Limits...
A novel approach for the detection and genetic analysis of live melanoma circulating tumor cells.

Xu MJ, Cooke M, Steinmetz D, Karakousis G, Saxena D, Bartlett E, Xu X, Hahn SM, Dorsey JF, Kao GD - PLoS ONE (2015)

Bottom Line: Circulating tumor cell (CTC) detection and genetic analysis may complement currently available disease assessments in patients with melanoma to improve risk stratification and monitoring.Tumor cells are then identified via an image processing system.These promising findings support further studies, including towards integrating into the management of patients with melanoma receiving multimodality therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: Circulating tumor cell (CTC) detection and genetic analysis may complement currently available disease assessments in patients with melanoma to improve risk stratification and monitoring. We therefore sought to establish the feasibility of a telomerase-based assay for detecting and isolating live melanoma CTCs.

Methods: The telomerase-based CTC assay utilizes an adenoviral vector that, in the presence of elevated human telomerase activity, drives the amplification of green fluorescent protein. Tumor cells are then identified via an image processing system. The protocol was tested on melanoma cells in culture or spiked into control blood, and on samples from patients with metastatic melanoma. Genetic analysis of the isolated melanoma CTCs was then performed for BRAF mutation status.

Results: The adenoviral vector was effective for all melanoma cell lines tested with sensitivity of 88.7% (95%CI 85.6-90.4%) and specificity of 99.9% (95%CI 99.8-99.9%). In a pilot trial of patients with metastatic disease, CTCs were identified in 9 of 10 patients, with a mean of 6.0 CTCs/mL. At a cutoff of 1.1 CTCs/mL, the telomerase-based assay exhibits test performance of 90.0% sensitivity and 91.7% specificity. BRAF mutation analysis of melanoma cells isolated from culture or spiked control blood, or from pilot patient samples was found to match the known BRAF mutation status of the cell lines and primary tumors.

Conclusions: To our knowledge, this is the first report of a telomerase-based assay effective for detecting and isolating live melanoma CTCs. These promising findings support further studies, including towards integrating into the management of patients with melanoma receiving multimodality therapy.

No MeSH data available.


Related in: MedlinePlus

Integration of the viral probe with a semi-automated, computer-driven image acquisition and analysis system.(A) Melanoma cells (A375P cells shown here as a representative example) incubated with the probe for 24 hours were visualized under fluorescent microscopy. Tiled images were taken for each well. Hoechst dye was added before image acquisition to delineate nucleated cells, which take up the dye and emit blue fluorescent signal under UV light. Bar, 30um. (B) Scatter plots showing individual cells, plotting size (X-axis) and fluorescent intensity (standard deviations (SD) above background) (Y-axis) identified by the imaging program. Control blood studies from a single subject with spiked melanoma cells (middle graph) or without spiked cells (left graph) demonstrated that a stringent GFP intensity cutoff (black dotted line) excluded false positive signals and contributed to greater specificity for patient samples. The size and fluorescent intensify cutoffs were applied to the blood samples of patients with metastatic melanoma (data from one representative patient is shown in the right graph). Color choices are for aesthetic purposes only. Melanoma cells in culture (middle panel, showing 60% of the total cells identified by the imaging program, not meant to reflect recovery rate of spiked melanoma cells) rapidly adhere to the culture surface and hence show greater variability in size compared to the CTCs from patients with melanoma (right panel). (C) Confirmation of melanoma origin of CTCs. Patient samples were fixed after exposure to probe, and immunofluorescence then performed for Melan-A and DAPI. The DAPI+/GFP+/Melan-A+ (arrows) cells confirm the identification of melanoma CTCs while the adjacent DAPI+ only cells (arrowheads) consist of white blood cells. Bar, 30 um.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4373770&req=5

pone.0123376.g002: Integration of the viral probe with a semi-automated, computer-driven image acquisition and analysis system.(A) Melanoma cells (A375P cells shown here as a representative example) incubated with the probe for 24 hours were visualized under fluorescent microscopy. Tiled images were taken for each well. Hoechst dye was added before image acquisition to delineate nucleated cells, which take up the dye and emit blue fluorescent signal under UV light. Bar, 30um. (B) Scatter plots showing individual cells, plotting size (X-axis) and fluorescent intensity (standard deviations (SD) above background) (Y-axis) identified by the imaging program. Control blood studies from a single subject with spiked melanoma cells (middle graph) or without spiked cells (left graph) demonstrated that a stringent GFP intensity cutoff (black dotted line) excluded false positive signals and contributed to greater specificity for patient samples. The size and fluorescent intensify cutoffs were applied to the blood samples of patients with metastatic melanoma (data from one representative patient is shown in the right graph). Color choices are for aesthetic purposes only. Melanoma cells in culture (middle panel, showing 60% of the total cells identified by the imaging program, not meant to reflect recovery rate of spiked melanoma cells) rapidly adhere to the culture surface and hence show greater variability in size compared to the CTCs from patients with melanoma (right panel). (C) Confirmation of melanoma origin of CTCs. Patient samples were fixed after exposure to probe, and immunofluorescence then performed for Melan-A and DAPI. The DAPI+/GFP+/Melan-A+ (arrows) cells confirm the identification of melanoma CTCs while the adjacent DAPI+ only cells (arrowheads) consist of white blood cells. Bar, 30 um.

Mentions: These encouraging results enabled the integration of the viral probe with a semi-automated, computer-driven image acquisition and analysis system (Fig. 2A, S2A Fig.). This system has been described in previous publications and incorporates reproducible cell identification and imaging, as well as filters for size and fluorescence, so that that cellular debris and cells with weak fluorescence are excluded from analysis [25–27]. As an additional validation step prior to testing patient samples, we performed control experiments in which peripheral blood samples from healthy volunteers were analyzed alone or spiked with melanoma cells, and in each case exposed to the probe. In the control blood without melanoma cells, the viral probe resulted in almost no fluorescence (specificity of 99.9% [95%CI 99.8%-99.9%]) (Fig. 2B, left panel). In contrast, robust fluorescence was detected in the control blood spiked with melanoma cells (Fig. 2B, middle panel).


A novel approach for the detection and genetic analysis of live melanoma circulating tumor cells.

Xu MJ, Cooke M, Steinmetz D, Karakousis G, Saxena D, Bartlett E, Xu X, Hahn SM, Dorsey JF, Kao GD - PLoS ONE (2015)

Integration of the viral probe with a semi-automated, computer-driven image acquisition and analysis system.(A) Melanoma cells (A375P cells shown here as a representative example) incubated with the probe for 24 hours were visualized under fluorescent microscopy. Tiled images were taken for each well. Hoechst dye was added before image acquisition to delineate nucleated cells, which take up the dye and emit blue fluorescent signal under UV light. Bar, 30um. (B) Scatter plots showing individual cells, plotting size (X-axis) and fluorescent intensity (standard deviations (SD) above background) (Y-axis) identified by the imaging program. Control blood studies from a single subject with spiked melanoma cells (middle graph) or without spiked cells (left graph) demonstrated that a stringent GFP intensity cutoff (black dotted line) excluded false positive signals and contributed to greater specificity for patient samples. The size and fluorescent intensify cutoffs were applied to the blood samples of patients with metastatic melanoma (data from one representative patient is shown in the right graph). Color choices are for aesthetic purposes only. Melanoma cells in culture (middle panel, showing 60% of the total cells identified by the imaging program, not meant to reflect recovery rate of spiked melanoma cells) rapidly adhere to the culture surface and hence show greater variability in size compared to the CTCs from patients with melanoma (right panel). (C) Confirmation of melanoma origin of CTCs. Patient samples were fixed after exposure to probe, and immunofluorescence then performed for Melan-A and DAPI. The DAPI+/GFP+/Melan-A+ (arrows) cells confirm the identification of melanoma CTCs while the adjacent DAPI+ only cells (arrowheads) consist of white blood cells. Bar, 30 um.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4373770&req=5

pone.0123376.g002: Integration of the viral probe with a semi-automated, computer-driven image acquisition and analysis system.(A) Melanoma cells (A375P cells shown here as a representative example) incubated with the probe for 24 hours were visualized under fluorescent microscopy. Tiled images were taken for each well. Hoechst dye was added before image acquisition to delineate nucleated cells, which take up the dye and emit blue fluorescent signal under UV light. Bar, 30um. (B) Scatter plots showing individual cells, plotting size (X-axis) and fluorescent intensity (standard deviations (SD) above background) (Y-axis) identified by the imaging program. Control blood studies from a single subject with spiked melanoma cells (middle graph) or without spiked cells (left graph) demonstrated that a stringent GFP intensity cutoff (black dotted line) excluded false positive signals and contributed to greater specificity for patient samples. The size and fluorescent intensify cutoffs were applied to the blood samples of patients with metastatic melanoma (data from one representative patient is shown in the right graph). Color choices are for aesthetic purposes only. Melanoma cells in culture (middle panel, showing 60% of the total cells identified by the imaging program, not meant to reflect recovery rate of spiked melanoma cells) rapidly adhere to the culture surface and hence show greater variability in size compared to the CTCs from patients with melanoma (right panel). (C) Confirmation of melanoma origin of CTCs. Patient samples were fixed after exposure to probe, and immunofluorescence then performed for Melan-A and DAPI. The DAPI+/GFP+/Melan-A+ (arrows) cells confirm the identification of melanoma CTCs while the adjacent DAPI+ only cells (arrowheads) consist of white blood cells. Bar, 30 um.
Mentions: These encouraging results enabled the integration of the viral probe with a semi-automated, computer-driven image acquisition and analysis system (Fig. 2A, S2A Fig.). This system has been described in previous publications and incorporates reproducible cell identification and imaging, as well as filters for size and fluorescence, so that that cellular debris and cells with weak fluorescence are excluded from analysis [25–27]. As an additional validation step prior to testing patient samples, we performed control experiments in which peripheral blood samples from healthy volunteers were analyzed alone or spiked with melanoma cells, and in each case exposed to the probe. In the control blood without melanoma cells, the viral probe resulted in almost no fluorescence (specificity of 99.9% [95%CI 99.8%-99.9%]) (Fig. 2B, left panel). In contrast, robust fluorescence was detected in the control blood spiked with melanoma cells (Fig. 2B, middle panel).

Bottom Line: Circulating tumor cell (CTC) detection and genetic analysis may complement currently available disease assessments in patients with melanoma to improve risk stratification and monitoring.Tumor cells are then identified via an image processing system.These promising findings support further studies, including towards integrating into the management of patients with melanoma receiving multimodality therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: Circulating tumor cell (CTC) detection and genetic analysis may complement currently available disease assessments in patients with melanoma to improve risk stratification and monitoring. We therefore sought to establish the feasibility of a telomerase-based assay for detecting and isolating live melanoma CTCs.

Methods: The telomerase-based CTC assay utilizes an adenoviral vector that, in the presence of elevated human telomerase activity, drives the amplification of green fluorescent protein. Tumor cells are then identified via an image processing system. The protocol was tested on melanoma cells in culture or spiked into control blood, and on samples from patients with metastatic melanoma. Genetic analysis of the isolated melanoma CTCs was then performed for BRAF mutation status.

Results: The adenoviral vector was effective for all melanoma cell lines tested with sensitivity of 88.7% (95%CI 85.6-90.4%) and specificity of 99.9% (95%CI 99.8-99.9%). In a pilot trial of patients with metastatic disease, CTCs were identified in 9 of 10 patients, with a mean of 6.0 CTCs/mL. At a cutoff of 1.1 CTCs/mL, the telomerase-based assay exhibits test performance of 90.0% sensitivity and 91.7% specificity. BRAF mutation analysis of melanoma cells isolated from culture or spiked control blood, or from pilot patient samples was found to match the known BRAF mutation status of the cell lines and primary tumors.

Conclusions: To our knowledge, this is the first report of a telomerase-based assay effective for detecting and isolating live melanoma CTCs. These promising findings support further studies, including towards integrating into the management of patients with melanoma receiving multimodality therapy.

No MeSH data available.


Related in: MedlinePlus