Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow

BMC Cancer

Glenn Deng²  Sujatha Krishnakumar⁴  Ashley A Powell⁵  Haiyu Zhang³  Michael N Mindrinos⁴  Melinda L Telli¹  Ronald W Davis⁴  Stefanie S Jeffrey² 

[1] Division of Medical Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA

[2] Division of Surgical Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA

[3] Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA

[4] Stanford Genome Technology Center, Stanford University School of Medicine, Palo Alto, CA 94304, USA

[5] Division of Gynecologic Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA

关键词: Single cell analysis;    PIK3CA;    Mutation analysis;    Heterogeneity;    Disseminated tumor cells (DTCs);    Circulating tumor cells (CTCs);    Cancer cell culture;   

Others  :  855650
DOI  :  10.1186/1471-2407-14-456

 received in 2013-02-12, accepted in 2014-05-28,  发布年份 2014

【 摘 要 】

Background

Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes.

Methods

We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients’ primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations.

Results

Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture.

Conclusions

Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.

【 授权许可】

   
2014 Deng et al.; licensee BioMed Central Ltd.

【 图 表 】

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