Apoptotic Index Research Articles

Thymoquinone alleviates doxorubicin induced acute kidney injury by decreasing endoplasmic reticulum stress, inflammation and apoptosis

ABSTRACT Doxorubicin (DOX) is used as an anticancer drug despite its many side effects. Thymoquinone (THQ) is a plant-derived substance that exhibits antioxidant and anti-inflammatory properties. We investigated the protective effects of THQ on DOX induced nephrotoxicity in rats. Rats were divided into five groups of eight: group 1, untreated control; group 2, olive oil group given olive oil intraperitoneally (i.p.) for 14 days; group 3, THQ group given 10 mg/kg THQ i.p. for 14 days; group 4, DOX group given a single dose of 15 mg/kg DOX i.p. on day 7 of experiment; group 5, DOX + THQ given 10 mg/kg THQ i.p. for 14 days and 15 mg/kg DOX i.p. on day 7. Kidney tissues were evaluated for histopathology. Caspase-3, IL-17, GRP78 and TNF-α immunostaining was used to determine the expression levels of these proteins among the groups. The TUNEL method was used to determine the apoptotic index. Total antioxidant status (TAS), total oxidant status (TOS), and TNF-α and TGF-β1 levels in kidney tissue were measured using ELISA assay. Histopathologic damage, caspase-3, IL-17, GRP78 and TNF-α immunoreactivity, TUNEL positive cells, TOS, TNF-α and TGF-β1 levels were increased in group 4 compared to group 1. The TAS of group 4 decreased compared to group 1. We found decreased caspase-3, IL-17, GRP78 and TNF-α expressions and TUNEL positive cells in group 5 compared to group 4. In rats given DOX, THQ reduced kidney damage by suppressing endoplasmic reticulum stress, inflammation and apoptosis pathways.

Effect of Notch1 signaling on cellular proliferation and apoptosis in human laryngeal carcinoma

BackgroundThe occurrence and development of malignancies include excessive proliferation and apoptosis resistance in tumor cells. This study aimed to identify the effects of Notch1 signaling on proliferation and apoptosis of laryngeal cancer cells in a hypoxic microenvironment.MethodsNotch1 and Ki-67 expression in laryngeal squamous cell carcinoma (LSCC) tissues was detected by immunohistochemistry. The apoptotic index (AI) of LSCC was evaluated by the TUNEL method. Small interfering RNA (siRNA) was used to inhibit Notch1 expression in laryngeal cancer cells. Real-time PCR was used to measure Notch1, Hes1, and Hey1 mRNA expression, and Western blotting was used to measure Notch1 and Notch1 intracellular domain (N1ICD) protein expression. Annexin V-FITC/propidium iodide staining and Cell Counting Kit-8 assays were used to measure cell apoptosis and proliferation, respectively.ResultsNotch1 expression was significantly related to the proliferation index (PI) and AI in LSCC tissues. Hypoxia could induce proliferation and inhibit apoptosis in cancer cells. Notch1 expression and Notch1 signaling activity could be upregulated by hypoxia. Suppressing Notch1 signaling activity in hypoxic cells could decrease proliferation and increase apoptosis.ConclusionsOur study has demonstrated that hypoxia may promote proliferation and inhibit apoptosis of laryngeal cancer cells. Notch1 signaling may play a pivotal role in regulating the proliferation and apoptosis resistance of laryngeal cancer cells under hypoxic conditions.

Protective Effect of the Chinese Herb Draba Scabra on the Sepsis Myocarditis in Molecular Mechanism.

Objective The aim of this study was to observe the efficacy and mechanism of Draba scabra in sepsis myocarditis. Methods The efficacy and pathways of action of Draba scabra on septic myocarditis were evaluated by making a rat model of sepsis with appendix perforation, using Draba scabra for pharmacological intervention, and measuring serum inflammatory factors, cardiac function indexes and parameters, and P38 protein expression in each group of rats, respectively. Results The inflammatory factor level, apoptotic index of cardiomyocytes, and P38-MARK protein were significantly higher, while the cardiac function index and hemodynamic index were significantly decreased in group B, while the opposite was true in group E. The treatment was also found to be dose-dependent. Conclusion Draba scabra pretreatment effectively reduces the inflammatory response and improves hemodynamic indexes in septic rats. The mechanism may be via the P38-MARK pathway to protect the myocardium.

Combination Therapy Comprising Paclitaxel and 5-Fluorouracil by Using Folic Acid Functionalized Bovine Milk Exosomes Improves the Therapeutic Efficacy against Breast Cancer.

Paclitaxel (PAC) has been approved by FDA for clinical use (Taxol®), yet dose-dependent severe toxicity due to the adjuvant Cremophor EL® in combination with ethanol is a major drawback. The drawbacks of the current therapy can be overcome by (i) finding a suitable vehicle that cannot only bypass the above adjuvant but also be used to deliver drugs orally and (ii) combining the PAC with some other chemotherapeutics to have the enhanced therapeutic efficacy. In the current work, we have used folic acid (FA) functionalized bovine milk-derived exosomes for oral delivery of PAC in combination with 5-fluorouracil (5-FU). Exosomes before and after the drug loading were found to have a particle size in the range of 80–100 nm, polydispersity index (PDI ~0.20), zeta potential (~−25 mV), entrapment efficiency (~82%), practical drug loading (~28%) and sustained drug release for 48 h. Significant decreases in IC50 were observed in the case of exosomes loaded drugs which further improved following the FA functionalization. FA functionalized coumarin-6-loaded exosomes showed remarkably higher cellular uptake in comparison with free coumarin-6. Moreover, FA-functionalized drug-loaded exosomes showed a higher apoptotic index with better control over cell migration. Collectively, data suggested the enhanced efficacy of the combination following its loading to the folic acid functionalized exosomes against breast cancer.

Unfolding the Potency of Adenosine in Targeting Triple Negative Breast Cancer via Paclitaxel-Incorporated pH-Responsive Stealth Liposomes.

Triple-negative breast cancer (TNBC) belongs to the category of the most destructive forms of breast cancer. Being a highly potent chemotherapeutic agent, paclitaxel (PTX) is extensively utilized in the management of various cancers. Commercially available PTX formulations contain non-targeted drug carriers that result in low antitumor activity because of non-specific tissue distribution. Thus, to resolve this issue, we designed PTX-loaded pH-sensitive liposomes (pH Lipos) in the present investigation and used adenosine (ADN) as a targeting ligand. Further, d-α-tocopheryl polyethylene glycol succinate (TPGS) was incorporated into the liposomes to impart a stealth effect to the system. For the development of these pH Lipos, different conjugates were synthesized (ADN-CHEMS and TPGS-ADN) and further utilized for the preparation of ADN-PEG-pH Lipo and ADN-pH Lipo by a thin-film hydration method. DOPE:HSPC:CHEMS:cholesterol at a molar ratio of 3:3:2:2 was selected for the preparation of pH-Lipo possessing 7.5% w/w drug loading. They showed a particle size below 140 nm, a PDI below 0.205, and a % EE greater than 60%. All of the pH Lipos displayed a biphasic pattern of PTX release at pH 7.4 and 5.5. However, the percent drug release at pH 5.5 was substantially greater because of the pH-sensitive nature of the liposomes. The MDA MB 231 and 4T1 cell lines depicted improvement in the qualitative as well as quantitative cellular uptake of PTX ADN-PEG-pH Lipo with a substantial decrease in the IC50 value. Moreover, a higher apoptotic index was observed with pH Lipo compared to free PTX. PTX ADN-PEG-pH Lipo revealed a 3.98- and 3.41-fold rise in the AUC and t1/2 values of PTX compared to Intaxel, respectively. Overall, characteristic decreases in tumor volume and serum toxicity marker levels were observed, which confirmed the development of an efficient and safe formulation.

High-fat diet during sexual maturation induces hyperplastic differentiation of rat prostate and higher expression of AR45 isoform and ERα

We examined the consequences of high-fat diet (HFD) on prostate histophysiology in two periods along sexual maturation of rats and the impact on the gland in adulthood. After weaning, male Wistar rats were fed a balanced diet (4 % fat-C3, C6, C9) or a HFD (20 % fat- HF3, HF6, HF9) for 3, 6 or 9 weeks. Fat deposit weights, blood glucose and levels of serum testosterone and estrogen were measured. Prostate was evaluated for histology, proliferative and apoptotic cell index, and for the expression of androgen (AR), estrogen receptors type α (ERα) and aromatase. HFD did not affect estrogen levels and elevated serum testosterone only in HF9. HFD reduced prostate weight in HF6 and increased it in adulthood (HF9) but relative prostate weight was unchanged among groups. Cell proliferation, height and density were higher in epithelium of all HFD-groups, compared to controls, featuring the epithelial hyperplasia. Epithelial apoptosis was lower in HF9. HF3 and HF9 exhibited higher expressions of ERα, indicating that HFD triggers a new activation of ERα expression in the acinar epithelium. The content of prostatic aromatase was also elevated in HF9. Increased numbers of AR-positive cells were observed in all HFD groups, and western blotting analysis showed an increase in the truncated form of 45 kDa (AR45) and a reduction in the expression of 110 kDa-AR for HF3 and HF9. In conclusion, excessive dietary fats during sexual maturation of rats led to developmental programming of the prostate, inducing a hyperplastic status with perturbations in AR isoforms expression and reactivation of ERα in adulthood, whose implications for posterior prostatic health could be detrimental.

Moxibustion exhibits therapeutic effects on spinal cord injury via modulating microbiota dysbiosis and macrophage polarization.

In this study, we aimed to study the effect of moxibustion (MOX) on microbiota dysbiosis and macrophage polarization, so as to unveil the mechanism underlying the therapeutic effect of MOX in the management of spinal cord injury (SCI). SCI animal models were established to study the effect of MOX. Accordingly, it was found that MOX treatment significantly suppressed the Ace index and Shannon index in the SCI group. Moreover, the reduced relative levels of Lactobacillales and Bifidobacteriales and the elevated relative level of Clostridiales in the SCI animals were mitigated by the treatment of MOX. The body weight, food intake, energy expenditure (EE) index and respiratory quotient (RQ) index of SCI mice were all evidently decreased, but the levels of interleukin (IL)-17, interferon (IFN)-γ, monocyte chemoattractant protein-1 (MCP-1) and IL-1β were increased in the SCI group. Moreover, MOX treatment significantly mitigated the dysregulation of above factors in SCI mice. Accordingly, we found that the Basso Mouse Scale (BMS) score was negatively correlated with the level of Clostridiales while positively correlated with the level of Lactobacillales. The apoptotic index and caspase-3 level were both evidently increased in the SCI group, while the SCI+MOX group showed reduced levels of apoptotic index and caspase-3. Therefore, it can be concluded that the treatment with MOX can promote microbiota dysbiosis and macrophage polarization, thus alleviating spinal cord injury by down-regulating the expression of inflammatory cytokines.

Correlation of Apparent Diffusion Coefficient With Proliferation and Apoptotic Indexes in a Murine Model of Fibrosarcoma: Comparison of Four Methods for MRI Region of Interest Positioning.

Diffusion-weighted imaging (DWI) has demonstrated great potential in predicting the expression of tumor cell proliferation and apoptosis indexes. To evaluate the impact of four region of interest (ROI) methods on interobserver variability and apparent diffusion coefficient (ADC) values and to examine the correlation of ADC values with Ki-67, Bcl-2, and P53 labeling indexes (LIs) in a murine model of fibrosarcoma. Prospective, animal model. A total of 22 female BALB/c mice bearing intramuscular fibrosarcoma xenografts. A 3.0 T/T1-weighted fast spin-echo (FSE), T2-weighted fast relaxation fast spin-echo, and DWI PROPELLER FSE sequences. Four radiologists measured ADC values using four ROI methods (oval, freehand, small-sample, and whole-volume). Immunohistochemical assessment of Ki-67, Bcl-2, and P53 LIs was performed. Interclass correlation coefficient (ICC), one-way analysis of variance followed by LSD-t post hoc analysis, and Pearson correlation test were performed. The statistical threshold was defined as a P-value of <0.05. All ROI methods for ADC measurements showed excellent interobserver agreement (ICC range, 0.832-0.986). The ADC values demonstrated significant differences among the four ROI methods. The ADC values for oval, freehand, small-sample, and whole-volume ROI methods showed a moderately negative correlation with Ki-67 (r=-0.623; r=-0.629; r=-0.642, and r=-0.431) and Bcl-2 (r=-0.590; r=-0.597; r=-0.659, and r=-0.425) LIs, but no correlation with P53 LI (r=0.364, P=0.104; r=0.350, P=0.120; r=0.379, P=0.091; r=0.390, P=0.080). The ADC value can be used to evaluate cell proliferation and apoptosis indexes in a murine model of fibrosarcoma, employing the small-sample ROI as a reliable method. 1 TECHNICAL EFFICACY: Stage 3.

Protective effect of melatonin on alleviating early oxidative stress induced by DOX in mice spermatogenesis and sperm quality maintaining

Doxorubicin (DOX) is an effective chemotherapy drug, but its clinical use has adverse effects on male reproduction. However, there are few studies about the specific biological processes related to male reproduction or strategies for improving fertility protection. In this paper, we examined the effects of DOX on spermatogenesis and sperm function, and tested the possible protective role of melatonin (MLT) against DOX’s reproductive toxicity. DOX-treated mice showed signs of significantly impaired spermatogenesis, including vacuolated epithelial cells, decreased testis weights, and lowered sperm counts and motility. DOX also reduced germ cell proliferation (PCNA) and meiosis-related proteins (SYCP3), but this effect could be partially improved with MLT administration. HSPA2 expression was maintained, which indicated that although MLT did not improve sperm motility, it did have a significant protective effect on elongated sperm. IVF results showed that MLT could partially promote two-cell and blastocyte development that was restricted by DOX. MLT reversed DOX-driven changes in the testes, including the antioxidant indices of SOD1, CAT and PRDX6, and the apoptotic indices of BAX and Caspase3. These results suggest that MLT effectively prevents DOX-induced early reproductive toxicity, and increase our understanding of the molecular mechanisms underlying DOX’s effects on male reproduction and the protective mechanism of MLT.

IGF2 reduces meiotic defects in oocytes from obese mice and improves embryonic developmental competency

BackgroundMaternal obesity is a global issue that has devastating effects across the reproductive spectrum such as meiotic defects in oocytes, consequently worsening pregnancy outcomes. Different studies have shown that such types of meiotic defects originated from the oocytes of obese mothers. Thus, there is an urgent need to develop strategies to reduce the incidence of obesity-related oocyte defects that adversely affect pregnancy outcomes. Multiple growth factors have been identified as directly associated with female reproduction; however, the impact of various growth factors on female fertility in response to obesity remains poorly understood.MethodsThe immature GV-stage oocytes from HFD female mice were collected and cultured in vitro in two different groups (HFD oocytes with and without 50 nM IGF2), however; the oocytes from ND mice were used as a positive control. HFD oocytes treated with or without IGF2 were further used to observe the meiotic structure using different analysis including, the spindle and chromosomal analysis, reactive oxygen species levels, mitochondrial functional activities, and early apoptotic index using immunofluorescence. Additionally, the embryonic developmental competency and embryos quality of IGF2-treated zygotes were also determined.ResultsIn our findings, we observed significantly reduced contents of insulin-like growth factor 2 (IGF2) in the serum and oocytes of obese mice. Our data indicated supplementation of IGF2 in a culture medium improves the blastocyst formation: from 46% in the HFD group to 61% in the HFD + IGF2-treatment group (50 nM IGF2). Moreover, adding IGF2 to the culture medium reduces the reactive oxygen species index and alleviates the frequency of spindle/chromosome defects. We found increased mitochondrial functional activity in oocytes from obese mice after treating the oocytes with IGF2: observed elevated level of adenosine triphosphate, increased mitochondrial distribution, higher mitochondrial membrane potentials, and reduced mitochondrial ultrastructure defects. Furthermore, IGF2 administration also increases the overall protein synthesis and decreases the apoptotic index in oocytes from obese mice.ConclusionsCollectively, our findings are strongly in favor of adding IGF2 in culture medium to overcome obesity-related meiotic structural-developmental defects by helping ameliorate the known sub-optimal culturing conditions that are currently standard with assisted reproduction technologies.

Sperm-borne proteins improve rabbit cloning efficiency via regulating embryonic cleavage and epigenetics.

Somatic cell nuclear transfer (SCNT) shows great application value in the generation of transgenic animals, protection of endangered species, and therapeutic cloning. However, the cloning efficiency is still very low, which greatly restricts its application. Compared to fertilized embryos, cloned embryos lack the sperm proteins, which are considered to play an important role in embryonic development. Here, we compared the sperm proteome, with that of donor fibroblasts and oocytes, and identified 342 proteins unique to sperm, with 42 being highly expressed. The 384 proteins were mainly enriched in the categories of post-translational modification and cytoskeletal arrangement. Extracts of soluble sperm or fibroblast proteins were injected into cloned embryos, and the result showed that injection of sperm protein significantly inhibited abnormal embryonic cleavage, significantly decreased the level of trimethylated histone H3 Lys9 (H3K9me3) and the apoptotic index, and increased the inner cell mass (ICM)-to-trophectoderm (TE) ratio. More importantly, the sperm proteins also significantly enhanced the birthrate. The results of in vitro and in vivo experiments demonstrate that sperm-derived proteins improve embryo cloning efficiency. Our findings not only provide new insights into ways to overcome low cloning efficiency, but also add to the understanding of sperm protein function.

Running exercise training-induced impact on oxidative stress and mitochondria-related apoptosis in rat's testicles.

The current study has been designed to explore the effects of running exercise training protocols (ETPs), with different intensities, on testicular redox and antioxidant capacities. Moreover, the crosstalk between oxidative stress (OS) and mitochondria-related apoptosis was analysed. To this end, 24 Wistar rats were subdivided into sedentary control, low- (LICT), moderate- (MICT), and high (HICT)-intensity continuous running ETP groups. Following 8 weeks, the Johnsen score, sperm count, testicular malondialdehyde (MDA) content, total oxidant status (TOS), and redox biomarkers, including glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) levels were evaluated. Additionally, the expression levels of Bcl-2, Bax, caspase-3, proteins involving in the mitochondria-related apoptosis, and the apoptotic index were analysed. The LICT and MICT running ETPs did not affect the spermatogenesis development, sperm count, and antioxidant and redox capacities. Accordingly, no significant changes were revealed in Bcl-2, Bax, and caspase-3 expression levels and apoptosis index compared to sedentary rats. In contrast, the HICT-induced rats showed a significant (p< 0.05) reduction in spermatogenesis development, sperm count, antioxidant and redox capacities versus control, LICT, and MICT groups. Moreover, the expression of Bcl-2 was decreased, while the Bax and caspase-3 expression levels were increased in the HICT-induced group. Finally, the apoptosis index was increased in the HICT group. In conclusion, the suppressed redox system after HICT can trigger the mitochondria-mediated ROS overload, result in OS condition in the testicular tissue, and reversely target the mitochondrial membrane permeability. All of these molecular alterations are suspected to initiate progressive mitochondria-related apoptosis after HICT.

Screening the immunotoxicity of different food preservative agents on the model organism Galleria mellonella L. (Lepidoptera: Pyralidae) larvae

Immunotoxic effects of sodium benzoate (SB, E211), sodium nitrate (SNa, E251), and sodium nitrite (SNi, E250), a few of the most common food preservatives, on the model organism Galleria mellonella L. (Lepidoptera: Pyralidae) larvae were investigated in this study. The last instar larvae were used for all experimental analyses. For this purpose, median lethal doses of SB, SNa, and SNi were applied to the larvae by the force-feeding method. We found that force-feeding G. mellonella larvae with SB, SNa, and SNi significantly reduced the larval total hemocyte counts, prohemocyte, and granulocyte ratios but increased plasmatocyte, spherulocyte, and oenocyte ratios, as well as the hemocyte mitotic indices and micronucleus frequency. The spreading ability of hemocytes and hemocyte-mediated immune responses were lower in the SB, SNa-, and SNi-treated larval groups compared to controls. Apoptotic indices were higher in all larval groups treated with food preservatives, but increments in necrotic indices were only significantly higher in SNi-treated larvae compared to controls. Our research shows that SB, SNa, and SNi have immunotoxic and cytotoxic potential on G. mellonella larvae. Thus, we suggest that G. mellonella larvae can be used as preliminary in vivo models to screen the immunotoxic effects of food preservative agents.

Effect of refloated soil dust on oxidative stress and apoptosis in rat lung

Little study has illustrated the associations between inhalation of refloated soil dust and the morbidity of respiratory disease in animals. In this study, soil dust samples in arid and semiarid areas of China were collected for investigating the underlying biotoxicity and mechanisms of oxidative stress and apoptosis in rat lung induced by inhalation of refloated soil dust. The soil dust samples were collected from the arid and semiarid areas in Zhangjiakou city, located close to Inner Mongolia Plateau, China. After intratracheal instillation of soil dust, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities in rat lung were declined in a dose-dependent relationship, and pathology changes in lung tissue were also enhanced along with increased levels of soil dust. Meanwhile, apoptotic cells were scattered in the pulmonary epithelial and distributed in the mesenchyma, and the apoptotic index was significantly increased in rat lung instilled by soil dust. Besides, soil dust promoted Bax expression and inhibited Bcl2 expression, i.e., increased Bax/Bcl2 ratio in a concentration-dependent manner. In summary, short-term exposure to soil dust caused oxidative damage and induced apoptosis in rat lung through the regulation of Bax and Bcl2 expression.

The Open Cryotop System Is Effective for the Simultaneous Vitrification of a Large Number of Porcine Embryos at Different Developmental Stages.

The Superfine Open Pulled Straw (SOPS) system is the most commonly used method for vitrification of pig embryos. However, this system only allows the vitrification of four to seven embryos per straw. In this study, we investigated the effectiveness of the open (OC) and closed (CC) Cryotop® systems to simultaneously vitrify a larger number of porcine embryos. Morulae, early blastocysts and full blastocysts were vitrified with the open Cryotop® (n = 250; 20 embryos per device) system, the closed Cryotop® (n = 158; 20 embryos per device) system and the traditional superfine open pulled straw (SOPS; n = 241; 4–7 embryos per straw) method. Fresh embryos from each developmental stage constituted the control group (n = 132). Data expressed as percentages were compared with the Fisher's exact test. The Kruskal-Wallis test was used to analyze the effect of the different vitrification systems on the embryo quality parameters and two-by-two comparisons were accomplished with the Mann-Whitney U test. Differences were considered statistically significant when p < 0.05. Vitrified and control embryos were incubated for 24 h and examined for viability and quality. At the warming step, the embryo recovery rate for the CC system was 51%, while all embryos were recovered when using OC and SOPS. There were no differences between the vitrification and control groups in the postwarming viability of full blastocysts. In contrast, morulae and early blastocysts that were vitrified-warmed with the SOPS system had lower viability (p < 0.01) compared to those from the OC, CC and control groups. The embryonic viability was similar between the OC and control groups, regardless of the developmental stage considered. Moreover, the embryos from the OC group had comparable total cell number and cells from the inner cell mass and apoptotic index than the controls. In conclusion, the OC system is suitable for the simultaneous vitrification of 20 porcine embryos at different developmental stages and provides comparable viability and quality results to fresh embryos subjected to 24 h of in vitro culture.

Roles of early events in the modifications undergone by bovine corneal endothelial cells during wound healing.

A mechanical injury in bovine corneal endothelial (BCE) cells in culture induces: (1) a fast calcium wave (FCW); (2) slow increases in cytosolic sodium and calcium, critical for the healing process, and (3) a rise in the apoptotic rate with respect to quiescent cells. In order to investigate the nature of the stimuli that determine the ionic changes and apoptotic response, we performed here studies on a non-injury model of tissue restitution in BCE monolayers. For this, we employed cell cultures grown to confluence in the presence of a Parafilm strip. We observed that, previously to strip removal, most of the border cells had already developed the slow ionic modifications, while in the scratch wounds these changes gradually occur after several hours of healing. This finding suggests that, in BCE cells, the presence of a free edge is sufficient to trigger ionic modifications necessary for wound healing and to elicit an augmented apoptotic response. The apoptotic index of the migrating cells in the Parafilm model (PF) was determined to be approximately two-fold the one of scratch wounds, a result that, in agreement with our previous observations, we attributed to the absence of the FCW in the PF experiments. The findings of this work further contribute to the understanding of epithelial wound healing, a crucial adaptive, and homeostatic response.

Decreased CD11c-positive dendritic cells in the tumor microenvironment predict double-hit/triple-hit genotype and survival in diffuse large B-cell lymphoma.

Diffuse large B‐cell lymphoma (DLBCL) is the most common type of non‐Hodgkin lymphoma and is a potentially curable disease. However, it is heterogenous, and the prognosis is poor if the tumor cells harbor fusions involving MYC and BCL2 or MYC and BCL6 (double‐hit [DH] lymphoma), or fusions involving all three genes (triple‐hit [TH] lymphoma). Fluorescence in situ hybridization is currently the gold standard for confirming the presence of DH/TH genotypes. However, the test is laborious and not readily available in some laboratories. Germinal center B (GCB) signatures and dual expression of MYC and BCL2 are commonly used as initial screening markers (traditional model) in clinical practice. Our study proposes immunohistochemical markers for more conveniently and accessibly screening DH/TH genotypes in DLBCL. We retrospectively reviewed the clinical and pathological parameters of patients with DLBCL. We assessed the proliferative index, apoptotic index, and tumor microenvironment (TME), with regard to T cells and CD11c(+) dendritic cells, in formalin‐fixed paraffin‐embedded tissue. We then generated a decision tree as a screening algorithm to predict DH/TH genotypes and employed decision curve analysis to demonstrate the superiority of this new model in prediction. We also assessed the prognostic significance of related parameters. Our study revealed that GCB subtypes, a Ki67 proliferative index higher than 70%, and BCL2 expression were significantly associated with DH/TH genotypes. Decreased CD11c(+) dendritic cells in the TME indicated additional risk. Our proposed screening algorithm outperformed a traditional model in screening for the DH/TH genotypes. In addition, decreased CD11c(+) dendritic cells in the DLBCL TME were an independent unfavorable prognosticator. In conclusion, we provide a convenient, well‐performing model that predicts DH/TH genotypes in DLBCL. The prognostic significance of CD11c(+) dendritic cells in the TME might influence the classification and development of immunotherapy for DLBCL in the future.

Abstract 5699: Overexpression of KMT2A is associated with worse prognosis and specific immune signatures in patients with TP53-mutated hepatocellular carcinomas

Abstract Background: Aberrant expression of epigenetic regulators is often associated with pathogenesis. Histone H3K4 methyltransferase, known as KMT2A, has been implicated in regulation of chromosome segregation, mitosis and DNA replication in pediatric leukemia and myeloma. However, the role of KMT2A expression in solid tumors is under-investigated. Here we examine the implications of KMT2A overexpression in prognosis, gene pathway enrichment, aneuploidy and immune infiltration patterns using a large, real-world clinical HCC dataset. Methods: A total of 403 HCC samples underwent comprehensive molecular profiling at Caris Life Sciences, including DNA-(592 Gene Panel, NextSeq, or whole exome sequencing, NovaSeq) and RNA- (NovaSeq, whole transcriptome sequencing, WTS) sequencing. Wilcoxon, Fisher’s exact test were used to determine statistical significance (p value without and q value with multi comparison correction). Aneuploidy scores were generated from CNVkit. Apoptotic index (AI), GSEA were assessed using mRNA levels (FDR&amp;lt;0.25 as cutoff). Overall survival was calculated from date of tissue collection to date of last contact from insurance claims data and used for Kaplan-Meier method. Results: Overexpression of KMT2A predicts poor survival in patients with HCC (HR 2.6, 95% CI [1.4 - 5.2], p&amp;lt;.01). KMT2Ahigh HCC has higher TP53 (57.9% vs 28.8%, p&amp;lt;.001) and lower CTNNB1 (14.1% vs 34.6%, p &amp;lt;.01) mutation rates (mt). GSEA analysis showed that pathways such as mitotic spindle (NES = 2.1), DNA repair (NES = 2.0), E2F regulation (NES = 2.0) and MYC (NES = 1.9) are significantly enriched in KMT2Ahigh HCC. Interestingly, in KMT2Ahigh HCC, TP53 mutation status is a stratification factor for several HCC features: 1) TP53 mt HCC displayed significantly lower aneuploidy score (median 6 vs 13.5, p&amp;lt;.01) and higher apoptotic index (median 1.1 vs 1.2, p&amp;lt;.05); 2) inflammatory response pathways (NES = 2.1) and IL6 JAK STAT3 signaling (NES = 2.1) are specifically enriched in TP53 mt HCC and 3) TP53 mt HCC has more infiltration of B cells (5.3% vs 3.3%, q&amp;lt;.05), Macrophage M1 (6.4% vs 2.9%, q&amp;lt;.01), CD8 + T cells and myeloid dendritic cells (1.1% vs 1%, q&amp;lt;.01) while TP53 wt is associated with more infiltration of Macrophage M2 (5.5% vs 3.3%, q&amp;lt;.05) cells. Conclusions: KMT2A could act as an independent prognostic marker in HCC. The negative correlation between KMT2A expression and aneuploidy scores in TP53 mt indicates potential roles of KMT2A in maintaining genome stability. Furthermore, our results suggest TP53 status is an important stratification factor for HCC with KMT2A overexpression. Our results warrant further investigation on the impact of KMT2A level on immune modulation and may define a subset of HCC that responds most effectively to immune checkpoint inhibition. Citation Format: Liang Sha, Jun Yin, Sungming Kim, Woojin An, Jian Zhang, Alex Farrell, Joanne Xiu, David Spetzler, Shuanzeng Wei, Dave S. Hoon, Stephen V. Liu, Emil Lou, Misako Nagasaka, Wafik S. El-Deiry, Benedito A. Carneiro, Wolfgang Michael Korn, Heinz-Josef Lenz, Yali Dou. Overexpression of KMT2A is associated with worse prognosis and specific immune signatures in patients with TP53-mutated hepatocellular carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5699.

Abstract 2582: Regulatory T cells restrain efficacy of immunotherapy in murine liver tumors

Abstract Background: Checkpoint blockade immunotherapy (CBI) can induce a durable response for some patients with hepatic tumors, but many derive no or incomplete oncologic benefit for unclear reasons. Regulatory T cells (Tregs) in the liver play a central role in maintaining the tolerogenic local immune environment, both in homeostasis and disease. Here we sought to explore the impact of CBI on hepatic Tregs to determine if they play a role in CBI resistance of liver tumors in mice. Methods: High-dimensional flow cytometry was used to examine the ex vivo characteristics of lymphocytes from both tumor-free and orthotopic tumor-bearing livers of mice treated with CBI or other immunomodulatory agents. Results: All hepatic T cell subsets examined displayed higher proliferative and apoptotic indices compared with those of splenic T lymphocytes. We found that hepatic Tregs intensely proliferate and undergo apoptosis compared with splenic Tregs under homeostatic conditions. Hepatic Treg proliferation was enhanced after administration of CBI treatment. This effect was abrogated by co-treatment with sirolimus. CD8, macrophages, and the gut microbiome were found to be dispensable for the in vivo in response to αPD1. Co-treatment of mice with αPD1 and αCD25 sensitized MC38-bearing liver tumors. Conclusion: Murine liver Tregs naturally proliferate and undergo apoptosis due to the mTOR rheostat at homeostasis making them highly responsive to CBI. This behavior potentially explains liver-specific CBI-resistance in tumors. Citation Format: Benjamin L. Green, Chi Ma, Qianfei Zhang, Benjamin Ruf, Umberto Rosato, Jonathan Qi, Simon Wabitsch, Kylynda Bauer, Yuta Myojin, Justin McCallen, John C. McVey, Varun Subramanyam, Vanessa Catania, Amran Nur, Firouzeh Korangy, Changqing Xie, Tim F. Greten. Regulatory T cells restrain efficacy of immunotherapy in murine liver tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2582.

Evaluation of Different Doses of the Aromatase Inhibitor Letrozole for the Treatment of Ectopic Pregnancy and Its Effect on Villous Trophoblastic Tissue

Letrozole, an aromatase inhibitor, has recently been introduced as a favorable medical treatment for ectopic pregnancy. We aimed at evaluating the effects of different doses of letrozole for termination of ectopic pregnancy and study their effects on villous trophoblastic tissue. Sixty patients with undisturbed ectopic pregnancy were classified into three equal groups. Group I: the control group that contained women who underwent laparoscopic salpingectomy, Group II: patients who received letrozole (5 mg day−1) for 10 days, and Group III: patients who received letrozole (10 mg day−1) for 10 days. Subsequently, the β-hCG levels were determined on the first day and after 11 days of treatment. Group IV consisted of patients of GII and GIII; their β-hCG did not drop below 100 mIU/ml within 11 days, and underwent salpingectomy. Placental tissues from patients undergoing salpingectomy either from the control group or GIV were processed for the evaluation of estrogen (ER) and progesterone (PR) receptors, vascular endothelial growth factor (VEGF), and cleaved caspase 3 (CC-3) expression. Cases exposed to high dose letrozole 10 mg day−1 resulted in a higher ectopic pregnancy resolution rate of 85% (17/20), while the resolution rate of the low dose letrozole-treated group (5 mg day−1) was 65% (13/20), and also showed a significant reduction in β-hCG levels on the 11th day, 25.63 ± 4.29 compared to the low dose letrozole group 37.91 ± 7.18 (P < 0.001), Meanwhile, the letrozole-treated group GIV showed markedly reduced expression of ER, PR, and VEGF and a significant increase in the apoptotic index cleaved caspase-3 compared to the control group (P < 0.001). The utilization of letrozole at a dose of 10 mg day−1 for medical treatment of ectopic pregnancy results in a high-successful rate without any severe side effects. Letrozole depriving the placenta of estrogen that had vascular supporting signals resulted in destroying the vascular network with marked apoptosis.Graphical abstract