Assessing circulating tumor DNA (ctDNA) is a promising method to evaluate somatic mutations from solid tumors in a minimally-invasive way. In a group of twelve metastatic colorectal cancer (mCRC) ...patients undergoing liver metastasectomy, from each patient DNA from cell-free DNA (cfDNA), the primary tumor, metastatic liver tissue, normal tumor-adjacent colon or liver tissue, and whole blood were obtained. Investigated was the feasibility of a targeted NGS approach to identify somatic mutations in ctDNA. This targeted NGS approach was also compared with NGS preceded by mutant allele enrichment using synchronous coefficient of drag alteration technology embodied in the OnTarget assay, and for selected mutations with digital PCR (dPCR). All tissue and cfDNA samples underwent IonPGM sequencing for a CRC-specific 21-gene panel, which was analyzed using a standard and a modified calling pipeline. In addition, cfDNA, whole blood and normal tissue DNA were analyzed with the OnTarget assay and with dPCR for specific mutations in cfDNA as detected in the corresponding primary and/or metastatic tumor tissue. NGS with modified calling was superior to standard calling and detected ctDNA in the cfDNA of 10 patients harboring mutations in APC, ATM, CREBBP, FBXW7, KRAS, KMT2D, PIK3CA and TP53. Using this approach, variant allele frequencies in plasma ranged predominantly from 1 to 10%, resulting in limited concordance between ctDNA and the primary tumor (39%) and the metastases (55%). Concordance between ctDNA and tissue markedly improved when ctDNA was evaluated for KRAS, PIK3CA and TP53 mutations by the OnTarget assay (80%) and digital PCR (93%). Additionally, using these techniques mutations were observed in tumor-adjacent tissue with normal morphology in the majority of patients, which were not observed in whole blood. In conclusion, in these mCRC patients with oligometastatic disease NGS on cfDNA was feasible, but had limited sensitivity to detect all somatic mutations present in tissue. Digital PCR and mutant allele enrichment before NGS appeared to be more sensitive to detect somatic mutations.
•Plasma cfDNA was analyzed with three targeted assays.•Ion Torrent sequencing on cfDNA was feasible but had limited sensitivity.•Digital PCR & the OnTarget assay had superior sensitivity for detecting mutant ctDNA.•Low-frequency somatic mutations in tumor-adjacent tissue were frequently observed.•Choosing the most suitable cfDNA assay should be driven by the research question.
In breast cancer (BC), recurrent fusion genes of estrogen receptor alpha (ESR1) and AKAP12, ARMT1 and CCDC170 have been reported. In these gene fusions the ligand binding domain of ESR1 has been ...replaced by the transactivation domain of the fusion partner constitutively activating the receptor. As a result, these gene fusions can drive tumor growth hormone independently as been shown in preclinical models, but the clinical value of these fusions have not been reported. Here, we studied the prognostic and predictive value of different frequently reported ESR1 fusion transcripts in primary BC.
We evaluated 732 patients with primary BC (131 ESR1-negative and 601 ESR1-positive cases), including two ER-positive BC patient cohorts: one cohort of 322 patients with advanced disease who received first-line endocrine therapy (ET) (predictive cohort), and a second cohort of 279 patients with lymph node negative disease (LNN) who received no adjuvant systemic treatment (prognostic cohort). Fusion gene transcript levels were measured by reverse transcriptase quantitative PCR. The presence of the different fusion transcripts was associated, in uni- and multivariable Cox regression analysis taking along current clinico-pathological characteristics, to progression free survival (PFS) during first-line endocrine therapy in the predictive cohort, and disease- free survival (DFS) and overall survival (OS) in the prognostic cohort.
The ESR1-CCDC170 fusion transcript was present in 27.6% of the ESR1-positive BC subjects and in 2.3% of the ESR1-negative cases. In the predictive cohort, none of the fusion transcripts were associated with response to first-line ET. In the prognostic cohort, the median DFS and OS were respectively 37 and 93 months for patients with an ESR1-CCDC170 exon 8 gene fusion transcript and respectively 91 and 212 months for patients without this fusion transcript. In a multivariable analysis, this ESR1-CCDC170 fusion transcript was an independent prognostic factor for DFS (HR) (95% confidence interval (CI): 1.8 (1.2-2.8), P = 0.005) and OS (HR (95% CI: 1.7 (1.1-2.7), P = 0.023).
Our study shows that in primary BC only ESR1-CCDC170 exon 8 gene fusion transcript carries prognostic value. None of the ESR1 fusion transcripts, which are considered to have constitutive ER activity, was predictive for outcome in BC with advanced disease treated with endocrine treatment.
Molecular characterization of circulating tumor cells is of high clinical relevance. Since circulating tumor cell (CTC) detection and isolation often rely on cell dimensions, we determined the size ...of 71 612 CellSearch‐detected CTCs using accept software. Strikingly, CTC size differs between tumor types and significantly deviates from the size of cultured tumor cells, which is currently used in the development of CTC isolation methods.
Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient‐derived CTCs acquired on cartridges of the FDA‐cleared CellSearch® method were retrospectively collected and automatically re‐analyzed using the accept software package. The median CTC diameter (μm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch® cartridges containing 71 612 CTCs were included. In BC, the median computed diameter (CD) of patient‐derived CTCs was 12.4 μm vs 18.4 μm for cultured cell line cells. For PC, CDs were 10.3 μm for CTCs vs 20.7 μm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5 μm and 8.6 μm, respectively. Finally, leukocytes were 9.4 μm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size‐based isolation methods.
Abstract BACKGROUND: Preclinical and clinical studies have reported that human epidermal growth factor receptor 2 (HER2) overexpression yields resistance to endocrine therapies. Here the prevalence ...and prognostic impact of HER2-positive circulating tumor cells (CTCs) were investigated retrospectively in metastatic breast cancer (MBC) patients with a HER2-negative primary tumor receiving endocrine therapy. Additionally, the prevalence and prognostic significance of HER2-positive CTCs were explored in a chemotherapy cohort, as well as the prognostic impact of the estrogen receptor (ER) CTC status in both cohorts. METHODS: Included were MBC patients with a HER2-negative primary tumor, with ≥1 detectable CTC, starting a new line of treatment. CTCs were enumerated using the CellSearch system, characterized for HER2 with the CellSearch anti-HER2 phenotyping reagent, and characterized for ER mRNA expression. Primary end point was progression-free rate after 6 months (PFR6months) of endocrine treatment in HER2-positive versus HER2-negative CTC patients. RESULTS: HER2-positive CTCs were present in 29% of all patients. In the endocrine cohort ( n = 72), the PFR6months was 53% for HER2-positive versus 68% for HER2-negative CTC patients ( P = .23). In the chemotherapy cohort ( n = 82), no prognostic value of HER2-positive CTCs on PFR6months was observed either. Discordances in ER status between the primary tumor and CTCs occurred in 25% of all patients but had no prognostic value in exploratory survival analyses. CONCLUSION: Discordances regarding HER2 status and ER status between CTCs and the primary tumor occurred frequently but had no prognostic impact in our MBC patient cohorts.
Abstract Background Recent phase II and III clinical trials demonstrated anti-tumour activity of eribulin, a tubulin-interacting cytotoxic agent, in patients with metastatic soft tissue sarcoma ...(STS). In this exploratory study, we aimed to identify putative microRNA biomarkers that associate with eribulin sensitivity or resistance in STS. Materials and methods Archival tumour tissue from primary tumours or metastatic lesions was collected prior to eribulin treatment, from 65 consenting patients involved in the EORTC trial 62052. This phase II study ( ClinicalTrials.gov identifier NCT00413192 ) included multiple subtypes of STS. Tissue was available from 21 leiomyosarcomas, 14 adipocytic sarcomas, 9 synovial sarcomas and 21 other sarcoma histotypes. Total RNA was isolated from formalin-fixed, paraffin-embedded tumour samples and analysed using Taqman® Low Density Arrays to determine microRNA expression profiles. The expression of a total of 756 microRNAs was assessed. Progression-free survival at week 12 (RECIST 1.0) measured as a binary variable, was the primary end-point of the clinical trial and used as a primary response measure for correlative studies. Seventeen of 53 (32.1%) evaluable patients in the analysed subset had non-progressive disease at week 12 and were defined as responders. Results The expression of 26 individual microRNAs (p < 0.05) differed significantly between non-responders and responders. Additional microRNAs of potential relevance were identified when considering the different histological subgroups. Conclusions The expression level of particular microRNAs in STS tissue samples may predict response to eribulin. Further validation studies as well as a preclinical assessment of the underlying molecular mechanisms are required.
MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression and which have been implicated in cancer. We evaluated whether five candidate predictive miRNAs, derived from a pilot study in ...which 249 miRNAs were assayed, were associated with clinical benefit of tamoxifen therapy in advanced breast cancer. These five miRNAs were measured in an independent series of 246 estrogen receptor (ER)-positive primary breast tumors of patients who received tamoxifen for advanced disease by quantitative Real Time PCR. Univariate analysis showed that higher expression levels of hsa-miR-30a-3p, hsa-miR-30c, and hsa-miR-182 were significantly associated with benefit of tamoxifen treatment and with longer PFS (all
P
-values <0.01). In multivariate analysis, corrected for the traditional predictive factors, only hsa-miRNA-30c was an independent predictor (
P
-value <0.01). Finally, in an attempt to understand the biology connected to this miRNA, Global testing pathway analysis showed an association of hsa-miRNA-30c expression with HER and RAC1 signaling pathways. We identified hsa-miRNA-30c as an independent predictor for clinical benefit of tamoxifen therapy in patients with advanced breast cancer. Assessment of tumor levels and connected pathways could be helpful to improve treatment strategies.
Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived ...EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA).
For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR.
Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue.
We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients' blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.
Desmoid-type fibromatosis (DTF) is a rare, soft tissue tumor of mesenchymal origin which is characterized by local infiltrative growth behavior. Besides "wait and see," surgery and radiotherapy, ...several systemic treatments are available for symptomatic patients. Recently, targeted therapies are being explored in DTF. Unfortunately, effective treatment is still hampered by the limited knowledge of the molecular mechanisms that prompt DTF tumorigenesis. Many studies focus on Wnt/β-catenin signaling, since the vast majority of DTF tumors harbor a mutation in the
gene or the
gene. The established role of the Wnt/β-catenin pathway in DTF forms an attractive therapeutic target, however, drugs targeting this pathway are still in an experimental stage and not yet available in the clinic. Only few studies address other signaling pathways which can drive uncontrolled growth in DTF such as: JAK/STAT, Notch, PI3 kinase/AKT, mTOR, Hedgehog, and the estrogen growth regulatory pathways. Evidence for involvement of these pathways in DTF tumorigenesis is limited and predominantly based on the expression levels of key pathway genes, or on observed clinical responses after targeted treatment. No clear driver role for these pathways in DTF has been identified, and a rationale for clinical studies is often lacking. In this review, we highlight common signaling pathways active in DTF and provide an up-to-date overview of their therapeutic potential.
We are living in an exciting era in the treatment of cancer, using drugs that target specific proteins rather than agents that cause more general cytotoxic effects. The identification of proteins and ...signal transduction pathways that play crucial roles in the pathogenesis of cancer has allowed treatments to be designed that target these tumor-driven events. Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors and were among the first solid tumor types for which such a novel treatment (in this case imatinib) became available. The tyrosine kinase inhibitor imatinib targets the human KIT receptor and the platelet-derived growth factor receptor-alpha. This drug exhibits impressive antitumor effects against GIST and has become the first-line therapy for advanced disease. Major insights into the mechanism of action of this drug, drug resistance, and patient management issues have been gleaned. Additionally, new drugs developed for the treatment of GIST have been identified. As a consequence, lessons learned from GIST are widely applicable to other tumor entities, thereby rendering GIST the paradigm of solid tumors treated with tyrosine kinase inhibitors. This Review discusses the pathogenesis of GIST, treatment strategies, mechanisms accounting for drug resistance, and potential future perspectives.
Here, we compare the number of mutant molecules and the variant allele frequency as units of measurement for circulating tumor DNA. We conclude that when using digital droplet PCR, both units of ...measurement yield greater agreement than when using next generation sequencing. Low frequent mutations and molecular coverage are important factors affecting agreement. These results will improve circulating tumor DNA analysis, contributing to better cancer patient management.
Quantification of tumor‐specific variants (TSVs) in cell‐free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement. To study the agreement between VAF and mutant molecules in current clinical studies, we analyzed 1116 TSVs from 338 patients identified with next‐generation sequencing (NGS) or digital droplet PCR (ddPCR). On different study cohorts, a Deming regression analysis was performed and its 95% prediction interval was used as surrogate for the limits of agreement between VAF and number of mutant molecules per mL and to identify outliers. VAF and number of mutant molecules per mL plasma yielded greater agreement when using ddPCR than NGS. In case of discordance between VAF and number of mutant molecules per mL, insufficient molecular coverage in NGS and high cell‐free DNA concentration were the main responsible factors. We propose several optimization steps needed to bring monitoring of TSVs in cell‐free DNA to its full potential.
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