Combined Analysis of Disseminated Tumor Cells (DTCs) and Circulating Tumor DNA (ctDNA) in a Patient Suffering from Triple Negative Breast Cancer Revealed Elevated Risk

Abstract

Background: Disseminated tumor cells (DTCs) in bone marrow aspirates of patients with primary breast cancer may serve as independent prognostic markers associated with impaired survival. Due to limited therapy options and high risk of recurrence particularly, women diagnosed with the aggressive triple negative breast cancer (TNBC) require personalized treatment choices. Genetic profiling of circulating cell-free tumor DNA (ctDNA) might help to find individual treatment options and to monitor disease course. Methods: Here we report the case of a 66-year-old patient with TNBC. She received neoadjuvant chemotherapy (NACT) that had to be interrupted due to intolerance. Surgical resection of the residual tumor resulted in pathologic complete response (pCR), though. Results: Bone marrow aspiration during surgery revealed an unusual high number of DTCs and thus elevated risk for recurrence. Analysis of pre-surgical blood and urine samples revealed the presence of plasma-derived and urinary ctDNA after NACT and indicated poor prognosis. Subsequent targeted sequencing showed that pathogenic variants occurred in urinary and plasma-derived ctDNA emphasizing the potential of liquid biopsy usage for early detection of relapse. Despite the detection of residual molecular disease after NACT, the presented patient reached pCR and could benefit from standard treatment until present. Conclusions: In this case, liquid biopsy based biomarkers did not necessarily correlate to clinical outcome. Further, ctDNA analysis did not reveal approved therapeutic options to target the identified pathogenic variants. Adjuvant bisphosphonate treatment was applied based on the positive DTC status and may improve the patients' prognosis. Further investigations are required to identify TNBC patients at risk for recurrence.

Targeted Sequencing of Plasma-Derived vs. Urinary cfDNA from Patients with Triple-Negative Breast Cancer

“…Aliquots of plasma and urine samples were thawed immediately and only once prior to cfDNA extraction to minimize freeze–thaw effects [ 22 ]. Isolation and quantification of cfDNA was performed as described elsewhere [ 23 ]. Subsequently, cfDNA was isolated from 4 mL of plasma and 10 mL of matching urine supernatant for each patient using the QIAamp MinElute ccfDNA Midi Kit (QIAGEN, Hilden, Germany) according to the manufacturers’ instructions.…”

The Challenge to Stabilize, Extract and Analyze Urinary Cell-Free DNA (ucfDNA) during Clinical Routine

“…Most of them are based on either spin columns or magnetic beads. Based on our previous studies [11,21], we used the QIAamp MinElute ccfDNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturers' instructions. Briefly, pre-concentration of cfDNA on magnetic beads was followed by purification using silica-based spin columns.…”

“…In urine, the half-life time of cfDNA is shorter than in plasma due to high levels of nucleases like DNase I and DNase II, as well as contaminants present in urine [9,10]. In previous studies, we were able to show that targeted sequencing of ucfDNA from patients with breast cancer was feasible without using stabilizing buffers when samples were processed within 2-4 h after collection [11,21]. However, the cfDNA yield from 10 mL of urine appeared to be low.…”