A FEASIBILITY STUDY OF GENE GUN MEDIATED IMMUNOTHERAPY FOR RENAL CELL CARCINOMA

PURPOSE: Gene modified autologous tumor cell vaccines have demonstrated a protective and therapeutic effect in murine tumor model systems. The majority of trials to date have used viral methods of gene transfer for vaccine construction. An alternative approach to transfer genes into tumor cells is to use the gene gun, which is a physical method of gene transfection that produces high levels of gene expression without viral agents. We establish the feasibility of generating cytokine secreting autologous renal tumor cell vaccines for use in gene therapy for metastatic renal cell carcinoma. MATERIALS AND METHODS: We obtained 1 cm3 tumor tissue from 12 patients undergoing resection of primary or metastatic renal cell carcinoma. The tumor was disaggregated and placed in culture. The phenotype of the primary renal cell lines was established by microscopy and immunohistochemistry. The 1x10(7) lethally irradiated tumor cells were transfected with plasmid deoxyribonucleic acid containing the human (h) granulocyte-macrophage colony-stimulating factor (GM-CSF) gene under control of a cytomegalovirus promoter using the gene gun. The hGM-CSF production was assayed by enzyme-linked immunosorbent assay in the cell culture media 24 hours after transfection. RESULTS: Of 12 tumor samples 8 grew rapidly to produce a mean of 1.8x10(8) cells after 4 to 5 passages in culture, which was sufficient to produce between 24 and 32 vaccines. Immunocytochemistry confirmed that all cultures were almost exclusively renal tumor cells. Gene gun mediated transfection of lethally irradiated tumor cells resulted in high levels of hGM-CSF production (mean 330 ng./10(6) cells per 24 hours). CONCLUSIONS: We have demonstrated the feasibility of producing cytokine secreting tumor cell vaccines from primary and metastatic human renal tumors, and plan to use this approach in phase I clinical trials of gene therapy for metastatic renal cell carcinoma.     

CURRENT STATUS AND FUTURE PROSPECTS FOR ADJUVANT THERAPY OF MELANOMA

Advances in the treatment of melanoma have resulted mainly from improved surgical management of the primary tumour assisted by a greater appreciation of major prognostic factors in the natural history of the disease. Further improvement in the treatment of melanoma will depend largely on introduction of methods to prevent recurrence of the disease. The present review discusses criteria for selection of patients with a high risk of recurrent disease and the adjuvant treatment that has been used in past studies to prevent recurrences. With few exceptions various regimens of chemotherapy, non-specific immunotherapy with bacterial products or combinations of these treatments have not increased disease free or survival periods. Immunotherapy with various sources of melanoma antigens or with viral lysates of melanoma cells have produced encouraging results in uncontrolled studies and require further evaluation. Several advances appear to provide scope for new initiatives in immunotherapy. These include an appreciation of the role of suppressor cells in regulation of immune responses against tumour cells and possible methods to inhibit their activity. A second is the definition of various lymphokines involved in generation of immune responses (particularly interleukin 2) and development of in vitro methods for large scale production of these factors. Thirdly, methods are becoming available to define the heterogeneity of tumour cells in terms of cell surface antigens or their release of soluble factors which may help select treatments appropriate to each patient.     

携带Fas基因重组腺病毒治疗瘢痕疙瘩的体外研究

目的 应用成功制备的携带人Fas基因的两种重组腺病毒，感染瘢痕疙瘩(keroid，K)成纤维细胞(fibroblasts，FB)，使其稳定高效地表达以替换原有无功能的Fas蛋白，并恢复正常的重建后Fas信号传导通道。方法 腺病毒感染KFB后，检测及比较两种腺病毒转染前后，KFB内Fas蛋白的表达变化、蛋白功能以及细胞增殖-凋亡状况。结果 腺病毒感染后的KFB均能稳定提高Fas蛋白的表达，并且在Fas单克隆抗体的诱导下可以发生明显的细胞凋亡。结论 ①构建的两种重组腺病毒Ad-Fas在体外实验中能有效地提高KFB蛋白的表达，并可以重建原来阻断的死亡信号传导通道；②细菌内重组腺病毒体外治疗效果更佳；⑧再次估证了Fas基因突变与K之间的联系，为K基因治疗展示了一条新途径。     

转移性肝癌介入治疗的疗效及影响预后的因素

目的 探讨转移性肝癌介入治疗的疗效及影响预后的因素。方法 ３６例中,原发癌灶切除者１７例,未切除者１９例,肝动脉化疗栓塞治疗１９例,肝动脉化疗药物灌注治疗者１７例。结果 原发癌灶切除组半年生存率,较未切除者具有显著性差异。肝动脉化疗栓塞治疗组半年生存率,较肝动脉化疗药物灌注治疗组,具有高度显著性差异。结论转移性肝癌患者,应尽可能切除原发癌灶,并给全身化疗,免疫治疗及原发癌灶的选择性动脉化疗药物灌注     

肝细胞生长因子基因治疗周围动脉闭塞症研究进展

目的 综述利用肝细胞生长因子（hepatocyte growth factor，HGF）基因治疗周围动脉闭塞症（peripheral arterial disease，PAD）的实验研究和临床试验。方法 查阅近年来对大鼠、家兔、犬及糖尿病大鼠缺血肢体模型或临床患者缺血部位转染HGF质粒DNA后，评价缺血肢体血流量、血管密度、血压及缺血性溃疡等相关文献。结果 缺血肢体模型转染HGF质粒DNA后，缺血肢体血流量、血管密度及血压均有显著增加。肌肉注射携带HGF质粒DNA对荷瘤小鼠的肿瘤生长和转移无促进作用。Ⅰ／Ⅱa期临床试验表明，6例患者均未观察到与基因治疗相关的不良反应，踝部绝对血压、血压指数增高，可视模拟疼痛尺度减低，缺血性溃疡长径减小、愈合或明显改善。结论 携带HGF质粒DNA可能成为治疗PAD的一种新手段。     

腺病毒介导的基因治疗在骨创伤及骨病中的应用

目的 介绍腺病毒介导的基因治疗的特点及其在骨创伤和骨病中的应用。方法 广泛查阅有关重组腺病毒载体的制备原理、特点及应用研究进展等方面的文献，并总结腺病毒介导的基因治疗在促进骨愈合、治疗骨质疏松及类风湿性关节炎等骨病研究中的应用情况。结果 腺病毒载体具有高转染率、目的基因的高表达率及在宿主细胞内的表达有一定的时限等特点，能有效转染软骨细胞、成骨细胞、破骨细胞、骨髓基质细胞、关节滑膜细胞、椎间盘髓核细胞及成纤维细胞等，适用于骨折愈合、脊柱融合、骨质疏松及类风湿性关节炎等骨病的基因治疗。结论 腺病毒介导的基因治疗可望为骨创伤、骨病的治疗提供新的途径。     

骨形成蛋白2基因修复兔桡骨缺损的实验研究

目的观察携带人骨形成蛋白2（bone morphogenetic protein 2，BMP-2）基因的腺病毒载体（adenovirus carrying BMP-2 gene，Ad—BMP2），通过纤维蛋白凝胶与牛松质骨支架（bovine cancellous bone，BCB）复合，修复骨缺损的效果。方法将60只新西兰大耳白兔随机分为4组，每组15只。制成双侧桡骨中段1．5cm骨缺损模型，采用4种材料植入修复。A组：Ad-BMP-2＋BCB；B组：重组BMP-2＋BCB；C组：携带D-半乳糖酐酶基因的腺病毒对照载体（adenovirus carrying β—galgene，Ad—Lacz）＋BCB；D组：单纯BCB支架。修复术后各组于4、8和12周各处死动物5只取材，行X线片、组织学、生物力学和免疫组织化学染色检查。结果A、B两组骨缺损均得到了修复，但术后各时间点，A组在成骨活跃程度、新生骨量、力学强度及BMP-2表达等方面均明显优于B组；C、D两组均无新骨形成。结论BMP-2直接基冈治疗，操作简便、骨诱导能力强，是修复节段性骨缺损的有效方法。     

RHABDOMYOSARCOMA IN A PATIENT TREATED FOR METASTATIC GERM CELL TUMOUR OF THE TESTIS CONTAINING TERATOMA—A CASE REPORT

A patient who developed a rhabdomyosarcoma following apparently successful chemotherapy for metastatic germ cell testicular carcinoma is presented. This newly recognized association may be seen particularly in patients whose initial germ cell malignancy contains immature teratoma. Possible reasons for this are discussed. The findings in this patient suggest that re-biopsy of recurrent disease be undertaken wherever possible, particularly where immature teratoma was a feature of the initial histopathology. A proportion of relapsing patients as described may not in fact have recurrent germ cell malignancy, but may have developed high grade, and often chemoresistant sarcomas. These second tumours appear to have an extremely poor prognosis, unless amenable to complete surgical resection.     

PULMONARY RESECTION FOR METASTATIC BREAST CANCER

Background : A patient with a solitary pulmonary metastasis who had breast cancer in the past may benefit from pulmonary resection. Methods : Between 1984 and 1996, 17 patients underwent metastatectomy for metastatic breast cancer. There were 15 females and two males whose average age was 59 (range: 40–74 years). The median tumour-free interval after the primary breast-cancer operation was 5.1 years (range: 8 months-18.2 years). Sixteen patients had complete resections, which included six lobectomies and 10 lesser resections. Results : The postoperative mortality was nil and the morbidity rate was 6%. Follow-up was complete in all patients. Recurrent disease developed in four patients and two patients died of their disease. The 5-year survival was 62%. Conclusion : An aggressive surgical approach is warranted in patients with isolated resectable pulmonary metastases from breast cancer.     

T CELLS ACTIVATED IN VITRO AS IMMUNOTHERAPY FOR RENAL CELL CARCINOMA: CHARACTERIZATION OF 2 EFFECTOR T-CELL POPULATIONS

PURPOSE: Effector T cell populations generated using 2 methods of in vitro activation are currently being tested in separate clinical trials as immunotherapy for patients with advanced cancer, including renal cell carcinoma. To determine the most appropriate method of activation for cancer immunotherapy in vitro antitumor activity of the 2 effector T-cell populations were compared. METHODS AND METHODS: The effector T-cell populations were generated concurrently by activation of peripheral blood mononuclear cells from patients with advanced renal cell carcinoma or other cancer using soluble anti-CD3 monoclonal antibody (3T cells) or anti-CD3 and anti-CD28 monoclonal antibodies immobilized on beads (3/28T cells). After 14-day culture the phenotype and functional activity of the cells were tested. RESULTS: Fold expansion of CD4(+) cells for 3T cultures was lower than for 3/28T cultures but expansion of CD8(+) cells was similar for both cultures. Expression of CD69 was higher on 3T cells. 3T and 3/28T cells exhibited similar ability to kill various human tumor cell lines. Although both effector T-cell populations produced Th1-type cytokines upon re-stimulation, 3T cells secreted a higher level of interferon-gamma and tumor necrosis factor-alpha compared with 3/28T cells. Intracellular cytokine analysis demonstrated that the percent of cells producing interferon-gamma was higher in CD4(+), CD8(+), CD25(+), CD69(+) and CD45RO(+) 3T cells compared with 3/28T cells. CONCLUSIONS: These data suggest that 3T cells may have increased efficacy as immunotherapy for patients with cancer due to higher levels of tumoricidal cytokine production than 3/28T cells.     

Breast cancer and the immune system: opportunities and pitfalls

The identification of tumor-associated antigens, and advances in our understanding of human immunology, have resulted in renewed interest in tumor immunology. A variety of approaches have been utilized in recent years against different tumor types. The results from some of these studies have been encouraging, but it is not yet clear whether they will be applicable to patients with breast cancer.     

血管内皮细胞生长因子及有关肢体缺血基因治疗的研究进展

目的 探讨血管内皮细胞生长因子（vascular endothelial growth.VEGF）基因治疗严重慢性肢体缺血性疾病的可能性。方法 综述近年来对有关ＶＥＧＦ的分子生物学、ＶＥＧＦ的促血管生成机理及通过基因转移表达ＶＥＧＦ改善缺血肢体血供所进行的研究。结果 临床前的研究显示,ＶＥＧＦ能够刺激动物血肢体侧支动脉的形成,即所谓的“治疗性的血管生成”;临床结果证实,能过基因转移表达的ＶＥＧＦ能够促进新生血管形成,使有严重肢体     

IL—2术前皮下免疫治疗对晚期结肠癌病人免疫功能及生存期的影响

目的：观察术前IL－2皮下注射治疗对晚期实施姑息性手术的结肠癌病人免疫功能及生存期的影响。方法：共收集可供分析的50例Dukes分期为D的结肠癌病人,将其分为术前IL－2治疗组和非IL－2治疗组。术后两组病人给予相同的化疗。疗程结束后检测外周血T淋巴细胞亚群、NK细胞杀伤活性、可溶性IL－2受体（sIL-2R）,观察两组免疫功能的变化,对两组病人同时进行2年的随访,观察生存期。结果：术前IL－2治疗组外周血T淋巴细胞亚群CD3^＋、CD4^＋水平及CD4^＋／CD8^＋比值,NK细胞杀伤活性明显高于不用IL－2组;可溶性IL－2受体（sIL-2R）水平在使用IL－2组明显低于未用组。1年生存率用IL－2组也显著高于未用组。结论：术前IL－2皮下注射治疗可以提高晚期结肠癌病人的免疫功能,中和外科手术诱发的免疫抑制,延长患者的生存期.     

BACILLUS CALMETTE-GUERIN (BCG) IMMUNOTHERAPY FOR BLADDER CANCER: REVIEW OF COMPLICATIONS AND THEIR TREATMENT

Background: Intravesical bacillus Calmette–Guerin (BCG) is widely used in the management of bladder cancer but because it is a living organism, local and disseminated infection may result. Methods: A prospective assessment of complications of this therapy in 200 patients in Queensland was performed. A review of management of complications of intravesical BCG was also carried out. Results: Major side effects were rare. Cystitis was the most common side effect, being seen to some degree in all patients, although only forcing cessation of BCG therapy in two patients. Two patients developed persistent cystitis necessitating institution of isoniazid and rifampicin. Two patients had culture-proven bladder infection that presented several months after the BCG treatment. These patients also responded to two-drug antituberculous therapy. While low-grade fever is very common with this therapy, seven patients (3.5%) had fevers of > 39°C within 48 h of receiving BCG. Fevers may be an indication of severe disseminated mycobacterial infection, which has a high mortality, so it needs to be treated aggressively. Alternatively bacterial sepsis with Gram-negative bacterial pathogens or a hypersensitivity reaction to BCG may cause this degree of fever, and cannot be rapidly distinguished from fulminant mycobacterial infection. One patient in the present series developed pneumonia attributed to mycobacterial dissemination. Conclusions: The key to appropriate management of complications of BCG therapy is awareness of their possibility, even months or years after the therapy has been given. Appropriate empirical therapy in acute situations and mycobacterial culture in chronic situations can then be performed.     

METASTATIC RENAL CELL CARCINOMA WITH CONCURRENT INFERIOR VENA CAVAL INVASION: LONG-TERM SURVIVAL AFTER COMBINATION THERAPY WITH RADICAL NEPHRECTOMY, VENA CAVAL THROMBECTOMY AND POSTOPERATIVE IMMUNOTHERAPY

PURPOSE: We report our experience using aggressive multimodal therapy in a high risk group of patients with metastatic renal cell carcinoma and concurrent inferior vena caval extension. MATERIALS AND METHODS: We retrospectively reviewed the records of all patients in our kidney cancer database who had metastatic renal cell carcinoma and tumor thrombus extension into the inferior vena cava at the initial diagnosis. Patients were included in the study if they underwent radical nephrectomy and inferior venal caval thombectomy, and immunotherapy was planned for the postoperative period. Tumor size and grade, metastatic sites, level of vena caval extension, surgical complications and overall survival were obtained from the medical records. The primary end point analyzed was overall survival. RESULTS: We identified 31 cases of metastatic renal cell cancer with extensive disease and vena caval extension. Of the patients 23% had an isolated lung metastasis, and 53% had metastasis in the lung and at other sites. The remaining patients had involvement primarily at nonpulmonary metastatic sites, including lymph node in 38%, soft tissue in 13%, liver in 29% and bone in 10%. Average blood loss during nephrectomy was 3,200 cc (median 2,100) and the rate of major complications was 12%. Of the patients 80% underwent the full course of surgery and postoperative immunotherapy. At a mean followup of 18 months (34 for survivors) 26% of the patients are alive. Actuarial overall 5-year survival of the group was 17%. Tumor thrombus level did not correlate with overall survival, while immunotherapy, tumor grade and metastatic site provided significant prognostic information. In patients with an isolated pulmonary metastasis the 5-year survival rate was 43%, while in those with low grade tumors it was 52%. CONCLUSIONS: In contrast to the poor results of surgery only in patients with renal cell carcinoma and concurrent inferior venal caval invasion, reasonable 5-year survival may be achieved after combined aggressive surgery and immunotherapy. Patients in whom metastasis was limited to the lungs and those with grade 1 to 2 tumors had a better prognosis. With careful planning and experienced immunotherapists therapy may be completed in the majority of this high risk group of patients.     

RADIATION THERAPY FOR PROSTATE CANCER CONFINED TO PELVIS

To evaluate the usefulness of external beam radiotherapy for patients with prostate cancer confined to the pelvis, long-term results and prognostic factors were analyzed. During the period–1989, 44 cases were treated with staging pelvic lymphadenectomy followed by monotherapy using external beam irradiation by Linac X-ray and/or fast neutrons and observed without any treatment until relapse was evident. All patients were followed until death or for a mean of 78.6 mo (range:–113 mo) for patients still alive. Four cases died of prostate cancer at 26, 28, 54, and 83 mo from the start of radiation. Eleven cases died of other causes (10-72 mo, mean 36.4 mo). Fourteen cases (31.8%) manifested clinical relapse of cancer; 4 had local relapse, 7 developed bone metastases, and 3 relapsed at lymph nodes. After relapse, endocrine therapy was effective in most cases. The five-year disease-free survival rates of pNO (32 cases) and pN1 (8 cases) patients were 79.8% and 52.5%, respectively, but that of pN2 (4 cases) was worse. Cause-specific survival was similar between patients with pNO and pN1 disease, the rate at 5 yr being 92.5% in the former and 100% in the latter. Those with high levels of serum prostate specific antigen (PSA) before treatment and advanced local disease (clinical stage C) showed unfavorable prognoses. The number of argyrophilic nucleolar organizer regions (AgNORs) might be a predictive factor in patients treated with irradiation. In conclusion, prostate cancer patients with stage A₂-C, diagnosed as pN0-1 by staging pelvic lymphadenectomy, were successfully treated with external beam radiotherapy. The selection of candidates using prognostic factors such as N category, clinical stage, PSA, and AgNORs seems to be important for monotherapy with radiation.     

基因治疗脊髓损伤的研究现状及应用前景

基因治疗是近年来生物医学发展较快的领域，其基础研究和临床试验都表现出巨大的前景，但在中枢神经系统损伤的研究还处于实验阶段。现以脊髓损伤为代表，介绍基因治疗技术在中枢神经系统损伤中的研究现状和前景。目前，基因治疗脊髓损伤的基本策略是将神经营养因子转至一定的受体细胞后移植到损伤区，让其在体内表达并发挥生物学效应而刺激轴索再生。转基因载体以病毒载体转染率高而常用。受体细胞包括成纤维细胞、肌母细胞等。介绍了当前国际上基因治疗脊髓损伤的研究情况，并对其存在的问题和前景作了分析和展望     

重组腺病毒相关病毒作为肝癌基因治疗载体的初步研究

目的 探讨重组腺病毒相关病毒（rAAV）的制备以及作为肝癌基因治疗载体的可行性。方法 重组报告基因加强的绿色荧光蛋白（EGFP）基因至质粒载体pSub201,用双质粒共转染方法制备重组腺病毒相关病毒颗粒（rAAV/EGFP),观察其对肝癌细胞系的感染性。结果 用常规的双质粒共转染方法制备高产量的rAAV(10^7-10^8感染单位／10cm板）;当病毒感染多重性（MOI）＝100时,rAAV/EGFP对肝癌细胞HepG2的转染效率可达100％。结论 重组腺病毒相关病毒可有效地转导外源基因至肝癌细胞,其作为肝癌的基因治疗载体具有较好的研究前景。