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

OBJECTIVETo analyze the clinicopathological characteristics of patients with anaplastic lymphoma kinase (ALK) rearrangements in lung adenocarcinoma, and the clinical therapy and prognosis of the patients.

METHODSClinicopathological data of 34 cases of ALK-positive patients treated in the Beijing Chest Hospital from 2005 to 2014 were reviewed. The expression of ALK proteins in the resected tumors was detected by immunohistochemistry, and EGFR mutations were examined by polymerase chain reaction and a direct DNA sequencing method.

RESULTSAmong the 34 patients, 20 were male and 14 were female, the median age was 49, and 11 were smokers and 23 were never smokers. The clinical stages of the patients were stage IA in 5 patients, IB in one patient, IIA in two patients, IIIA in 16 patients, IIIB in 5 patients, IV in 4 patients, and one patient of unknown stage. ALK-positive tumors showed strong granular staining in cell cytoplasm by immunohistochemistry. Forteen patients were solid predominant subtype with mucin production, 10 of acinar predominant subtype, 6 of papillary predominant subtype, 3 of micropapillary predominant subtype, and one was of colloid variant. There were 18 cases with mucin production, 6 cases had signet-ring cell morphology, and 10 cases showed cribriform pattern. Only one patient had coexistence of ALK rearrangement and EGFR mutation (L858R at exon 21). Of the 34 patients, 24 patients were followed up. The median follow up of the 24 patients was 11.0 months (1.7-48.7 months).

CONCLUSIONSALK-positive tumors as a molecular subtype of lung adenocarcinoma have distinct clinicopathological features. The histological findings of ALK-positive tumors are characterized by solid predominant subtype with mucin production, acinar predominant subtype, signet-ring cells and cribriform structures. They were rarely co-mutated with EGFR mutation.

Adenocarcinoma ; enzymology ; pathology ; therapy ; Exons ; Female ; Gene Rearrangement ; Genes, erbB-1 ; Humans ; Immunohistochemistry ; Lung Neoplasms ; enzymology ; pathology ; therapy ; Male ; Middle Aged ; Mucins ; biosynthesis ; Mutation ; Neoplasm Proteins ; genetics ; Polymerase Chain Reaction ; Prognosis ; Receptor Protein-Tyrosine Kinases ; genetics ; Sequence Analysis, DNA

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 explore the effect of Buzhong Yiqi decoction on PI3K/AKT signaling pathway in spleen, stomach and lung of nude mice with lung adenocarcinoma transplantation tumor.

METHODTotally 60 nude mice were randomly divided into the blank control group, the tumor-bearing control group, the cisplatin group, the low-dose Buzhong Yiqi decoction group, the middle-dose Buzhong Yiqi decoction group and the high-dose Buzhong Yiqi decoction group. After the corresponding interventions, efforts were made to measure the transplanted tumor volume and calculate the tumor inhibiting rate. The immunohistochemical method and real time PCR were used to detect the expression of PI3K and AKT level in nude mice spleen, stomach and lung.

RESULTBuzhong Yiqi decoction of different concentrations combined with cisplatin could inhibit the growth of the transplanted tumor, with the strongest inhibitory effect in the middle-dose Buzhong Yiqi decoction group and the high-dose Buzhong Yiqi decoction group. All of the expressions of PI3K and AKT protein and gene in the spleen, stomach and lung increased, with the most significant increase in the tumor-bearing group. Along with the increase of the concentration of cisplatin and Buzhong Yiqi decoction, the expressions of PI3K and AKT gradually reduced. Compared with the tumor-bearing control group, there were statistical differences in spleen and stomach tissues (P < 0.05). Compared with the cisplatin group, the middle-dose Buzhong Yiqi decoction group and the high-dose Buzhong Yiqi decoction group showed statistical differences (P < 0.05), but without statistical difference compared with the blank control group.

CONCLUSIONAmong nude mice with lung adenocarcinoma transplantation tumor, the PI3K and AKT protein and gene expressions in spleen, stomach and lung tissues increased, which might indicated the effect of cisplatin and Buzhong Yiqi decoction in reducing PI3K and AKT expressions and the relations between the reduction degree and the concentrations of Buzhong Yiqi decoction. Cisplatin combined with Buzhong Yiqi decoction could decrease the PI3K and AKT protein and gene expression in spleen, stomach and lung, and make the pathway closer to normal, so as to protect the functions of spleen, stomach and lung, there may be target spots of Buzhong Yiqi decoction in PI3K/AKT signal pathway.

Adenocarcinoma ; drug therapy ; enzymology ; genetics ; Animals ; Cell Line, Tumor ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Lung ; drug effects ; enzymology ; Lung Neoplasms ; drug therapy ; enzymology ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Oncogene Protein v-akt ; genetics ; metabolism ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Signal Transduction ; drug effects ; Spleen ; drug effects ; enzymology

BACKGROUND/AIMS: Doublecortin and CaM kinase-like-1 (DCAMKL1) is a marker of stem cells expressed predominantly in the crypt base in the intestine. However, DCAMKL1-positive cells have been shown to be differentiated tuft cells rather than quiescent progenitors. Tuft cells are the only epithelial cells that express cyclooxygenase 2 (COX-2) in the normal intestinal epithelium. We previously generated Cdx2-transgenic mice as model mice for intestinal metaplasia and gastric carcinoma. In the current study, we investigated the association between COX-2 and DCAMKL1 in gastric carcinoma. METHODS: We examined the association between COX-2 and DCAMKL1 expression in gastric carcinomas in clinical samples (early gastric well-differentiated adenocarcinoma) and Cdx2-transgenic mice; and the DCAMKL1-transgenic mouse stomach using immunohistochemistry and quantitative real-time polymerase chain reaction. RESULTS: The COX-2-expressing cells were scattered, not diffusely expressed, in gastric carcinomas from humans and Cdx2-transgenic mice. DCAMKL1-positive cells were also scattered in the gastric carcinomas, indicating that tuft cells could still be present in gastric carcinoma. COX-2 was expressed in DCAMKL1-positive tuft cells in Cdx2- and DCAMKL1-transgenic mouse stomachs, whereas the Sox9 transcription factor was ubiquitously expressed in gastric carcinomas, including COX-2-positive cells. CONCLUSIONS: COX-2 is expressed in DCAMKL1-expressing quiescent tuft cells in gastric carcinoma.
Adenocarcinoma/metabolism ; Animals ; Cyclooxygenase 2/genetics/*metabolism ; Epithelial Cells/metabolism ; Gastric Mucosa/metabolism ; Humans ; Intestinal Mucosa/cytology/*enzymology/metabolism ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Mice ; Mice, Transgenic ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; SOX9 Transcription Factor/genetics/metabolism ; Stomach Neoplasms/*enzymology/genetics

BACKGROUND/AIMS: To investigate the differential expression of RING finger (RNF) proteins in Barrett esophagus (BE) and esophageal adenocarcinoma (EAC). METHODS: The differential expression of RNFs in normal esophagus (NE), BE, and EAC was screened using microarray assay. Real-time quantitative polymerase chain reaction (PCR), tissue microarray assay, and Western blot analysis were independently performed to detect the mRNA and protein expression of screened RNFs. RESULTS: The expression of nine RNFs in the BE or EAC was 2-fold higher than those in NE. Among these proteins, the RNF32 and RNF121 expression in BE was 20.3-fold and 16.4-fold higher, respectively, than that in NE, and the expression of RNF24, RNF130, RNF141, RNF139, RNF11, RNF14, and RNF159 was upregulated more than 2-fold compared with NE. The expression of nine RNFs was not only upregulated in the EAC but was also positively related to the RNF expression in BE. The PCR results also indicated increased expression of these RNFs in BE and EAC compared to NE. Furthermore, the mRNA expression of all RNFs, except for RNF141 in EAC, was dramatically higher than those in the BE. Similar results were also obtained from the Western blot analysis. CONCLUSIONS: A total of nine RNFs play critical roles in the progression of BE to EAC.
Adenocarcinoma/*enzymology/genetics ; Adult ; Aged ; Barrett Esophagus/*enzymology/genetics ; Carrier Proteins/genetics ; Disease Progression ; Esophageal Neoplasms/*enzymology/genetics ; Female ; Gene Expression Profiling ; Humans ; Intracellular Signaling Peptides and Proteins/genetics ; Male ; Middle Aged ; Proteins/genetics ; *RING Finger Domains ; Receptors, Cell Surface/genetics ; Ubiquitin-Protein Ligases/genetics/*metabolism

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 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

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