The proteome of perilymph in patients with vestibular schwannoma. A possibility to identify biomarkers for tumor associated hearing loss?

Abstract

BackgroundDue to the surrounding bone, the human inner ear is relatively inaccessible and difficult to reach for cellular and molecular analyses. However, these types of investigations are needed to better understand the etiology, pathophysiology and progression of several inner ear disorders. Moreover, the fluid from the inner ear cannot be sampled for micro-chemical analyses from healthy individuals in vivo. Therefore, in the present paper, we studied patients with vestibular schwannoma (VS) undergoing trans-labyrinthine surgery (TLS). Our primary aim was to identify perilymph proteins in patients with VS on an individual level. Our second aim was to investigate the proteins identified at a functional level and our final aim was to search for biological markers for tumor-associated hearing loss and tumor diameter.Methods and findingsSixteen patients underwent TLS for sporadic VS. Perilymph was aspirated through the round window before opening the labyrinth. One sample was contaminated and excluded resulting in 15 usable samples. Perilymph samples were analyzed with an online tandem LTQ-Orbitrap mass spectrometer. Data were analyzed with MaxQuant software to identify the total number of proteins and to quantify proteins in individual samples. Protein function was analyzed using the PANTHER Overrepresentation tool. Associations between perilymph protein content, clinical parameters, tumor-associated hearing loss and tumor diameter were assessed using Random Forest and Boruta. In total, 314 proteins were identified; 60 in all 15 patients and 130 proteins only once in 15 patients. Ninety-one proteins were detected in at least 12 out of 15 patients. Random Forest followed by Boruta analysis confirmed that alpha-2-HS-glycoprotein (P02765) was an independent variable for tumor-associated hearing loss. In addition, functional analysis showed that numerous processes were significantly increased in the perilymph. The top three enriched biological processes were: 1) secondary metabolic processes; 2) complement activation and 3) cell recognition.ConclusionsThe proteome of perilymph in patients with vestibular schwannoma has an inter-individual stable section. However, even in a cohort with homogenous disease, the variation between individuals represented the majority of the detected proteins. Alpha-2-HS-glycoprotein, P02765, was shown to be an independent variable for tumor-associated hearing loss, a finding that needs to be verified in other studies. In pathway analysis perilymph had highly enriched functions, particularly in terms of increased immune and metabolic processes.

Biomarkers in Vestibular Schwannoma–Associated Hearing Loss

“…On the other hand, of the 91 commonly identified perilymph proteins of patients with VS on an individual level, Rasmussen et al described four proteins that were significantly associated with tumor-related deafness: Immunoglobulin (Ig) γ-4 chain C region, Ig κ chain C region, complement C3, and immunoglobulin heavy constant γ 3. These 91 proteins were identified in 12 out of 15 samples they used in the study (15), which was confirmed by analogy with data from previous MS research on perilymph (20, 24).…”

“…During an apoptotic or necrotic episode inside the inner ear, the proteins that are secreted can be found at high concentrations in this fluid (20). The knowledge of the perilymph proteome may shed some light on the mechanisms of tumor-associated hearing loss, which are mostly unknown to date (15). Stankovic et al speculated that because of an unknown toxic substance generated by the VS, hearing loss in VS could be linked to a high protein concentration in the perilymph (7).…”

“…Rasmussen et al hypothesized that VS may excrete alpha-2-HS-glycoprotein to the perilymph, where its inflammatory activity may lead to SNHL. Factors elicited from the VS may also affect the inner ear, inducing an upregulation of alpha-2-HS-glycoprotein within the perilymph (15).…”

“…As more and more patients are diagnosed and need to be managed, we are more than ever in need of searching for biomarkers associated with these tumors, in order to help with the choice of selecting between a “wait-and-scan” approach and surgery (15) aimed at reducing morbidity and increasing the hearing outcomes (4).…”

An LCMS-based untargeted metabolomics protocol for cochlear perilymph: highlighting metabolic effects of hydrogen gas on the inner ear of noise exposed Guinea pigs

“…As a consequence of the difficulty in obtaining samples, perilymph is a relatively unknown sample material in terms of bioanalysis. Most of the literature on the composition of human perilymph has been focused on its proteome content and is limited to patients undergoing surgical interventions (Lysaght et al 2011; Schmitt et al 2017; Edvardsson Rasmussen et al 2018; Palmer et al 2018). Some attention has been directed at characterizing the small metabolites of perilymph using mainly HPLC with fluorescence detection (Drescher, Medina and Drescher 1981; Medina and Drescher 1981; Scheibe and Haupt 1985; Bobbin and Fallon 1992).…”