Circulating Biomarkers From the Phase 1 Trial of Sirolimus and Autophagy Inhibition for Patients With Lymphangioleiomyomatosis

Abstract

Background: We have previously conducted the Sirolimus and Autophagy Inhibition in LAM (SAIL) trial, a phase 1 dose-escalation study of the combination of sirolimus and hydroxychloroquine in patients with lymphangioleiomyomatosis (LAM). The goal of the present study was to analyze sera from the SAIL trial to identify novel biomarkers that could shed light into disease pathogenesis and response to therapy.

Methods: We used the DiscoveryMAP platform from Rules Based Medicine to simultaneously measure 279 analytes in sera collected at each visit from subjects enrolled in the SAIL trial. We used longitudinal regression and pathway analysis to examine analyte rate of change and corresponding effect on lung function and to identify networks and potential nodes of interest.

Results: A total of 222 analytes were included in the analysis. We identified 32 analytes that changed over the treatment period of the study. Pathway analysis revealed enrichment in cytokine-receptor interaction and mechanistic/mammalian target of rapamycin-related pathways, in addition to seemingly unrelated processes such as rheumatoid arthritis. Search Tool for the Retrieval of Interacting Genes/Proteins analysis identified two hubs centered around acetyl-CoA carboxylase alpha and beta and coagulation factor II. In addition, we identified vascular endothelial growth factor receptor-3 and CCL21 as molecules significantly associated with changes in FEV1 during the study period.

Conclusions: We performed a large-scale analyte study in sera of women with LAM and identified potential markers that could be linked to disease pathogenesis, lung injury, and therapeutic response. These data will enable future investigation into the specific roles of these molecules in LAM.

Trial registry: ClinicalTrials.gov; No. NCT01687179; URL: www.clinicaltrials.gov).

Keywords: LAM; TOR; autophagy; biomarkers; lung function.

Copyright © 2018 American College of Chest Physicians. All rights reserved.

Figures

Figure 1

Study procedure. A, Trial design and timing of sample collection. B, Analytes selected for analysis. SAIL = Sirolimus and Autophagy Inhibition for Patients With Lymphangioleiomyomatosis.

Figure 2

Peripheral blood proteins changed with combination therapy with sirolimus and hydroxychloroquine. Heat map of analytes that significantly changed during the treatment period for subjects with analyte measurements at baseline, the end of treatment (week 24), and the end of observation (week 48). Columns: individual patient measurements at weeks 0, 24, and 48; rows: analytes that changed significantly between weeks 0 and 24 by repeated measures analysis of variance. Log10-transformed analyte levels were scaled separately for each patient across the three visits of interest. Increased shades of yellow, increased relative to subject mean; increased shades of purple, decreased relative to subject mean; gray, unchanged relative to subject mean. Analytes were clustered hierarchically and subjects were clustered hierarchically and separately within each visit.

Figure 3

Network analysis of treatment-dependent analytes. Analysis of 32 analytes differentially changed over time and multiple visits between baseline and end of treatment. STRING did not recognize the analyte PECAM1, so it was not included in the network. A, KEGG pathways. B, GO terms enriched in this group of analytes. C, STRING analysis shows a hub centered around acetyl-CoA carboxylase alpha and beta, and F2. F2 = coagulation factor II; GO = gene ontology; KEGG = Kyoto Encyclopedia of Genes and Genomes; PECAM1 = platelet endothelial cell adhesion molecule 1.

Figure 4

Analytes significantly associated with changes in % FEV1 over entire study. Estimated increase in % FEV1 given a 1-log unit change in the analyte when comparing two individuals with the same baseline FEV1 in a given week (mean ± standard error). Analytes are ordered from left to right on the basis of decreasing association significance. Analytes that increased during the study (red) and decreased during the study (blue) are included. MMP = matrix metalloproteinase 9; NGAL = neutrophil gelatinase-associated lipocalin; PARK = pulmonary and activation regulated chemokine; SHBG = sex hormone–binding globulin; SPD = pulmonary surfactant–associated protein D; VEGF-D = vascular endothelial growth factor-D.