utility of circulating cytokines vis-à-vis ctDNA in ALK-rearranged+ lung adenocarcinoma (ALK + NSCLC) and explored the optimal combination of molecular parameters that could indicate disease progression. METHODS: Longitudinal serum samples (n = 296) were collected from ALK + NSCLC patients (n = 38) under tyrosine kinase inhibitor (TKI) therapy and assayed to quantify eight cytokines: IFN-γ, IL-1ß, IL-6, IL-8, IL-10, IL-12p70, MCP1 and TNF-α. Generalised linear mixed-effect modelling was performed to test the performance of different combinations of cytokines and previously determined ctDNA parameters in identifying progressive disease. RESULTS: Serum IL-6, IL-8 and IL-10 were elevated at progressive disease, with IL-8 having the most significant impact as a biomarker. Integrating changes in IL-8 with ctDNA parameters maximised the performance of the classifiers in identifying disease progression, but this did not significantly outperform the model based on ctDNA alone. CONCLUSIONS: Serum cytokine levels are potential disease progression markers in ALK + NSCLC. Further validation in a larger and prospective cohort is necessary to determine whether the addition of cytokine evaluation could improve current tumour monitoring modalities in the clinical setting.

ITIH5 shows tumor suppressive properties in cervical cancer cells grown as multicellular tumor spheroids.

Cervical cancer (CC) arises from premalignant cervical intraepithelial neoplasia (CIN) induced by a persistent infection with human papillomaviruses. The multi-stepwise disease progression is driven by genetic and epigenetic alterations. Our previous studies demonstrated a clear downregulation of inter-α-trypsin-inhibitor-heavy chain 5 (ITIH5) at mRNA and protein levels in CC compared to CIN2/3 and normal cervical tissue. Initial in vitro functional analyses revealed a suppressive effect of ITIH5 on relevant mechanisms for cancer progression in conventional two dimensional (2D) cell culture model systems. Based on these studies, we aimed to investigate the functional relevance of ITIH5 in multicellular tumor spheroid (MCTS) models, which resemble in vivo tumors more closely. We successfully established CC cell line-derived MCTS using the hanging-drop technique. ITIH5 was ectopically overexpressed in HeLa and SiHa cells and its functional relevance was investigated under three dimensional (3D) culture conditions. We found that ITIH5 re-expression significantly suppressed tumor spheroid growth and spheroid invasiveness of both HeLa and SiHa spheroids. Immunohistochemical (IHC) analyses revealed a significant reduction in Ki-67 cell proliferation index and CAIX-positive areas indicative for hypoxia and acidification. Furthermore, we observed an increase in cPARP-positive cells suggesting a higher rate of apoptosis upon ITIH5 overexpression. An effect of ITIH5 expression on the susceptibility of cervical MCTS towards cytostatic drug treatment was not observed. Collectively, these data uncover pronounced anti-proliferative effects of ITIH5 under 3D cell culture conditions and provide further functional evidence that the downregulation of ITIH5 expression during cervical carcinogenesis could support cancer development.