Oncolytic Adenoviruses with Radiation Therapy for Treatment of Prostate Cancer

Prostate cancer is the most common cancer in males. Although many patients with localized disease can be cured with surgery and radiotherapy, advanced disease and especially castration resistant metastatic disease remains incurable, with a median life expectancy of less than 18 months. Oncolytic adenoviruses (Ads) are a new promising treatment against cancer due to their innate capacity to kill cancer cells. Viral replication in tumor cells leads to oncolysis and production of a multiplicity of new virions that are capable of further destroying cancerous tissue. Oncolytic Ads can be modified for tumor targeted infection and replication and be armed with therapeutic transgenes to maximize the oncolytic effect. Worldwide, clinical trials with oncolytic Ads have demonstrated good safety while the antitumor efficacy remains to be improved. Importantly, the best responses have been reported when oncolytic adenoviruses have been combined with standard cancer treatments, such as chemotherapy and radiation. Further, a challenge in many virotherapy approaches has been the monitoring of virus replication in vivo. Reporter genes have been extensively used as transgenes to evaluate the biodistribution of the virus and activity of specific promoters. However, these techniques are often limited to preclinical evaluation and not amenable to human use. The aim of the thesis was to find and develop new oncolytic Ads with maximum efficacy against metastatic, castration resistant prostate cancer and study them in vitro and in vivo combined to different forms of radiation therapy. Using combination therapy, we were aiming for better antitumor efficacy with reduced side effects. Capsid modified Ads for enhanced transduction were studied. Serotype 3 targeted chimera, Ad5/3, was found to have enhanced infectivity for prostate cancer and was used for developing new viruses for the study. Correlation between Ad-encoded marker peptide secretion and simultaneous viral replication was evaluated and the effects of radiotherapy on viral replication were studied in detail. We found that the repair of double strand breaks caused by ionizing radiation was inhibited by adenoviral proteins and led to autophagic cell death. Both subcutaneous models and intrapulmonary tumor models mimicking metastatic, aggressive disease were used in vivo. Virus efficacy was evaluated by intratumoral injections. Also, intravenous administration was evaluated to study the effectiveness in metastatic disease. Oncolytic adenovirus treatment led to significant tumor growth control and increased the survival rate of the mice. These results were further improved when oncolytic Ads were combined with radiation therapy. Oncolytic Ads expressing human sodium/iodide transporter (hNIS) as a transgene were evaluated for their oncolytic potency and for the functionality of hNIS in vitro and in vivo. Monitoring of viral replication was also assessed using different imaging modalities relative to clinical use. SPECT imaging of tumor-bearing mice was evaluated and combined with simultaneous CT-scanning to obtain important anatomical information on biodistribution, also in a three-dimensional form. It was shown that hNIS-expressing adenoviruses could harbour a bi-functional transgene allowing for localization and imaging of viral replication. Targeted radiotherapy was applied by systemic radioiodide administration and resulted in iodide accumulation into Ad-infected tumor. The combination treatment showed significantly enhanced antitumor efficacy in mice bearing prostate cancer tumors. In summary, the results presented above aim to provide new treatment modalities for castration resistant prostate cancer. Molecular insights were provided for better understanding of the benefits of combined radiation therapy and oncolytic adenoviruses, which will hopefully facilitate the translation of the approach into clinical use for humans.

Estramustine and its Derivatives in Potentiating Radiotherapy of Prostate Cancer

The purpose of this study was to deepen our knowledge of the combined use of estramustine and radiotherapy in the treatment of prostate cancer. Prostate cancer is a common disease, with a high variability between subjects in its malignant potential. In many cases, the disease is an incidental finding with little or no clinical significance. In other cases, however, prostate cancer may be an aggressive malignant disease, which, if the initial treatment fails, lacks an effective cure and may lead to severe symptoms, metastasis, and death despite all treatment. In many cases, the methods of treatment available at the moment provide cure or significant regression of symptoms, but often at the cost of considerable side effects. Estramustine, a cytostatic drug used for treating advanced cancer of the prostate, has been shown to inhibit prostate cancer progression and also to increase the sensitivity of cancer cells to radiotherapy. The goals of this study were, first, to find out whether it is possible to use either estramustine or an antibody against estramustine binding protein as carrier molecules for bringing therapeutic radioisotopes into prostate cancer cells, and, secondly, to gain more understanding of the mechanisms behind the known radiosensitising effect of estramustine. Estramustine and estramustine binding protein antibody were labelled with iodine-125 to study the biodistribution of these substances in mice. In the first experiment, both of the substances accumulated in the prostate, but radioiodinated estramustine also showed affinity to the liver and the lungs. Since the radiolabelled antibody was found out to accumulate more selectively to the prostate, we studied its biodistribution in nude mice with DU-145 human prostate cancer implants. In this experiment, the prostate and the tumour accumulated more radioactivity than other organs, but we concluded that the difference in the dose of radiation compared to other organs was not sufficient for the radioiodinated antibody to be advocated as a carrier molecule for treating prostate cancer. Mice with similar DU-145 prostate cancer implants were then treated with estramustine and external beam irradiation, with and without neoadjuvant estramustine treatment. The tumours responded to the treatment as expected, showing the radiation potentiating effect of estramustine. In the third experiment, this effect was found without an increase in the amount of apoptosis in the tumour cells, despite previous suggestions to the contrary. In the fourth experiment, we gave a similar treatment to the mice with DU-145 tumours. A reduction in proliferation was found in the groups treated with radiotherapy, and an increased amount of tumour hypoxia and tumour necrosis in the group treated with both neoadjuvant estramustine and radiation. This finding is contradictory to the suggestion that the radiation sensitising effect of estramustine could be attributed to its angiogenic activity.

Nanomaterial based Magnetic Resonance Imaging of Cancer

Magnetic Resonance Imaging (MRI) is a widely used non-invasive medical tool for detection and diagnosis of cancer. In recent years, MRI has witnessed significant contributions from nanotechnology to incorporate advanced features such as multimodality of nanoparticles, therapeutic delivery, specific targeting and the optical detectability for molecular imaging. Accurate composition, right scheme of surface chemistry and properly designed structure is essential for achieving desired properties of nanomaterials such as non-fouling surface, high imaging contrast, chemical stability, target specificity and/or multimodality. This review provides an overview of the recent progress in theranostic nanomaterials in imaging and the development of nanomaterial based magnetic resonance imaging of cancer. In particular, targeted theranostics is a promising approach along with its targeting strategy in cancer treatment using MRI and multimodal imaging. We also discuss recent advances in integrin mediated targeted MRI of cancer.

Rrp1B gene polymorphism (1307T > C) in metastatic progression of breast cancer

Rrp1B (ribosomal RNA processing1 homolog B) is a novel candidate metastasis modifier gene in breast cancer. Functional gene assays demonstrated that a physical and functional interaction existing between Rrp1b and metastasis modifier gene SIPA1 causes reduction in the tumor growth and metastatic potential. Ectopic expression of Rrp1B modulates various metastasis predictive extra cellular matrix (ECM) genes associated with tumor suppression. The aim of this study is to determine the functional significance of single nucleotide polymorphism (SNP) in human Rrp1B gene (1307 T > C; rs9306160) with breast cancer development and progression. The study consists of 493 breast cancer cases recruited from Nizam's Institute of Medical Sciences, Hyderabad, and 558 age-matched healthy female controls from rural and urban areas. Genomic DNA was isolated by non-enzymatic method. Genotyping was done by amplification refractory mutation system (ARMS-PCR) method. Genotypes were reconfirmed by sequencing and results were analyzed statistically. We have performed Insilco analysis to know the RNA secondary structure by using online tool m fold. The TT genotype and T allele frequencies of Rrp1B1307 T > C polymorphism were significantly elevated in breast cancer (chi (2); p = < 0.008) cases compared to controls under different genetic models. The presence of T allele had conferred 1.75-fold risk for breast cancer development (OR = 1.75; 95 % CI = 1.15-2.67). The frequency of TT genotype of Rrp1b 1307T > C polymorphism was significantly elevated in obese patients (chi (2); p = 0.008) and patients with advanced disease (chi (2); p = 0.01) and with increased tumor size (chi (2); p = 0.01). Moreover, elevated frequency of T allele was also associated with positive lymph node status (chi (2); p = 0.04) and Her2 negative receptor status (chi (2); p = 0.006). Presence of Rrp1b1307TT genotype and T allele confer strong risk for breast cancer development and progression.

A feasibility study evaluating docetaxel-based sequential and combination regimens in the adjuvant therapy of node-positive breast cancer

BACKGROUND AND PURPOSE: Docetaxel is an active agent in the treatment of metastatic breast cancer. We evaluated the feasibility of docetaxel-based sequential and combination regimens as adjuvant therapies for patients with node-positive breast cancer. PATIENTS AND METHODS: Three consecutive groups of patients with node-positive breast cancer or locally-advanced disease, aged < or = 70 years, received one of the following regimens: a) sequential A-->T-->CMF: doxorubicin 75 mg/m2 q 3 weeks x 3, followed by docetaxel 100 mg/m2 q 3 weeks x 3, followed by i.v. CMF days 1 + 8 q 4 weeks x 3; b) sequential accelerated A-->T-->CMF: A and T were administered at the same doses q 2 weeks; c) combination therapy: doxorubicin 50 mg/m2 + docetaxel 75 mg/m2 q 3 weeks x 4, followed by CMF x 4. When indicated, radiotherapy was administered during or after CMF, and tamoxifen started after the end of CMF. RESULTS: Seventy-nine patients have been treated. Median age was 48 years. A 30% rate of early treatment discontinuation was observed in patients receiving the sequential accelerated therapy (23% during A-->T), due principally to severe skin toxicity. Median relative dose-intensity was 100% in the three treatment arms. The incidence of G3-G4 major toxicities by treated patients, was as follows: skin toxicity a: 5%; b: 27%; c: 0%; stomatitis a: 20%; b: 20%; c: 3%. The incidence of neutropenic fever was a: 30%; b: 13%; c: 48%. After a median follow-up of 18 months, no late toxicity has been reported. CONCLUSIONS: The accelerated sequential A-->T-->CMF treatment is not feasible due to an excess of skin toxicity. The sequential non accelerated and the combination regimens are feasible and under evaluation in a phase III trial of adjuvant therapy.

3-year results of docetaxel-based sequential and combination regimens in the adjuvant therapy of node-positive breast cancer: a pilot study.

BACKGROUND: Docetaxel has proven efficacy in metastatic breast cancer. In this pilot study, we explored the efficacy/feasibility of docetaxel-based sequential and combination regimens as adjuvant therapy of node-positive breast cancer. PATIENTS AND METHODS: From March 1996 till March 1998, four consecutive groups of patients with stages II and III breast cancer, aged < or = 70 years, received one of the following regimens: a) sequential Doxorubicin (A) --> Docetaxel (T) --> CMF (Cyclophosphamide+Methotrexate+5-Fluorouracil): A 75 mg/m q 3 wks x 3, followed by T100 mg/m2 q 3 wks x 3, followed by i.v. CMF Days 1+8 q 4 wks x 3; b) sequential accelerated A --> T --> CMF: A and T administered at the same doses q 2 wks with Lenograstin support; c) combination therapy: A 50 mg/m2 + T 75 mg/m2 q 3 wks x 4, followed by CMF x 4; d) sequential T --> A --> CMF: T and A, administered as in group a), with the reverse sequence. When indicated, radiotherapy was administered during or after CMF, and Tamoxifen after CMF. RESULTS: Ninety-three patients were treated. The median age was 48 years (29-66) and the median number of positive axillary nodes was 6 (1-25). Tumors were operable in 94% and locally advanced in 6% of cases. Pathological tumor size was >2 cm in 72% of cases. There were 21 relapses, (18 systemic, 3 locoregional) and 11 patients (12%) have died from disease progression. At median follow-up of 39 months (6-57), overall survival (OS) was 87% (95% CI, 79-94%) and disease-free survival (DFS) was 76% (95% CI, 67%-85%). CONCLUSION: The efficacy of these docetaxel-based regimens, in terms of OS and DFS, appears to be at least as good as standard anthracycline-based adjuvant chemotherapy (CT), in similar high-risk patient populations.

c-Myc is required for maintenance of glioma cancer stem cells.

BACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.

Genetic Variants of the MDM2 Gene Are Predictive of Treatment-Related Toxicities and Overall Survival in Patients With Advanced NSCLC.

INTRODUCTION: Platinum agents can cause the formation of DNA adducts and induce apoptosis to eliminate tumor cells. The aim of the present study was to investigate the influence of genetic variants of MDM2 on chemotherapy-related toxicities and clinical outcomes in patients with advanced non-small-cell lung cancer (NSCLC). MATERIALS AND METHODS: We recruited 663 patients with advanced NSCLC who had been treated with first-line platinum-based chemotherapy. Five tagging single nucleotide polymorphisms (SNPs) in MDM2 were genotyped in these patients. The associations of these SNPs with clinical toxicities and outcomes were evaluated using logistic regression and Cox regression analyses. RESULTS: Two SNPs (rs1470383 and rs1690924) showed significant associations with chemotherapy-related toxicities (ie, overall, hematologic, and gastrointestinal toxicity). Compared with the wild genotype AA carriers, patients with the GG genotype of rs1470383 had an increased risk of overall toxicity (odds ratio [OR], 3.28; 95% confidence interval [CI], 1.34-8.02; P = .009) and hematologic toxicity (OR, 4.10; 95% CI, 1.73-9.71; P = .001). Likewise, patients with the AG genotype of rs1690924 showed more sensitivity to gastrointestinal toxicity than did those with the wild-type homozygote GG (OR, 2.32; 95% CI, 1.30-4.14; P = .004). Stratified survival analysis revealed significant associations between rs1470383 genotypes and overall survival in patients without overall or hematologic toxicity (P = .007 and P = .0009, respectively). CONCLUSION: The results of our study suggest that SNPs in MDM2 might be used to predict the toxicities of platinum-based chemotherapy and overall survival in patients with advanced NSCLC. Additional validations of the association are warranted.

Survival in 176 patients with inoperable non small cell lung cancer treated initially with chemotherapy in a district general hospital

In advanced non-small cell lung cancer (NSCLC) platinum based chemotherapy with second generation drugs improves median survival (MS) to 8 months and 29% and 10% at 1 and 2 years. Platinum with a third generation drug can improve survival further (BMJ 1995;311: 899) (Spiro et al. Thorax 2004;59:828 Big Lung Trial; N Engl J Med 2003;346:92 ECOG study). NICE now recommends chemotherapy with platinum and a third generation drug for inoperable NSCLC as the first treatment modality. Methods: We audited survival of 176/461 consecutive patients referred for at least 3 courses of platinum and either gemcitabine or vinorelbine from July 2001 to December 2005. Minimal follow up 17 months. Chemotherapy was given on site. Radical radiotherapy for stage IIIA, palliative radiotherapy and second line drugs were given as felt appropriate. Results: 64% were male. 30 (17%) were <55 years ; 66 (37.5%) age 55–65 years; 63 (35.8%) aged 66–75 and 16 (9.1%) >75 years. 5 (2.8%) were stage II; 46 (26%) stage IIIA; 68 (38%) stage IIIB and 55 (30.8%) stage IV. 68 (38%) had 0– 2 courses; 63 (36%) 3 courses and 44 (25%) had 4 or more.