TaqMan Gene Expression Assays Research Articles

Abstract 1024: Differential gene expression of colorectal cancer biomarkers in Hispanic individuals

Abstract Colorectal cancer (CRC) is a leading cause of cancer death among men and women in Puerto Rico and the United States. About 60-70% of CRC cases are found at advanced stages (III/IV), suggesting limited adherence to available screening methods. Several molecular biomarkers have been identified as discriminatory for CRC (e.g., KRAS, NRAS, MSI), improving CRC management and patient outcomes. This study evaluates the differential expression patterns of DKK3, PKM2, IGFBP2, and SEPT9 genes between matched CRC tumors and adjacent mucosa samples. The secondary outcome was to examine the association between the presence of the selected biomarkers in tissue compared to blood samples. Using a retrospective study design, we examined 112 tissue samples of Hispanic individuals with a pathology-confirmed CRC diagnosis recruited throughout the Puerto Rico Familial Colorectal Cancer Registry (PURIFICAR). We quantified expression levels of target genes by RT-qPCR normalized with endogenous control GAPDH, using TaqMan Gene Expression Assay in the QuantStudio 3 (Applied Biosystems). Wilcoxon signed-rank test was used to calculate differences between gene expression from tumor and adjacent mucosal tissue samples. Additionally, 39 paired blood and tissue samples were analyzed to explore the association between biomarkers in these two types of samples. Protein biomarkers were measured using a commercially available ELISA kit according to manufacturer protocols (Thermo Scientific Federick, MD and Biosource San Diego, CA). mSEPT9 was measured using the Epi proColon® 2.0 CE test. The association between biomarker presence in tissue and blood samples was assessed using the Chi-square test of independence, with biomarker concentrations dichotomized at the median value. Statistical analyses were performed using Stata 17 and R version 4.3.1. DKK3 and IGFBP2 expression levels were higher among CRC tumor samples. However, expression levels of PKM2 and SEPT9 genes were slightly lower. DKK3 was the only target gene significantly different (p<0.05) when comparing overall gene expression between the matched samples. Upon comparison between early and advanced CRC stages, DKK3 expression levels were higher in advanced-stage tumor samples when compared to adjacent mucosal tissue samples (p<0.05). Moreover, a significant association between IGFBG2 presence in tissue compared to blood was detected at a 90% confidence level (p-value<0.10). This association was evident in early-stage CRC cases; there is a relationship between IGFBG2 biomarker presence in tissue and IGFBG2 biomarker presence in blood (p<0.05). Our findings indicate DKK3 and IGFBP2 as potential biomarkers for CRC progression and early detection. The study’s insights into the differential expression patterns of these biomarkers can inform targeted screening strategies. Further research is needed to assess their potential to enhance early tumor detection in Hispanics. Citation Format: Elba V. Caraballo-Rivera, Hilmaris Centeno-Girona, Gabriel De Jesus-Astacio, Camille Zenon-Melendez, Brenda Torres-Velasquez, Madeline Martir-Ocasio, Marcia R. Cruz-Correa. Differential gene expression of colorectal cancer biomarkers in Hispanic individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1024.

The Association between Inefficient Repair of DNA Double-Strand Breaks and Common Polymorphisms of the HRR and NHEJ Repair Genes in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation affecting up to 2.0% of adults around the world. The molecular background of RA has not yet been fully elucidated, but RA is classified as a disease in which the genetic background is one of the most significant risk factors. One hallmark of RA is impaired DNA repair observed in patient-derived peripheral blood mononuclear cells (PBMCs). The aim of this study was to correlate the phenotype defined as the efficiency of DNA double-strand break (DSB) repair with the genotype limited to a single-nucleotide polymorphism (SNP) of DSB repair genes. We also analyzed the expression level of key DSB repair genes. The study population contained 45 RA patients and 45 healthy controls. We used a comet assay to study DSB repair after in vitro exposure to bleomycin in PBMCs from patients with rheumatoid arthritis. TaqMan SNP Genotyping Assays were used to determine the distribution of SNPs and the Taq Man gene expression assay was used to assess the RNA expression of DSB repair-related genes. PBMCs from patients with RA had significantly lower bleomycin-induced DNA lesion repair efficiency and we identified more subjects with inefficient DNA repair in RA compared with the control (84.5% vs. 24.4%; OR 41.4, 95% CI, 4.8-355.01). Furthermore, SNPs located within the RAD50 gene (rs1801321 and rs1801320) increased the OR to 53.5 (95% CI, 4.7-613.21) while rs963917 and rs3784099 (RAD51B) to 73.4 (95% CI, 5.3-1011.05). These results were confirmed by decision tree (DT) analysis (accuracy 0.84; precision 0.87, and specificity 0.86). We also found elevated expression of RAD51B, BRCA1, and BRCA2 in PBMCs isolated from RA patients. The findings indicated that impaired DSB repair in RA may be related to genetic variations in DSB repair genes as well as their expression levels. However, the mechanism of this relation, and whether it is direct or indirect, needs to be elucidated.

The association of Sort1 expression with LDL subfraction and inflammation in patients with coronary artery disease

Background Controversial effect of sortilin on lipoprotein metabolism in the development of atherosclerosis reveals the need for more extensive research Objectives The aim of this study was to investigate the association between Sort1 gene expression and lipids, lipoprotein subfractions, and inflammation in CAD. Methods The study population included 162 subjects with CAD and 49 healthy individuals. The Sort1 gene expression level was determined by qRT-PCR using Human Sortilin TaqMan Gene Expression Assays. Lipoprotein subclasses were analysed by the Lipoprint system. Serum levels of apolipoprotein and CRP were measured by autoanalyzer. Results Sort1 gene expression and atherogenic subfraction (SdLDL) levels were significantly higher (p < 0.001) while atheroprotective subfraction (LbLDL) was lower in the subjects with CAD (p < 0.050). Also, increased Sort1 gene expression levels were observed in those with higher CRP values. Conclusions Our findings reveal that the high Sort1 gene expression has a prominent linear relationship with both the atherogenic LDL phenotype and proinflammation, thereby might contribute to the occurrence of CAD.

The role of SOD2 and NOS2 genes in the molecular aspect of bladder cancer pathophysiology

Bladder cancer (BC) is a severe health problem of the genitourinary system and is characterised by a high risk of recurrence. According to the recent GLOBOCAN report, bladder cancer accounts for 3% of diagnosed cancers in the world, taking 10th place on the list of the most common cancers. Despite numerous studies, the full mechanism of BC development remains unknown. Nevertheless, precious results suggest a crucial role of oxidative stress in the development of BC. Therefore, this study explores whether the c. 47 C > T (rs4880)—SOD2, (c. 1823 C > T (rs2297518) and g.-1026 C > A (rs2779249)—NOS2(iNOS) polymorphisms are associated with BC occurrence and whether the bladder carcinogenesis induces changes in SOD2 and NOS2 expression and methylation status in peripheral blood mononuclear cells (PBMCs). In this aim, the TaqMan SNP genotyping assay, TaqMan Gene Expression Assay, and methylation‐sensitive high‐resolution melting techniques were used to genotype profiling and evaluate the expression of the genes and the methylation status of their promoters, respectively. Our findings confirm that heterozygote of the g.-1026 C > A SNP was associated with a decreased risk of BC. Moreover, we detected that BC development influenced the expression level and methylation status of the promoter region of investigated genes in PBMCs. Concluding, our results confirmed that oxidative stress, especially NOS2 polymorphisms and changes in the expression and methylation of the promoters of SOD2 and NOS2 are involved in the cancer transformation initiation of the cell urinary bladder.

Small‑molecule PKR‑like endoplasmic reticulum kinase inhibitors as a novel targeted therapy for Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in worldwide and remains a therapeutic challenge due to the low efficacy of current treatments. Numerous studies have demonstrated the pivotal role of endoplasmic reticulum (ER) stress in PD pathogenesis. ER stress, followed by activation of the protein kinase RNA‑like endoplasmic reticulum kinase (PERK)‑dependent branch of the unfolded protein response signaling pathway, ultimately leads to neural cell death and dopaminergic neurodegeneration in PD. Therefore, the present study evaluated the effectiveness of the small‑molecule PERK inhibitor LDN‑87357 in an invitro PD model using the human neuroblastoma SH‑SY5Y cell line. To assess the mRNA expression levels of the pro‑apoptotic ER stress markers, the TaqMan Gene Expression Assay was performed. Cytotoxicity was assessed using a colorimetric 2,3‑bis‑(2‑methoxy‑4‑nitro‑5‑sulfophenyl)‑ 2H‑tetrazolium‑5‑carboxanilide assay and apoptosis was assessed using a caspase‑3 assay. Moreover, cell cycle progression was evaluated using flow cytometry. The results indicated that LDN‑87357 treatment induced a significant decrease in ER stress markers gene expression in SH‑SY5Y cells exposed to ER stress. Furthermore, LDN‑87357 significantly increased viability, diminished apoptosis and restored the normal cell cycle distribution of SH‑SY5Y cells after ER stress induction. Therefore, the evaluation of small‑molecule PERK inhibitors, such as LDN‑87357, may lead to the development of novel therapeutic strategies against PD.

Variability, Expression, and Methylation of IL-6 and IL-8 Genes in Bladder Cancer Pathophysiology.

Bladder cancer (BC) is the 10th most common form of cancer globally, but its complete aetiology is still unknown. Nevertheless, there is evidence that chronic inflammation plays a role in the development and progression of BC. Therefore, the presented study aimed to detect a potential association between selected single nucleotide polymorphisms (SNPs)-rs1800797 and rs2069845 in IL-6 and rs2227307 in IL-8-and BC development, as well as to identify the impact of BC on the level of expression and methylation of IL-6 and IL-8 promoters in PBMCs with the use of the TaqMan SNP genotyping assay, TaqMan gene expression assay, and methylation-sensitive high-resolution melting techniques. We did not find any association between the genotypes and combined genotypes of all studied polymorphisms and the occurrence of BC. However, we found that BC patients were characterised by decreased IL-6 and IL-8 mRNA expression levels compared to the controls. Additionally, the methylation status of the IL-6 promoter was higher in controls than in BC patients. Our findings suggest that inflammation may be involved in the development and progression of BC.

Eosinophilic Esophagitis: Cytokines Expression and Fibrotic Markers in Comparison to Celiac Disease.

Introduction: Eosinophilic esophagitis (EoE) is now recognized as the main inflammatory condition that leads to fibrosis, unlike other chronic inflammatory gastrointestinal diseases, such as celiac disease. The aim of our study is to characterize the collagen deposition and cytokine expression involved in the fibrogenic response in patients affected by EoE in comparison to celiac disease. Materials and Methods: Consecutive patients with a clinical suspicion of untreated EoE or active celiac disease were enrolled. In the control group, patients with negative upper endoscopy were included. Total RNA was isolated from biopsy specimens using a commercial kit (SV Total RNA Isolation System, Promega Italia Srl). Quantitative real-time PCR (qRT-PCR) was performed in triplicate using a StepOne™ Real-Time PCR instrument (Thermo Fisher Scientific, Monza, Italy). mRNA encoding for inflammatory molecules: interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 13 (IL-13), and fibrotic markers: transforming growth factor beta 1 (TGF-β), mitogen-activated protein kinase kinase kinase 7 (MAP3K7), serpin family E member 1 (SERPINE1), were quantified using TaqMan Gene Expression Assays (Applied Biosystems). RESULTS. In EoE, the qPCR analysis showed an increase in all the inflammatory cytokines. Both IL-5 and Il-3 mRNA expression resulted in a statistically significant increase in oesophageal mucosa with respect to the celiac duodenum, while no differences were present in IL-4 expression. TGF-β expression was similar to the controls in the mid esophagus but reduced in the distal EoE esophagus (RQ: 0.46 ± 0.1). MAP3K7 expression was reduced in the mid esophagus (RQ: 0.59 ± 0.3) and increased in the distal esophagus (RQ: 1.75 ± 0.6). In turn, the expression of SERPINE1 was increased in both segments and was higher in the mid than in the distal esophagus (RQ: 5.25 ± 3.9, 1.92 ± 0.9, respectively). Collagen deposition was greater in the distal esophagus compared to the mid esophagus [18.1% ± 8 vs. 1.3% ± 1; p = 0.008]. Conclusions: The present study confirms the esophageal fibrotic involution involving the distal esophagus and shows that the inflammatory pathway in EoE is peculiar to this disease and different from other chronic inflammatory gastrointestinal disorders such as celiac disease.

Agomelatine Changed the Expression and Methylation Status of Inflammatory Genes in Blood and Brain Structures of Male Wistar Rats after Chronic Mild Stress Procedure.

The preclinical research conducted so far suggest that depression development may be influenced by the inflammatory pathways both at the periphery and within the central nervous system. Furthermore, inflammation is considered to be strongly connected with antidepressant treatment resistance. Thus, this study explores whether the chronic mild stress (CMS) procedure and agomelatine treatment induce changes in TGFA, TGFB, IRF1, PTGS2 and IKBKB expression and methylation status in peripheral blood mononuclear cells (PBMCs) and in the brain structures of rats. Adult male Wistar rats were subjected to the CMS and further divided into matched subgroups to receive vehicle or agomelatine. TaqMan gene expression assay and methylation-sensitive high-resolution melting (MS-HRM) were used to evaluate the expression of the genes and the methylation status of their promoters, respectively. Our findings confirm that both CMS and antidepressant agomelatine treatment influenced the expression level and methylation status of the promoter region of investigated genes in PBMCs and the brain. What is more, the present study showed that response to either stress stimuli or agomelatine differed between brain structures. Concluding, our results indicate that TGFA, TGFB, PTGS2, IRF1 and IKBKB could be associated with depression and its treatment.

Protein and functional isoform levels and genetic variants of the BAFF and APRIL pathway components in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is the prototype of an autoimmune disease. Belimumab, a monoclonal antibody targets BAFF, is the only biologic approved for SLE and active lupus nephritis. BAFF is a cytokine with a key-regulatory role in the B cell homeostasis, which acts by binding to three receptors: BAFF-R, TACI and BCMA. TACI and BCMA also bind APRIL. Many studies reported elevated soluble BAFF and APRIL levels in the sera of SLE patients, but other questions about the role of this system in the disease remain open. The study aimed to investigate the utility of the cytokine levels in serum and urine as biomarkers, the role of non-functional isoforms, and the association of gene variants with the disease. This case–control study includes a cohort (women, 18–60 years old) of 100 patients (48% with nephritis) and 100 healthy controls. We used ELISA assays to measure the cytokine concentrations in serum (sBAFF and sAPRIL) and urine (uBAFF and uAPRIL); TaqMan Gene Expression Assays to quantify the relative mRNA expression of ΔBAFF, βAPRIL, and εAPRIL, and next-generation sequencing to genotype the cytokine (TNFSF13 and TNFSF13B) and receptor (TNFRSF13B, TNFRSF17 and TNFRSF13C) genes. The statistical tests used were: Kruskal–Wallis (qualitative variables), the Spearman Rho coefficient (correlations), the Chi-square and SKAT (association of common and rare genetic variants, respectively). As expected, sBAFF and sAPRIL levels were higher in patients than in controls (p ≤ 0.001) but found differences between patient subgroups. sBAFF and sAPRIL significantly correlated only in patients with nephritis (rs = 0.67, p ≤ 0.001) and βAPRIL levels were lower in patients with nephritis (p = 0.04), and ΔBAFF levels were lower in patients with dsDNA antibodies (p = 0.04). Rare variants of TNFSF13 and TNFRSF13B and TNFSF13 p.Gly67Arg and TNFRSF13B p.Val220Ala were associated with SLE. Our study supports differences among SLE patient subgroups with diverse clinical features in the BAFF/APRIL pathway. In addition, it suggests the involvement of genetic variants in the susceptibility to the disease.

Association between metabolic disorders and seminal plasma miRNA levels: a pilot study

BackgroundExcess weight and metabolic disorders have a negative impact on male reproductive functions. The mechanisms involved are numerous and complex and epigenetic mechanisms may also be involved, notably through the small non-coding RNAs. Among them, microRNAs (miRNAs) are of particular interest. This preliminary study aimed to identify the miRNAs differentially enriched in seminal plasma related to metabolic disorders and if some are also associated with spermatic parameters alterations. One hundred and sixty men between 18 to 45 years, partners of infertile couple, were included in this cohort. The miRNAs associated with metabolism were selected from the literature and assayed by quantitative real-time PCR using TaqMan gene expression assays. A subset of those with an interesting profile in seminal plasma were secondarily tested in blood.ResultsAmong the 11 selected miRNAs, seven were detected in seminal plasma (miR10b, miR19a, miR19b, miR34b, miR34c, miR133b, miRlet7c). A negative correlation was observed between seminal miR19a levels and metabolic syndrome, blood glucose and C-peptide. Seminal miR19b levels were also negatively correlated with metabolic syndrome. Seminal miR34c levels were negatively correlated with body mass index (BMI) and waist circumference. Seminal miR133b levels were positively correlated with BMI, waist circumference and leptin levels. Interestingly, modifications of miRNAs in seminal plasma seem specific since highlighted above correlations were not retrieved in the blood plasma for the miR19a, 19b, 10b, 34c.ConclusionFew metabolic and anthropometric disorders are correlated with the level of specific miRNAs in seminal plasma. Further studies will be required to decipher if other small non-coding RNAs may also be correlated with metabolic and anthropometric disorders and to assess their potential implication in the alteration of reproductive functions in men with obesity or metabolic disorders.Clinical studyMetabolic Syndrome and Male Infertility (Metasperme): Trial registration: NCT01974947. Registered 18 July 2013.

Genetic variants, gene expression, and soluble CD36 analysis in acute coronary syndrome: Differential protein concentration between ST-segment elevation myocardial infarction and unstable angina.

BackgroundAtherosclerosis plays an important role in the pathophysiology of acute coronary syndrome (ACS). CD36 is a scavenger receptor involved in lipid metabolism. Some single‐nucleotide variants in the non‐coding region could indirectly alter the expression and the function of the protein.ObjectiveThe aim of this study was to investigate the gene and protein expression associated with CD36 variants (rs1194182;C > G; rs1049654;C > A, rs1334512;G > T, and rs3211892;G > A) in ACS patients from the western Mexican population.MethodsWe recruited 310 ACS patients and 308 subjects in the control group (CG). Genotyping was determined by TaqMan SNP genotyping assays. CD36 expression at the mRNA level was quantified by TaqMan gene expression assays. Soluble CD36 (sCD36) was measured by enzyme‐linked immunosorbent assay.ResultsWe show that rs1194182G > C variant provides a protective effect with a 1.7‐fold lower susceptibility to develop ACS (p = 0.03); however, this association was masked by diabetes and dyslipidemia. We observed a higher sCD36 concentration in patient with ST‐segment elevation myocardial infarction (STEMI) compared with patients with unstable angina (UA) (p = 0.038). Likewise, in diabetic patients versus non‐diabetic (p < 0.001). We observed in patients an increase in CD36 mRNA expression (1.91 times higher) than in the CG (p = 0.02).ConclusionThe rs1194182 seems to be associated with diabetes in a risky manner, in ACS patients and protective for dyslipidemia in both groups. The concentration of sCD36 seems to be associated with the clinical spectrum of the ACS patients and the presence of diabetes, since patients with STEMI present significantly elevated level compared with UA.

MO060: HIF1Α and Succinate Pathway: Role in Deceased Kidney Donors Inflammation

Abstract BACKGROUND AND AIMS The type of donor has an important impact on the renal prognosis of the recipient. Thus, it has been observed that the decline of renal function is higher in deceased donor (DD) than those observed in living donors (LD). These could be related with the pre-implantation inflammation state of the donor and the development of fibrotic processes. Own data indicate that the enzymatic complex succinate dehydrogenase (SDH) has a lower gene expression in DD grafts and is associated with inflammatory markers, macrophages and purinergic receptors and a worse renal function after transplantation. The aim of this study is to determine that in renal DD donation, ischemia/hypoxia can increase renal succinate levels, inducing infiltration of inflammatory cells. METHOD Preimplantation kidneys biopsies from 47 DD and 19 LD were collected in our institution. About 159 genes from these samples were analyzed by qRT–PCR with the TaqMan gene expression assay. Relative quantification of gene expression was performed with three internal controls. Genes with high positive correlation with HIF1α (Rho &amp;gt; 0.5) have been grouped in clusters using STRING database (http://string-db.org/). Succinate levels were measured in serum from 27 renal DD and in 10 healthy volunteers with the EnzyChromTM Succinate Assay Kit. All statistical data were analyzed using GraphPad Prism 5. HIF, SDH subunits and succinate levels in serum were analyzed with a Student's T-test (P-value of 0.001). Pearson correlation Test (Rho &amp;gt;0.5) was performed to analyze interdependence of HIF1α with the rest of genes. RESULTS To determine the hypoxia status of kidney donors, transcription levels of HIF1α and HIF2α were determined from renal tissue samples. In the graphic (Figure. 1A), a significant increase in HIF1α genic expression is observed in DD compared with LD (P &amp;lt; 0.001). The next figure (Figure. 2B) shows clusters in DD that are not present in LD and have a high correlation with HIF1a (Rho &amp;gt; 0.5). Twenty-four genes showed this strong correlation and can be grouped in three different clusters: Cluster 1: Transcription factors and metalloproteinases, Cluster 2: Extracellular Matrix (ECM) proteins and integrins and finally, Cluster 3: Inflammatory and Macrophages Markers. As is shown Figure 2A, we found reduced mRNA expression of all the SDH subunits (SDHA, SDHB, SDHC and SDHD) (P &amp;lt; 0.001) in DD samples compared with LD ones. As a consequence, succinate cannot be converted to fumarate in concordance with the greater succinate levels (P = 0.002) observed in sera of DD shown in the picture (Figure. 2B). CONCLUSION These results indicate that kidneys from DD undergo a greater hypoxia compared with LD, demonstrated by the increased transcription rate of HIF1α. Hypoxia not only increases HIF1α but also may disrupt the Krebs cycle, reducing the expression of SDH subunits, increasing extracellular levels of succinate that has a role in the maintenance of HIF1α levels, which could take part in a sustained inflammatory/anti-inflammatory process in kidney from DD.

Abstract P2-07-08: Identification of UGT2B15 as a potential biomarker in response to neoadjuvant therapy in HER2+ breast cancer

Abstract INTRODUCTION. In response to neoadjuvant therapy, pathological complete response (pCR; lack of residual disease in breast and lymph nodes), has been proposed as a prognostic marker for long-term clinical outcomes, such as disease-free (DFS) and overall survival (OS), in human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancer. In clinical practice, recognizing those patients likely to achieve such responses proves challenging to the oncologist, making the identification of new useful biomarkers vital. Here, we searched for potential biomarkers that anticipate pCR to neoadjuvant therapy in HER2-positive, hormone receptor-negative breast cancer tissue samples. METHODS. Patients with early and locally advanced breast cancer, diagnose as HER2-positive, hormone receptor-negative by immunohistochemistry (IHC) at the time of neoadjuvant treatment were included. All samples were collected from biobank at Hospital Universitario Virgen del Rocio. We defined two groups: responder (R) and non-responder (nR) and analyzed 18 samples in the discovery cohort (10 R vs 8 nR) and 12 samples in the validation cohort (6 R vs 6 nR).The RNA for the study was extracted from tissue fixed in formaldehyde and paraffin embedded. The extraction of the RNA was carried out using the commercial kit RecoverAll Total Nucleic Acid Isolation Kit from Ambion (Applied Biosystems). RNA was quantified by Qubit RNA HS Assay Kit (Molecular Probes). Before hybridization, RNA for the discovery cohort was amplified using GeneChip WT Pico Kit (Applied Biosystems).We analyzed transcript expression using ClariomD array. Differential expression between the two groups was analyzed using in-house R scripts (version 3.5.1). Data were corrected and normalized using Robust Multi-array Average method. Expression was summarized at gene level using the corresponding annotation for ClariomD BrainArray. Gene validation was performed by qPCR using TaqMan Gene Expression Assay (Applied Biosystem). RESULTS. Considering a fold change ≥ 2 and an adjusted p-value &amp;lt;0.05 as statistically significant, we found 53 differentially expressed transcripts: 51 downregulated transcripts (lower expression in R) and 2 upregulated transcripts (higher expression in R). The RNA was annotated as non-coding RNAs in over 25% of the cases. The distribution of such molecules was as followed: 8 long non-coding RNAs (56%); 3 non-coding RNAs (22%) and 3 pseudo-genes (22%). Regarding protein coding transcripts, gene ontology analysis revealed an enrichment of terms associated to metabolic processes and response to toxic substance.As expected, ERBB2, which encodes for HER2, appeared at the top of the list with a patent upregulation of expression in the responder group when compared with non-responder patients. To validate the data, we used qPCR and ERBB2 expression as a positive control of the results (p-value=0.0380). Our data showed a significant downregulation of UDP-glucuronosyltransferase 2B15 (UGT2B15; p-value= 0.0173), which encodes a glycosyltransferase, involved in the metabolism and elimination of toxic compounds. CONCLUSIONS. The ability to predict which patients will benefit from neoadjuvant therapy and achieve a pCR would allow for improved patient stratification and more personalized medicine. Here, we identified a set of transcript differentially expressed (FC&amp;gt;2; adjusted p-value&amp;lt;0.05) in patients that achieve pCR (R) when compared with tissue samples with residual disease (nR). We usedERBB2 expression as a positive control to validate the data and show that UGT2B15 is consistently upregulated in non-responder patients. Further work is needed to elucidate UGT2B15 role, but it is worth mentioning that an increased rate of glucuronidation has been associated with a loss of potency for the target drugs. Citation Format: Ana Gil-Torralvo, Marta Benavent, Maria A Dominguez-Cejudo, Alejandro Falcon, Begoña Vieites, Sonia Molina-Pinela, Manuel Ruiz, Álvaro Montaño, Rosario Gónzalez, Julia Martínez, Juan de la Haba, Antonio Rodríguez, Maria I Queipo, Begoña Jímenez, Javier Salvador-Bofill. Identification of UGT2B15 as a potential biomarker in response to neoadjuvant therapy in HER2+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-08.

Abstract P1-08-30: Validation of the AAGAB gene as a novel on-treatment biomarker predicting pCR in patients receiving NACT for early breast cancer

Abstract Background: Neoadjuvant chemotherapy (NACT) for breast cancer is given prior to surgery, aiming to reduce tumour burden and provide early information on response to treatment. Tumours with a pathological complete response (pCR) following NACT are less likely to recur than those in patients with residual disease following NACT, suggesting that pCR following NACT may be a surrogate of good prognosis. In addition, NACT provides a “window of opportunity” where sequential sampling of a tumour enables observation of changes occurring in response to treatment. We previously demonstrated that on-treatment biomarkers improve the accuracy of predicting response before resection, by performing differential gene expression analysis across sequential samples from 50 patients, across 3 time points; pre NACT, mid chemo and post NACT (Bownes et al, 2019). A single gene marker (AAGAB) was identified as a novel potential on-treatment biomarker for pCR, with greater predictive accuracy than currently established prognostic tests, supporting the potential value of on-treatment biomarker monitoring in breast cancer to improve the accuracy of tumour response prediction. Aim: To validate and test the finding that increased expression of AAGAB at mid-chemotherapy associates with pCR in ER+ve and HER+ve breast cancer patients receiving NACT. Method: mRNA extracted from tumour from the mid-chemotherapy time point during NACT, from 28 breast cancer patients, was analysed using real-time reverse transcription-PCR (real-time RT-PCR). Firstly, mRNA was reverse transcribed using Invitrogen SuperScript IV Reverse Transcriptase (RT), then cDNA was used in an Applied Biosystems real-time RT-PCR Taqman Gene Expression Assay to measure AAGAB gene expression. GAPDH and SYMPK Taqman assays were used as endogenous controls, to allow calculation of (ΔΔCt) using the Comparative CT Method. Quantitative PCR was carried out on the Applied Biosystems StepOnePlusTM Real-Time PCR System and Relative Quantitation (RQ) was calculated using 2^-(ΔΔCt). For each patient RCB (residual cancer burden) was plotted against AAGAB expression and statistical significance calculated using the Wilcoxon rank sum exact test. Results: As a group, patients with RCB scores of 0 and I demonstrated a significantly higher level of expression that patients with RCB II and RCB III. We also observed that patients with pCR after NACT demonstrated higher levels of AAGAB expression. This increased expression was also seen in patients with a decrease in tumour cellularity of more than 85%. Conclusion: Our data suggest that measurement of AAGAB expression may provide an informative on-treatment biomarker of response to NACT. Citation Format: Fiona Semple, Alastair Ironside, Richard Bownes, Carlos Martinez-Perez, Natalie Wilson, Arran Turnbull, Olga Oikonomidou. Validation of the AAGAB gene as a novel on-treatment biomarker predicting pCR in patients receiving NACT for early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-30.

Changes In Activity of HSPA1A and HSPA1L is Associated with Early Acute Pancreatitis Severity

Introduction: Genome assessment shows promise as a potential method for predicting acute pancreatitis (AP) severity. We hypothesized that Heat Shock Protein 70 (HSP70) single nucleotide polymorphisms (SNPs) and expression changes may play a role in early detection of acute pancreatitis severity. Methods: A total of 57 AP patients and 52 age- and sex-matched healthy controls were studied. Peripheral blood samples from pancreatitis patients were collected upon admission. Two SNPs of the HSP70-gene family were selected. RNA was extracted parallel to genomic DNA from AP patients (N=12) and healthy controls (N=21).Gene expression of two HSP70 family members HSPA1A and HSPA1L was measured using TaqMan gene expression assays via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: Major allele frequency in HSPA1A gene was 1 (A>G) for AP patients and 1 (A>G) for controls. For HSPA1A, the gene major allele frequency was 0.672 (A>C) for patients and 0.7 (A>C) for controls, the major allele frequencies were 0.638 (A>G) and 0.672 (G>C) for patients and 0.85 (A>G) and 0.65 (G>C) for controls, respectively. The allele frequencies differed between AP patients with organ failure and those with mild disease. Following gene expression of HSPA1L and SNP relationship analysis, AP patients with heterozygous genotype had higher expression levels as compared to non-AP patients (p=0.014). Conclusions: Our data suggest that polymorphism of the HSP70 promoter region may be a risk factor for developing severe acute pancreatitis. Further study to determine the urine levels of HSP70 protein expression is needed to confirm the protective mechanism of HSP70.

GPR56 gene down-regulation in patients with Klinefelter Syndrome: a candidate for infertility?

The etiology of azoospermia in patients with Klinefelter Syndrome (KS) is still unknown. The protein codified by the G protein-couple receptor 56 (GPR56) belongs to the adhesion family of G protein-coupled receptors (GPRs). Its mutations are involved in the pathogenesis of intellectual disability and, according to animal studies, infertility. As the expression of GPR56 in patients with KS has not been investigated so far, this study was undertaken with the purpose of evaluating its expression in peripheral blood mononuclear cells (PBMCs) of patients with KS and normal controls. This age-matched case-control study was performed in 10 patients with KS and 10 controls. Patients and controls underwent to blood sampling for next-generation sequencing (NGS) analysis, and differentially expressed mRNAs were identified using DESeq2 v.1.12. QRT-PCR was used to validate the results obtained by NGS analysis. TaqMan Gene Expression Assay primers were used to carry out the real-time PCR analysis for GPR56. GPR56 was down-regulated by -2081-fold (q-value <0.05) in PBMCs of patients with KS compared to controls. NGS data were confirmed by QRT-PCR. The possible contribution of the GPR56 gene down-regulation in the pathogenesis of spermatogenic failure in patients with KS is worthy to be further explored.

The Association of Hif-1α Expression with Primary Resistance to Azacytidine in Patients with High Risk MDS — Preliminary Data of the Hellenic MDS Study Group

Introduction : Azacitidine (AZA) is the standard of care for patients with high-risk myelodysplastic syndromes (MDS) (IPSS intermediate-2 or high) and patients with AML not candidate for treatment with intensive chemotherapy. Nevertheless, approximately 40% of patients fail to respond, whereas even responders will inevitably relapse. Although some clinical parameters and genetic mutations have been reported to correlate weakly with favorable AZA response, the exact mechanisms underlying primary azacitidine resistance are largely unknown and there is no approach to circumvent it. Several reports have demonstrated the significance of hypoxia in the regulation of both physiological and malignant hematopoiesis. In MDS, Hif-1α has been proposed to participate in the development of the disease, while its up-regulated expression has been correlated with poor prognosis.Aims : The objective of the current study was to elucidate the expression level and clinical significance of Hif-1α in both AZA responders and non-responders patients with high risk MDS, before and after treatment.Methods : Bone marrow samples from 44 de novo MDS patients, 3 CMML patients and 4 AML (40 M/11 F) with a median age of 78 (60-89) and 10 healthy donors were collected. MDS patients were classified according to WHO as RCMD (7/43), RAEB I (9/43) and RAEB II (28/43) and according to IPSS as low (3/43), intermediate-1 (4/43), intermediate-2 (20/43) and high (17/43). All patients received AZA treatment at the dose of 75mg/m2 x7 days SC. Low and inter-1 MDS patients received azacitidine as off label therapy. Bone marrow samples from 10 MDS [RCMD (1/15), RAEB I (3/15), RAEB II (6/15)], 1 CMML and 2 AML of the aforementioned patients were also collected 6 months after AZA therapy [median age of 80 (62-89), 11 M/2 F]. BM mononuclear cells were isolated from patients before and 6-months after treatment using the Ficoll-paque method, followed by RNA extraction using TRIzol reagent and cDNA preparation using Superscript II reverse transcriptase. Hif-1α expression was estimated by real time PCR TaqMan gene expression assay, using the appropriate primers and probes. Relative gene expression was calculated by comparative threshold cycle (ΔΔCt) method. β-actin was used as a housekeeping gene. The Mann-Whitney U test was performed for statistical analysis of the results.Results : Out of the 51 patients examined, 25 responded to AZA treatment (including CR, PR and HI) while 26 failed to respond. Using Rt-PCR we found that the ΔΔCt ratio of Hif-1α/β-Actin median expression for control samples was 0,82, for responders to AZA treatment 1,36, while for non-responders 0,83. Interestingly, in MDS patients, a trend towards increasing Hif-1α expression, although not statistically significant, was observed across IPSS risk groups, with Hif-1α median expression varying from 1,09 in low risk group to 1,12 in intermediate-1, 1,31 in intermediate-2 and 1,63 in high risk group of patients.Moreover, 10 responders and 3 non-responders were also examined after treatment. The ΔΔCt ratio of Hif-1α/β-Actin median expression after 6-months therapy presented an increasing trend for both responders [from 1,427 to 1,474 (3,29% increase)] and non-responders [from 0,425 to 0,598 (40,7% increase)], with more than a 10-fold difference in the up-regulation of Hif-1α after therapy between the two groups.Conclusions : Our data indicate that Hif-1α expression gradually increases across IPSS groups, suggesting differences among MDS patients' IPSS risk groups in terms of hypoxia signalling, and an association of Hif-1α with a more aggressive disease. Our data also suggest that patients with increased pretreatment expression of Hif-1α, which has been previously associated with poor prognosis, seem to better respond to AZA therapy. Most importantly, our findings indicate that AZA non-responders seem to present with up-regulated Hif-1α expression after therapy, which might be associated with their failure to respond to azacitidine. These early stage observations imply that the molecular mechanisms underlying patients' response to AZA treatment might be associated with hypoxia and Hif-1α expression and although they need to be further confirmed in a larger number of MDS patients in order to establish a statistical significance, they appear very encouraging for better understanding primary resistance to AZA, Hif-1α and hypoxia- wise, and for improving current MDS therapy. DisclosuresNo relevant conflicts of interest to declare.

Chronic Mild Stress and Venlafaxine Treatment Were Associated with Altered Expression Level and Methylation Status of New Candidate Inflammatory Genes in PBMCs and Brain Structures of Wistar Rats.

Preclinical studies conducted to date suggest that depression could be elicited by the elevated expression of proinflammatory molecules: these play a key role in the mediation of neurochemical, neuroendocrine and behavioral changes. Thus, this study investigates the effect of chronic mild stress (CMS) and administration of venlafaxine (SSRI) on the expression and methylation status of new target inflammatory genes: TGFA, TGFB, IRF1, PTGS2 and IKBKB, in peripheral blood mononuclear cells (PMBCs) and in selected brain structures of rats. Adult male Wistar rats were subjected to the CMS and further divided into matched subgroups to receive vehicle or venlafaxine. TaqMan gene expression assay and methylation-sensitive high-resolution melting (MS-HRM) were used to evaluate the expression of the genes and the methylation status of their promoters, respectively. Our results indicate that both CMS and chronic treatment with venlafaxine were associated with changes in expression of the studied genes and their promoter methylation status in PMBCs and the brain. Moreover, the effect of antidepressant administration clearly differed between brain structures. Summarizing, our results confirm at least a partial association between TGFA, TGFB, IRF1, PTGS2 and IKBKB and depressive disorders.

The Potential Role of Small-Molecule PERK Inhibitor LDN-0060609 in Primary Open-Angle Glaucoma Treatment.

Primary open-angle glaucoma (POAG) constitutes the most common type of glaucoma. Emerging evidence suggests that Endoplasmic Reticulum (ER) stress and the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated Unfolded Protein Response (UPR) signaling pathway play a key role in POAG pathogenesis. Thus, the main aim of the study was to evaluate the effectiveness of the PERK inhibitor LDN-0060609 in cellular model of glaucoma using primary human trabecular meshwork (HTM) cells. To evaluate the level of the ER stress marker proteins, Western blotting and TaqMan gene expression assay were used. The cytotoxicity was measured by XTT, LDH assays and Giemsa staining, whereas genotoxicity via comet assay. Changes in cell morphology were assessed by phase-contrast microscopy. Analysis of apoptosis was performed by caspase-3 assay and flow cytometry (FC), whereas cell cycle progression by FC. The results obtained have demonstrated that LDN-0060609 triggered a significant decrease of ER stress marker proteins within HTM cells with induced ER stress conditions. Moreover, LDN-0060609 effectively increased viability, reduced DNA damage, increased proliferation, restored normal morphology, reduced apoptosis and restored normal cell cycle distribution of HTM cells with induced ER stress conditions. Thereby, PERK inhibitors, such as LDN-0060609, may provide an innovative, ground-breaking treatment strategy against POAG.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314