Molecular Expression Differences in Blood Mononuclear Cells in Patients with AL Amyloidosis
Authors: Kaplan David , Christian Eric , Valent Jason , Anwer Faiz , Raza Shahzad , Mazzoni Sandra , Samaras Christy J. , Williams Louis S. , Dima Danai , Shepherd Kelly , Fennema Jared , Fada Sherry , Nakashima Megan , Hanna Mazen , Lazarus Hillard M. , Khouri Jack 
Published: December 2023

Background
Diagnosis of AL amyloidosis requires tissue biopsy using different surrogate and target biopsy sites with variable sensitivity leading to delay in diagnosis and worse patient outcomes. Mononuclear cells (MNC's) circulate in the peripheral blood and possess receptors for components in the tissue depositions of AL. Receptor triggered signaling is expected to result in characteristic changes in molecular expression levels and may be detected with a sensitive cell-specific technology. In this way, blood MNC's and receptor-mediated signaling could be used as a sensitive indicator of the presence of the AL deposits. The CellPrint™ platform, with the ability to assign molecular expression to specific cell lineages but with 10-100-fold enhanced sensitivity, is ideal for identifying the patterns of molecular expression in MNC's that are characteristic of AL. Herein, we are proposing a novel approach to the development of a biomarker for AL.
Methods
Peripheral blood samples were collected from patients with AL (n = 27) and multiple myeloma (MM) (n = 41) at the Cleveland Clinic. The samples were processed within four hours of venipuncture by isolating the mononuclear cell fraction via discontinuous gradient separation over ficoll/hypaque. The mononuclear cells were suspended in medium containing dimethyl sulfoxide, viably frozen, and stored in liquid nitrogen. The laboratory personnel were blinded to the clinical data associated with the samples. The code was not broken until the expression level dataset was finalized. Once accrual of the samples was completed, the cells were thawed, stained with antibodies to a lineage marker, fixed, permeabilized, and stained for the expression of selected analytes with signal amplification by CellPrint™. The expression levels of these analytes were assessed in CD4+ T lymphocytes and monocytes.
Results
Both differences in cell-type specific levels of expression and intermolecular organization, as indicated by bivariate correlations, were observed between samples from MM patients and AL. These differences were most pronounced in CD4+ T cells, but they also occurred in monocytes. CD4+ T cells from patients with AL demonstrated decreased expression of BDNF, calmodulin, and phospho-TBK1 as compared to those from patients with MM (figure 1). BDNF and phospho-TBK1 were found to be highly correlated in the CD4+ T cells from the patients with AL (r=0.75). In CD4+ T cells of patients without AL (MM patients), Vav expression correlated with phospho-Erk, phospho-GSK3β, phospho-cJun and other molecules. However, in these cells from patients with AL, Vav did not correlate with any of these molecules (table 1). Similar results were found with phospho-cJun. Thus, the contribution of Vav and phospho-cJun to various signal transduction pathways is altered in patients with AL. These analytes are known to be essential for the activation of T cells and the overall effect of the presence of amyloid on CD4+ T cells may be suppressive. Both cJun and Vav are known to be involved in the balance between T cell activation and exhaustion (Cells 9:2470, 2020). Alterations in Vav activation have also been observed because of PD1 ligation on T cells (Science 355:1428, 2017). Our findings show that both activated cJun (phospho-cJun) and Vav were disconnected to the major signaling pathways in CD4+ T cells.
Conclusion
Our results show molecular expression level and bivariate correlational changes in both CD4+ T cells and monocytes in patients with AL; the changes were more pronounced in the T cells. The finding of a possible exhaustion-type phenotype in CD4+ T cells in AL patients but not in MM patients requires further investigation and may have therapeutic implications. It will also be important to look at MNC molecular phenotype in patients with MGUS which could possibly serve as a predictor of progression to AL.