Keyhole limpet hemocyanine can enhance the natural killer activity of patients with transitional cell carcinoma of the bladder

It has been shown that natural killer (NK) cells are involved in the immunosurveillance of tumors. In patients with transitional cell carcinoma (TCC) of the bladder, there is a negative correlation between the levels of NK activity in peripheral blood mononuclear cells (PBMNCs) and both the clinical evolution and the pathological stages of the disease. We have investigated the immunoregulatory effect of keyhole limpet hemocyanin (KLH) on the NK activity of patients with TCC of the bladder. We found that KLH enhances the cytotoxic activity of PBMNC from patients with superficial TCC against NK-sensitive target cells in a time-dependent manner. This KLH-enhanced cytotoxicity is not directed against NK-resistant target cells. However, KLH fails to normalize the depressed NK activity of PBMNCs from patients with infiltrative TCC. The present in vitro studies suggest that this immunoregulatory effect of KLH on NK cytotoxicity may be implicated in its therapeutic effect on superficial TCC of the bladder.     

Effect of cardiopulmonary bypass on circulating lymphocyte function.

Extracorporeal cardiopulmonary bypass (CPB) has been associated with a wide variety of immunological derangements, including a transient postoperative impairment of lymphocyte function. We examined changes in phenotypic and nonspecific cytotoxicity of peripheral blood mononuclear cells after extracorporeal CPB. The peripheral blood samples obtained from 10 patients were subjected to natural killer and cytotoxic T lymphocyte activity assay before and at intervals after CPB. Phenotypic analysis of peripheral blood lymphocytes was performed in 5 patients before and immediately after CPB. We observed a significant increase in peripheral blood CD8+ cells (cytotoxic/suppressor T lymphocytes) (16.1% +/- 2.5% versus 22.5% +/- 2.1%; p less than .005) and a decrease in CD4+ cells (helper/inducer T lymphocytes) (46.1% +/- 3.5% versus 36.1% +/- 3.5%; p less than 0.02) immediately after extracorporeal circulation. The CD8/CD4 ratio in peripheral blood was significantly increased immediately after bypass (0.53 versus 0.80; p less than 0.001). No significant changes in percentages of other leukocyte subsets in peripheral blood were noted. The activity of cytotoxic T lymphocytes and natural killer cells in peripheral blood was impaired on postoperative days 1 and 3 but was restored to preoperative values by removal of mononuclear phagocytes from these cells. The decrease in natural killer cell and cytotoxic T lymphocyte activity in peripheral blood may signify a temporary impairment of the effector arm of the cell-mediated immunity in the post-operative period. The observed changes in peripheral blood phenotype and function may be involved in early organ injury and infectious complications after CPB.     

Increased expression of activation markers in renal cell carcinoma infiltrating lymphocytes

PURPOSE: As manifested by the presence of immune competent cells, failure to control the progression of renal cell carcinoma by a local immune response attests to impaired local cell mediated immunity. To test this hypothesis we compared the expression of T-cell activation markers in renal cell carcinoma infiltrating lymphocytes with the expression of activation markers of peripheral blood lymphocytes in the same patients. MATERIALS AND METHODS: Tumor infiltrating lymphocytes were harvested from a patient with renal cell carcinoma undergoing radical nephrectomy. Peripheral blood was obtained before surgery. Tumor infiltrating and peripheral blood lymphocytes were incubated with monoclonal antibodies defining specific differentiation and activation markers on the cell surface, and analyzed by flow cytometry. Cell subsets are expressed as a fraction of the total number of mononuclear cells. RESULTS: The T-cell subset level was significantly higher in peripheral blood than in renal cell carcinoma tissue of the same patient. However, the level of activated T-cell subset expressing HLA-DR was significantly higher in renal cell carcinoma tissue than in peripheral blood. The levels of interleukin-2 receptor and transferrin receptors expressing T-cell subsets were also significantly higher in carcinoma tissue than in peripheral blood. Natural killer cells were found in significantly higher proportions in renal cell carcinoma than in peripheral blood. CONCLUSIONS: These results point to significant activation of T, B and natural killer tumor infiltrating lymphocytes. The inability of tumor infiltrating lymphocytes to mount an effective immune response to renal cell carcinoma may be secondary to the presence of suppressive factors in the tumor that prevent tumor infiltrating lymphocytes from transforming into effector cells. These factors may be particularly valuable for the further study of renal cell carcinoma-host interactivity.     

The prognostic value of peripheral blood lymphocyte subsets in patients with bladder carcinoma treated using neoadjuvant M-VEC chemotherapy

OBJECTIVES: To assess the prognostic value of peripheral blood lymphocyte subsets in patients with bladder cancer who were treated with neoadjuvant chemotherapy. PATIENTS, SUBJECTS AND METHODS: Thirty patients with a histological diagnosis of invasive bladder transitional cell carcinoma and 30 age-matched controls with no evidence of cancer and immunological disorders were evaluated. Peripheral blood samples were assessed in both groups using monoclonal antibodies. Patients with bladder cancer who achieved complete or partial responses and those who had progression of the disease after systemic chemotherapy with methotrexate, vinblastine, epirubicin and cisplatin were compared according to the pretreatment values of the peripheral blood lymphocyte subsets. RESULTS: There were no significant differences in B lymphocyte levels between the groups. In patients with bladder cancer, the percentages of T lymphocytes (P<0.01), natural killer (NK) cells (P<0.05) and the CD4+/CD8+ ratio (P<0.05) were significantly lower than in the control group. In patients who responded to the chemotherapy regimen, the pretreatment values of T lymphocytes (P<0.001), the CD4+/CD8+ ratio (P<0.01) and NK cell levels (P<0.01) were significantly higher than in the patients who did not. CONCLUSION: In patients with invasive bladder carcinoma, cell-mediated immunity may have a role in the resistance to this malignancy and in these patients the pretreatment levels of T lymphocyte subsets may be an indicator of the potential response to chemotherapy.     

An investigation of factors influencing the in vitro induction of LAK activity against a variety of human bladder cancer cell lines.

We investigated the sensitivity of transitional cell carcinoma cells, derived from the human bladder, to lymphokine activated killer cells. Recombinant interleukin-2 activated peripheral blood mononuclear cells were studied for their ability to mediate the cytolysis of a panel of four established human bladder transitional cell carcinoma cell lines. Lymphokine activated killer activity was assessed using a standard four hour chromium release assay. All four bladder cancer cell lines proved to be susceptible to lymphokine activated killer mediated cytolysis. This was found to be dependent upon the dose of cytokine and upon the duration of the activation period. The four cell lines were differentially susceptible to lysis (specific cytotoxicity at effector to target ratio of 40:1; RT112 = 22.9%, RT4 = 49.2%, MGH-U1 = 49.1%, EJ18 = 62.3%). The varying susceptibility of lymphokine activated killer mediated cytotoxicity was found to be independent of the histological grade of the parent tumour or the donor of effector cells. Both interferon-alpha and tumour necrosis factor-alpha also elicited lymphokine activated killer cell activity, although the maximum specific cytotoxicity achieved was considerably lower than that obtained with interleukin-2 alone. Interleukin-2, at optimal concentration, and tumour necrosis factor-alpha were found to behave synergistically in the generation of lymphokine activated killer effectors. However, concentrations of tumour necrosis factor-alpha higher than 100 Uml.-1 resulted in a decrease in specific cytotoxicity. These findings suggest a possible use of adoptive immunotherapy in human bladder cancer and indicate the optimum conditions for the generation of such effector cells.     

Killer activities of peripheral blood mononuclear cells in patients with bladder cancer

Killer activity of peripheral blood mononuclear cells with or without adding interleukin-2 in vitro was measured in 12 patients with superficial bladder cancer, 12 patients with invasive bladder cancer and 13 adult healthy controls. Cultured cell lines, K562, Raji and T-24 (bladder carcinoma), were served as target cells. Killer activity was measured by a 4-hour 51Cr-release assay. Mononuclear cells from patients with superficial bladder cancer had a significantly higher natural killer activity against K562 and T-24 (50.8 +/- 6.2% and 15.0 +/- 9.5%, respectively) than those from patients with invasive bladder cancer (28.2 +/- 8.0%, 9.5 +/- 8.8%) and than those from controls (33.0 +/- 8.9%, 3.0 +/- 2.5%). When cultured in vitro with recombinant interleukin-2, mononuclear cells from patients with superficial bladder cancer developed a significantly higher killer activity against K562, Raji and T-24 (58.4 +/- 5.8%, 40.1 +/- 15.9% and 49.5 +/- 10.5%, respectively) than those from patients with invasive bladder cancer (48.1 +/- 5.9%, 27.9 +/- 13.8% and 40.1 +/- 7.4%) and than those from controls (48.9 +/- 6.7%, 30.3 +/- 10.5% and 39.5 +/- 3.8%). Flow cytometric analysis showed that there was no significant difference in surface markers between mononuclear cells from patients with superficial bladder cancer and those from patients with invasive bladder cancer. These results suggest that tumor immunity may participate in development and progression of bladder cancer.     

BCG induced killer cell activity

Summary To investigate the mechanism of Bacillus de Calmette Guérin (BCG) bladder instillation therapy, the killer cell activity induced in peripheral blood mononuclear cells (PBMNCs) after BCG instillation was examined. Significant cytotoxic activity against natural killer (NK) cell resistant target tumor cells was detected after 3 days of instillation. To characterize this BCG induced cytotoxic activity further, human PBMNCs were cultured with BCG in vitro. From 24 h maximum cytotoxicity was obtained and continued for 3 days, then decreased slightly. Neither a DNA synthesis inhibitor Cytosine-arabinoside (Ara-C) nor a cytotoxic T cell (CTL) generation inhibitor Cyclosporine A inhibited this killer cell activation. Monoclonal antibody treatment revealed that both precursor and effector cells are Leu1^-, 3a^-, 7⁺, 11b⁺. The recognition specificity from cold target competition experiments was selective. Taken together NK type precursor was activated with BCG into NK type effector which has wider spectrum of target cells than usual NK cell.     

Natural killer cell activity in patients with urologic cancer

Cell-mediated cytotoxicity in patients with urologic cancer was studied using the K562 cell line as target cell by a 4-hour chromium-51 release assay. Lysis of target cells by mononuclear cells of a healthy subject, over an 8-hour incubation period, demonstrated a linear function of incubation time and effector:target ratio. Natural killer (NK) cell activity was found decreased (mean 30.6%) in peripheral blood lymphocytes from 42 untreated patients with urologic cancer when compared to 20 healthy subjects (63.6%) and to 10 patients with varicocele, stone disease and benign prostatic hypertrophy (58.1%; p less than 0.05). There was no correlation between NK cell activity and the grade or stage status in bladder cancer patients. No age-dependent changes in NK cell activity could be found between young and aged groups of healthy subjects. Healthy male subjects have higher levels of NK cell activity (77.7%) than healthy female subjects (49.5%). Postoperative NK activity rose in 5 out of 6 cancer patients. It indicates that tumors may have an inhibitory effect on the surveillance activity of NK cells.     

Identification and expansion of human lymphokine-activated killer cells: implications for the immunotherapy of cancer

Immunotherapy with lymphokine-activated killer (LAK) cells and interleukin 2 (IL-2) has been demonstrated to cause the regression of some human tumors. Expansion of peripheral blood lymphocytes (PBL) in recombinant or naturally produced IL-2, mitogens, and feeder cells results in a progressive expansion of cell number but a decline in lytic activity with time. Lytic activity for fresh tumor is best generated in IL-2 alone from specific purified subpopulations of lymphocytes. The precursor of the LAK cell has been isolated from a nonadherent, sheep erythrocyte receptor negative (E-) lymphocyte subpopulation by the selection of Leu 11+ and Leu 15+ cells by cell sorting. Separation resulted in a significant increase (P2 less than 0.05) in lytic activity against fresh tumor by the separated 11+15+ lymphocytes compared to the 11-/15- lymphocytes or PBL. Leu 11+/15+ lymphocytes could be expanded up to eightfold in IL-2 alone with maintenance of lytic activity. We identified two surface antigens on the LAK precursor cell, Leu 15 and Leu 11, and demonstrated a technique for obtaining purified populations of these cells. Purified and expanded LAK cells when administered to patients may have a role in increasing the potency and decreasing the toxicity associated with this therapy.     

Cyclooxygenase-2 is highly expressed in carcinoma in situ and T1 transitional cell carcinoma of the bladder

PURPOSE: We describe cyclooxygenase-2 (COX-2) expression patterns in patients with carcinoma in situ and/or stage T1 transitional cell carcinoma. We determined whether expression is associated with clinical outcome in these patients. MATERIALS AND METHODS: Immunostaining for COX-2 was performed on paraffin embedded bladder biopsy specimens from 2 independent groups of patients without muscle invasive carcinoma, including 39 with carcinoma in situ and 34 with stage T1 tumors. Immunoreactivity was scored as the percent of carcinoma in situ cells with cytoplasmic staining for COX-2 in the carcinoma in situ group and as the percent of stage T1 cells expressing COX-2 in the stage T1 transitional cell carcinoma group. We evaluated other molecular alterations, including E-cadherin, p21 and p53, because evidence suggests a biological association of COX-2 with alterations in these molecular markers. RESULTS: In the carcinoma in situ group 5 patients (13%) had no immunoreactivity, while 2 (5%), 5 (13%) and 27 (69%) had 10%, 20% and 30% or greater carcinoma in situ cells positive for COX-2, respectively. In the transitional cell carcinoma group 1 (3%), 4 (12%) and 29 (85%) patients had 10%, 20% and 30% or greater positive cells, respectively. COX-2 expression was not associated with any clinical or pathological parameters, or with molecular markers regardless of the cutoff used. It was also not associated with clinical outcomes in the stage T1 transitional cell carcinoma group. In the carcinoma in situ group COX-2 expression was significantly associated with disease recurrence using cutoffs of 0% and greater than 10% positive cells, and with disease progression using a greater than 20% cutoff. However, it was not associated with bladder cancer related survival. CONCLUSIONS: COX-2 is expressed in a high percent of patients with carcinoma in situ and stage T1 transitional cell carcinoma, supporting the rationale for chemoprevention studies with selective COX-2. We could not substantiate a role for COX-2 immunohistochemistry for the staging and prognosis of carcinoma in situ and/or stage T1 transitional cell carcinoma.     

SUPERFICIAL BLADDER TUMORS AND INCREASED REACTIVITY AGAINST MYCOBACTERIAL ANTIGENS BEFORE BACILLUS CALMETTE-GUERIN THERAPY

Purpose

The precise mechanism of action of bacillus Calmette-Guerin (BCG) in bladder cancer treatment remains poorly understood. Whether bladder tumor cells are destroyed by nonspecific mechanisms or targeted by specifically activated lymphocytes recognizing cognate antigens is unclear. To investigate a possible cross-reactivity between BCG and bladder cell tumors, we tested before BCG treatment the lymphoproliferation of peripheral blood lymphocytes against several mycobacterial antigens, including the secreted fibronectin binding antigen 85 complex from BCG (AG 85) in patients with superficial bladder tumors compared to control matched patients.

Materials and Methods

Using a whole blood assay, T cell response against purified protein derivative, BCG extract, whole BCG, purified AG 85, and the nonspecific mitogens pokeweed and phytohemagglutinin was investigated in 79 patients with superficial bladder tumors before BCG and in 39 control subjects without malignancy matched for age and sex. Neither group had a history of tuberculosis. Lymphoproliferation was measured with a tritiated thymidine uptake assay on day 7 of culture.

Results

Of the 79 patients with superficial transitional cell carcinoma, a significant lymphoproliferative response before BCG against PPD, BCG extract, whole BCG and AG 85 was observed in 65 (82.2%), 67 (84.81%), 30 (37.97%) and 49 (62.02%) patients, respectively. Of the 39 controls only 26 (64.1%), 23 (58.9%), 3 (7.7%) and 3 (7.7%) patients, respectively, had a significant lymphoproliferation against PPD, BCG extract, BCG and AG 85 (p >0.05, p = 0.004, p = 0.00001 and p = 0.00001, respectively). In terms of lymphoproliferative levels, patients with superficial transitional cell carcinoma also showed a significantly higher response against PPD (p = 0.000012), BCG extract (p = 0.000001), AG 85 (p = 0.000001), whole BCG (p = 0.00001) and pokeweed (p = 0.01) than controls but not against phytohemagglutinin.

Conclusions

Patients with superficial transitional cell carcinoma demonstrate an increased lymphoproliferation against mycobacterial antigens before BCG compared to control subjects. Although a nonspecific activation of the immune system cannot be excluded at this stage, our data may suggest the possible existence of bladder cancer antigens cross-reactive with mycobacterial antigens responsible for boosting precursor cells witnessing previous contacts with mycobacteria. The implication of these findings in the antitumoral mechanism of action of BCG are under investigation.     

Cell-mediated cytotoxic activity of spleen cells from patients with gastric carcinoma

The cell-mediated cytotoxic activities of cells from the spleens (SP cells) of patients with gastric carcinoma were assayed in comparison with the activities of peripheral blood mononuclear cells (PBM cells) from the same patients, and from patients with benign lesions. The natural killer cell (NK) activity of the SP cells and their capacity to generate allogeneic cytotoxicity in mixed lymphocyte culture (MLC) were very similar to those of the PBM cells. The cytotoxic activity of SP cells induced by alloactivation in MLC, however, was significantly higher than that of the PBM cells from the same patient as well as from patients with benign lesions. The production of interleukin 2 (IL 2) and the ability to induce cytotoxic cells after activation with IL 2 (LAK) were therefore examined. Both the ability to produce IL 2 and to generate LAK cells were shown to be significantly increased in SP cells when compared to PBM cells. These results indicate that the spleen may be a potential reservoir for the precursors of these activated killer cells in patients with gastric carcinoma. Furthermore, it may play an important role in the defence against tumors in these patients.     

Natural killer (NK) activity of peripheral blood and regional lymph nodes in patients with genitourinary malignant tumors. 2. NK activity and T-lymphocyte subsets in peripheral blood and regional lymph nodes

We investigated natural killer (NK) activity and T-lymphocyte subsets in peripheral blood and regional lymph nodes in order to examine the host's defence mechanisms against cancer. The materials were obtained from 26 patients with genitourinary cancer and 9 patients with benign diseases, who were used as controls. NK activity was measured by ATP-chemiluminescence (ATP) assay, a new method which is well correlated to the 51Cr-release assay, and T-lymphocyte subsets were stained with anti-Leu-2, -3, -4, -7, and -11 monoclonal antibody. The NK activity of regional lymph nodes was lower than that of the peripheral blood in all 35 cases, and the percentage of Leu 7+ and Leu 11+ cells was in agreement with that of the NK activity detected by ATP assay. In the peripheral blood, the percentage of Leu 3+ cells was lower in high stage patients than it was in low stage patients and controls, but in the regional lymph nodes it was paradoxically higher. The NK activity of regional lymph nodes against autologous tumor cells was low in 8 patients with bladder cancer. It appears from this study that the regional lymph nodes have no function as immunological barriers for metastasis and we therefore conclude that they should be dissected during surgery.     

Immunoresponse of tissue infiltrating lymphocytes in bladder tumors

Local immunocompetence was evaluated immunohistochemically in patients with bladder tumors before and after local injections of an immunomodulator. The subpopulations of tissue infiltrating lymphocytes (TIL) were examined by staining six serial sections with Leu4, Leu7, Leu10, LeuM3, OKT4, and OKT8 antibodies. T cells predominated over B cells in 19 of 25 bladder tumors. T cell infiltration was prominent around tumor cells, and it was marked in non-invasive tumors. B cells were rare in the stroma. In patients with low-stage tumors, OKT8 cells were more prominent than OKT4 cells. NK cells accumulated within cancer nests but their infiltration was scanty in invasive bladder tumors. Before surgery, immunomodulators (OK-432, IL-2) were injected intratumorally. Their administration resulted in marked increase of T and NK cells, irrespective of the stage of disease; there was a slight increase in B cells. These findings suggest that local immunosurveillance plays a role against bladder tumors. Further studies are required to elucidate host immune responses in the microenvironment of the cancer site, as well as the systemic immune reaction.     

Cell-mediated cytotoxic activity of spleen cells from patients with gastric carcinoma

The cell-mediated cytotoxic activities of cells from the spleens (SP cells) of patients with gastric carcinoma were assayed in comparison with the activities of peripheral blood mononuclear cells (PBM cells) from the same patients, and from patients with benign lesions. The natural killer cell (NK) activity of the SP cells and their capacity to generate allogeneic cytotoxicity in mixed lymphocyte culture (MLC) were very similar to those of the PBM cells. The cytotoxic activity of SP cells induced by alloactivation in MLC, however, was significantly higher than that of the PBM cells from the same patient as well as from patients with benign lesions. The production of interleukin 2 (IL 2) and the ability to induce cytotoxic cells after activation with IL 2 (LAK) were therefore examined. Both the ability to produce IL 2 and to generate LAK cells were shown to be significantly increased in SP cells when compared to PBM cells. These results indicate that the spleen may be a potential reservoir for the precursors of these activated killer cells in patients with gastric carcinoma. Furthermore, it may play an important role in the defence against tumors in these patients.     

Prognostic implications of p53 gene mutations in bladder tumors

PURPOSE: Alterations in the p53 gene related to neoplastic progression were studied in tumor tissue samples from patients with transitional cell carcinoma and correlated with classic staging parameters. On this basis, biological characterization of the tumor was performed to establish subgroups of patients at high risk and those with a more favorable prognosis. MATERIALS AND METHODS: This observational, analytical and cross-sectional study included 115 patients divided into 4 homogeneous groups of 1-control, 2-primary superficial transitional cell carcinoma, 3-recurrent superficial transitional cell carcinoma, and 4-infiltrative transitional cell carcinoma. DNA was obtained from tumor tissue samples and polymerase chain reaction-single strand conformational polymorphism analysis was performed on exons 5 to 9 of the p53 gene. Samples showing mutations were submitted to automatic sequencing. Statistics included bivariate analysis and logistic regression. RESULTS: Of the tumors the 63.8% were superficial and 37.2% were infiltrative transitional cell carcinoma. Of the infiltrative tumors 23.5% (8 of 34) resulted from recurrent transitional cell carcinoma. Mutations were found in samples from 46.8% of patients, all with bladder tumors. There was a trend toward increasing appearance of mutations as the size of the tumor, number of tumor implants, degree of dedifferentiation and stage of local infiltration increased. The presence of mutations in p53 was 2.5 times greater in infiltrative tumors than in low stage and 4.3 times greater in moderate to high grade than in low grade tumors. All mutations found were point mutations and 79.25% provoked severe alterations in protein structure. CONCLUSIONS: Mutations in the p53 gene are mainly point mutations that aggregate in hot spots, and provoke genetic instability and substantial changes that alter p53 function, implying a trend to tumor progression and dissemination (with a greater proportion of mutations in high stage high grade tumors). Since a large percentage of bladder tumors are under staged, analysis of p53 gene mutations could be useful as a factor for prognosis and therapeutic decisions.     

Derivation of cells with lytic activity against fresh and cultured tumors from human bone marrow

We studied the derivation of cells with lymphokine-activated killer (LAK) cell activity from human bone marrow cultures. In these experiments, 26/26 specimens of human bone marrow obtained from sternum, iliac crest, rib, and humerus cultured with IL-2 (1000 U/cc) under varying conditions were able to generate LAK activity. LAK activity obtained from bone marrow mononuclear cell cultures (BMMC) was observed only with exposure to IL-2. Peak LAK activity against fresh melanomas and renal cell carcinoma and sarcomas was obtained usually by the second or third week in culture and could be maintained in some instances for greater than 28 days. Parallel experiments comparing autologous peripheral blood lymphocytes (PBL) cultured under identical conditions and tested simultaneously with BMMC demonstrated comparable LAK activity. T-cell depletion using sheep red blood cell rosetting and density separation on discontinuous gradients did not significantly enrich for a population with enhanced LAK activity. The use of irradiated Epstein-Barr virus-B-cell feeder lines significantly enhanced generation of LAK activity from BMMC when compared to controls (P less than 0.006). Conditioned media from PHA-stimulated 2-day and 4-day PBL cultures, 4-day mixed lymphocyte cultures, and 3-day LAK cultures did not consistently increase the growth or lytic activity of BMMC cultures grown in the presence of IL-2 (1000 U/cc). Phenotypic analysis of BMMC cultures after culture with IL-2 with lytic activity revealed mixed populations with mature T cells (CD3) and null cells (CD2+/CD4- and Leu 19+/Leu 7+) with decreased myeloid precursors (My 9+).     

Initial tumor stage and grade as a predictive factor for recurrence in patients with stage T1 grade 3 bladder cancer

PURPOSE: We evaluated whether the risk of progression and the recurrence rate were different in patients with primary and nonprimary stage T1 grade 3 transitional cell carcinoma of the bladder. MATERIALS AND METHODS: Between 1983 and 1997, 75 patients were treated for stage T1 grade 3 transitional cell carcinoma of the bladder. Of these patients 68 (primary and nonprimary tumor in 58 and 14, respectively) without carcinoma in situ who had not undergone complete cystectomy immediately after diagnosis were included in the study. No maintenance regimen was used. Median followup was 100 months (range 9 to 217). RESULTS: The incidence of multiple tumors in patients with nonprimary tumors was significantly higher than in patients with primary disease (p = 0.035). However, the recurrence-free survival rate in patients with primary T1 GIII bladder tumor was significantly lower than that of patients with nonprimary T1 GIII bladder tumor (p = 0.0016). Multivariate analysis using Cox's proportional hazard regression model revealed that only initial tumor status had statistically significant effects on tumor recurrence (p = 0.007) and no other factors had a significant influence on recurrence-free survival. Progression-free and cancer specific survival rates were also significantly different between the 2 groups (p = 0.036 and 0.0307, respectively). CONCLUSIONS: Our study indicates that patients with primary stage T1 grade 3 bladder cancers have higher recurrence and progression potential than those with nonprimary disease despite the higher incidence of multiple tumors in patients with nonprimary tumors.     

膀胱癌中bcl-2和p16基因的表达与预后的关系

目的 探讨ｂｃｌ－２和Ｐ１６基因蛋白在膀胱癌的表达与预后的关系。方法 采用ＳＡＢＣ法对５１例膀胱癌组织和５例正常膀胱粘膜行ｂｃｌ－２和Ｐ１６基因表达的检测。结果 ｂｃｌ－２在膀胱癌的阳性表达率为８０．２％,生存组和死亡组之间比较差异有显著性意义。Ｐ１６在膀胱癌在阳生表达率为５０．９％。５例正常膀胱粘膜均阳性,两者比较有显著性意义。在病理分级、临床分期和预后的总样本率中比较差异有显著性意义;即随病理     

In situ characterization, clonogenic potential, and antitumor cytolytic activity of T lymphocytes infiltrating human brain cancers

Mononuclear cells infiltrating human brain tumors were isolated from seven of nine surgical biopsy specimens. These cells were small T11+, T3+ lymphocytes that did not express DR antigens or the receptor for interleukin-2. In addition, large granular lymphocytes were recovered from two of these tumors. The clonogenic potential of tumor-infiltrating lymphocytes (TIL's) was assessed by limiting-dilution analysis (LDA) using a microculture system that permits proliferation of virtually 100% of normal peripheral blood T lymphocytes (PBL-T's). In comparison to normal and autologous PBL-T's, TIL's had a strikingly reduced proliferative potential revealed by a decrease in the frequency of proliferating T lymphocyte precursors calculated by LDA. On average, only one of every 100 T cells from TIL's was able to proliferate, as compared to one of every two or all of the T cells from the patient's peripheral blood or from normal donors. Furthermore, the TIL populations showed depressed proliferative responses to the lectins phytohemagglutinin and concanavalin A and to the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate. Clonal analysis performed on the proliferating microcultures from three tumors demonstrated that the majority of these clones possessed cytolytic activity against various tumor cell targets. Among clones tested for cytolytic activities with glioma cells, four lysed cultured autologous tumor cells, and the specific lysis was greater than 50% in all cases. Numerous clones with natural killer (NK)-like activity were obtained from two TIL preparations, and the frequency of cytolytic T lymphocyte precursors with NK-like activity was determined for one of these preparations and was found to be higher than that in the patient's peripheral blood. Glioma cells grown in culture and then mixed with normal peripheral blood lymphocytes (PBL's) were capable of inhibiting the PBL's response to lectins. This inhibitory property may account in part for the observed poor clonogenicity of TIL's from brain tumors. Nevertheless, nearly all proliferating clones displayed cytotoxicity against either autologous or allogeneic tumor cell targets and may imply selective accumulation of cytolytic effector cells at the tumor site.