Multi‐regional sequencing reveals intratumor heterogeneity and positive selection of somatic mtDNA mutations in hepatocellular carcinoma and colorectal cancer

Abstract

Recent studies have revealed significant intratumor heterogeneity (ITH) of nuclear genome mutations and highlighted its function in tumor progression and treatment resistance. However, the ITH of somatic mitochondrial DNA (mtDNA) mutations detected in cancers remains unknown. In this study, we performed multiregional mtDNA sequencing of tumor and paratumor tissue samples from 12 hepatocellular carcinoma (HCC) and 13 colorectal cancer (CRC) patients. A substantial level of mtDNA mutations was found in paired non-HCC inflammatory tissues, suggesting that these tissues might not be mtDNA-genetically "normal." Moreover, our data indicated that the ITH of somatic mtDNA mutations was a common feature in HCC and CRC patients. In addition, we found that shared mutations which were observed in at least 2 samples in each patient exhibited a significantly higher heteroplasmic level than mutations that were private to a specific tumor region from both HCC (p = 0.039) and CRC patients (p = 0.001). The heteroplasmic level of shared mutations was positively correlated with intratumoral recurrence of mtDNA mutations. We also found that shared mutations in tumor tissues with a higher degree of pathogenicity risk exhibited a higher heteroplasmic level and intratumoral recurrence in both HCC and CRC patients. These findings suggest that some mtDNA mutations may undergo positive selection during the clonal expansion. Taken together, our analyses identified various levels of ITH of somatic mtDNA mutations in HCC and CRC patients and provided evidence supporting the positive selection working on some somatic mtDNA mutations in tumor tissues.

Heterogeneity in Colorectal Cancer: A Challenge for Personalized Medicine?

“…ITH also affects mitochondrial DNA (mtDNA), known to play a critical role in the initiation, metastatization, and metabolic changes of cancer cells. In fact, different mtDNA mutations were detected in multiple regions of 13 CRCs, in particular in tissue samples from the peripheral part of the tumor [53].…”

An Innovative Data Analysis Strategy For Accurate NGS Detection of Tumor mtDNA Mutations

“…However, which sequencing depth can provide su cient information to detect lowfrequency mtDNA mutations remains to be investigated up to now. Since there are no consensus criteria for determining mtDNA heteroplasmy threshold, it is very common to arbitrarily select a constant heteroplasmy level (mainly 2% or 5%) in previous NGS analyses for mtDNA mutations (12,17). Here, we innovatively revealed the relationship of site sequencing depth and the minimum mutation threshold via establishing logarithmic functions in different sample types, which contributes to the discrimination of false-positive and false-negative mutations with ultra-low heteroplasmy level (up to 0.5%).…”

“…For each site, we rst counted the respective reads number of the major and minor allele and calculated site-speci c minor allele frequency (MAF). Then, mtDNA mutations were initially called using the default settings as we previously described (14,16,17): 1) at least 3 reads on each strand have the mutation site; 2) minimum MAF cutoff 1%; 3) remove heterogeneity sites in rCRS repeat regions (66-71, 303-311, 514-523, 12418-12425, 16184-16193).…”

Intratumor heterogeneity of prognostic DNA-based molecular markers in adrenocortical carcinoma

“…Intratumor heterogeneity of somatic mutations has been reported in many cancer types ( 12 , 13 , 14 ). In a small series of exome sequencing in 14 ACC patients, intratumor heterogeneity was reported in 43–63% of somatic mutations among different metastatic sites from the same patient ( 15 ).…”