Circulating Tumor DNA (ctDNA): 

 In cancer patients fragments of DNA from tumor cells are found in the blood along with short stretches of DNA from healthy cells (cell-free DNA, cfDNA) shed during cell turnover. In early-stage disease the ctDNA fraction of total cfDNA (circulating free DNA) may be <0.1%; in locally advanced disease, 0.1 to 1.0%; and in patients with advanced stage cancer the ctDNA fraction (ctDNA divided by cfDNA) may rise to 10% or substantially more. These DNA snippets are cleared rapidly from the blood with an estimate half-life of 30 – 120 minutes. (Data from Moser and Helzer, SCIENCE, Jan 2024). The quantity of ctDNA in the blood is proportional to the total tumor burden. Tumors with the most rapid proliferation shed the most ctDNA. Sequential measurements over time can reveal dynamic changes in tumor characteristics and burden and capture the heterogeneity among multiple sites of disease as opposed to the findings from a single site biopsy. Active research is positioning analysis of ctDNA to estimate prognosis, assess response to therapy and monitor therapy resistance. With improvement in analytic sensitivity, it may be possible to monitor minimal residual disease following initial treatment.

 Pascual et al. (Annals of Oncology, Aug 2022) reported “European Society for Medical Oncology recommendations on the use of circulating tumor DNA assays for patients with cancer.”  Based on the opinions of a panel of experts, some applications of ctDNA analysis are suitable for current clinical use: i.e., “For patients with advanced cancer, validated and sensitive ctDNA assays have utility in identifying actionable mutations [i.e., BRCA family mutations] to direct targeted therapy, and may be used in clinical practice.”

 An example of current ctDNA research is provided by Beltran and colleague:

“Noninvasive Detection of Neuroendocrine Prostate Cancer through Targeted Cell-free DNA Methylation” (Cancer Discovery, March 2024). This study addresses an unmet need. It Interrogates cfDNA to create a biomarker panel to assess and quantify trans-differentiation from conventional adenocarcinoma to neuroendocrine prostate cancer (NEPC).

 In late-stage disease under therapeutic pressure of extensive treatment 15 – 20% of patients experience a histologic transition toward neuroendocrine cancer – an aggressive variant currently only diagnosed by tissue biopsy. Based on analyses of plasma cfDNA of patients with NEPC Beltran identified highly specific markings (epigenetic alterations in patterns of methylation) on fragments of cfDNA and assembled a diagnostic panel, “NEMO” (NEuroendocrine detection and MOnitoring assay), that could quantify the proportion of NEPC relative to the total tumor burden down to the limit of 3% and rising to 30% or more in late disease.

 This data could suggest the timing to transition from hormone therapy to chemotherapy or do a follow-up biopsy. Beltran: “We hope that this blood test can be used by clinicians to determine if a patient is developing neuroendocrine prostate cancer” (Dana Farber). It is planned to make this test clinically available.

 Liquid biopsy of ctDNA to identify ‘actionable mutations’: The most clinically serviceable mutations are associated with defects in DNA damage repair genes, BRCA1, 2 and ATM — and several others. Patients harboring these mutations respond to PARP inhibition with olaparib, rucaparib and others. Mutated CDK12 confers a susceptibility to immune therapy. Assessment of germline (inherited) mutations on primary tissue yields ~5-8% actionable mutations, BRCA 1, 2 and ATM being the most common. Additional “actionable” somatic mutations develop under the pressure of treatment in advanced stage cancer and may be identified in ctDNA and suggest susceptibility to specific therapy or protocol eligibility.

 Mutations in genes termed mismatch repair genes (MLH1, MSH2, MSH6 and PMS2) occur only in up to 5% in germline and somatic profiles. The normal function of these gene is to maintain the stability of the genome by correcting errors occurring during DNA replication. Mutations in these genes confer susceptibility to the PD-L1 immune checkpoint inhibitor, Ketruda.

 Robinson, Chinnaiyan and 67 colleagues reported the results of biopsies of multiple metastatic sites in 150 men with CRPC and found a ~20% incidence of actionable mutations — BRCA 1, 2 and ATM (12.7% BRCA2) plus mutated CDK12 in 7% (“Integrative clinical genomics of advanced prostate cancer”) (Cell. 2015). Their clear message: in advanced disease assay ctDNA for actionable mutations in search for a therapeutic opportunity. Currently, liquid biopsy of ctDNA can efficiently capture somatic (and germline) mutations thus avoiding a tissue biopsy. An appropriate point to assay for somatic mutations could be after failure of the initial hormone treatment. if a BRCA family mutation were found, treatment with a PARP inhibitor might follow.

 Commercial companies offering cfDNA assays include Guardant Health (Guardant360 CDX), Foundation Medicine (FoundationOne LiquidCDX for mutations in BRCA 1,2 and ATM) and Personal Genome Diagnostics (Cancer Seek).

 Liquid biopsy of circulating free DNA and circulating tumor DNA as independent indicators of prognosis and response to therapy:

 Higher levels of pretreatment ctDNA% (ctDNA/cfDNA) in men with mCRPC predict shorter progression-free (PFS) and overall survival. Norgaard et al (Clinical Chemistry. Feb. 2023) assessed the tumor genome of 200 men with mCRPC at the start of second-line hormone therapy (i.e., Zytiga or Xtandi) and found that the baseline ctDNA% ranged from < 3% to 73%. In follow-up, men having PFS of <6 vs >18 months had a median baseline ctDNA% of 24.4% vs 3.0%, respectively. 

 The Future: Applications under study awaiting validation from clinical trials:

  •  Identification of Minimal Residual Disease (MRD) after treatment in early-stage disease might indicate the need for further treatment consolidation or argue for or against adjuvant therapy. To accurately assess MRD, however, will require greater sensitivity for detecting ctDNA than is currently available. The same limitation of sensitivity currently prevents using ctDNA to identify disease progression during active surveillance, although both applications are under study.
  •  Additional research and validation are needed to assess early response to treatment to guide an earlier change of therapy compared to decisions based on conventional biomarkers.

 In these situations, clinical trials will be needed to establish whether action taken on the basis of ctDNA data improves outcome and warrants the potential adverse effects of earlier intervention.

 BOTTOM LINE:

 Liquid Biopsies assessing ctDNA are being integrated into clinical practice. Validated applications include pretreatment estimation of prognosis, evaluation of response to therapy and detection of ‘actionable mutations.” Clinical implementation will require clinical trials to demonstrate improvement in patient outcome.