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Bianca Mostert,
Yuqiu Jiang,
Anieta M Sieuwerts,
Haiying Wang, Joan Bolt-de Vries,
Katharina Biermann,
Jaco Kraan,
Zarina Lalmahomed,
Anne van Galen,
Vanja de Weerd,
Petra van der Spoel,
Raquel Ramírez-Moreno,
Cornelis Verhoef,
Jan N M Ijzermans,
Yixin Wang,
Jan-Willem Gratama,
John A Foekens,
Stefan Sleijfer,
John W M Martens
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ABSTRACT: Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch™. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature (COLD)-PCR (Transgenomic™), real-time PCR (EntroGen™), and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior. © 2012 Wiley Periodicals, Inc.
International Journal of Cancer 12/2012; · 5.44 Impact Factor
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Bianca Mostert,
Jaco Kraan,
Anieta M Sieuwerts,
Petra van der Spoel, Joan Bolt-de Vries,
Wendy J C Prager-van der Smissen,
Marcel Smid,
Annemieke M Timmermans,
John W M Martens,
Jan W Gratama,
John A Foekens,
Stefan Sleijfer
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ABSTRACT: Circulating tumor cells (CTCs) can be enumerated using CellSearch, but not all breast cancer subtypes, specifically those with epithelial-mesenchymal transition (EMT) characteristics, sufficiently express the enrichment (EpCAM) and selection (CK8/18/19) markers used in this method. While CD146 can detect EpCAM-negative CTCs, we here evaluated the value of various cytokeratins and CD49f to detect CK8/18/19-negative CTCs. The tested cytokeratins provided no substantial benefit, but adding CD49f to CK8/18/19 as a selection marker resulted in improved recovery of normal-like cell lines. Combined staining of CK8/18/19 and CD49f after CD146/EpCAM enrichment is likely to further improve CTC detection in breast cancer.
Cancer letters 12/2011; 319(1):49-55. · 4.86 Impact Factor
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Anieta M Sieuwerts,
Bianca Mostert, Joan Bolt-de Vries,
Dieter Peeters,
Felix E de Jongh,
Jacqueline M L Stouthard,
Luc Y Dirix,
Peter A van Dam,
Anne Van Galen,
Vanja de Weerd, [......],
Raquel Ramírez-Moreno,
Carolien H M van Deurzen,
Marcel Smid,
Jack X Yu,
John Jiang,
Yixin Wang,
Jan W Gratama,
Stefan Sleijfer,
John A Foekens,
John W M Martens
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ABSTRACT: Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer.
CTCs were isolated with the epithelial cell adhesion molecule-based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors.
We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels.
Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs.
Clinical Cancer Research 06/2011; 17(11):3600-18. · 7.74 Impact Factor
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Bianca Mostert,
Jaco Kraan, Joan Bolt-de Vries,
Petra van der Spoel,
Anieta M Sieuwerts,
Mieke Schutte,
Annemieke M Timmermans,
Renée Foekens,
John W M Martens,
Jan-Willem Gratama,
John A Foekens,
Stefan Sleijfer
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ABSTRACT: Most assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells. Recently, our group reported that in contrast to other molecular breast cancer subtypes, "normal-like" cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology [J Natl Cancer Inst 101(1):61-66, 2009]. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. CD146 and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch technology, 7.5 ml whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. Eight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146. Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. Cells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients.
Breast Cancer Research and Treatment 04/2010; 127(1):33-41. · 4.43 Impact Factor
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ABSTRACT: Detection of circulating tumor cells (CTCs) in whole blood from metastatic cancer patients by the CellSearch CTC Test (Veridex LLC, Warren, NJ, USA) has been shown to have clinical relevance. In addition to enumeration, there is great interest in molecular characterization of these CTCs. We aimed to establish a robust method to perform mRNA expression analysis of multiple genes by a real-time reverse transcriptase (RT)-PCR on small numbers of CTCs enriched from whole blood by the CellSearch system. Despite the 4 log depletion of leukocytes after CellSearch enrichment, the CTC-enriched fractions still contained leukocytes, in particular B-lymphocytes, which severely interfered with our CTC-specific gene expression profiling. After extensive washing and leukocyte-specific depletion by anti-CD45 coated magnetic beads prior to CellSearch enrichment, the number of leukocytes present in the enriched fraction was still high (range 60-929). However, by using a set of genes with no or minor expression by leukocytes, we succeeded to perform quantitative gene expression profiling specific for as little as one breast cancer CTC present in a CTC-enriched environment typically containing over 800 contaminating leukocytes. Our method allows molecular characterization specific for as little as one CTC, and can be used to expand the understanding of the biology of metastasis and, potentially, to improve patient management.
Breast Cancer Research and Treatment 01/2009; 118(3):455-68. · 4.43 Impact Factor
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Inko Nimmrich,
Anieta M Sieuwerts,
Marion E Meijer-van Gelder,
Ina Schwope, Joan Bolt-de Vries,
Nadia Harbeck,
Thomas Koenig,
Oliver Hartmann,
Antje Kluth,
Dimo Dietrich,
Viktor Magdolen,
Henk Portengen,
Maxime P Look,
Jan G M Klijn,
Ralf Lesche,
Manfred Schmitt,
Sabine Maier,
John A Foekens,
John W M Martens
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ABSTRACT: In this study, we evaluated if PITX2 DNA methylation is a marker for disease recurrence in lymph node-negative (LNN), steroid hormone receptor-positive (HR+) breast cancer patients. In addition, we studied the association between PITX2 DNA methylation and PITX2 gene expression.
PITX2 DNA-methylation was measured in tumor tissue from 412 LNN/HR+ breast cancer patients who had not received any adjuvant systemic treatment. In addition, PITX2 DNA-methylation and mRNA expression was evaluated in 32 breast cancer cell lines.
In univariate Cox regression analysis, DNA-methylation of PITX2 as a continuous variable was associated with early distant metastasis (HR = 1.71; P < 0.01) and poor overall survival (HR = 1.71; P < 0.01). In multivariate analysis together with the established prognostic factors age, tumor size and tumor grade, and steroid hormone receptor levels, both associations retained their significance (for MFS, HR = 1.74; P < 0.01; for OS, HR = 1.46; P = 0.02). In the breast cancer cell lines, PITX2 DNA methylation was inversely association with PITX2A and PITX2B mRNA expression (P < 0.01).
Hypermethylation of PITX2 is, in cell lines, negatively associated with PITX2 mRNA expression and, in clinical specimens, positively associated with breast cancer disease progression.
Breast Cancer Research and Treatment 11/2007; 111(3):429-37. · 4.43 Impact Factor
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John W M Martens,
Inko Nimmrich,
Thomas Koenig,
Maxime P Look,
Nadia Harbeck,
Fabian Model,
Antje Kluth, Joan Bolt-de Vries,
Anieta M Sieuwerts,
Henk Portengen,
Marion E Meijer-Van Gelder,
Christian Piepenbrock,
Alexander Olek,
Heinz Höfler,
Marion Kiechle,
Jan G M Klijn,
Manfred Schmitt,
Sabine Maier,
John A Foekens
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ABSTRACT: To understand the biological basis of resistance to endocrine therapy is of utmost importance in patients with steroid hormone receptor-positive breast cancer. Not only will this allow us prediction of therapy success, it may also lead to novel therapies for patients resistant to current endocrine therapy. DNA methylation in the promoter regions of genes is a prominent epigenetic gene silencing mechanism that contributes to breast cancer biology. In the current study, we investigated whether promoter DNA methylation could be associated with resistance to endocrine therapy in patients with recurrent breast cancer. Using a microarray-based technology, the promoter DNA methylation status of 117 candidate genes was studied in a cohort of 200 steroid hormone receptor-positive tumors of patients who received the antiestrogen tamoxifen as first-line treatment for recurrent breast cancer. Of the genes analyzed, the promoter DNA methylation status of 10 genes was significantly associated with clinical outcome of tamoxifen therapy. The association of the promoter hypermethylation of the strongest marker, phosphoserine aminotransferase (PSAT1) with favorable clinical outcome was confirmed by an independent quantitative DNA methylation detection method. Furthermore, the extent of DNA methylation of PSAT1 was inversely associated with its expression at the mRNA level. Finally, also at the mRNA level, PSAT1 was a predictor of tamoxifen therapy response. Concluding, our work indicates that promoter hypermethylation and mRNA expression of PSAT1 are indicators of response to tamoxifen-based endocrine therapy in steroid hormone receptor-positive patients with recurrent breast cancer.
Cancer Research 06/2005; 65(10):4101-17. · 7.86 Impact Factor
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ABSTRACT: Purpose: To evaluate whether cathepsin D, uroki- nase-type plasminogen activator (uPA), its inhibitor, plasminogen activator inhibitor-1 (PAI-1), or clinical factors can predict which patients are at risk for develop- ing distant metastases after local recurrence (LR). Patients and Methods: Of 1,630 patients treated with breast-conserving surgery and radiotherapy of the breast between 1980 and 1992, LR developed in 171 as a first event. From the available primary tumor tissues, we determined the cytosolic levels of cathepsin D, uPA and PAI-1. Results: In patients with LR, a short (I 2 years) disease-free interval (DFI) and skin involvement of LR were associated with poor postrelapse distant metasta- sis-free survival (PR-DMFS, P 5 .001, both) and postre- lapse overall survival (PR-OS; P F.0001 and P F.0002, respectively). The primary tumor levels of uPA and PAI-1 were elevated for patients with a short DFI (P F.01), but such a relation was not observed for patients with skin involvement. In univariate analyses, high levels of uPA and PAI-1 in the primary tumor were associated with poor PR-OS (P 5 .038 and P 5 .040, respectively) but not PR-DMFS. In Cox multivariate analyses for PR- DMFS and PR-OS, only a short DFI and skin involvement of the LR were independently associated with a poor clinical outcome. Conclusion: In patients treated with breast-conserv- ing therapy who had LR as a first event, a short DFI and skin involvement were strong indicators for poor PR- DMFS and PR-OS. The proteases studied did not contrib- ute significantly to the final multivariate model. J Clin Oncol 17:1449-1457. r 1999 by American Society of Clinical Oncology.
