[Research and exploration of salivary biological markers for hepatitis B-related hepatocellular carcinoma].

Objective: To study using isotope-labeled relative and absolute quantitative proteomics methodologies to screen for salivary biological markers as a simple, non-invasive tool for identifying hepatitis B-related HCC at an early stage. Methods: Saliva samples were collected to extract salivary proteins. Isotope-labeled relative and absolute quantitative proteomics were used to analyze the differentially expressed proteins between the hepatocellular carcinoma (HCC) and non-HCC groups. Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays were used to verify differential proteins and identify markers in liver cancer tissues and saliva. Statistical analysis was used to analyze the diagnostic efficiency of salivary biomarkers. Results: 152 differentially expressed salivary proteins were screened out between the HCC and non-HCC groups. Western blot, immunohistochemistry, and enzyme-linked immunosorbent assays validated that the expressions of α-1-acid glycoprotein 1 (ORM1) and alpha-fetoprotein (AFP) were significantly increased in HCC (P < 0.05). There was a significant correlation between salivary AFP and serum AFP (P < 0.05). HCC was diagnosed when salivary α-1-acid glycoprotein 1 combined with AFP. The area under the receiver operating characteristic curve was 0.8726 (95% confidence interval: 0.8104 ~ 0.9347), the sensitivity was 78.3%, and the specificity was 88%. Conclusion: Salivary AFP and α-1-acid glycoprotein 1 can serve as potential biomarkers for hepatitis B-related hepatocellular carcinoma.

[Correlation between daily fluid intake behavioral habits and pathological characteristics of upper tract urothelial carcinoma].

OBJECTIVE: To investigate the correlation between drinking habits and pathological characteristics of patients with upper tract urothelial carcinoma (UTUC). METHODS: A preoperative questionnaire survey was conducted to understand the drinking habits of UTUC patients who were admitted to the Department of Urology, Peking University First Hospital for radical nephroureterectomy within one year from August 2020 to July 2021, and statistical analysis was performed in combination with their postoperative pathological characteristics. The statistical procedure was performed using SPSS 22.0 software, and firstly, the preliminary analysis was performed one by one using the columnar χ2 test on the pathological characteristics of UTUC tumors as the dependent variable and the factors related to patients' general information, past history and drinking habits as the independent variables, and the independent variables that met P < 0.2 between the case and control groups for each dependent variable were specified for screening. The screened variables were included in the binary Logistic regression analysis. A difference of P < 0.05 was used to indicate a statistically significant difference. RESULTS: A total of 239 patients, 134 males and 105 females, with a mean age of (68.1±9.98)years and a median disease duration of 4.8 months, were included in this study. Multifactorial Logistic regression results suggested that after adjusting for the effects of other variables, UTUC patients who had the habit of drinking at least once every hour during the daytime had a significantly increased risk of high grade (G3) tumors(OR=1.941, 95%CI: 0.352-1.029, P < 0.01); these patients also had a significantly decreased risk of multifocal UTUC tumors (OR=0.344, 95% CI: 1.18-5.582, P=0.004). The patients who had the habit of drinking over 100 mL water each time had a significantly decreased risk of mutifocal UTUC incidence (OR=0.477, 95%CI: 0.225-1.012, P=0.046). Patients who pay attention to daily water intakes had a significantly increased risk of renipelvic carcinoma (OR=2.530, 95%CI: 1.434-4.463, P=0.001) and a significantly decreased risk of ureteral carcinoma (OR=0.314, 95%CI: 0.172-0.573, P < 0.01). Other variables included in the regression model did not differ significantly in their effects on the occurrence of tumor pathological characteristics. CONCLUSION: Having the awareness of drinking water every 1 h during the day, drinking over 100 mL water each time, having the awareness of daily drinking habits correlated significantly with pathological characteristics of UTUC such as the presence of G3 tumor, multifocal tumors and location of the tumor. This conclusion still needs to be verified by subsequent trials with higher levels of evidence.

[Expression and significance of fibroblast growth factor receptor 2 in clear cell renal cell carcinoma].

OBJECTIVE: To investigate the expression of fibroblast growth factor receptor 2 (FGFR2) in clear cell renal cell carcinoma (ccRCC; or kidney renal clear cell carcinoma, KIRC), to analyze the relationship between the expression of FGFR2 and the clinical pathological features and prognosis of ccRCC, to study the relationship between the expression of FGFR2 and other molecules, and to explore its role in the development of ccRCC. METHODS: Gene expressional and clinical information of ccRCC patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus(GEO) database. Next, the data were transformed and collated. In the study, 104 clinical ccRCC samples and corresponding paracancerous normal tissue samples were collected from Department of Urology, Peking University First Hospital. Immunohistochemistry (IHC) was performed and the staining results were scored, so as to compare the expression of FGFR2 in ccRCC and paracancerous normal tissues. Besides, quantify real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of FGFR2 in normal renal epithelial cell lines (293) and ccRCC cell lines (786-O, 769-P, OSRC-2, Caki-1, ACHN, and A498). In addition, the relationship between FGFR2 expression and clinical pathological characteristics (including TNM staging and pathological grading) and survival prognosis in ccRCC patients was further analyzed. Furthermore, the relationship between FGFR2 expression and B cells, T cells, natural killer (NK) cells and neutrophil infiltration in the ccRCC patients was analyzed, and the Biological General Repository for Interactionh Datasets (BioGRID) was used to builds protein-protein interaction (PPI) networks to study molecules that interacted with the FGFR2 protein. RESULTS: In the TCGA database, the expression of FGFR2 was down-regulated in ccRCC tissue samples compared with normal tissue samples, and the expression in the GEO database also showed this differences. Furthermore, FGFR2 expression was downregulated in ccRCC clinical samples and ccRCC cell lines, compared with corresponding paracancerous normal tissue or normal renal epithelial cell lines. In addition, FGFR2 high expression was associated with earlier, lower-level ccRCC and was associated with a better prognosis in the patients with ccRCC. Moreover, FGFR2 expression was not significantly related to B cells, T cells, NK cells and neutrophil infiltration, and the PPI network showed that FGFR2 protein interacted with certain molecules. CONCLUSION: Our work sheds light on the potential role of FGFR2 in the development of ccRCC, suggesting that FGFR2 may serve as a prognostic marker and potential therapeutic target for patients with ccRCC.

Unique genetic profiles from cerebrospinal fluid cell-free DNA in leptomeningeal metastases of EGFR-mutant non-small-cell lung cancer: a new medium of liquid biopsy.

Background: Leptomeningeal metastases (LM) are more frequent in non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. Due to limited access to leptomeningeal lesions, the purpose of this study was to explore the potential role of cerebrospinal fluid (CSF) as a source of liquid biopsy in patients with LM. Patients and methods: Primary tumor, CSF, and plasma in NSCLC with LM were tested by next-generation sequencing. In total, 45 patients with suspected LM underwent lumbar puncture, and those with EGFR mutations diagnosed with LM were enrolled. Results: A total of 28 patients were enrolled in this cohort; CSF and plasma were available in 26 patients, respectively. Driver genes were detected in 100% (26/26), 84.6% (22/26), and 73.1% (19/26) of samples comprising CSF cell-free DNA (cfDNA), CSF precipitates, and plasma, respectively; 92.3% (24/26) of patients had much higher allele fractions in CSF cfDNA than the other two media. Unique genetic profiles were captured in CSF cfDNA compared with those in plasma and primary tissue. Multiple copy number variations (CNVs) were mainly identified in CSF cfDNA, and MET copy number gain identified in 47.8% (11/23) of patients was the most frequent one, while other CNVs included ERBB2, KRAS, ALK, and MYC. Moreover, loss of heterozygosity (LOH) of TP53 was identified in 73.1% (19/26) CSF cfDNA, which was much higher than that in plasma (2/26, 7.7%; P < 0.001). There was a trend towards a higher frequency of concomitant resistance mutations in patients with TP53 LOH than those without (70.6% versus 33.3%; P = 0.162). EGFR T790M was identified in CSF cfDNA of 30.4% (7/23) of patients who experienced TKI progression. Conclusion: CSF cfDNA could reveal the unique genetic profiles of LM and should be considered as the most representative liquid biopsy medium for LM in EGFR-mutant NSCLC.

[Precision treatment after resistance to first-generation EGFR-TKI in patients with non-small cell lung cancer].

Recently, with the research progress in molecular classification, the treatment of advanced non-small cell lung cancer (NSCLC) has been established as a model of anti-tumor treatment of precision medicine. The discovery of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) has transformed the treatment of NSCLC from platinum based doublet chemotherapy into era of target therapy. EGFR-TKI, such as erlotinib and gefitinib, have been recommended as standard first-line treatment of patients with EGFR mutation. However, acquired resistance, defined as tumor progression after initial response, seems to be an inevitable consequence of this treatment approach. Clinical modes of EGFR-TKI failure are classified into three types: dramatic progression, gradual progression and local progression. A threonine-to-methionine substitution (T790M) in exon 20 of the EGFR gene is the most common mechanism of resistance. Other mechanisms of resistance include MET amplification, epithelial to mesenchymal transition, small cell transformation, and PIK3CA mutation. This brief comment will provide an overview of the complex and heterogeneous problem of acquired resistance to EGFR-TKI therapy in NSCLC, and the clinical treatment options and new targeted drugs overcoming EGFR-TKI acquired resistance.

Predictors of liver histological changes and a sustained virological response to peginterferon among chronic hepatitis B e antigen-positive patients with normal or minimally elevated alanine aminotransferase levels.

A proportion of chronic hepatitis B patients with normal or only minimally elevated alanine aminotransferase (ALT) levels display significant histologic changes and would benefit from antiviral therapy. We aim to evaluate the histologic abnormalities seen in these patients and then determine which of them would most likely respond to peginterferon therapy. One hundred and thirteen hepatitis B e antigen (HBeAg)-positive patients with a normal or minimally elevated ALT level and moderate-to-severe histologic changes in their liver tissue were selected to receive peginterferon monotherapy and participate in a follow-up analysis. A multiple logistic regression analysis indicated that increasing age (P=.049) and lower hepatitis B virus (HBV) DNA levels (P=.038) were associated with significant histological abnormalities in patients with a normal or minimally elevated ALT. Our predictive model which incorporated HBeAg testing at treatment week 12 combined with hepatitis B surface antigen (HBsAg) testing at treatment week 24 was able to identify which patients with a normal ALT level would achieve a sustained virological response (SVR) (positive predictive value [PPV]: 66.7%, negative predictive value [NPV]: 90.0%). Lower HBsAg and HBeAg levels at treatment week 24 were associated with a SVR in patients with a minimally elevated ALT level (PPV: 100.0%, NPV: 100.0%). A liver biopsy and antiviral therapy should be strongly considered when treating HBeAg-positive patients with a normal or minimally elevated ALT level, low HBV DNA level, and aged >35 years. On-treatment quantification of combined HBsAg and HBeAg test results may be useful for predicting a SVR to peginterferon monotherapy in these patients.

A phase III randomised controlled trial of erlotinib vs gefitinib in advanced non-small cell lung cancer with EGFR mutations.

BACKGROUND: A phase III trial was conducted to compare the safety and efficacy of erlotinib with that of gefitinib in advanced non-small cell lung cancer harbouring epidermal growth factor receptor mutations in exon 19 or 21. METHODS: Eligible patients were randomised to receive erlotinib (150 mg per day) or gefitinib (250 mg per day) orally until disease progression or unacceptable toxicity. We aimed to determine whether erlotinib is superior to gefitinib in efficacy. The primary end point was progression-free survival. RESULTS: A total of 256 patients were randomised to receive erlotinib (N=128) or gefitinib (N=128). Median progression-free survival was not better with erlotinib than with gefitinib (13.0 vs 10.4 months, 95% confidence interval (CI) 0.62-1.05, P=0.108). The corresponding response rates and median overall survival were 56.3% vs 52.3% (P=0.530) and 22.9 vs 20.1 months (95% CI 0.63-1.13, P=0.250), respectively. There were no significant differences in grade 3/4 toxicities between the two arms (P=0.172). CONCLUSIONS: The primary end point was not met. Erlotinib was not significantly superior to gefitinib in terms of efficacy in advanced non-small cell lung cancer with epidermal growth factor receptor mutations in exon 19 or 21, and the two treatments had similar toxicities.

Complete mitochondrial genome of the Southern catfish (Silurus meridionalis Chen) and Chinese catfish (S. asotus Linnaeus): Structure, phylogeny, and intraspecific variation.

The complete mitochondrial genome of the Southern catfish (Silurus meridionalis) and the Chinese catfish (S. asotus), was determined using the long and accurate polymerase chain reaction (LA-PCR) method. The mitochondrial DNA nucleotide sequences of S. meridionalis and S. asotus were compared with those of 47 other catfish species in the same order. The total length of mitochondrial DNA for S. meridionalis and S. asotus was 16,526 and 16,525 bp, respectively, and included 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a non-coding control region. This mitochondrial gene arrangement is identical to that observed in other Siluriformes. To determine the relative phylogenetic positions of S. meridionalis and S. asotus, and to discover phylogenetic relationships among 24 families of Siluriformes, analyses were conducted, based on mitochondrial DNA, 12S ribosomal RNA, 16S ribosomal RNA, and 13 protein-coding gene sequence data sets. Phylogenetic analyses were congruent with a basal split of the order into Clupeiformes, Characiformes, Cypriniformes, and Siluriformes, and supported a closer relationship of the Southern catfish (family Siluridae) and the Chinese catfish (family Siluridae) to Pimelodidae than to Bagridae. We concluded that these two species are part of a molecular clade that is different from that proposed in recent studies, in which Amblycipitidae appears as a sister group. Our results showed Amblycipitidae appearing as the most basal extant, and Bagridae appearing as a sister group of Cranoglanididae and Pangasiidae. The Siluriformes showed close phylogenetic relationship to the Characiformes.

Pemetrexed versus gefitinib as a second-line treatment in advanced nonsquamous nonsmall-cell lung cancer patients harboring wild-type EGFR (CTONG0806): a multicenter randomized trial.

BACKGROUND: CTONG0806 assessed the efficacy of pemetrexed versus gefitinib as second-line treatment in advanced nonsquamous nonsmall-cell lung cancer (NSCLC) harboring wild-type epidermal growth factor receptor (EGFR). PATIENTS AND METHODS: Patients with locally advanced or metastatic nonsquamous NSCLC harboring wild-type EGFR, detected by direct sequencing, and previously treated with platinum-based chemotherapy were randomized to receive gefitinib (250 mg/day) orally or pemetrexed (500 mg/m(2)) i.v. on day 1 of a 21-day cycle until disease progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). The Independent Review Committee (IRC) evaluated all pictorial data. RESULTS: From February 2009 to August 2012, 161 patients were enrolled, and 157 were assessable (81 in the gefitinib arm, 76 in the pemetrexed arm). Baseline characteristics were balanced between the two arms. The median PFSs were 4.8 versus 1.6 months in the pemetrexed and gefitinib arms, respectively [hazard ratio (HR) 0.54, 95% confidence interval (CI) 0.40-0.75, P < 0.001] as confirmed by IRC evaluation (5.6versus 1.7 months, HR 0.53, 95% CI 0.38-0.75, P < 0.001). The median overall survival (OS) showed a trend of superiority in the pemetrexed arm (12.4 versus 9.6 months, HR 0.72, 95% CI 0.49-1.04, P = 0.077). Quality-of-life assessment showed no marked difference between the arms. No unexpected adverse events were found. Of 108 patients with sufficient DNA samples, EGFR mutation status was re-tested by Scorpion amplification refractory mutation system (ARMS); 32 (29.6%) tested positive (19 in the pemetrexed arm, 13 in the gefitinib arm; median PFS: 8.1 versus 7.0 months, HR 0.94, 95% CI 0.43-2.08, P = 0.877). CONCLUSIONS: CTONG0806 is the first trial to show significant improvement in PFS and an improved OS trend with pemetrexed compared with gefitinib as second-line setting treatment of EGFR wild-type advanced nonsquamous NSCLC. ARMS is superior to direct sequencing in excluding false-negative patients. CLINICALTRIALSGOV IDENTIFIER: NCT00891579.

A dendritic cell-based tumour vaccine for lung cancer: full-length XAGE-1b protein-pulsed dendritic cells induce specific cytotoxic T lymphocytes in vitro.

XAGE-1b is regarded as one of the most immunogenic antigens and the most promising targets for lung adenocarcinoma immunotherapy. In this study, we sought to determine whether monocyte-derived dendritic cells (DCs) pulsed with purified full-length XAGE-1b could induce specific cytotoxic T lymphocytes (CTLs) against tumour cells from patients with non-small cell lung cancer (NSCLC) in vitro. XAGE-1b mRNA expression was examined in primary cultures of lung cancer cells and normal lung epithelial cells established from fresh tissues surgically resected from 30 patients with NSCLC using reverse transcription-polymerase chain reaction (RT-PCR). XAGE-1b mRNA expression was observed in 11 of 18 (61.1%) adenocarcinomas and one of 12 (8.3%) lung cancers of other histological types (P = 0.015). The 246-base pairs XAGE-1b gene was inserted into a recombinant expression vector. Full-length XAGE-1b was then expressed in BL21 (DE3) Escherichia coli and purified by AKTA-fast performance liquid chromatography (FPLC). DCs generated from peripheral blood mononuclear cells were pulsed with XAGE-1b by incubation with the protein at an immature stage. The XAGE-1b-pulsed DCs induced CTLs following 14 days of co-culture. Finally, an adherent target detachment (ATD) assay was performed to test the cytotoxicity of the XAGE-1b-specific CTLs against cancer cells and normal lung epithelial cells. The XAGE-1b-specific CTLs had a stronger lytic effect on autologous XAGE-1b mRNA-positive cancer cells than on autologous XAGE-1b mRNA-negative cancer cells or allogenous XAGE-1b mRNA-positive cancer cells. The CTLs had no lytic activity against normal lung epithelial cells. These results can be used to develop simple and effective cancer/testis antigen-based immunotherapies for NSCLC.

Epidermal growth factor receptor double activating mutations involving both exons 19 and 21 exist in Chinese non-small cell lung cancer patients.

AIMS: It has been shown that the introduction of a second mutation into the already mutated epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) will alter the sensitivity to tyrosine kinase inhibitors (TKIs). EGFR double activating mutations involving both exons 19 and 21 were previously detected in Asian patients, but the sensitivity to TKIs had not yet been characterised. Our objective was to profile the status of EGFR double mutations in Chinese NSCLC patients and to further ascertain the biological properties. MATERIALS AND METHODS: In total, 145 NSCLC tumour samples from unselected Chinese NSCLC patients were sequenced to screen mutations in exons 18, 19 and 21 of EGFR. Five patients were detected to harbour the delE746-A750+L858R double activating mutations. Subcloning experiments were carried out, expression vectors inserted with corresponding full-length EGFR were constructed, and in vitro transient transfections were performed in 293T cells. Whole cell lysates were collected to assess the sensitivity to TKIs using immunoblotting. RESULTS: All five patients had adenocarcinoma. The frequency of double mutations was 3.4% (5/145). Three patients received and responded to gefitinib treatment. Subcloning experiments showed that all the subclones were either wild type or double mutated. At a concentration of TKIs of 0.1 microM, the autophosphorylation of the double mutant was inhibited greater than that of either single mutated EGFR. However, the difference disappeared when the concentration increased to 1 microM. CONCLUSIONS: delE746-A750+L858R double activating EGFR mutations exist in Chinese NSCLC patients and both locate on the same allele. These patients tend to respond well to TKIs and the sensitivity to TKIs of this double mutated EGFR is enhanced compared with either single mutant. Nonetheless, the alteration in downstream signal transduction of the double mutant remains to be determined.

Syntheses of cyclic prodrugs of RGD peptidomimetics with various macrocyclic ring sizes: evaluation of physicochemical, transport and antithrombic properties.

The objective of this work was to synthesize cyclic prodrugs 1a-d of RGD peptidomimetics 2a-d with various ring sizes (n[CH2] = 1, 3, 5 and 7) and to evaluate the effect of ring size on their transport, physicochemical, enzymatic stability, and antithrombic properties. The syntheses of cyclic prodrugs 1a-d were achieved by converging two key intermediates, Boc-Phe-O-CH2-OCO-OpNP (5) and H2N-(CH2)n-CO-Asp(OBzl)-OTce (8a-d), to give linear precursors Boc-Phe-O-CH2-OCO-HN-(CH2)n-CO-Asp(OBzl)-OTce (9a-d). The N- and C-terminus protecting groups were removed from 9a-d to give 10a-d. Linear precursors 10a-d were cyclized, and the remaining Bzl-protecting group was removed to produce cyclic prodrugs 1a-d in around 20% overall yield. The linear RGD peptidomimetics (2a-d) were synthesized using standard Boc-amino acid chemistry by solution-phase method. Increasing the ring size by adding methylene groups also increases the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics. The transport properties of cyclic prodrugs 1c and 1d were 2.6- and 4.4-fold better than those of parent compounds 2c and 2d, respectively. These results suggest that increasing the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics enhanced their transport properties. The hydrodynamic radii of the cyclic prodrugs were also smaller than those of their respective parent compounds, suggesting that the change in size may contribute to their transport properties. The chemical stability of the cyclic prodrugs was affected by the ring size, and the cyclic prodrug with the larger ring size (i.e. 1d) was more stable than the smaller one (i.e. 1a). All the cyclic prodrugs were more stable at pH 4 than at pH 7 and 10. Prodrug-to-drug conversion could be induced by isolated esterase as well as esterase found in human plasma. An increase in the length of methylene group (n[CH2] = 1, 3, 5, 7) enhanced the antithrombic activity of the prodrugs and the parent compounds. In summary, the ring size of cyclic prodrugs affected their transport, physicochemical, and antithrombic properties.

Cyclocariosides II and III: two secodammarane triterpenoid saponins from Cyclocarya paliurus.

Two new secodammarane triterpenoid saponins were isolated from the leaves of Cyclocarya paliurus. Based on chemical and spectral analysis they were assigned as (20S)-20-hydroxy-24-O-alpha-L-arabino-pyranosyl-3,4- secodammar-4(28),11,25-trien-3-oic acid, named cyclocarioside II and 25-hydroxy-(20S,24R)-epoxy-12R-O-beta-D- quinovopyranosyl-3,4-secodammar-4(28)-en,3-oic acid, named cyclocarioside III.