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Anemia is common in older adults, but often unexplained. Previously, we conducted a randomized, controlled trial of intravenous (IV) iron sucrose to study its impact on the 6-minute walk test and hemoglobin in older adults with unexplained anemia and ferritin levels of 20-200 ng/mL. In this report, we present for the first time the response of hemoglobin, as well as the dynamic response of biomarkers of erythropoiesis and iron indices, in a pooled analysis of the initially IV iron-treated group of 9 subjects and the subsequently IV iron treated 10 subjects from the delayed treatment group. We hypothesized that there would be a reproducible hemoglobin response from IV iron, and that iron indices and erythropoietic markers would reflect appropriate iron loading and reduced erythropoietic stress. To investigate the biochemical response of anemia to IV iron, we studied the dynamics of soluble transferrin receptor (STfR), hepcidin, erythropoietin (EPO), and iron indices over 12 weeks after treatment. In total, all 19 treated subjects were evaluable: 9 from initial treatment and 10 after cross-over. Hemoglobin rose from 11.0 to 11.7 g/dL, 12 weeks after initiating IV iron treatment of 1000 mg divided weekly over 5 weeks. We found early changes of iron loading after 1-2 IV iron dose: serum iron increased by 184 mcg/dL from a baseline of 66 mcg/dL, ferritin by 184 ng/mL from 68 ng/mL, and hepcidin by 7.49 ng/mL from 19.2 ng/mL, while STfR and serum EPO declined by 0.55 mg/L and 3.5 mU/mL from 19.2 ng/mL and 14 mU/mL, respectively. The erythroid response and evidence of enhanced iron trafficking are consistent with the hypothesis that IV iron overcomes iron deficient or iron-restricted erythropoiesis. These data provide new insight that iron-restricted erythropoiesis is a potential and targetable mechanism for patients diagnosed with unexplained anemia of the elderly and offers support for larger prospective trials of IV iron among anemic older adults of low to normal ferritin.

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-μm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted β-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Vascular endothelial growth factor (VEGF)-D was identified as a potential predictive biomarker for ramucirumab efficacy in second-line metastatic colorectal cancer using a research use only (RUO) assay. We describe results with a new assay for detecting VEGF-D in human plasma., Methods: In RAISE (Clinical Trial Registration: NCT01183780), 1072 patients were randomized 1:1 to ramucirumab or placebo plus FOLFIRI. All patients were then randomized 1:2 to marker exploratory (ME) and marker confirmatory (MC) groups, and those with plasma samples were analyzed accordingly. A new assay validated for investigational use only (IUO) was used to measure VEGF-D levels in plasma, which were analyzed for correlation with overall and progression-free survival (OS/PFS). IUO assay data were compared with historical RUO assay data., Results: ME subset analyses determined the optimal cutpoint of 5.4 ng/mL for defining high/low VEGF-D subgroups. In the combined ME/MC placebo arms, OS/PFS were numerically greater for patients with low vs high VEGF-D (OS: 12.8 vs 11.1 months; PFS: 5.6 vs 4.2 months). In patients with high VEGF-D, ramucirumab vs placebo demonstrated a numerically greater improvement in OS and PFS. Differential efficacy by VEGF-D level was statistically significant for PFS, but not OS., Conclusion: In patients with high VEGF-D, ramucirumab demonstrated a greater improvement in OS and PFS vs placebo; however, baseline VEGF-D level was not predictive of ramucirumab OS benefit using VEGF-D assay for IUO. The RAISE intent-to-treat results remain valid.

New therapeutics targeting immune checkpoint proteins have significantly advanced treatment of non-small cell lung cancer (NSCLC), but protein level quantitation of drug targets presents a critical problem. We used multiplexed, targeted mass spectrometry (MS) to quantify immunotherapy target proteins PD-1, PD-L1, PD-L2, IDO1, LAG3, TIM3, ICOSLG, VISTA, GITR, and CD40 in formalin-fixed, paraffin-embedded (FFPE) NSCLC specimens. Immunohistochemistry (IHC) and MS measurements for PD-L1 were weakly correlated, but IHC did not distinguish protein abundance differences detected by MS. PD-L2 abundance exceeded PD-L1 in over half the specimens and the drug target proteins all displayed different abundance patterns. mRNA correlated with protein abundance only for PD-1, PD-L1, and IDO1 and tumor mutation burden did not predict abundance of any protein targets. Global proteome analyses identified distinct proteotypes associated with high PD-L1-expressing and high IDO1-expressing NSCLC. MS quantification of multiple drug targets and tissue proteotypes can improve clinical evaluation of immunotherapies for NSCLC.

Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, posttranslational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed-death-ligand-1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using four antibodies (22C3, 28-8, E1L3N, and SP142), raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3041 protein groups (24,737 distinct peptides). PD-L1 quantitation correlated well with IHC expression on unaged sections (R 2  = 0.744; P < 0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal (56%, 58%, and 33% reduction respectively; p < 0.05). Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.

Background: The transforming growth factor β (TGF-β)-signaling pathway has emerged as a promising therapeutic target for many disease states including hepatocellular carcinoma (HCC). Because of the pleiotropic effects of this pathway, patient selection and monitoring may be important. TGF-β1 is the most prevalent isoform, and an assay to measure plasma levels of TGF-β1 would provide a rational biomarker to assist with patient selection. Therefore, the objective of this study was to analytically validate a colorimetric ELISA for the quantification of TGF-β1 in human plasma., Methods: A colorimetric sandwich ELISA for TGF-β1 was analytically validated per Clinical and Laboratory Standards Institute protocols by assessment of precision, linearity, interfering substances, and stability. A reference range for plasma TGF-β1 was established for apparently healthy individuals and potential applicability was demonstrated in HCC patients., Results: Precision was assessed for samples ranging from 633 to 10822 pg/mL, with total variance ranging from 28.4% to 7.2%. The assay was linear across the entire measuring range, and no interference of common blood components or similar molecules was observed. For apparently healthy individuals, the average TGF-β1 level was 1985 ± 1488 pg/mL compared to 4243 ± 2003 pg/mL for HCC patients. Additionally, the TGF-β1 level in plasma samples was demonstrated to be stable across all conditions tested, including multiple freeze-thaw cycles., Conclusions: The ELISA described in this report is suitable for the quantification of TGF-β1 in human plasma and for investigational use in an approved clinical study., (© 2018 American Association for Clinical Chemistry.)

Assessing the pharmacodynamics (PD) of a potential therapeutic through the use of a downstream biomarker is essential. This is traditionally performed in the target tissue but limited volume and invasiveness of sampling pose challenges with solid tumours. Currently, there are several small molecule receptor kinase inhibitors and large molecule therapeutic antibodies in clinical trials that interfere with TGFbeta signalling to treat various forms of cancer. With the advent of these new therapies, there is a need for a surrogate tissue that is easily accessible and indicative of tumour response. We propose the use of an ex vivo TGFbeta1 stimulation of peripheral blood mononuclear cells (PBMCs) coupled with the measurement of phosphorylated SMAD2 (Sma/Mothers Against dpp, a downstream transcriptional activator) using a sandwich ELISA. TGFbeta is involved in many different cellular responses, such as proliferation, angiogenesis, migration, invasion and immunomodulation. SMAD2 and SMAD3 are phosphorylated as a result of the canonical cascade through ligand binding and receptor kinase activation. These phosphorylated SMADs (pSMAD) associate with SMAD4, a co-SMAD, and transcriptionally activate TGFbeta-mediated genes. This paper describes the novel method for measuring the downstream effects of inhibiting canonical TGFbeta signalling using ex vivo stimulation of surrogate tissue to predict tumour response. In addition, we present the assay validation rationale and data. This novel, validated assay can be used to gain insight into clinical trials regarding TGFbeta signal modulation by multiple inhibitor platforms for both large and small molecules.

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/alpha(1)-antiprotease complex concentration or P(a)O(2)/F(i)O(2) ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.

The identification of Clostridium species can be problematic for the diagnostic microbiology laboratory. The introduction of the MIDI Microbial Identification System (MIS) has enabled personnel in diagnostic laboratories to perform cellular fatty acid analyses on a variety of microorganisms. We used the Virginia Polytechnic Institute (Blacksburg, VA) Anaerobe Library (Moore Version 3.8) to perform analyses on 216 strains representing 18 species of Clostridium. The organisms were characterized with use of traditional biochemical methods that employ prereduced anaerobically sterilized media and other reference diagnostic methods. The MIS identified 86% of the strains correctly to the species level without the need for supplemental tests, and identified an additional 6% of the strains to species levels with the aid of a few supplemental tests. Only 3% of strains were identified by genus incorrectly. The cellular fatty acid patterns of selected, medically important clostridia were so distinctive that 100% of these species were identified correctly. The MIS has many practical benefits, including speed of identification, and few disadvantages (such as equipment cost) for the clinical microbiology laboratory.