Proteomics of Plasma from Patients with Sickle Cell Disease with and without Chronic Pain: A Pilot Biomarker Discovery Study

Background Acute, severe, episodic nociceptive pain, or vaso-occlusive crises (VOCs) have been the hallmark of sickle cell disease (SCD), and responsible for >90% of health care encounters for this population. The concept that SCD patients experience a period of "normalcy" without any pain between these episodes of varying frequency has been challenged by several studies in the past two decades. It has been shown that ~30% of adults with SCD have chronic pain, defined as pain >50% of time for at least 6 months (Smith et al, 2008). The evolution of chronic pain is age dependent, and likely involves several different mechanisms. Studies in transgenic mouse models have shown that one mechanism involves neurogenic inflammation, triggered by mast cell activation (Vincent et al, 2013), through mediators such as tryptase and substance P. In a previous study (Albo et al, 2021) it was shown that patients with SCD and chronic pain had higher plasma levels of Tryptase and substance P, consistent with mast cell activation and neurogenic inflammation. Additionally, nerve growth factor (NGS), which has been incriminated in the pathophysiology of several chronic pain syndromes, was also found to be significantly elevated in the plasma of SCD patients with chronic pain. In this study, we conducted a pilot proteomics study of plasma samples from patients with and without chronic pain collected during the Albo et al study, and stored at -80 C.

Methods IRB approval was obtained, and informed consent was obtained from 72 patients enrolled in the study, whose plasma were stored. Discovery proteomics was conducted on 20 plasma samples from two groups (10 with chronic pain, 10 without chronic pain) through semiquantitative LC/MS/MS proteomics on the Thermo Fisher Orbitrap Mass Spectrometer. Samples were subjected to affinity depletion of major proteins to discern out lower concentration proteins. Filters were then utilized to remove excess proteins using two rules; a protein that only showed up in one patient and proteins that were not present in at least 5 patients. Differential expression was then used to yield proteins significantly different between the chronic pain and non-chronic pain groups

Results Table 1 shows the demographics, hydroxyurea therapy, opioid use, and pain scores of the 20 subjects included in the pilot study. Mass spectrometry of 20 samples revealed 5219 individual proteins. The filters cut down the protein count to 417, and differential expression yielded 16 proteins that were significantly different between the two groups. These included alpha-1 antitrypsin, Hepatocyte growth factor receptor, creatine kinase M-type, serine/threonine protein kinase SMG1, F-box only protein, Alstrom syndrome protein, beta transforming growth factor, serine/threonine protein mTOR, apolipoprotein C-1, keratin gylceraldehyde 3 phosphate dehydrogenase, Nesprin 1, retinol binding protein 3, Protein Wnt 16, multiple inositol polyphosphate phosphatase, immunoglobulin J-chain. Figure 1 demonstrates a heat map of these proteins down regulated and upregulated expression.

Conclusion This pilot study has yielded 16 differentially expressed proteins in the plasma of SCD patients with and without chronic pain. Validation of these findings in a larger cohort are under way. It is hoped that if confirmed, these findings will lead to the identification of biomarkers for chronic pain in SCD. In addition to the potential utility of these biomarkers in differential diagnosis of pain syndromes in SCD, targets for therapeutic intervention could also be identified.

Kutlar:Novartis Pharmaceuticals: Consultancy, Honoraria, Research Funding; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Vertex Pharmaceuticals, Inc.: Consultancy, Honoraria; bluebird bio Inc.: Consultancy, Honoraria; Graphite Bio Inc.: Consultancy, Honoraria; Forma Therapeutics, Inc.: Honoraria, Research Funding; Novo Nordisk Pharmaceuticals: Honoraria, Research Funding; Guidepoint Consulting Services: Consultancy, Honoraria; PeerView Institute: Honoraria; Eradigm Consultancy: Honoraria; MJH Life Sciences: Honoraria; American Society of Hematology: Consultancy, Honoraria, Research Funding; ORIC Pharmaceuticals, Inc.: Consultancy, Honoraria.