Study on the effect of different durations of decalcification and depigmentation on programmed death ligand 1 immunohistochemical staining.

Background: Programmed death ligand 1 (PD-L1) score is an important companion diagnosis to predict the response to immunotherapy. Immunohistochemistry can accurately assess the expression of PD-L1 in routine paraffin-embedded tissue. However, whether decalcified or depigmented tissue is still accurate and can be used as a companion diagnosis is controversial. This study attempts to resolve this controversy by analyzing the effects of decalcification and depigmentation at different times on PD-L1 expression. Methods: Placental tissues were selected for tissue microarray, decalcification was performed according to time gradients of 6, 12, 24, 36, and 48 h, and depigmentation was performed according to time gradients of 1, 5, 15, 30, and 60 min. The intensity of PD-L1 expression at different time points was observed and quantified. Ten PD-L1-positive esophageal squamous carcinoma samples were selected for decalcification treatment, and the PD-L1. Combined Positive Score (CPS), Tumor Proportion Score (TPS) and Immunocyte Proportion Score (IPS) and the positivity rates were compared before and after decalcification. Results: After the placenta was decalcified, the intensity of PD-L1 positivity diminished, and the average optical density (AOD) value decreased with the prolongation of decalcification time and decreased significantly (P<0.05) at 24 h compared with the control group, and significantly (P<0.01) at 36 and 48 h compared with the control group. The intensity of PD-L1 positivity was weakened considerably after the treatment with potassium permanganate depigmentation. In addition, the AOD value decreased significantly (P<0.01) after the depigmentation time reached 5 min compared with the control group. Ten cases of PD-L1 positive esophageal squamous carcinoma were treated with 24 h decalcification, although the PD-L1 score decreased to a certain degree (P>0.05), and the positivity rate could reach 90%. After 36 h treatment, PD-L1 scores decreased, the CPS and IPS scores decreased significantly (P<0.05), and the positive rate was only 50%. Conclusions: Potassium permanganate depigmentation significantly reduces PD-L1 expression, even for a shorter time, affecting the accuracy of the results. The accuracy of PD-L1 remained high within 24 h decalcification. The above results have certain reference value for clinical selection of immunotherapy.

Liquid biopsy in T-cell lymphoma: biomarker detection techniques and clinical application.

T-cell lymphoma is a highly invasive tumor with significant heterogeneity. Invasive tissue biopsy is the gold standard for acquiring molecular data and categorizing lymphoma patients into genetic subtypes. However, surgical intervention is unfeasible for patients who are critically ill, have unresectable tumors, or demonstrate low compliance, making tissue biopsies inaccessible to these patients. A critical need for a minimally invasive approach in T-cell lymphoma is evident, particularly in the areas of early diagnosis, prognostic monitoring, treatment response, and drug resistance. Therefore, the clinical application of liquid biopsy techniques has gained significant attention in T-cell lymphoma. Moreover, liquid biopsy requires fewer samples, exhibits good reproducibility, and enables real-time monitoring at molecular levels, thereby facilitating personalized health care. In this review, we provide a comprehensive overview of the current liquid biopsy biomarkers used for T-cell lymphoma, focusing on circulating cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), Epstein-Barr virus (EBV) DNA, antibodies, and cytokines. Additionally, we discuss their clinical application, detection methodologies, ongoing clinical trials, and the challenges faced in the field of liquid biopsy.

Primary proximal epithelioid sarcoma of the lung: A case report and review of the literature.

Epithelioid sarcoma (ES) is a rare mesenchymal tumor that can be divided into proximal/axial type and classical/distal type. Primary proximal epithelioid sarcoma of the lung is extremely rare. So far, no more than five cases have been reported. We reported a case of primary pulmonary ES and reviewed the literature to summarize its clinicopathological features. A 51-year-old man presented with hemoptysis and cough. Chest computed tomography (CT) showed a nodule located in the apical and posterior segments of the left upper lobe of the lung. The patient underwent a lobectomy, and a pathologic diagnosis of epithelioid sarcoma was made. Histologically, most tumors are composed of epithelioid cells with evidence of bidirectional expression of epithelium and mesenchyma. The SMARCB1 stain of tumor cells was negative, and a pathogenic mutation of SMARCB1 p.E115* (exon 3) were identified by the next-generation sequencing. Two months after surgery, positron emission tomography/computed tomography (PET/CT) indicated tumor recurrence, and the patient received a round of adjuvant chemotherapy combined with immunotherapy. After 11 months of follow-up, the patient died. We reported in detail the primary proximal epithelioid sarcoma of the lung treated with immunotherapy for the first time, providing ideas for diagnosis and treatment.

Development and Validation of a Recurrence-Free Survival Prediction Model for Locally Advanced Esophageal Squamous Cell Carcinoma with Neoadjuvant Chemoradiotherapy.

BACKGROUND: A recurrence-free survival (RFS) prediction model was developed and validated for patients with locally advanced esophageal squamous cell carcinoma treated with neoadjuvant chemoradiotherapy (NCRT) in combination with surgery. PATIENTS AND METHODS: We included 282 patients with esophageal squamous cell carcinoma who received neoadjuvant chemoradiotherapy (NCRT) combined with surgery, constructed three models incorporating pathological factors, investigated the discrimination and calibration of each model, and compared the clinical utility of each model using the net reclassification index (NRI) and the integrated discrimination index (IDI). RESULTS: Multivariable analysis showed that pathologic complete response (pCR) and lymph node tumor regression grading (LN-TRG) (p < 0.05) were independent prognostic factors for RFS. LASSO regression screened six correlates of LN-TRG, vascular invasion, nerve invasion, degree of differentiation, platelet grade, and a total diameter of residual cancer in lymph nodes to build model three, which was consistent in terms of efficacy in the training set and validation set. Kaplan-Meier (K-M) curves showed that all three models were able to distinguish well between high- and low-risk groups (p < 0.01). The NRI and IDI showed that the clinical utility of model 2 was slightly better than that of model 1 (p > 0.05), and model 3 was significantly better than that of model 2 (p < 0.05). CONCLUSIONS: Clinical prediction models incorporating LN-TRG factors have high predictive efficacy, can help identify patients at high risk of recurrence after neoadjuvant therapy, and can be used as a supplement to the  AJCC/TNM staging system while offering a scientific rationale for early postoperative intervention.

The Diagnostic and Prognostic Value of EBUS-TBNA for Intrathoracic Metastasis in Previously Treated Patients With Head and Neck Cancer.

OBJECTIVE: The present study assessed the diagnostic and prognostic significance of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for suspected intrathoracic metastasis after HNC treatment. METHODS: A retrospective analysis was conducted on 75 patients with a prior history of head and neck cancer treatment who underwent EBUS-TBNA for suspected intrathoracic metastases between March 2012 and December 2021. RESULTS: A total of 126 targeted lesions, including 107 mediastinal/hilar lymph nodes and 19 intrapulmonary/mediastinal masses, were sampled. The metastatic head and neck cancer (HNC) cases detected by EBUS-TBNA consisted of nasopharyngeal carcinoma (n = 24), oropharyngeal carcinoma (n = 3), hypopharynx carcinoma (n = 6), laryngeal carcinoma (n = 6), and oral cavity carcinoma (n = 6). Cases with negative EBUS-TBNA results consisted of tuberculosis (n = 9), sarcoidosis (n = 3), anthracosis (n = 9), and reactive lymphadenitis (n = 9). Six false-negative cases were found among the 75 patients with suspected intrathoracic metastases. The diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the EBUS-TBNA procedure for metastatic HNC were 88.2, 100.0, 100.0, 80, and 92.0%, respectively. The diagnosis of HNC intrathoracic metastasis by EBUS-TBNA correlated with an adverse prognosis in terms of overall survival (OS) (P = .008). The log-rank univariate analysis and Cox regression multivariate analysis results indicated that the detection of metastatic HNC through EBUS-TBNA was a significant independent prognostic factor for patients with HNC who had received prior treatment. CONCLUSIONS: Endobronchial ultrasound-guided transbronchial needle aspiration is a safe, effective, and minimally invasive procedure for assessing suspected intrathoracic metastasis in HNC patients after treatment. The intrathoracic metastasis detected by EBUS-TBNA has crucial prognostic significance in previously treated HNC patients.

PARG Promotes Esophagus Cancer Cell Metastasis by Activation of the Wnt/ß-Catenin Pathway.

Esophagus cancer (EC) is a highly malignant and metastatic cancer. Poly(ADP-ribose) glycohydrolase (PARG), a DNA replication and repair regulator, inhibits cancer cell replication defects. This study aimed to explore the role of PARG in EC. The biological behaviors were analyzed using MTT assay, Transwell assay, scratch test, cell adhesion assay, and western blot. PARG expression was detected using quantitative PCR and immunohistochemical assay. The regulation of the Wnt/ß-catenin pathway was assessed using western blot. The results showed that PARG was highly expressed in EC tissues and cells. Knockdown of PARG suppressed cell viability, invasion, migration, adhesion, and epithelial-mesenchymal transition. Conversely, overexpression of PARG promoted the biological behaviors mentioned above. Moreover, overexpression of PARG promoted the activation of the Wnt/ß-catenin pathway rather than the STAT and Notch pathways. XAV939, the Wnt/ß-catenin pathway inhibitor, partly abolished the biological behaviors mediated by PARG overexpression. In conclusion, PARG promoted the malignant advancement of EC via activating the Wnt/ß-catenin pathway. These findings suggested that PARG might be a new therapeutic target for EC.

CCDC106 promotes the proliferation and invasion of ovarian cancer cells by suppressing p21 transcription through a p53-independent pathway.

Ovarian cancers are the major cause of mortality for women worldwide. This study was aimed to elucidate the biological activities of CCDC106 in the proliferation and invasion of mutant p53 and of wild-type p53 ovarian cancer cells. CAOV3 (mutant p53) cells showed high expression levels of CCDC106, but it was expressed at low levels in SKOV3 (mutant p53) and in A2780 (wild-type p53) cells. The overexpression of CCDC106 promoted the expression of proliferation markers (cyclin family members), invasion and Epithelial-to-mesenchymal transition (EMT) markers (claudin-1, claudin-4, N-cadherin, snail, slug) while the knockdown of CCDC106 inhibited their expression in mutant p53 cells but not in wild-type p53 cells. Treatment with a CK2 inhibitor blocked the translocation of CCDC106 into the nuclei of mutant p53 cells. Immunoprecipitation assays confirmed that ATF4 is a potential binding partner of CCDC106. The overexpression of CCDC106 reduced p21 and p27 protein expression levels while treatment with an ATF4 siRNA rescued their expression. The overexpression of CCDC106 promoted colony formation and invasion of mutant p53 cells, which was suppressed by treatment with an ATF4 siRNA. Immunohistochemistry results showed that CCDC106 and ATF4 are expressed at high levels but p21 is expressed at low levels in FIGO III-IV stage and in mutant p53 ovarian cancer samples. A significant association between poor overall survival and high CCDC106 and ATF4 expression levels was observed in human ovarian cancer samples. In conclusion, CCDC106 promotes proliferation, invasion and EMT of mutant p53 ovarian cancer cells via the ATF4 mediated inhibition of p21.

Alleviation of neoadjuvant immunochemotherapy for esophageal squamous cell carcinoma and its relationship with expression and changes of PD-L1.

The alleviation of neoadjuvant immunochemotherapy (NICT) and neoadjuvant chemoradiotherapy (NCRT) was compared for esophageal squamous cell carcinoma (ESCC), and the correlation between the expression and changes of PD-L1 and the efficacy of NICT was evaluated in this study. Fourteen patients with ESCC who received preoperative NICT were included in group A, and fourteen patients with ESCC who received preoperative NCRT were included in group B. Next, group A was divided into CR (complete response), PR (partial response), and NR (no response) according to the degree of pathological response. Also, the expression and changes of PD-L1 (CPS, TPS, IPS) before and after treatment were compared between the groups. We observed that after the treatment, the expression of PD-L1 in both groups was higher than before treatment. In group B, the expression of PD-L1 was elevated in 92.8% of patients, which was higher than that in group A, which had significantly increased IPS (p<0.05). In group A, 9 (64.2%) patients with CPS <10 achieved partial or complete response. There was no significant difference in pathological response and reduction of tumor thickness between the two groups or significant differences in CPS and TPS among CR, PR, and NR groups before treatment. The IPS was the highest in the CR group; however, the difference was not statistically significant. The differences in IPS change were significant among the three groups (p<0.05). In conclusion, NICT and NCRT could upregulate the expression of PD-L1. NCRT more significantly upregulated the expression of PD-L1, mainly of PD-L1 in immune cells in the tumor microenvironment. NICT was not less effective than NCRT in pathological response and tumor thickness changes. The preoperative CPS and TPS scores of PD-L1 did not effectively predict the degree of pathological response, but the high IPS and high IPS downregulation could be related to the degree of pathological response. Some patients with low preoperative expression of PD-L1 could still achieve a good response by NICT. As NCRT can upregulate the expression of PD-L1, the low preoperative expression of PD-L1 is no contraindication for immunotherapy, which provides a new basis and prognostic indexes for chemoradiotherapy combined with immunotherapy.

The Novel Role of Cytomorphology from Ultrasound-Guided Fine Needle Aspiration Cytology in Evaluating the Status of Prognostic Factors including Estrogen Receptor, Progesterone Receptor and HER2 in Breast Cancer.

It is well established that estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2) could be regarded as prognostic factors in breast cancer. Ultrasound-guided fine needle aspiration cytology (FNAC) has revolutionized the management of cancers, providing less invasive and quick diagnostic method. There are hardly any studies on the correlation between cytomorphology and prognostic biomarkers. We retrospectively analyzed the immunohistochemistry and the fluorescence in situ hybridization of breast cancer specimens from 252 patients, who have been diagnosed as breast cancer at our hospital. Morphological features of cytology smears were scored. The relationship between cytological features and three biomarkers were analyzed. Based on this, we developed a system to predict the status of biomarkers. The results indicated that some cytological parameters, especially the features of nucleoli, were distinctively related to the makers' expression. In the novel scoring system, a cutoff of 12.0 provided a statistical discrimination for cytological grading. We concluded that cytomorphological features were associated with prognostic factors. The HR+ neoplasms showed scattered micronucleoli, while HER2+ neoplasms demonstrated centered macronucleoli. We summarized a scoring system to predict the status of three factors. This may help us to broaden the application of breast cancer cytology.

Centrosomal protein 290 is a novel prognostic indicator that modulates liver cancer cell ferroptosis via the Nrf2 pathway.

Ferroptosis is an iron-dependent form of cell death. In spite of its significance in pathogenesis and disease progression, ferroptotic signal transduction in HBV-HCC has not been fully explained. Here, four HCC open-source datasets were downloaded from the GEO repository. Cox regression and LASSO models were established to prioritize novel prognostic candidate biomarkers, and the results were verified in vitro and in vivo. We identified 633 common DEGs in both of the bulk RNA-Seq expression profiles. Next, based upon the TCGA-LIHC cohort, a prognostic signature consisting of nine genes was extracted from 633 shared DEGs, and the specificity and sensitivity of the signature were evaluated in both training and validation datasets. This signature showed that the high-risk group had a worse prognosis than the low-risk group. CEP290 was discovered among the prognostic signature genes, and its expression notably correlated with survival, AFP level, TNM stage and vascular invasion. We confirmed expression of CEP290 in eight pairs of HCC tissues and diverse liver cancer cell lines. CEP290 knockdown reduced proliferation, migration and invasion in Hep3B liver cancer cells while Fe2+ and malondialdehyde levels were elevated. Mechanically, co-immunoprecipitation showed an interaction between CEP290 and Nrf2 proteins, and biological phenotypes of Hep3B cells under CEP290 interference were rescued by Nrf2 activator. Furthermore, CEP290 silencing considerably blocked protein expression of Nrf2 pathway members. Finally, suppression of CEP290 effectively inhibited tumor growth in vivo. The above results shed light on the important role of CEP290 in ferroptosis and present an important implication for HCC progression.

Nuclear Morphological Characteristics in Breast Cancer: Correlation with Hormone Receptor and Human Epidermal Growth Factor Receptor 2.

BACKGROUND: Hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) are the common diagnostic/prognostic markers in breast cancer. Few articles have recently reported the correlation between cytology and molecular subtypes. We combined nuclear morphological characteristics with HR and HER2 status to observe the relationship and provide ideas for machine learning. METHODS: We reanalyzed fine-needle aspiration cytology samples and core-needle puncture histological specimens from 142 patients with invasive breast cancer between March 2019 and December 2019, and the findings were compared with the two groups (HR+/HER2- and HR-/HER2+) following nuclear cytomorphological features: nuclear/cytoplasmic ratio, difference of nuclear size, nuclear pleomorphism, chromatin feature, nuclear membrane and nucleoli, and Nottingham grading. RESULTS: Two groups were significantly associated with the difference of nuclear size, nuclear pleomorphism, and nucleoli (P < 0.001) and consistent with histological grading (P < 0.001). Moreover, nucleolar characteristics of size and number had obviously statistical significance (P < 0.001). Multiple micro-nucleoli were frequently seen in the HR+/HER2- group compared with the HR-/HER2+ group which mostly were observed centered medium-large nucleoli. We described four interesting nuclear morphologies in the experiment. CONCLUSIONS: There were significant differences in nuclear characteristics between two groups. HR and HER2 status not only might be predicted in cytological samples, but some specific nuclear morphological features might have potential value to help us understand molecular function and predict more information.

Ubiquitin-Like Modifier Activating Enzyme 1 as a Novel Diagnostic and Prognostic Indicator That Correlates With Ferroptosis and the Malignant Phenotypes of Liver Cancer Cells.

PURPOSE: Ferroptosis is a type of cell death that is iron dependent, a characteristic that distinguishes it from necrosis, apoptosis, and autophagy. However, the ferroptotic mechanisms for hepatitis B virus-associated hepatocellular carcinoma (HCC) remain incompletely described. METHODS: Two hepatitis B virus-associated HCC public datasets, GSE22058 (n=192) and GSE54238 (n=23), were obtained from the NCBI Gene Expression Omnibus (GEO) database. Bioinformatics methods, including weighted gene coexpression network analysis (WGCNA), Cox regression, and LASSO analysis, were used to identify signature markers for diagnosis and prognosis. CCK8, wound healing, Transwell migration/invasion, and ferroptosis assays were employed to explore the biological function of novel candidate markers weight gene coexpression network analysis. RESULTS: In total, 926 differentially expressed genes (DEGs) were common between the GSE22058 and GSE54238 datasets. Following WGCNA, 515 DEGs derived from the MEturquoise gene module were employed to establish diagnosis and prognosis models in The Cancer Genome Atlas (TCGA) HCC RNA-Seq cohort (n=423). The score of the diagnostic model was strikingly upregulated in the TCGA HCC group (p<2.2e-16). The prognostic model exhibited high specificity and sensitivity in both training and validation (AUC=0.835 and 0.626, respectively), and the high-risk group showed dismal prognostic outcomes compared with the low-risk group (training: p=1.416e-10; validation: p=4.495e-02). Ubiquitin-like modifier activating enzyme 1 (UBA1) was identified among both diagnosis and prognosis signature genes, and its overexpression was associated with poor survival. We validated the expression level of UBA1 in eight pairs of HCC patient tissues and liver cancer cell lines. UBA1 silencing decreased proliferation, migration, and invasion in Huh7 cells while elevating the Fe2+ and malondialdehyde (MDA) levels. Additionally, these biological effects were recovered by oltipraz (an Nrf2 activator). Furthermore, blocking UBA1 strikingly repressed the protein expression levels of Nrf2, HO-1, NQO1, and FTH1 in the Nrf2 signal transduction pathway. CONCLUSION: Our findings demonstrated that UBA1 participates in the development of HCC by modulating Huh7 phenotypes and ferroptosis via the Nrf2 signal transduction pathway and might be a promising diagnostic and prognostic indicator for HCC.

[Grape seed proanthocyanidins inhibits the invasion and migration of A549 lung cancer cells].

OBJECTIVE: To explore the effect of grape seed proanthocyanidins (GSPs) on the invasion and migration of A549 lung cancer cells and the underlying mechanism. METHODS: Trypan blue dye exclusion assay was used to determine the cytotoxic effect of varying doses of GSPs on the BEAS-2B normal human pulmonary epithelial cells. After treated with 0, 10, 20, 40, 80 µg/mL GSP, the proliferation of A549 cells was detected by MTT assay; the invasion and migration of A549 cells were determined by Transwell(TM) assay and scratch wound assay, respectively. The levels of epithelial growth factor receptor (EGFR), E-cadherin, N-cadherin in A549 cells treated with GSPs were detected by Western blotting. RESULTS: (0-40) µg/mL GSPs had no significant toxic effect on BEAS-2B cells, while 80 µg/mL GSPs had significant cytotoxicity to BEAS-2B cells. The proliferation of A549 cells was significantly inhibited within limited dosage in a dose-dependent manner, and the abilities of invasion and migration of A549 cells were also inhibited. Western blotting showed that the expression of EGFR and N-cadherin decreased, while E-cadherin increased after GSPs treatment. CONCLUSION: GSPs could inhibit the abilities of proliferation, invasion and migration of A549 cells, which might be related to the dow-regulation of EGFR and N-cadherin and the up-regulation of E-cadherin.

Resveratrol provides neuroprotection by inhibiting phosphodiesterases and regulating the cAMP/AMPK/SIRT1 pathway after stroke in rats.

Dysfunction of energy metabolism can be a significant and fundamental pathophysiological basis for strokes. In studies of both humans and rodents, resveratrol, a natural polyphenol, has been reported to provide protection from cerebral ischemic injury by regulating expression of silent mating type information regulation 2 homolog 1 (SIRT1). However, direct evidence demonstrating that resveratrol exerts neuroprotection from cerebral ischemia injury by decreasing energy consumption is still lacking. Therefore, the aim of this study was to elucidate the mechanisms and signaling pathways through which resveratrol regulates energy metabolism in the ischemic brain, and to identify potential targets of resveratrol. ATP levels in brain tissues were detected by high performance liquid chromatography. SIRT1 and the phosphorylation of adenosine-monophosphate-activated protein kinase (P-AMPK) expressiones were evaluated by western blot. Levels of phosphodiesterase (PDEs) and cAMP were quantitated by real-time PCR and ELISA, respectively. Results showed that resveratrol significantly reduced the harmful effects of cerebral ischemic injury in vivo. Moreover, levels of ATP, p-AMPK, SIRT1, and cAMP were increased by resveratrol and PDE inhibitors. In conclusion, our findings indicate that resveratrol provides neuroprotection by inhibiting PDEs and regulating the cAMP/AMPK/SIRT1 pathway, which reduces ATP energy consumption during ischemia.