Tumor Biomarker In-Solution Quantification, Standard Production, and Multiplex Detection

The diagnosis and monitoring of cancer have been facilitated by discovering tumor “biomarkers” and methods to detect their presence. Yet, for certain cancers, we still lack sensitive and specific biomarkers or the means to quantify subtle concentration changes successfully. The identification of new biomarkers of disease and improving the sensitivity of detection will remain key to changing clinical outcomes. Patient liquid biopsies (serum and plasma) are the most easily obtained sources for noninvasive analysis of proteins that tumor cells release directly and via extracellular microvesicles and tumor shedding. Therefore, an emphasis on creating reliable assays using serum/plasma and “direct, in-solution” ELISA approaches has built an industry centered on patient protein biomarker analysis. A need for improved dynamic range and automation has resulted in the application of ELISA principles to paramagnetic beads with chemiluminescent or fluorescent detection. In the clinical testing lab, chemiluminescent paramagnetic assays are run on automated machines that test a single analyte, minimize technical variation, and are not limited by serum sample volumes. This differs slightly from the R&D setting, where serum samples are often limiting; therefore, multiplexing antibodies to test multiple biomarkers in low serum volumes may be preferred. This review summarizes the development of historical biomarker “standards”, paramagnetic particle assay principles, chemiluminescent or fluorescent biomarker detection advancements, and multiplexing for sensitive detection of novel serum biomarkers.

Noninvasive Analysis Using Data-Independent Acquisition Mass Spectrometry: New Epidermal Proteins That Reveal Sex Differences in the Aging Process

The development of mass spectrometry has provided a method with extremely high sensitivity and selectivity that can be used to identify protein biomarkers. Epidermal proteins, lipids, and cornified envelopes are involved in the formation of the skin epidermal barrier. The epidermal protein composition changes with age. Therefore, quantitative proteomic changes may be indicative of skin aging. We sought to utilize data-independent acquisition mass spectrometry for noninvasive analysis of epidermal proteins in healthy Chinese individuals of different age groups and sexes. In our study, we completed high-throughput protein detection, analyzed protein differences with MaxQuant software, and performed statistical analyses of the proteome. We obtained interesting findings regarding ceruloplasmin (CP), which exhibited significant differences and is involved in ferroptosis, a signaling pathway significantly associated with aging. There were also several proteins that differed between sexes in the younger group, but the sex differences disappeared with aging. These proteins, which were associated with both aging processes and sex differences, are involved in signaling pathways such as apoptosis, oxidative stress, and genomic stability and can serve as candidate biomarkers for sex differences during aging. Our approach for noninvasive detection of epidermal proteins and its application to accurately quantify protein expression can provide ideas for future epidermal proteomics studies.

Utility of Circulating Tumor DNA in the Management of Patients With GI Stromal Tumor: Analysis of 243 Patients

PURPOSE GI stromal tumor (GIST) is the most common sarcoma of the GI tract. Management of patients with GIST is determined by KIT, PDGFRA, or other genomic alterations. Tissue-based next-generation sequencing (NGS) analysis is the standard approach for diagnosis, prognosis, and treatment selection. However, circulating tumor DNA (ctDNA)–based NGS is a novel and noninvasive alternative. METHODS ctDNA sequencing results were evaluated in blood samples from 243 de-identified patients within the Guardant360 database. Under an approved institutional review board protocol, a retrospective analysis was performed on 45 single-institution patients. RESULTS Of 243 patients, 114 (47%) were women, and the median age was 59 years (range, 17-90 years). Patients with no alterations and variations of uncertain significance were excluded. Of the 162 patients with known pathogenic mutations, KIT was the most common (56%), followed by NF (7%), PDGFRA (6%), PI3KCA (6%), KRAS (5%), and others (6%). Most tumors harbored an actionable KIT or PDGFRA mutation. Our institutional cohort (n = 45) had 16 (35%) KIT exon 11 mutations, 3 (6%) KIT exon 9 mutations, and 1 (2%) PDGFRA mutation detected on ctDNA. Resistance mutations were observed in KIT exon 17 (8 patients), exon 13 (3 patients), and in both (3 patients). Our comparison of ctDNA with tissue NGS revealed a positive predictive value (PPV) of 100%. Failure of concordance was observed in patients with localized or low disease burden. From the time of ctDNA testing, the median overall survival was not reached, whereas the median progression-free survival was 7 months. CONCLUSION ctDNA provides a rapid, noninvasive analysis of current mutations with a high PPV for patients with metastatic GIST. ctDNA-based testing may help to define the optimal choice of therapy on the basis of resistance mutations and should be studied prospectively.

The Noninvasive Analysis of Paint Mixtures on Canvas Using an EPR MOUSE

Many artists create the variety of colors in their paintings by mixing a small number of primary pigments. Therefore, analytical techniques for studying paintings must be capable of determining the components of mixtures. Electron paramagnetic resonance (EPR) spectroscopy is one of many techniques that can achieve this, however it is invasive. With the recent introduction of the EPR mobile universal surface explorer (MOUSE), EPR is no longer invasive. The EPR MOUSE and a least squares regression algorithm were used to noninvasively identify pairwise mixtures of seven different paramagnetic pigments in paint on canvas. This capability will help art conservators, historians, and restorers to study paintings with EPR spectroscopy.

Adaptation of lung, chest wall, and respiratory muscles during pregnancy: preparing for birth

A plethora of physiological and biochemical changes occur during normal pregnancy. The changes in the respiratory system have not been as well elucidated, in part because radioimaging is usually avoided during pregnancy. We aimed to use several noninvasive methods to characterize the adaptation of the respiratory system during the full course of pregnancy in preparation for childbirth. Eighteen otherwise healthy women (32.3 ± 2.8 yr) were recruited during early pregnancy. Spirometry, optoelectronic plethysmography, and ultrasonography were used to study changes in chest wall geometry, breathing pattern, lung and thoraco-abdominal volume variations, and diaphragmatic thickness in the first, second, and third trimesters. A group of nonpregnant women were used as control subjects. During the course of pregnancy, we observed a reorganization of rib cage geometry, in shape but not in volume. Despite the growing uterus, there was no lung restriction (forced vital capacity: 101 ± 15% predicted), but we did observe reduced rib cage expansion. Breathing frequency and diaphragmatic contribution to tidal volume and inspiratory capacity increased. Diaphragm thickness was maintained (1st trimester: 2.7 ± 0.8 mm, 3rd trimester: 2.5 ± 0.9 mm; P = 0.187), possibly indicating a conditioning effect to compensate for the effects of the growing uterus. We conclude that pregnancy preserved lung volumes, abdominal muscles, and the diaphragm at the expense of rib cage muscles. NEW & NOTEWORTHY Noninvasive analysis of the kinematics of the chest wall and the diaphragm during resting conditions in pregnant women revealed significant changes in the pattern of thoracoabdominal breathing across the trimesters. That is, concomitant with the progressive changes of chest wall shape, the diaphragm increased its contribution to both spontaneous and maximal breathing, maintaining its thickness despite its lengthening due to the growing uterus. These results suggest that during pregnancy the diaphragm is conditioned to optimize its active role provided during parturition.