Abstract P3-03-03: DNA Mutation as a Biomarker for Tracking Circulating Tumor Cells and Disseminated Tumor Cells in Breast Cancer Metastases
Authors: Deng G , Powell AA , Zhang H , Krishnakumar S , Telli ML , Herrler M , Mindrinos M , Davis RW , Jeffrey SS. 
Published: April 2011

Introduction: Most cancer deaths are caused by metastatic disease. Tumor cells exit the primary tumor and travel through blood and/or lymphatic channels to distant sites. Circulating tumor cells (CTCs) from blood and disseminated tumor cells (DTCs) from bone marrow may play key roles in the metastatic process. Primary tumor heterogeneity and how the metastatic process evolves over time may confound the study of metastatic biology. To better distinguish the cells of origin in metastases, we developed new methodologies to isolate single tumor cells from tissues, blood, and bone marrow for DNA and RNA analyses. We compared these analyses in individual cells from primary and metastatic tumors, CTCs and DTCs. Materials and Methods: Single tumor cell suspensions from primary and metastatic tissues were prepared from fresh human specimens or mouse xenografts. Single cells were isolated from blood, bone marrow, or single tumor cell suspensions from tissue using EpCAM-conjugated microbeads and the MagSweeper, a device invented by our group. Unbounded microbeads were removed and the single cells were placed into individual PCR tubes for further nucleic acid analyses. Live CTCs and DTCs from some breast cancer patients were cultured for further testing. Single cell DNA mutational analyses were performed by multiplex PCR sequencing. Single cell transcriptional profiling was performed by multiplex qRT-PCR. Single tumor cells with mutations were compared among multiple compartments: primary tumor, metastatic tumor, blood and bone marrow. Array CGH analysis was used to focus single cell analysis on genes in potential areas of DNA deletion or amplification. Xenograft mouse models were generated using primary or tumor tissues and single cells were compared.

Results: Single tumor cells from fresh or fixed solid tumor tissues were successfully isolated for nucleic acid analyses. CTCs and DTCs isolated by MagSweeper from breast cancer patients could be isolated live and cultured or injected for growth in xenografts. Single cell DNA mutation analysis of the PIK3CA gene was identified in 3/27 breast cancer patients, showing the mutation in only 45/178 single tumor cells. Sequential PIK3CA mutational analyses of CTCs, DTCs, and metastases showed that DTCs better reflected the mutational status of metastases at time points earlier in the metastatic process.

Discussion: Our results indicate that isolating single tumor cells from tumor tissues, blood, and bone marrow provides detailed information about different tumor compartments in individual cancer patients. Single cell DNA mutational analysis can provide a biomarker for tracking CTCs and DTCs over time during the metastatic process. Based on our initial findings, we postulate that the bone marrow serves as a tumor cell reservoir for early metastatic spread in breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-03-03.