Androgen deprivation therapy for the treatment of prostate cancer: consider both benefits and risks

CONTEXT: Androgen deprivation therapy (ADT) is increasingly used for the treatment of prostate cancer (PCa), even in clinical settings in which there is no evidence-based proof of prolonged overall survival (OS). ADT, however, may be associated with numerous side effects, including an increased therapy-related cardiovascular mortality. OBJECTIVE: To discuss different clinical settings in which ADT is currently used and to critically weigh the benefits of ADT against its possible side effects. EVIDENCE ACQUISITION: A MEDLINE search was conducted to identify original articles and review articles addressing the efficacy and side effects of ADT for the treatment of PCa. Keywords consisted of prostate cancer, hormonal therapy, adverse effects, radical prostatectomy, and radiotherapy. The articles with the highest level of evidence for the various examined end points were identified with the consensus of all authors and were reviewed. EVIDENCE SYNTHESIS: Even short-term use of ADT may lead to numerous side effects, such as osteoporosis, obesity, sarcopenia, lipid alterations, insulin resistance, and increased risk for diabetes and cardiovascular morbidity. Despite these side effects, ADT is commonly used in various clinical settings in which a clear effect on improved OS has not been shown. CONCLUSIONS: ADT is associated with an increased risk of multiple side effects that may reduce quality of life and/or OS. Consequently, these issues should be discussed in detail with patients and their families before initiation of ADT. ADT should be used with knowledge of its potential long-term side effects and with possible lifestyle interventions, especially in settings with the highest risk-benefit ratio, to alleviate comorbidities.

Risk of Second Malignant Neoplasms Following Proton Arc Therapy and Volumetric Modulated Arc Therapy for Prostate Cancer

The risk of second malignant neoplasms (SMNs) following prostate radiotherapy is a concern due to the large population of survivors and decreasing age at diagnosis. It is known that parallel-opposed beam proton therapy carries a lower risk than photon IMRT. However, a comparison of SMN risk following proton and photon arc therapies has not previously been reported. The purpose of this study was to predict the ratio of excess relative risk (RRR) of SMN incidence following proton arc therapy to that after volumetric modulated arc therapy (VMAT). Additionally, we investigated the impact of margin size and the effect of risk-minimized proton beam weighting on predicted RRR. Physician-approved treatment plans were created for both modalities for three patients. Therapeutic dose was obtained with differential dose-volume histograms from the treatment planning system, and stray dose was estimated from the literature or calculated with Monte Carlo simulations. Then, various risk models were applied to the total dose. Additional treatment plans were also investigated with varying margin size and risk-minimized proton beam weighting. The mean RRR ranged from 0.74 to 0.99, depending on risk model. The additional treatment plans revealed that the RRR remained approximately constant with varying margin size, and that the predicted RRR was reduced by 12% using a risk-minimized proton arc therapy planning technique. In conclusion, proton arc therapy was found to provide an advantage over VMAT in regard to predicted risk of SMN following prostate radiotherapy. This advantage was independent of margin size and was amplified with risk-optimized proton beam weighting.

Gene transfer by viral vectors

To initiate our clinical trial for chemotherapy protection, I established the retroviral vector system for human MDR1 cDNA gene transfer. The human MDR1 cDNA continued to be expressed in the transduced bone marrow cells after four cohorts of serial transplants, 17 months after the initial transduction and transplant. In addition, we used this retroviral vector pVMDR1 to transduce human bone marrow and peripheral blood CD34$\sp+$ cells on stromal monolayer in the presence of hematopoietic growth factors. These data suggest that the retroviral vector pVMDR1 could modify hematopoietic precursor cells with a capacity for long-term self renewal. Thus, it may be possible to use the MDR1 retroviruses to confer chemotherapeutic protection on human normal hematopoietic precursor cells of ovarian and breast cancer patients in whom high doses of MDR drugs may be required to control the diseases.^ Another promising vector system is recombinant adeno-associated virus (rAAV) vector. An impediment to use rAAV vectors is that production of rAAV vectors for clinical use is extremely cumbersome and labor intensive. First I set up the rAAV vector system in our laboratory and then, I focused on studies related to the production of rAAV vectors for clinical use. By using a self-inactivating retroviral vector carrying a selection marker under the control of the CMV immediate early promoter and an AAV genome with the deletion of both ITRs, I have developed either a transient or a stable method to produce rAAV vectors. These methods involve infection only and can generate high-titer rAAV vectors (up to 2 x 10$\sp5$ cfu/ml of CVL) with much less work.^ Although recombinant adenoviral vectors hardly infect early hematopoietic precursor cells lacking $\alpha\sb v\beta\sb5$ or $\alpha\sb v\beta\sb3$ integrin on their surface, but efficiently infect other cells, we can use these properties of adenoviral vectors for bone marrow purging as well as for development of new viral vectors such as pseudotyped retroviral vectors and rAAV vectors. Replacement of self-inactivating retroviral vectors by recombinant adenoviral vectors will facilitate the above strategies for production of new viral vectors. In order to accomplish these goals, I developed a new method which is much more efficient than the current methods to construct adenoviral vectors. This method involves a cosmid vector system which is utilized to construct the full-length recombinant adenoviral vectors in vitro.^ First, I developed an efficient and flexible method for in vitro construction of the full-length recombinant adenoviral vectors in the cosmid vector system by use of a three-DNA fragment ligation. Then, this system was improved by use of a two-DNA fragment ligation. The cloning capacity of recombinant adenoviral vectors constructed by this method to develop recombinant adenoviral vectors depends on the efficiency of transfection only. No homologous recombination is required for development of infectious adenoviral vectors. Thus, the efficiency of generating the recombinant adenoviral vectors by the cosmid method reported here was much higher than that by the in vitro direct ligation method or the in vivo homologous recombination method reported before. This method of the in vitro construction of recombinant adenoviral vectors in the cosmid vector system may facilitate the development of adenoviral vector for human gene therapy. (Abstract shortened by UMI.) ^

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, through down-regulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and tumor development of HER-2/neu-overexpressing ovarian cancer cells. Here, we established stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. The expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle delay in the G1 phase independently of the HER-2/neu down-regulation. In an orthotopic breast cancer model, we showed that expression of PEA3 inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment in another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, which suggests that PEA3 possessed a strong growth inhibitory effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence that the PEA3 gene could function as an antitumor and gene therapy agent for human breast cancers. ^

Mechanism of anti-tumor activity of adenovirus type 5 E1A in ovarian cancer

The adenovirus type 5 E1A gene was originally developed as a gene therapy to inhibit tumorigenicity of HER-2-overexpressing cells by transcriptional downregulation of HER-2. Our goal is to improve the overall efficacy of E1A gene therapy. To achieve this goal, we have conducted two preclinical experiments. ^ First, we hypothesized that Bcl-2 overexpressing ovarian cancer is resistant to E1A gene therapy. This hypothesis is based on that the 19 kDa protein product of the adenoviral E1B gene which is homologous to Bcl-2 inhibits E1A-induced apoptosis. Treating high Bcl-2-xpressing cells with E1A in combination with an antisense oligonucleotide to Bcl-2 (Bcl-2-ASO) resulted in a significant decrease in cell viability due to an increased rate of apoptosis relative to cells treated with E1A alone. In an ovarian cancer xenograft model, mice implanted with low HER-2, high Bcl-2 cells, treated with E1A plus Bcl-2-ASO led to prolonged survival. Bcl-2 thus may serve as a predictive molecular marker enabling us to select patients with ovarian cancer who will benefit significantly from E1A gene therapy. ^ Second, we elucidated the molecular mechanism governing the anti-tumor effect of E1A in ovarian cancer to identify a more potent tumor suppressor gene. We identified PEA-15 (phospho-protein enriched in astrocytes) upregulated in E1A transfected low HER-2-expressing OVCAR-3 ovarian cancer cell, which showed decreased cell proliferation. PEA-15 moved ERK from the nucleus to the cytoplasm and inhibited ERK-dependent transcription and proliferation. Using small interfering RNA to knock down PEA-15 expression in OVCAR-3 cells made to constitutively express E1A resulted in accumulation of phosphoERK in the nucleus, an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. PEA-15 also independently suppressed colony formation in some breast and ovarian cancer cell lines in which E1A is known to have anti-tumor activity. We conclude that the anti-tumor activity of E1A depends on PEA-15. ^ In summary, (1) Bcl-2 may serve as a predictive molecular marker of E1A gene therapy, allowing us to select patients and improve efficacy of E1A gene therapy. (2) PEA-15 was identified as a component of the molecular mechanism governing the anti-tumor activity of E1A in ovarian cancer, (3) PEA-15 may be developed as a novel therapeutic gene. ^

Potential roles for bcl-2 in the pathogenesis, progression, and treatment of prostate cancer

The most common molecular alterations observed in prostate cancer are increased bcl-2 protein expression and mutations in p53. Understanding the molecular alterations associated with prostate cancer are critical for successful treatment and designing new therapeutic interventions. Hormone-ablation therapy remains the most effective nonsurgical treatment; however, most patients will relapse with hormone-independent, refractory disease. This study addresses how hormone-ablation therapy may increase bcl-2, develops a transgenic model to elucidate the role of bcl-2 multistep prostate carcinogenesis, and assesses how bcl-2 may confer resistance to cell death induction using adenoviral wild-type p53 gene therapy. ^ Two potential androgen response elements were identified in the bcl-2 promoter. Bcl-2 promoter luciferase constructs were transfected into the hormone- sensitive LNCaP prostate cell line. In the presence of dihydrotestosterone, the activity of one bcl-2 promoter luciferase construct was repressed 40% compared to control cells grown in charcoal-stripped serum. Additionally, it was demonstrated that both bcl-2 mRNA and protein were downregulated in the LNCaP cells grown in the presence DHT. This suggests that DHT represses bcl-2 expression through possible direct and indirect mechanisms and that hormone-ablation therapy may actually increases bcl-2 protein. ^ To determine the role of bcl-2 in prostate cancer progression in vivo, probasin-bcl-2 mice were generated where human bcl-2 was targeted to the prostate. Increased bcl-2 expression rendered the ventral prostate more resistant to apoptosis induction following castration. When the probasin-bcl-2 mice were crossed with TRAMP mice, the latency to tumor formation was decreased. The expression of bcl-2 in the double transgenic mice did not affect the incidence of metastases. The double transgenic model will facilitate the study of in vivo effects of specific genetic lesions during the pathogenesis of prostate cancer. ^ The effects of increased bcl-2 protein on wild-type adenoviral p53-mediated cell death were determined in prostatic cell lines. Increased bcl-2 protected PC3 and DU145 cell lines, which possess mutant p53, from p53-mediated cell death and reductions in cell viability. Bcl-2 did not provide the same protective effect in LNCaP cell line, which expresses wild-type p53. This suggests that the ability of bcl-2 to protect against p53-mediated cell death is dependent upon the endogenous status of p53. ^

Effects of exogeneous p53 on growth suppression, apoptosis, and differentiation in oral cancer cell lines

The p53 gene is known to be one of the most commonly mutated genes in human cancers. Many squamous cell carcinomas of the head and neck (SCCHNs) have been shown to contain nonfunctional p53 as well. The use of p53-mediated gene therapy to treat such cancers has become an intensive area of research. Although there have been varied treatment responses to p53 gene therapy, the role that endogenous p53 status plays in this response has not been thoroughly examined. Because of this, the hypothesis of this study examined the role that the endogenous p53 status of cells plays in their response to p53 gene therapy. To test this, an adenoviral vector containing p53 (p53FAd) was administered to three squamous cell carcinoma lines with varied endogenous p53. The SCC9 cell line demonstrates no p53 protein expression, the SCC4 cell line displays overexpression of a mutant p53 protein, and the 1986LN cell line displays low to no expression of wild-type p53 protein as a consequence of human papillomavirus infection. After treatment with p53FAd, the cells were examined for evidence of exogenous p53 expression, growth suppression, alterations in cellular proteins, G1 growth arrest, apoptosis, and differentiation state. Each cell line exhibited exogenous p53 protein. Growth suppression was seen most prominently in the SCC9 cells, to some extent in the 1986LN cells, and little was seen with the SCC4 cells. WAF1/p21 protein was induced in all three cell lines, while PCNA, bcl-2, and bax expression was not significantly affected in any of the lines. Apoptosis developed first in SCC9 cells, next in 1986LN cells, with little seen in the SCC4 cells. The SCC9 line was the only line to show significant GI growth arrest. No significant differences were observed in the overall expression of differentiation markers, aside from increased keratin 13 mRNA levels in all three lines indicating a possible tendency toward differentiation. This study indicates that the endogenous p53 status of squamous cell carcinomas appears to play a critical role in determining the response to p53 adenoviral gene therapy. ^

Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts

Several groups have attempted to develop gene therapy strategies to treat cancer via introduction of the wild-type (wt) p53 cDNA into cancer cells. Unfortunately, these approaches do not result in regulated expression of the p53 gene and do not reduce expression of the mutant p53 that is overexpressed in cancerous cells. These shortcomings may greatly limit the utility of this gene replacement approach. We describe an alternative strategy with trans-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wt p53 activity in various human cancers. The ribozyme accomplished such conversion by repairing defective p53 mRNAs with high fidelity and specificity. The corrected transcripts were translated to produce functional p53 that can transactivate p53-responsive promoters and down-modulate expression of the multidrug resistance (MDR1) gene promoter. The level of wt p53 activity generated was significant, resulting in a 23-fold induction of a p53-responsive promoter and a 3-fold reduction in MDR1 promoter expression in transfected cancer cells. Once efficient delivery systems are developed, this strategy should prove useful for making human cancers more responsive to p53 activity and more sensitive to chemotherapeutic agents.

Converting cancer genes into killer genes.

Over the past decade, it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death. Theoretically, the proteins encoded by these genes provide excellent targets for new therapeutic agents. Here, we describe a gene therapy approach to specifically kill tumor cells expressing such oncoproteins. In outline, the target oncoprotein binds to exogenously introduced gene products, resulting in transcriptional activation of a toxic gene. As an example, we show that this approach can be used to specifically kill cells overexpressing a mutant p53 gene in cell culture. The strategy may be generally applicable to neoplastic diseases in which the underlying patterns of genetic alterations or abnormal gene expression are known.

Ligand-directed retroviral targeting of human breast cancer cells.

We explored the feasibility of designing retroviral vectors that can target human breast cancer cells with characteristic receptors via ligand-receptor interaction. The ecotropic Moloney murine leukemia virus envelope was modified by insertion of sequences encoding human heregulin. Ecotropic virus, which normally does not infect human cells, when pseudotyped with the modified envelope protein now crosses species to infect human breast cancer cell lines that overexpress HER-2 (human epidermal growth factor receptor; also called ERBB2) and HER-4 (also called ERBB4), while human breast cancer cell lines expressing low levels of these receptors remain resistant to infection. Since about 20% of human breast cancers overexpress HER-2 and some of breast cancer cell lines overexpress both HER-2 and HER-4, cell-specific targeting of retroviral vectors may provide a different approach for in vivo gene therapy of this type of breast cancer.

In vivo gene expression: DNA electrotransfer

The use of electric pulses to deliver therapeutic molecules to tissues and organs in vivo is a rapidly growing field of research. Electrotransfer can be used to deliver a wide range of potentially therapeutic agents, including drugs, proteins, oligonucleotides, RNA and DNA. Optimization of this approach depends upon a number of parameters such as target organ accessibility, cell turnover, microelectrode design, electric pulsing protocols and the physiological response to the therapeutic agent. Many organs have been successfully transfected by electroporation, including skin, liver, skeletal and cardiac muscle, male and female germ cells, artery, gut, kidney, retinal ganglion cells, cornea, spinal cord, joint synovium and brain. Electrotransfer technology is relevant in a variety of research and clinical settings including cancer therapy, modulation of pathogenic immune reactions, delivery of therapeutic proteins and drugs, and the identification of drug targets by the modulation of normal gene expression. This, together with the capacity to deliver very large DNA constructs, greatly expands the research and clinical applications of in vivo DNA electrotransfer.

Rapid screening of 4000 individuals for germ-line variations in the BRAF gene

Background: The BRAF gene is frequently somatically altered in malignant melanoma. A majority of variations are at the valine 600 residue leading to a V600E substitution that constitutively activates the kinase. We screened 4000 patient and control DNAs for germ-line variations at the valine 600 residue. Methods: We developed a novel assay by adapting single-base variation assays and software for MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry to screen for all 5 reported variants at codon 600 of the BRAF gene. We screened a case-control collection comprising samples from 1082 melanoma patients and 154 of their unaffected relatives from 1278 families and from 2744 individuals from 659 unselected twin families with no history of melanoma. A panel of 66 melanoma cell lines was used for variation-positive controls. Results: All melanoma cell lines that we had found previously to carry a codon 600 variation were verified in this study. Three of the 4 possible variants (V600E n = 47, V600K n = 2, V600R n = 1) were detected, but no case of V600D was available. No germ-line variants were found in the samples from the 3980 melanoma patients or from the control individuals. Conclusions: This new assay is a high-throughput, automated alternative to standard sequencing and can be used as a rapid initial screen for somatic variants associated with melanoma. Germ-line variants at valine 600 are unlikely to exist and do not contribute to the reported role of the BRAF gene in melanoma predisposition. (c) 2006 American Association for Clinical Chemistry.

The Feasibly of Functionally Guided Intensity Modulated Radiation Therapy Treatment Planning Using Perfluoropropane-Enhanced MRI for Lung Cancer

Radiotherapy is commonly used to treat lung cancer. However, radiation induced damage to lung tissue is a major limiting factor to its use. To minimize normal tissue lung toxicity from conformal radiotherapy treatment planning, we investigated the use of Perfluoropropane(PFP)-enhanced MR imaging to assess and guide the sparing of functioning lung. Fluorine Enhanced MRI using Perfluoropropane(PFP) is a dynamic multi-breath steady state technique enabling quantitative and qualitative assessments of lung function(1).

Imaging data was obtained from studies previously acquired in the Duke Image Analysis Laboratory. All studies were approved by the Duke IRB. The data was de-identified for this project, which was also approved by the Duke IRB. Subjects performed several breath-holds at total lung capacity(TLC) interspersed with multiple tidal breaths(TB) of Perfluoropropane(PFP)/oxygen mixture. Additive wash-in intensity images were created through the summation of the wash-in phase breath-holds. Additionally, model based fitting was utilized to create parametric images of lung function(1).

Varian Eclipse treatment planning software was used for putative treatment planning. For each subject two plans were made, a standard plan, with no regional functional lung information considered other than current standard models. Another was created using functional information to spare functional lung while maintaining dose to the target lesion. Plans were optimized to a prescription dose of 60 Gy to the target over the course of 30 fractions.

A decrease in dose to functioning lung was observed when utilizing this functional information compared to the standard plan for all five subjects. PFP-enhanced MR imaging is a feasible method to assess ventilatory lung function and we have shown how this can be incorporated into treatment planning to potentially decrease the dose to normal tissue.