The potential of ctDNA analysis in breast cancer

Breast cancer is a highly heterogeneous and dynamic disease, exhibiting unique somatic alterations that lead to disease recurrence and resistance. Tumor biopsy and conventional imaging approaches are not able to provide sufficient information regarding the early detection of recurrence and real time monitoring through tracking sensitive or resistance mechanisms to treatment. Circulating tumor DNA (ctDNA) analysis has emerged as an attractive noninvasive methodology to detect cancer-specific genetic aberrations in plasma including DNA mutations and DNA methylation patterns. Numerous studies have reported on the potential of ctDNA analysis in the management of early and advanced stages of breast cancer. Advances in high-throughput technologies, especially next generation sequencing and PCR-based assays, were highly important for the successful application of ctDNA analysis. However, before being integrated into clinical practice, ctDNA analysis needs to be standardized and validated through the performance of multicenter prospective and well-designed clinical studies. This review is focused on the clinical utility of ctDNA analysis, especially at the DNA mutation and methylation level, in breast cancer patients, incorporating the latest advances in technological approaches and involving key studies in the early and metastatic setting. © 2019, © 2019 Informa UK Limited, trading as Taylor & Francis Group

The potential of liquid biopsy in the management of cancer patients

Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine, since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of Circulating Tumor Cells (CTCs), and circulating tumor-derived material like circulating tumor DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTCs and ctDNA analysis has already an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients, while recent data show also its potential for early cancer diagnosis (Figure 1). Numerous clinical trials include now a liquid biopsy arm, and functional studies mainly based on CTC derived cell-lines and CTC derived explants (CDx) provide important insight on the metastatic process. The recent findings in the field of liquid biopsy and the benefits and main clinical applications of CTC and ctDNA analysis in solid tumors are summarized in this review. © 202

Nuclease-Assisted Minor Allele Enrichment Using Overlapping Probes-Assisted Amplification-Refractory Mutation System: An Approach for the Improvement of Amplification-Refractory Mutation System-Polymerase Chain Reaction Specificity in Liquid Biopsies

Allele-specific polymerase chain reaction (PCR) (amplification-refractory mutation system, ARMS) is one of the most commonly used methods for mutation detection. However, a main limitation of ARMS-PCR is the false positive results obtained due to nonspecific priming that can take place with wild-type (WT) DNA, which often precludes detection of low-level mutations. To improve the analytical specificity of ARMS, we present here a new technology, NAPA: NaME-PrO-assisted ARMS, that overcomes the ARMS deficiency by adding a brief enzymatic step that reduces wild-type alleles just prior to ARMS. We performed this technology for the simultaneous detection of two hot-spot PIK3CA mutations (E545 K and H1047R) in circulating tumor cells (CTCs) and cell free DNA (cfDNA). The developed protocol could simultaneously detect mutation-allelic-frequency of 0.5% for PIK3CA exon 9 (E545 K) and 0.1% for PIK3CA exon 20 (H1047R) with high specificity. We further compared the developed NAPA assay with (a) ddPCR considered as the gold standard and (b) our previous assay based on the combination of allele-specific, asymmetric rapid PCR, and melting analysis. Our data show that the newly developed NAPA assay gives consistent results with both these assays (p = 0.001). The developed assay resolves the false positive signals issue derived through classic ARMS-PCR and provides an ideal combination of speed, accuracy, and versatility and should be easily applicable in routine diagnostic laboratories. Copyright © 2019 American Chemical Society

Evaluation of preanalytical conditions and implementation of quality control steps for reliable gene expression and DNA methylation analyses in liquid biopsies

BACKGROUND: Liquid biopsy provides important information for the prognosis and treatment of cancer patients. In this study, we evaluated the effects of preanalytical conditions on gene expression and DNA methylation analyses in liquid biopsies. METHODS: We tested the stability of circulating tumor cell (CTC) messenger RNA by spiking MCF-7 cells in healthy donor peripheral blood (PB) drawn into 6 collection-tube types with various storage conditions. CTCs were enriched based on epithelial cell adhesion molecule positivity, and RNA was isolated followed by cDNA synthesis. Gene expression was quantified using RT-quantitative PCR for CK19 and B2M. We evaluated the stability of DNA methylation in plasma under different storage conditions by spiking DNA isolated from MCF-7 cells in healthy donor plasma. Two commercially available sodium bisulfite (SB)-conversion kits were compared, in combination with whole genome amplification (WGA), to evaluate the stability of SB-converted DNA. SB-converted DNA samples were analyzed by real-time methylation-specific PCR (MSP) for ACTB, SOX17, and BRMS1. Quality control was assessed using Levey-Jennings graphs. RESULTS: RNA-based analysis in CTCs is severely impeded by the preservatives used in many PB collection tubes (except for EDTA), as well as by time to analysis. Plasma and SB-converted DNA samples are stable and can be used safely for MSP when kept at 80 °C. Downstream WGA of SB-converted DNA compensated for the limited amount of available sample in liquid biopsies. CONCLUSIONS: Standardization of preanalytical conditions and implementation of quality control steps is extremely important for reliable liquid biopsy analysis, and a prerequisite for routine applications in the clinic. © 2018 American Association for Clinical Chemistr

PIM-1 is overexpressed at a high frequency in circulating tumor cells from metastatic castration- resistant prostate cancer patients

PIM-1 is an oncogene involved in cell cycle progression, cell growth, cell survival and therapy resistance, activated in many types of cancer, and is now considered as a very promising target for cancer therapy. We report for the first time that PIM-1 is overexpressed in circulating tumor cells (CTCs) from metastatic castration-resistant prostate cancer patients (mCRPC). We first developed and validated a highly sensitive RT-qPCR assay for quantification of PIM-1 transcripts. We further applied this assay to study PIM-1 expression in EpCAM(+) CTC fraction isolated from 64 peripheral blood samples of 50 mCRPC patients. CTC enumeration in all samples was performed using the FDA-cleared CellSearch® system. PIM-1 overexpression was detected in 24/64 (37.5%) cases, while in 20/24 (83.3%) cases that were positive for PIM-1 expression, at least one CTC/7.5 mL PB was detected in the CellSearch®. Our data indicate that PIM-1 overexpression is observed at high frequency in CTCs from mCRPC patients and this finding, in combination with androgen receptor splice variant 7 (AR-V7) expression in CTCs, suggest its potential role as a very promising target for cancer therapy. We strongly believe that PIM-1 overexpression in EpCAM(+) CTC fraction merits to be further evaluated and validated as a non-invasive circulating tumor biomarker in a large and well-defined patient cohort with mCRPC. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

PIK3CA hotspot mutations in circulating tumor cells and paired circulating tumor DNA in breast cancer: a direct comparison study

Liquid biopsy analysis, mainly based on circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides an extremely powerful tool for the molecular profiling of cancer patients in real time. In this study, we directly compared PIK3CA hotspot mutations (E545K, H1047R) in EpCAM-positive CTCs and paired plasma-ctDNA in breast cancer (BrCa). PIK3CA hotspot mutations in CTCs and ctDNA were analyzed using our previously developed highly sensitive (0.05%), specific, and validated assay in plasma-ctDNA from 77 early and 73 metastatic BrCa patients and 40 healthy donors. We further analyzed and directly compared PIK3CA hotspot mutations in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma-ctDNA, in 56 cases of early and 27 cases of metastatic breast cancer, and 16 corresponding primary tumors. In plasma-ctDNA, PIK3CA hotspot mutations were identified in 30/77(39.0%) early and 35/73(47.9%) metastatic BrCa cases; none (0/40, 0%) of the healthy donors’ plasma-ctDNA samples were positive. Our direct comparison study in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma-ctDNA from the same blood draws has shown a lack of concordance in early BrCa (27/56, 48.2%), while the concordance in the metastatic setting was higher (18/27, 66.6%). Our results were validated by ddPCR methodology, and the concordance between our assay and ddPCR for PIK3CA E545K hotspot mutation was 30/37 (81.1%). In many cases, PIK3CA hotspot mutations were detected in samples found to be negative for CTCs in CellSearch®. Our data demonstrated for the first time that (a) PIK3CA hotspot mutations are present at high frequencies in CTCs isolated from CellSearch® cartridges and paired plasma-ctDNA both in early and metastatic BrCa, (b) the detection and concordance of PIK3CA hotspot mutations between plasma-ctDNA and CTCs are higher in the metastatic setting, (c) PIK3CA mutational status significantly changes after therapeutic intervention, and (d) PIK3CA mutation detection in CTCs and plasma-ctDNA provides complementary information. © 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd

Esr1 napa assay: Development and analytical validation of a highly sensitive and specific blood‐based assay for the detection of ESR1 mutations in liquid biopsies

A considerable number of estrogen receptor‐positive breast cancer (ER+ BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of ESR1 mutations. We developed and analytically validated a highly sensitive and specific NaME‐PrO‐assisted ARMS (NAPA) assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G) in circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA) in patients with ER+ BrCa. The analytical specificity, analytical sensitivity and reproducibility of the assay were validated using synthetic oligos standards. We further applied the developed ESR1 NAPA assay in 13 ER+ BrCa primary tumour tissues, 13 non‐cancerous breast tissues (mammoplasties) and 64 liquid biopsy samples: 32 EpCAM‐positive cell fractions and 32 paired plasma ctDNA samples obtained at different time points from 8 ER+ metastatic breast cancer patients, during a 5‐year follow‐up period. Peripheral blood from 11 healthy donors (HD) was used as a control. The developed assay is highly sensitive (a detection of mutation‐allelic‐frequency (MAF) of 0.5% for D538G and 0.1% for Y537S, Y537C, Y537N), and highly specific (0/13 mammoplasties and 0/11 HD for all mutations). In the plasma ctDNA, ESR1 mutations were not identified at the baseline, whereas the D538G mutation was detected in five sequential ctDNA samples during the follow‐up period in the same patient. In the EpCAM‐isolated cell fractions, only the Y537C mutation was detected in one patient sample at the baseline. A direct comparison of the ESR1 NAPA assay with the drop‐off ddPCR using 32 identical plasma ctDNA samples gave a concordance of 90.6%. We present a low cost, highly specific, sensitive and robust assay for blood-based ESR1 profiling. The clinical performance of the ESR1 NAPA assay will be prospectively evaluated in a large number of well‐characterized patient cohorts. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Evaluation of Monocarboxylate Transporter 4 (MCT4) Expression and Its Prognostic Significance in Circulating Tumor Cells From Patients With Early Stage Non-Small-Cell Lung Cancer

Purpose: Monocarboxylate transporter 4 (MCT4) can influence the amount of lactate in the tumor microenvironment and further control cancer cell proliferation, migration, and angiogenesis. We investigated for the first time the expression of MCT4 in circulating tumor cells (CTCs) derived from early stage Non-Small Cell Lung Cancer patients (NSCLC) and whether this is associated with clinical outcome. Experimental Design: A highly sensitive RT-qPCR assay for quantification of MCT4 transcripts was developed and validated and applied to study MCT4 expression in CTC isolated through the Parsortix size-dependent microfluidic device from 53 and 9 peripheral blood (PB) samples of NSCLC patients at baseline (pre-surgery) and at relapse, respectively, as well as the “background noise” was evaluated using peripheral blood samples from 10 healthy donors (HD) in exactly the same way as patients. Results: MCT4 was differentially expressed between HD and NSCLC patients. Overexpression of MCT4 was detected in 14/53 (26.4%) and 3/9 (33.3%) patients at baseline and at progression disease (PD), respectively. The expression levels of MCT4 was found to increase in CTCs at the time of relapse. Kaplan-Meier analysis showed that the overexpression of MCT4 was significantly (P = 0.045) associated with progression-free survival (median: 12.5 months, range 5-31 months). Conclusion: MCT4 overexpression was observed at a high frequency in CTCs from early NSCLC patients supporting its role in metastatic process. MCT4 investigated as clinically relevant tumor biomarker characterizing tumor aggressiveness and its potential value as target for cancer therapy. We are totally convinced that MCT4 overexpression in CTCs merits further evaluation as a non-invasive circulating tumor biomarker in a large and well-defined cohort of patients with NSCLC

ESR1 methylation: A Liquid biopsy-based epigenetic assay for the follow up of patients with metastatic breast cancer receiving endocrine treatment: Short running title: Liquid biopsy: ESR1 methylation in CTCs and paired ctDNA

Purpose: Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of CTCs and plasma-ctDNA. ESR1 epigenetic silencing potentially affects response to endocrine treatment. We evaluated ESR1 methylation in CTCs and paired plasma-ctDNA. We evaluated ESR1 methylation in CTCs and paired plasma-ctDNA as a potential biomarker for response to everolimus/exemestane treatment. Experimental Design: A highly sensitive and specific real-time MSP assay for ESR1 methylation was developed and validated in: a) 65 primary breast tumors (FFPEs), b) EpCAM+ CTC-fractions (122 patients and 30 healthy donors; HD), c) plasma-ctDNA (108 patients and 30HD), d) in CTCs (CellSearch®) and in paired plasma-ctDNA for 58 BrCa patients. ESR1 methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER+/HER2-advanced BrCa receiving everolimus/exemestane. Results: ESR1 methylation was detected in: a) 25/65(38.5%) FFPEs, b) EpCAM+ CTC-fractions: 26/112(23.3%) patients and 1/30(3.3%) HD, c) plasma-ctDNA: 8/108(7.4%) patients and 1/30(3.3%) HD. ESR1 methylation was highly concordant in 58 paired DNA samples, isolated from CTCs (CellSearch®) and corresponding plasma. In serial peripheral blood samples of patients treated with everolimus/exemestane, ESR1 methylation was observed in 10/36(27.8%) CTC-positive samples, and was associated with lack of response to treatment (p=0.023 Fisher's Exact Test). Conclusions: We report for the first time the detection of ESR1 methylation in CTCs and a high concordance with paired plasma-ctDNA. ESR1 methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. ESR1 methylation should be further evaluated as a potential liquid biopsy-based biomarker. © 2017 American Association for Cancer Research

Comprehensive liquid biopsy analysis as a tool for the early detection of minimal residual disease in breast cancer

Liquid biopsy (LB) provides a unique minimally invasive tool to follow-up cancer patients over time, to detect minimal residual disease (MRD), to study metastasis-biology and mechanisms of therapy-resistance. Molecular characterization of CTCs offers additionally the potential to understand resistance to therapy and implement individualized targeted treatments which can be modified during the disease evolution and follow-up period of a patient. In this study, we present a long-term follow-up of operable breast cancer patients based on a comprehensive liquid biopsy analysis. We performed a comprehensive liquid biopsy analysis in peripheral blood of 13 patients with early-stage operable breast cancer at several time points for a period of ten years, consisting of: (a) CTC enumeration using the CellSearch system, (b) phenotypic analysis of CTCs using Immunofluorescence, (c) gene expression analysis, in EpCAM(+) CTCs for CK-19, CD24,CD44, ALDH1, and TWIST1, (d) analysis of PIK3CA and ESR1 mutations in EpCAM(+) CTCs and corresponding plasma ctDNA and (e) DNA methylation of ESR1 in CTCs. 10/13 (77%) patients were found negative for LB markers in PB during the whole follow-up period, and these patients did not relapse during the follow-up. However, 3/13(18%) patients that were positive for at least one LB marker relapsed within the follow-up period. The molecular characteristics of CTCs were highly different even for the same patient at different time points, and always increased before the clinical relapse. Our results indicate that liquid biopsy can reveal the presence of MRD at least 4 years before the appearance of clinically detectable metastatic disease demonstrating that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients. © 2023, The Author(s)