In recent years, the number of advanced non-small cell lung cancer (NSCLC) patients has gradually increased, and the treatment methods have also been significantly increased. However, there are no standard treatment plans at home and abroad for third-line and above patients who are refractory to targeted therapy epidermal growth factor receptor (EGFR)/anaplastic lymphoma kinase (ALK) or chemotherapy. The clinical treatment effect is also not satisfactory. Anlotinib is a novel TKI targeting the vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and c-Kit. ALTER0303 trail, phase III study has demonstrated that Anlotinib significantly prolonged overall survival (OS) and progression-free survival (PFS) in advanced NSCLC patients as 3rd line treatment.Here we report a case of advanced lung adenocarcinoma harboring KRAS mutation treated with Anlotinib.
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Adenocarcinoma ; drug therapy ; enzymology ; genetics ; pathology ; Adenocarcinoma of Lung ; Aged ; Antineoplastic Agents ; therapeutic use ; Humans ; Indoles ; therapeutic use ; Lung Neoplasms ; drug therapy ; enzymology ; genetics ; pathology ; Male ; Mutation ; Proto-Oncogene Proteins p21(ras) ; genetics ; metabolism ; Quinolines ; therapeutic use

OBJECTIVEThe aim of this study was to establish a standardized protocol for detection of ALK protein expression and gene fusion in cytologic specimens.

METHODSLung adenocarcinoma cytologic specimens were collected from seven hospitals in Beijing city. A detection protocol for ALK protein expression and gene fusion was designed according to the results of comparative experiment. Ventana immunohistochemical (IHC) ALK(D5F3) detecting ALK protein expression was performed in 203 prepared formalin-fixed paraffin-embedded (FFPE) cell blocks. ALK gene fusion in 98 EGFR gene wild type cytologic specimens and in 4 bronchoalveolar lavage fluid (BL) samples was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). ALK gene fusion in the Ventana IHC ALK (D5F3) positive samples was further tested by fluorescence in situ hybridization (FISH). Six patients with ALK IHC-positive result were followed up to analyze the responses of crizotinib therapy. Comparative experiments: (1) Comparison of the results of 4% neutral buffered formalin fixed for different time (24 h, 48 h, 72 h) on the Ventana IHC ALK (D5F3) staining was conducted in two cases of IHC ALK positive FFPE cell blocks; (2) Comparing qRT-PCR results for ALK fusion in samples from FFPE cell blocks and cytospin prepared slides in 10 cases of lung adenocarcinoma cytologic specimens.

RESULTSAmong the specimens examined using the standardized protocol recommended by this study, 229 cases of cytologic specimens met the diagnostic criteria of lung adenocarcinoma. Among them, 207 cases obtained ALK gene test results (by at least one method), with an ALK test ratio of 90.4% (207/229). FFPE cell blocks were successfully prepared in 203 cases, Ventana IHC ALK (D5F3) were successfully performed in all the 203 FFPE cell blocks (100%), and the ALK protein positive detection rate was 10.3% (21/203). ALK fusion was tested in 98 FFPE cytologic samples of EGFR wild types by qRT-PCR, and 96 out of 98 (97.96%) cytologic samples were successfully performed.18 out of 19 IHC ALK-positive cases were verified to be of ALK fusion status by qRT-PCR. The concordance rate was 94.7% (Kappa=0.967, P<0.001) between Ventana IHC ALK (D5F3) and qRT-PCR, and the sensitivity of the Ventana IHC ALK (D5F3) assay compared with qRT-PCR was 100% and the specificity was 98.7%. FISH assay was used to verify the positive cases detected by Ventana IHC ALK (D5F3) staining. Two cases of low tumor cell content FFPE samples obtained indefinite results by FISH test. The six patients with positive ALK protein expression received crizotinib therapy, and 5 paitents got treated effectively. For two ALK IHC positive cases, which were 4% neutral buffered formalin fixed for 72 h, the result of Ventana IHC ALK (D5F3) staining became weakened obviously and uneven. In 10 cases of samples, total RNA was extracted from FFPE cytologic sections and cytospin prepared slides, and the results of qRT-PCR test and ALK gene fusion showed good concordance.

CONCLUSIONSThe standardized protocol recommended in this study expands the detection types and quantity of cytologic specimens for ALK protein expression and gene fusion and increased the detection rate. Ventana IHC ALK (D5F3) is a reliable method for detecting ALK protein expression in FFPE cell blocks. The pathologic quality control procedure prior to Ventana IHC ALK (D5F3) is crucial for the accuracy of testing the ALK gene status. When FFPE cell blocks could not be prepared or prepared unsuccessfully from the cytologic specimens, qRT-PCR may be an alternative option for the detection of ALK gene fusion.

Adenocarcinoma ; drug therapy ; enzymology ; genetics ; pathology ; Alkaline Phosphatase ; genetics ; metabolism ; Gene Fusion ; Genes, erbB-1 ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; drug therapy ; enzymology ; genetics ; pathology ; Protein Kinase Inhibitors ; therapeutic use ; Proteomics ; Pyrazoles ; therapeutic use ; Pyridines ; therapeutic use ; Sensitivity and Specificity

OBJECTIVETo investigate the expression of tripartite-motif protein 25 (TRIM25) and pyruvate kinase M2 (PKM2) protein in non-small cell lung cancer (NSCLC) and explore their role in the occurrence and progression of NSCLC.

METHODSThe expressions of TRIM25 and PKM2 protein were detected in 60 NSCLC specimens and 20 adjacent normal lung tissue (>5 cm from the lesions) with immunofluorescence histochemical method and in 10 fresh specimens of NSCLC with Western blotting. The results were analyzed in relation with the clinicopathological features of the patients.

RESULTSThe positivity rates of TRIM25 expression was 45% in the 60 lung carcinoma specimens, significantly higher than that in the 20 normal lung tissues (10%, P=0.005). TRIM25 protein was expressed in 28.6% of lung adenocarcinoma tissues and in 59.4% of squamous carcinoma tissues (P=0.017). TRIM25 protein expression was positively correlated with the TNM stages and lymph node metastasis of NSCLC (P<0.05). The expressions of PKM2 protein in 60 cases of lung carcinoma was 73.3%,while in 20 cases of normal lung tissues the expressions was 30%(P=0.001). The positivity rates of PKM2 expression differed significantly between lung adenocarcinoma and squamous carcinoma (57.1% vs 87.5%, P=0.008). An inverse correlation was noted between TRIM25 and PKM2 expressions (P=0.026).

CONCLUSIONTRIM25 and PKM2 protein may participate in the occurrence and progression of NSCLC, and their expressions are inversely correlated.

Adenocarcinoma ; enzymology ; Carcinoma, Non-Small-Cell Lung ; enzymology ; Carcinoma, Squamous Cell ; enzymology ; Carrier Proteins ; metabolism ; Humans ; Lung ; pathology ; Lung Neoplasms ; enzymology ; Lymphatic Metastasis ; Membrane Proteins ; metabolism ; Thyroid Hormones ; metabolism ; Transcription Factors ; metabolism ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; metabolism

OBJECTIVETo study the role of arginase-1 (Arg-1) expression in differential diagnosis of hepatocellular carcinoma (HCC), Arg-1 staining pattern in clear cell neoplasm (HCC and non-HCC) and Arg-1 expression in non-hepatocellular tumors.

METHODSSeventy-eight cases of HCC (including 8 cases of clear cell type and 70 cases of non- clear cell type) and 246 cases of non-hepatocellular neoplasms (including 29 cases of metastatic tumors such as breast cancer, nasopharyngeal carcinoma and neuroendocrine carcinoma, 77 cases of tumors with clear cell changes such as malignant melanoma, clear cell renal cell carcinoma and alveolar soft part sarcoma, and 140 cases of other types of tumors such as ovarian endometrioid adenocarcinoma, pituitary tumor and thyroid papillary carcinoma) were studied.Immunohistochemical study for Arg-1 was performed on the paraffin-embedded tumor tissue.

RESULTSIn HCC, Arg-1 demonstrated both cytoplasmic and nuclear staining, with an overall sensitivity of 96.2% (75/78).In well, moderately and poorly differentiated HCC, the sensitivity was 15/15, 100% (41/41) and 86.4% (19/22), respectively. That was in contrast to negative staining for Arg-1 in all the 29 cases of metastatic tumors studied. The sensitivity, specificity, positive predictive value and negative predictive value of Arg-1 in distinguishing HCC from metastatic tumors was 96.2%, 100%, 100% and 90.6%, respectively. Cytoplasmic and membranous staining was observed in clear cell type of HCC. The overall sensitivity of Arg-1 expression in the 77 cases of tumors with clear cell changes was 14.3% (11/77), including 8/15 for malignant melanoma, 2/4 for ovarian clear cell carcinoma and 1/1 gall bladder adenocarcinoma with clear cell component.In malignant melanoma and ovarian clear cell carcinoma, only cytoplasmic staining was demonstrated. There was no expression of Arg-1 in the 140 cases of other tumor types studied.

CONCLUSIONSArg-1 is a sensitive and specific marker for HCC.It is a potentially useful immunohistochemical marker in distinguishing HCC from metastatic tumors. Though also expressed in malignant melanoma and ovarian clear cell carcinoma, Arg-1 shows a different staining pattern as compared with that in HCC.

Adenocarcinoma ; enzymology ; Adult ; Aged ; Arginase ; metabolism ; Carcinoma, Hepatocellular ; enzymology ; pathology ; secondary ; Cell Differentiation ; Diagnosis, Differential ; Female ; Gallbladder Neoplasms ; enzymology ; Humans ; Liver Neoplasms ; enzymology ; pathology ; secondary ; Male ; Melanoma ; enzymology ; Middle Aged ; Ovarian Neoplasms ; enzymology ; Stomach Neoplasms ; enzymology ; pathology

OBJECTIVETo study the effects of neuron specific enolase (NSE) gene silencing on the cell proliferation and apoptosis of lung cancer cells in vitro.

METHODSNSE protein expression was detected in human small cell lung cancer cell line NCI-H446 and non-small cell lung cancer cell line A549 by immunocytochemistry, and a small interference RNA (siRNA) was transfected into the cells to inhibit NSE gene expression. The changes in the cell cycle, apoptosis, Ki67 protein and caspase-3 activity in the transfected cells were observed by flow cytometry, Western blotting and colorimetric assay, respectively.

RESULTSBoth A549 and NCI-H446 cells expressed NSE protein. Transfection of siRNA for NSE gene significantly inhibited the expression of NSE gene in the cells, resulting in an inhibition rate exceeding 90%. NSE gene silencing caused significantly decreased cell percentage in S phase and the expression of Ki67 protein, and increased the cell apoptotic rate and caspase-3 activity.

CONCLUSIONNSE gene expression promotes the cell proliferation and inhibits the cell apoptosis in lung cancer cells with neuroendocrine differentiation, which might be a causative factor contributing to increased malignancy of the cells.

Adenocarcinoma ; enzymology ; genetics ; pathology ; Apoptosis ; genetics ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Ki-67 Antigen ; metabolism ; Lung Neoplasms ; enzymology ; genetics ; pathology ; Phosphopyruvate Hydratase ; genetics ; RNA Interference ; Small Cell Lung Carcinoma ; enzymology ; genetics ; pathology

BACKGROUND/AIMS: Oxidative stress results in protein oxidation and is implicated in carcinogenesis. Sulfiredoxin (Srx) is responsible for the enzymatic reversal of inactivated peroxiredoxin (Prx). Nuclear factor E2-related factor 2 (Nrf2) binds to antioxidant responsive elements and upregulates the expression of Srx and Prx during oxidative stress. We aimed to elucidate the biological functions and potential roles of Srx in lung cancer. METHODS: To study the roles of Srx and Prx III in lung cancer, we compared the protein levels of Nrf2, Prxs, thioredoxin, and Srx in 40 surgically resected human lung cancer tissues using immunoblot and immunohistochemical analyses. Transforming growth factor-beta1, tumor necrosis factor-alpha, and camptothecin treatment were used to examine Prx III inactivation in Mv1Lu mink lung epithelial cells and A549 lung cancer cells. RESULTS: Prx I and Prx III proteins were markedly overexpressed in lung cancer tissues. A significant increase in the oxidized form of a cysteine sulfhydryl at the catalytic site of Prxs was found in carcinogenic lung tissue compared to normal lung tissue. Densitometric analyses of immunoblot data revealed significant Srx expression, which was higher in squamous cell carcinoma tissue (60%, 12/20) than in adenocarcinoma (20%, 4/20). Also, Nrf2 was present in the nuclear compartment of cancer cells. CONCLUSIONS: Srx and Prx III proteins were markedly overexpressed in human squamous cell carcinoma, suggesting that these proteins may play a protective role against oxidative injury and compensate for the high rate of mitochondrial metabolism in lung cancer.
Adenocarcinoma/*enzymology/genetics/mortality/pathology ; Animals ; Antineoplastic Agents, Phytogenic/pharmacology ; Blotting, Western ; Camptothecin/pharmacology ; Carcinoma, Squamous Cell/*enzymology/genetics/mortality/pathology ; Cell Line, Tumor ; Humans ; Immunohistochemistry ; Lung Neoplasms/*enzymology/genetics/mortality/pathology ; Mink ; NF-E2-Related Factor 2/*metabolism ; Oxidoreductases Acting on Sulfur Group Donors/genetics/*metabolism ; Peroxiredoxin III/*metabolism ; Peroxiredoxins/metabolism ; Prognosis ; RNA Interference ; Reactive Oxygen Species/metabolism ; Transfection ; Transforming Growth Factor beta1/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Up-Regulation

OBJECTIVETo investigate the expression of indoleamine 2, 3-dioxygenase (IDO) in breast cancer and its correlation with clinicopathologic factors and prognosis.

METHODSThe expression of IDO, CD31, CD105 proteins in 40 specimens of breast cancer were assessed by immunohistochemistry.

RESULTSThe overexpression rate of IDO in breast cancer was 67.5% (27/40), and expression of IDO was closely associated with clinical stage and lymph nodes metastasis. The disease-free survival rate in patients with IDO overexpression was not significantly lower than that in patients with negative or low expression of IDO (P > 0.05). Moreover, the expression of IDO was positively correlated with CD105-labeled microvessel density (r = 0.659, P < 0.05).

CONCLUSIONSExpression of IDO is associated with clinical stage and lymph nodes metastasis, and microvessel densitty. IDO expression may promote the growth and metastasis of breast cancer, probably via the increased agiogenesis. A larger sample study is needed to verify whether the prognosis of beast cancer is significantly correlated with IDO expression.

Adenocarcinoma ; enzymology ; immunology ; pathology ; Adult ; Aged ; Antigens, CD ; metabolism ; Breast Neoplasms ; enzymology ; immunology ; pathology ; Carcinoma, Ductal, Breast ; enzymology ; immunology ; pathology ; Carcinoma, Medullary ; enzymology ; immunology ; pathology ; Disease-Free Survival ; Endoglin ; Female ; Follow-Up Studies ; Humans ; Immunohistochemistry ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Lymphatic Metastasis ; Microvessels ; enzymology ; immunology ; Middle Aged ; Neoplasm Staging ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Receptors, Cell Surface ; metabolism ; Survival Rate

OBJECTIVETo investigate the effects of silencing heparanase (HPA) on growth, angiogenesis and metastasis of human gastric carcinoma transplanted in nude mice.

METHODSHuman gastric carcinoma SGC-7901 cells and those cells with silenced HPA (gastric carcinoma SGC-7901-HPA(-)) were separately transplanted subcutaneously in 6 nude mice. The time, size and speed of tumor growth were recorded. RT-PCR and Western-blot were used to detect the expression of HPA mRNA and protein in the subcutaneous tumors of the two groups. Immunohistochemical staining was used to detect microvessel density (MVD) in the subcutaneous tumors of the two groups. Cells of the subcutaneous transplanted tumors of the two groups were separately injected into the peritoneal cavity of nude mice, 6 mice each. The growth of metastatic tumors in nude mice was observed.

RESULTSHuman gastric carcinoma SGC-7901 cells and SGC-7901-HPA(-) cells were subcutaneously inoculated in nude mice, and tumors appeared at 4 days and 7 days after inoculation, respectively. The MVD was (20.69 ± 1.20)/HP and (11.35 ± 1.94)/HP, respectively (P < 0.05). The expressions of HPA mRNA and protein of the subcutaneously transplanted SGC-7901-HPA(-) tumor were decreased. Four voluminous metastatic tumors caused by SGC-7901 cells occurred in 3 mice in the liver, right kidney, omentum and intestine. Two smaller abdominal metastatic tumors of SGC-7901-HPA(-) cells were found in the liver and right kidney.

CONCLUSIONSilencing HPA can inhibit the tumor growth, angiogenesis and metastasis of human gastric cancer in nude mice. It suggests that HPA might become a new target for prevention and treatment of gastric cancer.

Adenocarcinoma ; blood supply ; enzymology ; genetics ; pathology ; secondary ; Animals ; Cell Line, Tumor ; Gene Silencing ; Glucuronidase ; biosynthesis ; genetics ; physiology ; Humans ; Kidney Neoplasms ; secondary ; Liver Neoplasms ; secondary ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microvessels ; pathology ; Neoplasm Transplantation ; Neovascularization, Pathologic ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; blood supply ; enzymology ; genetics ; pathology

OBJECTIVETo investigate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) content and activity in lung adenocarcinoma cell lines and its correlation with radiosensitivity.

METHODSThe content and activity of DNA-PKcs were analyzed in two lung adenocarcinoma cell lines A549 and H1299 by Western blotting and the Signa TECT DNA-PK assay kit. The dose-survival relationship for two cell lines was analyzed using clonogenic formation assay.

RESULTSA549 was more radiosensitive than H1299. The survival fractions at 2 Gy (SF2) were 0.7412 in A549 cell line and 0.2473 in H1299 cell line. The content of DNA-PKcs was significantly higher in A549 cells than in H1299 cells (t=10.37, P<0.001). The integrated optical densities were 3.29-/+0.44 in A549 cells and 0.50-/+0.17 in H1299 cells. DNA-PKcs activities in A549 and H1299 cells were 8.29-/+1.37 and 2.47-/+1.09, respectively, showing a significant difference between them (t=5.76, P=0.005).

CONCLUSIONDNA-PKcs is an important factor to affect the radiosensitivity of lung adenocarcinoma cell lines.

Adenocarcinoma ; enzymology ; pathology ; Calcium-Binding Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Humans ; Lung Neoplasms ; enzymology ; pathology ; Radiation Tolerance

OBJECTIVETo investigate the effects of heparanase expression inhibition on the proliferation, invasiveness and apoptosis of human lung adenocarcinoma cell line A549 cells.

METHODSRecombinant eukaryotic expression plasmid pshRNA-Hpa targeting human heparanase gene was constructed. A549 cells were cultured in DMEM and transfected with pshRNA-Hpa. The expression of heparanase mRNA and protein were examined by RT-PCR and Western blot. The proliferation, invasiveness and apoptotic rates of A549 cells were determined by MTT method, matrigel invasion assays and flow cytometry respectively.

RESULTSThe expression levels of heparanase mRNA and protein were down-regulated in A549 transfected with pshRNA-Hpa. The number of cells penetrating matrigel and the proliferation ability of A549 cells transfected with pshRNA-Hpa were reduced significantly compared to the control cells. The apoptotic rate of A549 cells transfected with pshRNA-Hpa was 12.53% +/- 0.34%, being significantly higher than that of the control cells (both P < 0.01). Western-blot showed that inhibition of heparanase expression led to reduced Akt phosphorylation.

CONCLUSIONSThe recombinant plasmid pshRNA-Hpa effectively inhibited the expression of heparanase, thus suppressing the proliferation and invasion and inducing apoptosis of A549 cells. The effects may be due to the down-regulation of Akt phosphorylation level.

Adenocarcinoma ; pathology ; Apoptosis ; drug effects ; Carcinoma, Non-Small-Cell Lung ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; Glucuronidase ; antagonists & inhibitors ; metabolism ; Humans ; Lung Neoplasms ; enzymology ; pathology ; RNA Interference ; immunology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Transfection