Abstract
Accurate assessment of tumour heterogeneity is an important issue that influences prognosis and therapeutic decision in molecular pathology. Due to the shortage of protective histones and a limited DNA repair capacity, the mitochondrial (mt)-genome undergoes high variability during tumour development. Therefore, screening of mt-genome represents a useful molecular tool for assessing precise cell lineages and tracking tumour history. Here, we describe a highly specific and robust multiplex PCR-based ultra-deep sequencing technology for analysis of the whole mt-genome (wmt-seq) on low quality-DNA from formalin-fixed paraffin-embedded tissues. As a proof of concept, we applied the wmt-seq technology to characterize the clonal relationship of non-small cell lung cancer (NSCLC) specimens with multiple lesions (N = 43) that show either different histological subtypes (group I) or pulmonary adenosquamous carcinoma as striking examples of a mixed-histology tumour (group II). The application of wmt-seq demonstrated that most samples bear common mt-mutations in each lesion of an individual patient, indicating a single cell progeny and clonal relationship. Hereby we show the monoclonal origin of histologically heterogeneous NSCLC and demonstrate the evolutionary relation of NSCLC cases carrying heteroplasmic mt-variants.
Highlights
Tumour tracking and evolution analysis to identify the intra-tumour clonal structure or history of multiple tumour lesions within the same patient are currently evolving into important diagnostic tools for the precision treatment of malignant neoplasias[1,2,3]
Hereby we clearly show that an ultra-deep sequencing technology of the entire mt-genome is a suitable molecular tool for tumour history tracking on pathologically processed formalin-fixed paraffin-embedded (FFPE) material
In order to track tumourigenesis on FFPE archived material, we developed a novel approach for comprehensive mtDNA mutation analysis using a multiplex PCR-based ultra-deep sequencing approach on multifocal non-small cell lung cancer (NSCLC) lesions of different histological growth patterns
Summary
In order to track tumourigenesis on FFPE archived material, we developed a novel approach for comprehensive mtDNA mutation analysis using a multiplex PCR-based ultra-deep sequencing approach on multifocal NSCLC lesions of different histological growth patterns. Though a limited number of private and branch variants, which we identified in different nodules, did not allow us to describe the complete evolutionary dynamics of tumor clonal networks[2, 3], this technology provides for the first time a highly specific and sensitive approach to study the clonal relationship and tumour history on FFPE material of the pathology routine processing. This is of particular importance in terms of an appropriate tumour-specific treatment strategy when de-novo primary tumours and recurrent cancers have to be differentiated[12]. Due to the tolerance of low DNA quantity and quality, the multiplex PCR-based wmt-seq technology might be applicable to cell-free DNA approaches as a novel option to detect mitochondrial DNA alterations in various body fluids and to monitor cancer progression and mitochondrial disorders
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