Human Biospecimens Research Articles

Current aspects in biobanking for personalized oncology investigations and treatments.

A biobank is an organization that gathers, refines, preserves and provides access to biospecimens along with relevant clinical data that can be used in applied or clinical research. Biobanking is a critical component of the scientific foundation for personalized medicine; this implies the accessibility of high-quality human biospecimens, such as blood, tissue, and other body fluids, along with the patient clinical data that goes with them. This paper summarizes the function of biobanks in oncology and the requirements for biobank development in translational and clinical research. Biobanks raise numerous ethical issues that government agencies address by enacting particular laws. To develop personalized medicine, biobanks are crucial, given that the availability of an extensive collection of patient samples with thoroughly annotated clinical and pathological data is an essential necessity. Also, data related to biobanking raises complex ethical, legal, and social issues, particularly concerning the protection of donor privacy and the appropriate use of collected samples. International standards have been developed to address these issues to ensure biobanking practices' quality, safety, and integrity. Biobanking is vital in advancing biomedical research, supporting clinical applications, and enhancing our understanding of human health and disease. Using real-world data and biobanking can accelerate medical research, support personalized medicine initiatives, and improve patient care.

Inflammatory-associated proteomic predictors of cognitive outcome in subjects with ELVO treated by mechanical thrombectomy

BackgroundEmergent Large Vessel Occlusion (ELVO) stroke causes devastating vascular events which can lead to significant cognitive decline and dementia. In the subset of ELVO subjects treated with mechanical thrombectomy (MT) at our institution, we aimed to identify systemic and intracranial proteins predictive of cognitive function at time of discharge and at 90-days. These proteomic biomarkers may serve as prognostic indicators of recovery, as well as potential targets for novel/existing therapeutics to be delivered during the subacute stage of stroke recovery.MethodsAt the University of Kentucky Center for Advanced Translational Stroke Sciences, the BACTRAC tissue registry (clinicaltrials.gov; NCT 03153683) of human biospecimens acquired during ELVO stroke by MT is utilized for research. Clinical data are collected on each enrolled subject who meets inclusion criteria. Blood samples obtained during thrombectomy were sent to Olink Proteomics for proteomic expression values. Montreal Cognitive Assessments (MoCA) were evaluated with categorical variables using ANOVA and t-tests, and continuous variables using Pearson correlations.ResultsThere were n = 52 subjects with discharge MoCA scores and n = 28 subjects with 90-day MoCA scores. Several systemic and intracranial proteins were identified as having significant correlations to discharge MoCA scores as well as 90-day MoCA scores. Highlighted proteins included s-DPP4, CCL11, IGFBP3, DNER, NRP1, MCP1, and COMP.ConclusionWe set out to identify proteomic predictors and potential therapeutic targets related to cognitive outcomes in ELVO subjects undergoing MT. Here, we identify several proteins which predicted MoCA after MT, which may serve as therapeutic targets to lessen post-stroke cognitive decline.

A multiplexed assay for quantifying immunomodulatory proteins supports correlative studies in immunotherapy clinical trials.

Immunotherapy is an effective treatment for a subset of cancer patients, and expanding the benefits of immunotherapy to all cancer patients will require predictive biomarkers of response and immune-related adverse events (irAEs). To support correlative studies in immunotherapy clinical trials, we are developing highly validated assays for quantifying immunomodulatory proteins in human biospecimens. Here, we developed a panel of novel monoclonal antibodies and incorporated them into a novel, multiplexed, immuno-multiple reaction monitoring mass spectrometry (MRM-MS)-based proteomic assay targeting 49 proteotypic peptides representing 43 immunomodulatory proteins. The multiplex assay was validated in human tissue and plasma matrices, where the linearity of quantification was >3 orders of magnitude with median interday CVs of 8.7% (tissue) and 10.1% (plasma). Proof-of-principle demonstration of the assay was conducted in plasma samples collected in clinical trials from lymphoma patients receiving an immune checkpoint inhibitor. We provide the assays and novel monoclonal antibodies as a publicly available resource for the biomedical community.

Isolation and Differentiation of Neurons and Glial Cells from Olfactory Epithelium in Living Subjects

The study of psychiatric and neurological diseases requires the substrate in which the disorders occur, that is, the nervous tissue. Currently, several types of human bio-specimens are being used for research, including postmortem brains, cerebrospinal fluid, induced pluripotent stem (iPS) cells, and induced neuronal (iN) cells. However, these samples are far from providing a useful predictive, diagnostic, or prognostic biomarker. The olfactory epithelium is a region close to the brain that has received increased interest as a research tool for the study of brain mechanisms in complex neuropsychiatric and neurological diseases. The olfactory sensory neurons are replaced by neurogenesis throughout adult life from stem cells on the basement membrane. These stem cells are multipotent and can be propagated in neurospheres, proliferated in vitro and differentiated into multiple cell types including neurons and glia. For all these reasons, olfactory epithelium provides a unique resource for investigating neuronal molecular markers of neuropsychiatric and neurological diseases. Here, we describe the isolation and culture of human differentiated neurons and glial cells from olfactory epithelium of living subjects by an easy and non-invasive exfoliation method that may serve as a useful tool for the research in brain diseases.

Variant biomarker discovery using mass spectrometry-based proteogenomics.

Genomic diversity plays critical roles in risk of disease pathogenesis and diagnosis. While genomic variants-including single nucleotide variants, frameshift variants, and mis-splicing isoforms-are commonly detected at the DNA or RNA level, their translated variant protein or polypeptide products are ultimately the functional units of the associated disease. These products are often released in biofluids and could be leveraged for clinical diagnosis and patient stratification. Recent emergence of integrated analysis of genomics with mass spectrometry-based proteomics for biomarker discovery, also known as proteogenomics, have significantly advanced the understanding disease risk variants, precise medicine, and biomarker discovery. In this review, we discuss variant proteins in the context of cancers and neurodegenerative diseases, outline current and emerging proteogenomic approaches for biomarker discovery, and provide a comprehensive proteogenomic strategy for detection of putative biomarker candidates in human biospecimens. This strategy can be implemented for proteogenomic studies in any field of enquiry. Our review timely addresses the need of biomarkers for aging related diseases.

Abstract 4479: Targeting of eIF4A1 curtails lung metastases in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer. Patients diagnosed with TNBC benefit less or none from any current targeted therapies and also the standard-of-care neoadjuvant chemotherapies (NACT) which is cytotoxic. TNBC shows an increased risk of tumor relapse, therapy failure, and high proclivity to develop distant metastases. Intrinsic resistance to NACT is mainly due to a small population of breast cancer cells that acquire stem cell-like features (BCSCs). BCSCs are capable of self-renewal, proliferation, plasticity, immune regulation and chemoresistance. These properties mediate tumorigenesis, metastasis, and resistance to NACT and sometimes even to targeted therapy. Overall, FDA-approved drugs that are effective for advanced TNBC are currently unavailable. The eukaryotic translation initiation factor (eIF4A1) is an mRNA helicase that catalyzes the ATP-dependent unwinding of many oncogenic mRNAs which harbor secondary structures in the 5’ leader region. There is preferential reliance on eIF4A1 by tumor cells as many oncogenic mRNAs require the helicase activity of eIF4A1 for their efficient translation. Several eIF4A1-dependent mRNAs (downstream effectors) include: survivin (survival), c-MYC, STAT1 (immunoevasion through transcription and subsequent translation of PD-L1), Rho kinase1 (invasion), and Cyclins D1 and D3. Bioinformatic analyses revealed that higher gene expression of eIF4A1 significantly impacts patient survival. Clinically, an enhanced protein expression of eIF4A1 was evident in histopathologic analyses of drug-resistant human TNBC biospecimens. Importantly, our in vitro data indicated that the viability of BCSCs was significantly reduced when eIF4A1 is targeted by nanomolar levels of Rocaglamide A (RocA). Furthermore, in paclitaxel-resistant TNBC cells, there is an upregulation in the total levels of eIF4A1 and its downstream effectors. Pluripotency transcription factors (e.g., SOX2, OCT4 and NANOG) and drug transporters (ABCB1, ABCG2 and ABCC1) were also increased in an eIF4A1-dependent manner. Genetic ablation and pharmacological targeting of eIF4A1 with RocA reversed the drug-resistant profile. Our in vitro data implied a key role for eIF4A1 in chemoresistance/therapy failure. Targeting of eIF4A1 may be an effective anti-cancer strategy to overcome drug resistance. To this end, we orthotopically implanted CRISPR-control (CC) and eIF4A1-KO (KO) human TNBC cells and evaluated the primary tumor progression and metastases in NOD/SCID-beige mice. Longitudinal tumor progression was followed by IVIS imaging. The tumor volume and the wet weight was significantly higher in CC than KO tumors (n=7 mice). Similarly, the lung metastatic burden is significantly low in KO than CC mice (n=7 mice; p<0.002)). Taken together, we propose that eIF4A1 is an actionable molecular target for drug-resistant TNBC using chemotherapy/immunotherapy. Citation Format: Sangita Sridharan, Shobhit Srivastava, David Terrero, Saloni Malla, Amit K. Tiwari, Dayanidhi Raman. Targeting of eIF4A1 curtails lung metastases in triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4479.

Abstract 4435: A causal modeling platform for testing lifestyle interventions on the microbiome and response to immunotherapy

Abstract Rational manipulation of the gut microbiome by diet or other interventions is a promising approach to improving the efficacy and safety of cancer immunotherapy. However, linking lifestyle variables to specific microbial populations and the host immune response to cancer therapies is challenging due to their complexity. We established an experimental system to examine such complexity by linking a human dietary intervention and fecal biospecimens to mouse models to test the response to immunotherapy. Longitudinal human microbiome samples collected before and after a specific dietary or other intervention allow us to test the hypothesis that an intervention alters the response to immunotherapy via impact on the host microbiome. One set of samples was taken from the BEWELL study, which examined the impact of 2 × 80 mL black raspberry (BRB) drink boxes per day for 4 weeks in people at high risk for lung cancer. Participant samples were chosen based on enrichment of specific taxa. Pre- and post-BRB intervention samples were gavaged into mice. Mouse mc38 cells were injected subcutaneously and treated with anti-PD1 Ab or isotype control. Tumor size was monitored, and at the end of the study, tumor immune cell composition data were collected. Tumor growth over time was modeled using a linear mixed-effects model with tumor volume as the outcome variable and the predictor variables of time and treatment (PD1 vs. IgG) interacting with gavage and quadratic time to accommodate non-linear tumor growth and including random effects by mouse. For sample 68, enriched for Roseburia CAG 309, the interaction between time, gavage post-BRB, and Anti-PD1 treatment significantly affected tumor volume (p < 0.05) relative control. The same significant effect (p < 0.05) was also seen for sample 79, enriched for Lachnospira pectinoschiza, and samples 84 and 85, enriched for Blautia obeum. To determine the mechanism by which this might occur, we analyzed the tumor's immune cell composition. Post-BRB mice treated with Anti-PD1 showed a significant increase in tumor-infiltrating CD8+ immune cells relative control, as compared to mice gavaged with pre-BRB stool. Modeling results showed samples whose response was improved slightly after the BRB dietary intervention. These samples were associated with the enrichment of the taxon Roseburia CAG 309. A black-raspberry dietary intervention in humans modified the microbiomes of several participants in a way that is hypothesized to improve response to PD1 treatment. Future directions include supplementing individual microbes into pre-intervention gavages to confirm which taxa improve response. These results suggest that this modeling platform is an effective system for testing microbiome modification on tumor growth and assessing mechanisms by which this might occur and is extendable to other lifestyle-based interventions where pre- and post-intervention specimens are collected. Citation Format: Aaditya Pallerla, Bailey Conrad, Amna Bibi, Nyelia Williams, Caroline Wheeler, Rebecca Hoyd, Shankar Suman, Joseph Amann, Yangyang Liu, Marisa Bittoni, Shiqi Zhang, Madison Grogan, Alvin Anand, Najma Afrah, Carolyn Presley, Fred K. Tabung, Lang Li, Yael Vodovotz, Jiangjiang Zhu, David P. Carbone, Steven K. Clinton, Daniel Spakowicz. A causal modeling platform for testing lifestyle interventions on the microbiome and response to immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4435.

Abstract P4-08-03: Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC

Abstract Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC Background and Premise: Triple-negative breast cancer (TNBC) is an aggressive, metastatic disease with high mortality. The standard-of-care neoadjuvant chemotherapy (NACT) in TNBC shows an initial response but followed by an increase in tumor relapse and distant metastases. Furthermore, metastatic TNBC (mTNBC) patients often develop resistance to NACT and targeted therapies. Immunotherapy with therapeutic antibodies such as anti-programmed death ligand-1 (PD-L1) has shown efficacy in treating a subset of TNBC patients, but currently only 20% of patients qualify for that treatment. Hence, there is an unmet need for developing a precision approach against novel actionable targets along with immunotherapy to treat mTNBC. Eukaryotic initiation factor 4A1 (eIF4A1) is an integral part of the translational machinery for many oncogenic mRNAs including survivin, c-MYC, Rho kinase1, and Cyclins D1 and D3, which all require the helicase activity of eIF4A1 for their translation. Our lab previously established that the eIF4A1 is a vulnerable target in breast cancer stem-like cells (BCSCs) and bulk tumor cells. We further demonstrated a key role for eIF4A1 in cancer stemness and drug resistance. Interestingly, we also observed a reduction in levels of PD-L1 when eIF4A1 is pharmacologically inhibited or genetically ablated. Many downstream effectors of eIF4A1 such as c-MYC, STAT1 and ARF6 (involved in recycling of PD-L1 back to the plasma membrane) are indeed upstream regulators of PD-L1 gene expression and cell surface expression of PD-L1. In this study, we examine human TNBC biospecimens for total eIF4A1 level, levels of STAT1, c-MYC, ARF6 and PD-L1. This will help establish the differential operation of the eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant primary and mTNBC. Approach: Human biospecimens were obtained from CHTN NIH repositories and now we have a collection of more than 100 TNBC tumor samples as well as some lymph node, lung and brain metastases and one sample of male breast primary tumor. These specimens are analyzed for role of eIF4A1 in translation of oncogenic mRNAs that contribute to the gene expression of immune checkpoint PD-L1 and other emerging immune checkpoint markers such as VISTA, TIGIT, and LAG3. To mimic the inclusion criteria for current PD-L1 monoclonal antibody treatment, TNBC tumors are examined for PD-L1 expression via immunohistochemistry after subjecting the samples to deglycosylation for effective detection and also by immunoblotting. Results: The levels of total eIF4A1 from 16 matched, therapy naive TNBC tissue were compared to adjacent normal breast tissue from the same patient via immunoblotting. Total eIF4A1 levels were found to be significantly elevated in TNBC compared to adjacent normal tissue. The downstream effectors of eIF4A1 such as STAT1 and ARF6 were also significantly elevated indicating a robust helicase activity of eIF4A1 in tumor samples. Importantly, PD-L1 levels was found to be elevated. Additionally, the total eIF4A1 level from tumor lysates from TNBC patients that had received a variety of NACT (i.e., patients with demonstrated drug resistance) were compared against normal untreated breast tissue from volunteers who underwent reduction mammoplasty by immunoblotting. The total eIF4A1 was found to be elevated in drug-resistant TNBC specimens compared to control, therapy-naïve TNBC samples. Conclusion: The eIF4A1-STAT1-PD-L1 axis is highly active in TNBC especially in drug-resistant situations. Currently, we are evaluating emerging immune checkpoints such as VISTA, TIGIT, and LAG3. Citation Format: Andrew Boring, Dharmindra Dulal, Dayanidhi Raman. Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-03.

Abstract PD2-08: Targeting of eIF4A1 along with anti-PD-L1 therapy limits lung metastases efficaciously in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of metastatic breast cancer with poor clinical prognosis. Currently used neoadjuvant chemotherapy for TNBC, such as taxanes, and/or anthracyclines demonstrate an initial treatment response but is often followed by drug resistance, tumor relapse, and high proclivity to develop metastases. There is an unmet need to develop effective combination therapies against metastatic TNBC (mTNBC). eIF4A1 is an mRNA helicase that help translate many oncogenic mRNAs including PD-L1 (through STAT1 translational regulation) that are involved in tumor growth, chemoresistance and metastases. Our prior studies pointed out that eIF4A1 is intricately linked to cancer stemness and drug resistance in TNBC. Emerging evidence has revealed that the tumor immune microenvironment (TIME) of TNBC do consist of mononuclear cells and lymphocytes. Combining chemotherapy with immune checkpoint inhibition had demonstrated a significant benefit for high-risk TNBC patients. In this study, we examine the efficacy of targeting eIF4A1 along with anti-PD-L1 therapy as an effective therapeutic strategy to limit cancer stemness, primary tumor progression and metastasis in an immunocompetent murine model of TNBC. Approach: Initially, we examined the human TNBC biospecimens and found that eIF4A1 protein level is upregulated in drug-resistant cases. STAT1 is an upstream regulator of PD-L1 and a downstream effector of eIF4A1. The protein level of STAT1 was significantly elevated as assessed by immunoblotting of human tumor lysates. In human TNBC cells, when eIF4A1 is pharmacologically or genetically targeted, PD-L1 was downregulated at the protein level. Based on this premise, we targeted eIF4A1 with the small molecule inhibitor, Rocaglamide A (RocA), along with anti-PD-L1 immunotherapy in orthotopically implanted 4T1 mouse TNBC tumors in BALB/c mice. The treatment arms were 1) Control (vehicle for RocA + control IgG) 2) RocA 3) RocA + anti-PD-L1 antibodies 4) Control IgG only 5) Anti-PD-L1 antibodies only with 10 mice in each group. Results: The combination treatment comprising of RocA and anti-PD-L1 antibodies worked out efficaciously as the primary tumor volume, wet tumor weight, bioluminescent tumor imaging and quantitation were significantly lower than the combination vehicle control. Furthermore, metastasis to the lungs were significantly impaired (1 out of 10 mice only had metastasis and that too it was small). On the contrary, ‘RocA single arm’ group had 6 out of 10 mice with lung metastasis. The lung metastases were observed in 4 out of 10 mice in the PD-L1 single arm and 6 out of 10 mice in control IgG single arm group. The RocA was tolerated well at 0.6 mg/kg with no significant change in the body weight and the liver and kidney function test values were within the normal range. Importantly, the combination treatment with RocA and PD-L1 was also tolerated well. Conclusions: The combination therapy involving RocA and PD-L1 had the significant impact in reducing lung metastases and may pave the way for phase I/II clinical trials. Citation Format: Dharmindra Dulal, Andrew Boring, Dayanidhi Raman. Targeting of eIF4A1 along with anti-PD-L1 therapy limits lung metastases efficaciously in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-08.

A novel isolation method for spontaneously released extracellular vesicles from brain tissue and its implications for stress-driven brain pathology

BackgroundExtracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, exhibit great potential for the diagnosis and treatment of brain disorders, representing a valuable tool for precision medicine. The latter demands high-quality human biospecimens, especially in complex disorders in which pathological and specimen heterogeneity, as well as diverse individual clinical profile, often complicate the development of precision therapeutic schemes and patient-tailored treatments. Thus, the collection and characterization of physiologically relevant sEVs are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV fractions with intracellular vesicles.MethodsBased on multiscale analytical platforms such as cryo-EM, label-free proteomics, advanced flow cytometry, and ExoView analyses, we compared and characterized the EV fraction isolated with this novel method with a classical digestion-based EV isolation procedure. Moreover, EV biogenesis was pharmacologically manipulated with either GW4869 or picrotoxin to assess the validity of the spontaneous-release method, while the injection of labelled-EVs into the mouse brain further supported the integrity of the isolated vesicles.ResultsWe hereby present an efficient purification method that captures a sEV-enriched population spontaneously released by mouse and human brain tissue. In addition, we tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated, as well as under animals’ exposure to chronic stress, a clinically relevant precipitant of brain pathologies, such as depression and Alzheimer’s disease. Our findings show that the released method monitors the drug-evoked inhibition or enhancement of sEVs secretion while chronic stress induces the secretion of brain exosomes accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and related stress-driven brain pathology.ConclusionsOverall, the spontaneous release method of sEV yield may contribute to the characterization and biomarker profile of physiologically relevant brain-derived sEVs in brain function and pathology.7SKHktGsByupRjRZYNwNfMVideo

Viral genome prediction from raw human DNA sequence samples by combining natural language processing and machine learning techniques

Infection with a virus can lead to a range of illnesses in humans, including cancer. When viruses infect a host, they may disrupt normal host function and cause deadly diseases. Understanding complicated viral illnesses requires novel viral genome prediction. Since many of the sequences in assembled contigs from human samples are not identical to known genomes, many assembled contigs are labeled “unknown” by conventional alignments. In this study, sequences from 19 metagenomic investigations were used to create the model proposed here, and these sequences were examined and classified using BLAST. We implemented k-mer counting and the bag-of-words technique using CountVectorizer. As far as we are aware, this work represents the first framework that combines natural language processing (NLP) along with traditional ML classification approaches on raw metagenomic contigs to automatically identify viruses in a variety of human biospecimens. The suggested models are general rather than specialized to a particular viral family. Since the proposed methodology is precise and simple, we may incorporate it into computer-aided diagnosis (CAD) systems to make day-to-day hospital activities easier. In the last stage, binary classification of deoxyribonucleic acid (DNA) with normal and viral genomes was performed using traditional ML classifiers. Using the KNN classifier, the suggested model achieved 98.6% classification accuracy along with 98.5% precision, 98.6% recall, 0.984 F1 score, 0.896 Matthews correlation coefficient, 0.895 kappa, 0.97 classification success index and detection rate of 98.6% for the prediction of viral genomes in DNA. Compared to previously developed ML techniques, the model achieved a significantly greater performance for viral genome prediction.

Ensuring Procedural Guidance for Research Ethics and Research Integrity in Proposals using Human Biospecimens in Resource-limited Settings

Ensuring Procedural Guidance for Research Ethics and Research Integrity in Proposals using Human Biospecimens in Resource-limited Settings

Addressing the quality challenge of a human biospecimen biobank through the creation of a quality management system.

The objective of the COMET (COllection of MEtabolic Tissues) biobank project is to create a high-quality collection of insulin-sensitive tissues (liver, muscle, adipose tissues, and epiploic artery) and blood sample derivatives (plasma, serum, DNA and RNA), collected from 270 grade 2-3 obese patients undergoing bariatric surgery. Relevant data on patient such as clinical/biological characteristics and sample handling are also collected. For this, our aim was to establish a Quality Management System (QMS) to meet the reliability and quality requirements necessary for its scientific exploitation. The COMET QMS includes: (1) Quality Assurance to standardize all stages of the biobanking process, (2) Quality Controls on samples from the first patients included in order to validate the sample management process and ensure reproducible quality; and 3) "in process" Quality Controls to ensure the reliability of the storage procedures and the stability of the samples over time. For serum and plasma, several corrective actions, such as temperature handling and centrifugation conditions, were made to the protocol and led to improvement of the volume and quality of samples. Regarding DNA, all samples evaluated achieved a satisfactory level of purity and integrity and most of them yielded the required DNA quantity. All frozen tissue samples had RNAs of good purity. RNA quality was confirmed by RIN, achieving values in most cases over 7 and efficient amplification of housekeeping genes by RT-qPCR, with no significant differences among samples from the same tissue type. In the "in process" Quality Controls, DNA, RNA, and histological integrity of tissues showed no differences among samples after different preservation times. Quality Control results have made it possible to validate the entire biobank process and confirm the utility of implementing QMS to guarantee the quality of a biospecimen collection.

Collection of biospecimens from parent-child dyads in a community garden-based nutrition intervention: protocol and feasibility

BackgroundNon-invasive human biospecimens, including stool, urine, and hair, are important in understanding the relationship between diet and changes in human physiologic processes that affect chronic disease outcomes. However, biospecimen collection can be difficult when collecting samples for research studies that occur away from a centralized location. We describe the protocol and feasibility in collecting stool, urine, and hair biospecimens from parents and their children at a remote location as a part of a summer community garden-based intervention.MethodsStool, urine, and hair were collected as a part of the Summer Harvest Adventure (SHA) study, a randomized controlled, community garden-based intervention targeting children (ages 8–11 years) and their parents from low-resource neighborhoods. Biospecimens were collected from willing children and/or their parent/adult caregivers at baseline and post-intervention for evaluation of microbiome, metabolomics, and hair analyses among both intervention and control groups at a location distant from the academic laboratories conducting the analysis. The protocol used to assemble, deliver, collect, and process biospecimens are presented along with the frequencies with which specimens were successfully obtained.ResultsOne hundred forty six participants (73 parent-child dyads) were part of the larger SHA study and thus eligible to provide a biospecimen. A total of 126 participants, 115 participants, and 127 participants consented to provide their hair, stool and urine samples, respectively. Of the participants that consented to provide a sample, 44 children (69.8%) and 38 parents (60.3%) provided at least one hair sample, 27 children (48.2%) and 37 parents (62.7%) provided at least one stool sample, and 36 children (57.1%) and 42 parents (65.6%) provided at least one urine sample. Sample collection at the offsite location, transport, and handling at the academic center were successful and all biospecimens were deemed adequate for analyses. DNA and metabolomics yield on a subset of stool samples obtained provided excellent results in terms of an abundance of species and metabolities, as would be predicted. Urine and hair analyses are underway.ConclusionOur work is one of the first to describe the feasibility of collecting human biospecimens, specifically stool, urine, and hair, from both parents and their children from low-resourced neighborhoods in a non-traditional garden research setting. Future work will report findings related to mechanisms between diet, microbiome, metabolites, and clinical outcomes.

Systematic dissection, preservation, and multiomics in whole human and bovine hearts

ObjectivesWe sought to develop a rigorous, systematic protocol for the dissection and preservation of human hearts for biobanking that expands previous success in postmortem transcriptomics to multiomics from paired tissue. BackgroundExisting cardiac biobanks consist largely of biopsy tissue or explanted hearts in select diseases and are insufficient for correlating whole organ phenotype with clinical data. MethodsWe demonstrate optimal conditions for multiomics interrogation (ribonucleic acid (RNA) sequencing, untargeted metabolomics) in hearts by evaluating the effect of technical variables (storage solution, temperature) and simulated postmortem interval (PMI) on RNA and metabolite stability. We used bovine (n=3) and human (n=2) hearts fixed in PAXgene or snap-frozen with liquid nitrogen. ResultsUsing a paired Wald test, only two of the genes assessed were differentially expressed between left ventricular samples from bovine hearts stored in PAXgene at 0 and 12 hours PMI (FDR q<0.05). We obtained similar findings in human left ventricular samples, suggesting stability of RNA transcripts at PMIs up to 12 hours. Different library preparation methods (mRNA poly-A capture vs. rRNA depletion) resulted in similar quality metrics with both library preparations achieving >95% of reads properly aligning to the reference genomes across all PMIs for bovine and human hearts. PMI had no effect on RNA Integrity Number or quantity of RNA recovered at the time points evaluated. Of the metabolites identified (855 total) using untargeted metabolomics of human left ventricular tissue, 503 metabolites remained stable across PMIs (0, 4, 8, 12 hours). Most metabolic pathways retained several stable metabolites. ConclusionsOur data demonstrate a technically rigorous, reproducible protocol that will enhance cardiac biobanking practices and facilitate novel insights into human CVD. Condensed abstractCardiovascular disease (CVD) is the leading cause of mortality worldwide. Current biobanking practices insufficiently capture both the diverse array of phenotypes present in CVDs and the spatial heterogeneity across cardiac tissue sites. We have developed a rigorous and systematic protocol for the dissection and preservation of human cardiac biospecimens to enhance the availability of whole organ tissue for multiple applications. When combined with longitudinal clinical phenotyping, our protocol will enable multiomics in hearts to deepen our understanding of CVDs.

Targeting the ALK–CDK9-Tyr19 kinase cascade sensitizes ovarian and breast tumors to PARP inhibition via destabilization of the P-TEFb complex

Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated promising clinical activity in multiple cancers. However, resistance to PARP inhibitors remains a substantial clinical challenge. In the present study, we report that anaplastic lymphoma kinase (ALK) directly phosphorylates CDK9 at tyrosine-19 to promote homologous recombination (HR) repair and PARP inhibitor resistance. Phospho-CDK9-Tyr19 increases its kinase activity and nuclear localization to stabilize positive transcriptional elongation factor b and activate polymerase II-dependent transcription of HR-repair genes. Conversely, ALK inhibition increases ubiquitination and degradation of CDK9 by Skp2, an E3 ligase. Notably, combination of US Food and Drug Administration-approved ALK and PARP inhibitors markedly reduce tumor growth and improve survival of mice in PARP inhibitor-/platinum-resistant tumor xenograft models. Using human tumor biospecimens, we further demonstrate that phosphorylated ALK (p-ALK) expression is associated with resistance to PARP inhibitors and positively correlated with p-Tyr19-CDK9 expression. Together, our findings support a biomarker-driven, combinatorial treatment strategy involving ALK and PARP inhibitors to induce synthetic lethality in PARP inhibitor-/platinum-resistant tumors with high p-ALK–p-Tyr19-CDK9 expression.

Custodianship and Biobanks ―A New Framework for the Provision of Human Biospecimens and Data

Custodianship and Biobanks ―A New Framework for the Provision of Human Biospecimens and Data

International Spinal Cord Injury Biobank: A Biorepository and Resource for Translational Research.

Over the past few decades, tremendous advances have been made in our understanding of the biological changes underpinning the devastating impairment of traumatic spinal cord injury (SCI). Much of this scientific research has focused on animal models of SCI, and comparatively little has been done in human SCI, largely because biospecimens from human SCI patients are not readily available. This paucity of scientific enquiry in human SCI represents an important void in the spectrum of translational research, as biological differences between animal models and the human condition need to be considered in the pre-clinical development of therapeutic approaches. The International Spinal Cord Injury Biobank (ISCIB) is a multi-user biorepository with the mission of accelerating therapeutic development in traumatic SCI through improved biological understanding of human injury, and the vision of serving as a global research resource where human SCI biospecimens are shared with researchers around the world. Aligned with internationally recognized best practices, ISCIB's formal governance structure and standard operating procedures have earned it official biobank certification through the Canadian Tissue Repository Network. Herein, we describe the translational research gap that ISCIB is helping to fill; its structure, governance and certification; how data and samples are accrued, processed and stored; and finally, the process through which samples and data are shared with global researchers. The purpose of this paper describing ISCIB is to serve as an introductory guidance document for the wider community of SCI researchers. By helping researchers understand the contents of ISCIB and the process of accessing biospecimens, we seek to further ISCIB's vision as being a resource for human and translational research in SCI, with the ultimate goal of finding disease-modifying therapies for this disabling condition.

Opportunities and Challenges in Understanding Atherosclerosis by Human Biospecimen Studies.

Over the last few years, new high-throughput biotechnologies and bioinformatic methods are revolutionizing our way of deep profiling tissue specimens at the molecular levels. These recent innovations provide opportunities to advance our understanding of atherosclerosis using human lesions aborted during autopsies and cardiac surgeries. Studies on human lesions have been focusing on understanding the relationship between molecules in the lesions with tissue morphology, genetic risk of atherosclerosis, and future adverse cardiovascular events. This review will highlight ways to utilize human atherosclerotic lesions in translational research by work from large cardiovascular biobanks to tissue registries. We will also discuss the opportunities and challenges of working with human atherosclerotic lesions in the era of next-generation sequencing.

Circular and Fusion RNAs in Medulloblastoma Development.

Simple SummaryExpression of circular RNAs is known to be deregulated in cancer. Here the most comprehensive set of differentially expressed RNA circles in medulloblastoma compared to cerebellum is provided. Additionally, fusion RNAs are also identified in both cancerous and normal cerebellar tissue. Some of the fusions detected in medulloblastoma are generated by genomic rearrangements that link different genes. However, fusion RNAs are also detected in normal cerebellum. In fact, there are cases where the same fusion RNA is also found in medulloblastoma. This observation underscores that the formation of fusion transcripts may not be limited to chromosomal events but could also result from mechanisms that act at the RNA level. These include read-through transcription of neighboring genes and intermolecular splicing of pre-mRNAs from different genes Importantly, these RNA “recombination” events occur not only in normal but also in cancerous tissue.Background. The cerebellar cancer medulloblastoma is the most common childhood cancer in the brain. Methods. RNA sequencing of 81 human biospecimens of medulloblastoma using pipelines to detect circular and fusion RNAs. Validation via PCR and Sanger sequencing. Results. 27, 56, 28 and 11 RNA circles were found to be uniquely up-regulated, while 149, 7, 20 and 15 uniquely down-regulated in the SHH, WNT, Group 3, and Group 4 medulloblastoma subtypes, respectively. Moreover, linear and circular fusion RNAs containing exons from distinct genes joined at canonical splice sites were also identified. These were generally expressed less than the circular RNAs, however the expression of both the linear and the circular fusions was comparable. Importantly, the expression of the fusions in medulloblastoma was also comparable to that of cerebellum. Conclusions. A significant number of fusions in tumor may be generated by mechanisms similar to the ones generating fusions in normal tissue. Some fusions could be rationalized by read-through transcription of two neighboring genes. However, for other fusions, e.g., a linear fusion with an exon from a downstream gene joined 5′ to 3′ with an exon from an upstream gene, more complicated splicing mechanisms, e.g., trans-splicing, have to be postulated.