Olympus Molecular Biology Products Revolutionises Cancer Care

Enrich
The bottleneck in analysing circulating tumour cells (CTC) is the ability to enrich them in a way that allows for their molecular analysis (PCR) in comparison to “normal” cells. With existing techniques a 100% enrichment is not possible. Researchers usually end up with a ratio of 1 CTC to every 20-50 normal cells. This does not allow for traditional molecular analysis using PCR as there has to be more than one cell to produce enough starting template for reliable results in a high volume (10-25µL) microplate system. Therefore analysis of these mixtures would be of no prognostic value and an alternative approach has to be used.

With the Advalytix AmpliGrid system it is now possible to run systematic studies on CTCs, as one single cell provides sufficient template for consistently successful PCR. To do this, 48 single cells are deposited in separate positions on one AmpliGrid AG480Fslide and analysed individually. The relevant reagents for PCR or RT-PCR as well as sealing solution are added and the slide is placed on the AmpliSpeed amplification system (a thermal cycler for glass slides) with the required temperature profile. The unique surface coating and 2D structure of the AmpliGrid slide enable accurate and controllable placement of the cells, making it the only platform available for running reliable and successful single cell amplifications.

Analyse
Using single cell PCR analysis, the cells deposited on the AmpliGrid can be ‘typed’ (e.g. via epithelial antigens or cytokeratins) and screened quantitatively for diagnostic and/or prognostic markers at the DNA and RNA level. In addition to marker detection and analysis of expression levels, the number of CTCs per mL of blood also has a prognostic importance. With the AmpliGrid system, the ratio of CTCs to normal cells can be rapidly determined in native samples of peripheral blood.

Test
The functional testing of cells (e.g. morphological analysis) requires live cells after the CTC selection/enrichment process. The AmpliGrid system provides the ability to perform such tests even after cultivation and expansion on the chip. Advalytix offers the Automated cell detection software upgrade module for existing Olympus microscopes running the cell^P software. This enables automated screening for cells on the reaction sites of the AmpliGrid.

In the Clinic
Prof. Dr. med. Katharina Pachmann, from the Experimental Haematology and Oncology group at the University Hospital of Jena, has been investigating circulating tumour cells in breast cancer patients for a long time. It has become clear that nearly all patients with this disorder show circulating epithelial cells in their peripheral blood and that these can still be detected years after successful treatment. In addition, the AmpliGrid system is being used to characterise single cells on the molecular level even further than previously possible, with the goal to distinguish between aggressive and non-aggressive forms of those circulating cells.

Presently, several hundred patients have consented to have their CTCs monitored during their chemotherapy. This is done by taking an additional blood sample which is processed by Prof Pachmann’s group. The first single cell analysis takes place before the initial chemotherapy session. Depending on the number of CTCs detected (between 10 and 10000/mL) several hundred single cells will be labelled by a fluorescently tagged antibody and sorted to the AmpliGrid slide via the CellManipulator system from Olympus. Those cells are analysed using single cell multiplex PCR for the expression of selected genes. During chemotherapy, this process will be repeated periodically.

The results of this study (several 1000 single cells) will then be integrated in the routine therapy monitoring at the University Hospital of Jena.