Targeted Mass Spectrometry Enables Multiplexed Quantification of Immunomodulatory Proteins in Clinical Biospecimens.

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.

A clinical trial of IL-15 and IL-21 combination therapy for COVID-19 is warranted.

Previous studies of SARS-CoV-2 viral infection suggest that both the humoral and cytotoxic arms of the immune system are weak in patients with severe COVID-19 disease when compared to mild disease. A cytokine storm is also induced in severe disease. IL-15 has been shown to support the cytotoxic arm of the immune response. IL-21 has been shown to support both the cytotoxic and humoral arms of the immune response. In addition, in some settings, Il-21 has been shown to actually decrease IL-6 and TNF-alpha production, reducing the inflammatory proteins involved in the cytokine storm. Furthermore, in other settings, the combination of IL-15 and IL-21 has been shown to be more effective than either interleukin alone in promoting an effective immune response. Therefore, a clinical trial that examines the use of the combination of IL-15 and IL-21 for COVID-19 patients is warranted.

Development of a test to identify bladder cancer in the urine of patients using mass spectroscopy and subcellular localization of the detected proteins.

The survival rate for bladder cancer is much better when the disease is detected early, so improvements in methodology for early detection would be beneficial. When urine contains neoplastic urothelial cells, it carries biomarkers of the disease. This study aims to develop a test for the detection of urothelial carcinoma in the urine. The sediments from urines of ten patients with carcinoma and ten randomly selected normal controls were tested for cancer biomarkers using high-resolution mass spectroscopy. 212 unique individual proteins were identified. Most of them occurred only once or twice in the entire cohort of cases. When sorting the detected proteins by their subcellular compartments, we were able to develop a test that differentiates between the two sets. When the combination of nuclear and red blood cell proteins was used as the discriminating function, the level of statistical significance was p=0.003, the sensitivity was 90%, the specificity 67% and the area under the Receiver-Operating Characteristic curve (ROC) was 94%. When the lack of any detectible proteins, which includes nuclear proteins, was included as a criterion indicating benign urine, the specificity increased to 80%. This use of cellular compartment localization of the detected proteins in the discriminating function is less restrictive than requiring the presence of specific proteins, and we were able to develop a screening test with this less stringent criterion. This approach can be applied to other tumors, such as breast, lung and colon cancers, where the need for a simple screening test is even greater.