Heterogeneous mutation pattern in tumor tissue and circulating tumor DNA warrants parallel NGS panel testing

Abstract

Liquid biopsy by genotyping circulating tumor DNA (ctDNA) has provided a non-invasive approach in assessing tumor genomic alterations in clinical oncology. However, emerging evidence in clinical settings has shown significant discordance in the genomic alterations between matched tumor tissue and blood ctDNA samples, and even between the same set of blood samples analyzed on different testing platforms. Thus, it is necessary to study underlying causes of discrepancies in these studies by genotyping tumor tissue and ctDNA in parallel using next generation sequencing (NGS) panels based on the same technology. Here we enrolled 56 non-small-cell lung cancer (NSCLC) patients and evaluated tumor tissue genotyping and ctDNA based liquid biopsy by parallel NGS panel testing and compared different sample preparation conditions. Somatic mutations in plasma cell-free DNA (cfDNA) were detected in 63.6% patients with early-stage NSCLC and 60% patients with advanced-stage NSCLC. The overall concordance between matched formalin-fixed paraffin-embedded sample and cfDNA was 54.6% in early-stage NSCLC patients and 80% in advanced-stage NSCLC patients. The positive concordance rate was 44.4% and 71.4% in early-stage and advanced-stage patients, respectively. Using fresh frozen tumor samples did not improve the overall concordance rate between matched tumor tissue and cfDNA. Processing blood samples beyond 4 h after blood draw significantly decreased the detection rate of somatic mutations in cfDNA. Thus, the concordance rate between tumor tissue-based and ctDNA-based genotyping in clinical samples can be affected by multiple pre-analytical, analytical and biologic factors. Parallel NGS panel testing on both sample types for each patient may be warranted for effective guidance of cancer targeted therapies and possible early detection of cancer.Electronic supplementary materialThe online version of this article (10.1186/s12943-018-0875-0) contains supplementary material, which is available to authorized users.

Detection of mutations in circulating cell‐free DNA in relation to disease stage in colorectal cancer

“…Guo et al used panel sequencing to detect tissue‐matched mutations in cfDNA of 56 early‐stage and advanced‐stage patients with nonsmall cell lung cancer (NSCLC). They reported an overall concordance rate of 54.6% and 80%, respectively . Of particular importance is their observation that the concordance rate can be strongly affected by multiple pre‐analytical, analytical, and biological factors.…”

“…They reported an overall concordance rate of 54.6% and 80%, respectively. 21 Of particular importance is their observation that the concordance rate can be strongly affected by multiple pre-analytical, analytical, and biological factors. Regarding that, we might explain the sporadic mutation detection in our patient cohort with limitations, such as sample age and inconsistent processing, storing, and delivery conditions at two different hospitals.…”

Assessing the Concordance of Genomic Alterations between Circulating-Free DNA and Tumour Tissue in Cancer Patients

“…Specifically, all patients (n = 10) with metastatic disease had detectable mutations, while this rate was lower for patients in less advanced stages of the disease [80]. Consequently, higher tumour fraction in metastatic diseases may contribute to higher concordance rates observed between cfDNA and metastatic tumour compared to cfDNA and primary tumour [72,81], in addition to indicating poor prognosis [82]. Xie et al, tested 35 pairs of NSCLC primary tumour tissues or metastatic tumours and plasma from treatment-naïve patients using targeted sequencing for a custom panel of 56 lung cancer genes.…”

“…Tumour fraction can be impacted by technical practices used to extract cfDNA from plasma or serum. Guo et al, evaluate the effect of blood sample processing on cfDNA concentration and found that delaying processing beyond four hours significantly decreased detection rate of somatic mutations in cfDNA [71,72]. Release of genomic DNA from white blood cells, resulting in contamination of cfDNA, can be a consequence of delayed processing.…”

“…With regards to tumour tissue processing methods, the use of fresh frozen (FF) samples for tumour tissue instead of formalin-fixed paraffin-embedded (FFPE) marginally increased from 57.1% to 66.7%, suggesting fragmentation of DNA in FFPE processing may be significant, especially when the detection assay relies on amplicon-based amplification [72,100,148].…”