Abstracts of Presentations at the Association of Clinical Scientists 143rd Meeting Louisville, KY May 11-14,2022.

[1] Abraham J. Gitlitz Memorial Lecture: Development of a BroadSpectrum Antiviral-based Intranasal Spray as a Pandemic Preparedness Strategy. Kenneth E. Palmer, Ph.D, Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases and Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY In early 2020, the public health emergency prompted many researchers to contemplate how they might contribute to control of the COVID-19 pandemic. This presentation will detail the research steps taken to bring a novel broad spectrum antiviral protein into a first in humans clinical trial. We had been developing a broad spectrum antiviral protein, the lectin Q-Griffithsin (Q-GRFT), as a topical microbicide for HIV-1 prevention. We knew that Q-GRFT also inhibited replication of many members of the Coronavirus family of pathogens, so initiated a rapid preclinical and clinical development program of a Q-GRFT intranasal spray as a prophylaxis modality against SARS-CoV-2, the virus that causes COVID-19. We finalized a suitable formulation to deliver Q-GRFT to the nasopharynx, the initial site of replication of SARS-CoV-2. Non-clinical toxicology studies supported first-in-human clinical studies. Efficacy studies in mice and hamsters provided proof of concept that Q-GRFT can protect animals against challenge. We filed an investigational new drug (IND) application, and received study may proceed authorization from the FDA. We conducted a randomized, placebo-controlled single dose safety and pharmacokinetics clinical study in 18 volunteers, all of whom had been vaccinated against SARS-CoV-2. The product was safe, and enhanced the levels of SARS-CoV-2 as well as MERS-CoV inhibitory activity present in the nasal and naso-pharyngeal swabs. The safety and pharmacokinetics profile of our Q-GRFT intranasal spray supports a multi-dose Phase 1b clinical study, and further development as a general pandemic preparedness strategy. Upon completion of this activity, participants should be able to: describe the preclinical and clinical strategies employed to support a first in humans clinical trial of a novel broad-spectrum antiviral protein containing nasal spray; discuss planning, design and execution of a first-in-humans Phase 1a clinical trial for evaluation of safety, and selection of a dosing strategy for a Phase 1b multiple dose safety and pharmacokinetics study. [2] Acetylation pharmacogenomics: paradigm for informed individual risk assessment following environmental carcinogen exposure. David W. Hein, Ph.D, University of Louisville, Louisville, KY Human epidemiological studies associating chemical exposures to cancer risk often are inconsistently validated across studies. Examples include the effect of smoking on cancer etiology other than the lung, such as urinary bladder and breast. Research findings from the laboratory have improved the understanding of arylamine carcinogen metabolism leading to improved design and interpretation of human molecular epidemiology investigations. Laboratory studies that infer and test biological plausibility, including cancer risks modified by differential metabolism of arylamine carcinogens in rapid and slow arylamine N-acetyltransferase (NAT2) acetylators, have been critical for investigating the role of smoking in the etiology of human cancers. This chapter will illustrate these concepts with an example of a cancer in which the role of smoking has largely been validated (urinary bladder cancer) and examples where a consensus has yet to be achieved. Portions of this work were funded by the following NIH grants: R01-CA034627; T32-ES011564; P42-ES023716; P20-GM113226; R25-CA134283; and P30-ES030283. Upon completion of this activity, participants should be able to: 1. Assess the role of genetic polymorphisms in individual risk assessments following exposures to environmental carcinogens. 2. Recognize the importance of laboratory-based data in the biological plausibility of individual risk assessments. 3. Recognize the genetic complexity inherent in human epidemiological studies. [3] Rolling with the punches: A biosafety program at a research university addresses COVID-19. Allen Helm, PhD, The University of Chicago, Chicago, IL This presentation describes the biosafety program at a research university and medical center, demonstrating how the program was modified during the COVID-19 pandemic. Biosafety professionals are tasked with facilitating biomedical research by enacting coordinated efforts to protect research staff and students, the larger research community, and the environment and ecosystem from biological hazards used in the laboratory. These hazards include recombinant organisms ranging from microorganisms to animals, microbial pathogens, human-derived material, and biological toxins. The University of Chicago (UChicago) is an academic research institution affiliated with an urban medical center. There are approximately 300 biomedical principal investigator-led laboratories conducting investigations in basic, translational, and clinical science. The biosafety program at UChicago is part of the Office of Research Safety and consists of a director and four biosafety officers who perform a variety of duties, including: 1) Assisting researchers in planning experiments that utilize biohazards; 2) Developing and delivering biosafety training courses; 3) Performing annual inspections of laboratories; 4) Working with the university's Institutional Biosafety Committee; 5) Ensuring compliance with local, state, and federal agencies. The COVID-19 pandemic affected the biosafety program in several ways, as follows: 1) Altering administrative operations; 2) Developing biosafety standards for investigators working with SARS-CoV-2; 3) Establishing a "COVID Core" to handle influx of said researchers; 4) Enhanced biosafety interactions with clinical laboratories due to a demand for vaccines and treatments. Upon completion of this activity, participants should be able to: 1. Identify the role of biosafety professionals in basic, translational, and clinical research. 2. Recognize how a pandemic can alter the way biosafety services are delivered to research programs. 3. Recognize the need for flexibility in a successful biosafety program. [4] An interdisciplinary Post Operative Personalized Pain Management Clinical Trial. Loralie J. Langman1, Jeremy Gaskins2, Gwendolyn A. McMillin3, Paul J. Jannetto1, Brandi Hartley4, Arthur Malkani4, and Saeed A. Jortani5, department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, 2Departments of Bioinformatics and Biostatistics, University of Louisville School of Medicine, Louisville, KY, 3Department of Pathology, University of Utah, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 4Departments of Orthopedic Surgery, University of Louisville School of Medicine, Louisville, KY, and 5Departments of Pathology, and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY Various sources of variability in response and toxicity to hydrocodone were investigated. In a cohort of orthopedic surgery patients, we interrogated the associations between genetic, intrinsic and extrinsic patient factors, plasma concentrations of hydrocodone and metabolites, common side effects, and pain score. Data for each patient was collected by the review of the electronic medical record and a patient interview at the time of sample collection. Patients with trauma or undergoing scheduled elective surgery for total knee or total hip replacement at the University of Louisville, Baptist East, and Jewish Hospitals, Louisville, KY. Plasma opiate concentrations and a targeted genotyping panel were performed. We observed statistically significant correlations for daily (p<0.001) and total dose (p=0.002) of hydrocodone. Duration of in-hospital and duration of opioid therapy for patients were also significantly different based on their genotypes. The length of opioid administration was significantly shorter in CYP2D6 EM/UM compared to patients with CYP2D6 PM/IM genotypes (p=0.018). Subjects with the OPRM1 c.118G polymorphism were also on opioids for a longer period of time (p=0.022). Co-administration of medications with CYP2D6 inhibitor activity had a significant effect on the length of opioid therapy (P<0.001). Both the hospital stay period and days of opioid use post hospital discharge were greater in patients with the inhibitor-adjusted CYP2D6 phenotype (p<0.001). This trial showed that patients should be evaluated for the use of inhibitors of CYP2D6. Interaction with these therapeutics while administering hydrocodone therapy can alter the phenotype of the patient (phenocopy) and result in longer opioid therapy duration. Upon completion of this activity, participants should be able to: 1. to discuss various sources of variability to response and toxicity of analgesics. 2. to present the post-operative pain management trial in a cohort of women just undergone Cesarean Section. 3. to present the post-operative pain management trial in a cohort of lower extremity orthopedic patients. [6] Claude P. Brown Memorial Lecture: The Many Roads to Steatohepatitis and its Treatment. Craig J. McClain, MD, University of Louisville School of Medicine,Louisville, KY Hepatic steatosis and steatohepatitis are common histologic findings that can be caused by multiple etiologies. The three most frequent causes for steatosis/ steatohepatitis are alcohol (alcohol-associated steatohepatitis, ASH), obesity/metabolic syndrome (nonalcoholic steatohepatitis, NASH), and environmental toxicants (toxicant-associated steatohepatitis, TASH). Hepatic steatosis is an early occurrence in all three forms of liver disease, and they often share common pathways to disease progression/severity. Disease progression is a result of both direct effects on the liver as well as indirect alterations in other organs/tissues such as intestine, adipose tissue, and the immune system. Although the three liver diseases (ASH, NASH, and TASH) sharemany common pathogenic mechanisms, they also exhibit distinct differences. Both shared and divergent mechanisms can be potential therapeutic targets. I will provide an overview of selected important mechanistic similarities and differences in ASH, NASH, and TASH, and discuss the importance of a multidisciplinary and personalized approach. Upon completion of this activity, participants should be able to: 1. understand the mechanisms including similarities and differences between ASH, NASH and TASH. 2. understand targets to treat ASH, NASH and TASH. 3. recognize tests to help distinguish between ASH, NASH and TASH. [7] Bioactive lipid metabolites: biomarkers and therapeutic targets in alcohol-associated liver disease. Dennis Warner, Josiah Hardesty, Jeff Warner, Craig McClain, Irina Kirpich, University of Louisville, Louisville, KY Alcohol-associated liver disease (ALD) is a spectrum of liver disorders ranging from steatosis to steatohepatitis, fibrosis and cirrhosis. The mechanisms and mediators of ALD progression are not well understood and effective therapeutic options are limited. Various bioactive oxidized lipid mediators (oxylipins) have recently emerged as important factors in ALD pathogenesis. The current study aimed to examine plasma linoleic acid (LA)-derived lipid metabolites in heavy drinking individuals and to evaluate associations between these molecules and markers of liver injury. Analysis of plasma LA-derived metabolites was performed by HPLC/MS on 66 heavy drinking individuals and 29 socially drinking but otherwise healthy volunteers. Based on plasma ALT levels, 15 patients had no liver injury (ALT ≤ 40 U/L), 33 patients had mild liver injury (ALT > 40 U/L), and 18 were diagnosed with moderate Alcohol-associated Hepatitis (mAH). Statistically significant differences (set at p<0.05) were determined by One-way ANOVA. Lipoxygenase-derived LA metabolites, 13-HODE and 13-oxoODE, were markedly elevated only in mAH patients. The CYP450-derived LA epoxides, 9,10-EpOME and 12.13-EpOME were decreased in all patients regardless of the presence or the absence of liver injury. LA-derived diols, 9,10-DiHOME and 12.13-DiHOME, were elevated only in the mAH group. The current study provides evidence that specific changes in LA-metabolites in heavy drinking individuals can distinguish individuals with mAH from those with mild ALD. Upon completion of this activity, participants should be able to: 1. identify effects of alcohol consumption on circulating oxylipins; 2. determine the role of oxylipins in ALD pathogenesis; 3. associate severity of ALD with the specific changes in oxylipins [8] Hepatic protein and phosphoprotein signatures of alcohol-associated hepatitis. Josiah Hardesty, Jeffrey Warner, Dennis Warner, Craig McClain, and Irina Kirpich, University of Louisville, Louisville, KY The objective of the current study was to identify hepatic proteome and phosphoproteome signatures of alcohol-associated hepatitis (AH). AH is a clinical manifestation of ALD characterized by compromised liver function contributing to a 6-month mortality rate as high as 50%. Proteomic and phosphoproteomic analyses were conducted on explant liver tissue from AH patients (n=6) and non-AH controls (n=12). Data were statistically compared by an unpaired Student's t-test and a p < 0.05 was considered statistically significant. Alterations the expression of multiple proteins involved in various biological processes were observed in AH. Among significant findings in AH included elevated expression of pro-fibrotic transcription factors, reduced albumin (ALBU) phosphorylation, and diminished expression of functional mitochondria proteins. One transcription factor involved in fibrogenesis, yes1-associated transcriptional regulator (YAP1) was elevated in AH (p=0.003), along with increased phosphorylation at pS105 (p=0.01). In addition, expression of hepatic ALBU was elevated in AH (p=0.04) concomitant with diminished ALBU phosphorylation (p=0.02), which may prevent ALBU release leading to hypoalbuminemia. Lastly, we found a loss in the expression of mitochondria proteins in AH, including enzymes essential for mitochondria function and biogenesis (e.g., hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, [ECHA] p=0.04). This study identified hepatic protein and phosphoprotein signatures of AH which may facilitate the development of novel therapeutic strategies. Upon completion of this activity, participants should be able to: 1. understand the hepatic proteomic changes that occur in AH, 2. recognize hepatic protein and phosphoprotein signatures of AH and 3. identify novel mechanisms and pathways implicated in AH. [9] Soluble epoxide hydrolase inhibition in alcohol-associated liver disease: liver-specific drug delivery. Jeffrey Warner, Josiah Hardesty, Ying Song, Philip Bauer, Chirag Soni, Claudio Maldonado, Craig McClain, Dennis Warner, Irina Kirpich, University of Louisville, Louisville, KY Alcohol-associated liver disease (ALD) is a prevalent condition resulting from excessive alcohol consumption. Advanced stages of ALD, such as alcohol-associated hepatitis (AH), cause significant mortality and lack effective therapies. Previous data established that soluble epoxide hydrolase (sEH, an enzyme which degrades beneficial lipid epoxides) is induced in clinical/ experimental ALD, and that sEH inhibition may be an effective treatment for this disease. This study aimed to improve this approach by using liverspecific drug delivery via fusogenic lipid vesicles (FLVs) to increase efficacy and avoid extra-hepatic side effects. We prepared fluorescent-labeled FLVs loaded with the sEH inhibitor t-TUCB (t-TUCB-FLVs) at various doses. t-TUCB-FLV preparations had an appropriate size and charge and were confirmed to target the liver by fluorescent microscopy. Flow cytometry demonstrated that hepatocytes and macrophages were most responsible for t-TUCB-FLV uptake. In a dose response experiment using a chronic-binge ethanol feeding model mimicking AH, mice receiving ethanol+3.0 mg/kg t-TUCB-FLVs had the greatest reduction in liver injury by plasma ALT. This treatment was more efficacious than systemically delivered (non-FLV-encapsulated) t-TUCB at the same dose. t-TUCB-FLVs also decreased liver cell death and ER stress but had no effect on steatosis or neutrophil infiltration. These data demonstrated that liver-specific delivery of t-TUCB was more efficacious than systemic delivery. This drug delivery platform may help increase the efficacy of sEH inhibition in ALD while reducing extra-hepatic side effects, improving translation to humans. Upon completion of this activity, participants should be able to: 1.recognize the pathogenic role of sEH in ALD; 2. Describe basic concepts in liver-specific drug delivery by nanoparticle systems; and 3. Evaluate the beneficial effects of a liver-targeted sEH inhibitor in experimental ALD in mice. [10] COVID-19 and early post-primary TB: commonalities of pathobiology in pneumonitis and therapies. Robert L. Hunter and Robert E.Brown, UTHealth McGovern Medical School Houston, TX Concurrent infection with COVID-19 and M. tuberculosis has been reported to be more severe than either alone, resulting in increased mortality. Our objective was to define the shared pathobiology of COVID-19 and the developmental stage of TB in the lung and explore adjunctive therapies to treat such commonalities. We used similar morphoproteomic analyses to study lung tissues of patients with early postprimary tuberculosis or COVID-19 infection.These studies showed colocalization of the COVID-19 virus and M. tuberculosis antigens with cyclo-oxygenase-2 and fatty acid synthase in the reactive alveolar pneumocytes and with programmed death-ligand 1 expression on the alveolar interstitium and alveolar pneumocytes.This was associated with accumulation of pro-infectious M2 polarized macrophages in the alveolar spaces.The commonalities in these pathways suggest that they might be susceptible to adjunctive therapies with metformin and vitamin D3. This is supported by published studies that metformin and vitamin D3 could reduce the severity of both COVID-19 and early post-primary TB infections. Upon completion of this activity, participants should be able to identify and define the commonalities in the pathobiology of COVID-19 and early post-primary TB pneumonitis and describe the potential targets for therapy with metformin and vitamin D3. [11] Plasma ctDNA for Monitoring Response to Immune Check Point Inhibitors. Mark W. Linder PhD, DABCC, FAACC. Professor Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY Circulating tumor DNA (ctDNA) measurements from a variety of malignancies are aggressively being investigated in the context of a "liquid biopsy" to provide for a minimally invasive means of monitoring tumor status and therapeutic response. This session will discuss what is known about the biologic and physiological characteristics of plasma ctDNA. We will then describe the what is known regarding the relationship between plasma ctDNA and tumor characteristics such as tumor burden, proliferative activity and therapeutic response . Understanding of these relationships is central to developing a fundamental framework for interpretation longitudinal plasma ctDNA measurements in light of other routine measurements such as radiographic assessments and other blood based biomarkers. Upon completion of this activity, participants should be able to: 1. Explain the sources and methods of measurement of ctDNA. 2. Describe the relationships between plasma ctDNA, disease burden and therapeutic response. 3. Discuss how these relationships influence the clinical utility of routine plasma ctDNA testing. [12] Quantitative Methods of Monitoring Circulating Tumor DNA. Evan M. Alexander, PhD, Roland Valdes Jr., PhD, DABCC, Mark Linder, PhD, DABCC, Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY Circulating tumor DNA (ctDNA) is emerging as a new, robust biomarker that can be used for early cancer diagnosis, disease prognosis and even guiding treatment through precision medicine. This non-invasive tumor monitoring tool can be performed on the scale of a single gene mutation to an entire sequenced genome. Quantitation of ctDNA is complicated by two factors: wide ranges and low concentrations (0.003%-95% mutant allele frequency (MAF); 5-1500 ng/mL). Currently, the field of monitoring ctDNA is dominated by next-generation sequencing (NGS) and polymerase chain reaction (PCR) based technologies. Understandably, these different monitoring techniques require specialized analytical instrumentation. Furthermore, the sensitivity and specificity of these methods are not universal. There are many considerations a clinician needs to be mindful of when utilizing these technologies to monitor ctDNA (known vs unknown mutation, absolute quantitation vs MAF). Understanding the strengths and weaknesses of ct DNA quantification methods in a given clinical situation is paramount in utilizing this biomarker to its fullest and most appropriate potential. Upon completion of this activity, participants should be able to: 1. Identify which quantitation technique for ctDNA is most appropriate to use in a given clinical scenario 2. Recognize the difference between quantitation of mutant allele frequency and absolute quantitation of ctDNA 3. Recognize the importance of assay sensitivity when serially monitoring patient cancer progression. [13] The Value of Next Generation Sequencing in Myeloid Neoplasia. Mustafa Al-Kawaaz, University of Louisville, Louisville, KY This presentation will demonstrate some of the most important utilities of next generation sequencing (NGS) in a spectrum of acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and overlap syndromes. Interrogation of nucleic acid (DNA and/or RNA) to look for certain genomic alterations is essential for diagnostic, prognostic, and therapeutic purposes. Classification of myeloid neoplasms continues to evolve by including entities defined by specific genomic alterations. NGS is an essential utility to guide management decisions. Upon completion of this activity, participants should be able to: 1. Recognize changes to classification of myeloid neoplasms and some categories definite by genetic alterations. 2. Identify the utility of different vendors/ platforms offering certain advantages in next generation sequencing. 3. Recommend testing utility by evaluating the targeted genetic alteration as well as clinical scenario. [14] Forecasting clinical behavior and therapeutic response of breast carcinoma using gene expression. James L. Wittliff and Michael W. Daniels, University of Louisville, Louisville, KY Our goal is to associate expression of nuclear and peptide hormone receptor genes with biomarker status of breast carcinoma and risk of recurrence, to advance clinical management. Cellular heterogeneity of tissue specimens is a complicating factor in determining analyte (protein or gene) levels of specific cell types. A unique deidentified database was analyzed that contained microarray results of 22,000 genes derived only from total RNA extracted from breast carcinoma cells procured by laser capture microdissection (Pixcell IIe:Arcturus®/ Thermo Fisher) of 247 de-identified primary tissue biopsies. Relative expression levels of each gene candidate for 49 nuclear receptors as well as 61 peptide/protein hormones and 81 of their cognate receptor proteins were selected for this retrospective investigation. Assessment of a patient's risk of recurrence primarily utilizes estrogen (ER) and progestin receptor proteins (PR), quantified by radio-ligand binding (NEN/DuPont) and/or enzyme immunoassay (Abbott Labs). Parameters and clinical outcomes were analyzed by univariable and multivariable Cox regressions, Fisher's Exact Test, Kaplan Meier plots and with R software v4.0.0. Examples of multivariable Cox regression models of candidates of nuclear receptor genes, fit to predict disease-free survival (DFS) and overall survival (OS), revealed that only NR4A2, PGR, PPARA and THRB were required to predict DFS and NR3C2, PGR and THRB were necessary to predict OS. Of 142 candidate genes for peptide hormones and their cognate receptors, 30 exhibited expression levels that individually predicted DFS and/or OS. When pairs of genes for a peptide hormone and its receptor were evaluated by multivariable Cox Regression with interaction, complexes were identified that predicted DFS and OS (EDN1-ENDRA, GHRL-GHSR, INHBB-ACVR2B, NPY-NPY1R, INHBB-ACVR1B, RLN2-RXFP3 and NPY-NPY6R) based on unadjusted p-value for the interaction term. These investigations also revealed numerous over-expressed genes in carcinomas with poor clinical outcomes suggesting candidates for development of novel therapeutics. Collectively, use of small molecular signatures (gene subsets) with quantified ER/PR protein or ESR1/ PGR expression in a breast carcinoma with clinical outcomes enhanced prediction of risk of recurrence and identification drug development candidates. Upon completion of this activity, participants should be able to: 1. recognize the power of LCM to non-disruptively capture populations of specific cell types for genomics testing, 2. differentiate gene expression patterns based upon protein tumor marker status groups and 3. predict risk of recurrence for patients based upon expression of gene molecular sign. [15] Imaging Mass Spectrometry: Applications in Biomedical Research and Clinical Diagnosis. Yusheng Zhu, PhD; Pennsylvania State University College of Medicine Hershey Medical Center, Hershey, PA Mass spectrometry tissue imaging is a technology used in mass spectrometry to visualize the spatial distribution of molecules in tissues by their molecular masses. Compared to traditional tissue imaging methods such as immunohistochemistry, mass spectrometry imaging does not need any antibodies and tracers; it can detect and map multiple analytes including proteins, peptides, nucleic acids, lipids, carbohydrates, metabolites, drugs, toxins, and even elements simultaneously; researchers do not need prior knowledge of molecules in the samples; the analysis can be qualitative and/or quantitative. Therefore, mass spectrometry imaging has become a powerful technology for biomedical research and biomarker discovery. It is widely used in proteomic, peptidomic, lipidomic, glycomic, metabolomic, pharmacological and toxicological studies. This session will introduce common mass spectrometry imaging techniques including matrix assistant laser desorption ionization-time of flight (MALDI-TOF) imaging, time of flight-secondary ion mass spectrometry (TOF-SIMS) imaging, and desorption electrospray ionization (DESI)-mass spectrometry imaging. In addition, the basic principle, procedure, and application of these methods in biomedical research and clinical diagnosis will be discussed. Upon completion of this activity, participants should be able to: 1. Define tissue imaging mass spectrometry. 2. Explain basic principle of common types of imaging mass spectrometry. 3. Describe application of tissue imaging mass spectrometry in biomedical research and clinical diagnosis. [16] Immune Landscape in Sentinel Lymph Nodes from Melanoma Patients by Single-Cell Mass Cytometry (CyTOF) Analysis. Kavitha Yaddanapudi, University of Louisville, Louisville, KY We describe the development of a multiscale immune profiling strategy to map the immune landscape of sentinel lymph nodes (SLN) in our search for tumor-driven immune changes that can guide the design of novel immunotherapeutic strategies for patients with early-stage melanoma. We used mass cytometry by time-of-flight (CyTOF), flow cytometry, and T cell receptor immunosequencing to conduct simultaneous single-cell analyses of immune cells in the SLNs of melanoma patients. We identified unique tumordriven T, NK, and innate immune cell signatures that are present in stage III melanoma-bearing SLNs, but absent in stage I/II non-melanoma-bearing SLNs. We found increased effector-memory T cells and TCR clonality selectively in the melanoma-bearing SLNs relative to non-melanoma-bearing SLNs, consistent with possible activation of an anti-tumor immune response. However, we also observed a markedly immunotolerant environment in the melanoma-bearing SLNs indicated by reduced and impaired NK cells and increased levels of CD8+CD57+PD-1+ cells which are known to display low melanoma killing capabilities. Other changes observed in melanoma-bearing SLNs when compared to non-melanoma bearing SLNs include reduced CD8+CD69+ T cells/T regulatory cells ratio and high PD-1 expression on CD4+ and CD8+ T cells. Our data suggests that these immunological changes compromise anti-melanoma immunity and contribute to a high relapse rate. Upon completion of this activity, participants should be able to describe mass cytometry analysis of melanoma patient sentinel lymph node samples, identify new immunologic and therapeutic targets for preventing melanoma recurrence and identify unique melanoma-driven immune cell signatures. [17] Application of MassARRAY System in Molecular Diagnostics - Pharmacogenomics and Oncology. Shuko Harada and Alexander C. Mackinnon, University of Alabama at Birmingham, Birmingham, AL In molecular diagnostic laboratories, multiplex genetic analysis, for example Next Generation Sequencing (NGS), is increasingly utilized. NGS is expensive, laborious, and requires complicated bioinformatics analysis. The MassARRAY system provides accurate, low cost, facile, multiplexed analysis of hundreds of clinically relevant mutations with relatively simple analytics. In this study, we evaluated