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Supplementary Tables S1-2, Figure 1. Supplementary Table S1. Matrix view of Part A-2 dose titration and PK sampling scheme. Supplementary Table S2. Efficacy outcomes by investigator review per RECIST v1.1. Supplementary Figure 1. Seventeen patients were tested with IFN-γ ELISPOT assay.

Figure S1. Seventeen patients were tested with IFN-γ ELISPOT assay.

Purpose: This phase I study evaluated the safety, maximum tolerated dose, antitumor activity, and pharmacokinetics and pharmacodynamics of pembrolizumab in patients with advanced solid tumors.Experimental Design: In a 3 + 3 dose escalation study, 10 patients received pembrolizumab 1, 3, or 10 mg/kg intravenously every 2 weeks until progression or intolerable toxicity. Seven additional patients received 10 mg/kg every 2 weeks. Thirteen patients participated in a 3-week intrapatient dose escalation (dose range, 0.005–10 mg/kg) followed by 2 or 10 mg/kg every 3 weeks. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1.Results: No dose-limiting toxicities were observed. Maximum administered dose was 10 mg/kg every 2 weeks. One patient with melanoma and one with Merkel cell carcinoma experienced complete responses of 57 and 56+ weeks' duration, respectively. Three patients with melanoma experienced partial responses. Fifteen patients with various malignancies experienced stable disease. One patient died of cryptococcal infection 92 days after pembrolizumab discontinuation, following prolonged corticosteroid use for grade 2 gastritis considered drug related. Pembrolizumab exhibited pharmacokinetic characteristics typical of humanized monoclonal antibodies. Maximum serum target engagement was reached with trough levels of doses greater than or equal to 1 mg/kg every 3 weeks. Mechanism-based translational models with a focus on intratumor exposure prediction suggested robust clinical activity would be observed at doses ≥2 mg/kg every 3 weeks.Conclusions: Pembrolizumab was well tolerated and associated with durable antitumor activity in multiple solid tumors. The lowest dose with full potential for antitumor activity was 2 mg/kg every 3 weeks. Clin Cancer Res; 21(19); 4286–93. ©2015 AACR.See related commentary by van Elsas et al., p. 4251

The Global Lab Quality Initiative (GLQI), formerly known as the Emerging Countries program, was funded through a generous endowment from the Wallace H. Coulter Foundation. The aims of GLQI are to develop and implement innovative programs to promote education and training in laboratory medicine for low- or lower middle-income countries worldwide. From its inception in 2010, the GLQI was focused solely on the Latin America and Caribbean (LAC) region under the purview of AACC’s Latin American Working Group (LAWG), the members of which have strong ties to the region thereby facilitating the partnerships with national societies. The LAWG has provided in-person workshops in the LAC countries, at the AACC Annual Scientific Meeting, and on-demand webinars. The LAWG aims to implement the GLQI aims in the LAC region. In-person workshops are based on best-practice recommendations and sources such as Clinical Laboratory Standard Institute guidelines and supplemented with professional experiences of the LAWG’s lecturers and local experts of the countries visited. In 2015, the GLQI expanded to other regions of the world. Here we report the experience of the LAWG workshops, results of participant surveys, in-person visits to laboratories post-workshop, and the lessons learned throughout the years across different geographic areas. We are hopeful this report provides insights into the challenges and successes of the LAWG in LAC to help support the expansion of the GLQI.

Decades of discussion and publication have gone into the guidance from the scientific community and the regulatory agencies on the use and validation of pharmacokinetic and toxicokinetic assays by chromatographic and ligand binding assays for the measurement of drugs and metabolites. These assay validations are well described in the FDA Guidance on Bioanalytical Methods Validation (BMV, 2018). While the BMV included biomarker assay validation, the focus was on understanding the challenges posed in validating biomarker assays and the importance of having reliable biomarker assays when used for regulatory submissions, rather than definition of the appropriate experiments to be performed. Different from PK bioanalysis, analysis of biomarkers can be challenging due to the presence of target analyte(s) in the control matrices used for calibrator and quality control sample preparation, and greater difficulty in procuring appropriate reference standards representative of the endogenous molecule. Several papers have been published offering recommendations for biomarker assay validation. The situational nature of biomarker applications necessitates fit-for-purpose (FFP) assay validation. A unifying theme for FFP analysis is that method validation requirements be consistent with the proposed context of use (COU) for any given biomarker. This communication provides specific recommendations for biomarker assay validation (BAV) by LC-MS, for both small and large molecule biomarkers. The consensus recommendations include creation of a validation plan that contains definition of the COU of the assay, use of the PK assay validation elements that support the COU, and definition of assay validation elements adapted to fit biomarker assays and the acceptance criteria for both.

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid ligand binding assay (LBA)/LCMS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for biotherapeutics, biomarkers and immunogenicity assays using hybrid LBA/LCMS and regulatory agencies' inputs. Part 1 (LCMS for small molecules, peptides and small molecule biomarkers) and Part 3 (LBA: immunogenicity, biomarkers and pharmacokinetic assays) are published in Volume 9 of Bioanalysis, issues 22 and 24 (2017), respectively.

Background : A new total bilirubin (B T ) method, based on multiple wavelength absorbance measurements, and an algorithm to calculate concentration, were evaluated for accuracy in specimens containing variable amounts of unconjugated bilirubin (B U ), conjugated bilirubin (B C ) and delta (protein-bound) bilirubin (B D ). Methods : Quantitation of B U , B C , and B T (with calculation of B D ) using a Vitros 250 analyzer served as the comparison method. Results : Analysis of neonatal specimens using a preliminary algorithm yielded good overall agreement with the Vitros B T method, but there was considerable variation in the agreement for individual specimens. When specimens from adults selected to yield a range of B C and B D levels were analyzed, the preliminary algorithm underestimated B T . Refinement of the method in the form of a finalized algorithm resulted in elimination of the negative bias seen with specimens with high B D and B C levels, and better agreement for individual neonatal specimens. Conclusions : This new method overcomes the limitations observed in earlier spectrophotometric methods, and provides accurate results in specimens containing a range of bilirubin forms.

Background: Glomerular filtration rate (GFR) is routinely assessed by measuring the concentrations of endogenous serum markers such as blood urea nitrogen and serum creatinine (SCr). Although widely used, these endogenous markers are not ideal and do not perform optimally in certain clinical settings. The purpose of this review is to critically review the potential utility of cystatin C (CysC), especially in patient populations in which CysC may have an advantage over routinely used endogenous markers of GFR.Approach: In a narrative approach, we extensively review publications, primarily from the last 5 years, that address the development of methods to measure CysC, reference intervals, and the diagnostic accuracy of CysC to assess GFR. Between June 2000 and September 2001 Medline was searched using “cystatin c” as a textword, and articles that examined >75 individuals (except for renal transplant studies) and/or used accepted “gold standards” for assessing GFR were selected for inclusion. A total of 17 studies are reviewed that provide reference interval data for several populations. A total of 24 studies make conclusions about the utility of CysC vs SCr and/or creatinine clearance, with 20 providing data on the sensitivity and specificity of CysC for detecting impaired GFR. These publications are organized into subgroups that deal with specific patient populations or clinical situations.Content: This review focuses on two areas: (a) the evolution of immunoassays used to determine the concentration of CysC in serum, their analytic sensitivity, and reference intervals; and (b) the diagnostic performance of CysC against other renal markers in the general population and in specific subpopulations of patients.Summary: Studies of reference intervals for CysC overwhelmingly demonstrated that CysC values in blood are independent of age and sex. Of the 24 studies that examined clinical utility, 15 concluded that CysC is superior to SCr, whereas 9 concluded that CysC is equivalent but provides no advantage. Summary ROC plot analysis of 20 studies that provide sensitivity and specificity data strongly suggests that CysC will be superior to SCr for detecting impaired GFR. Taken together, it is clear that CysC performs at least as well as SCr in the population at large and that it is likely to be superior to SCr in specific patient populations.

Apolipoprotein(a) is a polymorphic glycoprotein covalently bound to apoB100 in Lp(a) particles and has been described to be both atherogenic and prothrombotic, although its exact mechanism of action is not well defined. Apolipoprotein(a) is routinely measured by immunoassays. Unfortunately, the accuracy of the measurement can be affected by the apolipoprotein(a) size (number of kringles) polymorphism in Lp(a) particles. Here we describe an ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) assay that is capable of measuring apolipoprotein(a) concentrations while simultaneously determining the number of kringles present per protein.Plasma samples were diluted and proteins de-lipidated with deoxycholate prior to tryptic digestion. Distinct tryptic peptides from different regions of apolipoprotein(a) were measured to determine both concentration and the number of kringles present per protein. Separation and quantitation of tryptic peptides is carried out at 700 μL/min using a 1.7 µm C18 column (2.1 × 100 mm) coupled to a Thermo Vantage triple quadrupole (QQQ) mass spectrometer with a heated electrospray ionization (HESI) source.This method was compared to established methods for measuring concentration (monoclonal antibody based ELISA) and size (gel-electrophoresis) using 80 plasma samples proved by NWLRL. The slope and r(2) value for the correlation of concentrations were determined to be 0.96 and 0.98, demonstrating excellent agreement of absolute values between the UPLC/MS and ELISA methods. As measured by UPLC/MS, the average kringle number or size is smaller than determined by the electrophoretic method.A single UPLC/MS method was developed capable of measuring apolipoprotein(a) concentration and size (by measuring the number of kringles per protein). This assay passes criteria required for 'fit for purpose' assays including sensitivity, intra and interday reproducibility and freeze/thaw stability. While the agreement between UPLC/MS and ELISA is excellent for concentration and may provide researchers with additional tools for studying apolipoprotein(a), the dissimilarities between UPLC/MS and the electrophoretic method may also be exploited for understanding apolipoprotein(a) structure and function.

Background: Interferences in immunometric assays as a result of human anti-immunoglobulin antibodies frequently have been described in the literature. The etiology of these interfering antibodies is usually not known but has been associated with rheumatoid factors in some assays. It is known that microorganisms in experimental settings can induce anti-immunoglobulin antibodies.Methods: Following Escherichia coli septicemia, a 56-year-old male patient had increased immunoassay results for cardiac troponin I, thyrotropin, human chorionic gonadotropin, α-fetoprotein, and CA-125 that were consistent with myocardial infarction, hyperthyroidism, and pregnancy, and suggestive of an occult neoplasm such as hepatic or ovarian cancer. None of these diagnoses were consistent with the rest of his medical exam. In addition, the patient had a restricted IgM λ paraprotein by immunofixation. Plasma from the patient was incubated with Sepharose-conjugated protein A, irrelevant murine monoclonal antibodies, and formalin-killed E. coli organisms from his infection to determine whether these immunoassay values were falsely increased.Results: Incubation of the patient’s plasma with irrelevant murine monoclonal antibodies or the E. coli organism produced normal immunoassay values and removed the IgM λ paraprotein.Conclusions: The patient produced a very restricted IgM λ antibody response to the E. coli infection that had anti-immunoglobulin activity and caused falsely increased values in numerous immunometric assays. Microorganism-induced anti-immunoglobulin antibodies are discussed in the context of this patient.

The role of leukotrienes (LTs) in airway inflammatory diseases, such as asthma, has been extensively reported. The measurement of LTs in sputum supernatants, which is commonly done via enzyme immunoassays (EIAs), may prove to be useful for assessing airway inflammation. Despite the many advantages of EIA, these methods suffer from a lack of selectivity. Therefore, a selective and reliable method for the analysis of LTs in human sputum is needed. In this study we developed and validated a sensitive and specific method using ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), to measure simultaneously cysteinyl leukotrienes (CysLTs) and leukotriene B₄ (LTB₄) in human sputum. Sputum supernatants obtained by ultracentrifugation were stabilized by protease inhibitors, spiked with stable isotopic internal standards, and subjected to solid phase extraction (SPE) and UHPLC separation. Multiple reaction monitoring (MRM) transitions were optimized and measured on a mass spectrometer. The limit of detection (LOD) for LTE₄ and LTB₄ was 9.8 and 19.5 pg/mL, respectively. The lower limit of quantitation (LLOQ) for LTE₄ and LTB₄ was 19.5 and 39.0 pg/mL, respectively. The dynamic range of the LTE₄ assay was from 9.8 to 5000 pg/mL, whereas for the LTB₄ assay was from 19.5 to 10,000 pg/mL. The intra- and inter-day % coefficient of variation (%CV) was6.5% and10%, for both LTE₄ and LTB₄, respectively. Spike recovery ranged from 105% to 111% for both analytes. In addition, twenty-two sputum samples were analyzed for cysLTs and LTB₄. Fourteen of these samples were purchased commercially and eight were collected during the course of a clinical trial. LTB₄ was detectable in all samples tested and it ranged from 79 to 7220 pg/mL. LTE₄ was detectable in most of the sputum samples (12.3-891 pg/mL), whereas LTC₄ and LTD₄ were below limit of detection for majority of sputum samples. The in vitro conversion of LTC₄ and LTD₄ into LTE₄ was observed. The measurement of LTB₄ was sensitive to low pH and high temperature. The use of UHPLC-MS/MS method will allow a more accurate and reliable quantitation of LTs in human sputum, which in turn, may lead to a better understanding of the role of LTs in airway disease pathways and the application in associated clinical treatments.

background: Definitive diagnosis of Alzheimer disease (AD) can be made only by histopathological examination of brain tissue, prompting the search for premortem disease biomarkers. We sought to determine if the novel brain injury biomarker, visinin-like protein 1 (VLP-1), is altered in the CSF of AD patients compared with controls, and to compare its values to the other well-studied CSF biomarkers 42-amino acid amyloid-β peptide (Aβ1–42), total Tau (tTau), and hyperphosphorylated Tau (pTau). methods: Using ELISA, we measured concentrations of Aβ1–42, tTau, pTau, and VLP-1 in CSF samples from 33 AD patients and 24 controls. We compared the diagnostic performance of these biomarkers using ROC curves. results: CSF VLP-1 concentrations were significantly higher in AD patients [median (interquartile range) 365 (166) ng/L] compared with controls [244 (142.5) ng/L]. Although the diagnostic performance of VLP-1 alone was comparable to that of Aβ, tTau, or pTau alone, the combination of the 4 biomarkers demonstrated better performance than each individually. VLP-1 concentrations were higher in AD subjects with APOE ε4/ε4 genotype [599 (240) ng/L] compared with ε3/ε4 [376 (127) ng/L] and ε3/ε3 [280 (115.5) ng/L] genotypes. Furthermore, VLP-1 values demonstrated a high degree of correlation with pTau (r = 0.809) and tTau (r = 0.635) but not Aβ1–42 (r = −0.233). VLP-1 was the only biomarker that correlated with MMSE score (r = −0.384, P = 0.030). conclusions: These results suggest that neuronal injury markers such as VLP-1 may have utility as biomarkers for AD.

Background: The diagnosis of diseases leading to brain injury, such as stroke, Alzheimer disease, and Parkinson disease, can often be problematic. In this study, we pursued the discovery of biomarkers that might be specific and sensitive to brain injury. Methods: We performed gene array analyses on a mouse model to look for biomarkers that are both preferentially and abundantly produced in the brain. Via bioinformatics databases, we identified the human homologs of genes that appeared abundant in brain but not in other tissues. We then confirmed protein production of the genes via Western blot of various tissue homogenates and assayed for one of the markers, visinin-like protein 1 (VLP-1), in plasma from patients after ischemic stroke. Results: Twenty-nine genes that were preferentially and abundantly expressed in the mouse brain were identified; of these 29 genes, 26 had human homologs. We focused on 17 of these genes and their protein products on the basis of their molecular characteristics, novelty, and/or availability of antibodies. Western blot showed strong signals in brain homogenates for 13 of these proteins. Tissue specificity was tested by Western blot on a human tissue array, and a sensitive and quantitative sandwich immunoassay was developed for the most abundant gene product observed in our search, VLP-1. VLP-1 was detected in plasma of patients after stroke and in cerebrospinal fluid of a rat model of stroke. Conclusions: The use of relative mRNA production appears to be a valid method of identifying possible biomarkers of tissue injury. The tissue specificity suggested by gene expression was confirmed by Western blot. One of the biomarkers identified, VLP-1, was increased in a rat model of stroke and in plasma of patients after stroke. More extensive, prospective studies of the candidate biomarkers identified appear warranted.

Background: Recent guidelines for use of cardiac troponin to detect cardiac damage and for cardiovascular risk stratification have made increasingly sensitive troponin assays important. Troponin assays continue to be plagued by interferences caused by heterophilic antibodies (HAs). We evaluated the performance of a revised cardiac troponin I (cTnI) assay designed to have increased analytical sensitivity and to minimize the effect of HAs.Methods: The revised Dade Behring Dimension® cTnI assay was evaluated according to NCCLS EP5-A at five institutions. Plasma samples from 14 309 patients were assayed by the original Dimension cTnI assay. To identify samples that may have interfering HAs, samples with values >1.4 μg/L were reanalyzed on the Dade Behring Stratus® CS cTnI assay. Samples with possible interfering antibodies were also analyzed before and after selective absorbance studies on the revised Dade Behring Dimension cTnI assay.Results: The limit of quantification in the revised method was 0.1 μg/L with imprecision (CV) of 11–17% at 0.1 μg/L. Values correlated well with the Stratus CS cTnI method: revised = 1.06(original) + 0.01; r = 0.98, Sy|x = 0.25 μg/L). Falsely increased results consistent with myocardial infarction by the original Dimension cTnI assay and presumably attributable to HAs were identified in 0.17% of all patients with samples submitted for cTnI analysis. The revised Dimension cTnI assay eliminated the interference in 17 of 25 samples identified and greatly decreased the interference in the other 8.Conclusions: The revised Dimension cTnI method greatly minimizes the effect of interfering HAs. It also exhibits analytical performance characteristics consistent with recent guidelines for use of this assay to detect cardiac damage.