Johannes Vieweg

Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells

In this study, we investigated whether elimination of CD4+/CD25+ Tregs using the recombinant IL-2 diphtheria toxin conjugate DAB(389)IL-2 (also known as denileukin diftitox and ONTAK) is capable of enhancing the immunostimulatory efficacy of tumor RNA-transfected DC vaccines. We show that DAB(389)IL-2 is capable of selectively eliminating CD25-expressing Tregs from the PBMCs of cancer patients without inducing toxicity on other cellular subsets with intermediate or low expression of CD25. DAB(389)IL-2-mediated Treg depletion resulted in enhanced stimulation of proliferative and cytotoxic T cell responses in vitro but only when DAB(389)IL-2 was omitted during T cell priming. DAB(389)IL-2 significantly reduced the number of Tregs present in the peripheral blood of metastatic renal cell carcinoma (RCC) patients and abrogated Treg-mediated immunosuppressive activity in vivo. Moreover, DAB(389)IL-2-mediated elimination of Tregs followed by vaccination with RNA-transfected DCs significantly improved the stimulation of tumor-specific T cell responses in RCC patients when compared with vaccination alone. Our findings may have implications in the design of immune-based strategies that may incorporate the Treg depletion strategy to achieve potent antitumor immunity with therapeutic impact.

Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors

Autologous dendritic cells (DCs) transfected with mRNA encoding prostate-specific antigen (PSA) are able to stimulate potent, T cell-mediated antitumor immune responses in vitro. A phase I trial was performed to evaluate this strategy for safety, feasibility, and efficacy to induce T cell responses against the self-protein PSA in patients with metastatic prostate cancer. In 13 study subjects, escalating doses of PSA mRNA-transfected DCs were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope PSA in six of seven subjects; three patients that could be analyzed exhibited a transient molecular clearance of circulating tumor cells. The demonstration of vaccine safety, successful in vivo induction of PSA-specific immunity, and impact on surrogate clinical endpoints provides a scientific rationale for further clinical investigation of RNA-transfected DCs in the treatment of human cancer.

Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells.

The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance, where a tumor cell line was successfully established in culture from a patient with renal cancer, the patients tumor cells were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional tumor antigens.

Cancer immunotherapy with mRNA‐transfected dendritic cells

Summary:  Bone marrow‐derived dendritic cells (DCs) are the most potent antigen‐presenting cells capable of activating naïve T cells. Loading DCs ex vivo with tumor antigens can stimulate potent antitumor immunity in tumor‐bearing mice. This review describes the use of mRNA‐encoded tumor antigens as a form of antigen loaded onto DCs, including our early experience from clinical trials in urological cancers. Transfection of DCs with mRNA is simple and effective. Comparative studies suggest that mRNA transfection is superior to other antigen‐loading techniques in generating immunopotent DCs. The ability to amplify RNA from microscopic amounts of tumor tissue extends the use of DC vaccination to virtually every cancer patient. The striking observation from two phase I clinical trials, in patients with prostate cancer immunized with prostate‐specific antigen mRNA‐transfected DCs and patients with renal cancer immunized with autologous tumor RNA‐transfected DCs, was that the majority of patients exhibited a vaccine‐induced T‐cell response. Suggestive evidence of clinically related responses was seen in both the trials. Immunization with mRNA‐transfected DCs is a promising strategy to stimulate potent antitumor immunity and could serve as a foundation for developing effective treatments for cancer.

Injection of Immature Dendritic Cells into Adjuvant-Treated Skin Obviates the Need for Ex Vivo Maturation

A key and limiting step in the process of generating human monocyte-derived dendritic cells (DC) for clinical applications is maturation. In the setting of immunotherapy, DC are matured ex vivo by culturing them with various agents that mimic the conditions encountered at a site of inflammation. This study examined whether the ex vivo DC maturation step could be replaced by maturing DC in situ by injecting immature DC into sites pre-exposed to agents that induce a microenvironment conducive to in situ maturation of the injected DC. The hypothesis was that recapitulation of the physiological conditions occurring during pathogen infection would lead to optimal conditions for DC maturation, migration, and function. Murine immature DC injected into adjuvant (Adjuprime, poly-arginine, or Imiquimod)-pretreated skin exhibited lymph node migratory capacity comparable to and immunostimulatory capacity equal to or exceeding that of ex vivo matured DC. Acquisition of migratory capacity did not always correlate with enhanced immunostimulatory capacity. Immunostimulatory capacity was not enhanced when mature DC were injected into adjuvant-pretreated sites and remained below that seen with immature DC matured in situ. Immature DC injected into adjuvant-pretreated sites were more effective than mature DC in stimulating antitumor immunity in mice. 111Indium-labeled human monocyte-derived immature DC injected into adjuvant (Imiquimod)-pretreated sites in cancer patients acquired lymph node migratory capacity comparable to ex vivo matured DC. This study shows that in situ maturation offers a simpler and potentially superior method to generate potent immunostimulatory DC for clinical immunotherapy.

UNENHANCED HELICAL COMPUTERIZED TOMOGRAPHY FOR THE EVALUATION OF PATIENTS WITH ACUTE FLANK PAIN

PURPOSE We determined the value of unenhanced helical computerized tomography (CT) in the diagnosis of acute flank pain in 105 patients evaluated for suspected stone disease. MATERIALS AND METHODS Noncontrasted spiral CT was done in 105 consecutive patients seen in our emergency department to evaluate acute flank pain. All CT studies were reviewed for the presence of ureteral or renal calculi, perinephric or periureteral stranding, presence and degree of pelvicalicectasis or other radiological findings. If necessary, an excretory urogram was performed to confirm the presence or absence of urinary stones. Patients were followed to determine clinical outcome including the need for urological intervention. RESULTS Of the 49 patients determined to have stones 24 (49%) had spontaneous stone passage, 10 (20%) had improved symptoms without documented stone passage and 14 (29%) required surgical intervention. In 29 of 51 patients (57%) with negative CT readings for stone disease a diagnosis was established by other intra-abdominal findings. In 21 patients (41%) no clinical diagnosis could be established, and 1 scan in a patient with a distal ureteral calculus was interpreted as falsely positive. These findings yielded a sensitivity of 98%, specificity 98% and overall accuracy 96% for diagnosing ureteral stones. CONCLUSIONS Despite the limitations of helical CT in evaluating renal function and nonobstructing ureteral calculi, noncontrasted CT is a sensitive imaging modality for the detection of urinary tract calculi and obstruction. The majority of our patients required no further imaging to determine the need for urological intervention. At our institution spiral CT has become the standard method to evaluate patients with acute flank pain leading to more rapid turnover in the emergency department at similar or even reduced cost to conventional excretory urography.

Outcome analysis in patients with primary necrotizing fasciitis of the male genitalia

OBJECTIVES To characterize patients with primary necrotizing fasciitis of the male genitalia (Fourniers gangrene) and to identify risk factors and prognostic variables of survival. METHODS Fifty consecutive patients with primary necrotizing fasciitis of the male genitalia treated at our institution during a 15-year period between 1984 and 1998 were retrospectively analyzed. Of these patients, 44 (88.0%) were found to be eligible for analysis of the outcome parameters. Univariate survival analysis was performed using the Kaplan-Meier algorithm followed by multivariate analysis of statistically significant variables. Six patients (12.0%) who were severely immunocompromised were studied separately. RESULTS Medical comorbidities were prevalent, with diabetes being the most common condition (50%). The overall mortality rate was 20% (10 of 50). Three statistically significant predictors of outcome were identified among the variables analyzed. These were the extent of the infection (P = 0.0262), the depth of the necrotizing infection (P = 0.0107), and treatment with hyperbaric oxygen (P = 0.0115). Multivariate regression analysis of these variables identified the extent of the infection (P = 0.0234) as the only statistically significant, independent predictor of outcome in the presence of other covariables. CONCLUSIONS The involved body surface area appears to be the most important prognostic variable, with a significant impact on outcome. Given the high mortality of the disease entity and a trend toward the improved survival of patients receiving hyperbaric oxygen, this treatment form appears indicated in more severe cases. Immunocompromised patients, who frequently have an atypical and fulminant clinical course, appear to constitute a separate group with a dismal prognosis.

DOES PROSTATE TRANSITIONAL CELL CARCINOMA PRECLUDE ORTHOTOPIC BLADDER RECONSTRUCTION AFTER RADICAL CYSTOPROSTATECTOMY FOR BLADDER CANCER

PURPOSE We determined if urethral preservation and orthotopic bladder replacement in patients with transitional cell carcinoma within the prostatic urethra or prostate placed these patients at risk for urethral recurrence or death. MATERIALS AND METHODS The clinical course of all patients undergoing urethral preservation and orthotopic bladder replacement was reviewed. The urethra was sacrificed only if the distal prostatic urethral margin was positive for transitional cell carcinoma. The pathological T stage and the grade of the primary malignancy, local recurrence, site of recurrence (urethral, pelvic, distant) and death were documented. RESULTS Of 81 patients 70 were evaluable (June 1996) with a mean followup of 35 months. Of the 70 patients 48 were alive without evidence of disease for a mean of 38 months (range 8 to 107) and 5 died without evidence of disease. Eight of these 53 patients (15%) had prostatic involvement (carcinoma in situ in 6, intraductal carcinoma in 1 and stromal invasive transitional cell carcinoma in 1). Of the 70 patients 17 had disease recurrence (13 died of disease and 4 are alive, 1 of whom had urethral recurrence without initial prostatic transitional cell carcinoma). Of the 17 patients (35%) 6 had transitional cell carcinoma prostatic involvement (carcinoma in situ in 4 and stromal invasion in 2), and 5 of these 6 died, none with or of urethral recurrence but of the primary bladder pathology. Of these 5 patients 1 had stromal invasive transitional cell carcinoma of the prostate and experienced a bulbar urethra recurrence at 1 month and a pelvic recurrence at 3 months, and died at 5 months. Death was not secondary to the urethral recurrence. Thus, of the 14 patients who had prostatic transitional cell carcinoma, only 1 had urethral recurrence (7%), and this recurrence did not present as the cause of death. CONCLUSIONS The guidelines for urethral resection can be relaxed, increasing the opportunities for orthotopic reconstruction, without placing the patients at increased risk for death of transitional cell carcinoma.

TRANSIENT LOWER EXTREMITY NEURAPRAXIA ASSOCIATED WITH RADICAL PERINEAL PROSTATECTOMY: A COMPLICATION OF THE EXAGGERATED LITHOTOMY POSITION

PURPOSE We assess the incidence and risk factors associated with lower extremity neurapraxia following radical perineal prostatectomy. MATERIALS AND METHODS The medical records of 111 consecutive patients undergoing radical perineal prostatectomy at Duke University Medical Center between June 1994 and June 1995 were retrospectively reviewed. Patients were interviewed by telephone to ascertain whether symptoms had resolved. RESULTS Neurapraxia developed in 23 patients (21%). Symptomatology was variable, including sensory and motor deficits of the lower leg and foot. Although lower extremity neurapraxia occurred in a significant number of patients undergoing radical perineal prostatectomy, it appeared to resolve in most. CONCLUSIONS Careful attention to detail when positioning the patient and limiting the time in the exaggerated lithotomy position appear to be the most critical aspects to prevent neurapraxia.

Considerations for the Use of Cytokine-Secreting Tumor Cell Preparations for Cancer Treatment

Limited efficacy of chemotherapy in most solid tumors has revived interest in immunotherapeutic approaches for cancer. One novel form of immunotherapy is the use of cancer vaccines consisting of tumor cells genetically engineered to secrete cytokines. The rationale for this immunization strategy is based on the existence of tumor-specific antigens, on the importance of the cellular arm of the immune system in mediating an effective antitumor response, and on the role of cytokines in regulating the cellular immune response. Such tumor vaccines showed considerable promise in various animal models and induced potent antitumor immunity in the host, which led to regression of established tumors and, moreover, produced immunological memory protecting animals from a subsequent tumor challenge at a distant site. Translated to the human patient, this implies that genetically modified tumor vaccines may be able to eradicate or reduce existing tumor deposits to subclinical levels as well as provide long-term protection from regrowth of tumor cells. This report will review and discuss the concept and rationale for the use of cytokine-secreting tumor vaccines for the treatment of human malignancies.