A Single Reference Interval for Interpreting Serum Free Light Chains across Patients with Varying Renal Function.

BACKGROUND: Serum free light chain (sFLC) assays are interpreted using a sFLC-ratio-based reference interval (manufacturer's interval) that was defined using a cohort of healthy patients. However, renal impairment elevates the sFLC-ratio, leading to a high false positive rate when using the manufacturer's interval. Prior studies have developed renal-specific reference intervals; however, this approach has not been widely adopted due to practical limitations. Thus, there remains a critical need for a renally robust sFLC interpretation method. METHODS: Retrospective data mining was used to define patient cohorts that reflect the spectrum of renal function seen in clinical practice. Two new reference intervals, one based on the sFLC-ratio and one based on a novel principal component analysis (PCA)-based metric, were developed for the FREELITE assay (Binding Site) on the Roche Cobas c501 instrument (Roche). RESULTS: Compared to the manufacturer's reference interval, both new methods exhibited significantly lower false positive rates and greater robustness to renal function while maintaining equivalent sensitivity for monoclonal gammopathy (MG) diagnosis. While not significantly different, the point estimate for sensitivity was highest for the PCA-based approach. CONCLUSION: Renally robust sFLC interpretation using a single reference interval is possible given a reference cohort that reflects the variation in renal function observed in practice. Further studies are needed to achieve sufficient power and determine if the novel PCA-based metric offers superior sensitivity for MG diagnosis. These new methods offer the practical advantages of not requiring an estimated glomerular filtration rate result or multiple reference intervals, thereby lowering practical barriers to implementation.

Single-Cell Discovery and Multiomic Characterization of Therapeutic Targets in Multiple Myeloma.

Multiple myeloma (MM) is a highly refractory hematologic cancer. Targeted immunotherapy has shown promise in MM but remains hindered by the challenge of identifying specific yet broadly representative tumor markers. We analyzed 53 bone marrow (BM) aspirates from 41 MM patients using an unbiased, high-throughput pipeline for therapeutic target discovery via single-cell transcriptomic profiling, yielding 38 MM marker genes encoding cell-surface proteins and 15 encoding intracellular proteins. Of these, 20 candidate genes were highlighted that are not yet under clinical study, 11 of which were previously uncharacterized as therapeutic targets. The findings were cross-validated using bulk RNA sequencing, flow cytometry, and proteomic mass spectrometry of MM cell lines and patient BM, demonstrating high overall concordance across data types. Independent discovery using bulk RNA sequencing reiterated top candidates, further affirming the ability of single-cell transcriptomics to accurately capture marker expression despite limitations in sample size or sequencing depth. Target dynamics and heterogeneity were further examined using both transcriptomic and immuno-imaging methods. In summary, this study presents a robust and broadly applicable strategy for identifying tumor markers to better inform the development of targeted cancer therapy. SIGNIFICANCE: Single-cell transcriptomic profiling and multiomic cross-validation to uncover therapeutic targets identifies 38 myeloma marker genes, including 11 transcribing surface proteins with previously uncharacterized potential for targeted antitumor therapy.

Murine models of human acute myeloid leukemia.

Primary human AML cells can be isolated and studied in vitro, but many experimental questions can only be addressed using in vivo models. In particular, tractable animal models are needed to test novel therapies. The genetic complexity of human AML poses significant challenges for the generation of reliable animal models. The hematopoietic systems of both zebrafish ( Danio rerio) and Drosophila have been well characterized ( reviewed in [5, 31]) . Both organisms are well suited to forward genetics mutagenesis screens. Although this approach has been useful for identification of mutants with hematopoietic phenotypes ( e.g., cloche), the impact on cancer biology and hematopoietic malignancies in particular has been limited. A zebrafish model of acute lymphoblastic leukemia has been generated [37] and Drosophila models have shed light on the biology of epithelial tumors ( reviewed in [60]). Nonetheless, in vivo modeling of human AML relies most heavily on mice. Most cellular, molecular, and developmental features of the hematopoietic system are well conserved across mammalian species. The availability of the human and mouse genome sequences and the capability of manipulating the mouse genome make mice the most valuable model organism for AML research. Mice have additional practical value because they have a short reproductive cycle and are relatively inexpensive to house.

NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase.

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells and CTLs. It has been localized to the cytolytic granules in NK cells and is up-regulated when cells are exposed to cytokines IL-2 or IFN-beta. We report in this study that NKLAM contains a cysteine-rich really interesting new gene (RING) in between RING-RING domain, and that this domain possesses strong homology to the RING domain of the known E3 ubiquitin ligase, Dorfin. To determine whether NKLAM functions as an E3 ligase, we performed coimmunoprecipitation binding assays with ubiquitin conjugates (Ubcs) UbcH7, UbcH8, and UbcH10. We demonstrated that both UbcH7 and UbcH8 bind to full-length NKLAM. We then performed a similar binding assay using endogenous NKLAM and UbcH8 expressed by human NK clone NK3.3 to show that the protein interaction occurs in vivo. Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. We confirmed that NKLAM and URKL-1 interact in mammalian cells by using both immunoprecipitation and confocal microscopy. We demonstrated decreased protein expression and enhanced ubiquitination of URKL-1 in the presence of NKLAM. These data indicate that NKLAM is a RING finger protein that binds Ubcs and has as one of its substrates, URKL-1, thus defining this cytolytic protein as an E3 ubiquitin ligase.