Efficacy of vaccination with plasmid DNA encoding for HER2/neu or HER2/neu-eGFP fusion protein against prostate cancer in rats.

Despite early diagnosis and improved therapy, 31,500 men will die from prostate cancer (PC) this year. The HER2/neu oncoprotein is an important effector of cell growth found in the majority of high-grade prostatic tumors and is capable of rendering immunogenicity. The antigenicity of this oncoprotein might prove useful in the development of PC vaccines. Our goal is to prove the principle that a single DNA vaccine can provide reliable immunity against PC in the MatLyLu (MLL) translational tumor model. The parental rat MatLyLu PC cell line expresses low to moderate levels of the rat neu protein. To simulate in vivo human PC, MatLyLu cells were transfected with a truncated sequence of human HER2/neu cDNA cloned into the pCI-neo vector. This HER2/neu cDNA sequence encodes the first 433 amino acids of the extracellular domain (ECD). MatLyLu cells were also transfected with the same HER2/neu cDNA sequence cloned into the N1-terminal sequence of EGFP reporter gene to produce a fusion protein. The partial ECD sequence of HER2/neu includes five rat major histocompatibility (MHC)-II-restricted peptides with complete human-to-rat cross-species homology. The HER2/neu protein overexpression was documented by Western Blot analysis, and the expression of fusion protein was monitored by confocal microscopy and fluorimetry. Vaccination with a single injection of HER2/neu cDNA protected 50% of animals against HER2/neu-MatLyLu tumors (P < 0.01). When the tumor cells were engineered to express HER2/neu-EGFP fusion protein, the antitumor immunity was enhanced, as following vaccination with HER2/neu-EGFP cDNA, 80% of these rats rejected HER2/neu-EGFP-MatLyLu (P<0.001). Both vaccines induced HER2/neu-specific antibody titers. Rats vaccinated with EGFP-cDNA rejected 80% of EGFP-MatLyLu tumors and, interestingly, 40% of HER2/neu-MatLyLu tumors. None of the cDNA vaccines induced immunity against parental MatLyLu cells. Our data clearly demonstrate that a single injection of HER2/neu-EGFP cDNA is a very effective vaccine against PC tumors expressing the cognate tumor-associated antigen (TA). The antitumor immunity is significantly more pronounced if the tumors express xenogeneic HER2/neu-EGFP fusion protein as opposed to only the syngeneic HER2/neu oncoprotein. Our data suggests that the HER2/neu-EGFP-MatLyLu tumor is a potential animal tumor model for investigating therapeutic vaccine strategies against PC in vivo and demonstrates the limitations of a cDNA vaccine only encoding for MHC-II-restricted HER2/neu-ECD sequence peptides.

Cytokine-mediated protection of human dendritic cells from prostate cancer-induced apoptosis is regulated by the Bcl-2 family of proteins.

Prostate cancer is the most common cancer in men in the United States, and second in cancer-induced mortality. It is likely that tumour-induced immunosuppression is one of the reasons for low treatment efficacy in patients with advanced prostate cancer. It has been recently demonstrated that prostate cancer tissue is almost devoid of dendritic cells (DC), the major antigen-presenting cells responsible for the induction of specific antitumour immune responses. In this study, we have tested the hypothesis that prostate cancer induces progressive suppression of the DC system. We found that co-incubation of human DC with three prostate cancer cell lines led to the high levels of premature apoptosis of DC, which were significantly higher than in DC cultures co-incubated with normal prostate cells or blood leucocytes. Stimulation of DC for 24 hours with CD40 ligand (CD154), IL-12 or IL-15 prior to their co-incubation with prostate cancer cells resulted in a significant increase in DC survival in the tumour microenvironment. Furthermore, activation of DC with these cytokines was also accompanied by increased expression of the anti-apoptotic protein Bcl-x(L) in DC, suggesting a possible mechanism involved in DC protection from apoptotic death. In summary, our data demonstrate that prostate cancer induces active elimination of DC in the tumour microenvironment. Stimulation of DC by CD154, IL-12 or IL-15 leads to an increased expression of the anti-apoptotic protein Bcl-x(L) and increased resistance of DC to prostate cancer-induced apoptosis. These results suggest a new mechanism of tumour escape from immune recognition and demonstrate the cytokine-based approaches which might significantly increase the efficacy of DC-based therapies for cancer.

Diagnostic value of voided urine and bladder barbotage cytology in detecting transitional cell carcinoma of the urinary tract.

In this article we report on our experience with the use of urine cytology for the screening and diagnosis of transitional cell carcinoma (TCC) of the bladder and upper urinary tracts at our institution between January 1987 and December 1995. A total of 76 patients were included in the study. All patients had voided urine cytology studies read as positive or highly suspicious for malignancy and no prior history of TCC of the urinary tract. All these patients subsequently underwent cystoscopy, bladder/ureteral barbotage cytology, random bladder biopsies, and radiographic studies of the upper tracts. Of the 76 patients with positive urine cytology, 53 also had barbotage urine cytologies which were positive. Six of these patients were found to have cystoscopically evident TCC of the bladder, and 1 patient had upper tract TCC. Three other patients subsequently went on to develop TCC of the bladder at 52, 89 and 111 months of follow-up. An additional patient was diagnosed with upper tract TCC at 12 months of follow-up. Among the 23 patients with negative bladder/ureteral barbotage cytology, 3 patients, 2 at the time of initial cystoscopy, and one 15 months later, showed evidence of TCC. Median patient follow-up was 97 (range 35-132) months. Thus of 76 patients with initial positive voided urine cytology studies, only 9 proved to have TCC at initial work-up, while 5 other patients were diagnosed with TCC during a median follow-up of 97 months. The statistical diagnostic values of the bladder/ureteral barbotage urine cytology studies at the time of cystoscopic work-up were: sensitivity 77%; specificity 31%; positive predictive value 13%, and negative predictive value 91%. Our data suggest that in patients without a previous history of TCC, the diagnostic value of bladder barbotage urine cytology is insignificant, and therefore not cost effective to be included as part of the routine work-up of TCC. Moreover, in patients with initially positive voided urine cytology and negative work-up, if the cytology subsequently becomes negative, the likelihood of the development of TCC is low. However, if the initially positive cytology continues to remain positive, there is a much higher probability of TCC being detected in this population.

Leiomyosarcoma with osteoclast-like giant cells of the spermatic cord. A case report and review of the literature.

We describe a case of leiomyosarcoma with osteoclast-like giant cells arising form the spermatic cord and involving the testis. This is a rare tumor which is histologically similar to the giant cell variant of malignant fibrous histiocytoma. No such cases have been reported arising from the external genitalia. The patient was initially treated with local excision. He had a local recurrence 7 years later which was treated with radical excision and adjuvant radiation. The indolent nature of the tumor, with a long survival of 7 years after initial diagnosis followed subsequently by a more aggressive clinical course, is emphasized.

Closure of large postradiation vesicovaginal fistula with rectus abdominis myofascial flap.

We report the case of a patient who developed a large radiation-induced vesicovaginal fistula. She also had a paucity of the omentum due to previous laparotomy and adhesions. We were able to treat this patient successfully with a pedicled myofascial rectus abdominis flap.

Chemoimmunotherapy of metastatic murine renal cell carcinoma using flavone acetic acid and interleukin 2.

Metastatic renal cell carcinoma (RCC) remains largely incurable. We used a murine RCC (Renca) in BALB/c mice to investigate the treatment possibilities with chemoimmunotherapy using in vivo boosters of natural killer (NK) activity. Diffuse pulmonary metastases were induced by intravenous (i.v.) inoculation with 100,000 Renca cells. All untreated control animals died within one month from pulmonary metastases. Chemoimmunotherapy using the NK immunostimulator flavonic-8-acetic acid (FAA) at 200 mg./kg. i.v. was given on the third day post tumor inoculation, followed by four consecutive days of twice daily intraperitoneal (i.p.) administration of 10,000 units human recombinant interleukin-2 (rIL-2). This chemoimmunotherapy regimen consistently cured 70% of tumor-bearing animals. Mice cured by this chemoimmunotherapy regimen did not reject subsequent reinoculation with Renca, indicating absence of specific antitumor immunity as a result of the treatment. While FAA and rIL-2 have no demonstrable in vitro cytotoxicity for Renca, they are excellent boosters of in vivo NK activity. These data suggest a potential alternative treatment method for metastatic RCC, a tumor type for which no efficient cytostatic drugs are available.

Improved lymphocyte cytotoxicity against murine renal cell carcinoma.

In this paper we describe the generation of antibody dependent cellular cytotoxicity against a murine renal cell carcinoma. Using human recombinant interleukin-2 and in vitro adherence to plastic, we generated lymphokine activated killer and adherent lymphokine activated killer cells. Adherent lymphokine activated killer cells had significant (p less than 0.05) higher unrestricted cytotoxicity than LAK cells. Using a rabbit antibody against Renca developed in our laboratory, we induced significant (p less than 0.01) antibody dependent cellular cytotoxicity using fresh spleen, lymphokine activated killer and adherent lymphokine activated killer cells. The strongest antibody dependent cellular cytotoxicity killing was mediated by adherent lymphokine activated killer cells and was restricted only to the renal cell carcinoma target. Using FACS cell surface analysis and antibody and complement depletion of selected effector cell subsets, we also demonstrate that the antibody dependent cellular cytotoxicity effector cell population consists of asialoGM1+ Lyt 2.1- natural killer cells. This first description of antibody dependent cellular cytotoxicity against renal cell carcinoma by activated natural killer cells suggests a novel method for more efficient use of cytotoxic effector cells against this type of cancer.

Flavone-8-acetic acid augments systemic natural killer cell activity and synergizes with IL-2 for treatment of murine renal cancer.

The investigational drug flavone-8-acetic acid (FAA) potently augments NK activity in the spleen, liver, lungs, and peritoneum in a dose-dependent manner after i.v. or i.p. administration. Augmented NK activity peaks by 24 h after FAA injection and returns to normal after 6 days. Combined treatment of established murine renal cancer with FAA and rIL-2 results in up to 80% long term survival whereas FAA or rIL-2 alone were unable to induce any long term survivors. The optimal dose of rIL-2 required for use with FAA was in the range of 10,000 to 30,000 U/day. Further studies demonstrated that the regimen of FAA plus rIL-2 administration that was effective in treating established murine renal cancer also induced a more potent augmentation of NK activity than did either FAA or rIL-2 alone. Subsequent studies revealed that the therapeutic effectiveness of FAA plus rIL-2 was significantly reduced when tumor-bearing mice were treated with anti-asialo GM1 serum. These results are consistent with a role for augmented NK activity in the therapeutic effects of FAA plus rIL-2 murine renal cancer. In addition, these studies demonstrate that FAA and rIL-2 is a useful approach for cancer treatment in that subtoxic doses of rIL-2 can be used and significant anti-tumor efficacy occurs even without accompanying adoptive immunotherapy.

Lymphokine-activated killer cells in rats: analysis of progenitor and effector cell phenotype and relationship to natural killer cells.

The progenitor and effector cell phenotype of lymphokine-activated killer (LAK) cells generated in F344 rats by recombinant human interleukin 2 (IL-2) (rIL-2) were analyzed. Highly purified populations of peripheral blood large granular lymphocytes (LGL) exhaustively depleted of T-cells were fully capable of generating high levels of LAK activity by 3 to 5 days in culture while purified populations of resting T-cells devoid of LGL could not generate LAK activity. This pure population of LGL expressed surface markers characteristic of rat natural killer (NK) cells [i.e., OX8+, asialomonoganglioside (asialo-GM1+), laminin+, OX19-, R1-3B3-, W3/25-, Ia-, surface immunoglobulin negative (SIg-)]. Further evidence that NK cells were the progenitors of cells with LAK activity was obtained by treatment of spleen or peripheral blood lymphocytes with anti-laminin or anti-asialo-GM1 antibodies plus complement or with the lysosomotropic agent L-leucine methyl ester. These treatments effectively depleted LGL/NK cell activity and the subsequent generation of rIL-2-induced LAK activity. Analysis of the LAK effector phenotype by cell sorting demonstrated that the majority of cells with LAK activity were OX8+, asialo-GM1+, laminin+, OX6+, OX19-, R1-3B3-, W3/25-, and SIg-. Furthermore, treatment of LAK cells with L-leucine methyl ester also significantly reduced their cytolytic activity. Thus, the LAK effector cells were also LGL and expressed surface marker characteristic of activated NK cells and not those of mature T- or B-cells. The proliferative response of rat spleen or blood lymphocytes to rIL-2 appeared to be primarily associated with LGL/NK cells since depletion of NK cells by anti-asialo-GM1 or anti-laminin antibody plus complement or by L-leucine methyl ester significantly (P less than 0.001) reduced the incorporation of [3H]thymidine into DNA. In contrast, depletion of T-cells (by anti-T-cell antibody plus complement) did not significantly affect rIL-2-induced proliferation. Similarly, T-cell-depleted, highly purified populations of LGL gave substantial proliferative responses to rIL-2. These studies clearly indicate that in the rat, the major cell population activated by rIL-2 is the LGL/NK cell and these cells appear to represent the major population of cells in blood or spleen which generate broad antitumor (LAK) cytotoxicity.

Precursor phenotype of lymphokine-activated killer cells in the mouse.

Lymphokine-activated killer (LAK) activity has been proposed to functionally differ from natural killer (NK) activity largely on the basis of a broader target cell spectrum and different kinetics of response to interleukin 2 (IL 2). Similarly, it has been proposed that the precursor cells for LAK activity are phenotypically distinct from NK cells. In most precursor studies, phenotype comparisons have been made between fresh NK cells and LAK cells which have been generated by 3 to 5 days of culture in IL 2. In the present study, we utilized positive selection with monoclonal antibodies to characterize the surface phenotype of precursor cells which give rise to rIL 2-augmented NK activity within 24 hr and to classically generated LAK activity which appears after 3 to 5 days of culture in rIL 2. The results demonstrated that highly purified (93 to 95%) Lyt-2+ or L3T4+ T lymphocytes were unable to generate appreciable amounts of either augmented NK activity or LAK activity when cultured with rIL 2, whereas the highly purified (98%) Lyt-2-, L3T4-, asialo GM1+ lymphocyte subset gave rise to both augmented NK and LAK activities. These findings demonstrate that both augmented NK and LAK activities can arise from precursors expressing the same phenotype. Overall, the results suggest that NK cells in mouse spleen constitute a major precursor component for the generation of LAK activity from that organ.

Successful treatment of advanced murine renal cell cancer by bicompartmental adoptive chemoimmunotherapy.

The most challenging aspect of cancer treatment remains the management of invasive and metastatic tumor growth. Recent progress in the development and use of biologic response modifiers for immunomodulation has raised the possibility that the immune system can be used as an additional antitumor treatment modality in conjunction with surgery, chemotherapy, and/or radiotherapy for the treatment of established tumors and their metastases. As a model for adoptive chemoimmunotherapy (ACIT) of renal cancer we have used a murine renal cancer (Renca) of spontaneous origin that mimics the tumor progression characteristically observed for human renal cell carcinoma. In the present study, we demonstrate that broadly cytotoxic lymphocytes, generated by in vitro culture with human recombinant interleukin 2, and used in conjunction with the chemotherapeutic drug doxorubicin hydrochloride, are effective in treating invasive and metastatic renal cell cancer. Administration of ACIT i.v. or i.p., alone, or after nephrectomy of the tumor-bearing kidney, did not cure mice with stage II (locoregional invasive tumor) or stage III (lymph node metastases) disease. In contrast, nephrectomy followed by simultaneous bicompartmental i.v. and i.p. ACIT administration cured 80% of mice with either stage II or stage III Renca. These data demonstrate that simultaneous bicompartmental ACIT affords dramatically improved cure rates for advanced and metastatic Renca. This effect most likely results from efficient control of both locoregional and metastatic tumor growth.

Adjuvant immunotherapy of established murine renal cancer by interleukin 2-stimulated cytotoxic lymphocytes.

We have used a transplantable experimental murine renal carcinoma (Renca) to evaluate the adjuvant immunotherapeutic potential of cytotoxic lymphocytes stimulated by in vitro incubation for 24 h with human recombinant interleukin 2 (rIL-2). The Renca tumor model is therapeutically challenging since, following intrarenal implant, it grows progressively with local invasion and development of spontaneous metastases in abdominal lymph nodes, lungs, and liver. Therefore, successful treatment requires control of both primary tumor, local and regional invasive growth, and systemic metastases. Our studies have shown that rIL-2-stimulated cytotoxic lymphocytes efficiently lyse Renca in vitro. Further, both doxorubicin hydrochloride and an active metabolite of cyclophosphamide also inhibited the growth of Renca in vitro. In vivo administration of doxorubicin hydrochloride, cyclophosphamide or rIL-2-stimulated cytotoxic lymphocytes and rIL-2 to mice bearing established Renca tumors (7-day disease) resulted in a significant (P less than 0.01) increase in short-term survivors (at 45 days), but only 17% cures. However, combination chemoimmunotherapy consisting of doxorubicin hydrochloride and rIL-2-stimulated cytotoxic lymphocytes plus rIL-2 results in the cure of 67% of mice bearing established Renca. These results demonstrate that chemotherapy and immunotherapy with adoptively transferred cytotoxic lymphocytes can function synergistically in the treatment of established murine renal carcinoma.

Treatment of adenocarcinoma in the peritoneum of mice: chemoimmunotherapy with IL-2-stimulated cytotoxic lymphocytes as a model for treatment of minimal residual disease.

We have used a transplantable murine adenocarcinoma of renal origin (Renca) introduced to the abdomen by i.p. injection of a tumor cell suspension, to study the therapeutic potential of adoptive immunotherapy and/or biological response modifiers (BRMs). This tumor model is therapeutically challenging since the tumor grows progressively resulting in extensive peritoneal carcinomatosis, with hemorrhagic ascites, metastases to abdominal lymph nodes, liver, most serous membranes, spleen, and in some animals, pulmonary metastases. Without therapy, death occurs invariably in 36 +/- 3 days. In vitro, the tumor is lysed by lymphocytes obtained from the peritoneal cavity of mice treated with human recombinant interleukin-2 (rIL-2) and by cytotoxic lymphocytes stimulated by in vitro culture with human rIL-2. Treatment of i.p. Renca with a single i.p. injection of the chemotherapeutic agent doxorubicin hydrochloride (DOX), or adoptive transfer of in vitro stimulated cytotoxic lymphocytes together with rIL-2 cured 50% and 20% of the tumor-bearing mice, respectively. In contrast, combined therapy with DOX and adoptive transfer of in vitro stimulated cytotoxic lymphocytes and rIL-2 cured the majority (90%) of tumor-bearing mice. These results suggest that administration of immunotherapy with in vitro activated cytotoxic cells together with human rIL-2 substantially enhances the effectiveness of chemotherapy.

Augmentation of NK activity and/or macrophage-mediated cytotoxicity in the liver by biological response modifiers including human recombinant interleukin 2.

Administration of several biological response modifiers (BRMs) to mice strongly augmented natural killer (NK) activity of leukocytes isolated from the liver. This augmentation of NK activity was induced by two synthetic molecules (MVE-2 and poly ICLC), by two BRMs of bacterial origin (formalin-fixed Propionibacterium acnes: P. acnes and a streptococcal cell wall preparation designated OK-432), as well as a single injection of human recombinant interleukin-2 (hrIL 2). All of these BRMs augmented NK activity in the liver to a greater degree than in the spleen. In addition, adherent leukocytes (greater than 90% macrophages) isolated from the liver following P. acnes administration also exhibited augmented macrophage-mediated cytotoxicity. This cytotoxicity was characterized as macrophage mediated and distinguished from NK activity, on the basis of adherence purification, kinetics of cytotoxicity, and target cell selectivity. The results demonstrate that a variety of BRMs induce augmented natural immunity in the liver and suggest that such organ-associated immune responses may play an important role in the antimetastatic effects of BRMs.

Role of natural killer activity in development of spontaneous metastases in murine renal cancer.

We have studied the role of natural killer activity during the growth and dissemination of a transplantable renal adenocarcinoma (Renca) of spontaneous origin in BALB/c mice. The pattern of growth of this tumor accurately mimics that of adult human renal cell carcinoma in terms of clinical stages I-IV, particularly with regard to spontaneous metastasis to lung and liver. Renca is moderately sensitive to lysis by natural killer cells from normal mice and is more efficiently lysed by natural killer cells from mice treated with the biological response modifier maleic anhydride divinyl ether, a pyran copolymer. Our studies demonstrate that selective depression of natural killer activity by administration of antiserum specific for the neutral glycosphingolipid asialo GM1 correlated with increased formation of spontaneous metastases in the lungs, liver, and lymph nodes. Conversely, augmentation of natural killer activity by the biological response modifier decreased the formation of spontaneous metastases in lungs, liver and lymph nodes. Further, the suppression of natural killer activity and subsequent increased formation of metastases were accompanied by a significantly reduced survival time, whereas the augmented natural killer activity and decreased incidence of metastases in biological response modifier-treated mice were accompanied by an increase in time of survival. These results demonstrate a significant role for natural killer cells in the control of spontaneous metastasis during growth of this murine renal cancer.

Immunomodulation of natural killer activity by polyribonucleotides.

Several synthetic polyribonucleotides have been examined for the ability to augment natural killer (NK) activity and induce interferon (IFN) production. The results demonstrate that the complex of polyinosinic-polycytidylic acid and poly-L-lysine, which has been stabilized in carboxymethylcellulose [poly(I,C)-LC], polyadenylic-polyuridylic acid [poly(A).poly(U)], and a labile poly (I,C) compound with mismatched bases, designated poly(I).poly(C12U), all augment NK activity. However, poly(A).poly(U) and poly(I).poly(C12U) are less efficient augmentors of NK activity on a milligram per kilogram basis, than is poly(I,C)-LC. Similarly, poly(I,C)-LC induces more serum IFN following administration of 1, 10, or 100 micrograms/mouse (1,000-10,000 U/ml) than does either poly(A).poly(U) (0-25 U/ml) or poly(I).poly(C12U) (0-250 U/ml) at the same doses. Further studies with poly(I,C)-LC demonstrated that this molecule is an excellent augmentor of liver-associated NK activity. In fact, administration of poly(I,C)-LC resulted in higher NK levels in lever (63% lysis) than in spleen (44%) or blood (36%), and the augmented NK response was maintained in the liver for up to 13 days, whereas levels of NK activity in both blood and spleen returned to normal by days 3-6.

Therapy of peritoneal murine cancer with biological response modifiers.

We have used a murine renal adenocarcinoma of spontaneous origin (Renca) inplanted in the peritoneal cavity to study the therapeutic potential of biological response modifiers (BRMs) used alone or in conjunction with chemotherapy. This tumor model is therapeutically challenging since following intraperitoneal (i.p.) injection, the tumor grows progressively with hemorrhagic ascites, abdominal metastases to lymph nodes, liver, spleen, most serous membranes, and, in some animals, metastases to extra-abdominal sites (lungs). In the absence of therapy, death invariably occurs within 36 +/- 2 days. The tumor is efficiently lysed in 4 hours by peritoneal cells isolated from mice treated with BRMs. Both MVE-2 and rIL-2 significantly increased the survival time of tumor-bearing mice, but only treatment with MVE-2 led to definite cures of i.p. Renca. A single i.p. injection of MVE-2 cured 20% of the tumor-bearing mice, while repeated i.p. administration of this drug at 12 day intervals cured 70% of i.p. Renca-bearing mice. Combined therapy with doxorubicin hydrochloride and a single dose of MVE-2 cured 90% of tumor-bearing animals. The superior therapeutic efficiency of MVE-2 compared to that of the rIL-2 may be due to its ability, after i.p. inoculation, to generate and maintain high levels of cytotoxic effector cell activity for an elevated period of time within the peritoneal cell population. Additionally, MVE-2 augments effector cell activity in the liver, lungs, spleen, and blood and may therefore more efficiently interfere with metastasis formation in those compartments. The additive effects of MVE-2 and the chemotherapeutic agent suggest that more effective therapy may be achieved by the combination of immunotherapy with BRMs with chemotherapeutic drugs.