Negative Expression Research Articles

Aberrant expression of UBE2C in endometrial cancer and its correlation to epithelial mesenchymal transition.

Ubiquitin-conjugating enzyme E2C (UBE2C), its overexpression promotes tumor progression, is a key component of the ubiquitin conjugating proteasome complex. Epithelial-mesenchymal transition, which is lost epithelial features and gained mesenchymal features in some epithelial cancers, is involved in epithelial cancers' invasiveness and metastasis. The aim of this study is to detect the expression of UBE2C, WNT5α, and E-cad in endometrial cancer (EC) and their clinical significance. The expression of UBE2C, WNT5α, and ZEB1 in 125 cases EC tissues were detected by immunohistochemistry. Patients clinicopathological, demography, and follow-up data were also collected. Positive rates of expression of UBE2C and ZEB1 were significantly higher in EC tissues when compared with the control tissues. The positive expression of UBE2C and ZEB1 were positively associated with tumor stages, local lymph node metastasis, and International Federation of Gynecology and Obstetrics (FIGO) stages. The positive rate of expression of WNT5a was significantly lower in EC tissues when compared with the control tissues. And positive expression of E-cad was inversely related to tumor stages, lymph node metastasis stages, and FIGO stages. Kaplan-Meier analyses demonstrated that positive expression of UBE2C or ZEB1 for EC patients had unfavorably overall survival time when compared with patients with negative expression of UBE2C or ZEB1. And EC patients with positive expression of WNT5a had favorably overall survival time when compared with EC patients with negative expression of WNT5a. Multivariate analysis demonstrated that positive expression UBE2C, WNT5α, and ZEB1, as well as FIGO stages were independent prognostic factors for EC patients. UBE2C, ZEB1, and WNT5a should be considered promising biomarkers for EC patients' prognosis.

Emotion recognition accuracy only weakly predicts empathic accuracy in a standard paradigm and in real life interactions.

The relationship between decoding ability (Emotion recognition accuracy, ERA) for negative and positive emotion expressions from only video, only audio and audio-video stimuli and the skill to understand peoples' unspoken thoughts and feelings (Empathic accuracy, EA) was tested. Participants (N = 101) from three groups (helping professionals with and without therapy training as well as non-helping professionals) saw or heard recordings of narrations of a negative event by four different persons. Based on either audio-video or audio-only recordings, the participants indicated for given time points what they thought the narrator was feeling and thinking while speaking about the event. A Bayesian regression model regressing group and ERA scores on EA scores was showing weak support only for the EA scores for ratings of unspoken feelings from audio only recordings. In a subsample, the quality of self-experienced social interactions in everyday life was assessed with a diary. The analysis of ERA and EA scores in relation to diary scores did not indicate much correspondence. The results are discussed in terms of relations between skills in decoding emotions using different test paradigms and contextual factors.

Ampulla of Vater carcinoma: advancement in the relationships between histological subtypes, molecular features, and clinical outcomes.

The incidence of ampulla of Vater carcinoma, a type of periampullary cancer, has been increasing at an annual percentage rate of 0.9%. However, patients with ampulla of Vater carcinoma have quite different prognoses due to the heterogeneities of the tissue origin of this carcinoma. In addition to TNM staging, histological subtypes and molecular features of ampulla of Vater carcinoma are the key factors for predicting the clinical outcomes of patients. Fortunately, with the development of testing technology, information on the histological subtypes and molecular features of ampulla of Vater carcinoma is increasingly being analyzed in-depth. Patients with the pancreaticobiliary subtype have shorter survival times. In immunohistochemical examination, high cutoff values of positive MUC1 staining can be used to accurately predict the outcome of patients. Mutant KRAS, TP53, negative SMAD4 expression, and microsatellite stability are related to poor prognosis, while the clinical value of BRCA1/BRCA2 mutations is limited for prognosis. Testing the histological subtypes and molecular characteristics of ampulla of Vater carcinoma not only is the key to prognosis analysis but also provides extra information for targeted treatment to improve the clinical outcomes of patients.

Proteogenomic and V(D)J Analysis of Human Decidual T Cells Highlights Unique Transcriptional Programming and Clonal Distribution.

Immunological tolerance toward the semiallogeneic fetus is one of many maternal adaptations required for a successful pregnancy. T cells are major players of the adaptive immune system and balance tolerance and protection at the maternal-fetal interface; however, their repertoire and subset programming are still poorly understood. Using emerging single-cell RNA sequencing technologies, we simultaneously obtained transcript, limited protein, and receptor repertoire at the single-cell level, from decidual and matched maternal peripheral human T cells. The decidua maintains a tissue-specific distribution of T cell subsets compared with the periphery. We find that decidual T cells maintain a unique transcriptome programming, characterized by restraint of inflammatory pathways by overexpression of negative regulators (DUSP, TNFAIP3, ZFP36) and expression of PD-1, CTLA-4, TIGIT, and LAG3 in some CD8 clusters. Finally, analyzing TCR clonotypes demonstrated decreased diversity in specific decidual T cell populations. Overall, our data demonstrate the power of multiomics analysis in revealing regulation of fetal-maternal immune coexistence.

Attention Training Program May Improve Social Anxiety Symptoms

Attention Training Program May Improve Social Anxiety Symptoms

Investigation of the Sox-9 and Cited-1 Immune Activity in Placentas of Women with Placenta Accreta

Aim: In this study, we investigated the immune activity of the Sox-9 and Cited-1 in women with placenta accreta. Material and Methods: 20 healthy and 20 placenta accreta were processed for routine histological tissue processing. Placentals samples were dissected and fixed in 10% formaldehyde solution. Samples were embedded in paraffin blocks. Clinical and biochemical parameters were recorded. Placental sections were cut from paraffin blocks and stained with Sox-9 and Cited-1 immunostaining. Results: In our study, control group showed negative Cited-1 expression in decidual cells, root villi and connective tissue areas in general. Placenta accreta group showed increased Cited-1 expression in degenerated decidual cells, fibroblastic cells and endothelium. In control group, Sox-9 expression was negative in the syncytial knots, in the vascular endothelial cells. In placenta accreta group, Sox-9 reaction was positive in the root villi, in the blood vessels, in the connective tissue. Conclusion: It was observed that the Sox-9 reaction was increased and inflammation was induced, depending on the differences in decidual cells, in the syncytial area and in the vascular endothelium in in placentas of women with placenta accreta. It is thought that Sox-9 signaling processes are being determined and Cited-1 may be stimulants that affect cell proliferation and angiogenesis regulation and affect placental development.

Development of a new biomaterial based on cashew tree gum (Anarcadium occidentale L.) enriched with hydroxyapatite and evaluation of cytotoxicity in adipose-derived stem cell cultures

Cashew tree gum is a polysaccharide material highly available in the Northeast region of Brazil. It has been explored for biocompatibility with human tissues. This research aimed to describe the synthesis and characterization of cashew gum/hydroxyapatite scaffold and evaluate the possible cytotoxicity in murine adipose-derived stem cells (ADSCs) cultures. ADSCs of the subcutaneous fat tissue of Wistar rats were collected, isolated, expanded, differentiated into three strains, and characterized immunophenotypically. The scaffolds were synthesized through chemical precipitation, lyophilized and characterized through scanning electron microscopy (SEM), infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermal analysis (TG and DTG), and mechanical testing. The scaffold presented a crystalline structure and pores with an average diameter of 94.45 ± 50.57 μm. By mechanical tests, the compressive force and modulus of elasticity were like the cancellous bone. The isolated adipose-derived stem cells (ADSCs) presented fibroblast morphology, adhesion capacity to plastic, differentiation in osteogenic, adipogenic and chondrogenic lineages, positive expression for the CD105 and CD90 markers and negative expression for the CD45 and CD14 markers. The MTT test showed increased cell viability, and the biomaterial showed a high level of hemocompatibility (<5 %). This study allowed the development of a new scaffold for future surgical applicability in tissue regeneration.

SPON1 is an independent prognostic biomarker for ovarian cancer

BackgroundOvarian cancer has the worst outcome among gynecological malignancies; therefore, biomarkers that could contribute to the early diagnosis and/or prognosis prediction are urgently required. In the present study, we focused on the secreted protein spondin-1 (SPON1) and clarified the prognostic relevance in ovarian cancer.MethodsWe developed a monoclonal antibody (mAb) that selectively recognizes SPON1. Using this specific mAb, we determined the expression of SPON1 protein in the normal ovary, serous tubal intraepithelial carcinoma (STIC), and ovarian cancer tissues, as well as in various normal adult tissues by immunohistochemistry, and verified its clinicopathological significance in ovarian cancer.ResultsThe normal ovarian tissue was barely positive for SPON1, and no immunoreactive signals were detected in other healthy tissues examined, which was in good agreement with data obtained from gene expression databases. By contrast, upon semi-quantification, 22 of 242 ovarian cancer cases (9.1%) exhibited high SPON1 expression, whereas 64 (26.4%), 87 (36.0%), and 69 (28.5%) cases, which were designated as SPON1-low, possessed the moderate, weak, and negative SPON1 expression, respectively. The STIC tissues also possessed SPON1-positive signals. The 5-year recurrence-free survival (RFS) rate in the SPON1-high group (13.6%) was significantly lower than that in the SPON1-low group (51.2%). In addition, high SPON1 expression was significantly associated with several clinicopathological variables. Multivariable analysis revealed that high SPON1 was an independent prognostic factor for RFS of ovarian cancer.ConclusionsSPON1 represents a prognostic biomarker for ovarian cancer, and the anti-SPON1 mAb could be valuable as an outcome predictor.

Effectiveness of Using Artificial Intelligence for Early Child Development Screening

This study presents a novel approach to recognizing emotions in infants using machine learning models. To address the lack of infant-specific datasets, a custom dataset of infants' faces was created by extracting images from the AffectNet dataset. The dataset was then used to train various machine learning models with different parameters. The best-performing model was evaluated on the City Infant Faces dataset. The proposed deep learning model achieved an accuracy of 94.63% in recognizing positive, negative, and neutral facial expressions. These results provide a benchmark for the performance of machine learning models in infant emotion recognition and suggest potential applications in developing emotion-sensitive technologies for infants. This study fills a gap in the literature on emotion recognition, which has largely focused on adults or children and highlights the importance of developing infant-specific datasets and evaluating different parameters to achieve accurate results.

Facial expressions capturing emotional engagement while learning with desktop VR: the impact of emotional regulation and personality traits

ABSTRACT Virtual reality (VR) has been shown to induce excessive affective processing, which in turn impacts the learning process and learning outcomes. Therefore, a better understanding of emotional dynamics and how emotions are instigated while learning with VR is needed. For this purpose, the students learning process with a VR simulation was captured using continuous measurement of facial expressions. In addition, personality traits and self-regulated emotion (SRE) characteristics were examined. The findings suggest that overall, joy was the most predominant emotion expressed by students, while intraindividual components of personality traits and SRE strategies were associated with negative emotional expressions. Specifically, students with high conscientiousness or agreeableness personality traits tended to experience less anger and/or sadness. In addition, the findings demonstrate that the experience of these negative emotions was mediated by the SRE strategies of reappraisal and suppression, implying a mechanism of emotional engagement while learning with VR. This suggests that while individual personality traits are relatively stable and immutable, SRE preferences can be supported as part of the VR instructional design and scaffolding. Future studies should explore how students’ emotion regulation preferences can be supported to benefit their emotional experiences while learning.

Emotionally focused couple therapy in cancer survivor couples with marital and sexual problems: a replicated single-case experimental design.

The current research examined the effect of Emotionally Focused Couples Therapy (EFCT) on perceived intimacy, affect, and dyadic connection in cancer survivor couples with relationship challenges. In this longitudinal replicated single-case study, positive and negative affect, intimacy, partner responsiveness, and expression of attachment-based emotional needs were reported every 3 days before and during treatment. Thirteen couples, with one partner having survived colorectal cancer or breast cancer, participated for the full duration of the study. Statistical analysis of the data was performed using randomization tests, piecewise regression, and multilevel analyses. Adherence to the therapeutic protocol was tested and found adequate. Compared with baseline, significant positive effects on affect variables were found during the therapeutic process. Positive affect increased and negative affect decreased. Partner responsiveness, perceived intimacy, and the expression of attachment-based emotional needs improved, but only in the later phase of treatment. Results at the group level were statistically significant, whereas effects at the individual level were not. This study found positive group-level effects of EFCT on affect and dyadic outcome measures in cancer survivors. The positive results warrant further research, including randomized clinical trials, to replicate these effects of EFCT in cancer survivor couples experiencing marital and sexual problems.

TFE3 gene rearrangement and protein expression contribute to a poor prognosis of renal cell carcinoma

BackgroundTFE3-rearranged renal cell carcinoma (TFE3-rearranged RCC) is a type of kidney cancer with a low incidence, with no consensus about whether it has a worse prognosis than clear cell renal cell carcinoma (ccRCC). This study attempted to elucidate the impact of TFE3-rearranged RCC by analyzing its clinical features and prognosis. MethodsPatients treated in Sun Yat-sen Memorial Hospital (SYSMH) who were suspected to be diagnosed with TFE3-rearranged RCC were divided into two groups, TFE3-rearranged RCC and ccRCC with positive TFE3 protein expression on immunohistochemistry [TFE3(+) ccRCC], by dual-color, break-apart fluorescence in situ hybridization (FISH). After balancing the baseline characteristics with TFE3(+) ccRCC using the propensity score matching (PSM) method in a ratio of 2, we selected patients diagnosed with ccRCC with negative TFE3 protein expression on immunohistochemistry [TFE3(−) ccRCC]. The impact of TFE3 gene rearrangement and protein expression on renal cell carcinoma was determined by feature comparison with a nonparametric test and survival analysis with the Kaplan‒Meier method. ResultsAmong 37 patients suspected of having TFE3-rearranged RCC, 13 patients were diagnosed with TFE3-rearranged RCC, and 24 patients had TFE3(+) ccRCC. The recurrence and new metastasis of TFE3-rearranged RCC was relatively common, even if the tumor stage was early at the first diagnosis. Through feature comparison and survival analysis, we found that TFE3-rearranged RCC was quite similar to TFE3(+) ccRCC. Compared with TFE3(−) ccRCC, TFE3(+) ccRCC tended to have a larger tumor diameter (P = 0.011), higher neutrophil/lymphocyte ratio (NLR) (P = 0.017) and metastatic potential (P = 0.022), and worse overall survival (OS) (P = 0.043) and PFS (P = 0.016). The survival analysis showed that TFE3-rearranged RCC had a worse PFS than ccRCC (P = 0.002), and TFE3(+) RCC had a worse PFS than TFE3(−) RCC (P = 0.001). According to the stratification system based on the combination of TFE3 and lymphovascular invasion (LVI), we further found that the prognosis from good to poor was TFE3(−) LVI(−), TFE3(+) LVI(−), TFE3(+) LVI(+) and TFE3(−) LVI(+), with statistically significant differences in both OS (P = 0.001) and PFS (P < 0.001). In addition, we also reported two cases with poor prognosis, of which one was TFE3-rearranged RCC and the other was TFE3(+) ccRCC. ConclusionsThis is a novel finding that both FISH confirmed TFE3 gene rearrangement-mediated TFE3-rearranged RCC and IHC confirmed positive TFE3 protein expression [TFE3(+)] contribute to a poor prognosis in RCC, suggesting more active treatment and careful follow-up for TFE3(+) RCC patients. The combination of TFE3 and LVI may be a new risk stratification system for RCC.

Clinical significance of serum-derived exosomal PD-L1 expression in patients with advanced pancreatic cancer

BackgroundInteractions between the programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) lead to immune evasion in various tumors and are associated with poor prognosis in patients with pancreatic cancer; however, the roles of PD-L1-containing exosomes in pancreatic cancer is poorly understood. Here, we investigated the correlation between circulating exosomal PD-L1 (exoPD-L1) and PD-L1 expression in tumor tissue, and survival outcomes in patients with advanced PDAC.MethodsExosomes were derived from pre-treatment serum samples isolated using ExoQuick kit from 77 patients with advanced pancreatic cancer. Exosomal PD-L1 (exoPD-L1) was detected by enzyme-linked immunosorbent assay, and matched tumor tissues PD-L1 expression were evaluated by PD-L1 immunohistochemistry (22C3) assay, described with combined positive score. Cutoff value of exoPD-L1 for survival was assessed with receiver operating characteristic curve analysis. Kaplan-Meier analysis was performed to obtain median overall survival (OS), and hazard ratio was estimated using a stratified Cox regression model.ResultsThe median exoPD-L1 serum concentration was 0.16 pg/mg, with undetected levels in seven patients. ExoPD-L1 levels were significantly higher in patients with systemic disease than in those with locally advanced disease (p = 0.023). There was a significantly higher proportion of elevated exoPD-L1 levels in patients with positive PD-L1 expression compared to patients with negative PD-L1 expression (p = 0.001). Patients were classified into groups with low and high exoPD-L1 levels using ROC curve-derived cutoffs (0.165 pg/mg; area under the curve, 0.617; p = 0.078). At a median follow-up of 8.39 months, the median OS was 13.2 (95% CI, 8.17–18.3) and 6.36 months (95% CI, 3.27–9.45) in the low and high exoPD-L1 groups, respectively (HR = 0.61; 95% CI, 0.35–1.04; p = 0.059). ExoPD-L1 levels did not affect the proportion of CD8+CD69+ effector cytotoxic T cells in either of the groups (p = 0.166).ConclusionsThe serum-derived exoPD-L1 levels were higher in metastatic pancreatic cancer than locally advanced disease. Collectively, higher serum exoPD-L1 levels in patients with advanced pancreatic cancer suggested worse survival outcomes and may have clinical implications.

The Relationship of Self Efficacy with Burnout Syndrome in Nursing Professional Students

Introduction: Burnout is a major problem for students as they face a variety of academic, social, and personal challenges. Burnout can cause negative behavior in students such as unreal in doing coursework, negative expression, not attending lectures, low motivation, dropping out and so on. The purpose of this study was to determine the relationship between self-efficacy and Burnout in professional studentsMethods: This study used a correlational analytical design with a cross sectional approach using the Simple Random Sampling (SRS) Technique, 89 respondents were obtained. The study data were taken using the General Self Efficacy Scale questionnaire. to measure self-efficacy and the Maslach Burnout Inventory questionnaire to measure Burnout. Data analysis using Spearman rhoResults: 82% of students had moderate self-efficacy and 73% had moderate burnout. The spearmans rho test showed indigo p = 0.609 with RS = -0.055, which means that there was no significant relationship between self-efficacy and Burnout in ners professional studentsConclusion: The results showed that the high and low Self Efficacy of professional students will not affect the level of student burnoutKeywords: Self Efficcay, Burnout, Nursing Students

K-POP을 활용한 한국어 어휘적 강조 표현 교육 연구

Objectives This study aims to analyze vocabulary emphasis using K-POP and find ways to learn about it, reflecting that this expression is also a characteristic of K-POP and that many foreign learners want to learn Korean through K-POP.&#x0D; Methods Since there are various meanings and uses of the Korean lexical emphasis expression, this paper examines the lexical characteristics and analyzes the vocabulary shown in the K-POP lyrics. In addition, an efficient educational plan was suggested by analyzing the error pattern of vocabulary emphasis and revealing the cause of the error based on the results.&#x0D; Results First, there is a problem that learners cannot accurately select infinitives according to the characteristics of each type for negative emphasis expression. Accordingly, there is a need to educate what infinitive is appropriate when the subject is different. Second, it is necessary to educate learners about adverbial emphasis so that they can use appropriate adverbs for each situation. Third, in the case of repeated and derivative emphasized expressions, there are many situations in which the meaning of the vocabulary itself is not known rather than incorrectly written, so education is needed.&#x0D; Conclusions It is necessary to use K-POP in order to efficiently educate Korean vocabulary emphasis. Accordingly, the Korean vocabulary emphasized expressions contained in K-POP were listed, and a teaching and learning model of negative emphasized expressions with many errors was presented.

Expression changes of ER, PR, HER2, and Ki-67 in primary and metastatic breast cancer and its clinical significance.

To explore the altered expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and cell proliferation index (Ki-67) in primary and metastatic breast cancer lesions and the correlation between the primary tumor size, lymph node metastasis, Tumor Node Metastasis (TNM) stage, molecular typing, and disease-free survival (DFS) and their clinical significance. A retrospective analysis was conducted on the clinical data of 130 patients with metastatic breast cancer biopsy admitted to the Cancer Center of the Second Affiliated Hospital of Anhui Medical University in Hefei, China, from 2014-2019. The altered expression of ER, PR, HER2, and Ki-67 in primary and metastatic lesions of breast cancer was analyzed with respect to the site of metastasis, size of the primary tumor, lymph node metastasis, disease progression, and prognosis. The inconsistent expression rates of ER, PR, HER2, and Ki-67 in primary and metastatic lesions were 47.69%, 51.54%, 28.10%, and 29.23%, respectively. The size of the primary lesion was not, but that accompanied by lymph node metastasis was related to the altered receptor expression. Patients with positive ER and PR expression in both primary and metastatic lesions had the longest DFS, while those with negative expression had the shortest DFS. Also, changes in HER2 expression in primary and metastatic lesions were not associated with DFS. Patients with low expression of Ki-67 in both primary and metastatic lesions had the longest DFS, while patients with high expression had the shortest DFS. Heterogeneity was detected in the expression levels of ER, PR, HER2, and Ki-67 in the primary and metastatic breast cancer lesions, which has a guiding significance for the treatment and prognosis of patients.

Prognosis value and positive association of Rab1A/IL4Rα aberrant expression in gastric cancer

Gastric cancer (GC) is the most common gastrointestinal cancer and the leading cause of worldwide cancer-associated mortality. Several GC patients are diagnosed at the advanced stage with an unsatisfactory 5-year survival rate. Rab1A was significantly associated with IL4Rα expression in non-small cell lung cancer. However, their potential correlation in expression and prognosis remains largely unknown in GC. In this study, Rab1A/IL-4Rα was significantly increased in GC than in para-cancerous tissues, and Rab1A/IL-4Rα overexpression caused poor prognosis among GC patients. Rab1A expression was significantly correlated with IL-4Rα expression in GC tissues, as determined by IHC analysis. In addition, the mRNA expression of Rab1A was closely linked with the IL-4Rα mRNA expression in GC tissue expressed by qPCR. Furthermore, the Kaplan–Meier analysis demonstrated that the group with negative Rab1A and IL-4Rα expression had longer 5-year survival rates than the other group. Besides, the group with positive Rab1A and IL-4Rα expression had a worse prognosis than the other group. Finally, nomograms revealed the overall 3 and 5-year survival determined crucial roles of Rab1A/IL-4Rα expression in predicting the prognosis of GC patients. Therefore, Rab1A/IL-4Rα is vital in GC, providing a novel perspective on targeted GC therapy.

Assessment of the Androgen Receptor in Older Women with Primary Breast Cancer: Association with a Panel of Biomarkers and Breast Cancer Specific Survival.

Breast cancer in older women tends to have more favourable biology, compared to younger women. Androgen receptor (AR) is significant for breast tumour carcinogenesis; however, the role of AR in older women has not been fully explored. Surgical specimens were obtained from an existing series of 1758 older women (≥ 70years) with primary breast cancer, treated in a single institution with long-term (≥ 37years) follow-up. As part of previous work, it was possible to construct good quality tissue microarrays (TMAs) in 575 surgical specimens and a panel of 24 biomarkers was measured by immunohistochemistry (IHC) in these TMAs. AR positivity was assessed by IHC and defined as H-score ≥ 40. The relationship between AR in this cohort was compared to an equivalent group of younger women (< 70years, n = 1708); the panel of 24 biomarkers and breast cancer specific survival (BCSS) in the older cohort. AR was assessed in 509 samples. Overall, 59% of the older women cohort had positive expression of AR, compared to 63% in the younger cohort. AR positivity (regardless of age) was associated with smaller size of tumour, lower grade of tumour, lower tubule formation, lower nuclear polymorphism and lower mitotic frequency. AR positivity was associated with positive expression of oestrogen receptor (ER), progesterone receptor (PR), breast cancer gene1 (BRCA1), cytokeratin (CK) 7/8, CK18, CK19, Bcell lymphoma (Bcl)2 and Mucin1 (Muc1) expression. Conversely, AR-positive expression was associated with negative expression of human epidermal growth factor receptor2 (HER2), Ki-67, CK5, CK17, epidermal growth factor receptor (EGFR), and CD44 expression. Older women with AR-positive tumours had better BCSS compared to AR-negative tumours (p = 0.009). There was no difference in AR expression between older and younger women with breast cancer. AR has prognostic potential in terms of BCSS. Further work is needed to investigate AR as a therapeutic target.

Study on construction of c-Met specific CAR-T cells and its killing effect on non-small cell lung carcinoma

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.

Acute myeloid leukemia with RAM immunophenotype: A new underdiagnosed entity.

Acute myeloid leukemia (AML) with RAM immunophenotype is a distinct subtype of AML, as described by the Children's Oncology Group (COG), with characteristic morphological and immunophenotypic properties. It is characterized by strong CD56 expression with dim to negative CD45, HLA-DR, and CD38 expression. It is an aggressive leukemia with a poor response to induction chemotherapy and/or frequent relapses. Seven cases with the characteristic RAM immunophenotype were identified in this retrospective analysis of newly diagnosed pediatric AML cases from January 2019 to December 2021. Herein, we have critically analyzed their clinical, morphological, cytochemical, immunophenotyping, cytogenetic, and molecular profiles. The patients were traced and followed for their current disease and treatment status. Of 302 cases of pediatric AML (age <18 years), seven cases (2.3%) with the distinct RAM phenotype were observed, with age ranging from 9 months to 5 years. Two patients were misdiagnosed earlier as small round cell tumor because of the strong CD56 positivity and the absence of leukocyte common antigen (LCA), but they were later correctly identified as granulocytic sarcoma. The bone marrow aspirate showed blasts with unusual cohesiveness and clumping with nuclear moulding, mimicking non-hematologic malignancies. Flow cytometry revealed blasts with low side scatter, dim to negative CD45 and CD38, negative cMPO, CD36, and CD11b; moderate to bright CD33, CD117, and bright CD56. The Mean fluorescence intensity (MFI) of CD13 expression was significantly lower as compared to the internal controls. Cytogenetic and molecular studies did not show any recurrent abnormalities. Reverse transcription polymerase chain reaction for CBFA2T3-GLIS2 fusion was performed in 5/7 cases, with one positive result. On clinical follow-up, two patients were refractory to chemotherapy. Six of the seven cases had succumbed to death (duration of survival: 3-343 days after initial diagnosis). AML with RAM immunophenotype, a distinct form of pediatric AML with a poor prognosis, may pose a diagnostic challenge if presented as a soft tissue mass. A comprehensive immunophenotypic evaluation, including stem cell and myeloid markers, is critical for an accurate diagnosis of myeloid sarcoma with the RAM-immunophenotype. Our data demonstrated weak CD13 expression as an additional immunophenotypic finding.