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.

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.

Direct and indirect effects of COVID-19 on short-term mortality of breast cancer patients.

We studied the COVID-19 impact in newly-diagnosed breast cancer (7,349 patients in 2019, and 5,563 in 2020). In 2020 there were two diagnostic drops: -37.2% (March-May), -15.8% (October-December). Early-stage at presentation (76.4% vs. 74.4%, p = 0.0013), conserving surgery (71.0% vs. 67.0%, p < 0.0001), chemotherapy (86.2% vs. 53.4%, p < 0.0001), and radiotherapy (65.7% vs. 42.1%, p < 0.0001) decreased in 2020 compared to 2019. COVID-19 occurred in 250 patients (4.49%). The time-dependent COVID-19 effect was associated with mortality (multivariable Cox analysis HR [95% CI] 2.26 [1.35-3.74]; p = 0.0018). Survival within the year of diagnosis was 97.6% in 2020 and 98.3% in 2019; 30-day mortality was 1.13% in 2020 (1.07 in uninfected patients), and 0.61% in 2019. The year of diagnosis lost its prognostic relevance after adjusting for stage and treatment. These findings emphasize the critical role of continuity of care, which was disrupted during the pandemic, and underscore the need for policies minimizing treatment initiation delay in newly diagnosed breast cancer patients.

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.

Comprehensive transcriptomic analysis to identify biological and clinical differences in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CC) is a rare and aggressive disease with limited therapeutic options and a poor prognosis. All available public records of cohorts reporting transcriptomic data on intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC) were collected with the aim to provide a comprehensive gene expression-based classification with clinical relevance. METHODS: A total of 543 patients with primary tumor tissues profiled by RNAseq and microarray platforms from seven public datasets were used as a discovery set to identify distinct biological subgroups. Group predictors developed on the discovery sets were applied to a single cohort of 131 patients profiled with RNAseq for validation and assessment of clinical relevance leveraging machine learning techniques. RESULTS: By unsupervised clustering analysis of gene expression data we identified both in the ICC and ECC discovery datasets four subgroups characterized by a distinct type of immune infiltrate and signaling pathways. We next developed class predictors using short gene list signatures and identified in an independent dataset subgroups of ICC tumors at different prognosis. CONCLUSIONS: The developed class-predictor allows identification of CC subgroups with specific biological features and clinical behavior at single-sample level. Such results represent the starting point for a complete molecular characterization of CC, including integration of genomics data to develop in clinical practice.

Moving HER2-low breast cancer predictive and prognostic data from clinical trials into the real world.

Background: Previous data, mostly from clinical trials, reported that HER2-low status is associated with low pathological complete response (pCR), and favourable prognosis. Since these findings suggest the existence of an additional breast cancer subtype, we questioned if the predictive/prognostic value of HER2-low was also relevant in the real world. Methods: Data from non-metastatic breast cancer patients treated with neoadjuvant chemotherapy and surgery (2009-2020) were retrieved from our institutional prospectively-maintained registry. Univariable and multivariable logistic models were implemented to study the association between pCR and baseline HER2 status. Univariable analysis of disease-free survival (DFS) was performed through Kaplan-Meier survival curves and log-rank tests. Results: Starting from a total of 790 consecutive cases, we identified 444 newly-diagnosed breast cancer patients featuring HER2 immunohistochemistry (IHC) 0 (HER2-0, n = 109), and 1 + or IHC 2+/in situ hybridization negative (HER2-low, n = 335) receiving anthracycline and taxane-based regimens in 88.9% of cases. Most of the patients were diagnosed with stage II (67.3%) and there was no difference of disease presentation according to HER2-status. pCR was attained by 71 (16.0%) patients and was significantly associated with increased DFS (p = 0.031). Compared to HER2-0, HER2-low cases were more likely hormone receptor-positive (81.2% vs. 43.1%, p < 0.001), well-differentiated (47.5% vs. 26.6%, p = 0.001), less proliferative (21.5% vs. 8.3%, p = 0.001) and less responsive to treatment (pCR 11.6% vs. 29.4%, p < 0.0001). There was no difference in DFS according to HER2 status, though hormone-receptor (HR) negative/HER2-low cases tended to have a worse prognosis compared to HR-negative/HER2-0. By pCR achievement, 3-years DFS was 87.5.% (75.1-100%) vs. 71.6% (65.9-77.8%) (p = 0.161) in HER2-low and 89.1% (75.8-100%) vs. 72.1% (59.7-87.0%) (p = 0.092) in HER2-0. Conclusion: Our real-world data show that HER2-low breast cancer patients represent roughly a half of the cases treated with neoadjuvant therapy, and have poor treatment response. In absence of pCR, HER2-low breast cancer patients have a dismal prognosis, especially when primary tumor hormone receptor status is negative. Studies are therefore needed to define the biology of these tumors for new therapeutic targets and to incorporate HER2-targeting agents in early-stage treatment.

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.

Gene signatures of circulating breast cancer cell models are a source of novel molecular determinants of metastasis and improve circulating tumor cell detection in patients.

BACKGROUND: Progression to stage IV disease remains the main cause of breast cancer-related deaths. Increasing knowledge on the hematogenous phase of metastasis is key for exploiting the entire window of opportunity to interfere with early dissemination and to achieve a more effective disease control. Recent evidence suggests that circulating tumor cells (CTCs) possess diverse adaptive mechanisms to survive in blood and eventually metastasize, encouraging research into CTC-directed therapies. METHODS: On the hypothesis that the distinguishing molecular features of CTCs reveal useful information on metastasis biology and disease outcome, we compared the transcriptome of CTCs, primary tumors, lymph-node and lung metastases of the MDA-MB-231 xenograft model, and assessed the biological role of a panel of selected genes, by in vitro and in vivo functional assays, and their clinical significance in M0 and M+ breast cancer patients. RESULTS: We found that hematogenous dissemination is governed by a transcriptional program and identified a CTC signature that includes 192 up-regulated genes, mainly related to cell plasticity and adaptation, and 282 down-regulated genes, involved in chromatin remodeling and transcription. Among genes up-regulated in CTCs, FADS3 was found to increases cell membrane fluidity and promote hematogenous diffusion and lung metastasis formation. TFF3 was observed to be associated with a subset of CTCs with epithelial-like features in the experimental model and in a cohort of 44 breast cancer patients, and to play a role in cell migration, invasion and blood-borne dissemination. The analysis of clinical samples with a panel of CTC-specific genes (ADPRHL1, ELF3, FCF1, TFF1 and TFF3) considerably improved CTC detection as compared with epithelial and tumor-associated markers both in M0 and stage IV patients, and CTC kinetics informed disease relapse in the neoadjuvant setting. CONCLUSIONS: Our findings provide evidence on the potential of a CTC-specific molecular profile as source of metastasis-relevant genes in breast cancer experimental models and in patients. Thanks to transcriptome analysis we generated a novel CTC signature in the MDA-MB-231 xenograft model, adding a new piece to the current knowledge on the key players that orchestrate tumor cell hematogenous dissemination and breast cancer metastasis, and expanding the list of CTC-related biomarkers for future validation studies.

Copy number alterations analysis of primary tumor tissue and circulating tumor cells from patients with early-stage triple negative breast cancer.

Triple negative breast cancer (TNBC) is characterized by clinical aggressiveness, lack of recognized target therapy, and a dismal patient prognosis. Several studies addressed genomic changes occurring during neoadjuvant chemotherapy (NAC) focusing on somatic variants, but without including copy number alterations (CNAs). We analyzed CNA profiles of 31 TNBC primary tumor samples before and after NAC and of 35 single circulating tumor cells (CTCs) collected prior, during and after treatment by using next-generation sequencing targeted profile and low-pass whole genome sequencing, respectively. In pre-treatment tissue samples, the most common gains occurred on chromosomes 1, 2 and 8, and SOX11 and MYC resulted the most altered genes. Notably, amplification of MSH2 (4/4 versus 0/12, p < 0.01) and PRDM1 and deletion of PAX3 (4/4 versus 1/12, p < 0.01) significantly characterized primary tumors of patients with pathological complete response. All patients with paired pre- and post-NAC samples reported a change in post-treatment CNAs compared to baseline, despite they showed at least one common alteration. CNAs detected after treatment involved genes within druggable pathways such as EGFR, cell cycle process and Ras signaling. In two patients, CTCs shared more alterations with residual rather than primary tumor involving genes such as MYC, BCL6, SOX2, FGFR4. The phylogenetic analysis of CTCs within a single patient revealed NAC impact on tumor evolution, suggesting a selection of driver events under treatment pressure. In conclusion, our data showed how chemoresistance might arise early from treatment-induced selection of clones already present in the primary tumor, and that the characterization of CNAs on single CTCs informs on cancer evolution and potential druggable targets.

End-of-neoadjuvant treatment circulating microRNAs and HER2-positive breast cancer patient prognosis: An exploratory analysis from NeoALTTO.

Background: The absence of breast cancer cells in surgical specimens, i.e., pathological complete response (pCR), is widely recognized as a favorable prognostic factor after neoadjuvant therapy. In contrast, the presence of disease at surgery characterizes a prognostically heterogeneous group of patients. Here, we challenged circulating microRNAs (miRNAs) at the end of neoadjuvant therapy as potential prognostic biomarkers in the NeoALTTO study. Methods: Patients treated within the trastuzumab arm (i.e., pre-operative weekly trastuzumab for 6 weeks followed by the addition of weekly paclitaxel for 12 weeks; post-operative FEC for 3 cycles followed by trastuzumab up to complete 1 year of treatment) were randomized into a training (n= 54) and testing (n= 72) set. RT-PCR-based high-throughput miRNA profile was performed on plasma samples collected at the end of neoadjuvant treatment of both sets. After normalization, circulating miRNAs associated with event free survival (EFS) were identified by univariate and multivariate Cox regression model. Results: Starting from 23 circulating miRNAs associated with EFS in the training set, we generated a 3-circulating miRNA prognostic signature consisting of miR-185-5p, miR-146a-5p, miR-22-3p, which was confirmed in the testing set. The 3-circulating miRNA signature showed a C-statistic of 0.62 (95% confidence interval [95%CI] 0.53-0.71) in the entire study cohort. By resorting to a multivariate Cox regression model we found a statistical significant interaction between the expression values of miR-194-5p and pCR status (p.interaction =0.005) with an estimate Hazard Ratio (HR) of 1.83 (95%CI 1.14- 2.95) in patients with pCR, and 0.87 (95%CI 0.69-1.10) in those without pCR. Notably, the model including this interaction along with the abovementioned 3-circulating miRNA signature provided the highest discriminatory capability with a C-statistic of 0.67 (95%CI 0.58-0.76). Conclusions: Circulating miRNAs are informative to identify patients with different prognosis among those with heterogeneous response after trastuzumab-based neoadjuvant treatment, and may be an exploitable tool to select candidates for salvage adjuvant therapy.

Integrated Molecular and Immune Phenotype of HER2-Positive Breast Cancer and Response to Neoadjuvant Therapy: A NeoALTTO Exploratory Analysis.

PURPOSE: Little is known about the efficacy of HER2-targeted therapy in patients with breast cancer showing different HER2-pathway dependence and immune phenotypes. Herein, we report a NeoALTTO exploratory analysis evaluating the clinical value of 22 types of tumor-infiltrating immune cells by CIBERSORT and 5 immune-related metagenes in the overall patient population, and in subgroups defined by the TRAR classifier as HER2-addicted (TRAR-low) or not (TRAR-high). PATIENTS AND METHODS: Association of baseline TRAR, immune-related metagenes, and CIBERSORT data with pathologic complete response (pCR) and event-free survival (EFS) were assessed using logistic and Cox regression models. Corrections for multiple testing were performed by the Bonferroni method. RESULTS: A total of 226 patients were analyzed: 80 (35%) achieved a pCR, and 64 (28%) experienced a relapse with a median follow-up of 6.7 (interquartile range 6.1-6.8) years; 108 cases were classified as TRAR-low, and 118 TRAR-high. Overall, γδ T-cell fraction [OR = 2.69; 95% confidence interval (CI), 1.40-5.18], and no immune-related metagenes were predictive of pCR. Notably, lymphocyte-specific kinase (LCK) predicted pCR to combination (OR = 2.53; 95% CI, 1.12-5.69), but not to single-agent trastuzumab or lapatinib [OR = 0.74; 95% CI, 0.45-1.22 (P interaction = 0.01)]. Integrating LCK with γδ T cells in a multivariate model added to the discriminatory capability of clinical and molecular variables with a shift in AUC from 0.80 (95% CI, 0.74-0.86) to 0.83 (95% CI, 0.78-0.89). In TRAR-low cases, activated mast cells, IFN and MHCII were reduced, and STAT1, HCK1, and γδ T cells were associated with pCR. STAT1 was broadly associated with improved EFS regardless of pCR, and nodal status in overall (HR = 0.68; 95% CI, 0.49-0.94) and in TRAR-low cases (HR = 0.50; 95% CI, 0.30-0.86). CONCLUSIONS: Immuno-phenotyping holds the promise to complement current predictive models in HER2-positive breast cancer and to assist in new therapeutic development.

Circulating Tumor Cell Clusters Are Frequently Detected in Women with Early-Stage Breast Cancer.

The clinical relevance of circulating tumor cell clusters (CTC-clusters) in breast cancer (BC) has been mostly studied using the CellSearch®, a marker-dependent method detecting only epithelial-enriched clusters. However, due to epithelial-to-mesenchymal transition, resorting to marker-independent approaches can improve CTC-cluster detection. Blood samples collected from healthy donors and spiked-in with tumor mammospheres, or from BC patients, were processed for CTC-cluster detection with 3 technologies: CellSearch®, CellSieve™ filters, and ScreenCell® filters. In spiked-in samples, the 3 technologies showed similar recovery capability, whereas, in 19 clinical samples processed in parallel with CellSearch® and CellSieve™ filters, filtration allowed us to detect more CTC-clusters than CellSearch® (median number = 7 versus 1, p = 0.0038). Next, samples from 37 early BC (EBC) and 23 metastatic BC (MBC) patients were processed using ScreenCell® filters for attaining both unbiased enrichment and marker-independent identification (based on cytomorphological criteria). At baseline, CTC-clusters were detected in 70% of EBC cases and in 20% of MBC patients (median number = 2, range 0-20, versus 0, range 0-15, p = 0.0015). Marker-independent approaches for CTC-cluster assessment improve detection and show that CTC-clusters are more frequent in EBC than in MBC patients, a novel finding suggesting that dissemination of CTC-clusters is an early event in BC natural history.

Detection of Genomically Aberrant Cells within Circulating Tumor Microemboli (CTMs) Isolated from Early-Stage Breast Cancer Patients.

Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known numbers of leukocytes into cancer cells (MDA-MB-453 and MDA-MB-36, displaying high and low DNA content, respectively) generating tumor fractions from 0-100%. After low-pass sequencing, ichorCNA was identified as the best algorithm to build a linear mixed regression model for tumor fraction (TF) prediction. We then isolated 53 CTMs from blood samples of six early-stage breast cancer patients and predicted the TF of all clusters. We found that all clusters harbor cancer cells between 8 and 48%. Furthermore, by comparing the identified CNAs of CTMs with their matched primary tumors, we noted that only 31-71% of aberrations were shared. Surprisingly, CTM-private alterations were abundant (30-63%), whereas primary tumor-private alterations were rare (4-12%). This either indicates that CTMs are disseminated from further progressed regions of the primary tumor or stem from cancer cells already colonizing distant sites. In both cases, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be explored in follow-up and mechanistic studies.