Mean Fold Change Research Articles

Artificial intelligence (AI) –powered spatial analysis of macrophages in tumor microenvironment and its association with interferon-gamma (IFNG) signature and immune phenotype (IP) in pan-cancer dataset.

2621 Background: Macrophages are known to play a crucial role in cancer progression, but few studies have reported macrophage spatial distribution and its association with genomic signatures and tumor-infiltrating lymphocytes (TIL). Here, we applied an AI-powered Whole Slide Image (WSI) analyzer in The Cancer Genome Atlas (TCGA) pan-cancer dataset to show correlation between specific localization of macrophage and anti-tumor activity, which is reflected by IFNG signature and IP based on spatial TIL density. Methods: For spatial macrophage analysis in TCGA pan-cancer dataset, we used Lunit SCOPE IO, an AI-powered H&E WSI analyzer, which identifies and quantifies various classes of cells such as tumor cell, macrophage, and TIL within cancer or stroma area. IPs were defined as follows: inflamed IP (IIP) as high intratumoral TIL (iTIL) and stromal TIL (sTIL); immune-excluded IP (IEP) as low iTIL and high sTIL; immune-desert IP (IDP) as low TIL overall. The immune cell profiling and quantification of the IFNG signature score were analyzed by using CIBERSORT and RSEM. Results: In TCGA dataset (N = 7,402), median macrophage density in total TME area (tMP) was 19.9 /mm2 (interquartile range [IQR] 9.0-44.9), and it was highly enriched in lung adenocarcinoma (median 65.1, IQR 34.3-110.0), lung squamous cell carcinoma (61, 32.4-101.3), and head and neck squamous cell carcinoma (39.4, 19.6-77.3). Based on IP classification, tMP was highly enriched in IIP (37.8, 18.7-73.8) compared to IEP (25.5, 13.8-48.6) and IDP (8.9, 4.4-17.1). Interestingly, spatial analysis showed that intratumoral macrophage density (iMP) in IIP was highly enriched compared to the others (mean fold change 2.57) and the difference of stromal macrophage density (sMP) among IPs was relatively smaller (mean fold change x1.85). Classified into four groups based on median values of iMP and sMP, high-iMP/high-sMP group had the highest proportion of IIP (47.3% vs 18.7%, p < 0.001), whereas low-iMP/high-sMP group had the highest one of IEP (54.9% vs 32.3%, p < 0.001) compared to the others, respectively. Moreover, the high-iMP/high-sMP group had the highest level of IFNG signature score among 4 groups (x2.13 times higher, p < 0.001). Notably, both iMP and sMP were significantly correlated with CD68 expression (rho 0.292 and 0.287) but CSF1R expression was more strongly correlated with iMP (rho 0.259) compared to sMP (rho 0.078). Conclusions: IIP, IFNG signature, and CSF1R expression are more strongly correlated with intratumoral macrophage density versus stromal macrophage density, suggesting a relationship between macrophage location and function in the TME warranting further investigation.

Deregulation of miR-10b and miR-21 Correlate with Cancer Stem Cells Expansion through the Apoptotic Pathway in Prostate Cancer.

MicroRNAs are small, non-coding RNA molecules that regulate important cellular processes such as tumorigenesis, cell proliferation, and apoptosis. Cancer stem cells are a subset of cells that control metastasis and cell proliferation. In this study, we focus on the roles of miR-10b, miR-21 and correlate with cancer stem cells through the apoptotic pathway in different stages of prostate cancer (PCa). In total, 45 patients, each group with Benign prostatic hyperplasia (BPH), localised PCa, and metastatic PCa, were recruited. MicroRNA and gene expression were estimated through quantitative polymerase chain reaction. Flow cytometry was used to characterise prostate cancer stem cells (PCSCs), estimate reactive oxygen species (ROS), apoptosis and chemiluminescent immunoassay was used to estimate interleukin 6 (IL-6), tumour necrosis factor (TNF-α), prostate-specific antigen (PSA), and testosterone. The fold change mean expressions of miR-21, miR-10b, Cytochrome C, and B-cell lymphoma 2 (BCL-2) were significantly upregulated in localised and metastatic PCa compared with BPH. In contrast, the mean fold change expressions of Bcl-2-associated X protein (BAX), Caspase-3, Caspase-9, and Second mitochondria-derived activator of caspase (SMAC) were lower in localised and metastatic PCa compared to BPH. The levels of IL-6, TNF-α, ROS, PSA and testosterone also showed a significant increase while apoptosis was decreased in both localized PCa and metastatic PCa as compared with BPH. In bioinformatics analyses, we found a similar pattern of miRNAs and gene expression in PCa databases. Our study also found a high expression of CD44+/CD24- and CD44+/CD133+ in localised and metastatic PCa compared with BPH. Our findings suggest miR-10b and miR-21 promote PCSCs and may target apoptotic genes involved in PCa pathogenesis; these miRNAs could be used as diagnosis biomarkers of PCa. In PCa pathogenesis and PCSCs regulation, the interaction between these two players is crucial and will help develop new PCa therapeutic targets.

Predictive value of tumor-infiltrating lymphocyte (TIL) dynamics in the tumor microenvironment (TME) during preoperative chemoradiotherapy (CRT) on pathologic complete response (pCR) in microsatellite-stable (MSS) locally advanced rectal cancer (LARC).

3608 Background: Preoperative CRT followed by consolidation nivolumab before surgery showed a promising pCR rate in MSS LARC; both high PD-L1 expression and an elevated CD8+ T cell/effector regulatory T cell ratio analyzed by pre-CRT samples were independently related to high pCR rate (Bando H, Clin Cancer Res. 2022). Here, we applied AI-powered spatial TIL analysis to the VOLTAGE study to investigate whether dynamic change of TIL in TME may predict pCR in MSS LARC. Methods: The VOLTAGE study is a multicenter phase I/II study to evaluate the efficacy of CRT followed by 5 cycles of nivolumab and surgery in patients (pts) with LARC. In this study, tumor samples were obtained at multiple time points; pre-CRT (B1), post-CRT/pre-nivolumab (B2), post-3 cycles of nivolumab (B3), and surgery. In this analysis, a total of 86 H&E whole-slide images (WSI) harvested at B1 and B2 time points from all the pts enrolled in the VOLTAGE study were included in the analysis. Lunit SCOPE IO (Lunit, Republic of Korea), AI-powered H&E-WSI analyzer developed based on 16,443 WSI with pathologists' annotations, was applied to quantify TIL density in TME. Results: In a total of 43 pts, pCR rate of MSS and microsatellite instability-high (MSI-H) were 28.9% (11/38) and 60% (3/5), respectively. In MSS subgroup (n = 38), which has a higher unmet need for pCR prediction, tumor samples with pCR (n = 11) had increased TIL density in TME (tTIL) in post-CRT (B2, median [IQR] 726 [249-1607] /mm2) compared to pre-CRT (B1, 598 [366-905] /mm2), whereas those without pCR (n = 27) had decreased tTIL in post-CRT (405 [148-748] /mm2), compared to pre-CRT (B1, 748 [460-1153] /mm2). Intratumoral TIL density changes were more prominent than stromal TIL density changes in MSS tumor samples with pCR (mean fold change x17.7 vs. x1.5). MSS Pts with tTIL change from B1 to B2 (during CRT) more than x1.8 times achieved 75% (6/8) pCR rate, whereas those with tTIL change less than x1.8 had 16.7% (5/30) pCR rate (p = 0.0035). Conclusions: Change of TIL density in TME during CRT was significantly correlated with favorable clinical outcome of preoperative CRT and consolidation nivolumab, resulting in high pCR rate in MSS LARC.

Levels of APE1 Repair Gene and CLEC4M in Lung Cancer Patients Receiving Cisplatin Chemotherapy.

This study aimed to detect the levels of apurinic/apyrimidinic endonuclease 1 (APE1) gene expression and C-type lectin domain family 4 member M (CLEC4M) and their association with cisplatin chemotherapy in lung cancer patients. Overall, 105 individuals who attended the Al-Amal National Hospital for Cancer Management, Baghdad, Iraq, were enrolled in the study and divided into three equal groups. The groups included the patients newly diagnosed with lung cancer, cancer patients who received cisplatin, and the healthy control group. All study groups were subjected to the sampling of the venous blood for molecular analysis by real-time polymerase chain reaction (RT-PCR) to detect the APE1 gene and enzyme-linked immunosorbent assay (ELISA) for serological testing to measure the concentration of CLEC4M protein. Significantly, the values of both cancer groups were higher than those reported in the control group. The relative index revealed a significant difference in the mean fold change level of APE1 in the newly diagnosed group (3 fold) and cisplatin therapy patients group (2 fold), compared to the control group (P=0.005). No significant differences were detected between the two cancer groups in terms of fold change mean of expression, demographic characteristics, and cancer histological type. Regarding human CLEC4M protein level, cases receiving cisplatin (139.2±25.9) and newly diagnosed patients (331.0±38.1) had a highly significant difference with the control group (100.3±47.5, P<0.001). There was no significant difference between the concentration level of CLEC4M and all parameters in demographic characteristics and cancer histological type. This was the first study to demonstrate that higher expression levels of new APE1, CLEC4M, and glutathione, especially after chemotherapy, are beneficial as diagnostic and prognostic markers for resistance to platinum chemotherapy in Iraqi lung cancer patients.

Systematic Investigation of Meta-Analysis Data on Treatment Effectiveness for Physical, Psychological, and Sexual Intimate Partner Violence Perpetration.

Intimate partner violence can lead to physical, economical, mental, and sexual well-being issues, and even death, and it is most commonly experienced by women. There exist a number of treatment models for the prevention and treatment of intimate partner violence (IPV). In this study, we provided a comprehensive meta-regression analysis of the effectiveness of batterer treatment programs, with a view to characterizing the interplay between different forms of IPV (physical, psychological, and sexual). Using meta-regression, we explore the effect sizes and whether IPV treatment methods have distinct impacts on the outcomes. We use the difference normalized by pretreatment mean and variance foldchange to uncover the relationship between different violence subtypes and how they drive each other. Specifically, our study found that studies with more pre-treatment psychological and/or sexual violence, lead to less favorable outcomes while the studies that start with more physical violence are able to demonstrate their effects more effectively. Results of this study can be used to help the clinician effectively select the treatment for the perpetrator based on the violence type and severity of violence in order to more effectively treat the needs for each specific relationship.

(184) A Novel, On-demand Therapy for Lifelong Premature Ejaculation using a Miniature Trans-Perineal Electrical Stimulator, the vPatch - a Randomized Controlled Trial

Abstract Introduction While Premature Ejaculation (PE) is a common and disturbing sexual dysfunction in men, currently available treatment modalities have limited efficacy and low treatment adherence. In this study, we assessed perineal Transcutaneous Electrical Stimulation (TES) as a new possibility to treat (PE) Objective To assess the feasibility, safety, and efficacy of vPatch, a miniaturized on-demand perineal (TES) device, for treating PE. Methods We designed a bi-center, international, prospective, randomized, double-blind, two-arm, sham-controlled, first-in-human, clinical study. Calculating for statistical power, 59 patients 21-56 (mean 39.8+9.28) y/o with lifelong PE were included. During the initial visit, intravaginal ejaculatory latency time (IELT) was measured over a 2-week run-in period. Eligibility was confirmed in Visit 2, based on IELT values, and medical/sexual history. Eligible patients tried the vPatch to determine the individualized sensory and motor activation thresholds. Patients were randomized to Active (vPatch) and Sham device groups in a 2:1 ratio. During Visit 3, IELTs, Clinical Global Impression of Change (CGIC) scores, and Premature Ejaculation Profile (PEP) questionnaire outcomes were recorded. The vPatch device safety profile was determined by comparing the incidence of treatment-emergent adverse events. Primary endpoints assessed: 1. vPatch device efficacy as mean fold change in geometric mean IELT; each person was compared to themselves, with and without the device, and the Sham group was compared to the Active Group. 2. vPatch safety profile. Secondary endpoints assessed changes in (PEP) before and after treatment and last visit (CGIC) scores. Results Of 59 patients, 51 completed the study, 34 in Active and 17 in Sham Group. The baseline geometric mean IELT significantly increased from 67 to 123 seconds (p&amp;lt;0.0001) in the Active Group, as compared to increasing insignificantly from 63 to 81 seconds (p=0.1653) in the Sham Group. IELT significantly increased by 3.22-fold in the Active as compared to the Sham group (p=0.0047). Geometric mean time fold increases of IELT in Active compared to the Sham group were 1.7 (95% CI 1.5-2.1) vs. 1.2 (95% CI 1.0-1.6), respectively. No serious adverse events were reported. According to the CGIC, patients in the active group were significantly more satisfied with the treatment (74% vs. 41%, p=0.0274). The subjects in the active arm reported significant improvement in all 4 PEP parameters (P&amp;lt;0.05). Conclusions We demonstrated the possibility to treat lifelong PE by prolonging coitus on-demand, using electric stimulation of ejaculation muscles with vPatch. This treatment may potentially become an on-demand, non-invasive, and drug-free treatment for PE. Disclosure Yes, this is sponsored by industry/sponsor: Virility LTD, Israel Clarification Industry funding only - investigator initiated and executed study Any of the authors act as a consultant, employee or shareholder of an industry for: Virility LTD, Israel.

P352 EXPLORATORY ANALYSES FROM APOLLO–B, A PHASE 3 STUDY OF PATISIRAN IN PATIENTS WITH ATTR AMYLOIDOSIS WITH CARDIOMYOPATHY

Abstract Introduction Transthyretin–mediated (ATTR) amyloidosis is a progressive and fatal disease. Patients with hereditary or wild–type ATTR amyloidosis frequently develop cardiomyopathy (CM). Patisiran, an IV RNAi therapeutic that inhibits synthesis of wt and variant TTR, is approved for the treatment of hATTR amyloidosis with polyneuropathy. Methods In APOLLO–B, patients were 18–85 yrs old with evidence of cardiac amyloidosis by echocardiography, ATTR amyloid deposition on tissue biopsy or fulfilling nonbiopsy diagnostic criteria for ATTR amyloidosis with CM, and had prior hospitalization for heart failure (HF) due to ATTR amyloidosis or clinical evidence of HF. Patients were randomized 1:1 to patisiran IV 0.3 mg/kg or placebo Q3W for 12 months. The primary endpoint was change from baseline in 6–MWT at Month 12 (M12) with patisiran vs placebo. Exploratory parameters at M12 included changes in cardiac biomarkers, echocardiography and Tc scintigraphy. Results APOLLO–B enrolled 360 patients (patisiran, n=181; placebo, n=179): median age, 76.0 yrs; male, 89%; wtATTR, 80%; on tafamidis at baseline, 25%. Patisiran showed a significant benefit compared with placebo in 6–MWT (median [95% CI] change from baseline: patisiran, –8.15 [–16.42, 1.50]; placebo, –21.35 [–34.05, –7.52]; HL estimate of median difference: 14.69 [0.69, 28.69]; p=0.0162). Patisiran demonstrated favorable trends in change from baseline of NT–proBNP (mean fold change ratio [95% CI]: 0.80 [0.73, 0.89]; p=1.825×10–5) and troponin I (mean fold change ratio [95% CI]: 0.87 [0.80, 0.95]; p=0.0011) at M12 vs placebo. Patisiran also demonstrated a trend towards benefit in change from baseline of most evaluated echocardiographic parameters at M12 vs placebo. Patisiran–treated patients evaluable for scintigraphy (n=37) experienced a reduction (37.8%) or no change (62.2%) in Perugini grade at M12 compared with baseline. Among placebo patients (n=28) at M12, none experienced a reduction from baseline in Perugini grade, and 1 (3.6%) increased in grade. Patisiran demonstrated an acceptable safety profile; AEs were mostly mild or moderate in severity, and there were no cardiac safety concerns. Conclusions Exploratory analyses after 12 months provide further evidence for the potential benefit of patisiran treatment on cardiac biomarkers and manifestations of cardiac amyloid involvement in patients with ATTR amyloidosis. The long–term impact of patisiran will be assessed in the ongoing APOLLO–B open–label extension.

Biological scaffold implants in a murine volumetric muscle loss trauma model cause time dependent CD62L and CD101 upregulation on “regulatory like” eosinophils.

Abstract Introduction: Previously, we reported a strong increase in the recruitment of eosinophils at injured muscle when treated with a biologic scaffold implant. As such, we sought to characterize the surface marker expression of these eosinophils to understand their role at the site of injury. Method: Volumetric muscle loss (VML) model was performed on C57BL/6 mice and was subsequently treated with a biologic scaffold (decellularized extracellular matrix). After 7 and 21 days, post-injury, implanted material along with injured tissue was dissected and converted to single cell suspension for flow cytometry. Results: Compared to uninjured mice, at both 7 and 21 days time point, mice that received biological scaffold featured significantly increased presence of eosinophils at the site of injury. These eosinophils had a ring-shaped nucleus which has been previously associated with regulatory eosinophils. Eosinophils at the site of injury showed time dependent upregulation of CD101 (Mean fold change from uninjured muscle after 7 days post implant was 2.73 and the same was 9.90 after 21 days, (p&amp;lt;0.0001)), and CD62L (Mean fold change from uninjured muscle after 7 days post implant was 3.65 and the same was 11.64 after 21 days(p&amp;lt;0.0001)) expression. Interestingly, the same pattern was not observed with blood eosinophils except that the blood eosinophils at 3-week timepoint were featuring a significant increase in CD62L expression compared with 1 week time point, suggesting potential return of homeostasis in circulatory compartment. Conclusion: Here, we describe time-dependent transformation in eosinophil phenotype in response to biologic scaffold implantation, including upregulation of adhesion molecule CD62L and a regulatory marker CD101.

Equivalence of plasma p-tau217 with cerebrospinal fluid in the diagnosis of Alzheimer's disease.

Plasma biomarkers are promising tools for Alzheimer's disease (AD) diagnosis, but comparisons with more established biomarkers are needed. We assessed the diagnostic performance of p-tau181 , p-tau217 , and p-tau231 in plasma and CSF in 174 individuals evaluated by dementia specialists and assessed with amyloid-PET and tau-PET. Receiver operating characteristic (ROC) analyses assessed the performance of plasma and CSF biomarkers to identify amyloid-PET and tau-PET positivity. Plasma p-tau biomarkers had lower dynamic ranges and effect sizes compared to CSF p-tau. Plasma p-tau181 (AUC=76%) and p-tau231 (AUC=82%) assessments performed inferior to CSF p-tau181 (AUC=87%) and p-tau231 (AUC=95%) for amyloid-PET positivity. However, plasma p-tau217 (AUC=91%) had diagnostic performance indistinguishable from CSF (AUC=94%) for amyloid-PET positivity. Plasma and CSF p-tau217 had equivalent diagnostic performance for biomarker-defined AD. Our results suggest that plasma p-tau217 may help reduce the need for invasive lumbar punctures without compromising accuracy in the identification of AD. p-tau217 in plasma performed equivalent to p-tau217 in CSF for the diagnosis of AD, suggesting the increased accessibility of plasma p-tau217 is not offset by lower accuracy. p-tau biomarkers in plasma had lower mean fold-changes between amyloid-PET negative and positive groups than p-tau biomarkers in CSF. CSF p-tau biomarkers had greater effect sizes than plasma p-tau biomarkers when differentiating between amyloid-PET positive and negative groups. Plasma p-tau181 and plasma p-tau231 performed worse than p-tau181 and p-tau231 in CSF for AD diagnosis.

Abstract 4822: Signaling pathways associated with stress factors in gastrointestinal cancers

Abstract Gastrointestinal (GI) cancer affects various significant organs along the gastrointestinal tract, which contains several of the leading cancer-related deaths in the United States. According to the American Cancer Society, colorectal and liver cancers are among the top ten leading deaths due to cancer, ranking third and sixth, respectively. In the Rio Grande Valley region with a majority Hispanic population, colorectal cancer and liver have a higher occurrence than in the entire state of Texas. However, for many, a cancer diagnosis can worsen with stress, which is also a common occurrence within the Rio Grande Valley region due to 30% of its population living in poverty. Stress can cause changes to the immune system's functionality and the inflammatory response that could lead to insufficient surveillance for any potential oncogenicity. When the Hypothalamic-Pituitary-Adrenal (HPA) axis becomes activated by stress, it will release several stress hormones, such as leptin and cortisol, in response that can play a role in the progression and growth of cancer tumors. To further explore this relationship, hepatocellular carcinoma (HCC) cells and colorectal cancer (CRC) cells were treated with cortisol and leptin at various time points to create models mimicking acute and chronic stress exposure. Afterward, the mRNA mean fold change levels were obtained utilizing the RT-PCR method to determine the levels of each gene in various liver and colorectal cancer cell lines.Additionally, a kinase assay will provide further insight and evidence into the signaling pathway based on the results of the RT-PCR. The current results have shown an upregulation of lncRNA MALAT1, ZNF384, and YB-1 genes at varying degrees depending on the cell line, stress hormone, and duration of treatment. Consequently, these findings have shown an upregulation of oncogenic long non-coding RNA and transcription factors, presenting a signaling oncogenic pathway model by stress factors in HCC and CRC cell lines. Therefore, understanding the role of stress-factors in the oncogenicity and progression of cancer can aid in creating therapeutics for a population where stress can play a role in cancer disparity. Citation Format: Amayrani Sanchez, Kyle D. Doxtater, Samantha Lopez, Sophia Leslie, Elias George, Vijian Dhevan, Meena Jaggi, Subhash Chauhan, Manish K. Tripathi. Signaling pathways associated with stress factors in gastrointestinal cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4822.

K-Ras4A Plays a More Significant Role than K-Ras4B in Ductal Carcinoma of Breast.

K-Ras mutations rarely occur in breast cancer. However, studies have supported that K-Ras upregulation is involved in breast cancer pathogenesis. Two main K-Ras transcript variants; K-Ras4A and K-Ras4B, originate from the alternative splicing of exon 4. In this study, we aimed to evaluate variations in the expression of K-Ras4A and K-Ras4B and their role in breast ductal carcinoma. Total RNA was extracted from breast tumors, and the NATs obtained via mastectomy. Patients were selected from new cases of breast cancer with no prior history of chemotherapy. Relative mRNA expression was calculated based on a pairwise comparison between the tumors and the NATs following normalization to the internal control gene. Predictive values of the transcript variants were examined by ROC curve analysis. A statistically significant increase was found in the K-Ras4A and K-Ras4B expression with the mean fold changes of 7.58 (p = 0.01) and 2.47 (p = 0.001), respectively. The K-Ras4A/K-Ras4B ratio was lower in the tumors than that of the normal tissues. ROC curve analysis revealed the potential of K-Ras4A (AUC: 0.769) and K-Ras4B (AUC: 0.688) in breast cancer prediction. There was also a significant association between K-Ras4B expression and HER2 statues (p = 0.04). Furthermore, a significant link was detected between K-Ras4A expression and pathological prognostic stages (p = 0.04). Our findings revealed that expression levels of K-Ras4A and K-Ras4B is higher in the tumor compared to the normal breast tissues. Increase in K-Ras4A expression was more significant than that of K-Ras4B.

The anti-caspase 1 inhibitor VX-765 reduces immune activation, CD4+ T cell depletion, viral load, and total HIV-1 DNA in HIV-1 infected humanized mice.

HIV-1 infection results in the activation of inflammasome that may facilitate viral spread and establishment of viral reservoirs. We evaluated the effects of the caspase-1 inhibitor VX-765 on HIV-1 infection in humanized NSG mice engrafted with human CD34+ hematopoietic stem cells. Expression of caspase-1, NLRP3, and IL-1β was increased in lymph nodes and bone marrow between day 1 and 3 after HIV-1 infection (mean fold change (FC) of 2.08, 3.23, and 6.05, p<0.001, respectively). IFI16 and AIM2 expression peaked at day 24 and coincides with increased IL-18 levels (6.89 vs 83.19 pg/ml, p=0.004), increased viral load and CD4+ T cells loss in blood (p<0.005 and p<0.0001, for the spleen respectively). Treatment with VX-765 significantly reduced TNF-α at day 11 (0.47 vs 2.2 pg/ml, p=0.045), IL-18 at day 22 (7.8 vs 23.2 pg/ml, p=0.04), CD4+ T cells (44.3% vs 36,7%, p=0.01), viral load (4.26 vs 4.89 log 10 copies/ml, p=0.027), and total HIV-1 DNA in the spleen (1 054 vs 2 889 copies /106 cells, p=0.029). We demonstrated that targeting inflammasome activation early after infection may represent a therapeutic strategy towards HIV cure to prevent CD4+ T cell depletion and reduce immune activation, viral load, and the HIV-1 reservoir formation.

Culture of leukocyte-derived cells from human peripheral blood: Increased expression of pluripotent genes OCT4, NANOG, SOX2, self-renewal gene TERT and plasticity.

There are few stem cells in human peripheral blood (PB). Increasing the population and plasticity of stem cells in PB and applying it to regenerative medicine require suitable culture methods. In this study, leukocyte populations 250 mL of PB were collected using a blood separator before that were cultured in optimal cell culture medium for 4 to 7 days. After culturing, stemness characteristics were analyzed, and red blood cells were removed from the cultured cells. In our results, stemness markers of the leukocyte populations Sca-1+ CD45+, CD117+ CD45+, and very small embryonic-like stem cells CD34+ Lin- CD45- and CXCR4+ Lin- CD45- were significantly increased. Furthermore, the expression of stem cell genes OCT4 (POU5F1), NANOG, SOX2, and the self-renewal gene TERT was analyzed by quantitative real-time polymerase chain reaction in these cells, and it showed a significant increase. These cells could be candidates for multi-potential cells and were further induced using trans-differentiation culture methods. These cells showed multiple differentiation potentials for osteocytes, nerve cells, cardiomyocytes, and hepatocytes. These results indicate that appropriate culture methods can be applied to increase expression of pluripotent genes and plasticity. Leukocytes of human PB can be induced to trans-differentiate into pluripotent potential cells, which will be an important breakthrough in regenerative medicine.

Autophagic-Related Anticancer Effect of Grapes Extract and Tomatoes Extract: Ex-Vivo Study

Cells undergo autophagy to save themselves from injury, but progressive autophagy can cause cell death. This study characterized and compared the effect of grape (resveratrol) and tomato (lycopene) extracts and their combination on modulating autophagy-related miRNA and its target gene in squamous cell carcinoma cell line. Docking analysis for extracts and selected genes was performed. Methyl Thiazol Tetrazolium assays were used to assess the cytotoxicity of extracts and their combination toward HEp-2 cells. qRT-PCR was used to quantify changes in gene expression. Data were statistically analyzed. miRNA-20a was identified as a potential effector in laryngeal cancer, and sequestosome-1 (SQSTM1) was its target gene. Docking analysis showed that resveratrol interacted with miRNA-20a and showed less affinity toward SQSTM1. Hydrogen bonds and hydrophobic interactions were predicted. In contrast, lycopene showed less affinity toward miRNA-20a than resveratrol. Increasing doses of resveratrol, lycopene, and their combination induced a statistically significant reduction in mean percent viability and mean fold changes of miRNA-20a and SQSTM1 expression in treated HEp-2 cells. Pearson’s correlation showed a statistically significant positive correlation between miRNA-20a and SQSTM1 (R=0.812, p≤0.001). Grape and tomato extracts and their combination display promising cytotoxicity against HEp-2 cells in a dose- and time-dependent fashion. Both extracts reduce the expression of miRNA-20a and SQSTM1 with subsequent inhibition autophagy and promotion of apoptosis in HEp-2 cells.&#x0D;

Correlation of NF-kB Serum Level with the Expression Pattern of Microrna-221 in a Sample of Hcv-Exposed Iraqi Patients

Background: Hepatitis C virus (HCV) has the ability to change cellular messenger RNA transcription and translation by stimulating the synthesis of cellular microRNAs (miRNAs) that impair immune response and facilitate viral reproduction. One of the most important members of the immune response against HCV is nuclear factor-kappa B (NF-κB), which is regulated by cellular miRNAs. Aims: we aimed to investigate the correlation of NF-κB serum level with circulatory miRNA-221 (miR-221) fold change in HCV-exposed individuals. Materials and Methods: Serum level of NF-κB in 88 samples (22 patients with persistent HCV infection, 22 individuals with spontaneous HCV virus clearance, 22 individuals treated with direct-acting antivirals (DAAs) drugs, and 22 uninfected apparently healthy blood donors as control) was measured by enzyme-linked immunosorbent assay. Reverse transcriptase–polymerase chain reaction was used to quantify the expression fold of circulatory miR-221. Results: The results showed that there was a significant decrease in the mean level of NF-κB at P &lt; 0.000 among HCV-exposed patients (2.0058 ng/ml) as compared to the control group (2.9841 ng/ml). The mean fold change of miR-221 was significantly upregulated about six more times among HCV-exposed patients (mean = 6.3545) compared to the control group (mean = 1.1864). Furthermore, the mean ± standard deviation of miR-221 fold change in patients with persistent HCV infection was significantly higher compared to patients cured after DAA therapy (P = 0.048), there was a weak negative correlation (r = −0.246, P = 0.021) between NF-κB serum level and miR-221 folding level. Conclusion: HCV infection disrupts NF-κB activation, resulting in dysregulation of miR-221 that persists long after the virus has been cleared. Thus, quantification of serum NF-κB and miR-221in HCV-exposed patients could be used as noninvasive prognostic marker during long-term follow-up. Furthermore, a miRNAs profile analysis can help distinguish HCV-exposed from healthy individuals.

Pre-Transplant Prediction of Acute Graft-versus-Host Disease Using the Gut Microbiome.

Gut microbiota is thought to influence host responses to allogeneic hematopoietic stem cell transplantation (aHSCT). Recent evidence points to this post-transplant for acute graft-versus-host disease (aGvHD). We asked whether any such association might be found pre-transplant and conducted a metagenome-wide association study (MWAS) to explore. Microbial abundance profiles were estimated using ensembles of Kaiju, Kraken2, and DeepMicrobes calls followed by dimensionality reduction. The area under the curve (AUC) was used to evaluate classification of the samples (aGvHD vs. none) using an elastic net to test the relevance of metagenomic data. Clinical data included the underlying disease (leukemia vs. other hematological malignancies), recipient age, and sex. Among 172 aHSCT patients of whom 42 developed aGVHD post transplantation, a total of 181 pre-transplant tool samples were analyzed. The top performing model predicting risk of aGVHD included a reduced species profile (AUC = 0.672). Beta diversity (37% in Jaccard's Nestedness by mean fold change, p < 0.05) was lower in those developing aGvHD. Ten bacterial species including Prevotella and Eggerthella genera were consistently found to associate with aGvHD in indicator species analysis, as well as relief and impurity-based algorithms. The findings support the hypothesis on potential associations between gut microbiota and aGvHD based on a data-driven approach to MWAS. This highlights the need and relevance of routine stool collection for the discovery of novel biomarkers.

Four Genes Predictive for the Severity of Hematological Damage Reveal a Similar Response after X Irradiation and Chemotherapy.

Radiological and especially nuclear accidents and incidents pose a threat to populations. In such events, gene expression (GE) analysis of a set of 4 genes (FDXR, DDB2, POU2AF1, WNT3) is an emerging approach for early and high-throughput prediction of the later manifesting severity degrees of the hematological acute radiation syndrome (H-ARS). Validation of this gene set on radiation victims is difficult since these events are rare. However, chemotherapy (CTX) is widely used e.g., breast cancer patient treatment and pathomechanisms, as well as blood cell count changes are comparable among both exposure types. We wondered whether GE changes are similarly deregulated after CTX, which would be interpreted as a confirmation of our already identified gene set for H-ARS prediction after irradiation. We examined radiation-induced differential GE (DGE) of our gene set as a positive control using in vitro whole blood samples from ten healthy donors (6 females, 4 males, aged: 24-40 years). Blood was incubated in vitro for 8 h after X irradiation with 0 and 4 Gy (1 Gy/min). These data were compared with DGE measured in vivo in blood samples of 10 breast tumor CTX patients (10 females, aged: 39-71 years) before and 4 days after administration of cyclophosphamide and epirubicin. RNA was isolated, reverse transcribed and quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to assess DGE of FDXR, DDB2, POU2AF1 and WNT3 relative to the unexposed samples using TaqMan assays. After X irradiation, we found a significant upregulation (irrespective of sex) with mean fold changes of 21 (P < 0.001) and 7 (P < 0.001) for FDXR and DDB2 and a significant down-regulation with mean fold changes of 2.5 (P < 0.001) and 2 (P = 0.005) for POU2AF1 and WNT3, respectively. After CTX, a similar pattern was observed, although mean fold changes of up-regulated FDXR (6-fold, P < 0.001) and DDB2 (3-fold, P < 0.001) as well as down-regulated POU2AF1 (1.2-fold, P = 0.270) and WNT3 (1.3-fold, P = 0.069) appeared lower corresponding to less altered blood cell count changes observed after CTX compared to historic radiation exposure data. However, a subpopulation of CTX patients (n = 6) showed on average a significant downregulation of POU2AF1 (1.8-fold, P = 0.04) and WNT3 (2.1-fold, P = 0.008). In summary, the pattern of up-regulated GE changes observed in all CTX patients and down-regulated GE changes observed in a subgroup of CTX patients appeared comparable with an already identified gene set predictive for the radiation-induced H-ARS. This underlines the significance of in vivo GE measurements in CTX patients, employed as a surrogate model to further validate already identified radiation-induced GE changes predictive for the H-ARS.

Elevated plasma α1-antichymotrypsin is a biomarker candidate for malaria patients

Advancements in the field of proteomics have provided opportunities to develop diagnostic and therapeutic strategies against various diseases. About half of the world's population remains at risk of malaria. Caused by protozoan parasites of the genus Plasmodium, malaria is one of the oldest and largest risk factors responsible for the global burden of infectious diseases with an estimated 3.2 billion persons at risk of infection. For epidemiological surveillance and appropriate treatment of individuals infected with Plasmodium spp., timely detection is critical. In this study, we used combinations of depletion of abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, LC-MS/MS and western blot analysis on the plasma of healthy donors (100 individuals) and vivax and falciparum malaria patients (100 vivax malaria patients and 8 falciparum malaria patients). These analyses revealed that α1-antichymotrypsin (AACT) protein levels were elevated in vivax malaria patient plasma samples (mean fold-change ± standard error: 2.83 ± 0.11, based on band intensities), but not in plasma from patients with other mosquito-borne infectious diseases. The results of AACT immunoblot analyses showed that AACT protein was significantly elevated in vivax and falciparum malaria patient plasma samples (≥ 2-fold) compared to healthy control donor plasma samples, which has not been previously reported. [BMB Reports 2022; 55(11): 571-576].

High-Throughput Proteomics Identifies THEMIS2 As Biomarker of Inferior Treatment-Free Survival in Chronic Lymphocytic Leukemia

Background Notwithstanding advances in the prognostication of chronic lymphocytic leukemia (CLL), it remains challenging to distinguish between patients with favorable and unfavorable treatment-free survival (TFS). In addition, the downstream protein correlates of well-known genomic features of CLL are not always clear. To address this, we performed large-scale, unbiased characterization of global intracellular protein expression in a cohort of previously untreated CLL patients. Materials and methods We selected 40 CLL cases (patients in need of treatment within 24 months of sampling) and 40 age- and sex-matched CLL controls (patients without need for treatment for at least 24 months since sampling) from the Dutch natural history CLL cohort. In this cohort, we characterized global intracellular protein expression of magnetically purified B cells using tandem-mass-tagging, followed by high-performance liquid chromatography and triple tandem mass-spectrometry. In addition, recurrent cytogenetic aberrations (del13q, del11q, del17p and trisomy 12), IGHV mutational status and TP53 mutations were characterized using targeted fluorescence in situ hybridization (FISH), Sanger sequencing and next-generation sequencing, respectively. Results Expression of 3268 proteins was quantified in the cohort, allowing for a comprehensive characterization of the CLL proteome. CLL with unmutated IGHV (U-CLL) was associated with significant overexpression of 32 proteins, including ZAP70 and proteins involved in B-cell receptor signaling, such as CD79b and immunoglobulin constant regions mu and delta (adjusted P-value <0.05). CLL with mutated IGHV (M-CLL) was associated with higher intracellular expression of 7 proteins, including both subunits of ferritin (FTH1 and FTL, adjusted P-value <0.05). Trisomy 12 was associated with increased intracellular expression of 32 proteins, 13 (40%) of which are encoded by genes on chromosome 12, and decreased expression of 8 proteins. Many of these hits are concordant with the results of independently performed mass-spectrometry screens. Interestingly, the intracellular concentration of THEMIS2 was significantly higher in cases, compared to controls. THEMIS2 is an protein that is constitutively bound to Grb2, Lyn and PLCγ2 and lowers the threshold for B-cell receptor activation by low-avidity antigens. THEMIS2 protein levels were higher in both U-CLL cases (mean fold change 2, P-value <0.0001) and M-CLL cases (mean fold change 1.7, P-value <0.01), compared to controls. Concordantly, Cox proportional hazards modelling demonstrated that higher concentration of THEMIS2 was significantly associated with shorter TTFT, independent of IGHV mutational status and TP53 aberrations (standardized HR 2.79 [95%CI 1.71-4.54], P<0.0001). ROC analysis indicated a good discriminatory capacity of elevated THEMIS2 concentration, with an AUC of 0.83. THEMIS2 protein quantification by ELISA in a cohort of 40 patients confirmed that higher expression of THEMIS2 was associated with inferior TTFT (median TTFT for patients with THEMIS2 levels above versus below the mean 11 months versus 66 months, P=0.04). Gene expression analysis by qPCR confirmed higher expression of THEMIS2 in cases, compared to controls (mean fold change of 3, P<0.01). Analysis of a publicly available, independently generated mass-spectrometry dataset of 68 CLL patients confirmed the association of elevated intracellular THEMIS2 expression and TFS (median TFS 3 months versus not reached, P<0.0001). Conclusions We present a comprehensive characterization of the proteome of untreated CLL. Of note, we identify elevated gene and protein expression of THEMIS2 as putative biomarker of inferior TTFT, independent of IGHV mutational status and TP53 aberrations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activina: A Key Factor Mediating Chemoresistance in B-Cell Type Acute Lymphoblastic Leukemia

B-Cell Acute Lymphoblastic Leukemia (B-ALL) is the most common cancer in children. Multidrug high-dose chemotherapy has increased the 5-year event free survival rate to more than 80%. However, the prognosis of refractory/relapsed (r/r) ALL remains dismal. Increasing evidence indicates that treatment failure could be linked to the protective role of leukemic bone marrow (BM) microenvironment. The identification of the molecules involved in the crosstalk between leukemic cells and the BM niche would be crucial to establish niche-targeted therapies to be combined with conventional drugs. Recently, we identified ActivinA (ActA) as a new potentially targetable leukemia-promoting factor, which is significantly overexpressed in the BM plasma of B-ALL patients. We demonstrated that ActA is able to potentiate the migratory and invasive ability of B-ALL cells in vitro and enhance both BM engraftment and metastatic potential of leukemic cells in a xenograft mouse model. Literature data indicate that ActA exerts a pro-tumoral role in several solid tumors, by promoting cancer cell migration, proliferation and chemoresistance. The aim of this work was to ascertain whether ActivinA could mediate resistance to standard chemotherapy agents routinely used in pediatric B-ALL, such as Vincristine (VCR), Dexamethasone (Dex) and Asparaginase (ASNase). To investigate the role of ActA on B-ALL cell chemoresistance, we stimulated or not 697 cells with ActA for 24h. Cells were then treated with increasing concentrations of Dex, ASNase and VCR for 72h to obtain a dose-response curve. Cell viability was evaluated by flow cytometry and used to calculate the inhibitory dose 50 (IC50). Interestingly, the IC50 of ActA stimulated 697 cells for Dex and ASNase was in both cases about ten-fold higher than NS controls. In the case of VCR-treated cells, we did not detect any difference in IC50. These data suggest that ActA is able to convey resistance to Dex and ASNase-induced cell death, while sparing VCR-based leukemic cell killing, possibly due to different mechanisms of action. Validation experiments, with selected doses of Dex and ASNase (7 µg/mL and 1 U/mL, respectively), confirmed the ability of ActA to induce chemoresistance. In detail, the median viability of ActA stimulated 697 cells after 3 days of treatment with Dex was 64.15% (range=51.95-73.75%). This percentage was significantly higher compared to NS cells (median viability=41.95%, range=31.95-60.93%, Wilcoxon test: p<0.0001). Alongside, the viability of ActA stimulated cells (median=53.40%, range=38.70-75.00%) was also significantly increased (median=31.40%, range=22.45-54.25%, p=0.001) after ASNase treatment. To understand the molecular mechanisms underlying Acta-mediated chemoprotection, we firstly evaluated in our experimental setting the percentage of 697 cells expressing the active form of Caspase-3, by flow cytometry. Our results highlight that the activation of Caspase-3 was significantly reduced in ActA pre-stimulated cells treated with Dex (mean fold change (FC) ActA/NS=0.48±0.22, p=0.0003, One-sample t test) and ASNase (mean FC=0.49±0.062, p<0.0001), compared to NS cells. Then, we focused on the impact of ActA on anti- and pro-apoptotic factors playing a key role in drug-mediated apoptosis. By western blot, we demonstrated that Bcl-2 was significantly increased upon Dex treatment in ActA pre-stimulated cells (median FC ActA+Dex/NS untreated cells=0.63, range=0.49-1.12, Wilcoxon test: p<0.05) compared to NS cells (median FC NS+Dex/NS untreated cells=0.43, range=0.26-0.74). In contrast, we did not observe any difference with ASNase. Furthermore, we demonstrated that ActA was able to decrease the drug-induced cleavage activation of pro-apoptotic factors BAX and BAK. Finally, we investigated by confocal microscopy the levels of intracellular Reactive Oxygen Species (ROS), well-known key elements of the drug-induced oxidative stress, resulting in the activation of apoptosis. Interestingly, upon Dex treatment we observed a reduction of ROS levels of 59.7%±4.6% (mean±sd) in ActA-stimulated cells compared to NS. In the case of ASNase treatment the mean reduction was 46.9%±8.8%. Overall, our data suggest that ActA plays a pivotal role in B-ALL cell chemoprotection and that its targeting may be a promising strategy to be used in combination with standard chemotherapy in r/r ALL patients.