Acquired activating ESR1 mutations in cell-free DNA and circulating tumor cells in estrogen receptor positive metastatic breast cancer
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Project description:Purpose: Early detection is essential for treatment plans before onset of metastatic disease. Our purpose was to demonstrate feasibility to detect and monitor estrogen receptor 1 (ESR1) gene mutations at the single circulating tumor cell (CTC) level in metastatic breast cancer (MBC).Experimental Design: We used a CTC molecular characterization approach to investigate heterogeneity of 14 hotspot mutations in ESR1 and their correlation with endocrine resistance. Combining the CellSearch and DEPArray technologies allowed recovery of 71 single CTCs and 12 WBC from 3 ER-positive MBC patients. Forty CTCs and 12 WBC were subjected to whole genome amplification by MALBAC and Sanger sequencing.Results: Among 3 selected patients, 2 had an ESR1 mutation (Y537). One showed two different ESR1 variants in a single CTC and another showed loss of heterozygosity. All mutations were detected in matched cell-free DNA (cfDNA). Furthermore, one had 2 serial blood samples analyzed and showed changes in both cfDNA and CTCs with emergence of mutations in ESR1 (Y537S and T570I), which has not been reported previously.Conclusions: CTCs are easily accessible biomarkers to monitor and better personalize management of patients with previously demonstrated ER-MBC who are progressing on endocrine therapy. We showed that single CTC analysis can yield important information on clonal heterogeneity and can be a source of discovery of novel and potential driver mutations. Finally, we also validate a workflow for liquid biopsy that will facilitate early detection of ESR1 mutations, the emergence of endocrine resistance and the choice of further target therapy. Clin Cancer Res; 23(20); 6086-93. ©2017 AACR.
| S-EPMC5641250 | biostudies-literature
Project description:The ESR1 ligand binding domain activating mutations are the most prevalent genetic mechanism of acquired endocrine resistance in metastatic hormone receptor-positive breast cancer. These mutations confer endocrine resistance that remains estrogen receptor (ER) dependent. We hypothesized that in the presence of the ER mutations, continued ER blockade with endocrine therapies that target mutant ER is essential for tumor suppression even with chemotherapy treatment. Here, we conducted comprehensive pre-clinical in vitro and in vivo experiments testing the efficacy of adding fulvestrant to fluorouracil (5FU) and the 5FU pro-drug, capecitabine, in models of wild-type (WT) and mutant ER. Our findings revealed that while this combination had an additive effect in the presence of WT-ER, in the presence of the Y537S ER mutation there was synergy. Notably, these effects were not seen with the combination of 5FU and selective estrogen receptor modulators, such as tamoxifen, or in the absence of intact P53. Likewise, in a patient derived xenograft (PDX) harboring a Y537S ER mutation the addition of fulvestrant to capecitabine potentiated tumor suppression. Moreover, multiplex immunofluorescence revealed that this effect was due to decreased cell proliferation in all cells expressing ER and was not dependent on the degree of ER expression. Taken together, these results support the clinical investigation of the combination of ER antagonists with capecitabine in patients with metastatic hormone receptor-positive breast cancer who have experienced progression on endocrine therapy and targeted therapies, particularly in the presence of an ESR1 activating mutation.
2024-06-01 | GSE266932 | GEO
Project description:BACKGROUND:The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. RESULTS:ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). METHODS:A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). CONCLUSIONS:We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information.
| S-EPMC5078029 | biostudies-literature
Project description:Purpose: Breast cancers with ESR1 mutations are resistant to antiestrogen therapy. We aimed to investigate the association of ESR1 mutations with resistance to CDK4/6 inhibitors (CDK4/6i) using real-world data analysis and experimental validation. Patients and Methods: A total of 3,958 patients with estrogen receptor-positive (ER+) metastatic breast cancer with DNA sequencing data were analyzed. Breast tumor DNA and circulating tumor (ct) DNA were sequenced using the Tempus xT tumor assay and Tempus xF liquid biopsy, respectively. Patients were stratified into either treated with CDK4/6i (breast tumors: 1,070; ctDNA: 1,885) or CDK4/6i naïve (breast tumors: 750; ctDNA: 253). Engineered MCF7 cells carrying ESR1 Y537S or D538G knock-in mutations were used to study antitumor efficacy of the CDK4/6i, palbociclib in vitro and in vivo. Results: In both xF and xT assays, ESR1 mutations were the only somatic alterations significantly more frequent in those who received CDK4/6i than those who did not. Knock-in of ESR1 Y537S or ESR1 D538G in MCF7 cells resulted in upregulation of cell cycle-related gene signatures upon treatment with CDK4/6i ± antiestrogen compared to cells with non-mutant ESR1. MCF7 xenografts harboring ESR1 Y537S and D538G mutations established in nude mice were resistant to palbociclib. Conclusions: We report herein real-world and preclinical evidence that ESR1 mutations, particularly Y537S and D538G, can drive resistance to CDK4/6 inhibitors.
2024-11-12 | GSE281302 | GEO
Project description:Medulloblastoma (MB) is an embryonal tumor of the cerebellum and a highly malignant childhood brain tumor. Cell-free circulating tumor DNA (ctDNA) from the cerebrospinal fluid (CSF) of patients with brain tumors faithfully represent genomic alterations of brain tumors. Distinct epigenetic signatures among subgroups of MB allow us to detect epigenetic alterations in CSF to aid classify and guide therapy of MB tumors. Here, we evaluate DNA methylation and hydroxymethylation of ctDNA derived from small amount of CSF (200 µL) and matched tumor DNA from 3 MB patients. We find highly concordance of DNA methylation and hydroxymethylation between CSF ctDNA and tumor DNA, especially in CpG islands. Importantly, CSF ctDNA can mostly recapitulate the dynamic changes of DNA methylation and hydroxymehtylation in tumor species compared to healthy cerebellums. Those MB tumor signature CpGs’ DNA methylation status are recovered in CSF ctDNA can clearly distinguish MB subgroups by utilizing public large cohort data. We further identified potential diagnostic and prognostic DNA methylation markers in CSF ctDNA. Our results show that CSF ctNDA methylation and hydroxymethylation can be a minimal invasive method to assess epigenetic alterations of MB, which is complementary to current diagnoses of MB tumors.
2020-10-28 | GSE142241 | GEO
Project description:Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tu-mor cells, CTCs) either alone or Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tu-mor cells, CTCs) either alone or as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell–cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils. Our results suggest that the EV proteome of CTCs contain signaling proteins that can modulate degranulation and granule mobilization in neutrophils and, also, contain tis-sue plasminogen activator and other proteins that can regulate cluster formation. By exposing naïve neutrophils to EVs isolated from CTCs, we further show how these changes are modulated in a dynamic fashion indicating evidence for a deeper EV based remodulatory effect on com-panion cells in clusters.as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell–cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils. Our results suggest that the EV proteome of CTCs contain signaling proteins that can modulate degranulation and granule mobilization in neutrophils and, also, contain tis-sue plasminogen activator and other proteins that can regulate cluster formation. By exposing naïve neutrophils to EVs isolated from CTCs, we further show how these changes are modulated in a dynamic fashion indicating evidence for a deeper EV based remodulatory effect on com-panion cells in clusters.
2021-06-07 | PXD026340 | Pride
Project description:Circulating tumor DNA (ctDNA) as a biomarker of disease activity in classic Hodgkin lymphoma (cHL) patients are still not well-defined. By profiling primary tumors and ctDNA, we identified common variants between primary tumors and longitudinal plasma samples in most of the cases, confirming high PBatial and temporal heterogeneity. Though ctDNA analyses mirrored HRS cell genetics overall, the prevalence of variants shows that none of them can be used as a single biomarker. Conversely, the estimation of hGE/mL, based in total ctDNA quantification, reflects disease activity and is almost perfectly correlated with standard parameters such as PET/CT that are associated with refractoriness.
2023-12-18 | GSE244989 | GEO
Project description:Circulating tumor cells (CTCs) and disseminated tumour cells with mesenchymal traits are difficult to detect by epithelial marker proteins. Particularly, triple negative breast cancers (TNBC) that are prone to therapy failure release a subpopulation of circulating tumour cells (CTCs) with mesenchymal traits. To provide tools that support their detection and analysis, the cell line BC-M1 established from disseminated tumour cells in the bone marrow of a breast cancer patient and a bone metastasis subline of MDA-MB-231 were analysed. Mass spectrometry analysis revealed high levels of CUB domain-containing protein 1 (CDCP1) in BC-M1. CDCP1 was found in other carcinoma cell lines (MDA-MB-231, MDA-MB-468) and other DTC cell lines (LC-M1, PC-E1) as well. Peripheral blood mononuclear cells were virtually negative for CDCP1 by Western Blot and immunofluorescent staining. Presence of CDCP1 in CTCs was confirmed by CellSearch. Here, CDCP1 positive CTCs were detected in eight of 30 analysed breast cancer patients. For the isolation of CTCs from the blood of breast cancer patients, we established a sandwich magnetic-activated cell sorting (MACS). The extracellular domain of CDCP1 served for cell catching and the cytoplasmic domain of CDCP1 for immunofluorescent detection of CDCP1 in CTCs. We showed that the MACS approach is suitable for the isolation of EpCam/keratin negative breast cancer cells from the blood and isolated CDCP1 positive CTCs from breast cancer patients by MACS. Hence, our approach is particularly suited for the detection and isolation of CTCs from TNBC when low EpCam or keratin levels limit the application of conventional approaches.
2023-03-20 | PXD035203 | Pride
Project description:Medulloblastoma (MB) is an embryonal tumor of the cerebellum and a highly malignant childhood brain tumor. Cell-free circulating tumor DNA (ctDNA) from the cerebrospinal fluid (CSF) of patients with brain tumors faithfully represent genomic alterations of brain tumors. Distinct epigenetic signatures among subgroups of MB allow us to detect epigenetic alterations in CSF to aid classify and guide therapy of MB tumors. Here, we evaluate DNA methylation of ctDNA derived from small amount of CSF (200 µL) and matched tumor DNA from four subtypes of MB patients. We find highly concordance of DNA methylation between CSF ctDNA and tumor DNA in a subtype manner. Our results show that CSF ctNDA methylation can be a minimal invasive precisely method to assess epigenetic alterations of MB in a subtype manner, which is complementary to current diagnoses of MB tumors.
2023-06-01 | GSE178169 | GEO
Project description:Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Highly parallel architecture of the chip allows deterministic screening of clinically relevant volumes of blood samples at slow, and hence, non-damaging flow rates. Using the Cluster-Chip, we identify CTC-clusters in 30-40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as being tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. We used the Cluster-Chip to capture CTC-clusters from the blood of a breast cancer patient with high CTC counts, released CTC-clusters in solution, stained them with TexasRed-conjugated antibodies against the leukocyte cell surface markers CD45, CD14 and CD16, and then isolated intact CTC-clusters individually using a micromanipulator. From a single time point, we retrieved 15 CTC-clusters, and each of those clusters was individually subjected to RNA-sequencing analysis using a next generation platform (SOLiD 5500). In addition, two leukocytes were isolated from the blood of a healthy donor were individually subjected to RNA-sequencing analysis using the same platform.
2015-05-17 | E-GEOD-67939 | biostudies-arrayexpress