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Classification of Cells in CTC-Enriched Samples by Advanced Image Analysis.

Sanne de Wit1, Leonie L Zeune2,3, T Jeroen N Hiltermann4

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Summary

This study used the ACCEPT program to analyze nucleated cells in blood samples from metastatic non-small cell lung cancer (NSCLC) patients. The analysis revealed significantly more nuclei in patients than controls, but the origin of many cells remains unknown.

Keywords:
ACCEPTCellSearch®EpCAMcirculating tumor cellsclassificationdeep Learningimage analysisleukocytesnon-small cell lung cancer

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Area of Science:

  • Oncology
  • Cell Biology
  • Medical Technology

Background:

  • The CellSearch system enriches for epithelial cell adhesion molecule (EpCAM)-positive cells but limits the analysis of other cell types.
  • Undetected circulating tumor cells (CTCs) may be missed by current detection methods, impacting patient prognostication.

Purpose of the Study:

  • To utilize the open-source Automatic CTC Classification, Enumeration and PhenoTyping (ACCEPT) program for comprehensive analysis of all nucleated cells in EpCAM-enriched blood samples.
  • To compare cell counts between metastatic non-small cell lung cancer (NSCLC) patients and healthy controls.
  • To investigate methods for improving the classification and identification of EpCAM-enriched cells.

Main Methods:

  • Analysis of 4',6-diamidino-2-phenylindole (DAPI)-positive nuclei in EpCAM-enriched blood samples from 192 metastatic NSCLC patients and 162 controls using the ACCEPT program.
  • Initial cell classification based on DAPI, Cytokeratin-PE (CK), and CD45-APC staining.
  • Subsequent experiments incorporated CD16-PerCP for granulocyte staining, LED excitation for CD45-APC, and wheat germ agglutinin for plasma membrane staining to enhance cell classification.

Main Results:

  • Significantly higher numbers of DAPI+ nuclei were detected in patient samples (73,570 ± 74,948) compared to controls (4191 ± 4463) (p < 0.001).
  • Leukocytes or CTCs constituted only 18% ± 21% in patients and 23% ± 15% in controls.
  • Enhanced staining protocols improved cell identification to 94% ± 5%, but the origin of unidentified cells, particularly in patients, remained unknown.

Conclusions:

  • The ACCEPT program enables a more comprehensive analysis of nucleated cells in EpCAM-enriched blood compared to the standard CellSearch system.
  • While cell counts are significantly higher in NSCLC patients, a substantial population of unidentified cells persists.
  • Further research is required to determine if undetected EpCAM+/DAPI+/CK-/CD45- CTCs are present within this unidentified cell population.