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Understanding the complex tumor microenvironment is key to the development of personalized therapies for the treatment of cancer including colorectal cancer (CRC). In the past decade, significant advances in the field of immunotherapy have changed the paradigm of cancer treatment. Despite significant improvements, tumor heterogeneity and lack of appropriate classification tools for CRC have prevented accurate risk stratification and identification of a wider patient population that may potentially benefit from targeted therapies. To identify novel signatures for accurate prognostication of CRC, we quantified gene expression of 12 immune-related genes using a medium-throughput NanoString quantification platform in 93 CRC patients. Multivariate prognostic analysis identified a combined four-gene prognostic signature (TGFB1, PTK2, RORC, and SOCS1) (HR: 1.76, 95% CI: 1.05-2.95, *p 0.02). The survival trend was captured in an independent gene expression data set: GSE17536 (177 patients; HR: 3.31, 95% CI: 1.99-5.55, *p 0.01) and GSE14333 (226 patients; HR: 2.47, 95% CI: 1.35-4.53, *p 0.01). Further, gene set enrichment analysis of the TCGA data set associated higher prognostic scores with epithelial-mesenchymal transition (EMT) and inflammatory pathways. Comparatively, a lower prognostic score was correlated with oxidative phosphorylation and MYC and E2F targets. Analysis of immune parameters identified infiltration of T-reg cells, CD8

Complex interactions in tumor microenvironment between ECM (extra-cellular matrix) and cancer cell plays a central role in the generation of tumor supportive microenvironment. In this study, the expression of ECM-related genes was explored for prognostic and immunological implication in clear cell renal clear cell carcinoma (ccRCC). Out of 964 ECM genes, higher expression (z-score > 2) of 35 genes showed significant association with overall survival (OS), progression-free survival (PFS) and disease-specific survival (DSS). On comparison to normal tissue, 12 genes (NUDT1, SIGLEC1, LRP1, LOXL2, SERPINE1, PLOD3, ZP3, RARRES2, TGM2, COL3A1, ANXA4, and POSTN) showed elevated expression in kidney tumor (n = 523) compared to normal (n = 100). Further, Cox proportional hazard model was utilized to develop 12 genes ECM signature that showed significant association with overall survival in TCGA dataset (HR = 2.45; 95% CI [1.78–3.38]; p

The long evolutionary battle between humans and pathogens has played an important role in shaping the current network of host-pathogen interactions. Each organ brings new challenges from the perspective of a pathogen to establish a suitable niche for survival while subverting the protective mechanisms of the host. Lungs, the organ for oxygen exchange, have been an easy target for pathogens due to its accessibility. The organ has evolved diverse capabilities to provide the flexibility required for an organism’s health and at the same time maintain protective functionality to prevent and resolve assault by pathogens. The pathogenic invasions are strongly challenged by healthy lung architecture which includes the presence and activity of the epithelium, mucous, antimicrobial proteins, surfactants, and immune cells. Competitively, the pathogens in the form of viruses, bacteria, and fungi have evolved an arsenal of strategies that can over-ride the host’s protective mechanisms. While bacteria such as Mycobacterium tuberculosis (M. tuberculosis) can survive in dormant form for years before getting active in humans, novel pathogens can wreak havoc as they pose a high risk of morbidity and mortality in a very short duration of time. Recently, a coronavirus strain SARS-CoV-2 has caused a pandemic which provides us an opportunity to look at the host manipulative strategies used by respiratory pathogens. Their ability to hide, modify, evade, and exploit cell’s processes are key to their survival. While pathogens like M. tuberculosis have been infecting humans for thousands of years, SARS-CoV-2 has been the cause of the recent pandemic. Molecular understanding of the strategies used by these pathogens could greatly serve in design of predictive, preventive, personalized medicine (PPPM). In this article, we have emphasized on the clinically relevant evasive strategies of the pathogens in the lungs with emphasis on M. tuberculosis and SARS-CoV-2. The molecular basis of these evasive strategies illuminated through advances in genomics, cell, and structural biology can assist in the mapping of vulnerable molecular networks which can be exploited translationally. These evolutionary approaches can further assist in generating screening and therapeutic options for susceptible populations and could be a promising approach for the prediction, prevention of disease, and the development of personalized medicines. Further, tailoring the clinical data of COVID-19 patients with their physiological responses in light of known host-respiratory pathogen interactions can provide opportunities to improve patient profiling and stratification according to identified therapeutic targets.

Nigella sativa is an annual plant from Ranunculaceae family and was traditionally used as medicine by ancient civilizations for its wide therapeutic values. N. sativa is not only the source of healthy nutrition but also has the potential to be an alternative treatment option for different pathologies, from microbial infection to metabolic disease. Nigella seed is composed of a number of biologically active components that are being used in traditional medicines from antimicrobial remedy to cardiovascular diseases This chapter aims to provide readers understanding of complex composition, biological activities, adverse effects, and therapeutic propertiesof N. sativa. Further, seeds from Nigella are rich in nutrients such as proteins, fats, carbohydrates, vitamins, oils, minerals, fibers, etc., and contain a wide variety of saponins and alkaloids. Alkaloids, specially thymoquinone and its derivatives in Nigella, are rich in medicinal activities and are of therapeutic importance in many diseases. Recent research has identified several important active components of N. sativa such as thymoquinone, α-hederin, thymol, and others. However, thymoquinone is the principle active ingredient of Nigella and shows disease-altering biological properties, to name few, antioxidant, antimicrobial, antiinflammatory, antiproliferative, anticancer, and antimetastatic effects, suggesting potential therapeutic roles in many human pathologies. In addition, Nigella has been shown to be effective in immunomodulatory, antidiabetic, hepatoprotective, and neuroprotective activities and in neurological disorders. Derived from its antioxidant and also antiinflammatory action, Nigella has also been associated with the treatment of cardiovascular diseases. In conclusion, Nigella is a good candidate to be developed as a natural therapeutic agent for many pathologies and extensive studies are therefore, recommended. In addition, a better understanding of the mechanisms of Nigella’s therapeutic medicinal effects is needed, as well as knowledge of the best dose to be used, resulting in high efficacy, low toxicity, and better therapeutic effect against different diseases.

Natural plants and compounds are nature’s precious gift to humankind that has solution of every health problem. Nigella sativa, also named as black cumin or black seed, is well known for its many health advantages in traditional medicinal system. N. sativa seed is rich in alkaloids and possesses healing ability in hormonal and endocrine dysfunction. Thymoquinone is the main active constituent of N. sativa and possesses antioxidant and antidiabetic properties and boost endocrine function. Compared to other biological properties, proendocrine properties of Nigella were not extensively studied. However, the scientific communities have started taking interests in Nigella’s healing ability to maintain hormonal homeostasis and other disease conditions. This chapter is summarizing the previous findings of Nigella in endocrine dysfunction and emphasizing on better understanding of mechanism behind proendocrine capabilities of Nigella plant or its active components to regulate body’s normal physiology.

Simple Summary Cancer is one of the leading causes of mortality around the globe. In the past decades, there has been rapid progress in the development of tools to detect, screen, and treat several cancers. For its benefit to reach a wider patient population, significant challenges such as tumor heterogeneity, resistance to therapies, and lack of biomarkers should be addressed. The immune system holds the key to a greater understanding of these complex barriers. Natural Killer cells are cytotoxic cells of innate immunity that can kill multiple tumorigenic cells. Dendritic cells link innate and adaptive immunity by processing and presenting tumor-derived antigens to initiate anti-tumor T cell response. These immune cells and associated gene signatures have emerged as potential biomarkers with prognostic and predictive potential in several cancers. In this review article, we have discussed the biological roles of NK cells and DCs along with their translational relevance in NSCLC. Abstract Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer that accounts for almost 85% of lung cancer cases worldwide. Although recent advances in chemotherapy, radiotherapy, and immunotherapy have helped in the clinical management of these patients, the survival rate in advanced stages remains dismal. Furthermore, there is a critical lack of accurate prognostic and stratification markers for emerging immunotherapies. To harness immune response modalities for therapeutic benefits, a detailed understanding of the immune cells in the complex tumor microenvironment (TME) is required. Among the diverse immune cells, natural killer (NK cells) and dendritic cells (DCs) have generated tremendous interest in the scientific community. NK cells play a critical role in tumor immunosurveillance by directly killing malignant cells. DCs link innate and adaptive immune systems by cross-presenting the antigens to T cells. The presence of an immunosuppressive milieu in tumors can lead to inactivation and poor functioning of NK cells and DCs, which results in an adverse outcome for many cancer patients, including those with NSCLC. Recently, clinical intervention using modified NK cells and DCs have shown encouraging response in advanced NSCLC patients. Herein, we will discuss prognostic and predictive aspects of NK cells and DC cells with an emphasis on NSCLC. Additionally, the discussion will extend to potential strategies that seek to enhance the anti-tumor functionality of NK cells and DCs.

SARS-CoV-2 is the cause of a recent pandemic that has led to more than 3 million deaths worldwide. Most individuals are asymptomatic or display mild symptoms, which raises an inherent question as to how does the immune response differs from patients manifesting severe disease? During the initial phase of infection, dysregulated effector immune cells such as neutrophils, macrophages, monocytes, megakaryocytes, basophils, eosinophils, erythroid progenitor cells, and Th17 cells can alter the trajectory of an infected patient to severe disease. On the other hand, properly functioning CD4+, CD8+ cells, NK cells, and DCs reduce the disease severity. Detailed understanding of the immune response of convalescent individuals transitioning from the effector phase to the immunogenic memory phase can provide vital clues to understanding essential variables to assess vaccine-induced protection. Although neutralizing antibodies can wane over time, long-lasting B and T memory cells can persist in recovered individuals. The natural immunological memory captures the diverse repertoire of SARS-CoV-2 epitopes after natural infection whereas, currently approved vaccines are based on a single epitope, spike protein. It is essential to understand the nature of the immune response to natural infection to better identify ‘correlates of protection’ against this disease. This article discusses recent findings regarding immune response against natural infection to SARS-CoV-2 and the nature of immunogenic memory. More precise knowledge of the acute phase of immune response and its transition to immunological memory will contribute to the future design of vaccines and the identification of variables essential to maintain immune protection across diverse populations.

Pesticides are globally used to eliminate pests from crops and plants. The increased use of pesticides has posed a serious threat to human health. This study evaluates the effects of pesticide exposure on pregnancy outcomes in tea garden workers (TGW). The acetylcholinesterase (AChE) activity was measured in the maternal blood, placenta, and cord blood of TGW and housewives (HWs). The placental structure and expression of hypoxia-inducible factor (HIF)-1α were also analyzed in TGW and HW groups delivering low birth weight (LBW) and normal birth weight (NBW) babies. A significantly decreased AChE activity was observed in maternal blood and cord blood in TGW as compared with HW in the LBW group. However, it did not change significantly in the NBW group (p < .05). The adjusted regression analysis of birth outcomes (birth weight, head circumference, infant's length, and ponderal index) revealed a significant and positive association with the levels of AChE activity in maternal blood, placenta, and cord blood in TGW (p < .05). The histological analysis showed significantly higher placental syncytial knots, chorangiosis, fibrinoid deposition, necrosis, and stromal fibrosis in the LBW group of TGW. Microinfarction, increased fibrinoid deposition, and atypical villi characteristics, such as mushroom-like structures, were observed during scanning electron microscopy along with increased HIF-1α expression in placental tissues of TGW exposed to pesticides. Results suggest that occupational pesticide exposure during pregnancy may decrease AChE activity and cause in utero pathological changes accompanied by an increased HIF-1α expression, which also contributes to placental insufficiency and fetal growth restriction.

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

Lung cancer is one of the leading causes of death worldwide. Cell death pathways such as autophagy, apoptosis, and necrosis can provide useful clinical and immunological insights that can assist in the design of personalized therapeutics. In this study, variations in the expression of genes involved in cell death pathways and resulting infiltration of immune cells were explored in lung adenocarcinoma (The Cancer Genome Atlas: TCGA, lung adenocarcinoma (LUAD), 510 patients). Firstly, genes involved in autophagy (n = 34 genes), apoptosis (n = 66 genes), and necrosis (n = 32 genes) were analyzed to assess the prognostic significance in lung cancer. The significant genes were used to develop the cell death index (CDI) of 21 genes which clustered patients based on high risk (high CDI) and low risk (low CDI). The survival analysis using the Kaplan&ndash, Meier curve differentiated patients based on overall survival (40.4 months vs. 76.2 months), progression-free survival (26.2 months vs. 48.6 months), and disease-free survival (62.2 months vs. 158.2 months) (Log-rank test, p &lt, 0.01). Cox proportional hazard model significantly associated patients in high CDI group with a higher risk of mortality (Hazard Ratio: H.R 1.75, 95% CI: 1.28&ndash, 2.45, p &lt, 0.001). Differential gene expression analysis using principal component analysis (PCA) identified genes with the highest fold change forming distinct clusters. To analyze the immune parameters in two risk groups, cytokines expression (n = 265 genes) analysis revealed the highest association of IL-15RA and IL 15 (&gt, 1.5-fold, p &lt, 0.01) with the high-risk group. The microenvironment cell-population (MCP)-counter algorithm identified the higher infiltration of CD8+ T cells, macrophages, and lower infiltration of neutrophils with the high-risk group. Interestingly, this group also showed a higher expression of immune checkpoint molecules CD-274 (PD-L1), CTLA-4, and T cell exhaustion genes (HAVCR2, TIGIT, LAG3, PDCD1, CXCL13, and LYN) (p &lt, 0.01). Furthermore, functional enrichment analysis identified significant perturbations in immune pathways in the higher risk group. This study highlights the presence of an immunocompromised microenvironment indicated by the higher infiltration of cytotoxic T cells along with the presence of checkpoint molecules and T cell exhaustion genes. These patients at higher risk might be more suitable to benefit from PD-L1 blockade or other checkpoint blockade immunotherapies.

Considering lack of target‐specific antiviral treatment and vaccination for COVID‐19, it is absolutely exigent to have an effective therapeutic modality to reduce hospitalization and mortality rate as well as to improve COVID‐19‐infected patient outcomes. In a follow‐up study to our recent findings indicating the potential of Cannabidiol (CBD) in the treatment of acute respiratory distress syndrome (ARDS), here we show for the first time that CBD may ameliorate the symptoms of ARDS through up‐regulation of apelin, a peptide with significant role in the central and peripheral regulation of immunity, CNS, metabolic and cardiovascular system. By administering intranasal Poly (I:C), a synthetic viral dsRNA, while we were able to mimic the symptoms of ARDS in a murine model, interestingly, there was a significant decrease in the expression of apelin in both blood and lung tissues. CBD treatment was able to reverse the symptoms of ARDS towards a normal level. Importantly, CBD treatment increased the apelin expression significantly, suggesting a potential crosstalk between apelinergic system and CBD may be the therapeutic target in the treatment of inflammatory diseases such as COVID‐19 and many other pathologic conditions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing has lagged in many countries because of test kit shortages and analytical process bottlenecks. This study investigated the feasibility and accuracy of a sample pooling approach for wide-scale population screening for coronavirus disease 2019. A total of 940 nasopharyngeal swab samples (934 negative and 6 positive) previously tested for SARS-CoV-2 were deidentified and assigned random numbers for analysis, and 94 pools of 10 samples each were generated. Automated RNA extraction, followed by RT-PCR, was performed in a 96-well plate. Positive pools were identified, and the individual samples were reanalyzed. Of the 94 pools/wells, four were positive [Ct values: N (22.7 to 28.3), ORF1ab (23.3 to 27.2), and internal control (34.4 to 35.4)]. The 40 samples comprising the four pools were identified and reanalyzed individually; six samples were positive, with Ct values of N gene, ORF1ab, and internal control comparable to their respective wells. Additional experiments were performed on samples with high Ct values, and overall results showed 91.6% positive and 100% negative agreement compared with individual testing approach. Thus, 940 samples were tested in 148 reactions compared with 940 reactions in routine screening. The sample pooling strategy may help catch up with testing needs and minimal turnaround times and facilitate enormous savings on laboratory supplies, extraction, and PCR kits currently in short supply.

The coronavirus disease-19 (COVID-19) pandemic is an unprecedented worldwide health crisis. COVID-19 is caused by SARS-CoV-2, a highly infectious pathogen that is genetically similar to SARS-CoV. Similar to other recent coronavirus outbreaks, including SARS and MERS, SARS-CoV-2 infected patients typically present with fever, dry cough, fatigue, and lower respiratory system dysfunction, including high rates of pneumonia and acute respiratory distress syndrome (ARDS); however, a rapidly accumulating set of clinical studies revealed atypical symptoms of COVID-19 that involve neurological signs, including headaches, anosmia, nausea, dysgeusia, damage to respiratory centers, and cerebral infarction. These unexpected findings may provide important clues regarding the pathological sequela of SARS-CoV-2 infection. Moreover, no efficacious therapies or vaccines are currently available, complicating the clinical management of COVID-19 patients and emphasizing the public health need for controlled, hypothesis-driven experimental studies to provide a framework for therapeutic development. In this mini-review, we summarize the current body of literature regarding the central nervous system (CNS) effects of SARS-CoV-2 and discuss several potential targets for therapeutic development to reduce neurological consequences in COVID-19 patients.

Studying the complex molecular mechanisms involved in traumatic brain injury (TBI) is crucial for developing new therapies for TBI. Current treatments for TBI are primarily focused on patient stabilization and symptom mitigation. However, the field lacks defined therapies to prevent cell death, oxidative stress, and inflammatory cascades which lead to chronic pathology. Little can be done to treat the mechanical damage that occurs during the primary insult of a TBI; however, secondary injury mechanisms, such as inflammation, blood-brain barrier (BBB) breakdown, edema formation, excitotoxicity, oxidative stress, and cell death, can be targeted by therapeutic interventions. Elucidating the many mechanisms underlying secondary injury and studying targets of neuroprotective therapeutic agents is critical for developing new treatments. Therefore, we present a review on the molecular events following TBI from inflammation to programmed cell death and discuss current research and the latest therapeutic strategies to help understand TBI-mediated secondary injury.

Chromosomal aberrations (CAs) are an important tool for assessment of exposure to pesticides. Genotoxic potential of pesticides is a principal risk factor for long-term health effects. The present study was aimed toward the assessment of CAs among agricultural workers exposed to pesticides and comparison with nonagricultural workers not exposed to pesticides. A total of 296 subjects were enrolled in the study: exposed (n = 148) and nonexposed subjects (n = 148) from Punjab. A significantly high frequency of aberrations was seen in peripheral blood lymphocytes of exposed subjects as compared with nonexposed ones. Most CAs were present as loss (aneuploidy) and were observed significantly in subjects having a history of alcohol consumption. It can be, thus, concluded that agricultural workers exposed to a mixture of pesticides, in addition to being alcoholic, are at a greater risk of genotoxic damage. It is highly recommended that the agricultural workers are educated regarding the potential hazards of occupational exposure to pesticides.

Cannabis-inspired medical products are garnering increasing attention from the scientific community, general public, and health policy makers. A plethora of scientific literature demonstrates intricate engagement of the endocannabinoid system with human immunology, psychology, developmental processes, neuronal plasticity, signal transduction, and metabolic regulation. Despite the therapeutic potential, the adverse psychoactive effects and historical stigma, cannabinoids have limited widespread clinical application. Therefore, it is plausible to weigh carefully the beneficial effects of cannabinoids against the potential adverse impacts for every individual. This is where the concept of “personalized medicine” as a promising approach for disease prediction and prevention may take into the account. The goal of this review is to provide an outline of the endocannabinoid system, including endocannabinoid metabolizing pathways, and will progress to a more in-depth discussion of the therapeutic interventions by endocannabinoids in various neurological disorders.

Mitochondria are dynamic organelles responsible for cellular energy production. Besides, regulating energy homeostasis, mitochondria are responsible for calcium homeostasis, signal transmission, and the fate of cellular survival in case of injury and pathologies. Accumulating reports have suggested multiple roles of mitochondria in neuropathologies, neurodegeneration, and immune activation under physiological and pathological conditions. Mitochondrial dysfunction, which occurs at the initial phase of brain injury, involves oxidative stress, inflammation, deficits in mitochondrial bioenergetics, biogenesis, transport, and autophagy. Thus, development of targeted therapeutics to protect mitochondria may improve functional outcomes following traumatic brain injury (TBI) and intracerebral hemorrhages (ICH). In this review, we summarize mitochondrial dysfunction related to TBI and ICH, including the mechanisms involved, and discuss therapeutic approaches with special emphasis on past and current clinical trials.

Colorectal cancer (CRC) is the third most common type of cancer affecting nearly 1.4 million individuals every year. In the past two decades, rapid advancements in genomics and transcriptomics have uncovered various molecular profiling methods that are assisting in the design of personalized treatment strategies for cancer patients. In this study, we attempted to identify differentially expressed genes in tumor and NAT for its clinical significance in CRC. We designed a novel 88 gene-expression panel consisting of immune and cancer driver genes based on literature. Under IRB approved protocol, 28 FFPE samples (tumor = 14, NAT = 14) were acquired from pathology archives. H&E slides were examined and by using macrodissection, regions with >95% of tumor or NAT region were identified by two expert pathologist. Total RNA was isolated from these tissue sections and quantified through Nanodrop method. The expression level of the genes was measured using NanoString digital quantification system. Pairwise comparisons by Wilcoxon rank-sum test identified significant gene perturbations in the NAT compared to the tumor. IL-15, GATA3, CCR4, CCL13 and IFNA1 genes were highly expressed in NAT (p Citation Format: Pankaj Kumar Ahluwalia, Ashis Mondal, Meenakshi Ahluwalia, Nikhil Sahajpal, Allan Njau, Nwogbo Okechukwu, Vamsi Kota, Ravindra Kolhe. Investigating the role immunogenomics in normal adjacent to tumor tissue (NAT) versus tumor in prognostication of stage matched colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-351.

Breast cancer has a high annual incidence among females in India, with an age adjusted rate of 25.8 cases per 100,000 women and a mortality rate of 12.7 per 100,000 women1. Approximately 75% of breast cancers are estrogen receptor positive (ER+), and unfortunately, a significant number will develop resistance to first line hormonal therapy. Although several mechanisms have been linked to hormonal therapy resistance, no biomarker in clinical use currently exists to predict hormonal therapy resistance in a reliable manner. Comprehensive genomic profiling (CGP) is not routinely performed for metastatic breast carcinoma (MBC) in a relapsed setting in India. This research highlights utility of CGP on a MBC sample combined with interpretation by Watson™ for Genomics (WfG), an artificial-intelligence (AI)-based decision-support system (DSS), when a patient in a middle-income country developed resistance to endocrine therapy. A 33-year-old Indian female was diagnosed with ER+, progesterone-receptor-negative (PR-), human-epidermal-receptor-2-negative (HER2-) breast cancer. Two years later, she noted an axillary mass which was biopsied and revealed an ER+, PR-, HER2- MBC metastasis. CGP was performed with a panel from Illumina designed to detect genomic alterations in 170 genes, including 148 genes for substitutions and indels, 55 genes for fusion and splice variants, and 59 genes for copy number variation detection. Variant annotation and interpretation performed using WfG revealed co-existing ESR1 D538G mutation and ESR1-CCDC170 fusion. In addition, alterations in CCND1 resulting in activation of the CDK signaling pathway, an oncogenic mutation in MTOR resulting in activation of the MTOR/PI3K signaling pathway, and amplification of FGFR1, a poor prognostic factor in MBC, were also identified. While ESR1 D538 and ESR1-CCDC170 fusion individually have been reported to confer resistance to endocrine therapy, to the best of our knowledge, our report is the first HR+ MBC case with these two oncogenic alterations co-occurring in a metastatic tumor suggesting a synergistic role. CGP in this patient revealed a possible mechanism of resistance to endocrine therapy informing the physician to change treatment to a generic mTOR inhibitor based on the oncogenic MTOR mutation. In conclusion, assessment of DNA/RNA tumor variants in one assay along with a robust AI-based DSS can provide relevant information for consideration by the clinician when developing a treatment plan, may create efficiencies in tumor sample usage, time, and cost of treatment. Further research is needed to explore the functional synergy of the oncogenic ESR1 mutation and fusion detected in this patient's tumor and the role of identifying these pathogenic variants through circulating tumor cell or cell-free DNA assays to detect minimal residual disease. Citation Format: Ravindra Kolhe, Ashis Mondal, Nikhil Sahajpal, Meenakshi Ahluwalia, Yull Arriaga, Allan Njau, David Brotman, Jane Snowdon, Gretchen Jackson, Dilhan Weeraratne. Co-existing ESR1 D538G mutation and ESR1-CCDC170 fusion in a patient with metastatic hormone receptor positive breast cancer with progression after hormonal therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4105.

A clinical next generation sequencing (NGS) assay comprises various factors taken into consideration including selection of appropriate markers thereby panel size, nucleic acid input, assessment and interpretation, and workflow. The downstream benefits of the development of such panels therefore involve improvement in prognosis, patients' outcome, and benefit from targeted therapy. Myeloid malignancies such as myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN, acute myeloid leukemia (AML) are associated with somatic mutations in around 25 to 50 genes. Herein, we describe clinical validation of a DNA based targeted NGS assay utilizing QIAact Myeloid DNA UMI panel in combination with QIAGEN GeneReader NGS system. The assay employs unique molecular index technology (UMI) into a gene specific, primer based target enrichment technology enabling sequencing of specific regions of interest providing an integrated solution by simultaneously assessing many candidate genes for actionable mutations. The QIAact Myeloid DNA UMI panel is a 25 gene panel for markers of known clinical significance, including single nucleotide variants (SNV), and large insertion/deletion mutations. At the lab, 40 individual AML specimens with known mutation profiles were tested. The limit of detection (LOD) was assessed by sequencing Seraseq myeloid mutation DNA mix as positive control. For accuracy, inter-run and intra-run controls were also implemented. All samples were previously characterized by using another 54 gene NGS panel. The samples were prepared using QIAact Myeloid DNA UMI panel kits, sequenced with QIAGEN GeneReader NGS system, and mutations identified using the QIAGEN Clinical Insight (QCI) Analyze software suite, adjusted specifically for variant calling. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection (LOD) studies also determined that input of 40ng DNA was optimal for high analytical sensitivity. High positive and negative percentage agreement with prior results was observed across all variant categories. The assay was also able to identify 52bp deletion CALR type 1 variant, and CEBPα amplicons mutations which require separate bi-directional Sanger sequencing for identification. Also, the assay was able to identify FLT3 ITDs up to 101bp insertion which are detected by performing PCR separately. Hence, the study presents the data establishing the QIAact Myeloid DNA UMI panel suitable for implementation as a routine clinical NGS test for myeloid malignancies. The optimized chemistry allows high-throughput analytical sensitivity for detection of highly relevant mutations including large Insertion/Deletion (InDel). Citation Format: Meenakshi Ahluwalia, Ashis Mondal, Nikhil Sahajpal, Allan Njau, Kimya Jones, Pankaj Kumar Ahluwalia, Yasmeen Jilani, Ravindra Kolhe. Utility and validation of a comprehensive cost-effective targeted DNA panel including FLT3-ITDs, CALR and CEBPA on a next-generation sequencing (NGS) platform for hematological malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 816.

Colorectal cancer (CRC) affects nearly 1.4 million individuals every year and is the third most common cause of deaths due to cancer. There is a need to identify novel prognostic biomarkers with accurate prognostic and therapeutic implications. In this study, we sought to identify prognostic gene expression signatures and explore their relationship with the tumor microenvironment. Under the IRB approved protocol, FFPE blocks of CRC patients were acquired from the Medical College of Georgia, Augusta, USA. Tumor regions and adjacent normal tissue regions were identified and marked by a board-certified pathologist. RNA isolation was performed from tumor sections (n = 88) and adjacent normal regions (n = 16). A panel of 17 genes with the highest perturbation in TCGA dataset was investigated on the FFPE tissues. The quantification of the mRNA molecules was performed using total RNA (300 ng) on the NanoString platform. It uses a specific molecular barcoding system to tag target nucleic acid molecules with very high sensitivity and specificity. The immunological dynamics of the prognostic genes were analyzed through TIMER (Tumor-Infiltrating Immune Cells) web portal using the TCGA-COAD dataset. In the clinical dataset, MCM4, YWHAB, LRRC59 and DPP7 were highly expressed in tumor tissue compared to adjacent control and were associated with worse prognosis (Log-rank test, p < 0.001*). However, PI4K2B, PCMT1 and PBX1P1 showed higher expression in adjacent normal tissue compared to tumor regions (Student's t-test, p < 0.001*). Further analysis using TIMER portal revealed that copy number alterations in YWHAB were significantly associated with decreased tumor infiltration of B cells, CD8+ T cells, neutrophils and dendritic cells (Wilcoxon test, p < 0.001*). The predominant alterations observed were arm-level gain and high amplification. Further, arm-level gain of MCM4 was associated with lower infiltration of B cells and CD8+ T cells (Wilcoxon test, p < 0.001*) and FBXO46 was associated with lower infiltration of B cells only (Wilcoxon test, p < 0.05*). Additional comparison between CRC tumor and adjacent normal tissue was explored using “DiffExp” module of TIMER web portal. The comparison showed that YWHAB, DPP7 and MCM4 were expressed at higher amounts in tumor tissues compared to adjacent normal tissue. These preliminary analyses, therefore reveal the significance of prognostic genes and their association with the infiltration of immune cells that in turn can be of therapeutic value. A comprehensive network of prognostic gene signatures and its perturbations in immune cell functioning can aid in efficient prognostic classification and design of new therapies. Citation Format: Pankaj Kumar Ahluwalia, Ashis Mondal, Meenakshi Ahluwalia, Nikhil Sahajpal, Kimya Jones, Yasmeen Jilani, Allan Njau, Ravindra Kolhe. Exploring prognostic and immunological impact of a novel 4 gene signature in colorectal cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4334.

Introduction: Next generation sequencing (NGS) based assays have revolutionized the field of genetic testing and paved their way into routine clinical practice. Owing to the complexity of the data, bioinformatics has become a necessary component for any laboratory implementing a clinical NGS test. However, there is an unmet need for accurate, efficient and standardized variant interpretation for actionable insights. In this effort, AMP/ASCO/CAP released a joint consensus recommendation proposing a four-tiered system to categorize somatic alterations based on their clinical significance in cancer diagnosis, prognosis, or therapeutics for standardization of interpretation and reporting of results among laboratories. Thus, the aim of the study was to assess and validate QIAGEN Clinical Insight (QCI) Interpret software for clinical laboratory test interpretation. Also, the AMP/ASCO/CAP guidelines were compared to an accredited laboratory approach for variant classification. Patients and Methods: 87 [BL-Q1] common and rare solid tumor cases were analyzed using the TrueSight170 NGS assay and secondary analysis platform (Illumina, San Diego, CA). The data was uploaded and analyzed using QCI Interpret. Patient case draft reports were produced using a TST170 customizable workflow with automated variant filtering, and variant classifications. Molecular geneticists compared the previous expert variant classification against the QCI Interpret computed AMP tier classification. Accuracy and specificity assessed as true positive, true negative, false positive and false negative rates were determined. Results: Preliminary analysis on 52 cases with 496 alterations (SNVs, in-frame deletions, CNVs, fusions) resulted in 44 expertly classified alterations with strong therapeutic, prognostic, or diagnostic actionability (Tier I), 147 expertly classified alterations of potential therapeutic prognostic or diagnostic actionability (Tier II), and 304 variants of unknown clinical significance (Tier III). The QCI interpret computed classification yielded 44 Tier I (Strong clinical significance), 153 Tier II, 299 Tier III [SNS2] variants. Expert and QCI Interpret computed Tier 1 classification are in 100% agreement. The accuracy for Tier II and Tier III classification were 96% (153 vs. 147) and 98% (299 vs. 304), respectively. However, the Manual interrogation of the 6 discrepant variants revealed that these were emerging biomarkers that were previously characterized as Tier III but due to recent biological evidence are now classified as Tier II. Conclusion: Systematic evaluation of an automated classification based on AMP/ASCO/CAP using an up to date knowledge base is critical to accurately classify actionable mutations and detect emerging relevant mutations. The guidelines seem to yield results sufficiently good for clinical use and are a big step forward regarding standardization and use for classifying somatic alterations. The QCI Interpret seems to be a critical bioinformatics tool that adheres to the AMP/ASCO/CAP classification system and can be used across clinical laboratories for accurate, efficient and standardized variant interpretation for actionable insights. Citation Format: Nikhil Sahajpal, Ashis Mondal, Meenakshi Ahluwalia, Allan Njau, Ravindra Kolhe. Validation of somatic variant interpretations from comprehensive cancer panels using QIAGEN Clinical Insight Interpret [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5482.

Next-generation sequencing (NGS) is increasingly used to inform diagnostic, therapeutic, and prognostic decisions in AML at the time of first presentation. We highlight the utility of NGS combined with Watson™ for Genomics (WfG), an artificial-intelligence-based decision-support system, in identifying new clinically actionable alterations as a result of clonal evolution in the relapsed disease setting. In less than 3 minutes, WfG identified an IDH1 R132H pathogenic mutation in the relapsed sample sequenced with the Illumina TruSight Tumor 170-gene panel leading to the compassionate use of ivosidenib. In addition, mutations in two genes resulting in increased sensitivity to PARP inhibitors and mutations in PTEN resulting in activation of the MTOR/PI3K signaling pathway were detected by WfG. In February 2018, a previously healthy 23-year old Caucasian female presented with AML consisting of 80% blasts with positive FLT3 mutation. She received induction cytarabine plus daunorubicin (7+3) followed by multikinase inhibitor therapy with midostaurin on days 8 to 21. A 28-day bone marrow biopsy showed persistent disease with 40% blasts. In March 2018, the patient underwent re-induction chemotherapy with mitoxantrone, etoposide, and cytarabine (MEC) followed by midostaurin on days 8 to 21. A bone marrow biopsy after completion of re-induction therapy showed complete remission with < 5% blasts (CR1). In May 2018, a matched donor was not found on a bone marrow registry, and the patient underwent a post-remission dual unrelated umbilical cord blood (UCB) hematopoietic stem cell transplantation (HSCT). Engraftment was not achieved. In July 2018, the patient had disease relapse with the presence of circulating blasts and 7% of blasts in the bone marrow. While NGS is typically not performed on relapsed samples, WfG identified IDH1 R132H and PTEN C78T pathogenic mutations using the 170-gene panel. Based on these results, the patient initiated azacytidine plus the IDH1 inhibitor ivosidenib. In August 2018, a bone marrow biopsy showed less than 5% blasts positive for a FLT3-ITD mutation. In September 2018, the patient underwent reduced-intensity conditioning with fludarabine, cyclophosphamide, and total body irradiation followed by haploidentical allogeneic HSCT from her mother. Maintenance therapy with azacytidine plus ivosidenib was continued until the present time. Currently, the patient is in remission for over 15 months without evidence of AML minimal residual disease. She has developed mild skin chronic graft-versus-host disease that is controlled with standard treatment. She works on a full-time basis and has excellent functional status. The combination of CGP, artificial intelligence, and expert care has resulted in an excellent outcome in a patient with relapsed AML. In conclusion, our experience suggests that CGP testing should be considered at different time points, at least in the relapsed setting, to help treating physicians alter or help improve clinical outcome. CGP testing in a relapsed setting is precluded because it is not covered by payers. In support of optimal care, we have initiated a new program for compassionate use of genomic testing, where such testing is medically necessary, but not covered by insurance or payer supported. Citation Format: Ravindra Kolhe, Ashis Mondal, Vamsi Kota, Nkhil Sahajpal, Meenakshi Ahluwalia, Allan Njau, Dilhan Weeraratne, Yull Arriaga, David Brotman, Gretchen Jackson, Jane Snowdon. Clinical utility of comprehensive genomic testing with artificial-intelligence-based analysis to identify targetable sub-clonal events in relapsed acute myeloid leukemia (AML) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6579.

e14051 Background: Adoption of next-generation sequencing (NGS) technology in routine clinical practice has enabled the detection of genetic aberrations such as single nucleotide variants, copy number alterations, and gene fusions. Pathway and network analyses (PNA) are key components for evaluation of NGS data in a clinical setting to explain findings involving thousands of altered genes and proteins with a smaller and more interpretable set of altered processes. Though PNA have been applied to identify driver genes and pathways in cohort-based analyses, its application in precision oncology remains unexplored. We investigate the potential utility of the Watson for Genomics (WfG) pathway analyses tool in interpreting complex and multiple genomic alterations in individual cancers. Methods: DNA and RNA isolated from 70 patient tumors across 30 different cancer types were processed with Illumina’s TST170 NGS platform. WfG’s feature of pathway analyses was used to identify gene variants, signaling pathways, networks, and the drugs targeting these alterations based on evidence in the clinical literature and FDA drug databases. Results: Analyses defined 5 different pathway/network models: 1) downstream therapeutic targets, 2) synthetic lethality, 3) combinatorial downstream targets + synthetic lethality, 4) two or more pathways converging to downstream targets, and 5) complex profile analyses. The five PNA models are illustrated by the following unique cases. 1) A thyroid cancer case with HRAS variant and activated RAF1 downstream pathway showed MAPK1/3 were suggestive of relevant targets. 2) An acute myeloid leukemia case with BRCA1, BRCA2 and PTEN variants, targeting a common synthetic lethal partner PARP1 was ideal for therapy. 3) A penile carcinoma case with BRAF, CDKN2A and TP53 variants, targeting the BRAF downstream pathway in combination with either CDKN2A or TP53 were the likely choice for therapy. 4) A glioma case with activated PI3K and MEK downstream pathway, targeting a common downstream marker would block both pathways. 5) A breast carcinoma case with a complex pathogenic variant profile provided relevant clinical information and levels of evidence for multiple drug targets. Conclusions: We discovered that the integrated WfG pathway analyses tool is ideal for visualization of the variants with levels of evidence from clinical literature and FDA drug databases that can help inform treatment options and provides a holistic understanding of a specific tumor profile allowing the treating clinician to select personalized targeted therapy.

e14048 Background: The rapid adoption of next-generation sequencing (NGS) methods into clinical diagnostic laboratories have revolutionized the field of genetic testing. To interpret the exhaustive NGS data, bioinformatics has become an inevitable component of any laboratory implementing a clinical NGS test. However, there is an unmet need for accurate, efficient and standardized variant interpretation for actionable insights. In this effort, AMP/ASCO/CAP released a joint consensus recommendation proposing a four-tiered system to categorize somatic alterations based on their clinical significance in cancer diagnosis, prognosis, or therapeutics for standardization of interpretation and reporting of results among laboratories. Thus, the aim of the study was to assess and validate QIAGEN Clinical Insight (QCI) Interpret software for clinical laboratory test interpretation. Methods: 67 malignant cases (myeloid neoplasm and lung) were analyzed using the TSO-500 NGS assay and secondary analysis platform (Illumina, San Diego, CA). The data was uploaded and analyzed using QCI Interpret. Patient case draft reports were produced using a TS0-500 customizable workflow with automated variant filtering, and variant classifications. Results: Data analysis on 67 cases resulted in a total of 251 alterations (SNVs and Indels). A total of 77 and 174 alterations were reported as pathogenic/ likely pathogenic into tier based classification by the QCI Interpret for myeloid and lung malignancies, respectively. For myeloid malignancies, the QCI Interpret was able to detect accurately all the clinically relevant variants and listed them into appropriate tier-based classification compared to the expertly reported alterations. For lung malignancies, 16 alteration were classified in tier 1, 96 alteration in tier 2, 61 alteration in tier 3, and 1 alteration in tier 4. The QCI interpret was able to detect 100% of the alterations being validated, and reported them into tier based classification. Conclusions: Systematic evaluation of an automated classification based on AMP/ASCO/CAP using an up to date knowledge base is critical to accurately classify actionable mutations and detect emerging relevant mutations. The QCI Interpret seems to be a critical bioinformatics tool that adheres to the AMP/ASCO/CAP classification system and incorporates TMB and MSI values from the TSO500 assay, which can be used across clinical laboratories for accurate, efficient and standardized variant interpretation for actionable insights.

Glutathione S-transferases are important detoxification enzymes involved in the metabolism of endogenous as well as exogenous compounds. Individuals differ in metabolic capacity due to inherited genetic variations. Due to the polymorphism exhibited by GSTT1 and GSTM1 that results in the complete loss of function, the present study was aimed towards the determination of the frequency distribution of GSTT1 and GSTM1 in agricultural workers in Punjab, India. The study aimed to investigate their contribution in susceptibility to increased disease risk. A total of 513 subjects were included in this study, out of which 250 were agriculture workers and 263 were non-exposed occupationally. GSTT1 and GSTM1 null-genotype distribution was analyzed through multiplex-PCR method. Complete gene deletion in either of the genes was strongly associated with an increased risk (OR = 1.8; 95% CI = 1.3–2.6; p