Publications by authors named "Maurice P H M Jansen"

32 Publications

Detection of tumor-derived extracellular vesicles in plasma from patients with solid cancer.

BMC Cancer 2021 Mar 24;21(1):315. Epub 2021 Mar 24.

Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus MC, Room Be400, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands.

Background: Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA).

Methods: For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR.

Results: Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue.

Conclusions: We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients' blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.
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http://dx.doi.org/10.1186/s12885-021-08007-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992353PMC
March 2021

Optimization of Pancreatic Juice Collection: A First Step Toward Biomarker Discovery and Early Detection of Pancreatic Cancer.

Am J Gastroenterol 2020 12;115(12):2103-2108

Department of Gastroenterology & Hepatology, Erasmus MC, University Medical Center, Rotterdam, the Netherlands.

Introduction: Imaging-based surveillance programs fail to detect pancreatic ductal adenocarcinoma at a curable stage, creating an urgent need for diagnostic biomarkers.

Methods: Secretin-stimulated pancreatic juice (PJ) was collected from the duodenal lumen during endoscopic ultrasound. The yield of biomarkers and organoids was compared for 2 collection techniques (endoscope suction channel vs catheter-based) and 3 periods (0-4 vs 4-8 vs 8-15 minutes).

Results: Collection through the endoscope suction channel was superior to collection with a catheter. Collection beyond 8 minutes reduced biomarker yield. PJ-derived organoid culture was feasible.

Discussion: The optimal protocol for secretin-stimulated PJ collection is through the endoscope suction channel for 8 minutes allowing biomarker detection and organoid culture.
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http://dx.doi.org/10.14309/ajg.0000000000000939DOI Listing
December 2020

Comparison of variant allele frequency and number of mutant molecules as units of measurement for circulating tumor DNA.

Mol Oncol 2021 01 31;15(1):57-66. Epub 2020 Oct 31.

Department of Medical Oncology, Erasmus MC Cancer Institute, University Medical Center, Rotterdam, The Netherlands.

Quantification of tumor-specific variants (TSVs) in cell-free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement. To study the agreement between VAF and mutant molecules in current clinical studies, we analyzed 1116 TSVs from 338 patients identified with next-generation sequencing (NGS) or digital droplet PCR (ddPCR). On different study cohorts, a Deming regression analysis was performed and its 95% prediction interval was used as surrogate for the limits of agreement between VAF and number of mutant molecules per mL and to identify outliers. VAF and number of mutant molecules per mL plasma yielded greater agreement when using ddPCR than NGS. In case of discordance between VAF and number of mutant molecules per mL, insufficient molecular coverage in NGS and high cell-free DNA concentration were the main responsible factors. We propose several optimization steps needed to bring monitoring of TSVs in cell-free DNA to its full potential.
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http://dx.doi.org/10.1002/1878-0261.12827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782075PMC
January 2021

Mutations in Serum Circulating Cell-Free Tumor DNA As Longitudinal Biomarker for High-Grade Serous Ovarian Cancer.

Biomolecules 2020 03 7;10(3). Epub 2020 Mar 7.

Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, 3015 CN Rotterdam, The Netherlands.

The aim of this study was to determine an optimal workflow to detect mutations in baseline and longitudinal serum cell free DNA (cfDNA) from high-grade serous ovarian carcinomas (HGSOC) patients and to define whether mutations are suitable as biomarker for disease. was investigated in tissue and archived serum from 20 HGSOC patients by a next-generation sequencing (NGS) workflow alone or combined with digital PCR (dPCR). AmpliSeq™-focused NGS panels and customized dPCR assays were used for tissue DNA and longitudinal cfDNAs, and Oncomine NGS panel with molecular barcoding was used for baseline cfDNAs. missense mutations were observed in 17 tissue specimens and in baseline cfDNA for 4/8 patients by AmpliSeq, 6/9 patients by Oncomine, and 4/6 patients by dPCR. Mutations in cfDNA were detected in 4/6 patients with residual disease and 3/4 patients with disease progression within six months, compared to 5/11 patients with no residual disease and 6/13 patients with progression after six months. Finally, mutations were detected at progression in 5/6 patients, but not during chemotherapy. NGS with molecular barcoding and dPCR were most optimal workflows to detect TP53 mutations in baseline and longitudinal serum cfDNA, respectively. mutations were undetectable in cfDNA during treatment but re-appeared at disease progression, illustrating its promise as a biomarker for disease monitoring.
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http://dx.doi.org/10.3390/biom10030415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175353PMC
March 2020

Whole exome sequencing of cell-free DNA - A systematic review and Bayesian individual patient data meta-analysis.

Cancer Treat Rev 2020 Feb 13;83:101951. Epub 2019 Dec 13.

Department of Medical Oncology, Erasmus MC Cancer Institute, Rotterdam, the Netherlands.

Molecular profiling of tumor derived cell free DNA (cfDNA) is gaining ground as a prognostic and predictive biomarker. However to what extent cfDNA reflects the full metastatic landscape as currently determined by tumor tissue analysis remains controversial. Though technically challenging, whole exome sequencing (WES) of cfDNA enables thorough evaluation of somatic alterations. Here, we review the feasibility of WES of cfDNA and determine the sensitivity of WES-detected single nucleotide variants (SNVs) in cfDNA on individual patient data level using paired tumor tissue as reference (sharedSNVsAlltissueSNVs×100%). The pooled sensitivity was 50% (95% credible interval (CI): 29-72%). The tissue mutant allele frequency (MAF) of variants exclusively identified in tissue was significantly lower (12.5%, range: 0.5-18%) than the tissue MAF of variants identified in both tissue and cfDNA (23.9%, range: 17-38%), p = 0.004. The overall agreement (sharedSNVsAllSNVs×100%)between SNVs in cfDNA and tumor tissue was 31% (95% CI: 15-49%). The number of detected SNVs was positively correlated with circulating tumor DNA (ctDNA) fraction (p = 0.016). A sub analysis of samples with ctDNA fractions ≥ 25% improved the sensitivity to 69% (95% CI: 46-89%) and agreement to 46% (95% CI: 36-59%), suggesting that WES is mainly feasible for patients with high ctDNA fractions. Pre- and post-analytical procedures were highly variable between studies rendering comparisons problematic. In conclusion, various aspects of WES of cfDNA are largely in its investigative phase, standardization of methodologies is highly needed to bring this promising technique to its clinical potential.
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http://dx.doi.org/10.1016/j.ctrv.2019.101951DOI Listing
February 2020

High ctDNA molecule numbers relate with poor outcome in advanced ER+, HER2- postmenopausal breast cancer patients treated with everolimus and exemestane.

Mol Oncol 2020 03 7;14(3):490-503. Epub 2020 Feb 7.

Department of Medical Oncology, Amsterdam UMC, Vrije Universiteit Amsterdam/Cancer Center Amsterdam, The Netherlands.

We determined whether progression-free survival (PFS) in metastatic breast cancer (MBC) patients receiving everolimus plus exemestane (EVE/EXE) varies depending on circulating tumour DNA (ctDNA) characteristics. Baseline plasma cell-free DNA (cfDNA) from 164 postmenopausal women with ER-positive, HER2-negative MBC refractory to a nonsteroidal aromatase inhibitor and treated with standard EVE/EXE (Everolimus Biomarker Study, Eudract 2013-004120-11) was characterised for 10 relevant breast cancer genes by next-generation sequencing with molecular barcoding. ctDNA molecule numbers, number of mutations and specific variants were related with PFS and overall survival (OS). Missense hotspot mutations in cfDNA were detected in 125 patients. The median of 54 ctDNA molecules per mL plasma distinguished patients with high and low/no ctDNA load. Patients with low/no ctDNA load (N = 102) showed longer median PFS of 5.7 months (P = 0.006) and OS of 124.8 months (P = 0.008) than patients with high ctDNA load (N = 62; 4.4 months and 107.7 months, respectively) in multivariate analyses. Patients with < 3 specific mutations (N = 135) had longer median PFS of 5.4 months compared to those with ≥ 3 mutations (3.4 months; P < 0.001). In conclusion, MBC patients with low/no ctDNA load or < 3 hotspot mutations experience longer PFS while treated with EVE/EXE.
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http://dx.doi.org/10.1002/1878-0261.12617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053245PMC
March 2020

RAS and BRAF mutations in cell-free DNA are predictive for outcome of cetuximab monotherapy in patients with tissue-tested RAS wild-type advanced colorectal cancer.

Mol Oncol 2019 11 30;13(11):2361-2374. Epub 2019 Sep 30.

Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, The Netherlands.

In metastatic colorectal cancer, RAS and BRAF mutations cause resistance to anti-EGFR therapies, such as cetuximab. Heterogeneity in RAS and BRAF mutations might explain nonresponse in a subset of patients receiving cetuximab. Analyzing mutations in plasma-derived circulating tumor DNA (ctDNA) could provide a more comprehensive overview of the mutational landscape as compared to analyses of primary and/or metastatic tumor tissue. Therefore, this prospective multicenter study followed 34 patients with metastatic colorectal cancer who were tissue-tested as RAS wild-type (exons 2-4) during routine work-up and received third-line cetuximab monotherapy. BRAF mutation status was also tested but did not exclude patients from therapy. At baseline and upon disease progression, cell-free DNA (cfDNA) was isolated for targeted next-generation sequencing (NGS). At 8 weeks, we determined that patients had benefited from treatment. NGS of cfDNA identified three patients with RAS mutations not detected in tumor tissue during routine work-up. Another six patients had a BRAF or rare RAS mutation in ctDNA and/or tumor tissue. Relative to patients without mutations in RAS/BRAF, patients with mutations at baseline had shorter progression-free survival [1.8 versus 4.9 months (P < 0.001)] and overall survival [3.1 versus 9.4 months (P = 0.001)]. In patients with clinical benefit (progressive disease after 8 weeks), ctDNA testing revealed previously undetected mutations in RAS/BRAF (71%) and EGFR (47%), which often emerged polyclonally. Our results indicate that baseline NGS of ctDNA can identify additional RAS mutation carriers, which could improve patient selection for anti-EGFR therapies. Acquired resistance, in patients with initial treatment benefit, is mainly explained by polyclonal emergence of RAS, BRAF, and EGFR mutations in ctDNA.
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http://dx.doi.org/10.1002/1878-0261.12550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822250PMC
November 2019

High-throughput isolation of circulating tumor DNA: a comparison of automated platforms.

Mol Oncol 2019 02 22;13(2):392-402. Epub 2018 Dec 22.

Department of Medical Oncology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, The Netherlands.

The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high-throughput method for fast, reproducible, and efficient isolation of ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study was to compare two competing automated DNA isolation platforms [Maxwell (MX) and QIAsymphony (QS)] to the current 'gold standard' QA to facilitate high-throughput processing of samples in prospective trials. We obtained blood samples from healthy blood donors and metastatic cancer patients for plasma isolation. Total cell-free DNA (cfDNA) quantity was assessed by TERT quantitative PCR. Recovery efficiency was investigated by quantitative PCR analysis of spiked-in synthetic plant DNA. In addition, a β-actin fragmentation assay was performed to determine the amount of contamination by genomic DNA from lysed leukocytes. ctDNA quality was assessed by digital PCR for somatic variant detection. cfDNA quantity and recovery efficiency were lowest using the MX platform, whereas QA and QS showed a comparable performance. All platforms preferentially isolated small (136 bp) DNA fragments over large (420 and 2000 bp) DNA fragments. Detection of the number variant and wild-type molecules was most comparable between QA and QS. However, there was no significant difference in variant allele frequency comparing QS and MX to QA. In summary, we show that the QS platform has comparable performance to QA, the 'gold standard', and outperformed the MX platform depending on the readout used. We conclude that the QS can replace the more laborious QA platform, especially when high-throughput cfDNA isolation is needed.
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http://dx.doi.org/10.1002/1878-0261.12415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360376PMC
February 2019

An Optimized Workflow to Evaluate Estrogen Receptor Gene Mutations in Small Amounts of Cell-Free DNA.

J Mol Diagn 2019 01 6;21(1):123-137. Epub 2018 Oct 6.

Department of Medical Oncology, Erasmus Medical Center Cancer Institute, Rotterdam, the Netherlands; Cancer Genomics Netherlands, Erasmus Medical Center Cancer Institute, Rotterdam, the Netherlands.

The detection of mutated genes in cell-free DNA (cfDNA) in plasma has emerged as an important minimally invasive way to obtain detailed information regarding tumor biology. Reliable determination of circulating tumor-derived DNA, often present at a low quantity amidst an excess of normal DNA in plasma, would be of added value for screening and monitoring of cancer patients and for hypothesis-generating studies in valuable retrospective cohorts. Our aim was to establish a workflow to simultaneously assess four hotspot estrogen receptor mutations (mESR1) in cfDNA isolated from only 200 μL of plasma by means of uniplex or multiplex pre-amplification combined with digital PCR. This workflow was then applied in metastatic breast cancer (MBC) patients receiving systemic therapies for MBC. In accordance with previous studies, estrogen receptor mutations were more frequently detected in endocrine-treated MBC patients at progressive disease [34.1% (15/44)] than before the start of endocrine therapy [3.9% (2/51); P = 0.001]. For a subset of samples, results were compared with analysis of these mutations by Oncomine-targeted next-generation sequencing, which, although requiring a higher cfDNA input, yielded concordant results. The data establish development and validation of a digital PCR workflow for the simultaneous detection of several tumor-derived mutations in minute amounts of cfDNA and show the potential of this workflow for use on archived volume-limited blood samples.
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http://dx.doi.org/10.1016/j.jmoldx.2018.08.010DOI Listing
January 2019

IGF1R signaling drives antiestrogen resistance through PAK2/PIX activation in luminal breast cancer.

Oncogene 2018 04 22;37(14):1869-1884. Epub 2018 Jan 22.

Division of Toxicology, Leiden Academic Center for Drug Research, Leiden University, Leiden, The Netherlands.

Antiestrogen resistance in estrogen receptor positive (ER) breast cancer is associated with increased expression and activity of insulin-like growth factor 1 receptor (IGF1R). Here, a kinome siRNA screen has identified 10 regulators of IGF1R-mediated antiestrogen with clinical significance. These include the tamoxifen resistance suppressors BMPR1B, CDK10, CDK5, EIF2AK1, and MAP2K5, and the tamoxifen resistance inducers CHEK1, PAK2, RPS6KC1, TTK, and TXK. The p21-activated kinase 2, PAK2, is the strongest resistance inducer. Silencing of the tamoxifen resistance inducing genes, particularly PAK2, attenuates IGF1R-mediated resistance to tamoxifen and fulvestrant. High expression of PAK2 in ER metastatic breast cancer patients is correlated with unfavorable outcome after first-line tamoxifen monotherapy. Phospho-proteomics has defined PAK2 and the PAK-interacting exchange factors PIXα/β as downstream targets of IGF1R signaling, which are independent from PI3K/ATK and MAPK/ERK pathways. PAK2 and PIXα/β modulate IGF1R signaling-driven cell scattering. Targeting PIXα/β entirely mimics the effect of PAK2 silencing on antiestrogen re-sensitization. These data indicate PAK2/PIX as an effector pathway in IGF1R-mediated antiestrogen resistance.
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http://dx.doi.org/10.1038/s41388-017-0027-9DOI Listing
April 2018

SNPitty: An Intuitive Web Application for Interactive B-Allele Frequency and Copy Number Visualization of Next-Generation Sequencing Data.

J Mol Diagn 2018 03 2;20(2):166-176. Epub 2018 Jan 2.

Cancer Computational Biology Center, Erasmus MC, University Medical Center, Rotterdam, the Netherlands; Department of Urology, Erasmus MC, University Medical Center, Rotterdam, the Netherlands. Electronic address:

Exploration and visualization of next-generation sequencing data are crucial for clinical diagnostics. Software allowing simultaneous visualization of multiple regions of interest coupled with dynamic heuristic filtering of genetic aberrations is, however, lacking. Therefore, the authors developed the web application SNPitty that allows interactive visualization and interrogation of variant call format files by using B-allele frequencies of single-nucleotide polymorphisms and single-nucleotide variants, coverage metrics, and copy numbers analysis results. SNPitty displays variant alleles and allelic imbalances with a focus on loss of heterozygosity and copy number variation using genome-wide heterozygous markers and somatic mutations. In addition, SNPitty is capable of generating predefined reports that summarize and highlight disease-specific targets of interest. SNPitty was validated for diagnostic interpretation of somatic events by showcasing a serial dilution series of glioma tissue. Additionally, SNPitty is demonstrated in four cancer-related scenarios encountered in daily clinical practice and on whole-exome sequencing data of peripheral blood from a Down syndrome patient. SNPitty allows detection of loss of heterozygosity, chromosomal and gene amplifications, homozygous or heterozygous deletions, somatic mutations, or any combination thereof in regions or genes of interest. Furthermore, SNPitty can be used to distinguish molecular relationships between multiple tumors from a single patient. On the basis of these data, the authors demonstrate that SNPitty is robust and user friendly in a wide range of diagnostic scenarios.
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http://dx.doi.org/10.1016/j.jmoldx.2017.11.011DOI Listing
March 2018

Estrogen receptor mutations and splice variants determined in liquid biopsies from metastatic breast cancer patients.

Mol Oncol 2018 01 17;12(1):48-57. Epub 2017 Nov 17.

Erasmus MC Cancer Institute, Department of Medical Oncology and Cancer Genomics Netherlands, Erasmus University Medical Center, Rotterdam, The Netherlands.

Mutations and splice variants in the estrogen receptor (ER) gene, ESR1, may yield endocrine resistance in metastatic breast cancer (MBC) patients. These putative endocrine resistance markers are likely to emerge during treatment, and therefore, its detection in liquid biopsies, such as circulating tumor cells (CTCs) and cell-free DNA (cfDNA), is of great interest. This research aimed to determine whether ESR1 mutations and splice variants occur more frequently in CTCs of MBC patients progressing on endocrine treatment. In addition, the presence of ESR1 mutations was evaluated in matched cfDNA and compared to CTCs. CellSearch-enriched CTC fractions (≥5/7.5 mL) of two MBC cohorts were evaluated, namely (a) patients starting first-line endocrine therapy (n = 43, baseline cohort) and (b) patients progressing on any line of endocrine therapy (n = 40, progressing cohort). ESR1 hotspot mutations (D538G and Y537S/N/C) were evaluated in CTC-enriched DNA using digital PCR and compared with matched cfDNA (n = 18 baseline cohort; n = 26 progressing cohort). Expression of ESR1 full-length and 4 of its splice variants (∆5, ∆7, 36 kDa, and 46 kDa) was evaluated in CTC-enriched mRNA. It was observed that in the CTCs, the ESR1 mutations were not enriched in the progressing cohort (8%), when compared with the baseline cohort (5%) (P = 0.66). In the cfDNA, however, ESR1 mutations were more prevalent in the progressing cohort (42%) than in the baseline cohort (11%) (P = 0.04). Three of the same mutations were observed in both CTCs and cfDNA, 1 mutation in CTCs only, and 11 in cfDNA only. Only the ∆5 ESR1 splice variant was CTC-specific expressed, but was not enriched in the progressing cohort. In conclusion, sensitivity for detecting ESR1 mutations in CTC-enriched fractions was lower than for cfDNA. ESR1 mutations detected in cfDNA, rarely present at the start of first-line endocrine therapy, were enriched at progression, strongly suggesting a role in conferring endocrine resistance in MBC.
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http://dx.doi.org/10.1002/1878-0261.12147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748489PMC
January 2018

Somatic mutation detection using various targeted detection assays in paired samples of circulating tumor DNA, primary tumor and metastases from patients undergoing resection of colorectal liver metastases.

Mol Oncol 2016 12;10(10):1575-1584

Assessing circulating tumor DNA (ctDNA) is a promising method to evaluate somatic mutations from solid tumors in a minimally-invasive way. In a group of twelve metastatic colorectal cancer (mCRC) patients undergoing liver metastasectomy, from each patient DNA from cell-free DNA (cfDNA), the primary tumor, metastatic liver tissue, normal tumor-adjacent colon or liver tissue, and whole blood were obtained. Investigated was the feasibility of a targeted NGS approach to identify somatic mutations in ctDNA. This targeted NGS approach was also compared with NGS preceded by mutant allele enrichment using synchronous coefficient of drag alteration technology embodied in the OnTarget assay, and for selected mutations with digital PCR (dPCR). All tissue and cfDNA samples underwent IonPGM sequencing for a CRC-specific 21-gene panel, which was analyzed using a standard and a modified calling pipeline. In addition, cfDNA, whole blood and normal tissue DNA were analyzed with the OnTarget assay and with dPCR for specific mutations in cfDNA as detected in the corresponding primary and/or metastatic tumor tissue. NGS with modified calling was superior to standard calling and detected ctDNA in the cfDNA of 10 patients harboring mutations in APC, ATM, CREBBP, FBXW7, KRAS, KMT2D, PIK3CA and TP53. Using this approach, variant allele frequencies in plasma ranged predominantly from 1 to 10%, resulting in limited concordance between ctDNA and the primary tumor (39%) and the metastases (55%). Concordance between ctDNA and tissue markedly improved when ctDNA was evaluated for KRAS, PIK3CA and TP53 mutations by the OnTarget assay (80%) and digital PCR (93%). Additionally, using these techniques mutations were observed in tumor-adjacent tissue with normal morphology in the majority of patients, which were not observed in whole blood. In conclusion, in these mCRC patients with oligometastatic disease NGS on cfDNA was feasible, but had limited sensitivity to detect all somatic mutations present in tissue. Digital PCR and mutant allele enrichment before NGS appeared to be more sensitive to detect somatic mutations.
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http://dx.doi.org/10.1016/j.molonc.2016.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423131PMC
December 2016

Increased MAPK1/3 Phosphorylation in Luminal Breast Cancer Related with PIK3CA Hotspot Mutations and Prognosis.

Transl Oncol 2017 Oct 5;10(5):854-866. Epub 2017 Sep 5.

Department of Medical Oncology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands. Electronic address:

Introduction: While mutations in PIK3CA are most frequently (45%) detected in luminal breast cancer, downstream PI3K/AKT/mTOR pathway activation is predominantly observed in the basal subtype. The aim was to identify proteins activated in PIK3CA mutated luminal breast cancer and the clinical relevance of such a protein in breast cancer patients.

Materials And Methods: Expression levels of 171 signaling pathway (phospho-)proteins established by The Cancer Genome Atlas (TCGA) using reverse phase protein arrays (RPPA) were in silico examined in 361 breast cancers for their relation with PIK3CA status. MAPK1/3 phosphorylation was evaluated with immunohistochemistry on tissue microarrays (TMA) containing 721 primary breast cancer core biopsies to explore the relationship with metastasis-free survival.

Results: In silico analyses revealed increased phosphorylation of MAPK1/3, p38 and YAP, and decreased expression of p70S6K and 4E-BP1 in PIK3CA mutated compared to wild-type luminal breast cancer. Augmented MAPK1/3 phosphorylation was most significant, i.e. in luminal A for both PIK3CA exon 9 and 20 mutations and in luminal B for exon 9 mutations. In 290 adjuvant systemic therapy naïve lymph node negative (LNN) breast cancer patients with luminal cancer, high MAPK phosphorylation in nuclei (HR=0.49; 95% CI, 0.25-0.95; P=.036) and in tumor cells (HR=0.37; 95% CI, 0.18-0.79; P=.010) was related with favorable metastasis-free survival in multivariate analyses including traditional prognostic factors.

Conclusion: Enhanced MAPK1/3 phosphorylation in luminal breast cancer is related to PIK3CA exon-specific mutations and correlated with favorable prognosis especially when located in the nuclei of tumor cells.
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http://dx.doi.org/10.1016/j.tranon.2017.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591392PMC
October 2017

Somatic Tumor Mutations Detected by Targeted Next Generation Sequencing in Minute Amounts of Serum-Derived Cell-Free DNA.

Sci Rep 2017 05 18;7(1):2136. Epub 2017 May 18.

Erasmus MC Cancer Institute, Department of Medical Oncology and Cancer Genomics Netherlands, Rotterdam, The Netherlands.

The use of blood-circulating cell-free DNA (cfDNA) as 'liquid-biopsy' is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment. In exploring cfDNA, valuable repositories are biobanks containing material collected over time, however these retrospective cohorts have restrictive resources. In this study, we aimed to detect tumor-specific mutations in only minute amounts of serum-derived cfDNA by using a targeted next generation sequencing (NGS) approach. In a retrospective cohort of ten metastatic breast cancer patients, we profiled DNA from primary tumor tissue (frozen), tumor-adjacent normal tissue (formalin-fixed paraffin embedded), and three consecutive serum samples (frozen). Our presented workflow includes comparisons with matched normal DNA or in silico reference DNA to discriminate germline from somatic variants, validation of variants through the detection in at least two DNA samples of an individual, and the use of public databases on variants. By our workflow, we were able to detect a total of four variants traceable as circulating tumor DNA (ctDNA) in the sera of three of the ten patients.
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http://dx.doi.org/10.1038/s41598-017-02388-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437051PMC
May 2017

Application of circulating tumor DNA in prospective clinical oncology trials - standardization of preanalytical conditions.

Mol Oncol 2017 03 22;11(3):295-304. Epub 2017 Feb 22.

Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands.

Circulating tumor DNA (ctDNA) has emerged as a potential new biomarker with diagnostic, predictive, and prognostic applications for various solid tumor types. Before beginning large prospective clinical trials to prove the added value of utilizing ctDNA in clinical practice, it is essential to investigate the effects of various preanalytical conditions on the quality of cell-free DNA (cfDNA) in general and of ctDNA in particular in order to optimize and standardize these conditions. Whole blood samples were collected from patients with metastatic cancer bearing a known somatic variant. The following preanalytical conditions were investigated: (a) different time intervals to plasma isolation (1, 24, and 96 h) and (b) different preservatives in blood collection tubes (EDTA, CellSave, and BCT). The quality of cfDNA/ctDNA was assessed by DNA quantification, digital polymerase chain reaction (dPCR) for somatic variant detection and a β-actin fragmentation assay for DNA contamination from lysed leukocytes. In 11 (69%) of our 16 patients, we were able to detect the known somatic variant in ctDNA. We observed a time-dependent increase in cfDNA concentrations in EDTA tubes, which was positively correlated with an increase in wild-type copy numbers and large DNA fragments (> 420 bp). Using different preservatives did not affect somatic variant detection ability, but did stabilize cfDNA concentrations over time. Variant allele frequency was affected by fluctuations in cfDNA concentration only in EDTA tubes at 96 h. Both CellSave and BCT tubes ensured optimal ctDNA quality in plasma processed within 96 h after blood collection for downstream somatic variant detection by dPCR.
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http://dx.doi.org/10.1002/1878-0261.12037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527445PMC
March 2017

LRG1 mRNA expression in breast cancer associates with PIK3CA genotype and with aromatase inhibitor therapy outcome.

Mol Oncol 2016 10 25;10(8):1363-73. Epub 2016 Jul 25.

Department of Medical Oncology, Erasmus MC - Cancer Institute, Rotterdam, The Netherlands. Electronic address:

Background: PIK3CA is the most frequent somatic mutated oncogene in estrogen receptor (ER) positive breast cancer. We previously observed an association between PIK3CA genotype and aromatase inhibitors (AI) treatment outcome. This study now evaluates whether expression of mRNAs and miRs are linked to PIK3CA genotype and are independently related to AI therapy response in order to define potential expressed biomarkers for treatment outcome.

Materials And Methods: The miR and mRNA expression levels were evaluated for their relationship with the PIK3CA genotype in two breast tumor datasets, i.e. 286 luminal cancers from the TCGA consortium and our set of 84 ER positive primary tumors of metastatic breast cancer patients who received first line AI. BRB Array tools class comparison was performed to define miRs and mRNAs whose expression associate with PIK3CA exon 9 and 20 status. Spearman correlations established miR-mRNA pairs and mRNAs with related expression. Next, a third dataset of 25 breast cancer patients receiving neo-adjuvant letrozole was evaluated, to compare expression levels of identified miRs and mRNAs in biopsies before and after treatment. Finally, to identify potential biomarkers miR and mRNA levels were related with overall survival (OS) and progression free survival (PFS) after first-line AI therapy.

Results: Expression of 3 miRs (miR-449a, miR-205-5p, miR-301a-3p) and 9 mRNAs (CCNO, FAM81B, LRG1, NEK10, PLCL1, PGR, SERPINA3, SORBS2, VTCN1) was related to the PIK3CA status in both datasets. All except miR-301a-3p had an increased expression in tumors with PIK3CA mutations. Validation in a publicly available dataset showed that LRG1, PGR, and SERPINA3 levels were decreased after neo-adjuvant AI-treatment. Six miR-mRNA pairs correlated significantly and stepdown analysis of all 12 factors revealed 3 mRNAs (PLCL1, LRG1, FAM81B) related to PFS. Further analyses showed LRG1 and PLCL1 expression to be unrelated with luminal subtype and to associate with OS and with PFS, the latter independent from traditional predictive factors.

Conclusion: We showed in two datasets of ER positive and luminal breast tumors that the expression of 3 miRs and 9 mRNAs associate with the PIK3CA status. Expression of LRG1 is independent of luminal (A or B) subtype, decreased after neo-adjuvant AI-treatment, and is proposed as potential biomarker for AI therapy outcome.
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http://dx.doi.org/10.1016/j.molonc.2016.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423197PMC
October 2016

An 8-gene mRNA expression profile in circulating tumor cells predicts response to aromatase inhibitors in metastatic breast cancer patients.

BMC Cancer 2016 Feb 18;16:123. Epub 2016 Feb 18.

Department of Medical Oncology and Cancer Genomics Netherlands, Erasmus MC - Cancer Institute, Erasmus University Medical Center, Room He 116, P.O. Box 2040, Rotterdam, 3000 CA, The Netherlands.

Background: Molecular characterization of circulating tumor cells (CTC) is promising for personalized medicine. We aimed to identify a CTC gene expression profile predicting outcome to first-line aromatase inhibitors in metastatic breast cancer (MBC) patients.

Methods: CTCs were isolated from 78 MBC patients before treatment start. mRNA expression levels of 96 genes were measured by quantitative reverse transcriptase polymerase chain reaction. After applying predefined exclusion criteria based on lack of sufficient RNA quality and/or quantity, the data from 45 patients were used to construct a gene expression profile to predict poor responding patients, defined as disease progression or death <9 months, by a leave-one-out cross validation.

Results: Of the 45 patients, 19 were clinically classified as poor responders. To identify them, the 75% most variable genes were used to select genes differentially expressed between good and poor responders. An 8-gene CTC predictor was significantly associated with outcome (Hazard Ratio [HR] 4.40, 95% Confidence Interval [CI]: 2.17-8.92, P < 0.001). This predictor identified poor responding patients with a sensitivity of 63% and a positive predictive value of 75%, while good responding patients were correctly predicted in 85% of the cases. In multivariate Cox regression analysis, including CTC count at baseline, the 8-gene CTC predictor was the only factor independently associated with outcome (HR 4.59 [95% CI: 2.11-9.56], P < 0.001). This 8-gene signature was not associated with outcome in a group of 71 MBC patients treated with systemic treatments other than AI.

Conclusions: An 8-gene CTC predictor was identified which discriminates good and poor outcome to first-line aromatase inhibitors in MBC patients. Although results need to be validated, this study underscores the potential of molecular characterization of CTCs.
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http://dx.doi.org/10.1186/s12885-016-2155-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759736PMC
February 2016

Decreased expression of ABAT and STC2 hallmarks ER-positive inflammatory breast cancer and endocrine therapy resistance in advanced disease.

Mol Oncol 2015 Jun 4;9(6):1218-33. Epub 2015 Mar 4.

Translational Cancer Research Unit, GZA Hospitals St-Augustinus, Oosterveldlaan 24, Antwerp B2610, Belgium; Department of Oncology, KU Leuven, Leuven, Belgium.

Background: Patients with Estrogen Receptor α-positive (ER+) Inflammatory Breast Cancer (IBC) are less responsive to endocrine therapy compared with ER+ non-IBC (nIBC) patients. The study of ER+ IBC samples might reveal biomarkers for endocrine resistant breast cancer.

Materials & Methods: Gene expression profiles of ER+ samples from 201 patients were explored for genes that discriminated between IBC and nIBC. Classifier genes were applied onto clinically annotated expression data from 947 patients with ER+ breast cancer and validated with RT-qPCR for 231 patients treated with first-line tamoxifen. Relationships with metastasis-free survival (MFS) and progression-free survival (PFS) following adjuvant and first-line endocrine treatment, respectively, were investigated using Cox regression analysis.

Results: A metagene of six genes including the genes encoding for 4-aminobutyrate aminotransferase (ABAT) and Stanniocalcin-2 (STC2) were identified to distinguish 22 ER+ IBC from 43 ER+ nIBC patients and remained discriminatory in an independent series of 136 patients. The metagene and two genes were not prognostic in 517 (neo)adjuvant untreated lymph node-negative ER+ nIBC breast cancer patients. Only ABAT was related to outcome in 250 patients treated with adjuvant tamoxifen. Three independent series of in total 411 patients with advanced disease showed increased metagene scores and decreased expression of ABAT and STC2 to be correlated with poor first-line endocrine therapy outcome. The biomarkers remained predictive for first-line tamoxifen treatment outcome in multivariate analysis including traditional factors or published signatures. In an exploratory analysis, ABAT and STC2 protein expression levels had no relation with PFS after first-line tamoxifen.

Conclusions: This study utilized ER+ IBC to identify a metagene including ABAT and STC2 as predictive biomarkers for endocrine therapy resistance.
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http://dx.doi.org/10.1016/j.molonc.2015.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528763PMC
June 2015

VAV3 mediates resistance to breast cancer endocrine therapy.

Breast Cancer Res 2014 May 28;16(3):R53. Epub 2014 May 28.

Introduction: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process.

Methods: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression.

Results: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy.

Conclusions: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.
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http://dx.doi.org/10.1186/bcr3664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076632PMC
May 2014

Hallmarks of aromatase inhibitor drug resistance revealed by epigenetic profiling in breast cancer.

Cancer Res 2013 Nov;73(22):6632-41

Authors' Affiliations: Department of Medical Oncology, Erasmus University Medical Center, Cancer Institute, Rotterdam; Departments of Molecular Carcinogenesis and Molecular Pathology, Central Genomic Facility, the Netherlands Cancer Institute; Agendia NV, Amsterdam, the Netherlands; and Translational Cancer Research Unit, Laboratory of Pathology, Antwerp University/Oncology Centre, GZA Hospitals St-Augustinus, Antwerp, Belgium.

Aromatase inhibitors are the major first-line treatment of estrogen receptor-positive breast cancer, but resistance to treatment is common. To date, no biomarkers have been validated clinically to guide subsequent therapy in these patients. In this study, we mapped the genome-wide chromatin-binding profiles of estrogen receptor α (ERα), along with the epigenetic modifications H3K4me3 and H3K27me3, that are responsible for determining gene transcription (n = 12). Differential binding patterns of ERα, H3K4me3, and H3K27me3 were enriched between patients with good or poor outcomes after aromatase inhibition. ERα and H3K27me3 patterns were validated in an additional independent set of breast cancer cases (n = 10). We coupled these patterns to array-based proximal gene expression and progression-free survival data derived from a further independent cohort of 72 aromatase inhibitor-treated patients. Through this approach, we determined that the ERα and H3K27me3 profiles predicted the treatment outcomes for first-line aromatase inhibitors. In contrast, the H3K4me3 pattern identified was not similarly informative. The classification potential of these genes was only partially preserved in a cohort of 101 patients who received first-line tamoxifen treatment, suggesting some treatment selectivity in patient classification.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-0704DOI Listing
November 2013

Hotspot mutations in PIK3CA associate with first-line treatment outcome for aromatase inhibitors but not for tamoxifen.

Breast Cancer Res Treat 2013 May 17;139(1):39-49. Epub 2013 Apr 17.

Department of Medical Oncology, Erasmus MC Cancer Institute, Room Be424, PO Box 2040, 3000 CA, Rotterdam, The Netherlands.

PIK3CA mutations occur frequently in breast cancer, predominantly in exons 9 and 20. The aim of this retrospective study is to evaluate the PIK3CA mutation status for its relationship with prognosis and first-line endocrine therapy outcome. PIK3CA exon 9 and 20 were evaluated for mutations in 1,352 primary breast cancer specimens by SnaPshot multiplex analyses. The mutation status was studied for their relationship with metastasis-free survival (MFS) in 342 untreated lymph node-negative (LNN) patients and to time to progression (TTP) in estrogen receptor (ER)-positive patients with metastatic disease treated with first-line tamoxifen (N = 447) or aromatase inhibitors (AIs; N = 84). We detected in 423 patients hotspot mutations for PIK3CA (31 %). Mutations in exon 20 were detected in 251 patients (59 %), with H1047L and H1047R mutations in 37 (15 %) and 214 (85 %) cases, respectively. Mutations in PIK3CA exon 9 were discovered in 173 patients (41 %), with E542K and E545K mutations in 57 (32 %) and 104 (60 %) cases as most prevalent ones. Evaluation of the untreated LNN patients for prognosis showed no relationship between MFS and PIK3CA mutations, neither for exon 9 [HR = 1.04 (95 % CI 0.57-1.89), P = 0.90] nor for exon 20 [HR = 0.98 (95 % CI 0.63-1.54); P = 0.94] when compared to wild-type. The PIK3CA mutation status was also not associated with treatment outcome after first-line tamoxifen. On the other hand, patients treated with first-line AIs showed a longer TTP when having a PIK3CA mutation in exon 9 [HR = 0.40 (95 % CI 0.17-0.95); P = 0.038] or exon 20 [HR = 0.50 (95 % CI 0.27-0.91); P = 0.024] compared to wild-types, both significant in uni- and multivariate analysis including traditional predictive factors. All results remained when only HER2-negative patients were evaluated for each cohort. PIK3CA mutations in ER-positive tumors were significantly associated with a favorable outcome after first-line AIs, which needs further confirmation in other datasets. Mutations were not associated with prognosis in untreated LNN patients nor predictive outcome after first-line tamoxifen therapy in advanced disease patients.
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http://dx.doi.org/10.1007/s10549-013-2529-7DOI Listing
May 2013

Pathway analysis of gene lists associated with platinum-based chemotherapy resistance in ovarian cancer: the big picture.

Gynecol Oncol 2010 May 4;117(2):170-6. Epub 2010 Feb 4.

Department of Medical Oncology, Erasmus MC-JNI-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.

Objective: Ovarian cancer is the leading cause of death from gynecological cancers in the Western world (Parkin et al., 2005). The overall 5-year survival is only 30% (Moss and Kaye, 2002), which is for a significant part due to platinum-based chemotherapy resistance. In this study, we performed a pathway analysis on nine published gene sets associated with platinum resistance in ovarian cancer, including a study by us. With this exploratory study, we aim to identify overlapping pathways associated with platinum-based chemotherapy resistance mechanisms in ovarian cancer.

Methods: Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA) were performed to determine which functional processes were differentially represented in the combined gene lists of nine studies (457 genes) compared to all Unigene identifiers or the Ingenuity knowledge base.

Results: The GO and IPA analysis resulted in the generation of 23 gene networks, and showed that 13 GO processes (>or=2 times enriched), 71 canonical pathways (p<0.05,), eight toxicity pathways (p<0.05) and 74 biological functions (p<0.005) are significantly associated with the 9-study gene set.

Conclusion And Recommendations: Several pathways identified have previously been shown to be associated with therapy resistance: these include 'oxidative stress response mediated by Nrf2,' 'TP53 signaling' and 'TGFbeta signaling.' The role of TGFbeta signaling and related miRNAs identified in the network analysis in epithelial-to-mesenchymal transition (EMT) and stemness as well as the possible relation with platin-based chemotherapy resistance are further discussed in detail. We propose that future international cooperation should aim at a uniform pooled analysis of the wealth of ovarian cancer array data already available. This will enhance the power of each separate ovarian cancer study and can lead to promising results.
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http://dx.doi.org/10.1016/j.ygyno.2010.01.010DOI Listing
May 2010

Integrated genomics of chemotherapy resistant ovarian cancer: a role for extracellular matrix, TGFbeta and regulating microRNAs.

Int J Biochem Cell Biol 2010 Jan 23;42(1):25-30. Epub 2009 Oct 23.

Department of Medical Oncology, Erasmus MC/JNI, Rotterdam, The Netherlands.

Epithelial ovarian cancer is the sixth most common cancer in women worldwide and the most important cause of death from gynaecological cancers in the Western world. Our explorative pathway analysis on seven published gene-sets associated with platinum resistance in ovarian cancer reveals TP53 and transforming growth factor beta as key genes. Furthermore, the extracellular matrix was associated with chemotherapy resistance in ovarian cancer as well as endocrine resistance in breast cancer. Pathway analysis again revealed transforming growth factor beta as a key gene regulating extracellular matrix gene expression. A model is presented based on literature linking transforming growth factor beta, extracellular matrix, integrin signalling, epithelial to mesenchymal transition and regulating microRNAs with a (bivalent) role in chemotherapy response.
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http://dx.doi.org/10.1016/j.biocel.2009.10.016DOI Listing
January 2010

Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response.

Clin Cancer Res 2008 Sep;14(17):5555-64

Department of Medical Oncology, Erasmus MC/Daniel den Hoed Cancer Center, Rotterdam, the Netherlands.

Purpose: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both.

Experimental Design: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors.

Results: TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node-negative untreated patients (P<0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of approximately 40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor-positive and lymph node-positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P=0.001), and (b) no clinical benefit (odds ratio, 0.81; P=0.035) and shorter progression-free survival (hazard ratio, 1.19; P=0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses.

Conclusion: FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-08-0555DOI Listing
September 2008

Downregulation of SIAH2, an ubiquitin E3 ligase, is associated with resistance to endocrine therapy in breast cancer.

Breast Cancer Res Treat 2009 Jul 16;116(2):263-71. Epub 2008 Jul 16.

Department of Medical Oncology, Erasmus MC, Josephine Nefkens Institute, Rotterdam, The Netherlands.

Purpose: In our microarray analysis we observed that Seven-in-Absentia Homolog 2 (SIAH2) levels were low in estrogen receptor (ER) positive breast tumors of patients resistant to first-line tamoxifen therapy. The aim of this study was to evaluate SIAH2 for its (a) predictive/prognostic value, and (b) functional role in endocrine therapy resistance.

Patients And Methods: SIAH2 expression was measured with quantitative Real-Time-PCR (qRT-PCR) in 1205 primary breast tumor specimens and related to disease outcome. The functional role of SIAH2 was determined in human breast cancer cell lines ZR-75-1, ZR/HERc, and MCF7. Cell lines were treated with estrogen (E2), anti-estrogen ICI164.384 or epidermal growth factor (EGF). Moreover, MCF7 was treated with ICI164.384 after silencing SIAH2 expression.

Results: SIAH2 was not prognostic in 603 lymph node negative patients who had not received adjuvant systemic therapy. In multivariate analysis of ER-positive tumors of 235 patients with recurrent disease, SIAH2 as continuous variable, significantly predicted first-line tamoxifen treatment failure (OR = 1.48; P = 0.05) and progression-free survival (PFS) (HR = 0.79; P = 0.007). Furthermore, in primary breast cancer patients treated with adjuvant tamoxifen, SIAH2 predicted metastasis-free survival (MFS) (HR = 0.73; P = 0.005). In vitro experiments showed that SIAH2 silencing in MCF7 cells resulted in resistance to ICI164.384-treatment when compared with mock silenced cells (P = 0.008). Interestingly, in ZR cells transfected with EGFR (ZR/HERc), SIAH2 expression was induced by E2 but downregulated by EGF.

Conclusion: In primary breast tumor specimens as well as in vitro low SIAH2 levels associated with resistance to endocrine therapy. Moreover, SIAH2 expression showed an opposite regulation by E2 and EGF.
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http://dx.doi.org/10.1007/s10549-008-0125-zDOI Listing
July 2009

Comparison of gene expression profiles predicting progression in breast cancer patients treated with tamoxifen.

Breast Cancer Res Treat 2009 Jan 4;113(2):275-83. Epub 2008 Mar 4.

Department of Pathology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Background: Molecular signatures that predict outcome in tamoxifen treated breast cancer patients have been identified. For the first time, we compared these response profiles in an independent cohort of (neo)adjuvant systemic treatment naïve breast cancer patients treated with first-line tamoxifen for metastatic disease.

Methods: From a consecutive series of 246 estrogen receptor (ER) positive primary tumors, gene expression profiling was performed on available frozen tumors using 44K oligoarrays (n = 69). A 78-gene tamoxifen response profile (formerly consisting of 81 cDNA-clones), a 21-gene set (microarray-based Recurrence Score), as well as the HOXB13-IL17BR ratio (Two-Gene-Index, RT-PCR) were analyzed. Performance of signatures in relation to time to progression (TTP) was compared with standard immunohistochemical (IHC) markers: ER, progesterone receptor (PgR) and HER2.

Results: In univariate analyses, the 78-gene tamoxifen response profile, 21-gene set and HOXB13-IL17BR ratio were all significantly associated with TTP with hazard ratios of 2.2 (95% CI 1.3-3.7, P = 0.005), 2.3 (95% CI 1.3-4.0, P = 0.003) and 4.2 (95% CI 1.4-12.3, P = 0.009), respectively. The concordance among the three classifiers was relatively low, they classified only 45-61% of patients in the same category. In multivariate analyses, the association remained significant for the 78-gene profile and the 21-gene set after adjusting for ER and PgR.

Conclusion: The 78-gene tamoxifen response profile, the 21-gene set and the HOXB13-IL17BR ratio were all significantly associated with TTP in an independent patient series treated with tamoxifen. The addition of multigene assays to ER (IHC) improves the prediction of outcome in tamoxifen treated patients and deserves incorporation in future clinical studies.
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http://dx.doi.org/10.1007/s10549-008-9939-yDOI Listing
January 2009

TSC22D1 and PSAP predict clinical outcome of tamoxifen treatment in patients with recurrent breast cancer.

Breast Cancer Res Treat 2009 Jan 26;113(2):253-60. Epub 2008 Feb 26.

Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Room Be 432, P.O. Box 2040, 3000 CA, Rotterdam, The Netherlands.

Purpose Two genes, TSC22 domain family, member 1 (TSC22D1) and prosaposin (PSAP) were identified in an in vitro functional screen for genes having a causative role in tamoxifen resistance. These genes were also present in our previously established 81-gene signature for resistance to first-line tamoxifen therapy. The aim of this study was to investigate the predictive value of these genes for tamoxifen therapy failure in patients with recurrent breast cancer. Experimental Design The mRNA levels of TSC22D1 and PSAP were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in 223 estrogen receptor-positive primary breast tumors of patients with recurrent disease treated with first-line tamoxifen therapy. The main objective of this study was the length of progression-free survival (PFS). Results High mRNA levels of TSC22D1 and PSAP were significantly associated with shorter PFS and both were independent of the traditional predictive factors (HR = 1.30, 95% CI = 1.04-1.64 P = 0.023; and HR = 1.40, 95% CI = 1.03-1.88, P = 0.029, respectively). In multivariate analysis, patients with high mRNA levels of both genes associated significantly with no clinical benefit (OR = 0.19, 95% CI = 0.06-0.62, P = 0.006) and had the shortest PFS (HR = 2.05, 95% CI = 1.29-3.25, P = 0.002). Conclusion These results confirm our previous in vitro and tumor-related findings and are indicative for the failure of tamoxifen treatment in breast-cancer patients. Both TSC22D1 and PSAP are associated with clinical outcome and may have a functional role in therapy resistance.
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http://dx.doi.org/10.1007/s10549-008-9934-3DOI Listing
January 2009

HOXB13-to-IL17BR expression ratio is related with tumor aggressiveness and response to tamoxifen of recurrent breast cancer: a retrospective study.

J Clin Oncol 2007 Feb;25(6):662-8

Department of Medical Oncology, Erasmus MC/Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.

Purpose: A HOXB13-to-IL17BR expression ratio was previously identified to predict clinical outcome of breast cancer patients treated with adjuvant tamoxifen. However, this ratio may predict a tumor's response to tamoxifen, its intrinsic aggressiveness, or both.

Patients And Methods: We have measured the HOXB13 and IL17BR expression levels by real-time polymerase chain reaction in 1,252 primary breast tumor specimens. Expression levels were normalized to housekeeper gene levels and related to clinicopathologic factors for all patients. The primary objective of this study was to determine the relationship of a HOXB13-to-IL17BR ratio with tumor aggressiveness and/or with response to tamoxifen therapy in estrogen receptor (ER) -positive disease. We selected ER-positive tumors, and clinical end points for the HOXB13-to-IL17BR ratio were disease-free survival (DFS) in patients with primary breast cancer (N = 619) and progression-free survival (PFS) in patients with recurrent breast cancer treated with first-line tamoxifen monotherapy (N = 193). The odds ratio (OR) and hazard ratio (HR) and their 95% CI were calculated, and all P values were two-sided.

Results: The HOXB13-to-IL17BR ratio was significantly associated with DFS and PFS. In multivariate analysis, HOXB13-to-IL17BR ratio expression levels were associated with a shorter DFS for node-negative patients only. Corrected for traditional predictive factors, the dichotomized HOXB13-to-IL17BR ratio was the strongest predictor in multivariate analysis for a poor response to tamoxifen therapy (OR = 0.16; 95% CI, 0.06 to 0.45; P < .001) and a shorter PFS (HR = 2.97; 95% CI, 1.82 to 4.86; P < .001).

Conclusion: High HOXB13-to-IL17BR ratio expression levels associate with both tumor aggressiveness and tamoxifen therapy failure.
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http://dx.doi.org/10.1200/JCO.2006.07.3676DOI Listing
February 2007