Development and validation of Multiplex Liquid Bead Array (MLBA

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Development and validation of Multiplex Liquid Bead Array (MLBA) assay for the simultaneous expression of fourteen genes in Circulating Tumor Cells (CTCs) Cleo A Parisi, Athina Markou, Areti Strati, Sabine Kasimir-Bauer, and Evi S. Lianidou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04975 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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Analytical Chemistry

Development and validation of Multiplex Liquid Bead Array (MLBA) assay for the simultaneous expression of fourteen genes in Circulating Tumor Cells (CTCs)

Cleo Parisi1, Athina Markou1, Areti Strati1, Sabine Kasimir-Bauer2 and Evi S. Lianidou1*

1Analysis

of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of

Chemistry, University of Athens, 15771, Greece 2Department

of Gynecology and Obstetrics, University Hospital of Essen, University of Duisburg-

Essen, D-45122 Essen, Germany

* Correspondence should be addressed to: Dr. E.S. Lianidou, Analysis of Circulating Tumor Cells lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771, Greece Tel: ++ 30 210 7274311, Fax: ++ 30 210 7274750, E-mail: [email protected]

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ABSTRACT

Liquid biopsy, based on the molecular information extracted from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), offers the possibility to characterize the evolution of a solid tumor in real time and is highly important for diagnostic and therapeutic purposes. The aim of the present study was the development and validation of a novel liquid bead array methodology for the molecular characterization of CTCs and its application in breast cancer. In the present study we developed and evaluated a multiplex PCR-coupled liquid bead array (MLBA) assay for studying simultaneously the expression of fourteen genes in CTCs. The 14-gene MLBA assay is characterized by high analytical specificity, sensitivity, and reproducibility. The analytical performance of the 14-gene MLBA assay was compared with a commercially available test (AdnaTest BreastCancer, Qiagen, Germany) and our previously described multiplex RT-qPCR assays. The developed assay has the potential to be further expanded in order to include up to 100 gene-targets. The assay is highly specific for each target gene and is not affected by the numerous primers and probes used for multiplexing hence it constitutes a sample-, cost-, and time-saving analysis.


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INTRODUCTION

Breast cancer (BrCa) is the most frequently diagnosed cancer among women worldwide1. Circulating tumor cells (CTCs) in patients’ blood are responsible for the hematogenous metastatic process and are nowadays well defined targets providing useful insights for tumor biology and cell dissemination2. CTCs are isolated through a minimally invasive blood draw, allowing the sampling at multiple time points during disease progression, and can thus serve as a real-time “liquid biopsy” for monitoring patients’ response to therapy3. The clinical significance of CTCs enumeration and detection has already been shown in patients with breast cancer4–8.

Although there is currently a great variety of CTCs isolation systems and a large number of highly sensitive methods for their detection, the CellSearch® system (Menarini Silicon Biosystems Inc, PA) remains the only FDA approved system7. Because of the great heterogeneity of CTCs – even in the blood sample of a single patient9–13 – and the epithelial-mesenchymal transition (EMT) through which CTCs lose partially or totally their epithelial characteristics13, CTCs subpopulations may escape detection by conventional enrichment approaches that are mainly based on the expression of surface epithelial markers, such as the epithelial cell adhesion molecule (EpCAM) and specific cytokeratins14,15. Therefore, CTCs enumeration alone is not enough in most cases and their molecular characterization is necessary to provide important information on their molecular and biological nature16.

Multiplexed PCR assays are widely used for the molecular characterization of CTCs12,17–19, since they can generate more data using a minimum amount of sample, less labor and lower consumption of reagents, analysis cost and time. Towards this direction, the commercially available AdnaTest BreastCancer (Qiagen, Germany), a combination of BreastCancerSelect with BreastCancerDetect, enables the immunomagnetic enrichment of CTCs via antibodies against EpCAM and MUC-1 (mucin 3 ACS Paragon Plus Environment


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1) followed by the detection of BrCa-associated gene expression in the enriched tumor cells by a multiplex RT-PCR for three tumor-associated transcripts: HER2, MUC-1 and GA733-2 (EpCAM). β-actin (beta-actin) is also amplified as a reference gene18.

Liquid bead array hybridization assays have been successfully used in a variety of molecular diagnostics applications20. We have previously developed a highly specific and sensitive hybridization assay for the molecular characterization of CTCs in BrCa by combining multiplex RTPCR with the liquid bead array technology and studied the expression of six genes in CTCs of BrCa patients21. The aim of the present study was to expand the potential of this liquid bead array assay by designing and validating a novel high throughput methodology for the simultaneous detection of the expression of 14 genes in genetic material isolated from CTCs. This is important, since in BrCa, there is an abundance of interesting gene-targets for the molecular characterization of CTCs including some already established markers or therapeutic targets. Detection of cytokeratin 19 (CK19), a specific epithelial marker, is of prognostic significance8,22-25, HER2 (erb-b2 receptor tyrosine kinase 2), a transmembrane receptor targeted by trastuzumab in HER2-postive early stage BrCa patients and many new agents are in clinical development26. hMAM (mammaglobin A), is a specific marker for the mammary gland27 while the expression of MAGEA3 (melanoma antigen family member A3) correlates with invasiveness28 and PTEN (phosphatase and tensin homolog) is a well-known tumor suppressor gene associated with resistance to trastuzumab29. Cancer stem cell markers are also of emerging importance in the field of molecular characterization of CTCs. ALDH1 (aldehyde dehydrogenase 1), correlates with cancer stem cell phenotype30 while the phenotype CD44high/CD24low/- is highly oncogenic in BrCa31. The process of EMT is also of interest in our effort to understand the metastatic cascade. SNAIL (snail family transcriptional repressor 1) plays a critical role in EMT mediation32 while TWIST1 (twist family bHLH transcription factor 1) and VIM (vimentin) are also expressed in CTCs of BrCa patients13. PDCD4 (programmed cell death 4), an apoptosis related gene, encodes for a tumor suppressor protein regulated by miR-21 in BrCa cells33. 4 ACS Paragon Plus Environment

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The developed 14-gene MLBA assay enables the simultaneous detection of these genes with high analytical sensitivity and specificity by combining the advantages of a multiplexed assay to those of the liquid bead array technology.

EXPERIMENTAL SECTION

Cell lines. For the development and optimization of the assay, the BrCa cell lines MCF-7, MDAMB-231 and SK-BR-3 were used. The analytical performance of the assay was then evaluated by using the following cancer cell lines: a) COLO-205 (human colon), b) HeLa (human cervix) and c) T-47D (human mammary gland). The cells were counted with a hemocytometer and their viability was assessed by trypan blue exclusion. For each cancer cell line total RNA was isolated, and serial dilutions were prepared in order to obtain cDNAs corresponding to 1-1,000 cells/μL. cDNA samples were kept in aliquots at -70°C and used for the analytical validation of the assay.

RNA extraction. RNA extraction was performed using the TRIzol® reagent (Invitrogen™, CA) as per manufacturer’s instructions. All RNA preparation and handling steps took place in a laminar-flow hood under RNase-free conditions.

cDNA synthesis. Reverse transcription was performed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems™, CA) according to manufacturer’s instructions. To ensure the isolated RNA integrity, two reference genes PBGD (porphobilinogen deaminase) and HPRT (hypoxanthine phosphoribosyltransferase 1) were included in the 14-genes panel.

Clinical samples. 30 healthy donors, 31 patients with operable BrCa and 27 BrCa patients with verified metastasis were included in the study. All patient samples were collected at the Department of Obstetrics and Gynecology in the University Hospital of Essen, Germany, after the approval by 5 ACS Paragon Plus Environment


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the ethics and scientific committees. Blood sampling, CTCs enrichment, mRNA isolation, and cDNA synthesis were performed as previously described using the commercially available assay AdnaTest BreastCancer (Qiagen) according to the manufacturer’s instructions.18,30 The present study complies with the principles stated in the Declaration of Helsinki. All participants in the study gave written informed consent for samples collection. Six samples from healthy donors and 4 metastatic samples were excluded from the subsequent analysis due to poor sample quality, as evaluated through the lack of expression of both HPRT and PBGD reference genes.

Primers and capture probes design. The following gene-targets, CK19, HER2, hMAM, MAGEA3, PBGD and TWIST were the same as previously described, so all primer pairs, the common biotinylated T7 (b-T7) primer, and respective capture probes were identical as before.21 Eight novel genes were added in this gene panel; for all these genes we designed in silico novel primer pairs using the Primer Premier 5.00 software (Premier Biosoft, CA) avoiding the formation of stable secondary primer structures (e.g. hairpins, dimers, cross-dimers), and false priming sites. Capture probes were also designed to be highly gene-specific using selected sequences of the corresponding biotinylated multiplex PCR products. All capture probes have a reactive amino group with a C12 spacer to provide a terminal amine-spacer for covalent coupling to xMAP® carboxylated beads (Luminex Corporation, TX). These new primers and probes were designed in a similar way as in our previously developed assay33 in terms of common extension sequences (T7 for upstream primers and T3 for downstream primers), amplicons size and melting temperature. The specificity of all primers and hybridization probes sequences was evaluated in silico using the Nucleotide Basic Local Alignment Search Tool (BLAST®) algorithm (National Centre for Biotechnology Information, NCBI) and further analyzed using the FastPCR 5.4.4 software (PrimerDigital, Finland) in order to eliminate any possible crosshybridization. Designed primers and capture probes sequences are available upon request. Nucleotides used in this study were synthesized by Integrated DNA Technologies (IDT, IL).


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Multiplex PCR. 2.0 μL cDNA in a final volume of 25 μL was used in each reaction, while a PCR negative control containing no cDNA but water was included in each assay run. After having optimized every experimental condition, each reaction was containing 12.5 μL Master Mix, 2.5 μL Q-Solution (Multiplex PCR Kit, Qiagen), 0.2 μL of 25 mM MgCl2, 0.4 μL of a solution containing 1 mM of each dNTP (Invitrogen™), and 0.1 μM of each primer for hMAM and 0.05 μM of each primer for the remaining 13 genes. Multiplex PCR was carried out in a Mastercycler® epgradient (Eppendorf, Germany) based on the temperature programme: denaturation at 95 °C for 15 min, 45 cycles of denaturation at 95 °C for 30 s, annealing at 65 °C for 1 min and extension at 72 °C for 30 s, and final extension at 72 °C for 10 min. Samples were kept at 4 °C until use.

Liquid bead array. Biotinylation of PCR products was performed as previously described21. Each gene-specific capture probe was coupled to a spectrally distinct set of carboxylated beads using a modified carbodiimide coupling method, as previously described32. Each capture probe-bead conjugate was kept separately in the dark at 4 °C and a fresh microspheres solution containing all conjugates was prepared for each run. Microsphere sets that were used in this assay are shown in Table S2. For each sample we prepared a bead solution as previously described21. Resuspended beads were placed in a 96-well microplate, analyzed with a Luminex® 200™ system (Luminex Corporation), and mean fluorescence intensities (MFIs) were computed.

Comparison with the commercially available AdnaTest BreastCancer kit (Qiagen). The developed assay was directly compared with the commercially available kit AdnaTest BreastCancer, using the same cDNAs, derived from CTCs that were isolated from blood samples using the AdnaTest BreastCancer kit, and analyzed as previously described in detail18,30,34.



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RT-qPCR. All cDNAs used in the present study were analyzed by our previously developed RTqPCR assays for selected gene-targets12 as well, in order to compare the developed MLBA assay through the common genes analyzed by RT-qPCR.

Statistical analysis. The independent samples t-test was used to determine the analytical sensitivity of the assay after a Shapiro-Wilk normality test was performed. The non-parametric Mann-Whitney test was used to detect the differentially expressed genes between healthy donors’ and patients’ samples. The agreement between the developed assay and our previously established RT-qPCR assays for PBGD, CK19, HER2, MAGEA3 and TWIST, as well as the AdnaTest BreastCancer for HER2 expression was assessed using χ2 test and Cohen’s kappa coefficient35. The tests were twosided referring to a confidence level (CL) of 95%, thus p ≤ 0.05 was considered statistically significant. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, release 23.0.0.0, IBM Analytics, NY). A sample is considered positive for the expression of each of the 14 genes when the S/N (signal-to-noise) ratio is > 2.0, where S=MFIsample and N=MFInegative control of multiplex PCR. The results obtained for each sample were further normalized using the following procedure: the ratios

(

𝑀𝐹𝐼𝑔𝑒𝑛𝑒 ― 𝑀𝐹𝐼𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑜𝑓 𝑚𝑢𝑙𝑡𝑖𝑝𝑙𝑒𝑥 𝑃𝐶𝑅 𝑀𝐹𝐼𝐻𝑃𝑅𝑇 ― 𝑀𝐹𝐼𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑜𝑓 𝑚𝑢𝑙𝑡𝑖𝑝𝑙𝑒𝑥 𝑃𝐶𝑅

) × 10 were calculated for each

analyzed sample. The mean value of these ratios was also calculated for all samples from the healthy control group. When the above calculated ratio for each sample was higher of the corresponding mean value for a specific gene, this gene is considered overexpressed. The heatmaps presenting the results of this study were generated using the “matrix2png interface”36 and the “Heatmap builder” software. The scatter dot plots were made using the “GraphPad Prism” (version 5.03, GraphPad Software, CA).

RESULTS

An outline of the MLBA assay is presented in Figure 1.


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Optimization of assay conditions. In order to achieve high specificity and sensitivity for the developed assay, the conditions of multiplex PCR were optimized for the primers concentration, the number of PCR cycles and the annealing temperature. The hybridization conditions were also optimized for the number of spectrally distinct beads used for each gene-target. To optimize the assay, we used a cDNA sample consisting of 1,000 cells/μL of each of the BrCa cell lines MCF-7, MDAMB-231 and SK-BR-3. Optimal experimental conditions in every step of the assay were selected based on the best S/N ratio (Figure S1).

Analytical specificity. The analytical specificity of the developed 14-gene MLBA assay was checked both in the presence and in the absence of each gene-target by using as controls cDNA derived from a mixture of BrCa cell lines (MCF-7, MDA-MB-231 and SK-BR-3). Firstly, the analytical specificity was checked when a single amplified gene-target per sample was hybridized in the presence of all conjugated fluorescent beads specific for all genes-targets. Therefore, a PCR for each single gene was carried out under the conditions described above. Then each single biotinylated PCR product was hybridized in the presence of all 14 beads sets. Secondly, the analytical specificity was checked in the absence of each single gene-target but in the presence of all remaining 13 targets and all 14 beads sets. The assay was highly specific since we detected the expression of each individual gene-target while we did not observe any of the 182 nonspecific interactions that theoretically could have occurred between the biotinylated multiplex PCR products and the specific oligonucleotides attached on the beads (Figures 2Α, 2Β).

Analytical sensitivity. The limit of detection (LOD) for the developed MLBA assay was evaluated by using synthetic controls prepared from serial dilutions of SK-BR-3 and MDA-MB-231 total RNA corresponding to 1, 10, 100 and 1,000 cells/μL cDNA. The LOD was found to correspond to one SKBR-3 cell for ALDH1, CD24, CD44, CK19, HER2, hMAM, HPRT, MAGEA3, PBGD, PDCD4 and PTEN, 100 cells for SNAIL and 1,000 cells for VIM (Figure 2C). The LOD was found to correspond 9 ACS Paragon Plus Environment


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to one MDA-MB-231 cell for CD24, CD44, CK19, HER2, HPRT, MAGEA3, PBGD, TWIST and VIM, 10 cells for PDCD4 and PTEN and 100 cells for SNAIL (Figure 2D).

Precision. The intra-assay (within-run) precision of the assay was evaluated by analyzing in triplicate a cDNA sample that consisted of 1,000 cells/μl from MCF-7, MDA-MB-231 and SK-BR-3 breast cancer cell lines, following the entire analytical procedure as described in Figure 1. Inter-assay (between-run) precision was evaluated by analyzing the same cDNA sample in different runs in 3 different days. Intra-assay CVs ranged from 1.5% to 13.4% (mean CV = 6,2% ± 3,9%) (Figure 3A) and inter-assay CVs ranged from 3.2% to 14.9%, (mean CV = 6,9% ± 3,4%) (Figure 3B, Table S1).

Evaluation of the 14-gene MLBA assay performance in cancer cell lines. Before application to clinical samples, the performance of the 14-gene MLBA assay was evaluated in 6 different cancer cell lines. We analyzed in triplicate in the same run and in 3 different days as well, cDNA samples derived from 106 cells from the cancer cell lines MCF-7, MDA-MB-231, SK-BR-3, COLO-205, HeLa and T-47D. Assay CVs ranged from 0.2% to 10.9% (Table S2) and 1.0% to 18.9% (Table S3) respectively. The S/N ratios obtained from the intra- and inter-assay precision evaluation are shown in Figures 3A and 3B respectively. The results for gene expression in cancer cell lines used in this study are shown as a heatmap (Figure 3C).

Application of the 14-gene MLBA assay in clinical samples. The developed 14-gene MLBA assay was applied in cDNA samples from 24 healthy donors, 31 patients with operable BrCa, and 23 BrCa patients with verified metastasis. The results for gene expression in these groups are presented in Table 1 and are also shown as a heatmap for each individual sample in Figure 4A. The expression ratios after normalization of the obtained MFI values for the 14 genes-targets (as described in Statistical Analysis) in the three groups of samples are shown as scatter dot plots in Figure 4B – excluding the reference genes. All 78 samples were found positive for HPRT. The genes that were 10 ACS Paragon Plus Environment

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found differentially expressed between healthy donors’ (n = 24) and BrCa patients’ samples (n = 54) were CK19 (p = 0.001), HER2 (p = 0.002), hMAM (p = 0.001), and MAGEA3 (p = 0.018).

Direct comparison of the 14-gene MLBA assay with other molecular assays. The developed MLBA assay was compared with our previously described RT-qPCR assays for the five common genes tested, by analyzing the same 78 cDNAs derived from patients with operable and metastatic BrCa, and healthy donors. According to our data there was a statistically significant agreement for PBGD (62/78, 79%), CK-19 (62/78, 79%), HER2 (58/78, 74%), TWIST1 (52/55, 95%), MAGEA3 (53/78, 68%) that were all satisfactory according to the Cohen’s kappa coefficient (Table 2). The developed MLBA assay was also compared with the AdnaTest BreastCancer but only for HER2 since this was the only common gene tested and the agreement was also very satisfactory (61/78, 78%) (Table 2).

DISCUSSION

Molecular characterization of CTCs at the gene expression level can provide valuable information for diagnostic and therapeutic purposes37–39. Gene expression profiling is also important to understand the mechanisms for early metastatic dissemination of cancer cells and their association with cancer stem cells15,30. In addition, finding potential target antigens on CTCs40 could enable a follow-up of immunophenotypic changes during the clinical course of disease progression41. Because of the low numbers of CTCs and the limited amount of peripheral blood that can be used for their analysis, the number of gene-targets that can be analyzed by non-multimarker methods is very limited. Therefore multi-parametric assays are essential for the molecular characterization of CTCs42. The application of CTCs molecular characterization in a routine clinical setting is also hampered by the general lack of automation, standardized protocols for isolating and detecting CTCs, cross-validation of findings



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between laboratories and accreditation of analytical methodologies used for their detection and enumeration43.

EpCAM-positive cells have also EMT characteristics since EMT is not an “on-off” procedure. It has been shown in various previous studies that CTCs can be partially epithelial and mensenchymal13,44, CTCs ranging from exclusively epithelial (E) to intermediate (E > M, E = M, M > E) and exclusively mesenchymal (M). We have already shown that EMT genes are highly expressed in EpCAM-positive CTCs after therapy45. CTCs heterogeneity is independent of their EMT status and is an inherent property, evident not only between different patients but in a blood sample of a single patient as well46. Furthermore, CTCs heterogeneity can be documented at multiple levels like gene expression, DNA mutations, and DNA methylation.

Molecular characterization of CTCs is a key step in the diagnosis and personalized treatment of cancer and is crucial to take full advantage of their potential as “liquid biopsy” biomarkers. Recent studies have shown that isolation of CTCs is feasible even at early disease stages. Moreover, molecular analyses, even at the single-cell level, can be performed on CTCs. The developed MLBA assay could be also used for the molecular characterization of CTCs in breast cancer patients at the single cell level or pools of CTCs, as these samples would be completely free of PBMC background. Moreover, although the CTC-enriched EpCAM-positive fractions have in most samples a huge excess of white blood cells, the specificity of our results is based on the analysis of healthy donors following the whole analytical procedure, to have a very good estimation of background noise.

The developed assay was validated in terms of analytical specificity, sensitivity, and precision. The assay is highly specific for each target gene and is not affected by the numerous primers and probes used for multiplexing. Regarding the analytical sensitivity, the assay presents a low LOD, a critical 12 ACS Paragon Plus Environment

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feature for CTCs analysis. The MLBA assay detects almost all the genes of the panel, when expressed in the cell lines used, at the single cell/μl cDNA level. More specifically, in SK-BR-3, a cell line of epithelial morphology overexpressing HER2, the LOD for the genes related with a dominant epithelial phenotype corresponded to 1 cell/μl cDNA. On the other hand, in MDA-MB-231, a highly aggressive and invasive cell line with mesenchymal properties, the genes-markers of the EMT process, were also detected at a low LOD. In terms of precision, both intra- and inter- assay CVs are very satisfactory. Moreover, the evaluation of the assay performance in cancer cell lines, provided a range of very satisfactory intra- and inter- assay CVs in accordance with the values resulting from the assay validation.

According to our results, the percentage of samples found positive for the differentially expressed genes CK19, HER2, hMAM and MAGEA3 clearly demonstrate the higher tumor burden present in the metastatic setting. The extremely low percentages of samples positive for the detection of SNAIL and TWIST suggest a dominant epithelial phenotype in these samples. On the contrary, although VIM is a mesenchymal marker, it was not found to be differentially expressed between patients’ samples and those of healthy donors. However, this is not a surprising observation, as VIM is also expressed in the co-isolated PBMCs in the immunomagnetically isolated EpCAM-positive cells fraction.

The stem cell phenotype CD44+/CD24low/- could not be determined using the 14-gene MLBA assay as CD24 and CD44 were not differentially expressed between healthy donors’ and patients’ samples. According to our results, ALDH1 positivity in CTCs using the 14-gene MLBA assay was higher than that reported in the literature; this could possibly be due to the lower number of PCR cycles used in the AdnaTest EMT-1/StemCell assay compared to the MLBA assay (35 and 45 cycles respectively) and the lower number of samples tested. PTEN was not differentially expressed between healthy donors’ and patients’ samples. Moreover, PTEN expression in patients’ CTCs samples was



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interestingly higher than in normal samples, despite the reported PTEN loss in tumor samples29,47. To the best of our knowledge this is the first time that PDCD4 expression in BrCa CTCs is reported.

The developed 14-gene MLBA assay gives comparable results to our previously established RTqPCR assays for the same genes. MLBA assay results when compared to our previously developed RT-qPCR assays were different for MAGEA3 expression although the same basis of primers was used. A higher rate of positive samples was also observed for MAGEA3 in our previous liquid bead array assay when compared to RT-qPCR21. This kind of discrepancies between the compared assays are partially explained because the MLBA assay represents an end-point approach. A sample that is low amplified is considered negative by RT-qPCR but the same amplification in MLBA assay provides a high MFI value clearly indicating a positive sample. When the developed 14-gene MLBA assay was compared with the AdnaTest for the only one common gene (HER2), there was a statistically significant concordance although a different set of primers was used in each assay.

The developed 14-gene MLBA assay for CTCs molecular characterization brings together the advantages of multiplex RT-PCR and the liquid bead microarray technology enabling the reliable gene expression analysis for 14 genes in parallel using a very limited amount of sample. The assay is highly specific for each target-gene and is not affected by the numerous primers and probes used for multiplexing hence it constitutes a sample-, cost-, and time-saving analysis. The developed liquid bead array assay has been successfully applied for the simultaneous detection of 14 genes with high analytical specificity, sensitivity and reproducibility. The assay produces fairly comparable results to those of our previously developed RT-qPCR assays when the same gene-targets are compared. The developed methodology has the potential to be further expanded in order to include up to 100 genetargets and can be applied for CTCs molecular characterization in many types of cancer.


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Our main aim was to show the potential of liquid bead array approach for the molecular characterization of CTCs. We strongly believe that this assay can be further expanded to include more analytes on CTCs, like biomarkers of therapy resistance or therapy response. Future studies will be performed including a larger number of clinical samples to examine the potential clinical value of the developed assay in breast cancer. We also plan to apply the assay in cDNA samples derived from CTCs isolated using a variety of different approaches, in order to study different CTCs subpopulations and single cells.

ACKNOWLEDGEMENTS

We would like to thank Dr. A. Pintzas (National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry & Biotechnology) for providing the COLO-205 cancer cell line used in this study, Prof. P. Moutsatsou (Department of Biological Chemistry, Medical School, University of Athens) for providing HeLa and MCF-7, Prof. V. Georgoulias (Laboratory of Tumor Biology, Medical School, University of Crete) for providing the MDA-MB-231 and SK-BR-3 cell lines and Dr. A. Efstratiadis (Biomedical Research Foundation of the Academy of Athens, Center of Clinical, Experimental Surgery and Translational Research) for providing the T-47D cell line. This work was supported by “Onco-Seed diagnostics” grant, under the “Sinergasia 2009” programme, co-funded by the European Regional Development Fund and National Resources (General Secretariat of Research and Technology in Greece).

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Table 1. Results of the MLBA assay in clinical samples. Positive and overexpressed samples in healthy donors and patients of the study. healthy donors

operable BrCa patients

verified metastasis patients

(n = 24)

(n = 31)

(n = 23)

positive

overexpressed

positive

overexpressed

positive

overexpressed

samples

samples

samples

samples

samples

samples

ALDH1

13 (54%)

10 (42%)

22 (71%)

21 (68%)

16 (70%)

14 (61%)

CD24

23 (96%)

12 (50%)

27 (87%)

8 (26%)

22 (96%)

9 (39%)

CD44

14 (58%)

10 (42%)

16 (52%)

14 (45%)

11 (48%)

8 (35%)

CK19*

3 (12%)

0 (0%)

14 (45%)

16 (70%)

HER2*

0 (0%)

0 (0%)

6 (19%)

6 (26%)

hMAM*

0 (0%)

0 (0%)

2 (6%)

8 (35%)

24 (100%)

-----

2 (8%)

0 (0%)

PBGD

19 (79%)

-----

29 (94%)

-----

17 (74%)

-----

PDCD4

13 (54%)

11 (46%)

21 (68%)

15 (48%)

17 (74%)

12 (52%)

PTEN

2 (8%)

0 (0%)

8 (26%)

8 (26%)

7 (30%)

6 (26%)

SNAIL

0 (0%)

0 (0%)

1 (3%)

0 (0%)

2 (9%)

0 (0%)

TWIST

0 (0%)

0 (0%)

2 (6%)

0 (0%)

0 (0%)

0 (0%)

24 (100%)

11 (46%)

31 (100%)

15 (48%)

HPRT

MAGEA3*

VIM

31 (100%)

-----

23

-----

(100%)

16 (52%)

6 (26%)

*differentially expressed genes


23 (100%)

15 (65%)

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Table 2. Direct comparison of the MLBA assay, RT-qPCR and AdnaTest BreastCancer (Qiagen, Germany), for the common gene-targets tested.

14-gene MLBA

RT-qPCR

PBGD

-

+

Total

CK19

-

+

Total

MAGEA3

-

+

Total

-

6

9

15

-

42

11

53

-

50

21

71

+

7

56

63

+

5

20

25

+

4

3

7

Total

13

65

78

Total

47

31

78

Total

54

24

78

Agreement

62/78 (79%),p = 0.015 a,b

62/78 (79%),p = 0.001 a,b

53/78 (68%),p = 0.670 a

kappa

k = 0.304

k = 0.557

k = 0.063

14-gene MLBA

value

RT-qPCR

HER2

-

+

Total

TWIST

-

+

Total

-

50

4

54

-

51

1

52

+

16

8

24

+

2

1

3

Total

66

12

78

Total

53

2

55

AdnaTest BreastCancer

HER2

-

+

Total

-

56

7

63

+

10

5

15

Total

66

12

78

Agreement

58/78 (74%),p = 0.006 a,b

52/55 (95%),p = 0.107 a

Agreement

61/78 (78%),p = 0.047 a,b

kappa

k = 0.301

k = 0.373

kappa value

k = 0.241

a chi-square value

test, b statistically significant



Page 24 of 30

FIGURES LEGENDS

Figure 1. Schematic representation of the experimental procedure of the 14-gene MLBA assay.

Figure 2. Analytical specificity and analytical sensitivity of the MLBA assay. A. All 14 microspheres sets are hybridized with only one individual gene-target in each sample. B. All 14 microspheres sets are hybridized with 13/14 gene-targets, so that one single gene-target is missing in each sample. C. LOD of the MLBA assay in samples prepared from serial dilutions of SK-BR-3 and D. MDA-MB231 total RNA corresponding to 1, 10, 100 and 1,000 cells/μL cDNA. (NC: Negative Control)

Figure 3. Evaluation of the expression of 14 genes-targets: A. Intra-assay precision: S/N ratios. B. Inter-assay precision: S/N ratios (all samples run in triplicate). C. Heatmap of 14 gene expression in cancer cell lines (n=3).

Figure 4. MLBA assay in clinical samples. A. Results of the 14 genes expression study in EpCAM(+) CTCs fractions in: 1) healthy donors, 2) early BrCa patients, and 3) metastasis verified BrCa patients in the form of a heatmap (red: positive, green: negative). B. Scatter dot plots of the normalized ratios for the expression of individual genes in 1) healthy donors (HD, n=24), 2) patients with operable breast cancer (early BrCa, n=31) and 3) metastasis verified (mBrCa, n=23) patients. Data are presented as the mean and range (minimum and maximum). Mean values are compared using Mann – Whitney test. **: p ≤ 0.001, *: p ≤ 0.05, n.s.: p-value not significant


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Supporting Information

SI-Figures. MLBA assay: optimization of experimental conditions of the multiplex PCR and the hybridization step.

SI-Tables. Intra-assay and inter-assay precision for the positive control and cancer cell lines.



For TOC only


Page 26 of 30

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Figure 1. Schematic representation of the experimental procedure of the 14-gene MLBA assay. 254x190mm (96 x 96 DPI)

ACS Paragon Plus Environment


Figure 2. Analytical specificity and analytical sensitivity of the MLBA assay. A. All 14 microsphere sets are hybridized with only one individual gene-target in each sample. B. All 14 microspheres sets are hybridized with 13/14 gene-targets, so that one single gene-target is missing in each sample. C. LOD of the MLBA assay in samples prepared from serial dilutions of SK-BR-3 and D. MDA-MB-231 total RNA corresponding to 1, 10, 100 and 1,000 cells/μL cDNA. (NC: Negative Control) 254x190mm (96 x 96 DPI)


Page 28 of 30

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Figure 3. Evaluation of the expression of 14 genes-targets: A. Intra-assay precision: S/N ratios. B. Interassay precision: S/N ratios (all samples run in triplicate). C. Heatmap of 14 gene expression in cancer cell lines (n=3). 254x190mm (96 x 96 DPI)



Figure 4. MLBA assay in clinical samples. A. Results of the 14 genes expression study in EpCAM(+) CTCs fractions in: 1) healthy donors, 2) early BrCa patients, and 3) metastasis verified BrCa patients in the form of a heatmap (red: positive, green: negative). B. Scatter dot plots of the normalized ratios for the expression of individual genes in 1) healthy donors (HD, n=24), 2) patients with operable breast cancer (early BrCa, n=31) and 3) metastasis verified (mBrCa, n=23) patients. Data are presented as the mean and range (minimum and maximum). Mean values are compared using Mann – Whitney test. **: p ≤ 0.001, *: p ≤ 0.05, n.s.: p-value not significant 254x190mm (96 x 96 DPI)


Page 30 of 30

Development and validation of Multiplex Liquid Bead Array (MLBA

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