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, Jun 2014

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.

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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

Glenn Deng 0 1 3 Sujatha Krishnakumar 2 3 Ashley A Powell 0 3 5 Haiyu Zhang 0 3 4 Michael N Mindrinos 2 3 Melinda L Telli 3 Ronald W Davis 2 3 Stefanie S Jeffrey 0 3 0 Division of Surgical Oncology, Stanford University School of Medicine , Stanford, CA 94305 , USA 1 College of Life Science and Chemistry, Wuhan Donghu University , Wuhan , P. R. China 2 Stanford Genome Technology Center, Stanford University School of Medicine , Palo Alto, CA 94304 , USA 3 of Medical Oncology, Stanford University School of Medicine , Stanford, CA 94305 , USA 4 Department of Pathology, Stanford University School of Medicine , Stanford, CA 94305 , USA 5 Division of Gynecologic Oncology, Stanford University School of Medicine , Stanford, CA 94305 , USA 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. (Continued on next page) - (Continued from previous page) 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. Background Clinical use of some newer or investigational drug therapies in cancer requires that primary tumors be assayed for specific mutations associated with response or lack of response [1-5]. However, not only are primary tumors known to be mutationally heterogeneous [6-8], new mutations may become apparent in recurrent tumors, emerging during disease progression [9-11]. Yet sequential biopsy and evaluation of molecular biomarkers and mutations in metastases is not routinely done, even during clinical trials [11], largely due to the multiplicity and internal location of many metastases (such as liver, lung, and/or brain metastases in breast cancer), and potential morbidity associated with sequential biopsy. An appealing alternative is a liquid biopsy with CTC capture and characterization [12,13]. As they are easily accessible by simple blood draw, CTCs can be sequentially sampled at multiple time points during the course of disease for biomarker or genotype determination. Moreover, it is hoped, but not ascertained, that CTCs represent mixtures of tumor cells that reflect the full spectrum of molecular phenotypes and genotypes present in multiple metastases. Following a slightly different tack, some groups are also investigating biomarker and genetic characteristics of DTCs from bone marrow [14-16], which are postulated to serve as a reservoir for active and dormant tumor cells [12,17]. However, genetic analyses of mutations in CTCs and DTCs are still in an early discovery stage, having been done on few patients, so clinical significance and utility is postulated but remains unproven. Moreover, it is not known whether CTC analysis can replace DTC analysis for there is, as yet, incomplete understanding of the relationship between these two populations [18]. In the present study, we used a previously des (...truncated)


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Glenn Deng, Sujatha Krishnakumar, Ashley A Powell, Haiyu Zhang, Michael N Mindrinos, Melinda L Telli, Ronald W Davis, Stefanie S Jeffrey. 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, 2014, pp. 456, 14, DOI: 10.1186/1471-2407-14-456