Dissemination of Mitochondrial DNA Variants: Looking at the 'Bigger' Picture of the Tumour Microenvironment in Rectal Cancer Patients.

The tumour microenvironment (TME) constitution is decisive for cancer outcome and is manifested in diffusion-weighted (DW) magnetic resonance imaging (MRI). We hypothesized that the TME metabolic state is reflected by mitochondrial DNA (mtDNA) secreted in extracellular vesicles (EVs) and examined whether plasma EV-mtDNA variants may divulge MRI-assessed TME attributes of rectal cancer aggressiveness. On the diagnostic MRI scans from 60 rectal cancer patients, the apparent diffusion coefficient (ADC) was calculated on DW images (n = 29), and tumour volume (n = 57) and extramural vascular invasion (EMVI; all patients) were determined on anatomical images. Plasma EVs (all patients) were isolated by size exclusion chromatography and verified for EV features. The EV-mtDNA was sequenced along with mtDNA in whole blood (WB; normal tissue) to calculate the EV/WB-mtDNA total variant number (TVN) and heteroplasmic variant number (HVN)-as a proxy for TME intracellular mtDNA variants expelled in EVs. Low EV/WB-mtDNA TVN and HVN, indicative of hampered clearance of mutated mtDNA via EVs, were associated with low ADC (high TME cell density; p = 0.018, p = 0.005) and a large tumour volume (p = 0.002, p = 0.003). Likewise, low EV/WB-mtDNA TVN and HVN were associated with positive EMVI (tumour infiltration in blood vessels; p = 0.002, p = 0.003) and histologic ypN stage 1-2 (lymph nodes with tumour cells surviving radiotherapy; p = 0.002, p = 0.005), both indicators of high tumour aggressiveness. High cellular density may hamper the clearance of pathogenic tumour mtDNA variants by EVs and thus promote rectal cancer aggressiveness. Trial Registration: ClinicalTrials.gov: NCT01816607. Registered 22 March 2013, https://clinicaltrials.gov/ct2/show/NCT01816607.