Identification Of Potential Candidate Biomarkers In Lymphangioleiomyomatosis Using ITRAQ Proteomic Technology

Study Rational: Lympangioleiomyomatosis (LAM) is a rare and progressive cystic lung condition that affects between 3-7/million women. It manifests primarily in women of child-bearing age, with an average lag time between symptom onset and definitive diagnosis of upwards of 4 years. Currently no reliable biomarker exists for patients with LAM, therefore, identifying a predictive or prognostic disease biomarker could reduce diagnosis lag time, as well as increase our knowledge of the underlying disease mechanisms in this multifactorial condition. This study hypothesized that there were significant alterations in the protein content in serum between LAM patients and healthy aged-matched controls. Study Aim: Identification of potential candidate biomarkers in LAM patient serum. Methods: Serum from LAM patient volunteers (n= 3, n= 5) and healthy aged matched control volunteers (n= 4, n= 5) were pooled and two runs of high-throughput protein analysis were carried out using 4-plex iTRAQ technology on a Triple TOF 5600 (AB Sciex). After network pathway analysis, using Ingenuity Pathway Analysis software™, a number of the differentially expressed proteins (Fibronectin, von Willebrand Factor and Kallikrein III) were validated using ELISA in a wider individual patient cohort (n= 18) and an age matched healthy control group (n= 12). Results: A total of 14 proteins were differentially expressed in LAM serum compared to healthy age-matched control serum (p<0.05). It was found that an intricate network of cell trafficking proteins was altered. Differential expression of the remodelling proteins fibronectin (decreased expression of 30% in LAM sera, p = 0.03), von Willebrand Factor (vWF, 40% reduction in LAM, p = 0.03) and the serum protease Kallikrein III (KLK3, 25% increase in LAM, p = 0.027) were identified in pooled circulating LAM sera and subsequently validated in individual samples. Conclusion: This study demonstrates, for the first time, that in LAM a substantial imbalance exists in protein networks important for remodelling, particularly in the circulating levels of the structural proteins; fibronectin, vWF and KLK3. We have also revealed an intricate network of significantly altered proteins involved in cell trafficking, an integral component of LAM disease progression. These results provide a set of novel candidate biomarkers which may be useful for the development of a feasible approach to diagnosis, and potentially lead to a new effective targeted treatment for LAM.