Characterization Of Disseminated Tumor Cells Research Articles

Microfluidic Isolation of Disseminated Tumor Cells from the Bone Marrow of Breast Cancer Patients.

Disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients are putative precursors of metastatic disease, and their presence is associated with an adverse clinical outcome. To achieve the personalization of therapy on a clinical routine level, the characterization of DTCs and in vitro drug testing on DTCs are of great interest. Therefore, biobanking methods, as well as novel approaches to DTC isolation, need to be developed. In this study, we established a protocol for the biobanking of BM samples and evaluated a microfluidic-based separation system (Parsortix®) for the enrichment of cryopreserved DTCs. We were able to successfully isolate viable DTCs after the prior cryopreservation of BM samples. We calculated a significant increase of up to 90-fold in harvested DTCs with the proposed method compared to the current standard techniques, opening up new analysis possibilities for DTCs. Our advanced method further presents options for 3D DTC cultures, enabling the individualized testing of targeted therapies for BC patients. In conclusion, we present a novel approach for DTC enrichment, with possibilities for future clinical implications.

Abstract 5578: Genomic and expression profiling reveals molecular heterogeneity of disseminated tumor cells in early breast cancer

Abstract BACKGROUND: The presence of disseminated tumor cells (DTCs) in bone marrow of early breast cancer (EBC) patients is a strong predictor of poor prognosis. We assessed heterogeneity in copy number, PIK3CA mutation, and gene expression in DTCs to shed light on the molecular biology of these cells. METHODS: We isolated EPCAM-positive DTCs in 71 EBC patients using immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). Isolated DTCs, along with corresponding primary tumors (n=16), were subjected to genome-wide copy number profiling (n=47) by array comparative genomic hybridization, and mutation screening of the PIK3CA gene (n=53) by Sanger sequencing. The expression of 64 cancer-related genes in DTCs was analyzed by microfluidic-based multiplexed RT-QPCR (n=30). DTC expression profiles were compared with available gene expression data from circulating tumor cells (CTCs) of metastatic breast cancer patients. RESULTS: Copy number profiles of DTCs were less aberrant and distinct from their corresponding primary tumors. PIK3CA mutations detected in 26% of DTCs were mutually exclusive to those found in matched primary tumors. Expression profiles of DTCs were distinguishable from marrow leukocytes, and displayed up-regulation of oncogenes MYC and CCNE1. Unsupervised hierarchical clustering analysis revealed two subtypes of DTCs: (1) luminal with dual epithelial-mesenchymal properties (high ESR1 and VIM/CAV1 expression), and (2) basal-like with proliferative/stem cell-like phenotype (low ESR1 and high MKI67/ALDH1A1 expression). DTCs possessed gene expression signatures that were unique from those of CTCs. ALDH1A1, CAV1 and VIM were up-regulated in DTCs relative to CTCs. ESR1/ER and ERBB2/HER2 status in DTCs vs. corresponding primary tumors showed high discordance (40% and 43%, respectively), suggesting shift in biomarker status in micrometastatic cells in the bone marrow. CONCLUSIONS: We demonstrate the feasibility of isolation and comprehensive molecular characterization of DTCs from bone marrow of EBC patients. Comparative genomic analysis suggests that DTCs disseminate early and acquire genomic aberrations independently of the primary tumor. Expression profiling revealed two distinct subpopulations of DTCs. Validation in larger cohorts is needed to confirm the presence of these molecular subtypes and to evaluate their biological and clinical significance. Citation Format: Mark Jesus M. Magbanua, Hope Rugo, Louai Hauranieh, Ritu Roy, Janet Scott, Jen Chieh Lee, Feng Hsiao, Eduardo Sosa, Denise Wolf, Laura van't Veer, Laura Esserman, John Park. Genomic and expression profiling reveals molecular heterogeneity of disseminated tumor cells in early breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5578.

Abstract 3082: Isolation and identification of disseminated tumor cells from bone marrow

Abstract Background: Disseminated tumor cells (DTCs), detected by immunohistological staining for cytokeratins, found in the bone marrow (BM) of breast cancer patients portend a poor prognosis. Study of these cells has been hindered by their rarity in BM specimens. We have tested a microfiltration technique for the enrichment of DTCs. Methods: BM aspirates from healthy volunteers were collected and mixed with defined numbers SKBR3 breast cancer cells at a concentration of 1,000 to 10,000 tumor cells per 1x10^6 nucleated BM cells., BM was then diluted 1:10 with PBS and 7 ml was placed in CellSaveTM tubes to simulate a patient BM collection. Each specimen was processed within 24 hours of collection. Specimens were pre-filtered using a 70 µm mesh sieve (BD cat#352350) to remove large particles. The effluent was filtered through CellSieveTM microfilters, which have 7 µm diameter pores in a uniform array, of 160,000 pores in a 9 mm diameter area. Filters containing cells were post-fixed, cells permeabilized, and stained with DAPI and fluorescent antibodies specific to epithelial cytokeratins 8, 18, and 19 (FITC), EpCAM (PE), and CD45, a negative control (Cy5). Five representative 1% areas of each filter were imaged and used to estimate total cell and tumor cell counts, based on fluorescent DAPI and cytokeratin antibody staining. Results: From each BM specimen (7 ml) containing a total of 14x10^6 nucleated BM cells, post filtration samples contained only 17-21x10^3 residual, nucleated BM cells. For specimens spiked at 10 tumor cells per 1,000 BM cells, approximately 30x10^3 positively staining tumor cells were recovered (20% tumor cell recovery with approximately 170 fold enrichment). For specimens spiked at 1 tumor cell per 1,000 BM cells, approximately 7x10^3 total tumor cells were recovered (50% tumor cell recovery with approximately 400-fold enrichment). Captured cells co-stained with cytokeratins and EpCAM, but not CD45. Control BM specimens with no added tumor cells demonstrated only rare, staining cells (4 per sample), likely due to background or epithelial cell contamination. Conclusions: We have tested a microfiltration technique for enrichment of BM DTCs. Microfiltration of BM is rapid without filter clogging. Recovered DTCs can be immuno-stained, and are easily identified and distinguished from BM cells. The DTC capture efficiency varied with cancer cell concentration and ranged from 20-50% with up to 400 fold enrichment when samples contained lower numbers of tumor cells. The microfiltration enrichment assay continues to be optimized for isolation and molecular characterization of DTCs from patient BM samples, where the concentration of DTCs seldom exceeds 10 cells per 1x10^6 BM cells. Citation Format: Peixuan Zhu, Daniel Adams, Rebecca L. Aft, Sreeraj G. Pillai, Mark A. Watson, Shuhong Li, Olga V. Makarova, Platte T. Amstutz, Cha-Mei Tang. Isolation and identification of disseminated tumor cells from bone marrow. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3082. doi:10.1158/1538-7445.AM2014-3082

P4-06-03: Multiplex Gene Expression of Disseminated Tumor Cells in the Bone Marrow of Breast Cancer Patients Identifies Novel Therapeutic Targets.

Abstract Background: Disseminated tumor cells (DTCs) are detected in the bone marrow (BM) of up to 40% of breast cancer patients at the time of diagnosis and are an independent prognostic factor for recurrent disease. Present techniques for detection of DTC are often laborious, and insensitive due to the molecular heterogeneity of the DTCs. We have previously optimized and validated a novel, multiplexed gene expression technology platform, Nanostring nCounter™ (NC) which counts single molecules of RNA, for the multi-marker detection of DTCs in BM at a sensitivity of 1 cancer cell per 1 million nucleated BM cells. We now validate a 36 gene panel for the detection and molecular characterization of DTCs in BM. Methods: Hybridization probes for 36 genes whose expression are associated with breast cancer, metastasis, and/or the cancer stem cell phenotype, and which exhibit no or low expression levels in normal bone marrow by qRT-PCR were developed for the NC assay. Total RNA was isolated from whole BM collected from the right and left iliac crest from breast cancer patients and healthy volunteers. 5 ug of RNA was analyzed, in duplicate with the NC assay. BM was scored positive for expression of an individual gene if expression in duplicate samples was 2 standard deviations above mean expression in a set of 11 independent normal BM samples. Results: Bilateral BM samples were analyzed prior to any therapy from 20 patients: 8 developed metastatic disease within 2–48 months (mean of 23 months) after diagnosis, and 12 had no evidence of metastatic disease with 3–5 years follow-up. Overall, expression of at least one gene in the 36-gene multi-marker panel was detected in 17 patients (85%). There was excellent correlation between individual gene expression in both the right and left iliac crest samples from the same patient. Six of the 8 patients (75%) who developed metastatic disease had detectable expression of 1–3 genes. Two genes were commonly associated with metastatic disease development. 50% (3 of 6) of the patients who had detectable expression of EBB2 in their BM developed metastatic disease, although this did not correlate with expression in the corresponding primary tumor from the same patient. 80% (4 of5) of the patients who expressed the hedgehog pathway gene, Ptch1, in their BM developed metastatic disease. Conclusions: Our data demonstrate the feasibility of using a 36-plex NC assay to detect gene expression associated with BM DTCs in breast cancer patients. We found expression of 2 targetable genes associated with the development of metastatic disease, ERBB2 and Ptch1. ERBB2 expression in BM did not correlate with expression in the primary tumor. The molecular diversity of gene expression observed underscores the need for a multiplexed gene expression panel. Ongoing studies are evaluating the clinical utility of this assay to detect DTCs relative to existing techniques, for predicting relapse-free survival, molecular classification, and selecting appropriate targeted therapeutics based on BM DTC profiles in breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-06-03.

Insights into minimal residual disease in cancer patients: Implications for anti-cancer therapies

Tumor cell dissemination appears even in patients with small solid tumors, and bone marrow (BM) is a common homing organ for disseminated tumor cells (DTC) derived from various types of primary epithelial tumors. Tumor cells are frequently detected in the BM of cancer patients without any clinical or even histopathological signs of overt metastases. It is crucial, however, to improve and standardize methods for the detection of DTC. The characterization of DTC has shed new light on the process underlying early tumor cell dissemination and metastatic progression in cancer patients. Characterization of DTC should help to identify novel targets for biological therapies aimed at preventing metastatic relapse and to monitor the efficacy of these therapies. Evidence has emerged that the detection of DTC and circulating tumor cells (CTC) in blood may provide important prognostic information and, in addition, might help to monitor the efficacy of therapy. In this article, we summarize the clinical background and the technical aspects of current methods used for the detection and characterization of DTC in BM and CTC in blood, with a special focus on breast cancer.

Review: Biological relevance of disseminated tumor cells in cancer patients

The prognosis of cancer patients is largely determined by the occurrence of distant metastases. In patients with primary tumors, this relapse is mainly due to clinically occult micrometastasis present in secondary organs at primary diagnosis but not detectable even with high resolution imaging procedures. Sensitive and specific immunocytochemical and molecular assays enable the detection and characterization of disseminated tumor cells (DTC) at the single cell level in bone marrow (BM) as the common homing site of DTC and circulating tumor cells (CTC) in peripheral blood. Because of the high variability of results in DTC and CTC detection, there is an urgent need for standardized methods. In this review, we will focus on BM and present currently available methods for the detection and characterization of DTC. Furthermore, we will discuss data on the biology of DTC and the clinical relevance of DTC detection. While the prognostic impact of DTC in BM has clearly been shown for primary breast cancer patients, less is known about the clinical relevance of DTC in patients with other carcinomas. Current findings suggest that DTC are capable to survive chemotherapy and persist in a dormant nonproliferating state over years. To what extent these DTC have stem cell properties is subject of ongoing investigations. Further characterization is required to understand the biology of DTC and to identify new targets for improved risk prevention and tailoring of therapy. Our review will focus on breast, colon, lung, and prostate cancer as the main tumor entities in Europe and the United States.

Micrometastatic spread in breast cancer: detection, molecular characterization and clinical relevance

Immunocytochemical or molecular assays allow the detection of single disseminated tumor cells (DTCs) in the bone marrow (BM) or the peripheral blood in 10% to 60% of breast cancer patients without signs of metastasis. Results from recently reported studies suggest that circulating tumor cell (CTC) levels may serve as a prognostic marker and be used for early assessment of therapeutic response in patients with metastatic breast cancer. In early stage breast cancer, however, the impact of CTCs is less well established than that of DTCs in BM, where several clinical studies demonstrated that such cells are an independent prognostic factor at primary diagnosis. The characterization of DTCs/CTCs has already shed new light on the complex process underlying early tumor cell dissemination and metastatic progression in cancer patients. Characterization of DTCs should help to identify novel targets for biological therapies aimed to prevent metastatic relapse. In addition, understanding tumor 'dormancy' and identifying metastatic stem cells might result in the development of new therapeutic concepts.