Besides the size of the primary tumor and the presence or absence of metastasis, another criterion in the staging of malignant tumors is lymph node colonization. When assessing the prognosis of a patient with malignant melanoma, the status of the so-called sentinel lymph node is currently considered the most important factor. Detection even of very small cell nests or single tumor cells will have an impact on the therapy decision and has, therefore, been included in the AJCC staging system. Standardized methods for the isolation and molecular characterization of such tumor cells, however, are still lacking. But indeed, molecular characterization of disseminated tumor cells can provide important patient-specific clues to the efficacy of treatment options.

Fraunhofer ITEM scientists in Regensburg, therefore, have collaborated with the University Hospital Regensburg to further develop the isolation method used already by the latter for manual isolation of single tumor cells. The result is a semi-automated method based on the DEPArray^TM technology. Lymph node tissue is first disaggregated and purified to a single-cell suspension. Potential tumor cells are then fluorescence-stained by using two markers that are highly specific of malignant melanoma (MCSP and gp100). After depletion of immune cells that are also present in the lymph node, the tumor cells are loaded into a DEPArray^TM cartridge, allowing single cells to be captured and systematically isolated via dielectric pulses. In addition, all cells are measured and precisely documented by means of the integrated microscope with camera. Different selection parameters enable detailed comparison of the individual cells with regard to staining and morphological criteria.

To enable downstream molecular analyses of the isolated tumor cells, global amplification of single-cell genomes is performed by using a special method (Ampli1^TM) developed by the two collaborating institutions. This allows the DNA of isolated single tumor cells to be investigated, for example, for disease-specific point mutations or typical chromosomal alterations.

Thanks to the development of this semi-automated method for the detection and isolation of single disseminated melanoma cells in sentinel lymph nodes, a standardized method is now available for reliable detection of the clinically relevant minimum tumor burden and further analysis of relevant factors for treatment decisions. This makes it possible to apply a targeted and personalized treatment to melanoma patients whose tumor cells have already disseminated to the sentinel lymph node.