Background

Multiple myeloma (MM) is a clear prototype of a bone marrow-infiltrating tumor for which a relatively small quantity of sample is available after the diagnostic procedure that involves morphological evaluation, immunophenotypic characterization by flow cytometry, and CD138⁺ plasma cell separation for routine fluorescence in situ hybridization analysis. Protein analysis in bone marrow samples from patients with MM has been limited by the low concentration of proteins obtained after CD138⁺ cell selection. In the last decades, genomics has dominated biomedical research. In fact, multiple myeloma has been comprehensively analyzed using high-throughput genomic technologies. Particularly, gene expression profiling has provided a molecular classification of MM, which is widely used in biological research. Despite the huge and relevant information obtained from this kind of analysis, it is surprising the very low number of biomarkers subsequently validated. The technological developments that have revolutionized the field of cancer genomics, and in particular of MM, have not progressed to the same extent in proteomics, mostly because the proteome is not as static as the genome, and can differ greatly between individuals and even cell to cell. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each myeloma sample in an automated manner. We have recently presented a method for the accurate and robust quantification of the expression of multiple proteins extracted from CD138-purified MM samples frozen in RLT+ buffer (QIAGEN), which is commonly used for nucleic acid preservation and isolation (Misiewicz-Krzeminska et al., 2018). Although previous data suggested the possibility of simultaneously extracting proteins and nucleic acids from those kinds of buffer (Chomczynski, 1993; Mathieson and Thomas, 2013; Vorreiter et al., 2016), this strategy has not been applied in the clinical setting. The capillary electrophoresis nano-immunoassay (CNIA) platform has the advantage of measuring protein expression with higher sensitivity and reproducibility compared to traditional Western blot. In addition, the high-throughput screening of samples, using low sample inputs, will allow detecting the relative abundance of protein isoforms in almost all MM patients with the consequent broad applications in MM biomarker discovery. Accordingly, we have demonstrated the biological and clinical value of this analysis for a panel of 12 proteins essential for MM pathogenesis (aiolos, calnexin, cereblon, c-myc, cyclin D1, cyclin D2, DDX21, HSP90, ikaros, PSME1, RIPK1 and XAF1) (Misiewicz-Krzeminska et al., 2018). Moreover, DEPTOR protein level in MM samples was successfully evaluated using this technique (Quwaider et al., 2017). Because of the low sample requirements, low complexity and rapidity of the protocol, these assays can be introduced in almost any laboratory with WES machine in order to evaluate potential protein biomarkers.