Abstract
Introduction: Although CTCs display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. A portion of ...CTCs may form clusters that contain two or more CTCs bound together which were reported to have up to 50-fold of potential of forming distant metastasis in MBC as compared to individual CTCs (Aceto N. Cell, 2015). However, genomic characterization of CTCs-clusters compared to single CTCs remain largely unknown. We previously reported single CTC sequencing for HER2+ CTCs (2020 AACR #3120). Herein, we report a new finding of heterogeneity profiling for CTC-clusters compared to single CTCs, which would be helpful to evaluate the MBC metastasis capability and treatment in clinic. Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV MBC patients before therapy. CTC enumeration was performed using the FDA-cleared CellSearch™ System (Menarini) targeting the EpCAM antigen for capturing CTCs which were then stained by Anti-CK-PE, DAPI, anti-CD45-APC and anti-HER2-FITC. The CTC-clusters and single CTCs were isolated using DEPArrayTM System (Menarini). DNA was isolated from CTC-clusters and single CTCs by ArcturusTM PicoPureTM DNA Extraction kit. The initial library was prepared by SMARTer® PicoPLEX® Gold Single Cell DNA-Seq Kit, and the exome capture was performed by Twist Human Core Exome EF Multiplex Complete Kit. The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina). It was a paired end run, 75×75 bps run with dual indexing. Results: We identified 107 CTCs by CellSearch™, including 93 single CTCs, 14 CTC-clusters and 145 WBCs. Autologous CTC-clusters (CK+DAPI+CD45-, Group 1), single CTCs (CK+DAPI+CD45-, Group 2), and leukocytes (CK-DAPI+CD45+, Group 3) were sequenced respectively. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 60,638 counts (6.77%) and 70,334 counts (8.20%) for exon variants in CTC-clusters and single CTCs respectively, 507,595 counts (56.69%) and 486,119 counts (56.69%) for intron variants, 194,026 (21.67%) and 175,819 counts (20.51%) for intergenic variants, 54,174 counts (6.05%) and 50,370 counts (5.87%) for downstream genes, 51,716 counts (5.78%) and 45,915 counts (5.36%) for upstream genes, and 3.04% and 3.37% of others variants in CTC-clusters and single CTCs respectively. Meanwhile, there was 0 count for exon and intron variants found in Group 3. There were 60 and 79 gene variants (SNP and Ins-Del) identified to have the highest impact effect (≥20) on CTC-clusters and single CTC exons respectively, which affect significantly on the functional proteins coding. Among the top 50 high impact gene variants in each group, there were 25 gene alteration sites were similar in Group 1 and 2, including XYLB, RAN, QPCT, HPGDS, HDAC8, GABBR2, CYP11B2 and CHKA. Specific to Group 1, there were 25 gene alterations which were primarily related to cellular proliferation and tumor promotion (AMD1), liver drug clearance (CES1), tissue remodeling (CHI3L1), immune cytokine signaling (JAK1) and metabolism (ASRGL1). Meanwhile, there are 25 specific gene alterations in Group 2 compared to Group 1, which were associated with nucleotide-excision repair (DDB1 and FAN1) chromosome positioning (KIF11), cell growth, differentiation, mitotic cycle, oncogenic transformation (PTPN3 and MAPK14), apoptosis (CASP1) and cell growth (CTNNB1). Conclusion: Genomic characterization of CTC-clusters compared to autologous single CTCs and leukocytes elucidated new specific gene alterations in CTC-clusters associated with most aggressive disease metastasis in MBC, which will help to gain new insights on the molecular mechanisms associated with the metastasis and find new molecularly driven therapies for disease metastasis.
Citation Format: Qiang Zhang, Lorenzo Gerratana, Paolo D'Amico, Andrew A. Davis, Saya Liz Jacob, Xinkun Wang, Zhe Ji, Zheng Cai, Elena Vagia, Wenan Qiang, Ami Shah, Youbin Zhang, Lisa Flaum, Firas Wehbe, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Genetic profiling for circulating tumor cell clusters to unveil molecular drivers of metastasis abstract. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-05.
With the rapid advancement of the new generation of the industrial revolution, the Industrial Internet of Things (IIOT) has placed higher demands on multidimensional indicators such as reliability, ...processing delay, energy consumption, and user experience in industrial applications. Mobile Edge Computing (MEC), as an important technology of IIOT, can satisfy the enormous resource demands of industrial mobile devices, including storage capacity, computing power, network bandwidth, and battery power. This can further provide corresponding solutions for the industrial Internet in terms of network performance, such as low latency, high reliability, and high security. Currently, there are many studies on independent task offloading in MEC. However, in the actual industrial Internet of Things scenarios, computing tasks typically consist of several interdependent subtasks. Therefore, we introduce hybrid workflows into the system model by considering the interdependence between computing tasks. The goal is to decrease the MEC system's computational time and resource consumption. This paper proposes an offloading method based on the Improved Bald Eagle Search Algorithm (IBES) to maximize system revenue. Simulation results show that compared with existing optimization algorithms, IBES can significantly reduce task completion time while consuming less energy. Therefore, it can significantly enhance user experience.
Abstract
Introduction: CTCs play a critical role in MBC, and a portion of CTCs may form clusters that contain two or more CTCs bound together which were reported to have up to 50-fold of potential of ...forming distant metastasis in MBC as compared to individual CTCs. However, genomic characterization of CTCs-clusters remain largely because the enrichment of CTC-clusters is technically challenging. Herein, we describe a novel isolation workflow to select CTC-clusters expressing specific biomarkers for patients with MBC.
Methods: Whole blood samples (7.5ml/each) were collected from 5 patients with stage III/IV BCa patients. CTC enumeration was performed with the FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit targeting the Epithelial Cell Adhesion Molecule antigen for capturing CTCs, and immunofluorescent staining reagents including Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocyte), and anti-HER-2/neu-FLU. Or, using CXC kit includes anti-CCR5-PE. After confirming CTCs-clusters, both CTCs and CTC-clusters were enriched from Celltracks cartridge and were loaded into DEPArrayTM NxT cartridge, and then were isolated and sorted by using fluorescent imaging based DEPArrayTM NxT System.
Results: All these samples were identified having CTCs (≥ 5, between 34 to 208 CTCs) with CTC-clusters (between 1 to 16) by CellSearch analysis. The images of CTCs and CTC-clusters were displayed in CellBrowserTM by the DEPArrayTM NxT System. According to multiple channels assigned for PE, DAPI, APC and FITC, the CTCs and CTC-clusters were classified and then sorted based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-, with additional markers of HER2 or CCR5. The targeted CTCs and CTC-clusters were routed, parked and then recovered under the direction of Recovery ManagerTM. By using this technique, we successfully recovered and harvested 27/34 (DEPArray harvest/CellSearch diagnosis), 54/151, 57/115, 75/196, and 115/208 CTCs from each CellSearch cartridge sample respectively. Meanwhile, we collected 1/1 (100%), 6/16 (37.5%), 1/1 (100%), 3/11 (27.3%), and 4/4 (100%) of CTC-clusters respectively, as each CTC-cluster includes average of 2.85 CTCs. CTC-clusters at diameter as much as 45µm could be routed and recovered smoothly. Furthermore, CTC-Clusters with biomarkers of interest such as HER2+ or CCR5+ expression were separated into different tubes. The white blood cells from the same samples were also selected successfully as the controls.
Conclusions: We first reported a new workflow for CTC-clusters isolation. With further optimization, this feasible and reliable strategy will help streamline isolation of single CTC and CTC-clusters in MBC patients for genomic analysis providing the opportunity to gain new insights on the molecular mechanisms associated with the metastasis process.
Citation Format: Qiang Zhang, Lorenzo Gerratana, Ami N. Shah, Andrew A. Davis, Lisa Flaum, Youbin Zhang, Amir Behdad, Firas Wehbe, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. A novel application of DEPArrayTM NxT System to isolate circulating tumor cell (CTC)-clusters from patients with metastatic breast cancer (MBC) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1351.
Abstract
Introduction: Molecular and genomic characterization of CTCs may help to understand MBC prognosis and predict treatment benefit. We previously reported that overexpression of HER2 in CTCs is ...associated with detection of clusters, more aggressive clinical features and prognosis in MBC (2018 AACR #5195). Herein, we report a new finding that a widespread variation on chromatin distribution in CTCs is associated with HER2 expression in MBC which may indicate more aggressive tumor.
Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV BCa patients before therapy. CTC enumeration were performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by targeting the Epithelial Cell Adhesion Molecule antigen for capturing CTCs. After confirming CTCs were positive by CellSearch system, live CTCs enrichment was performed by using Parsortix system (ANGLE) utilizes microfluidic based 10μm separation cassettes. The captured CTCs were stained by Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER2-FITC. CTCs chromatin packing density was scanned by Partial Wave Spectroscopic (PWS) microscopy which is a label-free spectroscopic microscopy method that resolves structures in cells and quantify the cell nucleus heterogeneity of chromatin packing density scaling between 20-350 nm, or from the kb-pair to 10 Mb-pair range. For each nuclei, the nanoscale heterogeneity of chromatin packing was analyzed as nuclear statistical parameter standard deviation Σ (sigma, RMS). Kruskal-Wallis test was used for statistics.
Results: We identified 400 CTCs by CellSearch system, including 115 HER2+ CTCs and 285 HER2- CTCs. The live CTCs were sorted by Parsortix system and were proceeded with multiple staining and scanning by PWS microscopy. CTCs were classified based on morphology and correct phenotype as CK+, DAPI+ and CD45-. There were 97 acquired nuclei that were scanned and evaluated successfully, including 17 HER2+ CTCs, 12 HER2- CTCs and 80 non-CTC cells. According to the images, stronger nanoscopic variations at each pixel in internal structure represents higher chromatin distribution heterogeneity quantified by RMS. The average RMS was 0.05125 in CTC cells which is significantly higher than the non-CTC population (RMS=0.0381, p<0.01). Moreover, the HER2+ CTCs demonstrated the highest nuclear sigma (RMS=0.05366) among all the subgroups when compared to HER2- CTCs (RMS=0.04546, p<0.01).
Conclusion: This is the first report of chromatin heterogeneity in CTCs, and the association between HER2 expression and high variations of chromatin distribution in MBC patients. This novel property of CTCs describing nanoscale heterogeneity of chromatin packing requires further evaluation and validation but, it may offer a novel dimension in the understanding of the metastatic process.
Citation Format: Qiang Zhang, Lorenzo Gerratana, Ami N. Shah, Andrew Adam Davis, Lisa Flaum, Xiang Zhou, Youbin Zhang, Vadim Backman, Amir Behdad, Firas Wehbe, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased chromatin heterogeneity in circulating tumor cells (CTCs) is associated with high levels of HER2 expression in metastatic breast cancer (MBC) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 429.
Abstract
Introduction: The Chemokine C-C motif ligand 5 (CCL5) and its receptor 5 (CCR5) play a significant role in solid tumors, particularly triple negative breast cancer (TNBC) and HER2+ subtypes ...with prognostic implications. CCL5-CCR5 axis was reported to govern cancer stem cells expansion and play key role in MBC progression. Our group recently reported that overexpression of HER2 was associated with CTC-clusters which caused poor prognosis of patients with MBC. Herein, we first reported the expression of CCR5 in CTCs of MBC patients, and described the correlation between CCR5 and HER2 expression in CTCs.
Methods: Whole blood samples (7.5ml/each) were collected from stage III/IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, and immunofluorescent staining reagents including anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. After confirming CTCs were positive by CellSearch system, CCR5 expression were evaluated by using CXC kit for multiple staining includes anti-CCR5-PE (R&D Systems), Anti-CK-FITC, DAPI and Anti-CD45-APC. The correlation between HER2 expression and CCR5 expression was analyzed by Kruskal-Wallis test was used for statistics.
Results: CTCs were found positive (≥5) in all seven MBC patients with a range of numbers between 124 and 442, and HER expression was identified in 6 out 7 cases (between 57 and 149 total cells). The ratios of HER2+ CTCs/total CTCs were varied between 28.5% and 88.1%. Meanwhile, CCR5 expression were found in 3 out 7 patients (Group 1), with CCR5+ CTCs/ total CTCs ratios were 4.45%, 43.1% and 59.1% respectively. There were 4 patients without CCR5 expression in CTCs (Group 2). The average HER2+ CTCs was 127.6 with HER2+ CTC/Total CTC as average of 59.4% in Group 1, which were significantly higher in compared with the corresponding numbers as 80.3 and 29.97% respectively in Group 2, which indicated that upregulation of CCR5 was positively associated with high level of HER2 expression in CTCs.
Conclusions: Our data provides the first evidence of strong expression of CCR5 in CTCs of MBC as potential new marker. The significant correlation between overexpression of HER2 in CTCs and high level of CCR5 indicated that CCR5 may contributes to more aggressive MBC subtypes with HER2 expression, which promotes carcinogenesis and metastasis at least partly by maintaining and increasing cancer stem cells leading to increased invasion. We conclude that the further understanding of the molecular interactions of CCR5 and HER2 in CTCs will be important to elucidate the mechanism of metastasis of MBC and predict prognosis.
Citation Format: Qiang Zhang, Lorezo Gerratana, Ami N. Shah, Andrew A. Davis, Lisa Flaum, Youbin Zhang, Richard G. Pestell, Firas Wehbe, Amir Behdad, Leonidas Platanias, William Gradishar, Massimo Cristofanilli. Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 408.
Abstract only
1036
Background: The monitoring of CTCs and cfDNA in metastatic BCa showed ability to predict treatment resistance and survival. Here we report a highly significant correlation between ...HER2 alterations and ESR1 mutations of cfDNA with CTCs and prognosis of BCa, which may help to predict disease recurrence and treatment benefit. Methods: A total of 85 blood samples (7.5ml/each) were collected from 85 patients with stage III/IV BCa who received treatments at Northwestern RHLCCC. CTC enumeration was performed in FDA approved CELLTRACKS ANALYZERII System (Menarini). Plasma cfDNA was analyzed using Guardant360 NGS-based assay including a 73-gene panel for genomic alterations or mutations. We previously reported cut-off of 5.7% (2018 ASCO) was used to dichotomize the prognostic value of cfDNA percentage. Kruskal-Wallis test was used for statistics. Results: Of the 85 whole samples analyzed, there were 72 samples and 67 samples without ESR1 mutations (ESR1
-
) and HER2 alterations ( HER2
-
) respectively, and there are 13 samples and 18 samples that had ESR1 mutations ( ESR1
+
) and HER2 alterations ( HER2
+
, 10 amplified, 7 mutated, 1 for both) respectively. CTC positive (≥5) were detected in 13/57 ESR1
-
HER2
-
samples (Group 1) and 5/15 ESR1
-
HER2
+
samples (Group 2), 7 /10 ESR1
+
HER2
-
samples (Group 3), 3/3 ESR1
+
HER2
+
samples (Group 4). The median CTCs number/sample in Group 3 (15 CTCs) and Group 4 (12 CTCs) were significantly higher than Group 1 (0 CTC) and Group 2 (2 CTCs) (P = 0.0020). There were a significant higher average metastasis sites in Group 3 (3 sites) and Group 4 (3 sites) in compared to Group 1 (2 sites) and Group 2 (1 site) (P = 0.0035). Furthermore, patients in Group 4 ( ESR1
+
HER2
+
) has the worst prognosis in compared to other groups (P = 0.0151) on overall survival. Conclusions: Both ESR1 mutations and HER2 alterations in cfDNA contribute to CTCs detection and disease metastasis sites independently, when ESR1 mutations plays a major role. The synergy of ESR1 mutation and HER2 alteration expands the predictive role of liquid biopsy tests monitoring the metastatic prognosis and endocrine resistance for clinical decision-making.
Abstract
Inflammatory breast cancer (IBC) is the most aggressive clinical manifestation of breast cancer (BC), with striking erythema of the overlying skin, and exhibiting features of early ...metastatic and treatment-refractory disease. Malignant cells and infiltrating leukocytes, e.g. tumor-associated microphages (TAMs) commonly designated "M2" macrophages, play a major part in metastasis and treatment resistance. TAMs help promote tumor growth and progression. There is a significant need for preclinical models that retain the characteristics of the original patient tumor for investigating mechanisms of IBC metastasis and for testing candidate therapies.
Six samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from five IBC or metastatic breast cancer (MBC) patients were obtained from Northwestern Memorial Hospital. IBC cells were used for 3D tumor spheroid cultures, and for developing PDX tumor models in immunodeficient mice. Short Tandem Repeat profiling against with original patient tumor DNA was conducted to establish patient DNA identify, and to serve as a reference for authenticating derivative tumor models.
The tumor genomic mutation analysis indicated the most common alterations of five IBC or MBC patients were TP53 (75%), CCND1 (75%), BRCA2 (50%), NF1 (50%), FGFR1 (50%), CDK6 (50%), EGFR (50%), MYC (50%), BRAF (50%). Examination IBC pleural effusion cellular composition indicated ~60% were tumor cells and ~40% were M2 TAMs (CD163+). In early 3D spheroid IBC culture, a symbiotic phenomenon was observed between tumor cells and attached TAMs; Anti-cancer drugs GW2580 and Pexidartinib inhibited 32% and 68% of cell proliferation, respectively, when added to spheroid cultures at 100 nanomolar.
Histopathologic analysis of derivative IBC and MBC PDX revealed highly heterogeneous characteristics and metastatic features characteristic of corresponding patient tumors. Subcutaneous tumors also demonstrated metastasis to liver and lung tissue. NSG mice engrafted with original IBC pleural effusion cells or derived spheroid cultures manifested a variety of inflammatory clinical symptoms. Mice engrafted with original pleural effusion cells developed palpable tumor and manifested most IBC-characteristic including reddish and ulcerated skin lesions necessitating euthanasia within 2 months of cell engraftment. Mice engrafted with > 6 passage or <4 passages of spheroid cultures (lower % of TAMs present) manifested lesser skin inflammation and lesion, and developed tumors of ~ 1 cm within 2-3 months. This may indicate the role of TAMs in the development of IBC skin lesion.
Our results indicate IBC 3D spheroid cultures and PDX tumor models recapitulate clinical characteristics observed in IBC patients, and will prove useful for studying mechanisms of IBC metastasis as well as responsiveness to investigational therapies.
Citation Format: Wenan Qiang, Youbin Zhang, Demirkan Gursel Gursel, Jian-Jun Wei, Charles David James, Thomas V. O'Halloran, Massimo Cristofanilli. Developing patient-derived xenograft tumor models that recapture clinical manifestation of inflammatory breast cancer patients abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1046.
Abstract only
1028
Background: MBC is a challenging clinical condition treated with palliative intent due to tumor heterogeneity. We reported in 2019 ASCO that the correlation of HER2 and ESR1 ...mutations of ctDNA with CTCs results in worse prognosis in MBC. Here we reported that ctDNA mutations is a key point which is different between Stage III and Stage IV, and it would be helpful to evaluate the MBC metastasis and outcome. Methods: This study included 33 Stage III and 204 Stage IV MBC patients who received systemic treatments at NMH (2016-2019). Plasma ctDNA before treatment was isolated from patients and then was analyzed by Guardant 360 Health NGS-based assay for a 73 genes panel for genomic alterations including single nucleotide variants, insertions/deletions, gene fusions/rearrangements and copy number variations. Causal Inference with Ensembel Learning was used for statistical analyses. Results: Among stage III patients, 40% are luminal, 44% are HER2
+
and 16% are TNBC, while in stage IV 50% patients are luminal, 20% are HER2
+
and 30% are TNBC. The major differences in ctDNA between two stages lie in several genes including PIK3CA, ERBB2 and KRAS. On the top of the list is PIK3CA, which is detected in 2 out of 33 stage III patients (1 luminal, 1 HER2
+
) (6.06%) in baseline, each of them carries 1 mutation on PIK3CA (E542K, E545G). In 43 out of 204 stage IV patients (21.57%) who carry this gene, 15 show 1 amplification, 34 have 1 mutation (mainly H1047R, E542K, E545K and H1047L), 11 have 2 mutations (E542K/E726K, D454N/D1029N, E545K/D1017H, E545K/L287L, H1047R/E453K, H1047R/N426S and P539R/H1047R), and 1 has 3 mutations (E542Q/D454N/D1029N and E545K/E726K/R93Q). On treatment effects, PIK3CA is found to be very detrimental on prognosis and on its effects on the treatment outcome. Patients without any mutation in PIK3CA live 2.65 times longer than those with more than one mutations on PIK3CA ( p-value = 4.47e-06, CI = 1.731, 3.926). PIK3CA, ESR1, TP53 and ARID1A are found to significantly affect liver metastasis when RET, FBXW7, ERBB2, CCND2, BRAF and MET are found to be associated with lung metastasis for both stages. EGFR, KIT and ARID1A are associated with CNS metastasis. Conclusions: We elucidated that ctDNA mutations on PIK3CA and other genes dramatically increased in Stage IV patients compared to Stage III patients which provides a new insight on the Stage III and Stage IV MBC determination. New set of genes especially PIK3CA are identified to correlate with metastasis and affect the outcome which may also be reliably used to monitor the response to therapy.
Abstract only
1059
Background: Metastatic breast cancer (MBC) is a heterogeneous disease associated with known somatic mutations of variable biological value in different subtypes. Furthermore, the ...clinical evolution of the disease demonstrates clonal evolution resulting in disease resistance more accurately detected using blood-based sequencing. Few studies have explored differences in genomic features of tumors across populations. Here, we performed circulating tumor DNA (ctDNA) sequencing to compare the genomic landscape of patients with hormone-receptor positive MBC at time of first recurrence or de-novo metastatic diagnosis in the United States (US) and China. Methods: Twenty-three US patients from Northwestern University and 65 Chinese patients from Peking University had ctDNA sequencing from plasma performed using the harmonized CLIA-certified, 152-gene PredicineCARE assay in laboratories in the US and China, respectively. The data analysis was conducted in China. Institutional Review Boards at each site approved the study. Fisher’s exact test was performed to compare mutational frequencies across populations. Results: Median age of patients at MBC diagnosis was 51 in the US cohort and 55 in the Chinese cohort. 87% of US patients and 82% of Chinese patients had received prior therapy for primary breast cancer, including endocrine therapy. Mutations were detected in 17 of 23 (74%) US patients and 59 of 65 (91%) Chinese patients. CNAs were observed in 57% of US patients and 58% of Chinese patients. The most common mutations detected in US patients were TP53 (26%), PIK3CA (22%), AKT1 (22%), CDH1 (17%), PTEN (13%), and ESR1 (9%) vs. PIK3CA (46%), TP53 (35%), ESR1 (12%), and BRCA2 (11%) in Chinese patients. Frequency of AKT1 and CDH1 mutations were significantly higher in the US population (P < 0.05), while PIK3CA mutations were higher in the Chinese population (P < 0.05). CNA gains in CCND3 and CDK4 were significantly higher in the US cohort, and FGFR1 was significantly more common in the Chinese cohort (all P < 0.05). Conclusions: To our knowledge, this is a first cross-regional comparison study in HR+ MBC patients in the US and China using a harmonized cfDNA NGS platform. At a population level, there were notable differences observed in somatic variants in two cohorts. Future sequencing efforts and clinical trials should include patients of diverse ethnic backgrounds to explore the impact of differences in genomic landscape on probability of benefit from treatments. A larger validation cohort is required to confirm these findings.
Most circulating tumor cells (CTC) are detected as single cells, whereas a small proportion of CTCs in multicellular clusters with stemness properties possess 20- to 100-times higher metastatic ...propensity than the single cells. Here we report that CTC dynamics in both singles and clusters in response to therapies predict overall survival for breast cancer. Chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of ST6GAL1-catalyzed α2,6-sialylation in glycoproteins. Dynamic hyposialylation in CTCs or deficiency of ST6GAL1 promotes cluster formation for metastatic seeding and enables cellular quiescence to evade paclitaxel treatment in breast cancer. Glycoproteomic analysis reveals newly identified protein substrates of ST6GAL1, such as adhesion or stemness markers PODXL, ICAM1, ECE1, ALCAM1, CD97, and CD44, contributing to CTC clustering (aggregation) and metastatic seeding. As a proof of concept, neutralizing antibodies against one newly identified contributor, PODXL, inhibit CTC cluster formation and lung metastasis associated with paclitaxel treatment for triple-negative breast cancer.
This study discovers that dynamic loss of terminal sialylation in glycoproteins of CTC clusters contributes to the fate of cellular dormancy, advantageous evasion to chemotherapy, and enhanced metastatic seeding. It identifies PODXL as a glycoprotein substrate of ST6GAL1 and a candidate target to counter chemoevasion-associated metastasis of quiescent tumor cells. This article is featured in Selected Articles from This Issue, p. 1949.
PDF
