Single-Cell Phenotypic and Molecular Characterization of Circulating Tumor Cells Isolated from Cryopreserved Peripheral Blood Mononuclear Cells of Patients with Lung Cancer and Sarcoma.

BACKGROUND: The isolation of circulating tumor cells (CTCs) requires rapid processing of the collected blood due to their inherent fragility. The ability to recover CTCs from peripheral blood mononuclear cells (PBMCs) preserved from cancer patients could allow for retrospective analyses or multicenter CTC studies. METHODS: We compared the efficacy of CTC recovery and characterization using cryopreserved PMBCs vs fresh whole blood from patients with non-small cell lung cancer (NSCLC; n = 8) and sarcoma (n = 6). Two epithelial cellular adhesion molecule (EpCAM)-independent strategies for CTC enrichment, based on Parsortix® technology or immunomagnetic depletion of blood cells (AutoMACS®) were tested, followed by DEPArray™ single-cell isolation. Phenotype and genotype, assessed by copy number alterations analysis, were evaluated at a single-cell level. Detection of target mutations in CTC-enriched samples from frozen NSCLC PBMCs was also evaluated by digital PCR (dPCR). RESULTS: The use of cryopreserved PBMCs from cancer patients allowed for the retrospective enumeration of CTCs and their molecular characterization, using both EpCAM-independent strategies that performed equally in capturing CTC. Cells isolated from frozen PBMCs were representative of whole blood-derived CTCs in terms of number, phenotype, and copy number aberration profile/target mutations. Long-term storage (≥3 years) did not affect the efficacy of CTC recovery. Detection of target mutations was also feasible by dPCR in CTC-enriched samples derived from stored PBMCs. CONCLUSIONS: Isolating CTCs from longitudinally collected PBMCs using an unbiased selection strategy can offer a wider range of retrospective genomic/phenotypic analyses to guide patients' personalized therapy, paving the way for sample sharing in multicenter studies.

The curious phenomenon of dual-positive circulating cells: Longtime overlooked tumor cells.

The presence in the blood of patients with solid tumors of circulating cells expressing both epithelial and leukocyte markers (dual-positive cells, DPcells), has often been reported, though it has never been investigated in detail. A recent study suggested that DPcells are hybrid cells derived from the fusion of tumor cells with macrophages. Such fusion hybrids acquire macrophage-associated features endowing them with accelerated growth, increased motility, enhanced invasion activity and thus, a higher efficiency in metastasis formation. However, no direct evidence proving the tumor origin of circulating DPcells was provided in patients. Here we contribute a review of literature data on DPcells and on the hybrid theory with the aim of putting the current evidence both in a biological and clinical perspective and to generate new hypotheses on the mechanisms underlying tumor progression. To add further biological and clinical context to our literature review, we also report some preliminary data from our laboratory on the identification of DPcells in several solid tumors and confirmation of their malignant genotype, thus classifying them as DP-CTCs.

A novel circulating tumor cell subpopulation for treatment monitoring and molecular characterization in biliary tract cancer.

In biliary tract cancer (BTC), tissue biopsies to guide treatment are rarely feasible, thus implementing liquid biopsy approaches to improve patient management represents a priority. So far, studies on circulating tumor cells (CTCs) in BTC are insufficient to promote their use in patient clinical management and are limited to EpCAM-enriched CTCs evaluated with the CellSearch. We applied a single-cell protocol allowing identification not only of epithelial CTCs (eCTCs), but also of nonconventional CTCs (ncCTCs) lacking epithelial and leukocyte markers, but presenting aberrant genomes as confirmed by copy number alterations and therefore representing a distinct subpopulation of bona fide CTCs. In 41 blood samples longitudinally collected from 21 patients with advanced-stage BTC, addition of ncCTC to classic eCTC led to a CTC-positivity increase from 19% to 83%. Patients presenting with at least 1 eCTC/10 ml of blood at baseline prior to treatment start had a significantly shorter median disease-specific survival (DSS) compared to those lacking eCTCs (9 months vs. 19 months, p = 0.03 by log-rank test). No differences in DSS were observed according to ncCTC-positivity, conversely, variations in ncCTC counts during, and at the end of treatment, were associated with the RECIST response supporting their role in treatment monitoring. Moreover, in 88 ncCTCs collected at different times during treatment, unsupervised clustering evidenced segregation of cells by patient's best response, allowing identification of genomic regions possibly involved in resistance mechanisms. The presence of ncCTCs beside eCTCs opens the way to exploiting liquid biopsy for optimizing clinical management in BTC.

Red clover and lifestyle changes to contrast menopausal symptoms in premenopausal patients with hormone-sensitive breast cancer receiving tamoxifen.

PURPOSE: To determine whether a red clover preparation plus dietary intervention administered to premenopausal women with breast cancer (BC), improves menopausal symptoms due to anti-oestrogen treatment, and hence promotes compliance with tamoxifen, prevents weight gain and is safe. METHODS: Surgically-treated premenopausal women with oestrogen receptor (ER) positive disease taking tamoxifen were recruited to a prospective double-blind randomized trial (NCT03844685). The red clover group (N = 42) received one oral tablet/day (Promensil® Forte) containing 80 mg red clover extract for 24 months. The placebo group (N = 39) received one oral tablet/day without active ingredient. All women were encouraged to follow a Mediterranean-type diet and keep active. Outcomes were Menopausal Rating Score (MRS), body mass index (BMI), waist and hip girth, insulin resistance, and levels of cholesterol, triglycerides, and sex hormones. As safety indicators, endometrial thickness, breast density, and effects of patient serum on ER-positive BC cell lines were investigated. RESULTS: MRS reduced significantly (p < 0.0001) with no between-group difference (p = 0.69). The red clover group had significantly greater reductions in BMI and waist circumference (p < 0.0001 both cases). HDL cholesterol increased significantly in both groups (p = 0.01). Hormone levels and insulin resistance changed little. Endometrial thickness remained constant (p = 0.93). Breast density decreased significantly in both groups (p < 0.0001). Proliferation and oestrogen-regulated gene expression didn't differ in cell lines treated with serum from each group. CONCLUSIONS: This is the first trial to assess red clover in BC patients on tamoxifen. The preparation proved safe clinically and in vitro, and was associated with reduced BMI and waist circumference, but the diet-lifestyle intervention probably improved the menopausal symptoms.

Circulating Tumor Cells (CTCs) Heterogeneity in Metastatic Breast Cancer: Different Approaches for Different Needs.

In metastatic breast cancer the role of circulating tumor cells (CTCs) enumeration for predicting clinical outcome is supported by many studies, most of them dealing with strictly epithelial cells. However, it is becoming clear that CTCs are a heterogeneous cell population characterized by plasticity and including also cells which have lost the epithelial phenotype. Here we review literature data on CTC heterogeneity both at phenotype and at molecular level and discuss the possible contribute of single cell analyses in precision medicine. We conclude with some remarks about the steps still necessary to achieve clinical validity and utility when considering also CTC phenotypic and molecular heterogeneity beyond a simple enumeration.

Analysis of Single Circulating Tumor Cells in Renal Cell Carcinoma Reveals Phenotypic Heterogeneity and Genomic Alterations Related to Progression.

Circulating tumor cells (CTCs) are promising biomarkers for prognosis, therapeutic response prediction, and treatment monitoring in cancer patients. Despite its epithelial origin, renal cell carcinoma (RCC) shows low expression of epithelial markers hindering CTC-enrichment approaches exploiting epithelial cell surface proteins. In 21 blood samples serially collected from 10 patients with metastatic RCC entering the TARIBO trial, we overcame this limitation using the marker-independent Parsortix™ approach for CTC-enrichment coupled with positive and negative selection with the DEPArray™ with single cell recovery and analysis for copy number alterations (CNA) by next generation sequencing NGS. Two CTC subpopulations were identified: epithelial CTC (eCTC) and non-conventional CTC (ncCTC) lacking epithelial and leukocyte markers. With a threshold ≥1CTC/10 mL of blood, the positivity rates were 28% for eCTC, 62% for ncCTCs, and 71% considering both CTC types. In two patients with detectable eCTCs at baseline, progression free survival was less than 5 months. In an index case, hierarchical structure by translational oncology (TRONCO) identified three clones among 14 CTCs collected at progression and at baseline, each containing cells with a 9p21.3loss, a well-known metastasis driving subclonal alteration. CTCs detection in RCC can be increased by marker-independent approaches, and CTC molecular characterization can allow detection of subclonal events possibly related to tumor progression.

How to study and overcome tumor heterogeneity with circulating biomarkers: The breast cancer case.

Breast cancer ranks first among female cancer-related deaths in Western countries. As the primary tumor can often be controlled by surgical resection, the survival of women with breast cancer is closely linked to the incidence of distant metastases. Molecular screening by next generation sequencing highlighted the spatial and temporal heterogeneity of solid tumors as well as the clonal evolution of cancer cells during progression and under treatment pressure. Such findings question whether an optimal assessment of disease progression and a screening for druggable mutations should be based on molecular features of primary or recurrent/metastatic lesions and therefore represent a crucial element for failure or success of personalized medicine. In fact, new targeted therapies may induce only short-term benefit annulled by the emergence of resistant clones with new driver mutations which would need to be rapidly and reliably identified. Serial tissue sampling is therefore essential but, unfortunately, also represents a problem since biopsies from solid lesions, which are invasive and potentially painful and risky, cannot be easily repeatedly sampled, are inaccessible or may not fully reflect tumor heterogeneity. The need to early detect and strike this "moving target" is now directing the scientific community toward liquid biopsy-based biomarkers, which include circulating tumor cells (CTC) and cell-free circulating tumor DNA (ctDNA), can be repeatedly assessed through non-invasive and easy-to-perform procedures and may act as reliable read-outs of functional and molecular features of recurrent/metastatic lesions. In this review we summarize the outcome of CTCs and ctDNA in breast cancer, with special reference on their role on unveiling and overcoming tumor heterogeneity, on their potential relevance for tumor surveillance and monitoring, and for the selection of therapeutic options. Finally, we propose integration between blood-based molecular and clinical approaches for monitoring disease progression according to the specific pattern of recurrence of the most aggressive breast cancer molecular subtypes.

Extracellular matrix proteins as diagnostic markers of breast carcinoma.

Changes in amount and composition of extracellular matrix (ECM) are considered a hallmark of tumor development. We tested the hypothesis that abnormal production of ECM components leads to blood-released ECM molecules representing tumor circulating biomarkers. Candidate genes were selected through class comparison in two publicly available datasets and confirmed in paired normal and tumor associated fibroblasts from breast carcinoma (BC) specimens. Production and release of ECM molecules were evaluated in normal human dermal fibroblasts (NHDFs) treated with conditioned media from three BC cell lines. Plasma samples from healthy donors and from patients with malignant or benign breast disease were tested by ELISA for the presence of collagen 11a1 (COL11A1), collagen oligomeric matrix protein (COMP), and collagen 10a1 (COL10A1). Selected ECM molecules were investigated by IHC in malignant and benign specimens. In silico analysis of gene expression profiles identified 11 ECM genes significantly up-regulated in tumor versus normal tissue. Western blot analyses revealed increased levels of molecules encoded by three of these genes, COL11A1, COMP, and COL10A1, in cell lysates and supernatants of conditioned NHDFs. Class comparison and class prediction analyses of two independent series of human plasma samples identified the combination of COL11A1, COMP, and COL10A1 as potentially informative in discriminating BC patients from those with benign disease. The three molecules resulted expressed in the stroma of BC tissue samples. Our results indicate that circulating COL11A1, COMP, and COL10A1 may be useful in diagnostic assessment of suspicious breast nodules and ECM molecules could represent an avenue to biomarker identification.

Dissecting Time- from Tumor-Related Gene Expression Variability in Bilateral Breast Cancer.

Metachronous (MBC) and synchronous bilateral breast tumors (SBC) are mostly distinct primaries, whereas paired primaries and their local recurrences (LRC) share a common origin. Intra-pair gene expression variability in MBC, SBC, and LRC derives from time/tumor microenvironment-related and tumor genetic background-related factors and pairs represents an ideal model for trying to dissect tumor-related from microenvironment-related variability. Pairs of tumors derived from women with SBC (n = 18), MBC (n = 11), and LRC (n = 10) undergoing local-regional treatment were profiled for gene expression; similarity between pairs was measured using an intraclass correlation coefficient (ICC) computed for each gene and compared using analysis of variance (ANOVA). When considering biologically unselected genes, the highest correlations were found for primaries and paired LRC, and the lowest for MBC pairs. By instead limiting the analysis to the breast cancer intrinsic genes, correlations between primaries and paired LRC were enhanced, while lower similarities were observed for SBC and MBC. Focusing on stromal-related genes, the ICC values decreased for MBC and were significantly different from SBC. These findings indicate that it is possible to dissect intra-pair gene expression variability into components that are associated with genetic origin or with time and microenvironment by using specific gene subsets.

Development of a Protocol for Single-Cell Analysis of Circulating Tumor Cells in Patients with Solid Tumors.

Genomic characterization of circulating tumor cells (CTCs) enables the monitoring of tumor progression and of adaption occurring during treatment. CTC molecular characterization represents indeed a precious tool to implement in the clinical practice for better dealing with acquired resistance to systemic treatment and tumor evolution. Unfortunately CTCs are very rare and enrichments from blood samples and subsequent identification of these cells are technically very challenging. We describe here the main steps leading to the development of a technical protocol for visualization, enumeration and recovery of single CTCs exploiting the recently developed DEPArray™platform. Our description of the technical workflow starts with evaluation of pre-analytical aspects related to blood sample collection warning about the possible effects on immunoreactivity profiles which may bias the interpretation. Subsequently, other CTC-enrichment approaches are critically discussed and compared in relation to their performances with the DEPArray™. Identification of CTCs represents another critical point due to their heterogeneity and due to the still-to-be clarified role of different subpopulations, typically epithelial, mesenchymal or mixed. Finally, issues related to single cell analysis are illustrated. The chapter ends with an overview of results obtained on real clinical samples which support the reliability of the protocol and its transferability to the daily clinical routine.

Cell cycle dependent oscillatory expression of estrogen receptor-α links Pol II elongation to neoplastic transformation.

Decades of studies provided a detailed view of the mechanism of estrogen receptor-α (ERα) regulated gene transcription and the physio-pathological relevance of the genetic programs controlled by this receptor in a variety of tissues. However, still limited is our knowledge on the regulation of ERα synthesis. Preliminary observations showed that the expression of ERα is cell cycle regulated. Here, we have demonstrated that a well described polymorphic sequence in the first intron of ERα (PvuII and XbaI) has a key role in regulating the ERα content in cycling cells. We have shown that the RNA Pol II (Pol II) elongation is blocked at the polymorphic site and that the proto-oncogene c-MYB modulates the release of the pausing polymerase. It is well known that the two SNPs are associated to an increased risk, progression, survival and mortality of endocrine-related cancers, here we have demonstrated that the c-MYB-dependent release of Pol II at a specific phase of the cell cycle is facilitated by the px haplotype, thus leading to a higher ERα mitogenic signal. In breast cancer, this mechanism is disrupted when the hormone refractory phenotype is established; therefore, we propose this oscillator as a novel target for the development of therapies aimed at sensitizing breast cancer resistant to hormonal treatments. Because PvuII and XbaI were associated to a broad range physio-pathological conditions beside neoplastic transformation, we expect that the ERα oscillator contributes to the regulation of the estrogen signal in several tissues.

Tumor-extracellular matrix interactions: Identification of tools associated with breast cancer progression.

Several evidences support the concept that cancer development and progression are not entirely cancer cell-autonomous processes, but may be influenced, and possibly driven, by cross-talk between cancer cells and the surrounding microenvironment in which, besides immune cells, stromal cells and extracellular matrix (ECM) play a major role in regulating distinct biologic processes. Stroma and ECM-related signatures proved to influence breast cancer progression, and to contribute to the identification of tumor phenotypes resistant to cytotoxic and hormonal treatments. The possible clinical implications of the interplay between tumor cells and the microenvironment, with special reference to ECM remodelling, will be discussed in this review.

Circulating tumor cells as a longitudinal biomarker in patients with advanced chemorefractory, RAS-BRAF wild-type colorectal cancer receiving cetuximab or panitumumab.

A still relevant number of patients with RAS-BRAF wild-type colorectal cancer (CRC) do not respond to treatment with antiepidermal growth factor receptor (EGFR) monoclonal antibodies cetuximab and panitumumab, suggesting that additional biomarkers to guide patient selection are urgently needed. Circulating tumor cells (CTCs) may represent such a biomarker. In this prospective study, 38 patients with advanced RAS-BRAF-wild-type CRC received third-line therapy with cetuximab-irinotecan or panitumumab. Peripheral blood samples for CTC status determination were collected at baseline, during treatment at early (2-4 weeks) and at later (8-10 weeks) times. CTC enrichment was done with the AdnaTest ColonCancerSelect kit, whereas CTC detection was done with the AdnaTest ColonCancerDetect kit. CTC status positivity was defined according to the kit manufacturer's thresholds. Fifty percent of patients were defined as CTC positive at baseline and the overall RECIST response rate was 26%. CTC baseline status was not associated with treatment response, whereas early CTC status and CTC status changes during treatment were significantly associated with tumor response. Kaplan-Meier analysis showed a significantly shorter progression-free survival (median, 2.0 versus 4.0 months, p = 0.004) and overall survival (4.7 versus11.4, p = 0.039) in patients with early CTC + status compared with CTC - ones. In multivariable analysis including classical prognostic factors, the CTC status changes profile during treatment was an independent predictor of both progression-free survival (p < 0.001) and overall-survival (p = 0.001). CTC status assessed early during treatment with anti-EGFR monoclonal antibodies may predict treatment failure in advance compared to imaging-based tools.

Gene expression profiling of circulating tumor cells in breast cancer.

BACKGROUND: Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells. METHODS: Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from whole blood with magnetic beads linked with antibodies against epithelial cell adhesion molecule (EpCAM) and the carcinoma-associated mucin, MUC1, designed to be used for CTC characterization in clinical samples. Low numbers of cells (5-200) from the MCF7 and MDA-MB-468 breast cancer cell lines were spiked in healthy donor blood samples and isolated with the AdnaTest EMT-1/Stem CellSelect kit. Gene expression profiles (GEPs) were obtained with the WG-DASL HT assay and compared with GEPs obtained from RNA isolated from cultured cell lines and unspiked samples. RESULTS: GEPs from samples containing 25 or more spiked cells correlated (r = 0.95) with cognate 100-ng RNA input samples, clustered separately from blood control samples, and allowed MCF7 and MDA-MB-468 cells to be distinguished. GEPs with comparable technical quality were also obtained in a preliminary series of clinical samples. CONCLUSIONS: Our approach allows technically reliable GEPs to be obtained from isolated CTCs for the acquisition of biologically useful information. It is reproducible and suitable for application in prospective studies to assess the clinical utility of CTC GEPs, provided that >25 CTCs can be isolated.

Isolation and Genomic Analysis of Circulating Tumor Cell Clusters in Cancer Patients.

The role of circulating tumor cell (CTC) clusters in the metastatic dissemination process is gaining increased attention. Besides homotypic clusters, heterotypic clusters that contain tumor cells admixed with normal cells are frequently observed in patients with solid tumors. Current methods used for cluster detection and enumeration do not allow an accurate estimation of the relative fractions of tumor cells. Here we describe a method for estimating tumor fraction of clusters including isolation and collection of single clusters, assessment of copy number alterations of single clusters by low-pass whole genome sequencing, and bioinformatic analysis of sequencing data.

A gene expression-based classifier for HER2-low breast cancer.

In clinical trials evaluating antibody-conjugated drugs (ADCs), HER2-low breast cancer is defined through protein immunohistochemistry scoring (IHC) 1+ or 2+ without gene amplification. However, in daily practice, the accuracy of IHC is compromised by inter-observer variability. Herein, we aimed to identify HER2-low breast cancer primary tumors by leveraging gene expression profiling. A discovery approach was applied to gene expression profile of institutional INT1 (n = 125) and INT2 (n = 84) datasets. We identified differentially expressed genes (DEGs) in each specific HER2 IHC category 0, 1+, 2+ and 3+. Principal Component Analysis was used to generate a HER2-low signature whose performance was evaluated in the independent INT3 (n = 95), and in the publicly available TCGA and GSE81538 datasets. The association between the HER2-low signature and HER2 IHC categories was evaluated by Kruskal-Wallis test with post hoc pair-wise comparisons. The HER2-low signature discriminatory capability was assessed by estimating the area under the receiver operating characteristic curve (AUC). Gene Ontology and KEGG analyses were performed to evaluate the HER2-low signature genes functional enrichment. A HER2-low signature was computed based on HER2 IHC category-specific DEGs. The twenty genes included in the signature were significantly enriched with lipid and steroid metabolism pathways, peptidase regulation, and humoral immune response. The HER2-low signature values showed a bell-shaped distribution across IHC categories (low values in 0 and 3+; high values in 1+ and 2+), effectively distinguishing HER2-low from 0 (p < 0.001) to 3+ (p < 0.001). Notably, the signature values were higher in tumors scored with 1+ as compared to 0. The HER2-low signature association with IHC categories and its bell-shaped distribution was confirmed in the independent INT3, TCGA and GSE81538 datasets. In the combined INT1 and INT3 datasets, the HER2-low signature achieved an AUC value of 0.74 (95% confidence interval, CI 0.67-0.81) in distinguishing HER2-low vs. the other categories, outperforming the individual ERBB2 mRNA AUC value of 0.52 (95% CI 0.43-0.60). These results represent a proof-of-concept for an observer-independent gene-expression-based classifier of HER2-low status. The herein identified 20-gene signature shows promise in distinguishing between HER2 0 and HER2-low expressing tumors, including those scored as 1+ at IHC, and in developing a selection approach for ADCs candidates.

A phase II study evaluating the efficacy of enzalutamide and the role of liquid biopsy for evaluation of ARv7 in mCRPC patients with measurable metastases including visceral disease (Excalibur study).

Background: Up to 30% of patients with metastatic castration-resistant prostate cancer (mCRPC) develop visceral metastases, which are associated with a poor prognosis. Objectives: Efficacy of enzalutamide in mCRPC patients with measurable metastases, including visceral and/or extra-regional lymph nodes. Methods: In this phase II multicenter study, patients with mCRPC and measurable metastases received enzalutamide as the first line. Primary endpoint: 3-month (mo) disease control rate (DCR) defined as the proportion of patients with complete (CR) or partial response (PR) or stable disease (SD) as per Response Evaluation Criteria in Solid Tumors 1.1. Secondary endpoint: safety. Exploratory endpoint: the association between ARv7 splicing variants in basal circulating tumor cell (CTC)-enriched blood samples and treatment response/resistance using the AdnaTest ProstateCancerSelect kit and the AdnaTest ProstateCancer Panel AR-V7. Results: From March 2017 to January 2021, 68 patients were enrolled. One patient never started treatment. Median age: 72 years. A total of 52 patients (78%) received enzalutamide as a first line for mCRPC. The median follow-up was 32 months. At the 3-month assessment, 24 patients presented an SD, 1 patient achieved a CR, and 23 patients had a PR (3-mo-DCR of 72%). Discontinuations due to adverse events (AEs), disease-related death, or disease progression occurred in 9%, 6%, and 48% of patients. All patients reported at least one grade (G) 1-2 AE: the most common were fatigue (49%) and hypertension (33%). Six G3 AEs were reported: two hypertension, one seizure, one fatigue, one diarrhea, and one headache. Basal detection of ARv7 was significantly associated with poor treatment response (p = 0.034) and a nonsignificant association (p = 0.15) was observed between ARv7 detection and response assessments. At month 3, ARv7 was detected in 57%, 25%, and 15% of patients undergoing progressive disease, SD, and PR, respectively. Conclusion: The study met its primary endpoint, showing the efficacy of enzalutamide in men with mCRPC and measurable metastatic lesions in visceral and/or lymph node sites. Trial registration: ClinicalTrials.gov Identifier: NCT03103724. First Posted: 6 April 2017. First patient enrollment: 19 April 2017.

Concomitant medications and circulating tumor cells: friends or foes?

The use of concomitant medications by patients with cancer is observed almost globally; however, little attention has been paid to this topic in the medical literature. Most clinical studies do not describe the type and duration of drugs used at the time of inclusion and during treatment or how these drugs may affect the experimental and/or standard therapy. Even less information has been published on the potential interaction between concomitant medications and tumor biomarkers. However, we do know that concomitant drugs can complicate cancer clinical trials and biomarker development, thus contributing to their interaction, leading to side effects, and resulting in suboptimal adherence to anticancer treatment. On the basis of these premises and moving from the study by Jurisova et al., which reported the effect of commonly used drugs on the prognosis of women with breast cancer and the detection of circulating tumor cells (CTCs), we comment on the role of CTCs as an emerging diagnostic and prognostic tool for breast cancer. We also report the known and hypothesized mechanisms of CTC interplay with other tumor and blood components, possibly modulated by widespread drugs, including over-the-counter compounds, and discuss the possible implications of commonly used concomitant medications on CTC detection and clearance. After considering all these points, it is conceivable that concomitant drugs are not necessarily a problem, but on the contrary, their virtuous mechanisms can be exploited to reduce tumor spread and enhance the effect of anticancer therapies.

Can we define breast cancer HER2 status by liquid biopsy?

Human Epidermal growth factor Receptor 2 (HER2) assessment is crucial for breast cancer treatment. Therapeutic decisions for recurrent cases often rely on primary tumor status. However, mounting evidence suggests that tumors show dynamic changes and up to 10% of breast cancer modify their initial status during progression. It is still debated whether these changes reflect a biological evolution of the disease or are secondary to primary tumor heterogeneity. Certainly, repeating HER2 assessment during breast cancer trajectory is important for the increasing availability of effective anti-HER2 drugs. In response to this need, circulating biomarkers such as circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) offer the potential to safely and repeatedly assess HER2 status over time. This chapter outlines current methods for testing HER2 in CTCs and ctDNA, and reviews clinical trials evaluating its prognostic and predictive value in patients with breast cancer, as well as recent advances in the field.