Relative Quantification of Proteins in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissue Using Multiplexed Mass Spectrometry Assays.

The identification of clinically relevant biomarkers represents an important challenge in oncology. This problem can be addressed with biomarker discovery and verification studies performed directly in tumor samples using formalin-fixed paraffin-embedded (FFPE) tissues. However, reliably measuring proteins in FFPE samples remains challenging. Here, we demonstrate the use of liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM/MS) as an effective technique for such applications. An LC-MRM/MS method was developed to simultaneously quantify hundreds of peptides extracted from FFPE samples and was applied to the targeted measurement of 200 proteins in 48 triple-negative, 19 HER2-overexpressing, and 20 luminal A breast tumors. Quantitative information was obtained for 185 proteins, including known markers of breast cancer such as HER2, hormone receptors, Ki-67, or inflammation-related proteins. LC-MRM/MS results for these proteins matched immunohistochemistry or chromogenic in situ hybridization data. In addition, comparison of our results with data from the literature showed that several proteins representing potential biomarkers were identified as differentially expressed in triple-negative breast cancer samples. These results indicate that LC-MRM/MS assays can reliably measure large sets of proteins using the analysis of surrogate peptides extracted from FFPE samples. This approach allows to simultaneously quantify the expression of target proteins from various pathways in tumor samples. LC-MRM/MS is thus a powerful tool for the relative quantification of proteins in FFPE tissues and for biomarker discovery.

Therapeutic Drug Monitoring of Orally Administered Letermovir Prophylaxis in Allogeneic Hematopoietic Stem Cell Transplant Recipients.

With balanced safety-efficacy profile, letermovir anti-cytomegalovirus (CMV) prophylaxis is used in hematopoietic stem cell transplant recipients (HSCTR). We assessed feasibility and usefulness of letermovir therapeutic drug monitoring (TDM) in HSCTR. We performed a prospective observational study on letermovir-TDM including 40 consecutive adult CMV-seropositive allogeneic-HSCTR who received orally (PO) administered letermovir. Minimal blood concentrations of letermovir (Ctrough) were measured on days 3 and 7 postletermovir initiation and weekly thereafter. Letermovir-Ctrough remained stable during the first 70 days post-HSCT at a median of 286 µg/L (interquartile range, 131 to 591 µg/L), with large interpatient/intrapatient variability. No associations between breakthrough clinically significant CMV infection or detectable CMV DNAemia and letermovir-Ctrough were observed. Patients with letermovir-associated adverse events had higher letermovir-Ctrough than patients without (400 versus 266 µg/L, P = 0.02). Letermovir-Ctrough was similar in patients with or without gastrointestinal symptoms (280 versus 300 µg/L, P = 0.49). Acute grade ≥2 GvHD was associated with higher letermovir-Ctrough (479 versus 248 µg/L, P = 0.001), including gastrointestinal GvHD (499 versus 263 µg/L, P = 0.004). Concomitantly administered posaconazole and cyclosporine were associated with higher letermovir-Ctrough (707 versus 259 µg/L, P < 0.001 and 437 versus 248 µg/L, P = 0.01, respectively). In multivariable analysis, both posaconazole (odds ratio [OR], 4.9; 95% confidence interval [CI], 2.4 to 9.7; P < 0.0001) and cyclosporine-adjusted letermovir dose at 240 mg daily (OR, 3.5; 95% CI, 1.4 to 9.0; P = 0.01) were independently associated with higher letermovir-Ctrough. In conclusion, administration of PO letermovir led to measurable and relatively stable letermovir-Ctrough, without noticeable associations with clinical efficacy. Letermovir exposure was not affected by gastrointestinal symptoms, but with posaconazole and cyclosporine administration. Associations between letermovir and concomitantly administered agents and adverse events warrant additional clinical studies.

[Vitamin deficiencies in the adult outpatient setting]. / Carences vitaminiques chez l'adulte en médecine ambulatoire.

Since their discovery more than a century ago to this day, vitamins went from misunderstood molecules with mysterious properties to fundamental components with undoubted clinical implications. Despite the scientific progresses in the understanding of their physiopathological role, vitamins raise to this day multiple interrogations in clinical practice. This article aims at answering questions that are frequently encountered in the outpatient setting regarding vitamin deficiencies: who to screen ? At what moment ? By which test ? How to interpret the results ? How to supplement ? By answering these questions, we hope to provide the general practitioners with a pragmatic tool to guide them in the management of issues related to vitamins.

Depuis leur découverte il y a plus d'un siècle à aujourd'hui, les vitamines sont passées de molécules méconnues et aux propriétés mystérieuses à des composants primordiaux et aux implications cliniques certaines. Malgré les progrès scientifiques dans la compréhension de leur rôle physiopathologique, les vitamines suscitent encore de nombreuses interrogations en pratique clinique. Cet article s'efforce de répondre aux questions fréquem ment rencontrées en médecine ambulatoire portant sur les carences vitaminiques: qui dépister ? À quel moment ? Par quel test ? Comment interpréter les résultats ? Comment supplémenter ? En répondant à ces questions, nous espérons fournir au médecin de premier recours un outil pragmatique pour l'orienter dans la prise en charge des problématiques vitaminiques.

Clinical method evaluation of hemoglobin S and C identification by top-down selected reaction monitoring and electron transfer dissociation.

BACKGROUND: Biological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Currently, hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Co-elution and co-migration may represent major issues for precise identification of Hb variants, even for the most common ones such as Hb S and C. METHODS: We adapted a top-down selected reaction monitoring (SRM) electron transfer dissociation (ETD) mass spectrometry (MS) method to fit with a clinical laboratory environment. An automated analytical process with semi-automated data analysis compatible with a clinical practice was developed. A comparative study between a reference HPLC method and the MS assay was performed on 152 patient samples. RESULTS: The developed workflow allowed to identify with high specificity and selectivity the most common Hb variants (Hb S and Hb C). Concordance of the MS-based approach with HPLC was 71/71 (100%) for Hb S and 11/11 (100%) for Hb C. CONCLUSIONS: This top-down SRM ETD method can be used in a clinical environment to detect Hb S and Hb C.

Biomarkers for acute kidney injury in decompensated cirrhosis: A prospective study.

AIM: Acute kidney injury (AKI) is a frequent complication in cirrhotic patients. As serum creatinine is a poor marker of renal function in this population, we aimed to study the utility of several biomarkers in this context. METHODS: A prospective study was conducted in hospitalized patients with decompensated cirrhosis. Serum creatinine (SCr), Cystatin C (CystC), NGAL and urinary NGAL, KIM-1, protein, albumin and sodium were measured on three separate occasions. Renal resistive index (RRI) was obtained. We analyzed the value of these biomarkers to determine the presence of AKI, its aetiology [prerenal, acute tubular necrosis (ATN), or hepatorenal (HRS)], its severity and a composite clinical outcome at 30 days (death, dialysis and intensive care admission). RESULTS: We included 105 patients, of which 55 had AKI. SCr, CystC, NGAL (plasma and urinary), urinary sodium and RRI at inclusion were independently associated with the presence of AKI. SCr, CystC and plasma NGAL were able to predict the subsequent development of AKI. Pre-renal state showed lower levels of SCr, NGAL (plasma and urinary) and RRI. ATN patients had high levels of NGAL (plasma and urinary) as well as urinary protein and sodium. HRS patients presented an intermediate pattern. All biomarkers paralleled the severity of AKI. SCr, CystC and plasma NGAL predicted the development of the composite clinical outcome with the same performance as the MELD score. CONCLUSIONS: In patients with decompensated cirrhosis, early measurement of renal biomarkers provides valuable information on AKI aetiology. It could also improve AKI diagnosis and prognosis.

Regulatory context and validation of assays for clinical mass spectrometry proteomics (cMSP) methods.

Clinical mass spectrometry proteomics (cMSP) assays are being increasingly used in clinical laboratories for analyzing peptides and proteins. It has therefore become urgent to characterize and validate the methods available for liquid chromatography-tandem mass spectrometry (LC/MS-MS) targeted quantification of peptide and protein biomarkers in biological fluids in the context of in vitro diagnostics. LC-MS/MS for the detection of peptides and proteins is currently the main approach used in the field of cMSP. As a result of their selectivity, low reagent costs and the fact that these methods can be used for absolute quantification and multiplexing, they will likely eventually replace immunoassays. Although LC-MS/MS is known to be the main reference method involved in reference measurement procedures (RMPs), it needs to meet the requirements of in vitro diagnostic (IVD) regulations and standards. This review shows that cMSP is fully compatible with the regulatory IVD requirements and provides an overview of the characterization and validation of the use of LC-MS/MS targeted quantification of clinical protein biomarkers in biological fluids.

IL8 and IL16 levels indicate serum and plasma quality.

BACKGROUND: Longer pre-centrifugation times alter the quality of serum and plasma samples. Markers for such delays in sample processing and hence for the sample quality, have been identified. METHODS: Twenty cytokines in serum, EDTA plasma and citrate plasma samples were screened for changes in concentration induced by extended blood pre-centrifugation delays at room temperature. The two cytokines that showed the largest changes were further validated for their "diagnostic performance" in identifying serum or plasma samples with extended pre-centrifugation times. RESULTS: In this study, using R&D Systems ELISA kits, EDTA plasma samples and serum samples with a pre-centrifugation delay longer than 24 h had an IL16 concentration higher than 313 pg/mL, and an IL8 concentration higher than 125 pg/mL, respectively. EDTA plasma samples with a pre-centrifugation delay longer than 48 h had an IL16 concentration higher than 897 pg/mL, citrate plasma samples had an IL8 concentration higher than 21.5 pg/mL and serum samples had an IL8 concentration higher than 528 pg/mL. CONCLUSIONS: These robust and accurate tools, based on simple and commercially available ELISA assays can greatly facilitate qualification of serum and plasma legacy collections with undocumented pre-analytics.

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.

Detection of busulfan adducts on proteins.

RATIONALE: Busulfan is a bifunctional alkyl sulfonate antineoplastic drug. This alkylating agent was described as forming covalent adducts on proteins. However, only limited data are available regarding the interaction of busulfan with proteins. Mass spectrometry and bioinformatics were used to identify busulfan adducts on human serum albumin and hemoglobin. METHODS: Albumin and hemoglobin were incubated with busulfan or control compounds, digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Thermo Fisher LTQ Orbitrap Velos Pro. MS data were used to generate spectral libraries of non-modified peptides and an open modification search was performed to identify potential adduct mass shifts and possible modification sites. Results were confirmed by a second database search including identified mass shifts and by visual inspection of annotated tandem mass spectra of adduct-carrying peptides. RESULTS: Five structures of busulfan adducts were detected and a chemical structure could be attributed to four of them. Two were primary adducts corresponding to busulfan monoalkylation and alkylation of two amino acid residues by a single busulfan molecule. Two others corresponded to secondary adducts generated during sample processing. Adducts were mainly detected on Asp, Glu, and His residues. These findings were confirmed by subsequent database searches and experiments with synthetic peptides. CONCLUSIONS: The combination of in vitro incubation of proteins with the drug of interest or control compounds, high-resolution mass spectrometry, and open modification search allowed confirmation of the direct interaction of busulfan with proteins and characterization of the resulting adducts. Our results also showed that careful analysis of the data is required to detect experimental artifacts. Copyright © 2016 John Wiley & Sons, Ltd.

Proteomics in mechanistic toxicology: history, concepts, achievements, caveats, and potential.

Toxicoproteomics can be defined as the application of proteomic approaches to the understanding of toxicology problems, and this review deals with the various types of applications that have been described in the literature. Toxicoproteomics has been applied to very different classes of toxicants, from drugs and natural products to metals, or from industrial chemicals to nanoparticles and nanofibers. It has also been applied to address questions at different levels, from the search of the primary molecular targets of toxicants to the deciphering of the molecular responses of cells and tissues to toxicants. Although restricted to mammalian cells and tissues, this paper reviews these two levels of investigation and the different application areas of toxicoproteomics, leading to the discussion of the advantages and drawbacks of the most popular proteomic platforms. Some of the pending questions in toxicoproteomics are also critically addressed, such as the specificity, validation, and result hierarchization issues. The question of shared mechanisms, which are encountered in many toxicoproteomic papers dealing with different toxicants, is also discussed. Finally, the future of toxicoproteomics is briefly outlined.

An integrated approach for comparative proteomic analysis of human bile reveals overexpressed cancer-associated proteins in malignant biliary stenosis.

Proteomics is a key tool in the identification of new bile biomarkers for differentiating malignant and nonmalignant biliary stenoses. Unfortunately, the complexity of bile and the presence of molecules interfering with protein analysis represent an obstacle for quantitative proteomic studies in bile samples. The simultaneous need to introduce purification steps and minimize the use of pre-fractionation methods inevitably leads to protein loss and limited quantifications. This dramatically reduces the chance of identifying new potential biomarkers. In the present study, we included differential centrifugation as a preliminary step in a quantitative proteomic workflow involving iTRAQ labeling, peptide fractionation by OFFGEL electrophoresis and LC-MS/MS, to compare protein expression in bile samples collected from patients with malignant or nonmalignant biliary stenoses. A total of 1267 proteins were identified, including a set of 322 newly described bile proteins, mainly belonging to high-density cellular fractions. The subsequent comparative analysis led to a 5-fold increase in the number of quantified proteins over previously published studies and highlighted 104 proteins overexpressed in malignant samples. Finally, immunoblot verifications performed on a cohort of 8 malignant (pancreatic adenocarcinoma, n=4; cholangiocarcinoma, n=4) and 5 nonmalignant samples (chronic pancreatitis, n=3; biliary stones, n=2) confirmed the results of proteomic analysis for three proteins: olfactomedin-4, syntenin-2 and Ras-related C3 botulinum toxin substrate 1. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Bile carcinoembryonic cell adhesion molecule 6 (CEAM6) as a biomarker of malignant biliary stenoses.

Differentiating malignant from nonmalignant biliary stenoses is challenging. This could be facilitated by the measurement of cancer biomarkers in bile. We aimed at (i) identifying new cancer biomarkers by comparative proteomic analysis of bile collected from patients with a malignant or benign biliary stenosis (exploratory phase) and (ii) verifying the accuracy of the newly identified potential biomarkers for discriminating malignant versus nonmalignant biliary stenoses in a larger group of patients (confirmation phase). Overall, 66 proteins were found overexpressed (ratio>1.5) in at least one cancer condition using proteomic analysis and 7 proteins were increased in all malignant/nonmalignant disease comparisons. Preliminary screening by immunoblot highlighted carcinoembryonic cell adhesion molecule 6 (CEAM6), a cell surface protein overexpressed in many human cancers, as an interesting candidate biomarker. ELISA subsequently confirmed CEAM6 as a potential bile biomarker for distinguishing malignant from benign biliary stenoses with a receiver operating characteristic (ROC) area under the curve (AUC) of 0.92 (specificity 83%, sensitivity 93%, positive predictive value 93%, and negative predictive value 83%). No significant difference in serum CEAM6 level was found between malignant and nonmalignant samples. Combining bile CEAM6 and serum CA19-9 in a panel further improved diagnostic accuracy for malignant stenoses (AUC 0.96, specificity 83%, sensitivity 97%, positive predictive value 93%, and negative predictive value 91%). CEAM6 measurement in bile could be clinically useful to discriminate between malignant and nonmalignant causes of biliary stenosis. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Identification of hemoglobin variants by top-down mass spectrometry using selected diagnostic product ions.

Hemoglobin disorder diagnosis is a complex procedure combining several analytical steps. Due to the lack of specificity of the currently used protein analysis methods, the identification of uncommon hemoglobin variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissociation mass spectrometry method was developed for hemoglobin ß chain analysis. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the hemoglobin ß chain sequence. The method was applied to the analysis of rare hemoglobin ß chain variants and an (A)γ-ß fusion protein. The results showed that the developed data analysis process allows fast and reliable interpretation of top-down electron transfer dissociation mass spectra by nonexpert users in the clinical area.

The proteomic to biology inference, a frequently overlooked concern in the interpretation of proteomic data: a plea for functional validation.

Proteomics will celebrate its 20th year in 2014. In this relatively short period of time, it has invaded most areas of biology and its use will probably continue to spread in the future. These two decades have seen a considerable increase in the speed and sensitivity of protein identification and characterization, even from complex samples. Indeed, what was a challenge twenty years ago is now little more than a daily routine. Although not completely over, the technological challenge now makes room to another challenge, which is the best possible appraisal and exploitation of proteomic data to draw the best possible conclusions from a biological point of view. The point developed in this paper is that proteomic data are almost always fragmentary. This means in turn that although better than an mRNA level, a protein level is often insufficient to draw a valid conclusion from a biological point of view, especially in a world where PTMs play such an important role. This means in turn that transformation of proteomic data into biological data requires an important intermediate layer of functional validation, i.e. not merely the confirmation of protein abundance changes by other methods, but a functional appraisal of the biological consequences of the protein level changes highlighted by the proteomic screens.

Applications of mass spectrometry for quantitative protein analysis in formalin-fixed paraffin-embedded tissues.

Proteomic analysis of tissues has advanced in recent years as instruments and methodologies have evolved. The ability to retrieve peptides from formalin-fixed paraffin-embedded tissues followed by shotgun or targeted proteomic analysis is offering new opportunities in biomedical research. In particular, access to large collections of clinically annotated samples should enable the detailed analysis of pathologically relevant tissues in a manner previously considered unfeasible. In this paper, we review the current status of proteomic analysis of formalin-fixed paraffin-embedded tissues with a particular focus on targeted approaches and the potential for this technique to be used in clinical research and clinical diagnosis. We also discuss the limitations and perspectives of the technique, particularly with regard to application in clinical diagnosis and drug discovery.

Assessment of the influence of the patient's inflammatory state on the accuracy of a haptoglobin selected reaction monitoring assay.

BACKGROUND: The use of targeted LC-MS/MS methods for protein quantitation in clinical laboratories implies a careful evaluation of potential sources of analytical interference. In this study, we investigated whether inflammation, which is associated with both the release of proteolytic enzymes and increased expression of acute phase protease inhibitors, is affecting the accuracy of a haptoglobin selected reaction monitoring (SRM) assay. RESULTS: A SRM assay was developed and used to quantify haptoglobin in 57 human serum samples. The SRM assay had CVs (n = 6) of 12.9% at 698 mg/L and 11.8% at 1690 mg/L. Results of the SRM assay were compared to those of a commercial immunonephelometric test. Passing-Bablok regression gave a proportional bias of 0.92 (95% CI: 0.82 to 1.04) and a constant bias of 75.40 (95% CI: -71.09 to 251.04), indicating that SRM and immunonephelometric assays provided comparable results. We then investigated whether the accuracy of the SRM assay was influenced by the patient's inflammatory state by assessing the relationship between the serum CRP concentration and the bias between the two methods. No correlation was found between the SRM/immunoassay bias and the CRP concentration (Pearson correlation coefficient r = 0.0898). CONCLUSIONS: These data indicate that neither the release of proteolytic enzymes nor the increased level of protease inhibitors occurring during inflammation processes have a significant impact on the haptoglobin SRM assay accuracy. Such studies provide important information about potential sources of analytical interferences in protein SRM assays.

Quantitative mass spectrometry analysis of intact hemoglobin A2 by precursor ion isolation and detection.

Precise and accurate quantification of proteins is essential in clinical laboratories. Here, we present a mass spectrometry (MS)-based method for the quantification of intact proteins in an ion trap mass spectrometer. The developed method is based on the isolation and detection of precursor ions for the quantification of the corresponding signals. The method was applied for the quantification of hemoglobin (Hb) A2, a marker used for the diagnosis of a ß-thalassemia trait. The α and δ globin chains, corresponding to total Hb and HbA2, respectively, were isolated in the ion trap at specific charge states and ejected without activation. Areas of the corresponding isolated precursor ions were used to calculate the δ to α ratio. Three series of quantifications were performed on 7 different days. The standard curve fitted linearly (R(2) = 0.9982) and allowed quantification of HbA2 over a concentration range from 3% to 18% of total Hb. Analytical imprecision ranged from 3.5% to 5.3%, which is enough to determine if the HbA2 level is below 3.5% or above 3.7%. In conclusion, our method reaches precision requirements that would be acceptable for the quantitative measurement of diagnostic proteins, such as HbA2, in clinical laboratories.

Therapeutic drug monitoring and clinical outcomes in severely ill patients receiving amoxicillin: a single-centre prospective cohort study.

Therapeutic drug monitoring (TDM) of ß-lactam antibiotics is increasingly used to overcome rising antimicrobial resistance and improve antibiotic exposure. However, there is little guidance on target amoxicillin plasma concentrations. We aimed to define these by evaluating associations between amoxicillin concentrations and clinical outcomes. This single-centre prospective cohort study enrolled severely ill and/or immunosuppressed adult patients receiving amoxicillin for suspected or confirmed bacterial infection. TDM with ≥1 intermediate and ≥1 trough level was performed 24 h after therapy initiation. Primary and secondary outcomes were incidence of adverse events (AEs) and clinical failure through Day 30, respectively. A total of 156 patients were included. Important variations were observed both for intermediate (mean 13 mg/L, S.D. 13) and trough (mean 7 mg/L, S.D. 9) amoxicillin levels. Of 111 patients, 33 (30%) had trough levels below the non-species-related breakpoint (2 mg/L). AEs occurred in 27/156 patients (17%); no intermediate- or trough-level threshold predicting toxicity could be established. Patients with the highest-quartile trough levels (9.07-51.5 mg/L) did not experience significantly increased AEs [6/28 (21%) vs. 13/83 (16%); P = 0.6]. Nearly one-third (48/156; 31%) experienced clinical failure; low trough levels did not correlate with failure. There were few amoxicillin AEs yet a relatively high incidence of clinical failure. While no toxicity threshold could be established, the absence of increased AEs among patients with the highest trough concentrations suggests that trough levels up to 40 mg/L may be safe, at least for limited durations. Larger trials must further define optimal amoxicillin concentrations. [ClinicalTrials.gov ID: NCT03790631].