Objective Studies show that the abnormal expression of EphB4 plays an important role in the development and progression of colon cancer .The present study aims to provide some experimental evidence for the gene therapy of colon cancer by con -structing a lentiviral expression vector carrying the homo EphB4 gene and further establishing colon cancer cell lines with stable overex-pression of EphB4. Methods A series of oligonucleotides (oligo) encoding the homo EphB4 gene were ligated together by PCR and then cloned into a lentiviral expression vector pLenti 6.3-MCS-IRES2-EGFP.After confirmed by sequencing , the vector pLenti6.3-EphB4-IRES2-EGFP and its helper vectors were mixed and co-transfected into 293 T cells to obtain recombinant virus containing the EphB4 gene.The lentiviral titer was detected and the resulting recombinant lentiviruses carrying EphB4 or control viruses only carrying green fluorescence protein (GFP) were used to infect the human colon cancer cell lines .The expression of GFP was determined under the inverted fluorescence microscope and the level of EphB 4 mRNA in the infected cells detected by qPCR . Results The lentiviral expression vector pLenti6.3-EphB4-IRES2-EGFP carrying correct homo EphB4 gene sequence was successfully constructed .The titer of the recombinant EphB4 lentiviral supernatant Lenti6.3-EphB4 was 1 ×108 TU/mL.The expression of GFP was observed in the trans-duced cells under the fluorescence microscope , and that of EphB4 mRNA in the transfected SW480 and Coca-2 cells was significantly up-regulated as compared with the control and blank groups . Conclusion The homo EphB4 gene was successfully amplified and cloned.A lentiviral expression vector was successfully constructed , and so were colon cancer cell lines stably overexpressing EphB 4, which may shed light on the lentivirus-mediated genetic therapy for colon cancer .

Objective To summarize the experience of nursing patients undergoing NovaSure endometrial ablation. Methods Two hundred and eleven patients with abnormal uterine bleeding claiming no need of giving birth,who were hospitalized in our hospital during August 2011 to April 2013,were managed with NovaSure endometrial ablation and nursing care was performed.Follow-ups were conducted for investigating the curative effects.Results All the patients lived through the operations,their life signs were stable.As for the adverse effects,124 of them had mild abdominal pains,7 medium-level or severe hypogastralgia,1 dizziness and nausea,10 uterial hematocele,and 1 cystitis.All of them got recovered after treatment.One-month follow-up after hospitalization showed a success rate of 98.0%.Conclusions NovaSure is a new approach to abnormal uterine bleeding.It is advantageous for its simplicity,shortness in operation duration,less trauma to patients and good effects.Meanwhile,preoperative mental care and cooperative care during operation may facilitate their recoveries.

Objective Studies show that the role of EphB 4 in the development and progression of cancer is correlated to its ligand EphrinB2.The present study was to observe the effect of the changes in EphB4 on the expression of EphrinB2 by constructing and identifying microRNA ( miRNA) interference vectors targeting the EphB4 gene in colon cancer cells . Mte hods According to the EphB4 gene sequence , 3 pairs of oligo DNA sequences of miRNA were designed .The single strand of oligo DNA was annealed to form double-strand DNA, and then connected with the plasmid pcDNA 6.2-GW/EmGFP-miRNA.The expression vector pcDNA6.2-GW/EmGFP-miR-EphB4 was linked to pDONR221 and pLenti6/V5-DEST to construct the lentiviral expression vector pLenti 6/V5-DEST-EphB4, which was cotransfected with packaging mix (pLP1, pLP2 and pLP/VSVG) into 293FT cells by lipofectamine 2000 transfec-tion to produce lentivirus , and the lentivirus titer was measured by infection of HEK 293 cells.The stable cell lines were selected and cultured.The expression levels of EphB4 and EphrinB2 were examined by qPCR. Results Three miRNA interference vectors SR-1, SR-2, and SR-3 targeting the EphB4 gene were successfully constructed , with SR-3 exhibiting the most significant interference efficien-cy.The constructed lentiviral vector pLenti 6/V5-DEST-EphB4 was successfully packaged in 293FT cells.The virus titer was 7 ×108 Caco-2 cells. Conclusion The exogenous EphB4 expression could be significantly inhibited by treatment with specific miRNA in co-lon cancer cells .The correlation of EphB4 and EphrinB2 may be effected by many factors and need further studies .

Purpose To explore the immunohistochemical ( IHC) expression of ALK antibodies with different clones in anaplastic lym-phoma kinase ( ALK) gene fusion non-small cell lung cancer ( NSCLC) . Methods ALK expression in 60 NSCLCs were detected by IHC including autostainer (D5F3, Ventana+BenchMark) and manual staining using 4 different antibodies of D5F3 (Ventana), D5F3 (Cell Signaling), 1A4/1H7 (OriGene), 5A4 (Abcam), and all cases were verified with ALK FISH. Their expressions with 4 anti-bodies were compared with those by D5F3 (Ventana+BenchMark). Results 32 ALK gene rearrangement NSCLCs and 28 negative cases were identified by FISH and D5F3 (Ventana+BenchMark). The sensitivity of D5F3 (Ventana), D5F3 (Cell Signaling), 1A4/1H7 (OriGene), 5A4 (Abcam) was 93. 8%, 84. 4%, 93. 8%, 56. 3%, and all the speciticity was 100%. The consistency with D5F3 (Ventana+BenchMark) was 96. 7%, 91. 7%, 96. 7% and 76. 7%, respectively. The validity of immunohistochemical staining in surgical resection specimens was better than in small biopsies. Conclusion Effective routine manual immunohistochemistry with high-affinity antibody clone may provide a more economic and widespread pre-screening technique.

Objective To summarize the operation period in the treatment of elderly patients with benign prostatic hyperplasia(BPH)clinical effect with inguinal hernia.Methods 60-96years old in elderly patients with BPH complicated with inguinal hernia in 53 cases,transurethral plasmakinetic resection of prostate (TUREP)or open operation for the treatment of benign prostatic hyperplasia,while using bard tension-free repair of inguinal hernia, disposable operation treatment of BPH complicated with inguinal hernia,the clinical effect and safety were observed. Results 53 cases were cured all operation,followed up for 6months to 2years,there were no recurrence of hernia operation,incision infection and other complications of operation.Micturition was apparent improvement.The average postoperative hospital stay was 7.6days,14 863 yuan for hospitalization.Conclusion It is safe and effective for sim-ultaneous TURP andinguinal hernia repair,which can avoid pain,and two times the risk of operation and anesthesia, especially has the positive significance in aged patients.

Objective To establish an external quality assessment ( EQA) system of genitourinary tract secretions routine testing in Guizhou Province and improve the overall testing level.Methods From 2009 to 2011, more than 50 clinical laboratories in different grade hospitals from Guizhou Province were enrolled as participating units every year.EQA was carried out twice a year.Each time, five slides of high quality Wright′s or Gram stain smear of the genitourinary tract secretions or photographs obtained from these smears were selected to send to the participating laboratories for testing, and the feedback results from each laboratory were analyzed.The qualification was judged by the coincidence rate equal to or more than 80%. The average coincidence rates of each time and each year were statistically analyzed by Chi-squared test. Results From 2009 to 2011, the number of EQA participating units increased from 55 to 96, with an average return rate of >80%.Coincidence rates <80%of the 6 EQA results in the 3 years were as follow:four times for coccobacteria (73.7%,77.8%,61.1%,77.1%), twice for bacillus (75.6%,79.3%) and coccobacillus (64.3%,52.1%), once for infusorian (79.7%), epithelial cells (76.1%), neutropenia (75.7%) and cleanliness (71.3%).There were six batches of 30 quality assessment controls (accounting for 20.0%) in the six EQAs had the coincidence rate of <80%.Eleven items of 30 quality assessment controls with 1 to 15 batches were unqualified ( average coincidence rate of<80%) respectively.The item with the highest total average coincidence rate was suspected gonococcus (94.2%), and two items with the lowest total average coincidence rates were coccus and coccobacillus ( 77.0%, 75.2%, respectively ) . Conclusions This EQA program carried out within a certain range of clinical laboratories achieved good results:participating units increased significantly;the total score of all the items showed an obviously upward trend;the quality awareness of clinical lab technicians has enhanced to a certain extent.In this study, EQA system of genitourinary tract secretion routine testing were preliminarily established in Guizhou province, which provided a reference model of internal quality control ( IQC ) and EQA for clinical laboratories and higher authorities, and will be bound to have a positive impact on improvement of the overall level of genitourinary tract secretion routine testing.

To explore the automated immunostainer screening anaplastic lymphoma kinase (ALK) gene fusion non-small cell lung cancer (NSCLC) and clinicopathological characteristics of the molecular subtype lung cancers. Methods Five hundred and sixty-six cases of NSCLC were collected over a 16 month period. The test for ALK was performed by Ventana automated immunostainer with anti-ALK D5F3. The histological features, treatment and outcome of patients were assessed. Results Thirty-eight cases (6.7%, 38/566) of NSCLC showed ALK gene fusion. The frequency of ALK gene fusion was higher in male (7.1%, 25/350) than that in female (6.0%, 13/216) patients, but not achieving statistical significance (chi2 = 0.270, P = 0.604). ALK + NSCLC was more significantly more frequent in patients < or = 60 years (9.9%, 28/282) than >60 years (3.5% , 10/284) of age. Histologically, the ALK + NSCLCs were mostly adenocarcinoma (81.6%, 31/38) , among which eighteen cases were solid predominant subtype with mucin production; nine cases were acinar predominant subtype; one case was papillary predominant subtype and three cases were invasive mucinous adenocarcinoma. The ALK + non-adenocarcinoma included three cases of squamous cell carcinoma, three cases of adenosquamous carcinoma and one case of pleomorphic carcinoma. Among the ALK + NSCLC patients, the number of non/light cigarette smokers (86. 8% , 33/38) was more than that of heavy smokers. Twenty-nine cases were stages III and IV; twenty-nine cases showed lymph node metastasis; twenty cases showed metastases mostly to brain and bone; and one case showed EGFR gene mutation coexisting with ALK gene fusion. Twelve of fifteen patients received crizotinib therapy and remained stable. Conclusions NSCLC with ALK gene rearrangement shows distinctive clinical and histological features. Ventana-IHC may he a feasible and valid technique for detection of ALK rearrangement in NSCLC.
Adenocarcinoma ; genetics ; pathology ; Carcinoma, Adenosquamous ; genetics ; pathology ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Carcinoma, Squamous Cell ; genetics ; pathology ; Female ; Gene Fusion ; Gene Rearrangement ; Humans ; Lung Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Protein Kinase Inhibitors ; therapeutic use ; Pyrazoles ; therapeutic use ; Pyridines ; therapeutic use ; Receptor Protein-Tyrosine Kinases ; genetics ; Sex Factors

Objective To explore the causes of Fra-1 high expression in breast cancer cells and molecular mechanism of Fra-1 high transcription activity .Methods Two human breast cancer cell lines MDA231 and MCF-7 and 1 human umbilical vein endothelial cell ECV304 were selected as the research objects .Real time fluorescent quantitative reverse transcription -polymerase chain re-action and Western blotting were used to detect the Fra-1 mRNA and protein expression ;the Fra-1 dual fluorescence reporter vector was constructed ,and the Fra-1 promotor transcription activity was detected ;the Fra-1 promotor short-cut reporter vector and mutation reporter vector at related loci were constructed ,and the transcription activity of short-cut reporter vector and mutation reporter vector was detected ;the binding situation of specific probe marked by biotin with activator protein-1(AP1) was observed by using the gel migration block test .Results The relative expression of mRNA and protein and promotor whole length reporter vector transcription activity of Fra-1 in human breast cancer cell lines MDA231 and MCF-7 cells were higher than that in human umbilical vein endothelial cell MCV304 ,the difference was statistically significant (P＜0 .05);in constructed series Fra-1 promotor shortcut reporter vectors ,only the activity of pGL3B-256 was obviously decreased ,the difference was statistically significant (P＜0 .05);the activity of AP1 mutation reporter vector pGL3B-M2 and SP1 and AP1 dual mutation reporter vector pGL3B-M3 was significantly lower than that of wild repoter vector pGL 3B-M1 ,the difference was statistically significant (P＜0 .05);the gel migration block test found that the binding of specific probe marked by biotin with AP1 had obvious blocking effect .Conclusion In in vitro cultured breast cancer cell lines MDA231 and MCF-7 ,the trans-acting factor AP1 up-regulates its expression by activating Fra-1 gene promoter .

Objective: To study the clinicopathologic features, immunophenotype, characteristic FISH pattern and prognosis of renal cell carcinoma (RCC) associated with chromosome X inversion harboring gene fusions involving TFE3. Methods: Ten cases of NONO-TFE3 RCC and four cases of RBM10-TFE3 RCC were investigated at Nanjing Jinling Hospital from 2009 to 2016 by clinicopathological findings, immunohistochemistry, and genetic analysis. Results: Morphologically, the distinct pattern of secretory endometrioid subnuclear vacuolization was overlapped with clear cell papillary RCC, and often accompanied by sheets of epithelial cells in NONO-TFE3 RCC. Most cases of RBM10-TFE3 RCC presented with the biphasic feature that acinar, tubular and papillary patterns of epithelioid cells combined with sheets of small cells with "pseudorosette-like" architectures. In addition, cytoplasmic vacuolization, nuclear groove, and psammoma bodies were also observed. Immunohistochemically, all NONO-TFE3 RCC cases were immunoreactive for TFE3, CD10, RCC markers, and PAX8, and negative for CK7, Cathepsin K, Melan A, HMB45, Ksp-cadherin, vimentin, and CD117. All 4 cases of RBM10-TFE3 RCC showed moderate to strong immunoreactivity for TFE3, Cathepsin K, CD10, Ksp-cadherin, E-cadherin, P504s, RCC marker, PAX8, and vimentin but negative for TFEB, HMB45 and CK7. CKpan and Melan A were at least focally expressed. The antibody to Ki-67 showed labeling of 3%-8% (mean 5%). There were some expression discrepancies of immunochemistry between different histological patterns. PAX8, CKpan, P504s, and Ksp-cadherin were expressed in epithelioid areas but not in small-cell areas. Ki-67 labeling index of epithelioid areas was higher than that in small-cell areas. In molecular analysis, NONO-TFE3 fusion transcripts were identified in 6 patients. The fusion points were between exon 7 of NONO and exon 6 of TFE3 in 5 patients and between exon 9 of NONO and exon 5 of TFE3 in one patient. All 4 cases of RBM10-TFE3 RCC demonstrated to have RBM10-TFE3 fusion transcripts and the fusion points were between exon 5 of TFE3 and exon 17 of RBM10. Using TFE3 break-apart FISH assay, all 10 cases of NONO-TFE3 RCC showed characteristic patterns of equivocal split signals with a distance of nearly 2 signal diameters. All 4 cases of RBM10-TFE3 RCC showed colocalized or subtle split signals with a distance of <1 signal diameter, which was considered as negative results. Long-term follow-up was available for 7 patients of NONO-TFE3 RCC and 4 patients of RBM10-TFE3 RCC. All patients were alive with no evidence of disease. Conclusions Two rare genotypes, NONO-TFE3 RCC and RBM10-TFE3 RCC, are reported in this study. Both of these two tumors show specific morphology and good prognosis, along with the positive TFE3 staining and the equivocal or false-negative TFE3 FISH results, which could be missed. PCR detection or next-generation sequencing can determine the genotype.

Objective: To investigate the clinicopathological, and molecular characteristics of myoepithelial tumors (MTs) of salivary glands. Methods: A total of 37 MTs cases including 13 malignant epithelial tumors (MMTs) and 24 benign epithelial tumors (BMTs) of salivary glands were identified from the archives of the Department of Pathology, General Hospital of Eastern Theater Command, dating from 2006 to 2016. Clinical features, histological patterns, immunohistochemical characteristics and status of EWSR1 gene rearrangement by fluorescence in situ hybridization (FISH) analysis were reviewed in all cases. Results: Clinically, 37 MTs cases mainly occurred in the parotid glands, when most of the patients presented with painless masses. Of the 13 MMTs cases, male to female ratio was 7∶6, and the median age was 62 years old. Of the 24 BMTs cases, male to female ratio was 5∶7, and the median age was 54 years old. Immunohistochemically, 37 MTs cases were positive for CKpan, and at least one myoepithelial marker. Twenty six of 37 MTs cases were analyzable for the EWSR1 gene break by FISH. Based on the previous evaluation criterion, the EWSR1 translocation was detected in 4 cases of 11 MMTs, and 4 cases of 15 BMTs. According to the main histological composition of tumor cells, 4 EWSR1-positive MMTs covered 2 clear-cell cases and 2 epithelioid-cell cases, when 4 EWSR1-positive BMTs covered 2 clear-cell cases, 1 plasmacytoid-cell case, and 1 spindle-cell case. Conclusions Males and females are affected equally. MTs express immunoreactivity for CKpan, and at least one myoepithelial marker. The EWSR1 rearrangement is present in a subset of MTs, with variable morphological characteristics, and has no statistical significance on clinical behavior.