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.

ObjectiveThe choice of neoadjuvant therapy for esophageal squamous cell carcinoma (ESCC) is controversial. This study aims to provide a basis for clinical treatment selection by establishing a predictive model for the efficacy of neoadjuvant immunochemotherapy (NICT).MethodsA retrospective analysis of 30 patients was conducted, divided into Response and Non-response groups based on whether they achieved major pathological remission (MPR). Differences in genes and immune microenvironment between the two groups were analyzed through next-generation sequencing (NGS) and multiplex immunofluorescence (mIF). Variables most closely related to therapeutic efficacy were selected through LASSO regression and ROC curves to establish a predictive model. An additional 48 patients were prospectively collected as a validation set to verify the model’s effectiveness.ResultsNGS suggested seven differential genes (ATM, ATR, BIVM-ERCC5, MAP3K1, PRG, RBM10, and TSHR) between the two groups (P < 0.05). mIF indicated significant differences in the quantity and location of CD3+, PD-L1+, CD3+PD-L1+, CD4+PD-1+, CD4+LAG-3+, CD8+LAG-3+, LAG-3+ between the two groups before treatment (P < 0.05). Dynamic mIF analysis also indicated that CD3+, CD8+, and CD20+ all increased after treatment in both groups, with a more significant increase in CD8+ and CD20+ in the Response group (P < 0.05), and a more significant decrease in PD-L1+ (P < 0.05). The three variables most closely related to therapeutic efficacy were selected through LASSO regression and ROC curves: Tumor area PD-L1+ (AUC= 0.881), CD3+PD-L1+ (AUC= 0.833), and CD3+ (AUC= 0.826), and a predictive model was established. The model showed high performance in both the training set (AUC= 0.938) and the validation set (AUC= 0.832). Compared to the traditional CPS scoring criteria, the model showed significant improvements in accuracy (83.3% vs 70.8%), sensitivity (0.625 vs 0.312), and specificity (0.937 vs 0.906).ConclusionNICT treatment may exert anti-tumor effects by enriching immune cells and activating exhausted T cells. Tumor area CD3+, PD-L1+, and CD3+PD-L1+ are closely related to therapeutic efficacy. The model containing these three variables can accurately predict treatment outcomes, providing a reliable basis for the selection of neoadjuvant treatment plans.

OBJECTIVE:To compare the application of different treatments in the diagnosis of melanoma with severe pigment interference, to solve the problem of pigment interference with immunohistochemical interpretation. METHODS:The pigment-rich melanomas were first depigmented with potassium permanganate using a concentration gradient (0.1%, 0.5%, 1%) and a time gradient (1, 5, 10, 15, 30 min, 6 h), and the optimal concentration and time were found. Then, 12 cases of pigment-rich melanoma tissues were collected, and the tissues were stained with diaminobenzidine (DAB), alkaline phosphatase-fast red (AP red), multiplex immunofluorescence (MIF), and 3-amino-9-ethylcarbazole (AEC), and ferrous sulfate, comparing different methods, positive expression of HMB45, MelanA, S100, SOX10, ki67. RESULTS:First, the concentration of 0.5% potassium permanganate after 15 min treatment of the pigment significantly faded, and the intensity of antibody positivity was better than other concentrations and time. Second, after depigmentation treatment, the antibody positivity rate was 41.7%-66.7% for DAB, 66.7%-91.7% for AP red, 83.3%-100% for multiplex immunofluorescence, 25%-33.3% for AEC, and 33.3% for ferrous sulfate. CONCLUSION:AP red staining and mIF are more suitable for the diagnosis of melanoma with severe pigment interference, and AP red staining is more economical and practical.

The outcomes of patients with diffuse large B-cell lymphoma (DLBCL) vary widely, and about 40% of them could not be cured by the standard first-line treatment, R-CHOP, which could be due to the high heterogeneity of DLBCL. Here, we aim to construct a prognostic model based on the genetic signature of metabolic heterogeneity of DLBCL to explore therapeutic strategies for DLBCL patients. Clinical and transcriptomic data of one training and four validation cohorts of DLBCL were obtained from the GEO database. Metabolic subtypes were identified by PAM clustering of 1,916 metabolic genes in the 7 major metabolic pathways in the training cohort. DEGs among the metabolic clusters were then analyzed. In total, 108 prognosis-related DEGs were identified. Through univariable Cox and LASSO regression analyses, 15 DEGs were used to construct a risk score model. The overall survival (OS) and progression-free survival (PFS) of patients with high risk were significantly worse than those with low risk (OS: HR 2.86, 95%CI 2.04-4.01, p < 0.001; PFS: HR 2.42, 95% CI 1.77-3.31, p < 0.001). This model was also associated with OS in the four independent validation datasets (GSE10846: HR 1.65, p = 0.002; GSE53786: HR 2.05, p = 0.02; GSE87371: HR 1.85, p = 0.027; GSE23051: HR 6.16, p = 0.007) and PFS in the two validation datasets (GSE87371: HR 1.67, p = 0.033; GSE23051: HR 2.74, p = 0.049). Multivariable Cox analysis showed that in all datasets, the risk model could predict OS independent of clinical prognosis factors (p < 0.05). Compared with the high-risk group, patients in the low-risk group predictively respond to R-CHOP (p = 0.0042), PI3K inhibitor (p < 0.05), and proteasome inhibitor (p < 0.05). Therefore, in this study, we developed a signature model of 15 DEGs among 3 metabolic subtypes, which could predict survival and drug sensitivity in DLBCL patients.

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.

Adenoid cystic carcinoma (AdCC) of the breast is a rare indolent carcinoma of salivary gland-type tumors, frequently associated with MYB genetic alteration. Solid and basaloid adenoid cystic carcinoma (SB-AdCC) is considered a sparse variant of AdCC. This study sought to search for clinicopathological and genomic features in SB-AdCC. Registered clinicopathological data on a cohort of 13 AdCC of the breast cases, including six conventional adenoid cystic carcinoma (C-AdCC) cases and seven SB-AdCC cases, were collected. MYB gene rearrangement via fluorescent in situ hybridization was investigated and MYB protein expression was evaluated by immunohistochemistry. Compared with C-AdCC, we found that the distribution of SB-AdCC cases were shifted to older age and were more frequently distant metastasis. Moreover, metastasis cases also showed a high (exceed 30%) Ki-67 index. Both groups showed MYB rearrangements and MYB protein expression, but they were less frequent in SB-AdCC than C-AdCC. To conclude, our results suggest that SB-AdCC is an aggressive variant of mammary AdCC with a higher incidence of distant metastases compared with C-AdCC, though they share common molecular features. A high Ki-67 index may be an adverse prognostic factor for metastasis.

e17513 Background: The differential diagnosis is challenging in clinical practice, especially among gastric-type AD of the cervix with ovarian metastases, multiple primary mucinous neoplasm in cervix and ovary, and synchronous mucinous metaplasia and neoplasia of the female genital tract (SMMN-FGT). Previous studies have revealed that next-generation sequencing might be a feasible tool in identifying the origin of tumor. Herein, whole exome sequencing (WES) was performed to distinguish patients with metastatic disease. Methods: WES analysis was performed on 28 tumors and 7 normal tissues derived from 8 patients. Each patient had one lesion of the cervix uteri. Most of them had lesions at other anatomical sites, including corpus uteri, ovary, oviduct, vermiform appendix and omentum. Mutations signatures and clonal evaluation inferred from single-nucleotide/copy number variants were explored. Results: Previous studies of multiple types of cancer have identified more than 40 single-base substitution (SBS) signatures. SBS3/6 referring to defective DNA mismatch repair and SBS10 reported in both uterus and colorectal AD were significantly enriched in this study. Next, we found no significant distribution of SBS3/6/10 among tumors at different anatomical sites. These findings indicate that primary cervical/ovarian AD with metastases other than multiple primary cancers was the predominant histology in 8 patients. The association of genomic alterations in POLE/ POLD1/HRR pathway with the distribution of SBS signatures was explored. SBS3 had a trend of significant distribution (p = 0.01) in four patients who had germline alterations in HRR pathway. Furthermore, SBS10 was significantly enriched (p = 0.009) in 2 patients with germline POLE/POLD1 alterations. Clonal evolution analyses revealed that 2 patients were identified as having SMMN-FGT, and other 6 patients had dominant clones in different tumors, which suggested a . Notably, besides the dominant clones and subclones, some tumors also exhibited extra independent clones, which might be the mixed histology of primary and metastatic tumor. In addition, we found the difference of overall survival in patients having gastric-type AD of the cervix with ovarian metastases and cervical AD patients with different FIGO stage disease based on SEER database data. Conclusions: Our study demonstrated that WES might be a feasible tool to distinguish patients having primary cervical AD with multiple metastases from those with multiple primary cancers of the female genital tract, which suggests that WES proved valuable in facilitating the diagnostic workup. A prospective, multi-center, large cohort study is needed to investigate the performance of WES in identifying the origin of tumors in patients with multiple tumors of the FGT.

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.

Objective To study the pharmacokinetics and tissue distribution of dihydro-salvianolic acid B and sal-vianolic acid B in mice after intravenous injection.Methods Mice were injected with dihydro-salvianolic acid B or salvianolic acid B through the caudal vein.Then at different time points,the concentration of dihydro-salvianolic acid B and salvianolic acid B in serum and in tissues of heart,liver,spleen,lung and kidney were determined by HPLC with protein precipitation method,and pharmacokinetic parameters were calculated with DAS2.0 practical pharma-cokinetics program.Results Half-life of elimination of dihydro-salvianolic acid B and salvianolic acid B in mice serum was(8.272±0.689)min and(9.844±1.946)min,and area under the curve(0-t) was(7482.991±1999.117)mg.L-1.min and(7407.538±1078.886)mg.L-1.min,respectively.The all distributed widely in mice tissues,particularly in liver,kidney and lung,and were eliminated rapidly.Conclusion The established HPLC methods are sensitive,simple,and accurate,and can be used to determine the concentration of dihydro-salvianolic acid B and salvianolic acid B in the biological samples.Distribution and elimination of dihydro-salvianolic acid B are similar to that of sal-vianolic acid B in mice after intravenous injection,distributed widely in the tissues and eliminated rapidly.

Ethnopharmacological relevance: Quality analysis and control of Chinese herbal medicines (CHM) or herbal medicines (HM) are being more and more investigated based on fingerprint analysis, and there are also some researches on correlating fingerprints of CHM to their efficacy. Multi-component analysis methods together with fingerprints are considered potential useful tools to select candidate herbal drugs from extracts of herbs in pharmacological/bio-prospecting investigations.Aim of the study: To explore a strategy for evaluating efficacy strength of CHM samples based on their spectra fingerprints and validate it.Methodology: Radix bupleuri (RB), a typical Chinese medicinal herb for relieving exterior syndrome, and Flos lonicerae (FL), Fructus forsythiae (FF), and Radix isatidis (RI) that are widely applied Chinese herbs for heat clearing and detoxifying, were selected as herbal sources. The aqueous extracts, volatile oils and mixtures of the extracts and oils of the four herbs, plus Ibuprofen suspension (IS). Shuanghuanglian oral liquid (SHL), mixture of SHL and the volatile oils of FL and FF, were used for subject samples to do antipyretic experiments on rats. Ultraviolet spectra were used as the spectra fingerprints to represent chemical characteristics of the samples. Principal component analysis (PCA) and canonical correlation analysis (CCA) were adopted as evaluation tools to establish the correlation between pharmacological and spectra data, from which a spectral index for evaluating antipyretic effects of CHM samples was constructed. Furthermore, four compound samples were designed by mixing 50% volatile oils and 50% aqueous extracts of the four herbs with different ratios to validate the strategy.Results: Efficacy sequence of the 15 calibrating and 4 validating CHM samples, defined by the first canonical correlative variable U-1 of their UV spectra, was consistent with that given by pharmacological experiments.Conclusions: The strategy proposed in this study could be applied to evaluate efficacy strength of CHM and helpful for screening candidate herbal drugs from different herbs or prepared by different technologies. (C) 2009 Elsevier Ireland Ltd. All rights reserved.