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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210440	1/3	Homo sapiens	Blood plasma	(d)(U)C ExoQuick	König L	2017	38%
Study summary Full title Elevated levels of extracellular vesicles are associated with therapy failure and disease progression in breast cancer patients undergoing neoadjuvant chemotherapy. All authors König L, Kasimir-Bauer S, Bittner AK, Hoffmann O, Wagner B, Santos Manvailer LF, Kimmig R, Horn PA, Rebmann V Journal Oncoimmunology Abstract Extracellular vesicles (EVs) have been discussed as a diagnostic tool for minimal residual disease ( (show more...)Extracellular vesicles (EVs) have been discussed as a diagnostic tool for minimal residual disease (MRD) evaluation in breast cancer (BC) in addition to the analysis of circulating tumor cells (CTCs). Therefore, we investigated circulating EV levels as surrogate markers for disease monitoring and prediction of prognosis in primary, non-metastatic, locally advanced BC patients. EVs were enriched from blood samples of BC patients before and after neoadjuvant chemotherapy (NACT) and from healthy females. EV marker expression analysis was performed and EV sizes and concentrations were determined by nanoparticle tracking analysis. The results were associated with disease status, outcome and CTC presence, evaluated by gene expression analysis after enrichment. We demonstrated that i) the EV concentration was 40-fold higher in BC patients compared to healthy females, ii) the EV concentration increased during therapy, iii) an increased EV concentration pre-NACT was associated with therapy failure and iv) an elevated EV concentration post-NACT was associated with a reduced three-year progression-free and overall survival. Of note, residual stem cell-like and/or resistant CTCs after therapy were associated with a lower EV concentration post-NACT. Our study highlights that the concentration of EVs within BC blood samples may serve as a complementary parameter reflecting the status of MRD as well as therapy and disease outcome in parallel with CTC investigation. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: TSG101/ Synthenin/ CD63/ CD81/ HSP70/ CD9 non-EV: Cytochrome c Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ Synthenin/ TSG101/ HSP70/ CD81 Not detected contaminants Cytochrome c Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV210440	2/3	Homo sapiens	Blood plasma	(d)(U)C ExoQuick	König L	2017	38%
Study summary Full title Elevated levels of extracellular vesicles are associated with therapy failure and disease progression in breast cancer patients undergoing neoadjuvant chemotherapy. All authors König L, Kasimir-Bauer S, Bittner AK, Hoffmann O, Wagner B, Santos Manvailer LF, Kimmig R, Horn PA, Rebmann V Journal Oncoimmunology Abstract Extracellular vesicles (EVs) have been discussed as a diagnostic tool for minimal residual disease ( (show more...)Extracellular vesicles (EVs) have been discussed as a diagnostic tool for minimal residual disease (MRD) evaluation in breast cancer (BC) in addition to the analysis of circulating tumor cells (CTCs). Therefore, we investigated circulating EV levels as surrogate markers for disease monitoring and prediction of prognosis in primary, non-metastatic, locally advanced BC patients. EVs were enriched from blood samples of BC patients before and after neoadjuvant chemotherapy (NACT) and from healthy females. EV marker expression analysis was performed and EV sizes and concentrations were determined by nanoparticle tracking analysis. The results were associated with disease status, outcome and CTC presence, evaluated by gene expression analysis after enrichment. We demonstrated that i) the EV concentration was 40-fold higher in BC patients compared to healthy females, ii) the EV concentration increased during therapy, iii) an increased EV concentration pre-NACT was associated with therapy failure and iv) an elevated EV concentration post-NACT was associated with a reduced three-year progression-free and overall survival. Of note, residual stem cell-like and/or resistant CTCs after therapy were associated with a lower EV concentration post-NACT. Our study highlights that the concentration of EVs within BC blood samples may serve as a complementary parameter reflecting the status of MRD as well as therapy and disease outcome in parallel with CTC investigation. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Breast cancer patients before neoadjuvant chemotherapy Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: TSG101/ Synthenin/ CD63/ CD81/ HSP70/ CD9 non-EV: Cytochrome c Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ Synthenin/ TSG101/ HSP70/ CD81 Not detected contaminants Cytochrome c Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV210440	3/3	Homo sapiens	Blood plasma	(d)(U)C ExoQuick	König L	2017	38%
Study summary Full title Elevated levels of extracellular vesicles are associated with therapy failure and disease progression in breast cancer patients undergoing neoadjuvant chemotherapy. All authors König L, Kasimir-Bauer S, Bittner AK, Hoffmann O, Wagner B, Santos Manvailer LF, Kimmig R, Horn PA, Rebmann V Journal Oncoimmunology Abstract Extracellular vesicles (EVs) have been discussed as a diagnostic tool for minimal residual disease ( (show more...)Extracellular vesicles (EVs) have been discussed as a diagnostic tool for minimal residual disease (MRD) evaluation in breast cancer (BC) in addition to the analysis of circulating tumor cells (CTCs). Therefore, we investigated circulating EV levels as surrogate markers for disease monitoring and prediction of prognosis in primary, non-metastatic, locally advanced BC patients. EVs were enriched from blood samples of BC patients before and after neoadjuvant chemotherapy (NACT) and from healthy females. EV marker expression analysis was performed and EV sizes and concentrations were determined by nanoparticle tracking analysis. The results were associated with disease status, outcome and CTC presence, evaluated by gene expression analysis after enrichment. We demonstrated that i) the EV concentration was 40-fold higher in BC patients compared to healthy females, ii) the EV concentration increased during therapy, iii) an increased EV concentration pre-NACT was associated with therapy failure and iv) an elevated EV concentration post-NACT was associated with a reduced three-year progression-free and overall survival. Of note, residual stem cell-like and/or resistant CTCs after therapy were associated with a lower EV concentration post-NACT. Our study highlights that the concentration of EVs within BC blood samples may serve as a complementary parameter reflecting the status of MRD as well as therapy and disease outcome in parallel with CTC investigation. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Breast cancer patients after neoadjuvant chemotherapy Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: TSG101/ Synthenin/ CD63/ CD81/ HSP70/ CD9 non-EV: Cytochrome c Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ Synthenin/ CD81 Not detected contaminants Cytochrome c Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

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EV-TRACK ID	EV210440
species	Homo sapiens
sample type	Blood plasma
condition	Control condition	Breast cancer patients before neoadjuvant chemotherapy	Breast cancer patients after neoadjuvant chemotherapy
separation protocol	dUC/ ExoQuick	dUC/ ExoQuick	dUC/ ExoQuick
Exp. nr.	1	2	3
EV-METRIC %	38	38	38