Biomarkers of erythropoiesis response to intravenous iron in a crossover pilot study in unexplained anemia of the elderly.

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.

Clinical development and evaluation of a VEGF-D assay in plasma from patients with metastatic colorectal cancer in the RAISE study.

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.

Analytical Characterization of an Enzyme-Linked Immunosorbent Assay for the Measurement of Transforming Growth Factor ß1 in Human Plasma.

BACKGROUND: The transforming growth factor ß (TGF-ß)-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-ß1 is the most prevalent isoform, and an assay to measure plasma levels of TGF-ß1 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-ß1 in human plasma. METHODS: A colorimetric sandwich ELISA for TGF-ß1 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-ß1 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-ß1 level was 1985 ± 1488 pg/mL compared to 4243 ± 2003 pg/mL for HCC patients. Additionally, the TGF-ß1 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-ß1 in human plasma and for investigational use in an approved clinical study.