Chapter 5 - Biomarker analysis of soil archives

Apportioning source of erosion-induced organic matter in the hilly-gully region of loess plateau in China: Insight from lipid biomarker and isotopic signature analysis

Machine learning analysis of metabolomic biomarkers for diagnosis of heart failure

Molecular markers, carbon isotopes, and rare earth elements of highly mature reservoir pyrobitumens from Sichuan Basin, southwestern China: Implications for PreCambrian-Lower Cambrian petroleum systems

A methodology for combined palynological and molecular geochemical high-resolution analysis of lake sediments

The future of personalized medicine depends on the ability to efficiently and rapidly elucidate a reliable set of disease-specific molecular biomarkers. High-throughput molecular biomarker analysis methods have been developed to identify disease risk, diagnostic, prognostic, and therapeutic targets in human clinical samples. Currently, high throughput screening allows us to analyze thousands of markers from one sample or one marker from thousands of samples and will eventually allow us to analyze thousands of markers from thousands of samples. Unfortunately, the inherent nature of current high throughput methodologies, clinical specimens, and cost of analysis is often prohibitive for extensive high throughput biomarker analysis. This review summarizes the current state of high throughput biomarker screening of clinical specimens applicable to genetic epidemiology and longitudinal population-based studies with a focus on considerations related to biospecimens, laboratory techniques, and sample pooling.

BackgroundHistopathology is the standard method for cancer diagnosis and grading to assess aggressiveness in clinical biopsies. Molecular biomarkers have also been described that are associated with cancer aggressiveness, however, the portion of tissue analyzed is often processed in a manner that is destructive to the tissue. We present here a new method for performing analysis of small molecule biomarkers and histology in exactly the same biopsy tissue.MethodsProstate needle biopsies were taken from surgical prostatectomy specimens and first fixed, each in a separate vial, in 2.5 ml of 80% methanol:water. The biopsies were fixed for 24 hrs at room temperature and then removed and post-processed using a non-formalin-based fixative (UMFIX), embedded, and analyzed by hematoxylin and eosin (H&E) and by immunohistochemical (IHC) staining. The retained alcohol pre-fixative was analyzed for small molecule biomarkers by mass spectrometry.ResultsH&E analysis was successful following the pre-fixation in 80% methanol. The presence or absence of tumor could be readily determined for all 96 biopsies analyzed. A subset of biopsy sections was analyzed by IHC, and cancerous and non-cancerous regions could be readily visualized by PIN4 staining. To demonstrate the suitability for analysis of small molecule biomarkers, 28 of the alcohol extracts were analyzed using a mass spectrometry-based metabolomics platform. All extracts tested yielded successful metabolite profiles. 260 named biochemical compounds were detected in the alcohol extracts. A comparison of the relative levels of compounds in cancer containing vs. non-cancer containing biopsies showed differences for 83 of the compounds. A comparison of the results with prior published reports showed good agreement between the current method and prior reported biomarker discovery methods that involve tissue destructive methods.ConclusionsThe Molecular Preservation by Extraction and Fixation (mPREF) method allows for the analysis of small molecule biomarkers from exactly the same tissue that is processed for histopathology.

Purpose: The goal of this study was to investigate two molecular biomarkers, beta-thromboglobulin (β-TG) and Thrombin-Antithrombin (TAT), for in vitro thrombogenicity testing of materials and to assess the impact of heparin concentration on test sensitivity. Methods: Four commonly used medical device materials: polytetrafluoroethylene (PTFE), high–density polypropylene (HDPE), 316 L stainless steel (SS), and silicone (Si), and two thrombogenic materials: Buna-N rubber (Buna) and glass beads, were investigated. Acid-Citrate-Dextrose Solution A (ACDA) anticoagulated human blood, drawn from healthy adult donors (n=6 and tested separately), was used within 4 hrs post blood draw. Immediately before starting each test, the blood was recalcified with calcium chloride and heparinized with a concentration range of 0.5- 1.5 U/mL heparin. Test materials were incubated in the heparinized blood at an exposure ratio of 6 cm2/ml in a polypropylene tube (Figure 1A) for 1 hr in a shaking water bath (37 °C, 60 rpm). After the incubation, additional ACDA was added to inhibit further blood reactions and the tube was placed on ice. Platelet (PLT) and leukocyte (WBC) counts were measured using a hematology analyzer (Hemavet 950 FS). Platelet free plasma was prepared by centrifuging the blood at 4 °C (in two steps: 1500g for 10min, then 10000g for 10 min) and then stored at −80 °C until enzyme-linked immunosorbent assay (ELISA) analysis for molecular biomarkers β-TG and TAT. Results: The results show that test sensitivity was improved when donor-specific heparin concentration (based on the heparin concentration that yielded Activated Clotting Time (ACT) between 200-235 sec) was used in the test for each donor blood compared to a fixed heparin concentration. As shown in Figure 1B-D, similar to PLT and WBC count based thrombogenicity markers, both molecular biomarkers (β-TG and TAT) could differentiate between the thrombogenic materials and commonly used biomaterials under the test conditions of the current study, which used 6 cm2/mL material surface area to blood volume ratio (lower than ASTM F2888-19 standard recommended ratio of 12 cm2/mL). In addition, compared to PLT and WBC count markers, the molecular biomarkers showed slightly better consistency in differentiating materials with intermediate thrombogenicity potential (e.g., Si and SS) from thrombogenic materials. Summary: Both molecular biomarkers (β-TG and TAT) can distinguish thrombogenic potentials of different materials at a surface area to volume ratio of 6 cm2/mL. The test sensitivity can be improved by using a donor-specific heparin concentration, which can be determined by ACT measurement for each donor blood. Additional testing is needed to further validate these results and to determine their regulatory and clinical relevance. Different molecular biomarkers will also be investigated in the future.Figure 1. (A) Experimental setup showing the test material segments were incubated in recalcified whole blood with a series of heparin concentrations for 1 hr at 37°C. For positive control: Test was run with 20 µM ADP in recalcified blood. For negative control: Test was run without any test material. (B-D) Test results for Platelet and WBC counts, Mean TAT and β-TG concentrations after 1 hour incubation of materials at ACT-based donor-specific heparin concentrations, respectively. Data are shown as Mean ± SD (n=6).

All inducible proteins which respond to known pollutants are potential molecular biomarkers. Quantitative competitive (qc) RT-PCR represents a uniquely sensitive tool for measuring the extent of induction of molecular biomarkers such as metallothionein, which is responsive to inducers that include a range of heavy metals. Using the yellowbelly flounder Rhombosolea leporina collected from sites in the Manukau Harbour and Hauraki Gulf (Auckland, New Zealand) as an indicator species, we describe the methodology underpinning the use of qcRT-PCR as a tool in biomarker analysis with reference to the induction of metallothionein. The results show reasonable correlation between the extent of metallothionein induction and the liver burden of Cu and Zn.

5526 Background: In the phase II KEYNOTE-199 study (NCT02787005), pembrolizumab monotherapy demonstrated antitumor activity in pts with docetaxel-refractory mCRPC (n = 258). Here we evaluated the association between prespecified molecular biomarkers and clinical outcomes. Methods: Cohorts 1 (C1) and 2 (C2) enrolled pts with RECIST-measurable PD-L1–positive (combined positive score [CPS] ≥1 using immunohistochemistry) and PD-L1–negative (CPS <1) disease, respectively. C3 enrolled pts with nonmeasurable, bone-predominant disease, irrespective of PD-L1 status. Biomarkers evaluated in this analysis were tumor mutational burden ([TMB; mutations/exome] n = 155), PD-L1 CPS (n = 255), tumor microenvironment–based 18-gene RNA expression profile ([GEP] n = 196), and microsatellite instability ([MSI] as determined by Promega PCR analysis; n = 147). Outcomes evaluated for C1 and C2 (n = 200) were ORR, disease control rate (DCR), and radiographic PFS (rPFS) per blinded, independent central review per PCWG-modified RECIST v1.1. Outcomes evaluated for C1-C3 (n = 258) were prostate-specific antigen (PSA) response, time to PSA progression, and OS. Significance of continuous biomarkers (CPS; TMB; GEP) was prespecified at 0.05 for one-sided P values from logistic (ORR; DCR; PSA response) and Cox proportional hazard regression (rPFS; OS; PSA progression) adjusted for Eastern Cooperative Oncology Group performance status. Binary biomarkers (MSI) were analyzed using Fisher’s exact test (ORR; DCR; PSA response). Clinical data cutoff date: Jun 24, 2019. Results: Median TMB was 53.0 (interquartile range [IQR], 40.5 to 78.0), median CPS was 1 (IQR, 0 to 5), and median GEP was –0.64 (IQR, –0.88 to –0.46); 6 pts (2.3%) had MSI-high tumors. In C1-C3, TMB was associated with PSA response (one-sided nominal P = 0.0016) and time to PSA progression (one-sided nominal P = 0.00092). In C1-C3, PD-L1 CPS was associated with PSA response (one-sided nominal P = 0.046) and time to PSA progression (one-sided nominal P = 0.021). In C1-C3, GEP was not significantly associated with response. In C1-C3, MSI was associated with PSA response (one-sided nominal P = 0.019). Conclusions: In this biomarker analysis from KEYNOTE-199 C1-C3, TMB and PD-L1 CPS were associated with better PSA response; however, small pt numbers limit definitive conclusions on ORR, DCR, and OS. Further evaluation of molecular biomarkers in pts with mCRPC treated with pembrolizumab is warranted. Clinical trial information: NCT02787005 .

Molecular biomarkers in endometrial hyperplasias predict cancer progression

An increasing burden of neurological diseases (NDs) has been a public health challenge in an aging society. Age, especially biological age, is the most important risk factor for NDs. Identification of biomarkers of aging to capture NDs might lead to a better understanding of the underlying mechanisms of pathological brain aging and the implementation of effective intervention. We conducted a comprehensive two-sample Mendelian Randomization (MR) study to investigate the association between various biomarkers of aging and three leading causes of NDs: Alzheimer’s disease (AD), vascular dementia (VaD), and ischemic stroke. Publicly available GWAS summary statistics on people from European ancestry were obtained for six molecular biomarkers, two physiological biomarkers, and eight functional biomarkers, and three NDs. Genetic variants serving as instrumental variables (IVs) were identified for each biomarker. The MR analysis included inverse variance weighted (IVW), weighted median, MR-Egger, and MR-PRESSO. We found that short telomere length and decrease in appendicular lean mass were associated with an increased risk for AD (OR IVW = 1.12 per 1SD decrease, 95% confidence interval 1.02–1.22, and OR IVW = 1.11, 1.06–1.16, respectively), whereas high frailty index showed a protective effect for AD. Accelerated BioAge appeared to be associated with increased risk for ischemic stroke (OR IVW = 1.3 per year in BioAge acceleration, 95% CI 1.19–1.41). Our findings implied a causal association of short telomere length and a decrease in appendicular lean mass with an increased risk for AD, while BioAge appeared to be a good biomarker for ischemic stroke. Further studies are needed to validate these associations and explore underlying mechanisms.

Chapter 7 - Land use and environmental changes in the Andean Paramo soils

Molecular biomarkers are mRNA transcripts that indicate the (nutrient) status of an organism or tissue. Molecular biomarker panels have the potential to readily and more accurately determine nutrient status than individual traditional biomarkers. To study the efficacy of molecular biomarker panels for predicting selenium (Se) status, we examined 30 biomarkers from rats fed graded levels of Se from deficient to eight times the minimum Se requirement, including four liver and four kidney traditional biomarkers, and 13 liver and nine kidney selenoprotein mRNA levels. Multiple regression analysis against liver and kidney Se and glutathione peroxidase-1 (Gpx1) activity, with stepwise single elimination of biomarkers that did not significantly contribute, was used to identify biomarker panels with significant (P < 0.05) regression coefficients. Resulting regression equations were then used to predict Se status, and compared with traditional Se biomarkers panels. Over the full spectrum of Se status from 0 to 0.8 microg Se/g diet, the resulting 4-selenoprotein mRNA biomarker panel predicted liver Se concentration with a correlation of 0.948, which was nominally higher and statistically the same as the correlation of 0.909 for the panel based on Gpx1 activity. The molecular biomarker panels for predicting kidney Se and liver and kidney Gpx1 activity were all comparable to predictions based on traditional biomarkers. These analyses show that molecular biomarker panels can be used to predict accurately two traditional biomarkers of Se status. The resulting analyses also illustrate that additional orthogonal biomarkers reflecting higher Se intakes are needed to better predict supernutritional Se status and further strengthen this approach.

Detecting vulnerability of humid tropical forests to multiple stressors

Warming promotes loss of subsoil carbon through accelerated degradation of plant-derived organic matter