Warren D. W. Heston

Tumor target Prostate specific membrane antigen (PSMA) and its regulation in Prostate cancer

Prostate specific membrane antigen (PSMA), is a unique membrane bound glycoprotein, which is overexpressed manifold on prostate cancer as well as neovasculature of most of the solid tumors, but not in the vasculature of the normal tissues. This unique expression of PSMA makes it an important marker as well as a large extracellular target of imaging agents. PSMA can serve as target for delivery of therapeutic agents such as cytotoxins or radionuclides. PSMA has two unique enzymatic functions, folate hydrolase and NAALADase and found to be recycled like other membrane bound receptors through clathrin coated pits. The internalization property of PSMA leads one to consider the potential existence of a natural ligand for PSMA. In this review we have discussed the regulation of PSMA expression within the cells, and significance of its expression in prostate cancer and metastasis.

High Energy Shock Waves Suppress Tumor Growth in Vitro and in Vivo

Exposure of the Dunning R3327AT-3 rat prostatic carcinoma and SK-Mel-28 human melanoma, in vitro, to high energy shock waves resulted in a reduction in cell viability as determined by trypan blue exclusion and a decrease in the number of colonies formed in a clonogenic assay. Flow cytometric determination of DNA content in R3327AT-3 cells treated in vitro indicated a selective diminution of cells in the G2 and M phases of the cell cycle. When R3327AT-3 cells exposed to high energy shock waves were subsequently injected into rats, or tumor bearing animals were treated by high energy shock waves targeted at the tumor, a delay in tumor growth was observed. These observations indicate that high energy shock waves are cytotoxic to tumor cells in vitro and in vivo. Additional research into the possible use of high energy shock waves in the non-invasive destruction of animal and human tumors is warranted.

Cloning, expression, genomic localization, and enzymatic activities of the mouse homolog of prostate-specific membrane antigen/NAALADase/folate hydrolase

Abstract. Human Prostate Specific Membrane Antigen (PSMA), also known as folate hydrolase I (FOLH1), is a 750-amino acid type II membrane glycoprotein, which is primarily expressed in normal human prostate epithelium and is upregulated in prostate cancer, including metastatic disease. We have cloned and sequenced the mouse homolog of PSMA, which we have termed Folh1, and have found that it is not expressed in the mouse prostate, but primarily in the brain and kidney. We have demonstrated that Folh1, like its human counterpart, is a glutamate-preferring carboxypeptidase, which has at least two enzymatic activities: (1) N-acetylated α-linked l-amino dipeptidase (NAALADase), an enzyme involved in regulation of excitatory signaling in the brain, and (2) a γ-glutamyl carboxypeptidase (folate hydrolase). The 2,256-nt open reading frame of Folh1 encodes for a 752-amino acid protein, with 86% identity and 91% similarity to the human PSMA amino acid sequence. Cells transfected with Folh1 gained both NAALADase and folate hydrolase activities. Examination of tissues for NAALADase activity correlated with the mRNA expression pattern for Folh1. Fluorescent in situ hybridization (FISH) revealed Folh1 maps to only one locus in the mouse genome, Chromosome 7D1-2.

Targeted Elimination of Prostate Cancer by Genetically Directed Human T Lymphocytes

The genetic transfer of antigen receptors is a powerful approach to rapidly generate tumor-specific T lymphocytes. Unlike the physiologic T-cell receptor, chimeric antigen receptors (CARs) encompass immunoglobulin variable regions or receptor ligands as their antigen recognition moiety, thus permitting T cells to recognize tumor antigens in the absence of human leukocyte antigen expression. CARs encompassing the CD3zeta chain as their activating domain induce T-cell proliferation in vitro, but limited survival. The requirements for genetically targeted T cells to function in vivo are less well understood. We have, therefore, established animal models to assess the therapeutic efficacy of human peripheral blood T lymphocytes targeted to prostate-specific membrane antigen (PSMA), an antigen expressed in prostate cancer cells and the neovasculature of various solid tumors. In vivo specificity and antitumor activity were assessed in mice bearing established prostate adenocarcinomas, using serum prostate-secreted antigen, magnetic resonance, computed tomography, and bioluminescence imaging to investigate the response to therapy. In three tumor models, orthotopic, s.c., and pulmonary, we show that PSMA-targeted T cells effectively eliminate prostate cancer. Tumor eradication was directly proportional to the in vivo effector-to-tumor cell ratio. Serial imaging further reveals that the T cells must survive for at least 1 week to induce durable remissions. The eradication of xenogeneic tumors in a murine environment shows that the adoptively transferred T cells do not absolutely require in vivo costimulation to function. These results thus provide a strong rationale for undertaking phase I clinical studies to assess PSMA-targeted T cells in patients with metastatic prostate cancer.

Metastatic renal cell carcinoma neovasculature expresses prostate-specific membrane antigen

OBJECTIVES To determine whether the tumor-associated neovasculature of metastatic prostate and metastatic conventional (clear cell) renal carcinoma express prostate-specific membrane antigen (PSMA). PSMA is a type II integral membrane glycoprotein highly expressed in prostate cancer cells and also recently discovered to be expressed in the neovasculature of non-prostatic primary malignancies. METHODS We examined metastatic prostate carcinoma (22 patients) and metastatic conventional (clear cell) renal carcinoma (20 patients) in various anatomic sites, including bone, lymph nodes, liver, lung, and soft tissue. Using the biotin-streptavidin method, we performed immunohistochemical reactions with the anti-PSMA monoclonal antibodies (mAbs) 7E11 and PM2J004.5 and with the anti-endothelial cell mAb CD34. RESULTS Metastatic conventional (clear cell) renal carcinoma consistently expressed PSMA. The PM2J004.5 mAb was positive in 20 of 20 specimens, and the 7E11 mAb was positive in 15 of 20. The anti-PSMA immunoreactions with the neovasculature were confirmed by similar staining by the anti-CD34 mAb (20 of 20). Although the metastatic prostatic cancer cells expressed PSMA in all the specimens, only 2 of 22 had neovasculature PSMA expression. CONCLUSIONS As in primary prostatic adenocarcinomas, the neovasculature of metastatic prostate cancer, regardless of site, rarely express PSMA. The neovascular endothelial cells of metastatic clear cell renal carcinoma, however, express PSMA. This expression may make PSMA an effective target for mAb-based antineovasculature therapy in metastatic renal carcinoma.

Histopathologic and ultrastructural correlates of tumor growth suppression by high energy shock waves

High energy shock waves (HESW) are cytotoxic to tumor cells as determined by vital staining and impaired ability of viable cells to form colonies in a clonogenic assay. In addition, direct exposure of tumor nodules to HESW results in suppression of tumor growth rate. In order to identify histopathologic and ultrastructural correlates of these observations, R3327AT-3 prostatic tumor cells were exposed to HESW in vitro and in vivo. Damage to cells in suspension was manifested by fragmentation of cells to form debris. At the ultrastructural level, mitochondria were swollen and contained distorted cristae following exposure of tumor cells to HESW. In vivo exposure of tumor nodules to HESW did not cause a distinct histopathologic or ultrastructural effect that could be qualitatively distinguished from spontaneously occurring cell death. Hemorrhage and necrosis were observed in muscle and fibroadipose tissue adjacent to tumor. The mechanism of HESW-induced cytotoxicity is not clear from our studies. Evidence of damage of normal tissues exposed in vivo and tumor cells in vitro is reflected in histomorphological changes.

Comparison of anti-prostate-specific membrane antigen antibodies and other immunomarkers in metastatic prostate carcinoma

OBJECTIVES To compare the immunohistochemical properties of the 7E11 anti-prostate-specific membrane antigen (anti-PSMA) monoclonal antibody (mAb) with the recently developed anti-PSMA mAb, PM2J004.5, and with other common immunomarkers in metastatic prostate cancer. PSMA is a type II integral membrane glycoprotein highly expressed in prostate cancer cells. The mAb 7E11 is currently used in the radioisotopic evaluation of prostate cancer, and its immunohistochemical properties have been examined in primary prostate cancer specimens. METHODS We examined 23 formalin-fixed, paraffin-embedded, metastatic prostate carcinoma specimens from various anatomic sites, including bone, lymph node, liver, lung, and soft tissue. Using the biotin-streptavidin method, we performed immunohistochemical reactions with the anti-PSMA mAbs 7E11 and PM2J004.5 and with antibodies to prostate-specific antigen and prostatic acid phosphatase. The immunoreactions were scored by pathologists unaware of the clinical and pathologic data according to a staining intensity scale and the percentage of cells stained. RESULTS All four mAbs consistently stained the metastatic prostate cancer specimens. In 2 (8.7%) of 23 cases, however, the prostate-specific antigen immunoreaction was negative but the anti-PSMA mAbs had positive staining. Although 7E11 and PM2J004.5 had a similar staining intensity and percentage of cells stained for most specimens, in 3 (13%) of 23 specimens, 7E11 had less intense staining. None of the specimens were negative for all four antibodies. CONCLUSIONS Anti-PSMA mAbs consistently immunoreacted with metastatic prostate cancer specimens and were positive in instances when prostate-specific antigen staining was negative. The anti-PSMA mAbs demonstrated similar staining patterns; however, in select cases, the PM2J004.5 mAb did show more intense staining. The anti-PSMA mAbs 7E11 and PM2J004.5 are useful in the pathologic evaluation of paraffin-embedded metastatic prostate cancer specimens.

Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer

Prostate‐specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases. In addition, unlike prostate‐specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer. Recently, both the promoter and the enhancer driving prostate‐specific expression of the PSMA gene were cloned. We describe here our analysis of the PSMA enhancer for the most active region(s) and present a way of using the enhancer in combination with the E. coli cytosine deaminase gene for suicide‐driven gene therapy that converts the nontoxic prodrug 5‐fluorocytosine (5‐FC) into the cytotoxic drug 5‐fluorouracil (5‐FU) in prostate cancer cells.

Intravesical oncolytic viral therapy using attenuated, replication-competent herpes simplex viruses G207 and Nv1020 is effective in the treatment of bladder cancer in an orthotopic syngeneic model

Attenuated, replication‐competent herpes simplex virus mutants are attracting interest because of their ability to replicate within and kill tumor cells while remaining of low pathogenicity to normal tissues. In this study we investigated the ability of two oncolytic candidates, G207 and NV1020, to infect and lyse human and murine transitional cell carcinoma (MBT‐2) cells in vitro and their in vivo efficacy in a well‐established immunocompetent animal model of bladder cancer. Both viruses were effective at infecting, replicating within, and achieving subsequent cell lysis for all four human bladder cancer cell lines and MBT‐2. We found a strong correlation between infection efficiency and subsequent cell death. In vivo studies demonstrated that these viruses were effective with a single intravesical instillation and even more effective with multiple instillations at reducing tumor burden and achieving cures of orthotopic bladder cancer in syngeneic C3h/Hej mice. Immunohistochemistry and histological studies demonstrated that viral replication and cell death were restricted to bladder cancer cells. These results suggest that both G207 and NV1020 hold particular promise for intravesical treatment of human bladder cancer and that ease of intravesical instillation facilitates efficient delivery of virus to tumor cells.

The indications, rationale, and results of neoadjuvant androgen deprivation in the treatment of prostatic cancer: Memorial Sloan-Kettering Cancer Center results**

OBJECTIVES The use of neoadjuvant chemotherapy prior to definitive surgery has been firmly established in other areas of oncology, most notably in the treatment to testis and Wilms tumors. The use of neoadjuvant androgen deprivation therapy (ADT) in conjunction with radical prostatectomy remains a source of controversy. We have conducted phase II and phase III studies to assess the effects of 3 months of preoperative ADT (goserelin and flutamide) on the pathologic staging and postsurgery prostate-specific antigen (PSA) relapse rate. We also reviewed the data confirming the understaging of clinically localized prostatic cancer and the experimental data providing the conceptual support for ADT. METHODS We report the results of 141 patients, Stage T0-T0, in a Phase II study with concurrent, nonrandomized controls (N = 72) versus a treatment arm (N = 69) of men receiving 3 months of ADT with 3.6 mg goserelin for 28 days and 750 mg flutamide daily. We also report the interim results in 114 men participating in a prospective, randomized study of ADT versus surgery alone. RESULTS The 69 patients who received 3 months of goserelin and flutamide followed by radical prostatectomy had a pathologic organ-confined cancer rate of 74%, versus 48% in the control group who received no ADT prior to surgery. The margin-positive rate was 10% in the ADT group versus 33% in the control group. In an interim analysis of 114 patients (59 ADT, 55 control), the organ-confined and margin-positive rates were 73% and 17% in the ADT group versus 56% and 36% in the control arm, respectively. The PSA disease-free rate at a mean follow-up of 28.6 months (range 6.2 to 49.5 months) was 89% in the ADT-treated patients (N = 98) and 84% in the control patients (N = 96). There was no statistical difference demonstrated between the arms with respect to biochemical failure. CONCLUSIONS While the pathologic staging of tumors following ADT treatment was improved compared with surgical controls, to date the PSA disease-free survival rates are similar. Patients with residual extracapsular (P3) disease after ADT manifest an increased PSA failure rate compared with those with P3 tumors treated by surgery alone. This suggests that ADT may identify a subset of patients with aggressive tumors that may be candidates for additional therapeutic interventions even before PSA failure occurs.