Expression of cellular oncogenes in human malignancies.

Cellular oncogenes have been implicated in the induction of malignant transformation in some model systems in vitro and may be related to malignancies in vivo in some vertebrate species. This article describes a study of the expression of 15 cellular oncogenes in fresh human tumors from 54 patients, representing 20 different tumor types. More than one cellular oncogene was transcriptionally active in all of the tumors examined. In 14 patients it was possible to study normal and malignant tissue from the same organ. In many of these patients, the transcriptional activity of certain oncogenes was greater in the malignant than the normal tissue. The cellular fes (feline sarcoma) oncogene, not previously known to be transcribed in mammalian tissue, was found to be active in lung and hematopoietic malignancies.

Acute effects of orally administered levamisole on random monocyte motility and chemotaxis in man.

Cellular immune functions were studied in patients with early bladder cancer 2 hours after ingestion of either levamisole or a placebo. Random monocyte motility was significantly increased (P less than 0.025) in 13 of 17 patients receiving levamisole. Monocyte chemotaxis was significantly increased (P less than 0.025) in 16 of the 17 patients. Random monocyte motility and monocyte chemotaxis did not change in either 8 patients on the placebo or in 15 normal controls. Monocytes from normal donors showed increased random motility and chemotaxis after incubation with levamisole in vitro. These results indicated that increases in peripheral blood monocyte motility followed oral administration of levamisole. Kinetic studies indicated that these effects were rapid in onset and short lived.

The chemotherapy of advanced bladder carcinoma.

This paper is a summary of the clinical chemotherapy of metastatic or recurrent bladder carcinoma. Mitomycin C, doxorubicin, methotrexate, and 5-fluorouracil appear to have cytotoxic activity against bladder carcinoma when used as single agents, although the reported objective responses to each agent vary greatly. Cyclophosphamide in intermittent high i.v. doses produced an objective response in 4 of 10 patients treated by the author, and other reports suggest that this drug also may have activity against bladder cancer. cis-Dichlorodiammineplatinum is a new drug that also deserves further study.

Serum-free medium for the in vitro growth of normal and malignant urinary bladder epithelial cells.

A serum-free medium, DH-S1, is described which is valuable for the establishment of primary cultures of normal and malignant transitional epithelium (transitional cell carcinoma of the bladder). Growth of epithelial cells in DH-S1 is facilitated but that of fibroblasts is suppressed. Another established human transitional cell carcinoma cell line, 647V, has grown continually in DH-S1 for over 36 passages. Morphological and antigenic studies comparing 647V cells growing in serum-containing and serum-free medium reveal differences which are pronounced at low cell density but which almost disappear at higher densities. A new human transitional cell carcinoma cell line, LA-B1, is described whose initial growth was supported by this serum-free medium.

Identification of a positive regulatory element responsible for tissue-specific expression of prostate-specific antigen.

The prostate-specific antigen (PSA) promoter (PSA-P) has been identified, characterized, and determined to be tissue specific. Compared with high expression of the genomic PSA gene in prostate cells, expression of the transgene driven by the putative PSA promoter is low. This suggests that the identified promoter may be incomplete or may function optimally with additional regulatory elements. To identify the presence of additional regulatory elements, we screened sequences upstream of the PSA promoter and identified a DNA fragment of 822 bp, which enhances PSA gene expression. Combining the newly identified PSA gene regulatory sequence (PSAR) with our previously identified PSA promoter (PCPSA-P) exhibited enhanced expressional activity in the PSA-producing LNCaP cell line. With the addition of 10 to 100 nM dihydrotestosterone, a more than 1000-fold increase in expression was observed as compared to androgen-negative controls. Furthermore, although the combined regulatory element (PSAR)-PSA promoter (PCPSA-P) sequence resulted in high transgene expression in LNCaP cell lines, the combined regulatory element-promoter sequence resulted in minimal expression in the non-PSA-producing prostate cell line PC-3, renal tumor cell line R11, and cervical adenocarcinoma cell line HeLa. The newly identified 822 bp alone could also function as a promoter. Compared with the combined promoter, however, the 822-bp fragment alone demonstrated lower activity and lower responsiveness to androgen stimulation. Our results suggest that coupling the PSA promoter with an upstream regulatory element results in a marked increase in PSA expression, suggesting that the complete PSA promoter contains two functional domains: a proximal promoter and a distal promoter, which can also function as an enhancer. The enhanced gene expression of the new construct, combined with its tissue specificity and androgen responsiveness, in turn provides a foundation for the development of tissue-specific vectors for prostate cancer gene therapy.

Efficacy of nephron-sparing surgery for renal cell carcinoma: analysis based on the new 1997 tumor-node-metastasis staging system.

PURPOSE: To analyze the experience with nephron-sparing surgery as a treatment modality for renal cell carcinoma (RCC). PATIENTS AND METHODS: Between 1980 and 1997, 146 patients underwent partial nephrectomy at the University of California-Los Angeles Medical Center. A matched group of 125 patients who underwent radical nephrectomy at the same institution between 1986 and 1997 were selected for comparison. Patients were monitored for an average period of 57 months. Patients were staged according to both the 1997 and 1987 tumor-node-metastasis (TNM) staging criteria. Survival data were calculated in terms of both staging criteria. RESULTS: When comparing cancer-specific survival rates for patients with T1 lesions under both the 1987 and 1997 TNM staging criteria, no statistically significant difference in survival was noted (P =.53), although most of the tumors in our series measured < or = 4 cm. Patients with T2 lesions (1997 TNM) demonstrated a significant decrease in survival (66%) when compared with patients with T1 lesions (100%; P <.001). No statistically significant difference in survival for patients with T1 RCC treated with either radical or partial nephrectomy was noted (P =.219). Survival rates of partial and radical nephrectomies for patients with unilateral T1 RCC and a normal contralateral kidney also were not significantly different (P =.53). In contrast, for patients with lesions greater than T1, survival rates were significantly higher with radical versus partial nephrectomy (P =.001). CONCLUSION: Partial nephrectomy has become an effective method of treating T1 RCC lesions as categorized by both the 1987 and the revised 1997 TNM staging criteria. Selected patients with localized unilateral RCC lesions less than 7 cm (ideally, < 4 cm) and a normal contralateral kidney will benefit from partial nephrectomy.

Recombinant interferon alfa-2a in metastatic renal cell carcinoma: assessment of antitumor activity and anti-interferon antibody formation.

Twenty-one patients with advanced, measurable, renal cell carcinoma (RCC) were administered recombinant interferon alfa-2a (rIFN-alpha 2a) (Roferon-A; Roche Laboratories, Nutley, NJ) intramuscularly beginning at 3 x 10(6) units and escalating to 36 x 10(6) units, 5 d/wk for a total induction period of 14 weeks. rIFN-alpha 2a antibody production was measured using an enzyme immunoassay (EIA). Those sera found to be positive for presence of antibody by the EIA were tested for the presence of neutralizing antibodies (NA) by an antiviral neutralization bioassay (ANB). All patients were evaluable for toxicity, and 19 were evaluable for response and for incidence of antibody formation. Five patients (26%; 95% confidence interval, 6% to 46%) had complete responses (CR) or partial responses (PR) with a median duration of 283 days. An additional ten patients (53%) had minor tumor regressions with a median duration of 86 days. Fifty-one percent of evaluable patients are alive at 18.6 months. Antibodies to rIFN-alpha 2a as measured by the EIA, were detected in 12 (63%) patients. NA were measured in the serum of six (50%) of those EIA-positive patients. Overall, six of 19 patients (32%) developed NA. Median time to the development of antibody as measured by EIA or NA was 8 and 14 weeks, respectively. Median NA titer was 1,200 IFN neutralizing U/mL. NA-positive and -negative patients had a median duration of response of 13.7 v 9.9 months, and survival of greater than 21.3 v 18.3 months, respectively. Clinical toxicity was mild and not therapeutically limiting. Autoantibody production (ANA, rheumatoid factor [RF], Coombs' direct/indirect) occurred in both NA-positive and -negative patients. The clinical significance of the antibodies to rIFN-alpha 2a and the associated autoantibody formation remain unclear; however, presence of antibody was not associated with adverse clinical sequelae.

Improved prognostication of renal cell carcinoma using an integrated staging system.

PURPOSE: To integrate stage, grade, and Eastern Cooperative Oncology Group (ECOG) performance status (PS) into a clinically useful tool capable of stratifying the survival of renal cell carcinoma (RCC) patients. PATIENTS AND METHODS: The medical records of 661 patients undergoing nephrectomy at University of California Los Angeles between 1989 and 1999 were evaluated. Median age was 61 years, male-to-female ratio was 2.2:1, and median follow-up was 37 months. Survival time was the primary end point assessed. Sixty-four possible combinations of stage, grade, and ECOG PS were analyzed and collapsed into distinct groups. The internal validity of the categorized was challenged by a univariate analysis and a multivariate analysis testing for the accountability of each UCLA Integrated Staging System (UISS) category against independent variables shown to have impact on survival. RESULTS: Combining and stratifying 1997 tumor-node-metastasis stage, Fuhrman's grade and ECOG PS resulted in five survival stratification groups designated UISS, and numbered I to V. The projected 2- and 5-year survival for the UISS groups are as follows for the groups: I, 96% and 94%; II, 89% and 67%; III, 66% and 39%; IV, 42% and 23%; and V, 9% and 0%, respectively. UISS accounted for the significant variables in the variate analysis. CONCLUSION: A novel system for staging and predicting survival for RCC integrating clinical variables is offered. UISS is simple to use and is superior to stage alone in differentiating patients' survival. Our data suggests that UISS is an important prognostic tool for counseling patients with various stages of kidney cancer. Further prospective large-scale validation with external data is awaited.

Prostate tissue specificity of the prostate-specific antigen promoter isolated from a patient with prostate cancer.

We have cloned and characterized a 620-bp fragment of DNA that flanks 5' of the prostate-specific antigen (PSA) gene from a prostate cancer patient. Using DNA transfection, the efficacy of this putative promoter in regulating gene expression was quantitated in several prostate and nonprostate tissue cell lines. Our results demonstrated that the 620-dp DNA fragment actively drives gene expression in LNCaP, a PSA-producing prostate tumor cell line. No promoter activity was detected in the non-PSA-producing prostate tumor lines, DU145 and PC-3, nor in a renal (R11) or breast (MCF-7) cancer cell line. Furthermore, the promoter activity could be regulated in vitro by androgen stimulation. Dihydrotestosterone (DHT) concentrations between 3 and 30 nM induced the highest promoter activity in the transfected LNCaP cells, which parallels the expression profile of the androgen receptor in LNCaP cells. In addition, our PSA promoter exhibited competitive inhibition of the endogenous genomic PSA promoter in transfected LNCaP cells, suggesting that prostate cell-specific DNA-binding proteins are required to activate the PSA promoter. increased its potency four- to five-fold while retaining tissue specificity. Our data suggest that a strong tissue-specific negative regulatory element capable of overriding the nonspecific CMV promoter is present in the PSA promoter and confers its tissue specificity. The use of a highly specific promoter-driven gene vector will allow selective expression of therapeutic genes within PSA-producing prostate cancer cells, providing a unique strategy for prostate cancer gene therapy.

Modulation of tumor-infiltrating lymphocytes derived from human renal cell carcinoma by interleukin-4.

Current methods of expanding tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma bulk cultures result in a heterogeneous population of cells with low tumor-killing specificity. To improve the yield of cells with higher autologous and lower nonspecific cytotoxicity, interleukin-4 (IL-4) was added to high (1,000 U/ml)- and low (20 U/ml)-dose IL-2 and compared to cultures grown without IL-4 for proliferation, phenotype, and cytotoxicity against targets including autologous and allogeneic tumors. When compared to culture in IL-2 alone, the addition of IL-4 improved overall expansion in both high-dose (mean fold expansion of 2,061 vs. 1,087) and low-dose (mean fold expansion of 1,904 vs. 262) IL-2. Enhancement of TIL proliferation was dependent on the timing of IL-4 addition to the culture; augmented growth occurred only when IL-4 was added with or following activation by IL-2. The phenotype consisted primarily of CD3+/CD4+ lymphocytes with a reciprocal reduction in CD56+/CD16+ cells. Finally, there was a significant reduction in nonspecific cytotoxicity against K-562, M-14, and allogeneic tumor targets, but no significant change against autologous tumor. We conclude that IL-4 has an important regulatory effect on the expansion of renal cell carcinoma TILs in IL-2 by the promoting growth of CD3+/CD4+ lymphocytes and inhibiting the growth and nonspecific cytotoxicity associated with LAK-like CD16+/CD56+ cells. These findings may be beneficial in extracting more potent effector cells from bulk TIL culture for use in clinical trials.

Autologous tumor-specific cytotoxicity of tumor-infiltrating lymphocytes derived from human renal cell carcinoma.

Conditions for generating and expanding cytotoxic tumor-specific, tumor-infiltrating lymphocytes (TIL) were studied to improve the efficacy of adoptive cancer immunotherapy. Thus, we have examined the growth and cytolytic patterns of bulk culture TIL from human renal cell carcinoma (RCC) cultured in low (20 U/ml) or high (1,000 U/ml) dose interleukin (IL)-2, with or without irradiated autologous tumor stimulation. By 55 days in culture, TIL grown in the presence of IL-2 without tumor stimulation lost their lytic activity, whereas those exposed to tumor stimulation maintained high levels of cytotoxicity against autologous and/or nonautologous tumor targets. Only TIL grown with low dose IL-2 and autologous tumor maintained long-term (over 4 months in culture) specific cytotoxicity against the autologous tumor, even upon cryopreservation and regrowth. These TIL were 88-97% and 80% positive for CD3 and CD8, with a persistent subset exhibiting CD4+ CD8+ double positive staining. Their specific cytotoxic activity was major histocompatibility complex Class I-restricted and inhibited by pretreating the TIL with anti-CD3 monoclonal antibody. TIL exposed to the four types of culture conditions, low or high dose IL-2, with or without irradiated autologous tumors, and exhibiting different lytic specificities, all expressed mRNA for interferon-gamma and tumor necrosis factor (TNF)-alpha, but not for IL-1-beta, IL-4, IL-6, and granulocyte-macrophage colony stimulating factor. The degree of TNF-alpha mRNA expression correlated with the degree of autologous tumor-specific cytotoxicity of these TIL. This initial report demonstrates that antigen-specific cytotoxicity against the autologous tumor does, in fact, exist within the RCC tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Metastatic renal cell cancer: interleukin-2 toxicity induced by contrast agent injection.

We describe a clinical syndrome occurring after contrast medium injection in patients who previously received recombinant interleukin-2 (rIL-2) and recombinant interferon-alpha 2A treatment for metastatic renal cancer. The clinical picture mimics many aspects of rIL-2-related systemic side effects. The patients were managed conservatively but the potential for severe manifestations can occur, requiring more intensive treatment.

Pathology, biology, and clinical staging of renal cell carcinoma.

The clinical features of renal cell carcinoma may include complex systemic presenting symptoms unrelated to the urogenital tract. A particular characteristic of the tumor is the presence of widespread and unusual metastatic sites due to the high frequency of extension of the tumor into the renal vein and to subsequent hematogenous invasion. The prognosis, in general, is poor. A contributing factor is that the silent nature of the primary tumor frequently results in far-advanced disease at the time of diagnosis. The overall 10-year survival rate after nephrectomy for renal cell carcinoma is 18% to 27%. The evidence that an immune mechanism regulates tumor growth is minimal. Paraneoplastic syndromes and ectopic hormone production result in multiple-systematic symptoms and abnormal clinical chemistries. Compared with other methods of staging, the new TNM system contains a greater number of separate categories for different levels of renal vein, vena caval, and lymph node metastases. Although the system is complicated, it allows for a more accurate determination of prognosis. Computerized tomography appears to be the most effective and accurate method for making staging determinations.

Renal cell carcinoma: basic biology and current approaches to therapy.

Renal cell carcinoma (RCC) represents an unusual solid tumor for which no treatment other than surgical therapy has been effective. The remarkable heterogenous behavior of this tumor and the documented rare spontaneous regressions suggest an unusual sensitivity to host immunologic control. In recent years, exciting developments in molecular genetics, growth factors, modulators of invasion of metastases, and cytokine-lymphocyte interactions have produced new hypotheses and a wealth of information regarding the origin, behavior, and control of RCC. Interest in the immunotherapy of metastatic RCC has recently increased with the demonstrated reproducible tumor responses obtained with recombinant human interferon-alpha or interleukin-2. Durable clinical remissions in some patients with advanced RCC can now be achieved by using cytokine therapy alone or in combination with activated killer cells. This article reviews the current understanding of the basic biology of RCC, surgical approaches to localized RCC, and biologic therapy for advanced disease.

The role of interferon and interleukin-2 in the immunotherapeutic approach to renal cell carcinoma.

Approximately 24,000 cases of renal cell carcinoma are expected in the United States in 1990. Although approximately 50% of patients with local disease are cured by surgery, in patients with metastatic disease the median survival is only approximately 10 months. Neither chemotherapy nor radiation therapy has been shown to be effective against metastatic renal cell carcinoma. Immunotherapy has come to the forefront of clinical research for the treatment of metastatic renal cell carcinoma. In the past decade, the development of recombinant DNA techniques has enabled the production of large quantities of biologic response modifiers such as the interferons and interleukins. Following initial reports in 1983 by the University of California-Los Angeles (UCLA) group and the investigators at M. D. Anderson Hospital, in Houston, TX, numerous trials have demonstrated a reproducible objective response rate to interferon of 15% to 20%. These responses are independent of the interferon preparation used, and optimal dosage/schedule has not been determined. In general, responses have been correlated with the following patient factors: previous nephrectomy, good performance status, a long disease-free interval, and lung-predominant disease. Median response durations of from 8 to 10 months can be expected. The addition of vinblastine, gamma-interferon, or aspirin has not improved the therapeutic index. Interleukin-2 therapy has produced encouraging results in 10% to 15% of patients. Although high-dose therapy is associated with substantive side effects, a small cohort of patients have been in continuous remission for extended periods of time, raising the possibility of "true" complete remissions of clinical significance. Recent trials, including our trials at UCLA, have combined the interleukins and interferons in this patient population. This combination has a sound scientific basis and the results are encouraging, especially when the toxicity profile is considered. Most patients receive these combinations as outpatients and have not required hospitalization nor suffered the toxicities of the high-dosage regimens. Complete pathologic remissions have been observed using this lower dosage, outpatient schedule. Clinical trials suggest that interferon and interleukin-2 may have an expanding role in metastatic kidney cancer both as single agents and in combination outpatient biologic therapy. The future clinical trials of kidney cancer will continue to incorporate these biologic response modifiers into the therapeutic strategies of the 1990s.

The in vitro effects of interleukin-12 upon tumor-infiltrating lymphocytes derived from renal cell carcinoma.

Clinical trials utilising interleukin (IL)-2 with tumor-infiltrating lymphocytes (TIL) have demonstrated efficacy in the treatment of metastatic renal cell carcinoma (RCC). Several cytokines, as well as growth factors have demonstrated modulatory effects upon the biological properties of TIL from RCC, suggesting a potentially important role for cytokines other than IL-2 in the development of active and tumor-specific TIL. IL-12 was recently characterized as a natural-killer-cell-stimulatory factor or cytotoxic-T-cell-maturation factor. These properties of IL-12 prompted us to investigate the impact of this cytokine upon the activation of TIL from human RCC. In an attempt to enhance the in vitro growth and activity of renal TIL, we have grown eight renal TIL cultures in varying concentrations of IL-2 (8, 40, 80, 400 U/ml) and IL-12 (200 U/ml). In addition, IL-12 (200 U/ml) was added to TIL cultures pre-activated with IL-2 (400 U/ml). Growth, cell expansion, and the ability of TIL to release certain cytokines upon tumor stimulation were determined. Proliferation assays, phenotypic analysis, and cytotoxicity assays were performed at an early and a late culture stage. IL-12, alone and when added to suboptimal concentrations of IL-2, failed to induce TIL growth. While the addition of IL-12 to optimal doses of IL-2 suppressed TIL culture expansion, sequential culture exposure first to IL-2 and then to IL-2+IL-12 increased the number of cells expressing CD3+/CD56+ and these cultures demonstrated enhanced in vitro lysis of autologous tumor. IL-12 clearly demonstrated a sequence-dependent impact of the biological behaviour of TIL from RCC. The optimal use of IL-12 in the in vitro expansion of renal TIL may result in cells with an enhanced specific anti-tumor effect.

Cell surface blood group antigens in prostatic carcinoma.

Surface blood group antigens are present to some degree in most epithelia. These antigens frequently are lost during neoplastic transformation. The authors looked for the presence or absence of surface blood group antigens in 52 cases of prostatic carcinoma of various histologic grades using the specific red blood cell adherence test. The normal prostatic tissue showed a 2+ reaction in patients with type A or B blood and 0-1+ in type O. The hyperplastic areas were 4+ for red blood cell adherence in patients with type A or B blood and 0-4+ in type O. In contrast, all malignant foci were negative for blood group antigens, no matter what the histologic grade or blood type.

Treatment of skeletal metastases from urologic malignancies.

Metastases from genitourinary tumors frequently involve the skeletal system and often produce the most disabling symptoms. Judicious early orthopedic management may prevent paralysis and maintain ambulation, thus significantly improving the quality of life. The role of prophylactic surgery for impending fractures and the symptomatic therapy of painful metastatic foci are discussed.

Pitfalls in preoperative staging in prostate cancer.

Clinical staging in 60 patients with adenocarcinoma of the prostate was compared with pathologic staging to identify factors which may contribute to staging errors. Understaging was directly related to tumor stage and was documented in 0 per cent of A2, 26.5 per cent of B1, and 66.7 per cent of B2 patients. Capsular invasion was found in 11.8 per cent of B1 and 52.4 per cent of B2 patients, seminal vesicle extension in 17.7 per cent of B1 and 52.4 per cent of B2 patients, and lymph node metastases in 2.9 per cent of B1 and 28.6 per cent of B2 patients. The majority of patients who had unnoticed gross extension of the tumor beyond the prostate underwent transurethral resection of the prostate or failed irradiation therapy prior to radical prostatectomy. The results suggest that intraprostatic or periprostatic changes caused by previous treatment to the prostate may interfere with the preoperative staging.

Salvage radical prostatectomy after failure of curative radiotherapy for adenocarcinoma of prostate.

Radical prostatectomy with curative intent was performed in 13 patients with prostate cancer after local failure of radiotherapy. Of these patients, 2 underwent cystoprostatectomy for bladder neck involvement by the prostatic tumor. Local recurrence had been diagnosed twenty-one to one hundred sixty-eight months (mean 65.4 months) after completion of radiotherapy (6,000-7,000 cGy; mean 6,136 cGy). Three patients had radioactive implants. Rising prostate-specific antigen (PSA) was part of the indication for surgery in 5 patients. Complications included minor rectal injury (1 patient) and total incontinence (2/13 patients). Two patients had positive surgical margins and 6/13 patients had involvement of seminal vesicles, 2 of whom also had positive lymph nodes. The authors conclude that salvage prostatectomy is feasible after radiation failure. Transrectal ultrasound and careful monitoring of PSA after irradiation treatment may improve patient selection and minimize the risk of complications and incomplete excision.