Circulating tumor DNA in patients with colorectal adenomas: assessment of detectability and genetic heterogeneity

Abstract

Improving early detection of colorectal cancer (CRC) is a key public health priority as adenomas and stage I cancer can be treated with minimally invasive procedures. Population screening strategies based on detection of occult blood in the feces have contributed to enhance detection rates of localized disease, but new approaches based on genetic analyses able to increase specificity and sensitivity could provide additional advantages compared to current screening methodologies. Recently, circulating cell-free DNA (cfDNA) has received much attention as a cancer biomarker for its ability to monitor the progression of advanced disease, predict tumor recurrence and reflect the complex genetic heterogeneity of cancers. Here, we tested whether analysis of cfDNA is a viable tool to enhance detection of colon adenomas. To address this, we assessed a cohort of patients with adenomas and healthy controls using droplet digital PCR (ddPCR) and mutation-specific assays targeted to trunk mutations. Additionally, we performed multiregional, targeted next-generation sequencing (NGS) of adenomas and unmasked extensive heterogeneity, affecting known drivers such as APC, KRAS and mismatch repair (MMR) genes. However, tumor-related mutations were undetectable in patients’ plasma. Finally, we employed a preclinical mouse model of Apc-driven intestinal adenomas and confirmed the inability to identify tumor-related alterations via cfDNA, despite the enhanced disease burden displayed by this experimental cancer model. Therefore, we conclude that benign colon lesions display extensive genetic heterogeneity, that they are not prone to release DNA into the circulation and are unlikely to be reliably detected with liquid biopsies, at least with the current technologies.

A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation

“…Validation of the PIK3CA p.H1047R mutation was performed using a Bio-Rad QX200 droplet digital PCR system as described previously 36 . Primer sequences included: forward, 5′-AGAGGCTTTGGAGTATTTCATG-3′; reverse, 5′-TGCATGCTGTTTAATTGTGTG-3′; probe sequences were wild-type VIC-MGB 5′-CCACCATGATGTGCA-3′; and mutant FAM-MGB 5′-CCACCATGACGTGCA-3′.…”

Colorectal Adenomas—Genetics and Searching for New Molecular Screening Biomarkers

“…The cfDNA analysis, known as the liquid biopsy approach, is cost-effective, minimally invasive, and its specificity can be increased by tailoring the assay to detect tumor-specific mutations [51,103,104]. Recently, liquid biopsies have been used to detect minimal residual disease and monitor relapse after surgical resection of a localized disease [105]. The use of liquid biopsies for the detection of benign tumors has proved to be challenging [106,107].…”

“…The use of liquid biopsies for the detection of benign tumors has proved to be challenging [106,107]. The probability of detecting cfDNA is low in early-stage CRC [107] and many groups showed different results in terms of diagnostic value of total ctDNA levels or analysis of KRAS mutations in the plasma of patients with adenomas [105,[108][109][110][111]. Encouraging studies reported an increase in total cfDNA or even detected tumor-related mutations in patients with benign adenomas [108,110].…”

The interplay of circulating tumor DNA and chromatin modification, therapeutic resistance, and metastasis

“…Apart from malignant tumor cells, it has been reported that benign tumors (e.g., adenomas) harboring overlapping malignant mutations can also release ctDNA in the early stage [190,191], which may lead to false-positive test results. Nevertheless, it has also been illustrated that benign lesions are unlikely to release significant quantities of ctDNA, making them less amenable for liquid biopsies [100,192]. Clonal hematopoiesis with somatic mutations, which is often related to aging and confers high relative risks to hematopoietic cancer in the elderly, does not actually translate into many hematopoietic cancers [193,194].…”