Objective:To investigate the intra-observer reproducibility of Ki-67　　　assessment in breast cancers using three methods based on digital slide.Methods:Thirty cases of invasive breast cancer tissues were immunostained for Ki-67 by automatic stainer, and then scanned into digital pathological slides. Ki-67 positive index was measured individually by three pathologists using size-set visual assessment of hot spot (SSVAHS), size-set semi-automatic counting of hot spot(SSSACHS), and size-set automatic counting of hot spot (SSACHS), respectively, and repeated for 10 times. Intraclass correlation coefficient (ICC) of each assessment method was calculated, and the intraobserver reliability was classified as excellent, good, fair and poor according to ICC.Results:The ICC by 3 pathologists using SSVAHS was 0.832, 0.843 and 0.826, respectively, The ICC using SSSACHS was 0.926,0.938,0.929, and the ICC using SSACHS was 0.964, 0.971 and 0.968.The intraobserver reliability level of all three methods was excellent.Conclusion:The three methods of Ki-67 assessment achieve satisfactory intraobserver reproducibility, and the order of reproducibility from high to low is SSACHS, SSSACHS, and SSVAHS.

Objective To investigate the mutation status of epidermal growth factor receptor ( EGFR) and KRAS gene in patients with non-small cell lung cancer ( NSCLC) in Xuanwei, Yunnan and to correlate the mutation status with clinicopathologic features.Methods Mutation status of exons 18, 19, 20 and 21 of EGFR, and codons 12, 13 of KRAS in 63 cases of NSCLC were analyzed by gene sequencing and ARMS-Taqman probe method.Correlation with patients′clinicopathological characteristics was performed. Results EGFR and KRAS mutations were present in 55.6% (35/63) and 6.3% (4/63), respectively. EGFR gene mutations were present, including exon 18 G719X in 14.3% (5/35), exon 19 in 14.3%(5/35), exon 20 S768I and T790M in 20.0% (7/35), exon 21 L858R in 31.4% (11/35), exon 18 G719X and exon 20 S768I double mutation in 17.1%(6/35), and exon 20 T790M and exon 21 L858R double mutation in 2.9%(1/35).KRAS mutations were seen in codon 12 in 3 of 4 cases, and codon 13 in 1 of 4 cases.EGFR mutations were mutually exclusive with KRAS mutations.According to statistic analysis, EGFR mutations were associated with the histological types of NSCLC ( P<0.05 ) , but without correlation with patient′s gender, age, smoking status and lymph node metastasis ( P >0.05 ) .KRAS mutations in NSCLC had no correlation with the clinical pathologic characteristics of the patients.Conclusions A higher frequency of EGFR exon 18 G719X and 20 exon S768I mutations are found in the patients in Xuanwei, Yunnan.EGFR mutations are associated with histologic types of NSCLC, but without correlation with patient′s gender, age, smoking status and lymph node metastasis.KRAS mutation in NSCLC has no correlation with the clinicopathologic characteristics of the patients.

Purpose To discuss the TCR gene rearrangements in the diagnosis of T-cell lymphomas. Methods Formalin-fixed and paraffin-embedded samples including 30 cases of T-cell lymphomas and 30 cases of reactive lymphoid hyperplasia were chosen for ex-tracting genomic DNA and PCR amplification using 56 BIOMED-2 primers. PCR products were analyzed by heteroduplex and polyacryl-amide gel electrophoresis. Results In all 30 cases of T-cell lymphomas, 25 cases (83. 3%) showed TCRβ gene monoclonal rear-rangements, 28 cases (93. 3%) of TCRγ gene monoclonal rearrangements, 4 cases (13. 3%) of TCRδ gene monoclonal rearrange-ments. 29 cases (96. 7%) with TCRβ+TCRγ+TCRδ gene monoclonal rearrangements were detected. but no clonal TCR gene rear-rangements were found in 30 cases of reactive lymphoid hyperplasia. Conclusions The detection of TCR gene rearrangements using BIOMED-2 primers is a useful assistant method for the diagnosis of T-cell lymphomas.

Objective To investigate the expressions of excision repair cross complementation group 1 ( ERCC1) and Ki67 in patients with breast cancer, and the relationships between their expressions and sensitivity of platinum-based chemotherapy. Methods Totally, 129 cases were pathologically diagnosed as breast cancer.Paclitaxel and carboplatin were used simultaneously. Chemotherapy regimen was as follows:Gemcitabine 1 000 mg??( m2 )-1 , IV drop on day 1 and 8;cisplatin 25 mg??( m2 )-1 , IV drop on day 1-3, for six cycles ( 21 days a cycle ) . ERCC1 and Ki67 expression in tumor tissue was observed by immunohistochemical analysis.Platinum-based chemotherapy sensitivity and survival of patients with different levels of ERCC1 and Ki67 expression were analyzed. Results In 129 patients, 18 cases were ERCC1 and Ki67 double-negative ( ERCC1-Ki67-) , and the clinical effective rate and 3-year cumulative survival rate were 88.89%and 83.33%, respectively.Twenty-four cases were ERCC1 positive but Ki67 negative ( ERCC1+Ki67-) , and the clinical effective rate and 3-year cumulative survival rate were 50. 00% and 62.50%, respectively.Thirty-three cases were ERCC1 negative but Ki67 positive (ERCC1-Ki67+), and the clinical effective rate and 3-year cumulative survival rate were 54. 55% and 60. 60%, respectively. Fifty-four patients were ERCC1 and Ki67 double-positive ( ERCC1+Ki67+) , and the clinical effective rate and 3-year cumulative survival rate were 22.78% and 31. 48%, respectively.Compared with ERCC1-Ki67- group, the clinical treatment efficiencies of cisplatin-based chemotherapy in ERCC1+Ki67- group, ERCC1-Ki67+ group, and ERCC1+Ki67+ group were significantly decreased ( P<0. 05 ) . The clinical treatment efficiency in patients of ERCC1+Ki67+ group with cisplatin-based chemotherapy was significantly decreased as compared with ERCC1+Ki67- group and ERCC1-Ki67+ group (P<0.05).Compared with ERCC1- Ki67- group, three-year cumulative survival rate in patients of ERCC1+ Ki67- group and ERCC1- Ki67+ group, ERCC1+Ki67+ group was significantly decreased ( P<0. 05 ) . Compared with ERCC1+Ki67-group and ERCC1-Ki67+group, three-year cumulative survival rate in patients of the ERCC1+Ki67+group was significantly decreased ( P<0.05) . Conclusion The expression levels of ERCC1 and Ki67 in breast cancer were high. Their expression levels are closely related with clinical efficiency of platinum-based chemotherapy.

Objective: To explore the relationship between ERCC 1, Ki67, PCNA expression with anthracycline chemotherapeutic drugs′sensitivities in breast cancer tissues.Methods:The ERCC1,Ki67,PCNA expression in 93 breast cancer tissue was detected by immunohistochemical staining.The efficacy of chemotherapy was observed and the difference of anthracycline chemotherapy effect among patients with different ERCC 1,Ki67,PCNA expression was compared.Results:The positive rate of ERCC1 was 65.59%,the positive rate of Ki67 was 69.89%,the positive rate of PCNA was 64.52%.The total effective rate of ERCC 1-positive group was 50.82%,and ERCC1-negative group was 84.38%.In Ki67-positive group,the effective rate of patients in 25%-50%intensity was 73.68%, the effective rate of patients in 50%-75% intensity was 85.71%, the effective rate of patients in >75%intensity was 88.89%, and Ki67-negative group was 60.71%.In PCNA-positive group , the effective rate of patients in 25%-50%intensity was 52.94%, the effective rate of patients in 50%-75% intensity was 62.07%, the effective rate of patients in >75%intensity was 71.43%, and PCNA-negative group was 81.82%.These differences were all statistically significant ( P<0.01 ,P<0.05 , P<0.05).Conclusion: There are correlations between ERCC1,Ki67,PCNA expression with anthracycline chemotherapeutic drugs′sensitivity of patients with breast cancer.Combined detection of multi-factor in clinical is more helpful for the selection of chemotherapy drugs and the formulation of chemotherapy regimen.

Purpose To investigate the sensitivity of BIOMED-2 primer system in T lymphoblastic lymphoma ( T-LBL) and acute lym-phoblastic leukemia ( ALL) patients immunoglobulin ( Ig) and T-cell receptor ( TCR) gene rearrangement, and to analyze the co-rear-rangement pattern. Methods Amplification of rearranged Ig and TCR gene was performed in standard PCR in 35 T-LBL/ALL pa-tients. PCR products were analyzed by heteroduplex and polyacrylamide gel electrophoresis. Results 16 cases (45. 7%) of 35 sam-ples were detected to have TCR gene rearrangements, including 6 cases (37. 5%) of TCRβgene monoclonal rearrangements, 4 cases (25. 0%) of TCRγ gene monoclonal rearrangements, 3 cases (18. 8%) of TCRβ and TCRγ gene double rearrangements, 2 cases (12. 5%) of TCRδ gene monoclonal rearrangements and 1 case (6. 3%) of TCRγand TCRδgene double rearrangements were detec-ted. 4 cases (11. 4%) of 35 samples detected to have clonal immunoglobulin and TCR gene rearrangements. 11 cases (39. 3%) of 28 T-LBL patients were detected to have TCR gene rearrangements, 6 cases (85. 7%) of 7 T-ALL have TCR gene rearrangements. Con-clusions BIOMED-2 multiplex PCR analysis strategy is a useful technique in the T-LBL patients.

Objective:To analyze the nuclear proliferation in breast cancer tissue related antigen (Ki-67) and proliferating cell nucleus antigen ( PCNA ) expression changes and the relationship between breast cancer and its relationship to breast cancer chemotherapy sensitivity, provide theoretical basis for clinical effective chemotherapy of breast cancer.Methods: Subjects from our hospital in recent years,by clinical examination,84 cases of patients diagnosed with breast cancer,breast cancer tissue were measured with immunohistochemical method of Ki-67 and PCNA content, compared different Ki-67 and PCNA expression levels of patients undergoing chemotherapy curative effect difference.Results:Ki-67 positive cases for 52 cases,PCNA positive cases of 62 cases.Ki-67 positive rate and the patients with lymph node metastasis and tumor classification stage were positively correlated,the difference was sta-tistically significant,P<0.05).The PCNA positive rate and the tumor was closely relative to lymph node metastasis,P<0.05,has nothing to do with tumor clinical classification stage(P>0.05).The total effective rate of Ki-67+was significantly higher than that of Ki-67-(80.8%and 56.2%,P<0.05).Effective rate of PCNA-significantly higher than that of PCNA+(72.7% to 45.2%,P<0.05).Conclusion:Ki-67 clinical data and PCNA expression is closely related to breast cancer and chemotherapy sensitivity.It can be used as a prediction index of curative effect of chemotherapy.

Purpose To study the relationship between Epstein-Barr virus ( EBV) and breast cancer. Methods 246 cases of breast lesions at different development stages were selected and EBV DNA, RNA and protein was used by polymerase chain reaction ( PCR) , in situ hybridization ( ISH) , laser capture microdissection ( LCM) , immunohistochemistry ( IHC) EnVision technology. Results No expression of EBV latent membrane protein LMP1 was detected in all 246 cases of benign and malignant breast lesions. In 12 cases of breast cancer of EBV DNA, carcinoma in situ and breast lesions not EBV DNA was detected by PCR. However, using digoxigenin la-beled EBV DNA probe for the 48 cases ( including 12 cases of breast cancer specimens of positive PCR amplification) of benign and malignant breast lesions, no positive hybridization signal was detected in cancer cells, mammary epithelial cells and stromal lympho-cytes. Using laser capture microdissection and PCR amplification, cancer cells and stromal cells were captured respectively from 12 ca-ses of PCR positive and 12 cases of PCR negative of breast cancer specimens, we found EBV DNA was only amplified in mesenchymal cells. In the detection of the EBER expression with EBV RNA probe and in situ hybridization, the results of 75 cases of benign and malignant breast lesions ( including 12 cases of breast cancer by positive PCR amplification) were all negative. Conclusions The re-sults indicate that the tumorigenesis and development of breast cancer have nothing to do with EBV infection in all cases were chosen.

Purpose To evaluate the tumor tropism of cytokine-induced killer ( CIK) cells, the movement track in nude mice bearing breast carcinoma and the influence on major organs of nude mice. Methods Separated and prepared CIK cells using human peripheral blood. The transwell migration assay was used to study the migratory response of CIK cells to human MDA-MB-435 breast carcinoma cells. A nude mouse xenograft model ( BALB/c) was established by injection of human MDA-MB-435 breast carcinoma cells. CIK cells labelled with DiI were injected into caudal vein of the nude mice bearing transplantation tumor. Movement track of CIK cells in vi-vo and influence on major organs were observed by living imaging technology, histopathology and immunohistopathology. Results When cultured in vitro during 14 ~20 days, CIK cells reached the peak level in proliferating stage with the maximum proportion of CD3 +CD56 + T cells. Transwell migration assay showed that the migrating number of CIK cells was increasing along with the increasing concentration of tumor cell cultural supernatants. Living imaging technology showed that the fluorescence signal began to appear 24 hours after injection of CIK cells and was strongest at 48 hours. Immunohistochemical technique and hematoxylin-eosin stain showed CIK cells tended to gather around tumor tissue 6 hours after injection, the most at 48 hours, and with some of the remaining cells on 14 day. In the meantime, no pathological damage caused by CIK cells was observed. Conclusion CIK cells have good tropism to the tumor tissue and safety to the normal tissue, and could be used as a promising cell vector for targeted therapy of cancer.

Purpose To observe the histopathologic changes of acquired immure deficiency syndrome (AIDS) in a Chinese Rhesus monkeys model and the pathogenesis that initiated the changes.Methods Chinese Rhesus monkeys were sacrificed after being inoculated SIVmac239 by Ⅳ(n=2)for four months.Autopsy was carried out by pathologic routine method.The lymph nodes, heart, liver, spleen, lung, kidney, digestive tract and other tissues were selected, the tissues fixed with 10% neutral formalin, and the pathologic sections were prepared by HE staining and immunohistochemical staining and special staining after paraffin imbedding.Results The main histopathological changes appeared in the immune system in different organs. The lymph nodes began to display the complex changes in a short period of time infected by the virus, including proliferation of lymphoid follicles, atrophy, or both; some lymphoid follicles of lymph nodes had few lymphocytes, with fibrous hyperplasia and immune complex (IC) deposition, displaying a burning down phenomenon.Splenomegaly and blood vessel and its endothelial cell proliferation in splenic corpuscles were noted with the immune complex deposition. Other parts of the mucosa-associated lymphoid tissue had different degrees of hyperplasia, or atrophy.Conclusion Histopathologic changes in Chinese Rhesus monkeys infected by SIVmac239 strain are very similar to human AIDS, which suggests that the model is a useful tool for the prevention and treatment study of AIDS.