Phenotypic Biomarkers Research Articles

Abstract 3642: Phenotypic biomarkers of aqueous humor extracellular vesicles from retinoblastoma eyes

Abstract Purpose: Retinoblastoma (RB) is the most common pediatric intraocular cancer, yet tumor biopsies are contraindicated. This challenge emphasizes the need for investigation of non-tissue biomarkers. Extracellular vesicles (EVs), which are secretory vesicles containing bioactive molecules, have emerged as promising biomarker candidates due to their ability to be phenotyped using known tetraspanin markers. Aqueous humor (AH) provides an exciting liquid biopsy source for intraocular tumor information, with EVs having recently been identified in AH. Our laboratory has previously proposed that CD63/81+EVs are associated with retinoblastoma, but specific tumor-derived markers have not been linked to AH EVs using traditional tetraspanin assays. In this study, we employed MACSPlex, a high-dimensional magnetic bead-based immunoassay, to further explore RB-EV. Methods: 7 AH samples consisting of two congenital glaucoma (GLC) samples and five RB samples taken at the time of primary enucleation were analyzed. We utilized Macsplex, a multiplex bead-based flow cytometry assay, to analyze the expression profiles of surface markers found on EVs. With 39 capture beads coated with monoclonal antibodies for 37 different EV surface antigens and two negative controls, the mean fluorescence intensity (MFI) of APC conjugated antibody cocktail from three tetrapanins (CD9, CD63 and CD81) on EVs captured by each surface antigen could be measured by flow cytometry. Enrichment ratios were then calculated by normalizing the fluorescence of each signal to the mean fluorescence of CD9, CD63, and C81. Results: Macsplex analysis of the 2 GLC AH samples demonstrated a strong CD63 dominance compared with RB AH. The mean CD63 enrichment ratio for GLC samples was 2.6 (S.D. = 0.34), and 1.3 (S.D. = 0.27) in RB samples, with a p-value of 0.069. In comparison to the GLC patients, the analysis of EVs in the AH of the 5 RB samples exhibited a greater co-dominance of CD63/CD81 positivity consistent with past research on RB EVs. The mean CD81 enrichment ratio for the 5 RB samples was 1.5 (S.D. = 0.26), in contrast to 0.3 (S.D. = 0.24) in GLC AH, with a p-value of 0.03. Interestingly, 4 out of the 5 RB AH samples demonstrated very strong CD133 signals, which is a known cancer marker. Deeper investigation into one RB AH sample utilizing single tetraspanin detection revealed higher coexpression of CD63 and CD81 with CD133 than seen with CD9. The CD133 enrichment ratio for the CD81, CD63, and CD9 single channel experiments were 6.6, 3.4, and 1.5 respectively. Conclusions: Comparison between this study and previous work done by our group demonstrate a high concordance between results utilizing Macsplex and Exoview. However, this is the first study identifying the expression of CD133, a cancer stem cell marker, on retinoblastoma-derived EVs in AH. Macsplex technology illustrates an incredible clinical potential of AH EVs for characterizing retinoblastoma phenotype in vivo. Citation Format: Anne Amacker, Chen-Ching Peng, Bibiana J. Reiser, Jesse L. Berry, Liya Xu. Phenotypic biomarkers of aqueous humor extracellular vesicles from retinoblastoma eyes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3642.

Characterization and applicability of a novel physiologically relevant 3D-tetraculture bronchial model for in vitro assessment of respiratory sensitization

Chemical Respiratory Allergy (CRA) is triggered after exposure to Low Molecular Weight (LMW) sensitizers and manifests clinically as asthma and rhinitis. From a risk/toxicity assessment point of view, there are few methods, none of them validated, for evaluating the respiratory sensitization potential of chemicals once the in vivo-based models usually employed for inhalation toxicity addressment do not comprise allergenicity endpoints specifically. Based on that, we developed, characterized, and evaluated the applicability of a 3D-tetraculture airway model reconstructed with bronchial epithelial, fibroblasts, endothelial and monocytic cell lines. Moreover, we exposed the tissue to maleic anhydride (MA) aerosols to challenge the model and subsequently assessed inflammatory and functional aspects of the tissue. The reconstructed tissue presented phenotypic biomarkers compatible with human bronchial epithelium, and MA aerosol exposure triggered an increased IL-8 and IL-6 production, reactive oxygen species (ROS) formation, and apoptosis of epithelial cells. Besides, augmented IL-8 production by monocytic cells was also found, correlating with dendritic cell activation within the co-culture model after MA exposure. Our results demonstrated that the 3D-tetraculture bronchial model presents hallmarks related to human airways' structure and function. Additionally, exposure to a respiratory sensitizer induced inflammatory and functional alterations in the reconstructed tissue, rendering it a valuable tool for exploring the mechanistic framework of chemically induced respiratory sensitization.

Genomic, Proteomic, and Phenotypic Biomarkers of COVID-19 Severity: Protocol for a Retrospective Observational Study.

Health organizations and countries around the world have found it difficult to control the spread of COVID-19. To minimize the future impact on the UK National Health Service and improve patient care, there is a pressing need to identify individuals who are at a higher risk of being hospitalized because of severe COVID-19. Early targeted work was successful in identifying angiotensin-converting enzyme-2 receptors and type II transmembrane serine protease dependency as drivers of severe infection. Although a targeted approach highlights key pathways, a multiomics approach will provide a clearer and more comprehensive picture of severe COVID-19 etiology and progression. The COVID-19 Response Study aims to carry out an integrated multiomics analysis to identify biomarkers in blood and saliva that could contribute to host susceptibility to SARS-CoV-2 and the development of severe COVID-19. The COVID-19 Response Study aims to recruit 1000 people who recovered from SARS-CoV-2 infection in both community and hospital settings on the island of Ireland. This protocol describes the retrospective observational study component carried out in Northern Ireland (NI; Cohort A); the Republic of Ireland cohort will be described separately. For all NI participants (n=519), SARS-CoV-2 infection has been confirmed by reverse transcription-quantitative polymerase chain reaction. A prospective Cohort B of 40 patients is also being followed up at 1, 3, 6, and 12 months postinfection to assess longitudinal symptom frequency and immune response. Data will be sourced from whole blood, saliva samples, and clinical data from the electronic care records, the general health questionnaire, and a 12-item general health questionnaire mental health survey. Saliva and blood samples were processed to extract DNA and RNA before whole-genome sequencing, RNA sequencing, DNA methylation analysis, microbiome analysis, 16S ribosomal RNA gene sequencing, and proteomic analysis were performed on the plasma. Multiomics data will be combined with clinical data to produce sensitive and specific prognostic models for severity risk. An initial demographic and clinical profile of the NI Cohort A has been completed. A total of 249 hospitalized patients and 270 nonhospitalized patients were recruited, of whom 184 (64.3%) were female, and the mean age was 45.4 (SD 13) years. High levels of comorbidity were evident in the hospitalized cohort, with cardiovascular disease and metabolic and respiratory disorders being the most significant (P<.001), grouped according to the International Classification of Diseases 10 codes. This study will provide a comprehensive opportunity to study the mechanisms of COVID-19 severity in recontactable participants. DERR1-10.2196/50733.

Logical design of synthetic cis-regulatory DNA for genetic tracing of cell identities and state changes.

Descriptive data are rapidly expanding in biomedical research. Instead, functional validation methods with sufficient complexity remain underdeveloped. Transcriptional reporters allow experimental characterization and manipulation of developmental and disease cell states, but their design lacks flexibility. Here, we report logical design of synthetic cis-regulatory DNA (LSD), a computational framework leveraging phenotypic biomarkers and trans-regulatory networks as input to design reporters marking the activity of selected cellular states and pathways. LSD uses bulk or single-cell biomarkers and a reference genome or custom cis-regulatory DNA datasets with user-defined boundary regions. By benchmarking validated reporters, we integrate LSD with a computational ranking of phenotypic specificity of putative cis-regulatory DNA. Experimentally, LSD-designed reporters targeting a wide range of cell states are functional without minimal promoters. Applied to broadly expressed genes from human and mouse tissues, LSD generates functional housekeeper-like sLCRs compatible with size constraints of AAV vectors for gene therapy applications. A mesenchymal glioblastoma reporter designed by LSD outperforms previously validated ones and canonical cell surface markers. In genome-scale CRISPRa screens, LSD facilitates the discovery of known and novel bona fide cell-state drivers. Thus, LSD captures core principles of cis-regulation and is broadly applicable to studying complex cell states and mechanisms of transcriptional regulation.

Chensinin-1b Alleviates DSS-Induced Inflammatory Bowel Disease by Inducing Macrophage Switching from the M1 to the M2 Phenotype.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder with an increasing prevalence worldwide. Macrophage polarization is involved in the pathogenesis of IBD. Repolarization of macrophage has thus emerged as a novel therapeutic approach for managing IBD. Chensinin-1b, derived from the skin of Rana chensinensis, is a derivative of a native antimicrobial peptide (AMP). It shows anti-inflammatory effects in sepsis models and can potentially modulate macrophage polarization. The objective of this research was to study the role of chensinin-1b in macrophage polarization and dextran sulfate sodium (DSS)-induced colitis. RAW264.7 macrophages were polarized to the M1 phenotype using lipopolysaccharide (LPS) and simultaneously administered chensinin-1b at various concentrations. The ability of chenisnin-1b to reorient macrophage polarization was assessed by ELISA, qRT-PCR, and flow cytometry analysis. The addition of chensinin-1b significantly restrained the expression of M1-associated proinflammatory cytokines and surface markers, including TNF-α, IL-6, NO, and CD86, and exaggerated the expression of M2-associated anti-inflammatory cytokines and surface markers, including IL-10, TGF-β1, Arg-1, Fizz1, Chil3, and CD206. Mechanistically, via Western Blotting, we revealed that chensinin-1b induces macrophage polarization from the M1 to the M2 phenotype by inhibiting the phosphorylation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK). In mouse models of colitis, intraperitoneal administration of chensinin-1b alleviated symptoms induced by DSS, including weight loss, elevated disease activity index (DAI) scores, colon shortening, colonic tissue damage, and splenomegaly. Consistent with our in vitro data, chensinin-1b induced significant decreases in the expression of M1 phenotype biomarkers and increases in the expression of M2 phenotype biomarkers in the mouse colitis model. Furthermore, chensinin-1b treatment repressesed NF-κB phosphorylation in vivo. Overall, our data showed that chensinin-1b attenuates IBD by repolarizing macrophages from the M1 to the M2 phenotype, suggesting its potential as a therapeutic candidate for IBD.

Adaptive, morphometric and productive responses of Brazilian hair lambs: Crossing between indigenous breeds - A machine learning approach

For the first time, the thermoregulatory, morphometric and productive responses of the Morada Nova breed and its crosses with the Santa Inês and Rabo Largo breeds in the Brazilian semi-arid region were evaluated. Subsequently, the relationship between thermoregulation and morphometric, productive and non-carcass components was verified. 30 lambs, weighing 2.26 ± 0.53 kg at birth and 7.31 ± 1.85 kg at weaning, were distributed into three genetic groups: Morada Nova (MN) and its F1 crossbred Morada Nova × Rabo Largo (RL×MN) and Morada Nova x Santa Inês (SI×MN) animals in a completely randomized factorial design (3 genetic groups × 2 sexes). Machine learning techniques were performed in order to understand the relationship between the variables and in order to identify phenotypic biomarkers. No interaction effect was observed for any variable under study (P > 0.05). Male lambs have higher heart rate (P = 0.03), are heavier (P < 0.001), have higher weight gain (P < 0.001) and higher skin weight (P = 0.01) when compared to females. MN ×SI lambs showed lower heart rate (P = 0.03) and rectal temperature (P = 0.02). Respiratory rate was similar (P = 0.26) between genetic groups. It was also observed that crossbred lambs had higher morphological measures, non-carcass components and productive responses (P < 0.001). Lung weight in purebred animals was lower (P = 0.01) when compared to MN × SI, but equal to MN × RL. The first component was characterized by morphological, productive and non-carcass components, and the second component by thermoregulatory response. The canonical correlation analysis showed that the thermoregulatory response is influenced by the morphological response (P = 0.002). Furthermore, the weight of non-carcass components does not have a relationship (P = 0.834) with thermoregulatory responses. Finally, the thermoregulatory responses did not influence (P = 0.717) the productive responses of sheep in the Brazilian semi-arid region. The redundancy index showed that 36.1% of the variation in thermoregulatory responses is explained by morphological responses, more specifically: the smaller the body length and chest circumference, the higher the animal's heart rate. The productive and morphometric responses and the non-carcass components are the main phenotypic biomarkers used to differentiate the genetic groups of lambs. The crossing of the native Brazilian semi-arid region breed led to higher body weight attributed to higher adaptability, manifested by lower rectal temperature and heart rate with no recorded increase of the morphological indicators.

Abstract A007: Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F)

Abstract Background: Although a range of therapeutic options are available in the management of invasive breast carcinomas, spatial segregation of tumour subclones has hampered biomarker identification in single-region samples. Representative sampling (RS) overcomes spatial bias by sampling from a homogenized and well-mixed cancer specimen. The Homogenization of Leftover Surgical Tissue Feasibility Study (HoLST-F - NCT03832062) is a prospective trial aiming to assess the feasibility of RS in tumor tissue leftover after pathology sampling. An interim analysis of the breast cancer cohort is presented. Methods: In the context of the HoLST-F study and pre-specified end-points, representative samples derived from 75 leftover invasive breast carcinoma specimens underwent flow cytometric analysis of CK8/18, CD3, Ki67 and DAPI. Tumor cell enrichment by CK8/18 positivity and ploidy (in aneuploid carcinomas) was performed for downstream DNA extraction and whole exome sequencing. Somatic variants were determined using a bespoke pipeline to remove artefacts associated with fixation. Variant oncogenicity and therapeutic evidence levels were assigned by OncoKB variant annotation. Quantitative image analysis was applied to tissue sections from diagnostic FFPE blocks stained with Ki67 immunohistochemistry (n=78) and H&amp;E for lymphocytic infiltration scores (n=175) to correlate with flow cytometry. Results: In enriched representative samples, oncogenic mutations were commonly identified at high variant allele frequency (VAF) in known breast carcinoma driver genes including PIK3CA, CDH1, TP53, KMT2C and GATA3. Other clinically relevant oncogenic mutations were identified in ESR1, PTEN, ERBB2, RB1, AKT1, BRCA1, NF1 and FOXA1 which have been associated with resistance to various anti-cancer therapies. These mutations occurred at low and high VAFs indicative of both of clonal and sub-clonal resistance mechanisms. Recurrent variants in ESR1 (e.g. D538G) and PIK3CA (e.g. H1047R/L) were associated with Level 1 evidence for use as predictive biomarkers, while variants in twelve other genes across the cohort were associated with Level 2-4 evidence. Lymphocyte infiltration scores and Ki67 expression varied by tumor region, however RS by flow cytometry showed strong correlation with a weighted average across multiregional quantification of Ki67 expression (R=0.81, p=2.8 × 10-8) and lymphocyte infiltration (R=0.61, p&amp;lt;2.2 × 10-16). Conclusions: RS of invasive breast carcinoma in the HoLST-F trial has recapitulated the expected genomic driver landscape in breast cancer, in addition to identifying both clonal and subclonal genomic mechanisms of therapy resistance. Many of these mutations are either current or emerging therapeutic targets. Flow quantification of cells expressing phenotypic biomarkers (Ki67 and CD3) is feasible through RS and early analysis indicates that RS correlates with a weighted average across multiple regions. Leftover surgical tissue is an underutilized resource for biomarker assessment in breast carcinomas and can be examined by RS. Citation Format: Brian Hanley, Lisa Gallegos, Lavinia Spain, Husayn Pallikonda, Zayd Tippu, Samantha Hill, Aoune Barhoumi, Fiona Byrne, Yulia Dogva, Ashley Gilchrist, Glenn Noel-Storr, Hannah Veloz, Stacey Stanislaw, Harold Sansano, Kim Edmonds, Eleanor Carlyle, Nicholas Turner, James Larkin, Nelson Alexander, Samra Turajlic. Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A007.

Establishment and characterization of DPC-X4: a novel mixed-type ampullary cancer cell line.

Mixed-type ampullary cancer is a distinct subtype of ampullary cancer that manifests a merging of the biological characteristics of both intestinal and pancreaticobiliary subtypes. The absence of established cell lines specific to this subtype has resulted in a concomitant scarcity of research on its tumorigenic mechanisms and the development of novel therapeutic modalities. The present study achieved the successful establishment of a novel mixed-type ampullary cancer cell line, designated DPC-X4 through primary culture techniques. Subsequent analyses pertaining to phenotypic characteristics, molecular profiling, biomarker identification, and histological features validated the DPC-X4 cell line as a potent model for delineating the pathogenesis of mixed-type ampullary cancer and facilitating the development of new pharmacological agents. This newly established cell line was subjected to continuous cultivation for 1year, with stable passaging for over 50 generations. Notably, the DPC-X4 cell line manifested typical morphological features associated with epithelial tumors. Furthermore, the population doubling time for the DPC-X4 cell line was determined at 70h. Short tandem repeat (STR) analysis confirmed that the DPC-X4 cell line exhibited a high genetic concordance with the primary tumor from the patient. Karyotypic profiling indicated an abnormal sub-triploid karyotype, with representative karyotypes of 57, XXY inv (9), 14p + , 15p + , der (17), + mar. The DPC-X4 cell line demonstrated a high capacity for efficient organoid formation under suspension culture conditions. In addition, the subcutaneous inoculation of DPC-X4 cells into NXG mice led to the formation of xenografted tumors. The results of drug sensitivity testing indicated that DPC-X4 cells were sensitive to paclitaxel and resistant to oxaliplatin, 5-fluorouracil, and gemcitabine. Immunohistochemistry revealed positive expression of CK7, CK19, and CK20 in DPC-X4 cells, while CDX2 demonstrated negative expression. In addition, positive expression of E-cadherin and vimentin was identified in DPC-X4 cells, with a proliferation index indicated by Ki-67 at 70%. The findings of our study establish DPC-X4 as a novel mixed-type ampullary cancer cell line, which can serve as a potential experimental model for exploring the pathogenesis of ampullary cancer and the development of therapeutic drugs.

Novel Serum Biomarkers for Patients with Allergic Asthma Phenotype.

In distinguishing the allergic asthma (AA) phenotype, it has been identified that specific biomarkers could assist; however, none of them are considered ideal. This study aimed to analyze three groups of biologically active substances in the serum. Twenty steroid-free AA patients, sensitized to Dermatophagoides pteronyssinus, and sixteen healthy subjects (HSs) were enrolled in this study. Blood samples were collected from all patients. Additionally, all AA patients underwent a bronchial allergen challenge (BAC) with Dermatophagoides pteronyssinus, all of which were positive, and blood samples were collected again 24 h later. The concentrations of ten biologically active substances were measured in the serum samples, using enzyme-linked immunosorbent assay (ELISA) and the Luminex® 100/200™ System technology for bead-based multiplex and singleplex immunoassays. Descriptive and analytical statistical methods were used. A p-value of 0.05 or lower was considered statistically significant. The soluble interleukin 5 receptor subunit alpha (sIL-5Rα) and thioredoxin 1 (TRX1) concentrations were significantly increased, whereas those of tyrosine-protein kinase Met (MET), pentraxin 3 (PTX3), and I C-telopeptide of type I collagen (ICTP) were decreased in the AA group compared with the HS group. A significant positive correlation was noted for sIL-5Rα with fractional exhaled nitric oxide (FeNO), blood eosinophil (EOS) count, and total immunoglobulin E (IgE) levels, and a negative correlation was noted with forced expiratory volume in 1 s (FEV1). Moreover, PTX3 showed negative correlations with blood EOS count and total IgE levels, whereas ICTP exhibited a negative correlation with the blood EOS count. In conclusion, this study demonstrated that the serum concentrations of MET, PTX3, TRX1, ICTP, and particularly sIL-5Rα could potentially serve as biomarkers of the AA phenotype.

Pharmacogenomic and pharmacometabolomic biomarkers of the efficacy and safety of antidepressants: focus on selective serotonin reuptake inhibitors

The efficacy and safety of psychopharmacotherapy with antidepressants is of great medical importance. The search for clinical and biological predictors for choosing the optimal psychopharmacotherapy with antidepressants is actively underway all over the world. Research is mainly devoted to searching for associations of polymorphic gene variants with the efficacy and safety of therapy. However, information about a patient's genetic polymorphism is often insufficient to predict the efficacy and safety of a drug. Modern research on the personalization of pharmacotherapy should include, in addition to genetic, phenotypic biomarkers. This is important because genotyping, for example, cannot accurately predict the actual metabolic activity of an isoenzyme. To personalize therapy, a combination of methods is required to obtain the most complete profile of the efficacy and safety of the drug. Successful treatment of depression remains a challenge, and inter-individual differences in response to antidepressants are common. About half of patients with depressive disorders do not respond to the first attempt at antidepressant therapy. Serious side-effects of antidepressant pharmacotherapy and discontinuation of treatment due to their intolerance are associated with ineffective therapy. This review presents the results of the latest studies of «omics» biomarkers of the efficacy and safety of antidepressants.

Rates of change of pons and middle cerebellar peduncle diameters are diagnostic of multiple system atrophy of the cerebellar type.

Definitive diagnosis of multiple system atrophy of the cerebellar type (MSA-C) is challenging. We hypothesized that rates of change of pons and middle cerebellar peduncle diameters on MRI would be unique to MSA-C and serve as diagnostic biomarkers. We defined the normative data for anterior-posterior pons and transverse middle cerebellar peduncle diameters on brain MRI in healthy controls, performed diameter-volume correlations and measured intra- and inter-rater reliability. We studied an Exploratory cohort (2002-2014) of 88 MSA-C and 78 other cerebellar ataxia patients, and a Validation cohort (2015-2021) of 49 MSA-C, 13 multiple system atrophy of the parkinsonian type (MSA-P), 99 other cerebellar ataxia patients and 314 non-ataxia patients. We measured anterior-posterior pons and middle cerebellar peduncle diameters on baseline and subsequent MRIs, and correlated results with Brief Ataxia Rating Scale scores. We assessed midbrain:pons and middle cerebellar peduncle:pons ratios over time. The normative anterior-posterior pons diameter was 23.6 ± 1.6 mm, and middle cerebellar peduncle diameter 16.4 ± 1.4 mm. Pons diameter correlated with volume, r = 0.94, P < 0.0001. The anterior-posterior pons and middle cerebellar peduncle measures were smaller at first scan in MSA-C compared to all other ataxias; anterior-posterior pons diameter: Exploratory, 19.3 ± 2.6 mm versus 20.7 ± 2.6 mm, Validation, 19.9 ± 2.1 mm versus 21.1 ± 2.1 mm; middle cerebellar peduncle transverse diameter, Exploratory, 12.0 ± 2.6 mm versus 14.3 ±2.1 mm, Validation, 13.6 ± 2.1 mm versus 15.1 ± 1.8 mm, all P < 0.001. The anterior-posterior pons and middle cerebellar peduncle rates of change were faster in MSA-C than in all other ataxias; anterior-posterior pons diameter rates of change: Exploratory, -0.87 ± 0.04 mm/year versus -0.09 ± 0.02 mm/year, Validation, -0.89 ± 0.48 mm/year versus -0.10 ± 0.21 mm/year; middle cerebellar peduncle transverse diameter rates of change: Exploratory, -0.84 ± 0.05 mm/year versus -0.08 ± 0.02 mm/year, Validation, -0.94 ± 0.64 mm/year versus -0.11 ± 0.27 mm/year, all values P < 0.0001. Anterior-posterior pons and middle cerebellar peduncle diameters were indistinguishable between Possible, Probable and Definite MSA-C. The rate of anterior-posterior pons atrophy was linear, correlating with ataxia severity. Using a lower threshold anterior-posterior pons diameter decrease of -0.4 mm/year to balance sensitivity and specificity, area under the curve analysis discriminating MSA-C from other ataxias was 0.94, yielding sensitivity 0.92 and specificity 0.87. For the middle cerebellar peduncle, with threshold decline -0.5 mm/year, area under the curve was 0.90 yielding sensitivity 0.85 and specificity 0.79. The midbrain:pons ratio increased progressively in MSA-C, whereas the middle cerebellar peduncle:pons ratio was almost unchanged. Anterior-posterior pons and middle cerebellar peduncle diameters were smaller in MSA-C than in MSA-P, P < 0.001. We conclude from this 20-year longitudinal clinical and imaging study that anterior-posterior pons and middle cerebellar peduncle diameters are phenotypic imaging biomarkers of MSA-C. In the correct clinical context, an anterior-posterior pons and transverse middle cerebellar peduncle diameter decline of ∼0.8 mm/year is sufficient for and diagnostic of MSA-C.

Ex-Vivo Testing Platform to Predict Clinical Response and Resistance to Proteosome Inhibitors, IMiDs and Daratumumab in Multiple Myeloma

Treatment of Multiple Myeloma (MM) relies on Standard of Care (SoC) schemes that include immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs) in combination with dexamethasone (Dexa). More recently, monoclonal antibodies such as Daratumumab (Dara) have become complementary therapeutic strategies to treat newly diagnosed and relapsed MM patients. Despite the multiple therapeutic schemes available, MM remains an incurable disease in which patients transit intermittent cycles of relapse and remission that ultimately lead to multi-drug resistance. It has therefore become clear that novel tools for personalized therapy selection are necessary to improve outcomes and reduce trial and error, particularly when selecting later lines of treatment. At OncoPrecision, we have developed a personalized ex-vivo platform that mimics the tumor microenvironment and promotes the survival of Patient-Derived Cells (PDCs), with the goal to predict the performance of the complete array of FDA-approved drugs, as well as experimental treatments, within 7 days. This technology, which we have named Patient Micro Avatars (PMAs), consists of the co-culture of PDCs with engineered neoplastic (System Control) and stromal (Tox Control) heterologous cells in combination with a multi-tagging approach coupled to high-throughput flow cytometry (Figure: Upper Panel). Our PMAs not only enable us to rank the activity of all approved treatments, but also unveil potential unspecific toxicities of novel treatments on non-tumoral cells, thus unlocking unique insights for early drug development programs. In this work, we introduce our PMA technology for MM by presenting differential response between patients in a Clinical Study performed in collaboration with two healthcare centers which, through approved IRBs, have provided MM patient samples from bone marrow aspiration. We present ex-vivo profiling results with a comprehensive MM drug matrix which includes SoC treatments used in several lines of therapy. We illustrate the potential of PMAs to identify differential activity from individual agents and synergistic drugs combinations in 13 patients (Figure: Lower Panel). By anticipating vulnerability/resistance to SoC treatments, our platform has the potential to become a first-in-class phenotypic biomarker to serve as a decision-support tool for Physicians to select optimal therapeutic regimes, as well as to elucidate valuable pre-clinical patient-derived insights for the development of innovative therapies for MM.

A Single Phenotypic Biomarker of Gemtuzumab Ozogamicin (GO) Response in Acute Myeloid Leukemia Using Ex-Vivo Testing

Background or Introduction: Gemtuzumab Ozogamicin (GO), a therapeutic antibody targeting CD33, gained FDA approval in 2017 for the treatment of patients with Acute Myeloid Leukemia (AML). Biomarkers to predict GO response were historically based on multiple indicators: CD33 expression levels, favorable cytogenetics and specific genetic alterations (NPM1, FLT3-internal tandem duplications, other mutations). Moreover, comprehensive analyses including stemness expression (LSC17 score) and molecularly derived scores such as the CD33_PGx6_Score (a composite score derived from six CD33 SNP of prognostic significance) have further refined the biomarkers to identify potential responders (Fenwarth et al, nt. J. Mol. Sci. 2020, 21, 5626). However, due to its toxicity profile and elevated cost, the identification of robust biomarkers of GO response in AML remains a challenge to optimize its therapeutic efficacy and worldwide clinical adoption. Methods: In this study, we utilized an innovative ex-vivo functional platform of Patient Micro Avatars (PMAs) developed at OncoPrecision (García et al, Blood 2022 140 (Supplement 1): 13027-13028) to assess the response to GO in a small cohort of AML patients. Patient-Derived Cells (PDCs) were obtained from five newly diagnosed AML patients and GO activity was assessed using high-throughput flow cytometry. The correlation between CD33 expression and the ex-vivo response to GO was also examined. Results: Our findings revealed a clear differential response to GO in AML patients, with contrasting outcomes observed within a dose-range that does not exceed its theoretical plasma concentration (Figure - Upper Panel). Interestingly, despite significant variations in CD33 expression levels among the evaluated patients, no obvious association was found between CD33 expression and the ex-vivo response to GO (Figure - Lower Panel). Conclusions: This study presents an innovative approach with the potential to predict GO responders versus non-responders in AML. A single phenotypic biomarker overcomes the need to determine CD33 expression levels in each patient or to assess the presence of specific polymorphic variants, as well as additional molecular biomarkers. Patient Micro Avatars present a compelling alternative to comprehensively identify patients who are most likely to benefit from GO treatment.

Relationships Between the Nicotine Metabolite Ratio and Laboratory Assessments of Smoking Reinforcement and Craving Among Adults in a Smoking Cessation Trial.

People who metabolize nicotine more quickly are generally less successful at quitting smoking. However, the mechanisms that link individual differences in the nicotine metabolite ratio (NMR), a phenotypic biomarker of the rate of nicotine clearance, to smoking outcomes are unclear. We tested the hypotheses that higher NMR is associated with greater smoking reinforcement, general craving, and cue-induced cigarette craving in a treatment-seeking sample. Participants were 252 adults who smoke cigarettes enrolled in a randomized controlled smoking cessation trial (NCT03262662) conducted in Buffalo, New York, USA. Participants completed the Choice Behavior Under Cued Conditions (CBUCC) paradigm, a laboratory choice procedure, ~1 week before the first cessation treatment visit, at which time a saliva sample was collected for NMR assessment. On each CBUCC trial, participants reported cigarette craving during cue presentation (cigarette, water) and spent $0.01-$0.25 for a chance (5%-95%) to sample the cue (one puff, sip), providing measures of smoking reinforcement (spending for cigarettes vs. water), general cigarette craving (averaged across cigarette and water cues), and cue-specific craving (cigarette craving during cigarette vs. water cues). As observed in prior work, the NMR was significantly higher among White and female participants. As expected, both spending and cigarette craving were significantly greater on cigarette compared to water trials. However, contrary to our hypotheses, higher NMR was not associated with greater smoking reinforcement, general craving, or cue-specific craving. The present data do not support that smoking reinforcement or craving is related to nicotine metabolism among individuals seeking to quit smoking. Though greater smoking reinforcement, general craving, and cue-specific craving are hypothesized to be linked to faster nicotine metabolism, there was no evidence of such relationships in the present sample of adults seeking to quit smoking. Further research, including replication and consideration of alternate hypotheses, is warranted to elucidate the mechanisms by which the NMR is related to smoking cessation.

Immunological and senescence biomarker profiles in patients after spontaneous clearance of hepatitis C virus: gender implications for long-term health risk

BackgroundAbout 25% of patients with acute hepatitis C virus (HCV) infection show spontaneous clearance within the first six months of infection but may remain at risk of inflammaging, aging, and liver and non-liver disease complications. This study evaluated the differences in the plasma levels of immune checkpoints (ICs) and senescence-associated secretory phenotype (SASP) biomarkers between patients who had spontaneously eliminated HCV infection (SC group) and individuals without evidence of HCV infection (C group).MethodsWe performed a multicenter retrospective study of 56 individuals: 32 in the SC and 24 in the C groups. ICs and SASP proteins were analyzed using a Luminex 200TM analyzer. The statistical analysis used Generalized Linear Models with gamma distribution (log-link) adjusted by significant variables and sex.Results13 ICs (BTLA, CD137(4-1BB), CD27, CD28, CD80, GITR, HVEM, IDO, LAG-3, PD-1, PD-L1, PD-L2, and TIM-3) and 13 SASP proteins (EGF, Eotaxin, IL-1alpha, IL-1RA, IL-8, IL-13, IL-18, IP-10, SDF-1alpha, HGF, beta-NGF, PLGF-1, and SCF) were significantly higher in SC group after approximately more than two years of HCV clearance. After stratifying by sex, differences remained significant for males, which showed higher levels for 13 ICs and 4 SASP proteins in SC. While only PD-L2 was significantly higher in SC women, and no differences in SASP were found.ConclusionsHigher plasma levels of different IC and SASP proteins were found in individuals after more than two years of HCV clearance, mainly in men. Alterations in these molecules might be associated with an increased risk of developing liver and non-hepatic diseases.

Biomarkers of Progressive Fibrosing Interstitial Lung Diseases

Despite adequate therapy, interstitial lung diseases (ILD) can cause progressive scarring of lung tissue. This type of ILD is known as progressive fibrosing ILD (PF­-ILD). The challenge in diagnosing PF-­ILD lies in the lack of uniformly accepted criteria for a progressive fibrosing phenotype. Most authors use criteria based on clinical features and assessment of functional imaging and radiological findings over time. However, forced vital capacity (FVC) measurement is limited by its variability, and the follow­up lasts 1­2 years. The above diagnostic challenges prevent from prescribing early adequate therapy in patients with progressive ILD, indicting the need to search for new biomarkers of the progressive fibrosing phenotype. We review the most studied and informative biomarkers of fibrosis progression in patients with ILD.

323 Rumen Fluid Amine/Phenol-Metabolome of Beef Steers with Divergent Residual Feed Intake Phenotype

Abstract The amine/phenol-metabolome of rumen fluid was analyzed to identify amino acid metabolism-related biomarkers associated with divergent residual feed intake (RFI) phenotype in beef cattle. Fourteen beef steers [most feed-efficient (HFE; RFI = −1.89 kg/d, n = 7) and least feed-efficient (LFE; RFI = +2.05 kg/d, n = 7)] were selected from a total of 56 crossbred growing beef steers (average BW = 261 ± 18.5 kg) after a 49-d feeding period in a dry lot equipped with two GrowSafe intake nodes. Rumen fluid samples were collected 4 h after feeding on d 56, 63, and 70 from the HFE and LFE beef steers. Metabolome analysis of the rumen fluid was performed using chemical isotope labeling/liquid chromatography-mass spectrometry to identify all metabolites containing amine/phenol chemical groups, which are mostly amino acid metabolites. A total of 493 metabolites were detected and identified in the rumen fluid. The partial least squares discriminant scores plot showed a slight separation between the two groups of steers, and a total of eight metabolites were found to be differentially abundant (FDR ≤ 0.05). Out of the eight differentially abundant metabolites, four metabolites (isomer 1 of cadaverine, baeocystin, 6-methyladenine, and N(6)-methyllysine) qualified as candidate biomarkers of divergent RFI phenotype based on area under the curve ≥ 0.70. The results of this study revealed that divergent RFI phenotype is associated with alteration in rumen amine/phenol-metabolome of beef steers.

Predicting Tumour Progression of ID-8 Syngeneic Mouse Ovarian Cancer with Blood Biomarkers of CA-125, IL-6 and VEGF: A Prospective Laboratory-Based Study

Background: A preclinical animal model is an imperative tool for uncovering and understanding the tumourigenic hallmarks of human ovarian cancer; the disease is often lethal because it is commonly diagnosed in the advanced stage, where widespread cancer nodules mainly reside within peritoneal regions. Mouse models as a xenograft tumour host or genetic manipulation ovarian cancer-derived mice are widely used for studying specific hypothesis rationale in ovarian cancer. However, limited information associated with disease progression is obtained from such studies; whether it is the best model to study advanced ovarian cancer phenotype or suitable preclinical biomarkers for detecting and monitoring ovarian cancer progression is under study. This study used an ID-8 syngeneic mouse ovarian cancer model with immunocompetence. We monitored cancer growth and development using combination modalities of cancer-specific cancer antigen-125 (CA-125), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) blood markers, which are well-known for their association with tumour progression in humans. Methods: Ten C57/BL6 female mice were intraperitoneally implanted with ID-8 Trp53 wild-type and monitored the progression of the tumour, until mice developed clinical ascites. Blood was taken at the time of intraperitoneal (IP) implantation (Day 0) and then collected weekly, and levels of biomarkers were analysed with enzyme-linked immunosorbent assay (ELISA). In addition, tumour tissue was collected and proceeded with histological staining. Results: We found that blood biomarkers CA-125, IL-6 and VEGF were not readily correlated with tumour progression. However, these biomarkers were markedly elevated in ascitic fluid at the advanced stage of the disease. Conclusions: We conclude that blood biomarkers in a syngeneic mouse model are, to some extent, not readily found in the blood as opposed to human ovarian cancer. Model anatomical and physiological differences between rodents and humans might explain this discrepancy.

Physical activity interventions for cancer-related fatigue: A scoping review of randomized controlled trials from a Nursing Science Precision Health Model perspective

BackgroundThe Nursing Science Precision Health (NSPH) Model has the potential to guide research on the development, testing, and targeting of interventions. PurposeThis scoping review examines the relationship between physical activity (PA) and cancer-related fatigue (CRF) within the context of the NSPH Model. MethodsThe Joanna Briggs Institute scoping review methodology and Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews guided this review. We included randomized controlled trials in people with cancer that investigated PA interventions and measured change in CRF as an outcome. DiscussionA total of 181 studies met the eligibility criteria. Over 20 different instruments were used to measure CRF. The most common PA interventions were strength training (48%), walking (36%), cycling (26%), and yoga (15%). A limited number of studies reported phenotypic characteristics (32/181, 17%) or biomarkers (31/181, 17%) associated with CRF. ConclusionThis scoping review identified the body of existing research exploring CRF and PA from a precision health perspective.

The translational potential of the lung microbiome as a biomarker and a therapeutic target for chronic obstructive pulmonary disease

AbstractChronic obstructive pulmonary disease (COPD) is a major chronic lung disease with a leading global morbidity and mortality. Emerging evidence suggests that the lung microbiome, the collection of the microorganisms in the lung, plays a crucial role in COPD pathogenesis. The lung microbiome alters in COPD during the acute exacerbations, associates with clinical phenotypes and inflammatory endotypes, and predicts treatment response, lung function decline and mortality, suggesting it could be a potential biomarker for COPD phenotyping, prognosis and personalized therapies. The lung microbiome produces metabolites that interact with host immunity and contribute to COPD pathogenesis, implying that it could be a possible novel target for therapeutic intervention. Despite these potentials, critical gaps exist for translational application of the lung microbiome in COPD clinical practice, starting with the lack of approaches in precisely measuring and manipulating specific members of the lung microbiome, highlighting the need for future collaborative efforts from bench to bedsides. In this perspective, we review existing knowledge and progress on the understanding of the lung microbiome as a possible COPD biomarker or a therapeutic target. We discuss current challenges and share our views on future directions toward fully realize the potential in translational application of the lung microbiome in the promise of COPD precision medicine.