Urine is a highly desirable biospecimen for biomarker analysis because it can be collected recurrently by non-invasive techniques, in relatively large volumes. Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect, at a given time point, an individual's metabolic and pathophysiologic state. High-resolution mass spectrometry, coupled with state of the art fractionation systems are revealing the plethora of diagnostic/prognostic proteomic information existing within urinary exosomes, glycoproteins, and proteins. Affinity capture pre-processing techniques such as combinatorial peptide ligand libraries and biomarker harvesting hydrogel nanoparticles are enabling measurement/identification of previously undetectable urinary proteins. Future challenges in the urinary proteomics field include a) defining either single or multiple, universally applicable data normalization methods for comparing results within and between individual patients/data sets, and b) defining expected urinary protein levels in healthy individuals.

An accurate urine test for pulmonary tuberculosis (TB), affecting 9.6 million patients worldwide, is critically needed for surveillance and treatment management. Past attempts failed to reliably detect the mycobacterial glycan antigen lipoarabinomannan (LAM), a marker of active TB, in HIV-negative, pulmonary TB–infected patients’ urine (85% of 9.6 million patients). We apply a copper complex dye within a hydrogel nanocage that captures LAM with very high affinity, displacing interfering urine proteins. The technology was applied to study pretreatment urine from 48 Peruvian patients, all negative for HIV, with microbiologically confirmed active pulmonary TB. LAM was quantitatively measured in the urine with a sensitivity of >95% and a specificity of >80% (n = 101) in a concentration range of 14 to 2000 picograms per milliliter, as compared to non-TB, healthy and diseased, age-matched controls (evaluated by receiver operating characteristic analysis; area under the curve, 0.95; 95% confidence interval, 0.9005 to 0.9957). Urinary LAM was elevated in patients with a higher mycobacterial burden (n = 42), a higher proportion of weight loss (n = 37), or cough (n = 50). The technology can be configured in a variety of formats to detect a panel of previously undetectable very-low-abundance TB urinary analytes. Eight of nine patients who were smear-negative and culture-positive for TB tested positive for urinary LAM. This technology has broad implications for pulmonary TB screening, transmission control, and treatment management for HIV-negative patients.

Generating molecular information in a clinically relevant time frame is the first hurdle to truly integrating precision medicine in health care. Reverse phase protein microarrays are being utilized in clinical trials for quantifying posttranslationally modified signal transduction proteins and cellular signaling pathways, allowing direct comparison of the activation state of proteins from multiple specimens, or individual patient specimens, within the same array. This technology provides diagnostic and therapeutic information critical to precision medicine. To enhance accessibility of this technology, two hurdles must be overcome: data normalization and data acquisition. Herein we describe an unamplified, dual-color signal detection methodology for reverse phase protein microarrays that allows multiplex, within spot data normalization, reduces data acquisition time, simplifies automated spot detection, and provides a stable signal output. This method utilizes Quantum Nanocrystal fluorophore labels (Qdot) substituted for organic fluorophores coupled with an imager (ArrayCAM) that captures images of the microarray rather than sequentially scanning the array. Streamlining and standardizing the data analysis steps with ArrayCAM high-resolution, dual mode chromogenic/fluorescent array imaging overcomes the data acquisition hurdle. The spot location and analysis algorithm provides certain parameter settings that can be tailored to the particular microarray type (fluorescent vs. colorimetric), resulting in greater than 99 % spot location sensitivity. The described method demonstrates equivalent sensitivity for a non-amplified Qdot immunoassay when using automated vs. manual immunostaining procedures.

Bone metastasis from primary cancer sites creates diagnostic and therapeutic challenges. Calcified bone is difficult to biopsy due to tissue hardness and patient discomfort, thus limiting the frequency and availability of bone/bone marrow biopsy material for molecular profiling. In addition, bony tissue must be demineralized (decalcified) prior to histomorphologic analysis. Decalcification processes rely on three main principles: (a) solubility of calcium salts in an acid, such as formic or nitric acid; (b) calcium chelation with ethylenediaminetetraacetic acid (EDTA); or (c) ion-exchange resins in a weak acid. A major roadblock in molecular profiling of bony tissue has been the lack of a suitable demineralization process that preserves histomorphology of calcified and soft tissue elements while also preserving phosphoproteins and nucleic acids. In this chapter, we describe general issues relevant to specimen collection and preservation of osseous tissue for molecular profiling. We provide two protocols: (a) one-step preservation of tissue histomorphology and proteins and posttranslational modifications, with simultaneous decalcification of bony tissue, and (b) ethanol-based tissue processing for TheraLin-fixed bony tissue.

Chemotherapy modulates proteomic pathways and neuronal metabolites in the brainChemotherapy saves lives but diminishes the quality of life and functional ability of cancer patients by impairing cognitive function in a phenomenon called “chemobrain”. Elucidating the neural substrates modulated by chemotherapy is critical for deciphering the molecular underpinnings of cognitive dysfunction. Animal models have been used to investigate the impact of numerous chemotherapy drugs on cognitive ability and revealed negative effects of nearly all drugs tested, with oxidative stress, inflammation, apoptosis, and a reduction in hippocampal neurogenesis observed after treatment. Still, the underlying proteomic and metabolic mechanisms of chemotherapy-related cognitive dysfunction is not fully understood. We developed an animal model to assess the effects of cyclophosphamide (CTX) and docetaxel (DTX), two chemotherapy agents commonly delivered to breast cancer patients. Our study goals were to determine the proteomic effects of chemotherapy in specific brain regions and the associated global metabolomic shifts in the brain. Healthy, female Long Evans rats were treated with CTX and/or DTX, in four weekly doses. Controls received injections of vehicle alone. Following chemotherapy, fresh brain tissue was dissected medially. The right brain region was frozen for global metabolomic analysis via tandem mass spectrometry. The left brain was fixed in TheraLin for proteomic analysis of post-translationally modified proteins using reverse phase protein microarrays and immunohistochemistry. Fixed brain tissue was sectioned, and the hippocampus, cerebellum, olfactory bulb, and cerebral cortex were procured using laser capture microdissection. We identified biochemical shifts associated with chemotherapy that impact neurobiological processes underlying memory dysfunction. Hierarchical clustering of signal transduction proteins revealed that chemotherapy resulted in elevated levels of Calpain 1 and 2 in the cerebellum, Calpain 2 in the cerebral cortex, and autophagy proteins in the hippocampus and cerebellum. Doublecortin, a neurogenesis protein, was elevated in the cerebellum. Docetaxel raised the abundance of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), while all chemotherapies diminished levels of glutathione, a neuromodulator of excitatory NMDA receptors. This animal study provides molecular information that offers empirical support for future intervention research that may help prevent cognitive decline and preserve cognitive health in cancer survivors. Citation Format: Justin B. Davis, Grace N. Lawrence, Michael G. Harpole, Robin D. Couch, Lance A. Liotta, Theodore C. Dumas, Virginia A. Espina. Chemotherapy modulates proteomic pathways and neuronal metabolites in the brain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3476.