A mass cytometry‐based lens on DNA damage repair in circulating tumor cells from breast cancer patients

As the global incidence and mortality of breast cancer continue to rise, better strategies for disease management and treatment are urgently needed. The development of liquid biopsy tools is anticipated to facilitate this progress by transforming the traditional "watchful waiting" into a clinically useful strategy for monitoring recurrence and guiding therapy selection. Current evidence supports the clinical validity of circulating tumor cell (CTC) enumeration in metastatic breast cancer (mBC).1 Serial monitoring of patients during treatment has demonstrated prognostic relevance, as changes in CTC numbers are associated with treatment outcomes (recently summarized in a review2). However, the clinical utility of CTCs (addressed in several completed clinical trials—for example, SWOG0500, CirCe01, CirCe T-DM1, STIC CTCs, DETECT III) is still debated.2 These studies used CellSearch®—the gold standard in CTC enumeration, which raises the question: does cancer's biological complexity and heterogeneity demand more diverse isolation methods and broader CTC characterization to capture clinically relevant information? As treatment exerts selective pressure on tumor cells, driving genomic and phenotypic adaptations,3 low-invasive and longitudinally applicable methods are essential to track these changes effectively. In the current issue, Nidermayer et al. present a mass cytometry (CyTOF®)-based approach for detecting and characterizing the DNA damage response in CTCs from mBC patients undergoing systemic therapy. This retrospective study utilized cryopreserved peripheral blood mononuclear cells (PBMCs), in which CTCs were identified using a marker panel (EpCAM+, CK+/CD45−), further expanded to include mesenchymal (vimentin), stem cell (CD44, CD24, ALDH1A3), and DNA damage/repair markers (RAD51–homologous recombination; 53BP1–non-homologous end joining; γH2AX and pRPA32-DNA damage/repair indicators). Concurrently, circulating tumor DNA (ctDNA) was detected using shallow whole-genome sequencing (sWGS), and CTCs were also evaluated with CellSearch®, enabling comprehensive assessment of tumor burden. CyTOF® detected CTCs in some samples that were negative by CellSearch®, in general though the CTC count obtained by CyTOF® (according to the same panel of markers as CellSearch) correlated with the CTC count by CellSearch® and ctDNA fraction. The study revealed dynamic changes in homologous recombination deficiency (HRD) in CTCs during genotoxic treatment. Detection of HRD markers in CTCs correlated with longer patient survival, emphasizing the importance of CTCs' functional profiling rather than just enumeration. Discordance in HRD status in ctDNA and CTCs further supports the notion of different clonal origin of the tested tumor material and strengthens the idea of CTCs and ctDNA providing non-orthogonal information regarding patient's prognosis4, 5 This reinforces the need for a multi-analyte liquid biopsy approach, which is especially important in genomically and phenotypically diverse tumors, such as breast cancer. Activating epithelial-mesenchymal transition (EMT) is an example of a process that drives such diversity. Due to the heterogeneity of breast cancer and the complexity of EMT itself, our understanding of EMT-related CTC biology and its clinical implications remains limited.6 Although EMT traits in CTCs tend to increase with disease progression7—leading to escape from detection by epithelial markers—there is no consensus on the optimal isolation methods and markers to detect these cells with high sensitivity and specificity.6 Nidermayer et al. demonstrated that CyTOF® could detect a broad EMT spectrum, as CTCs frequently expressed vimentin. Given EMT's association with genomic (in)stability8, 9 evaluating DNA damage repair in epithelial and mesenchymal CTCs is an apt question to be answered. The study showed that the percentage of mesenchymal CTCs correlated positively with γH2AX- and RAD51-positive cells and negatively with 53BP1-positive cells, suggesting that EMT status of a CTC might be associated with a different DNA damage repair pathway. This study exemplifies the next generation of CTCs analysis—shifting from enumeration to molecular characterization. With CyTOF®'s high-throughput capacity and ability to multiplex dozens of markers,10 deeper profiling of protein level, including EMT status, stem cell markers, DNA damage response, and immune evasion mechanisms, is achievable. Additionally, using cryopreserved samples enables flexibility in sample collection across multicenter trials, hopefully accelerating studies addressing the clinical utility of a wide spectrum of CTCs phenotypes and their molecular characteristics. The author's work is supported by the National Science Centre, Poland (grant no.2020/39/I/NZ5/03434). The author declares no conflicts of interest.