Abstract text (max. 300 words)Please use the following headings for the structure of your abstract: ObjectivesMethodsResultsConclusion
Objective: Minimal residual disease (MRD) after treatment is associated with reduced progression-free survival in myeloma. Detection of circulating tumor cells (CTC’s) could represent a non-invasive method to evaluate MRD. The aim of this study is to develop a sensitive and reliable technique to detect CTC’s.
Methods: In a series of spike-in experiments, we contaminated a fixed amount of peripheral blood mononuclear cells (PBMC’s) with LP-1 cells (human myeloma cell line) in decreasing concentrations. After a CD138-based double immunomagnetic enrichment-step (Auto MACS) we tried to detect the myeloma cells with allele-specific oligonucleotide polymerase chain reaction (qASO-PCR). We determined the efficiency of enrichment, the best method of DNA extraction and the optimal qASO-PCR conditions.
Results: Immunomagnetic cell separation allowed to enrich the tumor cells more than 150-fold. We used the QiAmp DNA extraction kit for all our samples. When paired samples (with equal starting numbers) were analyzed, there was no real significant difference between detection rates in unseparated versus enriched fractions (limit of detection 10-5). However, the tumor cell enrichment step depleted PBMC’s sufficiently to remain within the capacity limits of the Qiagen DNA extraction method and qASO-PCR. This allowed to start with higher cell numbers (108) and to increase the qASO-PCR detection level at least ten-fold (10-6).
Conclusion: Our data indicate that CTC enrichment prior to molecular tumor detection allows to analyze larger blood sample volumes while preserving the lower detection limit at the currently standard 10-6 level. Compared with the standard qASO-PCR technique with non-enriched cell suspensions, our method allows for more tumor cells to be analyzed. Future experiments have to reveal whether the enrichment efficiency of CTC (in terms of purity and yield) can be increased and whether the use of Ig targeted next-generation sequencing can further improve the tumor detection sensitivity in this already promising dual-platform strategy.

Immunomagnetic enrichment of circulating tumor cells prior to tumor Ig specific qASO-PCR enhances the sensivity of minimal residual disease detection in multiple myeloma.