The introduction of technologies such as mass spectrometry and protein and DNA arrays, combined with our understanding of the human genome, has enabled simultaneous examination of thousands of proteins and genes in single experiments, which has led to renewed interest in discovering novel biomarkers for cancer. The modern technologies are capable of performing parallel analyses as opposed to the serial analyses conducted with older methods, and they therefore provide opportunities to identify distinguishing patterns (signatures or portraits) for cancer diagnosis and classification as well as to predict response to therapies. Furthermore, these technologies provide the means by which new, single tumor markers could be discovered through use of reasonable hypotheses and novel analytical strategies. Despite the current optimism, a number of important limitations to the discovery of novel single tumor markers have been identified, including study design bias, and artefacts related to the collection and storage of samples. Despite the fact that new technologies and strategies often fail to identify well-established cancer biomarkers and show a bias toward the identification of high-abundance molecules, these technological advances have the capacity to revolutionize biomarker discovery. It is now necessary to focus on careful validation studies in order to identify the strategies and biomarkers that work and bring them to the clinic as early as possible.

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A "bottom-up" proteomics approach and a two-dimensional (strong cation exchange followed by reversedphase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracellular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identified in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 g/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellular localization was determined based on Genome Ontology terms for all the 1,139 proteins of which 34% were classified as extracellular and membrane-bound. Proteomics analysis of MDA-MB-468 cell lysate demonstrated that only 5% of all identified proteins were extracellular. This confirmed our hypothesis that examining the CM of cell lines, as opposed to the cell lysates, leads to a significant enrichment in secreted proteins. Tissue specificity, functional classifications, and spectral counting were performed. Elafin, a protease inhibitor, identified in the conditioned media of BT474 and MDA-MB-468 and the three kallikreins (KLK5, KLK6, and KLK10) were validated using an immunoassay on various serum and biological samples. Some of the secreted proteins identified have established roles in breast cancer development (cell growth, differentiation, and metastasis) and/or are linked to early onset breast cancer. Our approach to mining for low abundance molecules could identify proteins in various stages of breast cancer development. Many of the identified proteins are potentially useful to investigate as circulating serum breast cancer biomarkers. Molecular

Solid organ transplant recipients are at high risk of severe disease from COVID‐19. We assessed the immunogenicity of mRNA‐1273 vaccine using a combination of antibody testing, surrogate neutralization assays, and T cell assays. Patients were immunized with two doses of vaccine and immunogenicity assessed after each dose using the above tests. CD4+ and CD8+ T cell responses were assessed in a subset using flow‐cytometry. A total of 127 patients were enrolled of which 110 provided serum at all time points. A positive anti‐RBD antibody was seen in 5.0% after one dose and 34.5% after two doses. Neutralizing antibody was present in 26.9%. Of note, 28.5% of patients with anti‐RBD did not have neutralizing antibody. T cell responses in a sub‐cohort of 48 patients showed a positive CD4+ T cell response in 47.9%. Of note, in this sub‐cohort, 46.2% of patients with a negative anti‐RBD, still had a positive CD4+ T cell response. The vaccine was safe and well‐tolerated. In summary, immunogenicity of mRNA‐1273 COVID‐19 vaccine was modest, but a subset of patients still develop neutralizing antibody and CD4+T‐ cell responses. Importantly polyfunctional CD4+T cell responses were observed in a significant portion who were antibody negative, further highlighting the importance of vaccination in this patient population. IRB Statement: This study was approved by the University Health Network Research Ethics Board (CAPCR ID 20–6069).