Marko Velimirovic, MD
With our expanding understanding of complex cancer genetics and the emergence of personalized anticancer therapeutics tailored to driver mutations, there is a need for repeated tissue biopsies throughout the course of cancer treatment. Ideally, each time a patient clinically progresses, a repeat biopsy of the tissue could provide information regarding tumor evolution, but this is not practically feasible.
Although tissue biopsies are still the gold standard for tumor diagnosis and genotyping, core biopsies are invasive and associated with discomfort, high cost, long turnaround time, and complications. More so, it has been demonstrated that metastatic clones at different sites tend to harbor mutations that are not the same as those from their primary tumors and, as such, genetically represent a distinct disease that may not respond to the same therapy that the primary tumor does. This is known as genomic heterogeneity.
Tissue biopsies also contain only a small portion of the tumor and, therefore, may not be representative of all the mutational clones present within the biopsied tumor, which poses a problem of capturing intratumor heterogeneity.
Investigators gained a better insight into genomic variability across all tumor clones present in a patient with the discovery that tumors shed fragments of their DNA into the circulation. This occurs either passively, when cancer cells undergo necrosis or apoptosis, either spontaneously or as a response to therapy, or actively during hematogenous spreading. This valuable genetic information can be captured from circulating free tumor DNA (ctDNA), also referred to as a liquid biopsy, isolation of whole circulating tumor cells (CTCs), RNA, microRNAs/exosomes, and tumor-educated platelets.
Analysis of ctDNA has gained significant interest over the past decade in nearly all aspects of cancer treatment and across all tumor types. CtDNA consists of short fragments of double-stranded, short-lived (approximately 2 hours) DNA and is characterized by unique somatic mutations that are not present in normal cells, thereby allowing them to be differentiated from circulating free DNA (cfDNA) shed by noncancerous cells.1,2
CtDNA ranges from less than 0.1% to more than 10.0% of the total cfDNA depending on stage, tumor burden, vascularization of the tumor, biological features like apoptotic rate, and metastatic potential of the cancer cells.3,4
Timing of blood collection in relation to exposure to therapy can also be critical because tumors tend to undergo necrosis and, therefore, shed their DNA when exposed to cytotoxic agents. Numerous techniques for detection and processing of ctDNA have been developed and can be categorized either as polymerase chain reaction tests or nextgeneration sequencing (NGS) assays.
The main differences between these 2 techniques are that those in the first group can detect mutations in only a limited number of loci, usually within a single gene, whereas NGS techniques are able to detect a large number of mutations, including gene amplifications, fusions, rearrangements, and aneuploidy.5,6
These developments have led to several commercially available ctDNA test for detection of EGFR
-sensitizing mutations in lung cancer. Cellsearch (Menarini Silicon Biosystems) is used to detect CTCs. These tests carry high sensitivity and specificity of nearly 99%.
Advantages of ctDNA Testing
The most relevant advantages of ctDNA detection over traditional tissue genotyping include as follows7,8
Given the relatively high recurrence rates of breast cancer months to years following the curative treatment of non–stage IV cancer, significant efforts have been made to find applications of ctDNA for analyzing stored genetic material.9-11
Breast Cancer Subtypes
It is now clear that breast cancer is a heterogeneous disease, both phenotypically and genetically. Four luminal types of breast cancer have been identified based on hormonal expression; 10 subtypes, termed integrative clusters, have been classified by their distinct genetic signatures.12
Each type carries its own genetic specificities, which are likely responsible for the observed differences in sensitivity to chemotherapy and targeted treatments.
There are a number of genetic alterations, including mutations, amplifications, and translocations, detectable by ctDNA, which rises as the disease progresses. TP53
Y220C, Y220N, R273C, and R273H mutations have been associated with worse prognosis in basal-like triple-negative and other subtypes of breast cancer. Mutations in AR, ESR1
(Y537N, Y537S, Y537C, D538G), and clones harboring ESR1
gene mutations have been shown to emerge in patients who develop resistance to aromatase inhibitors, implying that this could be the driver mutation in this subset of patients.