Simultaneous VENTANA IHC and RT-PCR testing of ALK status in Chinese non-small cell lung cancer patients and response to crizotinib.

BACKGROUND: ALK rearrangement-advanced NSCLC patients respond to crizotinib. ALK rearrangement is currently determined with RT-PCR. VENTANA IHC is a standard method to identify ALK protein overexpression in NSCLC; however, VENTANA IHC has rarely been used to determine the response to crizotinib in Chinese patients with NSCLC and ALK overexpression. To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of Chinese patients with NSCLC undergoing screening for ALK rearrangements. METHODS: A total of 1720 patients with NSCLC who had ALK rearrangements detected by VENTANA IHC and/or RT-PCR were included in this analysis. We compared the efficacy and survival of ALK-positive patients detected by VENTANA IHC and RT-PCR. We used NGS to identify patients in whom the two methods were inconsistent. RESULTS: Among 1720 patients, 187 (10.87%) were shown to be ALK-positive by VENTANA IHC and/or RT-PCR, and 66 received crizotinib treatment. We identified 10.27% (172/1674) of patients as ALK-positive by the VENTANA IHC method, and 12.73% (41/322) of patients had ALK rearrangements by the RT-PCR method. Twenty-nine of 276 (10.51%) ALK-positive patients were simultaneously analyzed using VENTANA IHC and RT-PCR. The overall response rates were 65.90% (29/44) by VENTANA IHC and 55.88% (19/34) by RT-PCR. The disease control rates were 86.36% (38/44) by VENTANA IHC and 76.47% (26/34) by RT-PCR. In contrast, the median progression-free survival for VENTANA IHC and RT-PCR was 8.5 and 9.2 months, respectively. The VENTANA IHC and RT-PCR results obtained for 6 of 17 ALK-positive patients were inconsistent based on NGS; specifically, 4 patients had EML4-ALK fusions, 2 patients had non EML4-ALK fusions, 1 patient had a KCL1-ALK fusion, and one patient had a FBXO36-ALK fusion. CONCLUSIONS: VENTANA IHC is a reliable and rapid screening tool used in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy. VENTANA IHC has moderate sensitivity and a slightly higher association with response to therapy with ALK inhibitors, and some VENTANA IHC-positive, but RT-PCR-negative cases may benefit from crizotinib.

Maslinic acid protects against lipopolysaccharide/d-galactosamine-induced acute liver injury in mice.

Acute liver injury is a life-threatening syndrome that often caused by hepatocyte damage. In this study, we investigated the protective effects of maslinic acid (MA) on lipopolysaccharide (LPS)/d-galactosamine (D-gal)-induced acute liver injury and clarified its mechanism. Mice acute liver injury model was induced by given LPS and D-gal and MA was given intraperitoneally 1 h before LPS and D-gal. Our results showed that MA protected against liver injury by attenuating liver histopathologic changes, serum AST and ALT levels. The increased inflammatory cytokines TNF-α and IL-6 in serum and liver tissues were also inhibited by MA. The level of MDA and the activity of MPO in liver tissues were up-regulated by LPS/D-gal and dose-dependently inhibited by MA. Furthermore, MA attenuated hepatic NF-κB protein expression and increased hepatic Nrf2 and HO-1 protein expression. Taken together, MA offers a protective role against LPS/D-gal-induced liver injury through suppressing NF-κB and activating Nrf2 signaling pathways.

JAK/STAT3 Signaling Activation Related to Distinct Clinicopathologic Features in Systemic ALK - Anaplastic Large Cell Lymphomas : New Insights into Their Heterogeneity.

Systemic anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a group of heterogenous CD30 + T-cell non-Hodgkin lymphomas. Previous studies have highlighted the importance of JAK/STAT3 signaling activation in the molecular pathogenesis of ALK - ALCLs. In the present study, we aimed to establish a potential relationship between JAK/STAT3 signaling activation and clinicopathologic features in ALK - ALCLs, and further recognize the heterogenous nature of these neoplasms. Immunohistochemistry staining of the phosphorylated-STAT3 (p-STAT3) and dual-specificity protein phosphatase 22 ( DUSP22 ) gene rearrangement analysis were performed. Forty-five cases of ALK - ALCL were divided into 3 groups, including 9 DUSP22 -rearranged ALCLs, 21 p-STAT3 + double-negative (DN) ALCLs (both ALK and DUSP22 rearrangement negative), and 15 p-STAT3 - DN-ALCLs. Morphologically, p-STAT3 + DN-ALCLs exhibited sheet-like neoplastic cells and sometimes showed large pleomorphic cells scattered in a lymphocyte-rich background more frequently than those in other ALK - ALCLs subtypes. Phenotypically, the p-STAT3 + DN-ALCLs frequently expressed cytotoxic molecules, epithelial membrane antigen, and programmed death-ligand 1, whereas CD3 and CD5 expression was not observed. Clinically, patients with p-STAT3 + DN-ALCLs had a better prognosis than those with p-STAT3 - DN-ALCLs. These observations suggest that p-STAT3 + DN-ALCLs represent a distinct subtype of ALK - ALCLs. Identifying ALK - ALCL subtypes by using p-STAT3 staining and DUSP22 rearrangement is a promising approach that may contribute to risk stratification and better treatment decisions in the future clinical practice.

Molecular profiling of Chinese systemic anaplastic large cell lymphoma patients: novel evidence of genetic heterogeneity.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is a rare non-Hodgkin lymphoma. A comprehensive understanding of the genetic and clinical heterogeneity of ALCL may help to improve the clinical management of patients with ALCL. However, due to the rarity of the disease, the genetic heterogeneity of ALCL has not been well elucidated. This study aimed to comprehensively elucidate the mutational landscape of tumor tissue samples from patients with systemic ALCL. METHODS: Thirty-six patients with systemic ALCL were enrolled in this retrospective study. Immunohistochemistry (IHC) was performed on tumor tissues at baseline to identify anaplastic lymphoma kinase (ALK) fusions. Capture-based targeted next-generation sequencing (NGS) with a panel spanning 112 lymphoma-related genes, including ALK rearrangements, was also performed on tumor tissue samples. RESULTS: A total of 102 mutations were identified in the entire cohort. Among the 36 patients included in this analysis, 14 (38.8%) were ALK positive, as determined by IHC, while NGS showed 12 patients (33.3%) to harbor ALK rearrangements. Younger patients were more likely to have ALK-positive ALCL (P=0.011). Patients with wild-type (WT) ALK were more likely to have single-nucleotide variants (SNVs) and insertions or deletions (INDELs) than patients with ALK rearrangements (P=0.027). Among the 22 patients with WT ALK, the most commonly mutated genes were TP53 (n=6, 27.3%), followed by NOTCH1 (n=5, 22.7%), KMT2D (n=3, 13.6%), KRAS (n=3, 13.6%), TET2 (n=3, 13.6%), and JAK1 (n=2, 9.1%). Mutations in PRDM1, a commonly mutated gene in ALK-negative patients, were not detected in our ALK-negative cohort. Start-loss of beta-2-microglobulin (B2M) was detected in another patient; this patient had a favorable prognosis, with an overall survival exceeding 19 months. CONCLUSIONS: Our study revealed the unique genomic profiles of Chinese ALCL patients and represents an incremental step in deepening the understanding of the genetic heterogeneity of ALCL patients.

[Dynamic Changes in Hydrochemical Characteristics and Influencing Factors in the Karst Watershed Flood Process].

The hydrochemistry of river water in a karst basin has a rapid response to the rainstorm/flood process, which is an important process of the karst carbon cycle and should not be ignored. Based on the dynamic monitoring of the hydrochemical characteristics of the flood process in the Yangshuo section on November 8-12, 2015, the dynamic change in the main ions and the influencing factors were analyzed, and the concentration and flux of inorganic carbon from different sources were calculated. The results showed that the hydrochemistry types in different stages of the flood area belonged to the Ca-HCO3 type. The ions were mainly sourced from carbonate weathering, and affected by silicate weathering, rainfall, and human activities. Because of the hydrological process, the weathering strength of carbonate rocks sharply weakened at the beginning of the flood, and then gradually increased. The concentrations of HCO3-, Ca2+, and Mg2+ sharply decreased at the beginning of the flood, then gradually increased, and continued to increase in the second flood process because of the waterlogging in the karst system. Because of the waterlogging, the reaction time between water and rock become longer; thus, the concentrations are higher. The dynamic changes in SO42-, Cl-, Na+, and K+ were mainly affected by precipitation and human activities. At the beginning of the flood, the concentrations of SO42-, Cl-, Na+, and K+ increased because the runoff takes more ions sourced from activities. The concentrations of SO42-, Cl-, Na+, and K+ decreased with the decrease of easily transported substances. At the lowest point of concentration, SO42- and Cl- were mainly sourced from precipitation, and Na+ and K+ were mainly sourced from precipitation and silicate weathering. The weathering of carbonates by carbonic acid was the main source of inorganic carbon, accounting for 74.3% of total inorganic carbon on average. Because of the input of sulfuric/nitric acid, the contribution of the weathering of carbonates by sulfuric/nitric acid to the inorganic carbon cannot be ignored, and the contribution increased significantly in the flood, up to 31.7%. The geological carbon sinks before the flood, and during the first and second flood processes in the Yangshuo section were 1.28×108, 5.28×108, and 11.52×108 g·d-1, respectively. The geological carbon sink before the flood was equal to the annual average flux, whereas the geological carbon sink in the flood process was several times that of the annual average flux. Moreover, because of the significant difference in the weathering strength of carbonate rocks during the two floods, there was also a significant difference in the amount of geological carbon sink under the same discharge.

TP53 mutations predict for poor survival in ALK rearrangement lung adenocarcinoma patients treated with crizotinib.

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) patients who harbor anaplastic lymphoma kinase (ALK) rearrangement are sensitive to an ALK inhibitor (crizotinib), but not all ALK-positive patients benefit equally from crizotinib treatment. We analyze the impact of TP53 mutations on response to crizotinib in patients with ALK rearrangement NSCLC. METHODS: Sixty-six ALK rearrangement NSCLC patients receiving crizotinib were analyzed. 21 cases were detected successfully by the next generation sequencing validation FFPE before crizotinib. TP53 mutations were evaluated in 8 patients in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). RESULTS: TP53 mutations were observed in 2 (25.00%), 1 (12.50%), 1 (12.50%) and 4 (50.00%) patients in exons 5, 6, 7 and 8, respectively. The majority of patients were male (75.00%, 6/8), less than 65 years old (62.50%, 5/8) and never smokers (75.00%, 6/8). ORR and DCR for crizotinib in the entire case series were 61.90% and 71.43%, respectively. Statistically significant difference was observed in terms of PFS and OS between TP53 gene wild group and mutation group patients (P=0.038, P=0.021, respectively). CONCLUSIONS: TP53 mutations reduce responsiveness to crizotinib and worsen prognosis in ALK rearrangement NSCLC patients.

[Screening up-regulated genes in hepatic stellate cells treated with PDGF-BB using suppression subtractive hybridization technique].

OBJECTIVE: To construct a subtractive cDNAs library of up-regulated genes in rat hepatic stellate cells (HSCs) stimulated with platelet-derived growth factor (PDGF)-BB by suppression subtractive hybridization (SSH) technique, to clone the up-regulated genes associated with its regulation effects, and to elucidate the mechanism of the molecular biology of hepatic fibrosis involved in PDGF-BB. METHODS: The mRNA was isolated from HSCs stimulated with PDGF-BB and controlled with identical cells untreated with PDGF-BB, and then the cDNAs were synthesized. The cDNAs were designated as tester and driver. After being digested by restriction enzyme Rsa I, small-sized cDNAs were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, individually. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS: The subtractive cDNAs library of up-regulated genes in HSCs stimulated with PDGF-BB was constructed successfully. The amplified library contained 102 positive clones. Colony PCR showed that 93 clones contained 200-1000 bp inserts. Sequence analysis was performed in 31 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank; altogether 13 coding sequences were obtained, which were known ones. The genes mainly included voltage-dependent anion channel (VDAC), heat shock protein 47 and RAN-member RAS oncogene family genes. CONCLUSION: Acting as one of the most effective mitogens, PDGF-BB up-regulated some gene expressions during stimulation of the HSCs, including some cell growth associated proteins, some proteins participating in intracellular metabolism and some molecular chaperone proteins. This work brings some new clues for studying the molecular biological mechanism involved in the up-regulated genes in PDGF-BB transactivated HSCs in hepatic fibrosis.

Patients harboring ALK rearrangement adenocarcinoma after acquired resistance to crizotinib and transformation to small-cell lung cancer: a case report.

Anaplastic lymphoma kinase (ALK) rearrangement responds to ALK tyrosine kinase inhibitors (TKIs) in lung cancer. Many cases ultimately acquire resistance to crizotinib. Resistance, including ALK-dominant or ALK non-dominant, mechanisms have been described. Transformation to small-cell lung cancer is rare. Herein, we report a 49-year-old man diagnosed with adenocarcinoma, who was negative for EGFR and ALK genes as detected by reverse transcription polymerase chain reaction, and was treated with crizotinib. A new biopsy showed a small-cell lung cancer after disease progression. Then, next-generation sequencing (NGS) was carried out and detected a TP53 gene mutation, an ALK rearrangement, and no loss of the retinoblastoma gene (RB). Although a regimen for small-cell lung cancer may be one treatment option, a heterogeneous tumor may exist at the time of diagnosis and manifest during the course of disease.

[The relationship between serum HBsAg levels and liver inflammation and fibrosis in patients with chronic hepatitis B].

OBJECTIVE: To investigate the relation of hepatitis B surface antigen (HBsAg) level with chronic hepatitis B (CHB) and liver inflammation and fibrosis. METHODS: A total of 301 patients who diagnosed CHB and underwent liver biopsy were enrolled into the study. Meantimes, the biochemical markers, ferritin (FERR), serum HBsAg and HBV DNA quantitation were detected. The relation between HBsAg level and liver pathology were determined by spearman rank correlation analysis. The receiver operating characteristic curve was used to evaluate the accuracy of HBsAg level for liver inflammation and fibrosis. RESULTS: The body mass index (BMI), age, gender, genotype and family history had no effective on liver inflammation and fibrosis (P < 0.05). With the progressing of inflammation and fibrosis, the serum AST and ALT raise obviously (chi2 = 71.193, 96.344, 47.847, 63.981; P = 0.000, 0.000, 0.000, 0.000). When fibrosis reached to S4, the level of HBV DNA decreased obviously (chi2 = 33. 322; P = 0.000). With the aggravation of inflammation and fibrosis, the serum HBsAg gradually descended (chi2 = 68.173,15.719; P = 0.000, 0.000). The areas under operating characteristics curves of HBsAg predicted < or = G3 and < or = S3 were 0.732 and 0.793, and the specificity were 0.778, 0.891, and sensitivity were 0.685, and 0.633, respectively. CONCLUSION: The level of HBsAg of Chinese CHB patients descended gradually with the aggravation of liver inflammation and fibrosis. The serum HBsAg had a higher specificity to predict < or = G3 and < or = S3 of CHB patients. But there had superiority of predicting fibrosis than inflammation.

Specific sIgA antibodies against swine influenza virus H3N2 infection / 中国人兽共患病学报

To study different breed pigs reply the swine flu virus infections,specific antibody of sIgA secretion regularity of respiratory tract and the differences of sIgA antibody according to different antigen proteins were detected.A/swine/Nanjing/ 51/2010(H3N2) was intranasally infected pigs (1 × 107 TCID50/mL and 2 mL/pig),and then the nasal swab samples were collected at different time points within 21 days after infection.M1,NS1 and PB1 recombinant protein,respectively,were used to establish indirect ELISA method for detecting specific antibody of sIgA,and to analyze its secretion regularity.Results displayed that there was no significant difference among three kinds of recombinant protein in the whole test,characterizing by specificity sIgA antibody levels rising rapidly after 5 infection days and reaching peak at day 14,then began to decline.Among different varieties of pigs,sIgA antibody production of PB1 protein in Obama group was significantly higher than that in binary pigs at 14th and 21st day (P＜0.05).It had no significant difference between M1 group and the NS1 group (P＞0.05).This experiment preliminary explores the secretion regularity of specificity sIgA antibody after infected swine flu virus,which laid a foundation for further study of SIV mucosal antibody diagnostic reagents.

Differentially expressed genes identified by microarray analysis following leptin treatment of hepatic stellate cells.

BACKGROUND: Liver fibrosis is the process through which numerous chronic liver diseases develop into liver cirrhosis. Leptin can activate hepatic stellate cells (HSCs) and play an important role in the formation of liver fibrosis. However, the process by which leptin activates HSCs is complicated, and research on this process is limited. The aim of this study was to explore the related changes in gene expression and the control mechanisms involved in leptin activated HSCs to understand the overall mechanism of liver fibrosis development. METHODS: We cultivate rat HSCs, with and without stimulation by leptin, and extracted mRNA. Differentially expressed genes were detected by microarray analysis. RESULTS: The differentially expressed genes identified included six upregulated genes and six downregulated genes. The representative upregulated genes included short chain dehydrogenase (CY5/CY3 = 2.265) and pulmonary surfactant protein A1 (CY5/CY3 = 2.036). The significant downregulated gene encoded hepatic stearoyl coenzyme A desaturase 1 (SCD-1) (CY5/CY3 = 0.351). CONCLUSION: Leptin might mediate the molecular biological mechanisms of liver fibrosis.