title: Targeted MS Assay Predicting Tamoxifen Resistance in Estrogen-Receptor-Positive Breast Cancer Tissues and Sera.
abstract: We recently reported on the development of a 4-protein-based classifier (PDCD4, CGN, G3BP2, and OCIAD1) capable of predicting outcome to tamoxifen treatment in recurrent, estrogen-receptor-positive breast cancer based on high-resolution MS data. A precise and high-throughput assay to measure these proteins in a multiplexed, targeted fashion would be favorable to measure large numbers of patient samples to move these findings toward a clinical setting. By coupling immunoprecipitation to multiple reaction monitoring (MRM) MS and stable isotope dilution, we developed a high-precision assay to measure the 4-protein signature in 38 primary breast cancer whole tissue lysates (WTLs). Furthermore, we evaluated the presence and patient stratification capabilities of our signature in an independent set of 24 matched (pre- and post-therapy) sera. We compared the performance of immuno-MRM (iMRM) with direct MRM in the absence of fractionation and shotgun proteomics in combination with label-free quantification (LFQ) on both WTL and laser capture microdissected (LCM) tissues. Measurement of the 4-proteins by iMRM showed not only higher accuracy in measuring proteotypic peptides (Spearman r: 0.74 to 0.93) when compared with MRM (Spearman r: 0.0 to 0.76) but also significantly discriminated patient groups based on treatment outcome (hazard ratio [HR]: 10.96; 95% confidence interval [CI]: 4.33 to 27.76; Log-rank P < 0.001) when compared with LCM (HR: 2.85; 95% CI: 1.24 to 6.54; Log-rank P = 0.013) and WTL (HR: 1.16; 95% CI: 0.57 to 2.33; Log-rank P = 0.680) LFQ-based predictors. Serum sample analysis by iMRM confirmed the detection of the four proteins in these samples. We hereby report that iMRM outperformed regular MRM, confirmed our previous high-resolution MS results in tumor tissues, and has shown that the 4-protein signature is measurable in serum samples.
authors.affiliation: Erasmus University Medical Center Rotterdam , Erasmus MC Cancer Institute, Department of Medical Oncology, 3015 CN Rotterdam, The Netherlands.|Postgraduate School of Molecular Medicine, Erasmus University Medical Center , 3015 CN Rotterdam, The Netherlands.
authors.forename: Tommaso
authors.initials: T
authors.lastname: De Marchi
authors.affiliation: Broad Institute of MIT and Harvard , Cambridge, Massachusetts 02142, United States.
authors.forename: Erik
authors.initials: E
authors.lastname: Kuhn
authors.affiliation: Erasmus University Medical Center Rotterdam , Department of Neurology, 3015 CN Rotterdam, The Netherlands.
authors.forename: Lennard J
authors.initials: LJ
authors.lastname: Dekker
authors.affiliation: Erasmus University Medical Center Rotterdam , Department of Neurology, 3015 CN Rotterdam, The Netherlands.
authors.forename: Christoph
authors.initials: C
authors.lastname: Stingl
authors.affiliation: Erasmus University Medical Center Rotterdam , Erasmus MC Cancer Institute, Department of Medical Oncology, 3015 CN Rotterdam, The Netherlands.|Postgraduate School of Molecular Medicine, Erasmus University Medical Center , 3015 CN Rotterdam, The Netherlands.
authors.forename: Rene B H
authors.initials: RB
authors.lastname: Braakman
authors.affiliation: Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Division of Medical Oncology, 1066 CX Amsterdam, The Netherlands.
authors.forename: Mark
authors.initials: M
authors.lastname: Opdam
authors.affiliation: Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Division of Medical Oncology, 1066 CX Amsterdam, The Netherlands.
authors.forename: Sabine C
authors.initials: SC
authors.lastname: Linn
authors.affiliation: Radboud University Medical Center , Department of Laboratory Medicine, 6525 GA Nijmegen, The Netherlands.
authors.forename: Fred C G J
authors.initials: FC
authors.lastname: Sweep
authors.affiliation: Radboud University Medical Center , Department of Radiation Oncology, 6525 GA Nijmegen, The Netherlands.
authors.forename: Paul N
authors.initials: PN
authors.lastname: Span
authors.affiliation: Erasmus University Medical Center Rotterdam , Department of Neurology, 3015 CN Rotterdam, The Netherlands.
authors.forename: Theo M
authors.initials: TM
authors.lastname: Luider
authors.affiliation: Erasmus University Medical Center Rotterdam , Erasmus MC Cancer Institute, Department of Medical Oncology, 3015 CN Rotterdam, The Netherlands.|Broad Institute of MIT and Harvard , Cambridge, Massachusetts 02142, United States.
authors.forename: John A
authors.initials: JA
authors.lastname: Foekens
authors.affiliation: Erasmus University Medical Center Rotterdam , Erasmus MC Cancer Institute, Department of Medical Oncology, 3015 CN Rotterdam, The Netherlands.
authors.forename: John W M
authors.initials: JW
authors.lastname: Martens
authors.affiliation: Broad Institute of MIT and Harvard , Cambridge, Massachusetts 02142, United States.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Carr
authors.affiliation: Erasmus University Medical Center Rotterdam , Erasmus MC Cancer Institute, Department of Medical Oncology, 3015 CN Rotterdam, The Netherlands.|Postgraduate School of Molecular Medicine, Erasmus University Medical Center , 3015 CN Rotterdam, The Netherlands.
authors.forename: Arzu
authors.initials: A
authors.lastname: Umar
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D018931:Antineoplastic Agents, Hormonal / Q000627:therapeutic use*; D051017:Apoptosis Regulatory Proteins / Q000097:blood / Q000235:genetics; D014408:Biomarkers, Tumor / Q000097:blood* / Q000235:genetics; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000188:drug therapy* / Q000235:genetics / Q000401:mortality; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000097:blood / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D047468:Immunoprecipitation; D007201:Indicator Dilution Techniques; D008565:Membrane Proteins / Q000097:blood / Q000235:genetics; D008840:Microfilament Proteins / Q000097:blood / Q000235:genetics; D009363:Neoplasm Proteins / Q000097:blood / Q000235:genetics; D009587:Nitrogen Isotopes; D011379:Prognosis; D016601:RNA-Binding Proteins / Q000097:blood / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D016019:Survival Analysis; D013629:Tamoxifen / Q000627:therapeutic use*; D053719:Tandem Mass Spectrometry; D016896:Treatment Outcome
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018931:Antineoplastic Agents, Hormonal; D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; C061407:CGN protein, human; D002247:Carbon Isotopes; D002352:Carrier Proteins; C574606:G3BP2 protein, human; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D009587:Nitrogen Isotopes; C114487:PDCD4 protein, human; D016601:RNA-Binding Proteins; D011960:Receptors, Estrogen; C529235:ovarian cancer immuno-reactive antigen domain containing 1, human; D013629:Tamoxifen
keywords: MRM; SISCAPA; breast cancer; immunoaffinity capture; predictive biomarkers