Sensitive Molecular Biomarkers Research Articles

Evaluation of salivary biomarker interleukin-6 in oral squamous cell carcinoma and oral potentially malignant disorders − A comparative cross-sectional South Indian study

Abstract Background: Oral squamous cell carcinoma (OSCC) accounts for nearly 90% of oral malignancies and represents a major global health care problem. It is often preceded by oral potentially malignant disorders (OPMD). Although regular clinical examination forms the backbone for oral cancer screening, subtle lesions go unnoticed and there is a need for more sensitive and specific molecular biomarkers in mass screening of population. Salivary proteomics offer an attractive alternative to serum and tissue testing. Aims: To find the diagnostic utility of salivary interleukin-6 (IL-6) in the differential diagnosis of OSCC, OPMD from healthy controls. Study Design: In vivo study. Methods: After approval from the Institutional Review Board, unstimulated whole saliva was collected from 90 subjects, 30 in each group of OSCC, OPMD and controls after ethical clearance. Salivary IL-6 was measured by ELISA, and the results were statistically analysed. Results: Significant difference in salivary IL-6 was seen between OSCC, OPMD and controls. Receiver operating characteristic curve analysis showed the highest area under a curve of 0.982 in distinguishing OSCC from controls. It showed a sensitivity of 71% and specificity of 100% at a cut-off value of 33.4 pg/mL (P = 0.000). Moderately differentiated OSCC (MDSCC) showed a significant increase in salivary IL-6 concentration compared to well-differentiated OSCC (WDSCC). Conclusion: Results of the present study showed strong predictive power of salivary IL-6 in distinguishing OSCC from controls. Its levels also increased with tumor aggressiveness from WDSCC to MDSCC. Thus, salivary IL-6 could have a diagnostic and/or prognostic significance in identifying high-risk groups in mass screening of the population.

Molecular detection of exosomal miRNAs of blood serum for prognosis of colorectal cancer

Colorectal cancer (CRC) is the third most common cancer affecting people. The discovery of new, non-invasive, specific, and sensitive molecular biomarkers for CRC may assist in the diagnosis and support therapeutic decision making. Exosomal miRNAs have been demonstrated in carcinogenesis and CRC development, which makes these miRNAs strong biomarkers for CRC. Deep sequencing allows a robust high-throughput informatics investigation of the types and abundance of exosomal miRNAs. Thus, exosomal miRNAs can be efficiently examined as diagnostic biomarkers for disease screening. In the present study, a number of 660 mature miRNAs were detected in patients diagnosed with CRC at different stages. Of which, 29 miRNAs were differentially expressed in CRC patients compared with healthy controls. Twenty-nine miRNAs with high abundance levels were further selected for subsequent analysis. These miRNAs were either highly up-regulated (e.g., let-7a-5p, let-7c-5p, let-7f-5p, let-7d-3p, miR-423-5p, miR-3184-5p, and miR-584) or down-regulated (e.g., miR-30a-5p, miR-99-5p, miR-150-5p, miR-26-5p and miR-204-5p). These miRNAs influence critical genes in CRC, leading to either tumor growth or suppression. Most of the reported diagnostic exosomal miRNAs were shown to be circulating in blood serum. The latter is a novel miRNA that was found in exosomal profile of blood serum. Some of the predicted target genes of highly expressed miRNAs participate in several cancer pathways, including CRC pathway. These target genes include tumor suppressor genes, oncogenes and DNA repair genes. Main focus was given to multiple critical signaling cross-talking pathways including transforming growth factor β (TGFβ) signaling pathways that are directly linked to CRC. In conclusion, we recommend further analysis in order to experimentally confirm exact relationships between selected differentially expressed miRNAs and their predicted target genes and downstream functional consequences.

Up-regulation of GINS1 highlighted a good diagnostic and prognostic potential of survival in three different subtypes of human cancer.

Cancer is a fatal malignancy and its increasing worldwide prevalence demands the discovery of more sensitive and reliable molecular biomarkers. To investigate the GINS1 expression level and its prognostic value in distinct human cancers using a series of multi-layered in silico approach may help to establish it as a potential shared diagnostic and prognostic biomarker of different cancer subtypes. The GINS1 mRNA, protein expression, and promoter methylation were analyzed using UALCAN and Human Protein Atlas (HPA), while mRNA expression was further validated via GENT2. The potential prognostic values of GINS1 were evaluated through KM plotter. Then, cBioPortal was utilized to examine the GINS1-related genetic mutations and copy number variations (CNVs), while pathway enrichment analysis was performed using DAVID. Moreover, a correlational analysis between GINS1 expression and CD8+ T immune cells and a the construction of gene-drug interaction network was performed using TIMER, CDT, and Cytoscape. The GINS1 was found down-regulated in a single subtypes of human cancer while commonly up-regulated in 23 different other subtypes. The up-regulation of GINS1 was significantly correlated with the poor overall survival (OS) of Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), and Kidney renal clear cell carcinoma (KIRC). The GINS1 was also found up-regulated in LIHC, LUAD, and KIRC patients of different clinicopathological features. Pathways enrichment analysis revealed the involvement of GINS1 in two diverse pathways, while few interesting correlations were also documented between GINS1 expression and its promoter methylation level, CD8+ T immune cells level, and CNVs. Moreover, we also predicted few drugs that could be used in the treatment of LIHC, LUAD, and KIRC by regulating the GINS1 expression. The expression profiling of GINS1 in the current study has suggested it a novel shared diagnostic and prognostic biomarker of LIHC, LUAD, and KIRC.

Combined exposure of beta-cypermethrin and emamectin benzoate interferes with the HPO axis through oxidative stress, causing an imbalance of hormone homeostasis in female rats

The impact of pesticides on reproductive health has been increasingly recognized. β-cypermethrin (β-CYP) and emamectin benzoate (EMB) are commonly used with agricultural workers. There are few published studies on the effects of combined poisoning of these two pesticides on the reproductive system. This study investigated the toxic effects and mechanism of β-CYP and EMB on the reproductive system of female rats based on the hypothalamic-pituitary-ovarian (HPO) axis. The hypothalamic GnRH content tended to decrease, and Kiss-1 and GPR-54 mRNA and protein expression tended to increase in exposed rats. FSH content was elevated for the pituitary gland, and Kiss-1 and GPR-54 mRNA and protein expression were enhanced in all experimental groups compared with the control group. E2 content in rat ovaries and ERα mRNA and protein expression were reduced by β-CYP and EMB. Furthermore, there were interactive effects of β-CYP and EMB on FSH and E2 release, pituitary GPR-54 mRNA and protein, and ovarian ERα mRNA expression. To investigate causes of damage, oxidative damage indicators were tested and showed that exposure to β-CYP and EMB decreased GSH-Px and SOD activities in the HPO axis, increased MDA levels in the hypothalamus and ovary together with LDH activities in the HPO axis, with an interaction effect on GSH-Px and SOD activities in the hypothalamus and pituitary gland as well as on MDA in the ovary. The above results support the screening of sensitive molecular biomarkers and evaluation of the adverse effects of pesticide exposure in greenhouse operations on reproductive health.

Abstract P058: Rapid, noninvasive, office-based, esophageal cell collection for early detection of esophageal precancer and cancer

Abstract Purpose: Esophageal Adenocarcinoma (EAC) is the second most lethal (<20% five-year survival) and fastest growing (>500% increase in incidence over 40 years) cancer in the U.S. All EAC is believed to arise from a metaplastic esophageal precancer, Barrett’s Esophagus (BE), in at-risk patients with gastroesophageal reflux disease (GERD). Non-dysplastic BE can be monitored for progression to dysplastic BE, which can be ablated endoscopically, reliably halting progression to EAC. Since mortality is high even in early stage EAC (<50% five-year survival), clinical practice guidelines recommend BE screening in GERD patients with multiple risk factors. Since, endoscopic BE screening has failed (<10% compliance with guidelines), recently updated guidelines from both the American College of Gastroenterology and American Gastroenterological Association now support non-endoscopic biomarker screening as an acceptable alternative to endoscopy. EsoCheck®, a noninvasive swallowable balloon capsule catheter device, is the only FDA 510(k)-cleared esophageal cell collection device which permits anatomically targeted and protected sampling of distal esophageal mucosal cells. The sample is collected in a less than five-minute office procedure, without sedation or anesthesia, and sent to a central laboratory for molecular biomarker testing. We report our initial clinical experience using EsoCheck® for collection of esophageal cells in outpatient test centers. Experimental Design: 687 patients underwent EsoCheck® distal esophageal cell sampling by a trained nurse practitioner (average sampled length 5 cm). Patient tolerance was rated on a five-point scale for gag reflex (GR) severity: 1 = no GR; 2 = minimal GR; 3 = mild GR; 4 = severe GR; and 5 = worst GR therefore collection could not be completed. DNA was extracted using the QIAamp DNA Mini kit and quantitated using Qubit™ dsDNA Assay kit. Cytologic assessment using H&E staining was performed on 15 samples. Results: 98% of patients successfully completed the procedure. 88% tolerated it well (rating ≤ 3); 10% experienced severe gagging (rating 4); and only 2% were unable to complete the procedure (rating 5). Average procedure time was 3.5 min (range: 1 min to 15 min). Average DNA yield was 868 ng (range: 0 ng to 19 µg) and 92% yielded at least 100 ng. The samples undergoing cytologic assessment averaged 500,000 cells (range: 100,000 to 2.5 million). Conclusion: Rapid, noninvasive, office-based, anatomically-targeted, and protected sampling of distal esophageal mucosal cells using EsoCheck® can be successfully performed in nearly all patients and is broadly well-tolerated. Cellular and DNA yields were very good and are expected to improve with clinical experience and assay optimization. When combined with a highly sensitive molecular biomarker test, the EsoCheck® device has the potential to significantly improve BE screening compliance and prevent EAC deaths. Citation Format: Suman M. Verma, Ivy T. Le, Abhisek B. Ghosal, Marci L. Hageman, Victoria T. Lee, Brian J. deGuzman, Lishan Aklog. Rapid, noninvasive, office-based, esophageal cell collection for early detection of esophageal precancer and cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P058.

Genome-wide identification of seven superoxide dismutase genes in the marine rotifer Brachionusrotundiformis and modulated expression and enzymatic activity in response to microplastics and nutritional status.

Microplastics (MPs) pollution has attracted worldwide attention. Superoxide dismutase (SOD) is a sensitive indicator for assessing the toxic effects of MPs in aquatic organisms. However, few studies have been performed to identify all genes encoding SOD in aquatic invertebrates. Especially, effects of MPs on SOD activity and expression in aquatic organisms under starvation or a subsequent refeeding status are unclear. In the present study, all full-length genes encoding SOD were cloned and characterized from the marine rotifer Brachionus rotundiformis, including CuZnSOD1, CuZnSOD2, CuZnSOD3, CuZnSOD4, CuZnSOD5, MnSOD1, and MnSOD2. The CuZnSOD1, CuZnSOD2 and MnSOD2 are homologous to SODs from vertebrates and the other SOD proteins are rotifer-specific according to the results from the phylogenetic tree. The conserved signature sequences and binding sites of Cu2+, Zn2+and Mn2+ were also identified in the seven SOD proteins. Compared with feeding, starvation down-regulated SOD activity and mRNA expression of CuZnSOD2, CuZnSOD4, CuZnSOD5, MnSOD1 and MnSOD2 while refeeding maintained SOD activity comparable to the feeding level and up-regulated CuZnSOD5 and MnSOD2. Intake of MPs by B. rotundiformis was observed by examining fluorescence signals from the fluorescently-labeled microplastics under different nutritional status. Exposure to MPs reduced rotifer density and increased malondialdehyde (MDA) content and SOD activity in the rotifers under the refeeding condition, but did not affect these indicators under the feeding and starvation conditions. However, mRNA expression of some tested genes was responsive to MPs in the fed, starved and refed rotifers. The present study for the first time demonstrated a nutritional status-dependent effect of MPs on oxidative stress response, and provided more sensitive molecular biomarkers for assessing the toxicity of MPs using B. rotundiformis as a model animal.

Nutritional-status dependent effects of microplastics on activity and expression of alkaline phosphatase and alpha-amylase in Brachionus rotundiformis

Tissue-nonspecific alkaline phosphatase (ALPL) and alpha-amylase (AMY) are essential in the immune and digestive systems, respectively. Microplastics (MPs) pose a risk to zooplankton which may be in a state of feeding, starvation, or subsequent refeeding. However, molecular characterization of both enzymes and the regulated mechanisms affected by nutritional statuses and MPs remain unclear in zooplankton. In the present study, four full-length genes encoding ALPL and two genes encoding AMY were cloned and characterized from an isolated marine rotifer, Brachionus rotundiformis, including alplA, alplB, alplC, alplD, amy2a, and amy2al. AMY activity and expression of amy2a and amy2al were reduced by starvation and recovered after refeeding compared with feeding. ALPL activity remained unchanged among different statuses, while alplA, alplB and alplD were down-regulated by starvation and refeeding compared with feeding. ALPL activity was not affected by exposure to 10, 100 and 1000 μg/L MPs in rotifers subjected to feeding, starvation and refeeding, whereas AMY activity was significantly enhanced by 1000 μg/L MPs in rotifers subjected to refeeding. Gene expression of the tested genes, except amy2a, was significantly responsive to MPs, especially in the feeding rotifers, depending on MPs concentrations and nutritional statuses. Two-way ANOVA confirmed that these changes were strongly associated with the interaction between MPs concentrations and nutritional statuses. The present study is the first to demonstrate a nutritional status-dependent impact of MPs on immune and digestive responses, and provides more sensitive molecular biomarkers for assessing MPs toxicity using the species as model animals.

Diagnostic values of microRNA 27a in breast cancer patients

Breast cancer is one of the most malignant tumors in women across the globe. Diagnosis of breast cancer at early stages is essential to improve treatment outcomes and decrease mortality rates. There is a pressing need for new non-invasive biomarkers to improve early diagnosis of breast cancer. This study aims to assess plasma miR-27a for the early diagnosis of breast cancer. miR-27a was evaluated in a total of 95 blood samples, 40 newly diagnosed cancer patients, 20 patients with benign breast lesions, 20 females with positive family history for breast cancer and 15 apparently healthy controls, using quantitative real time polymerase chain reaction. Our results exhibited significantly higher expression level of plasma miR-27a in breast cancer patients (median= 8.3 and 19 fold change for early and late stages respectively) compared to controls, high risk group and benign group with (P <0.001) for each. Plasma miR-27a was significantly higher in late breast cancer (median=19 fold change) compared to early breast cancer (median= 8.3) with (P <0.001). There was no statistically significant difference of plasmamiR-27a levels in benign group (median=1.8 fold change) compared to both control group and high risk group. There was no statistically significant difference of plasma miR-27a levels in high risk group (median= 1.2 fold change) compared to control group (median= 1 fold change). We performed Receiver Operating Characteristic (ROC) analysis for discriminating malignant from non-malignant cases. Plasma miR-27a yielded an area under the curve (AUC) of 0.983 with sensitivity 97.5%, specificity 91% and accuracy 94%.We concluded that miR-27a expression level represents sensitive and specific non-invasive molecular biomarkers for diagnosis of breast cancer.

Effects of continuous and intermittent cadmium exposure on HPGL axis, GH/IGF axis and circadian rhythm signaling and their consequences on reproduction in female zebrafish: Biomarkers independent of exposure regimes

Typical biomarkers of cadmium (Cd) pollution have well been confirmed in fish from continuous exposure pattern. However, in a natural environment, fish may be exposed to Cd intermittently. In this study, juvenile female zebrafish were exposed for 48 days to 10 μg/L Cd continuously, 20 μg/L for 1 day in every 2 days or 30 μg/L for 1 day in every 3 days. The toxic effects were evaluated using 8 various physiological and biochemical endpoints like specific growth rate (SGR), 17β-estradiol (E2) and vitellogenin (VTG) concentrations in plasma, reproductive parameters (gonadosomatic index (GSI), egg-laying amount, spawning percentage, and hatching and mortality rate of embryos). Transcription of 59 genes related to hypothalamic-pituitary-gonadal-liver (HPGL) axis, circadian rhythm signaling and insulin-like growth factor (IGF) system was examined. SGR, spawning percentage, E2 and VTG levels declined in fish exposed to 10 and 20 μg/L Cd but remained relatively stable in fish exposed to 30 μg/L Cd. Exposure to 10, 20 and 30 μg/L Cd significantly reduced GSI, hatching rate and mortality rate. Similarly, mRNA expression of 27 genes were sensitive to both continuous and intermittent Cd exposure. Among these genes, expression levels of 10 genes had more than 5-fold increase or decrease, including mRNA levels of vtg1, vtg2, vtg3, esr1, igf2a, igf2b, igfbp5b, nr1d1, gnrh3 and gnrhr4. The most sensitive molecular biomarker was vtg3 expression with 1500–3100 fold increase in the liver. The present study, for the first time, provides effective candidate biomarkers for Cd, which are independent of exposure regimes.

Insulinoma-associated protein 1 (INSM1) may serve as a sensitive and specific immunohistochemical and molecular biomarker of neuroendocrine differentiation in pancreatic neuroendocrine tumors (PNETs)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Identifying conserved polychaete molecular markers of metal exposure: Comparative analyses using the Alitta virens (Annelida, Lophotrochozoa) transcriptome

Polychaetes are vital for evaluating the effects of toxic metals in marine systems, and sensitive molecular biomarkers should be integral to monitoring efforts. However, the few polychaete markers that exist are inconsistent, even within the same species, failing to identify gene expression changes in metal-exposed animals incurring clear metabolic costs. Comparing previously characterised polychaete metal-responsive genes with those of another carefully selected species could identify biomarkers applicable across polychaetes. The ragworm Alitta virens (Sars, 1835) is particularly suited for such comparisons due to its dominance of fully saline coastal areas, widespread distribution, large biomass, and its phylogenetic position relative to other polychaete ‘omic’ resources. A transcriptome atlas for A. virens was generated and an RNASeq-qPCR screening approach was used to characterise the response to chronic exposures of environmentally relevant concentrations of copper and zinc in controlled mesocosms. Genes presenting dramatic expression changes in A. virens were compared with known metal-responsive genes in other polychaetes to identify new possible biomarkers and assess those currently used. This revealed some current markers should probably be abandoned (e.g. Atox1), while others, such as GST-Omega, should be used with caution, as different polychaete species appear to upregulate distinct GST-Omega orthologues. In addition, the comparisons give some indication of genes that are induced by metal exposure across phylogenetically divergent polychaetes, including a suite of haemoglobin subunits and linker chains that could play conserved roles in metal-stress response. Although such newly identified markers need further characterisation, they offer alternatives to current markers that are plainly insufficient.

ANGPTL1 is a potential biomarker for differentiated thyroid cancer diagnosis and recurrence.

Differentiated thyroid cancer (DTC) is a common type of cancer among women with an increasing worldwide incidence rate. However, there are no specific and sensitive molecular biomarkers for DTC diagnosis or prognosis. Angiopoietin-like protein 1 (ANGPTL1) may be a novel tumor suppressor in lung, breast, colorectal and hepatocellular carcinoma. However, little is known about the influence of ANGPTL1 on the malignant properties of thyroid cancer cells or DTC recurrence in patients. Thus, the present study aimed to investigate the effects of ANGPTL1 on thyroid cancer malignancy or recurrence. The present study examined the mRNA levels of ANGPTL1 in thyroid cancer and paracancerous tissues using RNA sequencing data from The Cancer Genome Atlas. The present study also determined the effects of ANGPTL1 on thyroid cancer cell proliferation using the Cell Counting Kit-8 assay. Associations were identified among ANGPTL1 expression levels and thyroid cancer proliferation, migration and metastasis using The Cancer Genome Atlas data set and by Gene Set Enrichment Analysis. The expression of ANGPTL1 in patients with DTC and without recurrence was compared in order to assess its potential as a prognostic biomarker for DTC. In addition, ANGPTL1 concentrations in the serum of patients with DTC and individuals with benign thyroid nodules were compared to evaluate the sensitivity and specificity of ANGPTL1 as a predictive biomarker for DTC. The results of the present study demonstrated that ANGPTL1 expression levels were lower in thyroid cancer compared with those in adjacent normal thyroid tissues. ANGPTL1 expression was observed to decrease with thyroid cancer progression. In addition, ANGPTL1 was demonstrated to inhibit thyroid cancer cell proliferation, migration and invasion and ANGPTL1 expression levels were reduced in patients with DTC with recurrence compared with those in patients with non-recurrent DTC. Additionally, serum concentrations of ANGPTL1 in patients with DTC were decreased compared with those in individuals with benign thyroid nodules. In conclusion, ANGPTL1 may be a novel predictive biomarker for DTC diagnosis and recurrence in patients with DTC.

Functional annotation of genomic variation: DNA methylation episignatures in neurodevelopmental Mendelian disorders.

The breadth and complexity of genetic testing in patients with suspected Mendelian neurodevelopmental disorders has rapidly expanded in the past two decades. However, in spite of advances in genomic technologies, genetic diagnosis remains elusive in more than half of these patients. Epigenomics, and in particular genomic DNA methylation profiles, are now known to be associated with the underpinning genetic defects in a growing number of Mendelian disorders. These often highly specific and sensitive molecular biomarkers have been used to screen these patient populations, resolve ambiguous clinical cases and interpret genetic variants of unknown clinical significance. Increasing the diagnostic yield beyond genomic sequencing technologies has rapidly propelled epigenomics to clinical utilization, with recent introduction of DNA methylation 'EpiSign' analysis in clinical diagnostic laboratories. This review provides an overview of the principles, applications and limitations of DNA methylation episignature analysis in patients with neurodevelopmental Mendelian disorders, and discusses clinical implications of this emerging diagnostic technology.

Abstract IA35: Molecular characterization of sessile serrated adenoma/polyps: A risk factor for higher colorectal cancer that further associates with endometrial polyps in female African Americans

Abstract Colorectal cancer is the third leading cause of cancer-related deaths in the United States, and rates are highest among African Americans (AAs). Sessile serrated adenoma/polyps (SSA/Ps) may be precursors to up to 30% of all colorectal cancers. Flat and mucinous features make SSA/Ps difficult to detect and diagnose. As such, there is a need for specific sensitive molecular biomarkers for an accurate and reliable diagnosis. Our aim was to assess the diagnostic value of molecular biomarkers that may distinguish SSA/Ps from benign hyperplastic polyps (HPs) among AA SSA/P patients. We conducted a retrospective study of all colonoscopies (n=12,085) performed at Howard University Hospital (2010-2015), which confirmed 4,070 AA patients with polyps, including 252 with SSA/Ps. Gene expression and mutation frequency profiles were analyzed in a total of 47 patients (62 specimens: 29 SSA/Ps, 26 HPs, 3 tubular adenomas, and 4 normal tissues). We tested 4 transcripts (MUC6, FSCN1, SEMG1, and TRNP1) using qRT-PCR. MSI and BRAF mutations were analyzed. CIMP analysis was performed using CACNA1G, IGF2, NEUROG1, RUNX, SOCS, and MLH1. In a parallel study, we assessed the association between endometrial polyp occurrence in patients with different types of colorectal lesions. MUC6, SEMG1, TRNP1, and FSCN1 were significantly more expressed in SSA/Ps vs. HPs (P&amp;lt;0.05; fold differences of 37.2, 10.7, 5.8 and 2.5, respectively). BRAF mutation was found in 55.6% of SSA/Ps vs. 12.0% in HPs (P = 0.001). The frequency of CIMP was higher in SSA/Ps but not statistically significant, while MSI was more prevalent in HPs (P &amp;gt; 0.05). There was a higher loss of MLH1 expression in HPs than SSA/Ps (42.9% showing expression vs 70.3% IHC staining &amp;gt;=2 in HPs and SSA/Ps). The SSA/Ps in our AA study were primarily distal (67%). In female patients, SSA/Ps associated with the higher frequency of endometrial polyps (8% vs. 2% in controls, p=0.003). Our results show that MUC6-SEMG1-TRNP1 expression and BRAF mutation have the strongest correlation with SSA/Ps. The distal location might help explain why MSI and CIMP may not be optimal molecular biomarkers in African American patients with SSA/Ps. These markers may be of high relevance for the diagnosis of ambiguous lesions and will benefit patients’ management for scheduling follow-ups based on the nature of index lesions. Females with colon lesions of the SSA/Ps type might benefit from a screening for an endometrial polyp in an age-independent manner. Citation Format: Hassan Ashktorab, Saman Azam, Babak Shokrani, Edward Lee, Taraneh Arjomand, Priyanka Kanth, Don Delker, Adeyinka Laiyemo, John Caretheres, Mehdi Nouraie, Hassan Brim. Molecular characterization of sessile serrated adenoma/polyps: A risk factor for higher colorectal cancer that further associates with endometrial polyps in female African Americans [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr IA35.

Response Patterns of Biomarkers as Tools to Identify Toxic Effects of Cadmium and Lead on Bufo gargarizans Embryo.

Molecular biomarkers play an increasing crucial role in evaluating and predicting toxicity of metals. Expressions patterns of genes related to oxidative stress, apoptosis, immune and inflammation response in the Bufo gargarizans embryo exhibited a development dependent manner. The genes related to oxidative stress (HSP, GPx and SOD) are the first response in the development of embryo, followed by the apoptosis (Bax, BCLAF1 and TRAIL) and inflammation and immune response (SOCS3, IL-27 and IL-17D), respectively. Then, we have verified the HSP, Bax and SOCS3 IL-27 (expressed highest in their respective processes) exhibited the most significant changes in Cd-Pb mixed group compared with control. In addition, we found exposure of Cd-Pb mixed metals causes greater adverse effects than Cd, Pb alone on development and morphology of embryo. Overall, our results provide a useful tool to use the sensitive molecular biomarkers as indicators of developmental toxicity in amphibian embryo.

Drug Resistance Biomarkers and Their Clinical Applications in Childhood Acute Lymphoblastic Leukemia.

Biomarkers are biological molecules found in body fluids or tissues, which can be considered as indications of a normal or abnormal process, or of a condition or disease. There are various types of biomarkers based on their application and molecular alterations. Treatment-sensitivity or drug resistance biomarkers include prognostic and predictive molecules with utmost importance in selecting appropriate treatment protocols and improving survival rates. Acute lymphoblastic leukemia (ALL) is the most prevalent hematological malignancy diagnosed in children with nearly 80% cure rate. Despite the favorable survival rates of childhood ALL (chALL), resistance to chemotherapeutic agents and, as a consequence, a dismal prognosis develops in a significant number of patients. Therefore, there are urgent needs to have robust, sensitive, and disease-specific molecular prognostic and predictive biomarkers, which could allow better risk classification and then better clinical results. In this article, we review the currently known drug resistance biomarkers, including somatic or germ line nucleic acids, epigenetic alterations, protein expressions and metabolic variations. Moreover, biomarkers with potential clinical applications are discussed.

Integrated genomics-based approach to identify new therapeutic targets and cancer biomarkers for lung cancer.

e13019 Background: Because the number of lung cancer patients who show good response to standard therapies is still limited, development of new anti-cancer agents with minimum risk of adverse effects and highly sensitive molecular biomarkers is urgently required. Methods: We have been screening novel therapeutic targets and their companion biomarkers for lung cancer as follows; i) To identify up-regulated genes in lung cancers by the gene microarray analysis, ii) To verify the candidate genes for their low expression in normal organs, iii) To validate the clinicopathological significance of their protein expression by tissue microarray covering hundreds of lung cancers, and iv) To verify their function for the growth of lung cancer cells by siRNAs. Results: We identified dozens of candidate oncoproteins and selected a serine/threonine kinase LASK2 (lung cancer-associated kinase 2). Immunohistochemical analysis showed that strong LASK2 positivity was an independent prognostic factor for non-small cell lung cancer patients (P &lt; 0.0001). Suppression of LASK2 expression by its siRNAs inhibited proliferation of lung cancer cells. Introduction of LASK2 in mammalian cells also enhanced cellular growth in vitro and in mice model. Induction of LASK2 appeared to increase the levels of phosphorylation of oncogenic signal proteins for lung cancer. The data indicate that LASK2 is a prognostic biomarker and therapeutic target for lung cancers. Conclusions: Integrated genomics-based approach could facilitate the development of new cancer biomarkers as well as therapeutic targets for small molecules, monoclonal antibodies, nucleic acid drugs, and immunotherapies.

Molecular cloning, characterization of dax1 gene and its response to progesterone in Misgurnus anguillicaudatus

Progesterone (P4) are aquatic contaminants that can impair fish reproduction even in low concentrations. The aim of this study was to investigate the effects of P4 on the sex differentiation, by quantitative determination of transcriptional changes of a candidate target gene (dax1, has a function in the sex determination and gonadal differentiation of several vertebrate species) in Misgurnus anguillicaudatus. We first cloned and characterized the full-length cDNAs for the dax1 in M. anguillicaudatus (designated as Ma-dax1). Sequence analysis reveals that Ma-dax1 shares high homology with dax1 in other species. Quantitative real-time PCR (qRT-PCR) and in situ hybridization showed that Ma-dax1 gene was highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, central nervous system development and gonad development. For the P4 administration assay, groups of mature fish were exposed for 1, 7, 14, 21 and 28 days to nominal concentrations of 10, 100, and 1000 ng/L P4 in a flow-through system. Quantification of Ma-dax1 transcripts revealed the expression of Ma-dax1 mRNA is altered after P4 treatment in mature gonads. Those showed that P4 could influence the sexual development and sex differentiation in M. anguillicaudatus by disturbing sex differentiation-associated gene expression, and dax1 can be used as a sensitive molecular biomarker for early warning to monitor the environmental progestins chemicals in fresh water environment.

Radiation-induced bystander effect following hypo- fraction technique of grid therapy by use of sensitive molecular markers

Introduction: Radiation-induced bystander effect (RIBE) is a well-known response associated with the induction of radiation effects in non-irradiated cells via signaling of hit ones. As this so-called phenomenon has been largely investigated in low dose levels, few studies focused on higher dose levels such as grid therapy. Grid therapy or spatially fractionated grid radiation therapy (SFGRT) is a hypo-fraction technique for treatment of advance and bulky malignant tumors. Different advantages of SFGRT including minimal skin side effects and effective tumor control despite the small portion of exposure based on grid pattern, made it as an attractive therapy approach. SO the aim of this research was determination of bystander response following the mentioned therapy method. Materials and Methods: In this study, we evaluated RIBE via distinct protocols of media transfer and cell to cell contact following grid therapy. Two different human cell lines (SCC and Hela) were irradiated by challenging dose of 10 Gy with available grid block attached to the linac head in single radiation field. Irradiation with Grid pattern of 145 circular fields, 13mm in diameter with 17mm center to center distance created the cell to cell contact of directly irradiated and bystander cells. To fulfill the aim of media transfer of our study, bystander cells or grid adjacent cells were irradiated by 1.5Gy receiving the conditioned medium of hit cells. Based on RIBE DNA damage and DSB formation, phosphorylation of histone H2AX could be the novel sensitive marker for early detection of DSBs in intact cells. So Bystander effect has been assessed by use of sensitive molecular biomarkers of gamma h2ax as well as gene expression. Results& Conclusion: Our results indicated the occurrence of RIBE following the high dose hypo-fraction technique of grid therapy and emphasized on the role of sensitive molecular biomarkers in bystander DNA damage mechanism.

Molecular Nodal Staging Using miRNA Expression in Lung Cancer Patients by Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration

Background: The limited negative predictive value of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has often been discussed. Objective: The aim of this study was to identify a highly sensitive molecular biomarker for lymph node staging by EBUS-TBNA. Methods: Five microRNAs (miRNAs) (miR-200a, miR-200b, miR-200c, miR-141, and let-7e) were selected as biomarker candidates for the detection of nodal metastasis in a miRNA expression analysis. After having established a cutoff level of expression for each marker to differentiate malignant from benign lymph nodes among surgically dissected lymph nodes, the cutoff level was applied to snap-frozen EBUS-TBNA samples. Archived formalin-fixed paraffin- embedded (FFPE) samples rebiopsied by EBUS-TBNA after induction chemoradiotherapy were also analyzed. Results: The expression of all candidate miRNAs was significantly higher in metastatic lymph nodes than in benign ones (p < 0.05) among the surgical samples. miR-200c showed the highest diagnostic yield, with a sensitivity of 95.4% and a specificity of 100%. When the cutoff value for miR-200c was applied to the snap-frozen EBUS-TBNA samples, the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 97.4, 81.8, 95.0, 90.0, and 94.0%, respectively. For restaging FFPE EBUS- TBNA samples, the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 100, 60.0, 80.0, 100, and 84.6%, respectively. Among the restaged samples, 4 malignant lymph nodes were false negative by EBUS-TBNA, but they were accurately identified by miR-200c. Conclusions: miR-200c can be used as a highly sensitive molecular staging biomarker that will enhance nodal staging of lung cancer.