Targeted Therapy Possibilities for Metastatic Melanoma

The incidence of malignant melanoma of the skin has been steadily rising worldwide during the past decades. Most early detected primary tumors can be removed surgically and the prognosis is good. However, at the same time there still is no permanent cure for metastatic melanoma and its prognosis is poor, although lately new effective drugs have emerged. In this thesis, four different approaches of experimental therapy for metastatic melanoma were studied. Endogenous cis-Urocanic acid (UCA) is found in every individual’s skin, where exposure to UV light from the sun generates it from its inactive trans conformation. Cis- UCA was found to destroy malignant melanoma cells in culture under an acidified pH and sufficient concentration through caspase-3 mediated apoptosis. Furthermore, cis-UCA is able to considerably diminish the growth rate in human melanoma tumors on living SCID mice. Using replication-competent Semliki Forest viruses, human melanoma tumors grown in SCID mice were dramatically shrunken as the fulminant production of viruses in melanoma cells leads them to apoptosis within 72 hours. Small oligopeptides attaching to melanoma cells were identified using in vivo phage display. The melanoma-specific peptides found were further tested in vitro on adenoviruses. Ultimately, the adenoviral retargeting using the peptides was tested in vivo. One peptide homed to human transferring receptor upregulated on melanoma cells. In order to kill the malignant melanoma cells with the retargeted adenoviruses, the viruses should carry genetic material producing apoptotic proteins in the cancer tissue. TIMP-3 has been identified as a good candidate for such a protein, as it inhibits malignant cell adhesion as well as promotes apoptosis through a caspase-8 pathway. It is further shown here that adenovirally delivered TIMP-3 is even more potent, as it could kill non-adherent cancer cells, lacking the fully functional death receptor signalling pathway. Adenovirally delivered TIMP-2 also showed marked antitumor effects in human malignant melanoma xenografts on SCID mice both in ex vivo and systemic delivery.

Effect of estrogen receptor-alpha (ESR1) gene polymorphism on high density lipoprotein levels in response to hormone replacement therapy

Studies have shown that estrogen replacement therapy and estrogen plus progestin replacement therapy alter serum levels of total, LDL and HDL cholesterol levels. However, HDL cholesterol levels in women vary considerably in response to hormone replacement therapy (HRT). A significant portion of the variability of these levels has been attributed to genetic factors. Therefore, we investigated the influence of estrogen receptor-alpha (ESR1) gene polymorphisms on HDL levels in response to postmenopausal HRT. We performed a prospective cohort study on 54 postmenopausal women who had not used HRT before the study and had no significant general medical illness. HRT consisted of conjugated equine estrogen and medroxyprogesterone acetate continuously for 1 year. The lipoprotein levels were measured from blood samples taken before the start of therapy and after 1 year of HRT. ESR1 polymorphism (MspI C>T, HaeIII C>T, PvuII C>T, and XbaI A>G) frequencies were assayed by restriction fragment length polymorphism. A general linear model was used to describe the relationships between HDL levels and genotypes after adjusting for age. A significant increase in HDL levels was observed after HRT (P = 0.029). Women with the ESR1 PvuII TT genotype showed a statistically significant increase in HDL levels after HRT (P = 0.032). No association was found between other ESR1 polymorphisms and HDL levels. According to our results, the ESR1 PvuII TT genotype was associated with increased levels of HDL after 1 year of HRT.

Influence of apolipoprotein E genotype and dietary alpha-tocopherol on redox status and C-reactive protein levels in apolipoprotein E3 and E4 targeted replacement mice

The molecular basis of the positive association between apoE4 genotype and CVD remains unclear. There is direct in vitro evidence indicating that apoE4 is a poorer antioxidant relative to the apoE3 isoform, with some indirect in vivo evidence also available. Therefore it was hypothesised that apoE4 carriers may benefit from alpha-tocopherol (alpha-Toc) supplementation. Targeted replacement mice expressing the human apoE3 and apoE4 were fed with a diet poor (0 mg/kg diet) or rich (200 mg/kg diet) in alpha-Toc for 12 weeks. Neither apoE genotype nor dietary alpha-Toc exerted any effects on the antioxidant defence system, including glutathione, catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities. In addition, no differences were observed in mitogen-induced lymphocyte proliferation. alpha-Toc concentrations were modestly higher in plasma and lower in tissues of apoE4 compared with apoE3 mice, with the greatest differences evident in the lung, suggesting that an apoE4 genotype may reduce alpha-Toc delivery to tissues. A tendency towards increased plasma F-2-isoprostanes in apoE4 mice was observed, while liver thiobarbituric acid-reactive substances did not differ between apoE3 and apoE4 mice. In addition, C-reactive protein (CRP) concentrations were reduced in apoE4 mice indicating that this positive effect on CRP may in part negate the increased CVD risk associated with an apoE4 genotype.

Effect of apoE genotype and dietary quercetin on blood lipids and TNF-alpha levels in apoE3 and apoE4 targeted gene replacement mice

The aim of this study was to determine the effect of dietary quercetin supplementation on blood lipids and TNF-alpha levels according to the apoE genotype in apoE3 and apoE4 targeted gene replacement mice. In a two-factorial design female apoE3 and apoE4 mice were fed semi-synthetic diets without (controls) and with quercetin (2 mg/g diet) for 6 weeks. Feeding the quercetin-supplemented diets significantly increased plasma levels of quercetin and isorhamnetin both in apoE3 and apoE4 mice. There was no significant effect of apoE genotype on plasma quercetin levels. ApoE3 and apoE4 transgenic mice exhibited similar plasma levels of apoE and cholesterol which were not significantly affected by dietary quercetin supplementation. In mice receiving the basal diet without quercetin supplementation, levels of TNF-alpha in whole blood stimulated ex vivo with lipopolysaccharide were higher in apoE3 as compared to apoE4 transgenic mice. Dietary quercetin significantly lowered levels of TNF-alpha by 44% in apoE3 mice relative to apoE3 mice receiving the unsupplemented diets. In apoE4 mice a moderate (20%) but not significant decrease in TNF-alpha levels in response to the quercetin supplementation was evident. Following quercetin supplementation TNF-alpha levels were similar between apoE3 and apoE4 transgenic mice. Current findings indicate that apoE3 mice are more responsive to the TNF-alpha lowering properties of dietary quercetin supplementation as compared to apoE4 animals.

Keratinocyte growth factor: a new mesothelial targeted therapy to reduce postoperative pericardial adhesions

Background: Several methods have been utilized to prevent pericardial and retrosternal adhesions, but none of them evaluated the mesothelial regenerative hypothesis. There are evidences that the mesothelial trauma reduces pericardial fibrinolytic capability and induces an adhesion process. Keratinocyte growth factor (KGF) has proven to improve mesothelial cells proliferation. This study investigated the influence of keratinocyte growth factor in reducing post-surgical adhesions. Methods: Twelve pigs were operated and an adhesion protocol was employed. Following a stratified randomization, the animals received a topical application of KGF or saline. At 8 weeks, intrapericardial adhesions were evaluated and a severity score was established. The time spent to dissect the adhesions and the amount of sharp dissection used, were recorded. Histological sections were stained with sirius red and morphometric analyses were assessed with a computer-assisted image analysis system. Results: The severity score was lower in the KGF group than in the control group (11.5 vs 17, p = 0.005). The dissection time was lower in the KGF group (9.2 +/- 1.4 min vs 33.9 +/- 9.2 min, p = 0.004) and presented a significant correlation with the severity score (r = 0.83, p = 0.001). A significantly less sharp dissection was also required in the KGF group. Also, adhesion area and adhesion collagen were significantly tower in the KGF group than in the control group. Conclusion: The simulation of pericardial cells with KGF reduced the intensity of postoperative adhesions and facilitated the re-operation. This study suggests that the mesothelial regeneration is the new horizon in anti-adhesion therapies. (C) 2008 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

The results of a randomized trial looking at 24 weeks vs 48 weeks of treatment with peginterferon alpha-2a (40 kDa) and ribavirin combination therapy in patients with chronic hepatitis C genotype 1

Peginterferon-alpha plus ribavirin is the most effective therapy for chronic hepatitis C. This study was designed to evaluate the effect of peginterferon alpha-2a (40 kDa) plus ribavirin on sustained virological response (SVR) when administered for 24 vs 48 weeks in genotype 1 naive patients. One hundred and seventeen patients were enrolled in this controlled trial. Genotype 1 patients were randomized to 24 weeks treatment vs 48 weeks treatment. Genotype non-1 patients received 24 weeks treatment as an observational group. Outcomes were SVR (defined by hepatitis C virus-RNA-negative at week 24 of follow-up) and tolerability across the study period. The end-of-treatment response was 59% for genotype 1 (24 weeks treatment), 80% for genotype 1 (48 weeks treatment) and 92% for genotype non-1 (24 weeks treatment). The end-of-follow-up response was 19% (95% confidence interval (CI): 7.2-36.4) (genotype 1, 24 weeks) and 48% (95% CI: 30.2-66.9; P = 0.0175) (genotype 1, 48 weeks). Among genotype non-1, SVR was 76% (95% CI: 62.3-86.5). There were no unexpected adverse events.Almost half of the genotype 1 patients achieved an SVR after 48 weeks treatment with peginterferon alpha-2a (40 kDa) and low-dose ribavirin and confirmed that they should be treated for 48 weeks. Safety profile was acceptable.

Mucopolysaccharidoses in northern Brazil: targeted mutation screening and urinary glycosaminoglycan excretion in patients undergoing enzyme replacement therapy

Mucopolysaccharidoses (MPS) are rare lysosomal disorders caused by the deficiency of specific lysosomal enzymes responsible for glycosaminoglycan (GAG) degradation. Enzyme Replacement Therapy (ERT) has been shown to reduce accumulation and urinary excretion of GAG, and to improve some of the patients' clinical signs. We studied biochemical and molecular characteristics of nine MPS patients (two MPS I, four MPS II and three MPS VI) undergoing ERT in northern Brazil. The responsiveness of ERT was evaluated through urinary GAG excretion measurements. Patients were screened for eight common MPS mutations, using PCR, restriction enzyme tests and direct sequencing. Two MPS I patients had the previously reported mutation p.P533R. In the MPS II patients, mutation analysis identified the mutation p.R468W, and in the MPS VI patients, polymorphisms p.V358M and p.V376M were also found. After 48 weeks of ERT, biochemical analysis showed a significantly decreased total urinary GAG excretion in patients with MPS I (p < 0.01) and MPS VI (p < 0.01). Our findings demonstrate the effect of ERT on urinary GAG excretion and suggest the adoption of a screening strategy for genotyping MPS patients living far from the main reference centers.

Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-gamma cells (P<0.05), CD8-IFN-gamma cells (P<0.01) and CD49b-tumor necrosis factor-alpha cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.

Evaluation of Antitumoral activity of bone targeted drugs/conventional chemotherapies and identification of biomarkers for the selection of patients with breast cancer for the bone targeted therapy in adjuvant setting

Bone metastases are responsible for different clinical complications defined as skeletal-related events (SREs) such as pathologic fractures, spinal cord compression, hypercalcaemia, bone marrow infiltration and severe bone pain requiring palliative radiotherapy. The general aim of these three years research period was to improve the management of patients with bone metastases through two different approaches of translational research. Firstly in vitro preclinical tests were conducted on breast cancer cells and on indirect co-colture of cancer cells and osteoclasts to evaluate bone targeted therapy singly and in combination with conventional chemotherapy. The study suggests that zoledronic acid has an antitumor activity in breast cancer cell lines. Its mechanism of action involves the decrease of RAS and RHO, as in osteoclasts. Repeated treatment enhances antitumor activity compared to non-repeated treatment. Furthermore the combination Zoledronic Acid + Cisplatin induced a high antitumoral activity in the two triple-negative lines MDA-MB-231 and BRC-230. The p21, pMAPK and m-TOR pathways were regulated by this combined treatment, particularly at lower Cisplatin doses. A co-colture system to test the activity of bone-targeted molecules on monocytes-breast conditioned by breast cancer cells was also developed. Another important criticism of the treatment of breast cancer patients, is the selection of patients who will benefit of bone targeted therapy in the adjuvant setting. A retrospective case-control study on breast cancer patients to find new predictive markers of bone metastases in the primary tumors was performed. Eight markers were evaluated and TFF1 and CXCR4 were found to discriminate between patients with relapse to bone respect to patients with no evidence of disease. In particular TFF1 was the most accurate marker reaching a sensitivity of 63% and a specificity of 79%. This marker could be a useful tool for clinicians to select patients who could benefit for bone targeted therapy in adjuvant setting.

Serum anti-interferon alpha antibodies in chronic hepatitis C patients treated with pegylated interferon alpha containing therapy

The development of anti-IFNα antibodies is an occurrence described in chronic hepatitis C patients during treatment with Interferonα/PEG-Interferonα. However, its relevance, especially in difficult-to treat patients, has not been defined. Methods: We retrospectively measured the serum levels of anti-IFNα antibodies (baseline and week 12) and IFNα levels (week 12) by ELISA in 76 previous non-responders, and in 14 naive patients treated with Pegylated-IFNα and Ribavirin. A group of 57 healthy donors (HD) was also assessed as control. Positivity to anti-IFNα antibodies was established on the values of HD. Results: Baseline anti-IFNα antibodies were detected in 15.5% of patients and in 7% of HD, with significantly higher concentrations in patients than HD (181.5±389.9 vs 95.9±143.0 ng mL−1, p=0.0023). All positive patients were IFNα-experienced. At week 12, the prevalence of positivity increased to 22.3 and 28.5% in experienced and naïve patients, respectively, and the levels of anti-IFNα antibodies did not differ between the two groups (391±792.3 vs 384.7±662.6 ng mL−1, respectively). IFNα concentrations were significantly lower in antibody-positive patients than in antibody-negatives (988.2±1402 vs 3462±830.8 pg mL−1, p≤0.0001) and the levels of antibodies and IFNα were inversely correlated (r=-0.405, p=0.0001). The antibody-positive population clustered in null responders (67%) and 19/21 patients (90%) did not achieve SVR. Conclusions: The development of anti-IFNα antibodies is a non-negligible occurrence in patients treated with PEG-IFNα, is stable over time, and has a relevant clinical impact when associated with low levels of circulating PEG-IFNα. It should be considered in patients undergoing treatments including PEG-IFNα.

Cardiovascular complications of conventional and targeted adjuvant breast cancer therapy

Adjuvant therapy has improved the survival of women with early breast cancer (BC). Meta-analyses suggest that anthracycline-based regimens reduced the annual BC death rate by 40% in women below the age of 50 and 20% in older women. Novel agents designed to modulate abnormal growth factor signaling in and around the BC cell further increase patients' chances of survival. However, both conventional chemotherapeutic agents as well as some of the novel signaling inhibitors can induce important cardiovascular side-effects, potentially attenuating the progress made in recent years. The mechanism of cancer drug-induced cardiovascular complications varies greatly with some compounds inducing irreversible myocardial cell damage, while others lead to temporary cell dysfunction. The challenge of the future will be to prospectively discriminate between irreversible damage which can lead to progressive cardiovascular disease and reversible cardiovascular dysfunctions without further prognostic implications. Since adjuvant therapy for BC is potentially curative, emphasis must be placed on finding treatments combining maximum efficacy with the minimum of long-term side-effects in order to achieve survival with preserved quality of life.

Alpha- versus beta-particle radiopeptide therapy in a human prostate cancer model (213Bi-DOTA-PESIN and 213Bi-AMBA versus 177Lu-DOTA-PESIN)

Recurrent prostate cancer presents a challenge to conventional treatment, particularly so to address micrometastatic and small-volume disease. Use of α-radionuclide therapy is considered as a highly effective treatment in such applications due to the shorter range and exquisite cytotoxicity of α-particles as compared with β-particles. (213)Bi is considered an α-emitter with high clinical potential, due to its short half-life (45.6 minutes) being well matched for use in peptide-receptor radionuclide α-therapy; however, there is limited knowledge available within this context of use. In this study, two novel (213)Bi-labeled peptides, DOTA-PEG(4)-bombesin (DOTA-PESIN) and DO3A-CH(2)CO-8-aminooctanoyl-Q-W-A-V-G-H-L-M-NH(2) (AMBA), were compared with (177)Lu (β-emitter)-labeled DOTA-PESIN in a human androgen-independent prostate carcinoma xenograft model (PC-3 tumor). Animals were injected with (177)Lu-DOTA-PESIN, (213)Bi-DOTA-PESIN, or (213)Bi-AMBA to determine the maximum tolerated dose (MTD), biodistribution, and dosimetry of each agent; controls were left untreated or were given nonradioactive (175)Lu-DOTA-PESIN. The MTD of (213)Bi-DOTA-PESIN and (213)Bi-AMBA was 25 MBq (0.68 mCi) whereas (177)Lu-DOTA-PESIN showed an MTD of 112 MBq (3 mCi). At these dose levels, (213)Bi-DOTA-PESIN and (213)Bi-AMBA were significantly more effective than (177)Lu-DOTA-PESIN. At the same time, (177)Lu-DOTA-PESIN showed minimal, (213)Bi-DOTA-PESIN slight, and (213)Bi-AMBA marked kidney damage 20 to 30 weeks posttreatment. These preclinical data indicate that α-therapy with (213)Bi-DOTA-PESIN or (213)Bi-AMBA is more efficacious than β-therapy. Furthermore, (213)Bi-DOTA-PESIN has a better safety profile than (213)Bi-AMBA, and represents a possible new approach for use in peptide-receptor radionuclide α-therapy treating recurrent prostate cancer.