Targeting the epigenome in malignant melanoma: Facts, challenges and therapeutic promises.

Malignant melanoma is the most lethal type of skin cancer with high rates of mortality. Although current treatment options provide a short-clinical benefit, acquired-drug resistance highlights the low 5-year survival rate among patients with advanced stage of the disease. In parallel, the involvement of an aberrant epigenetic landscape, (e.g., alterations in DNA methylation patterns, histone modifications marks and expression of non-coding RNAs), in addition to the genetic background, has been also associated with the onset and progression of melanoma. In this review article, we report on current therapeutic options in melanoma treatment with a focus on distinct epigenetic alterations and how their reversal, by specific drug compounds, can restore a normal phenotype. In particular, we concentrate on how single and/or combinatorial therapeutic approaches have utilized epigenetic drug compounds in being effective against malignant melanoma. Finally, the role of deregulated epigenetic mechanisms in promoting drug resistance to targeted therapies and immune checkpoint inhibitors is presented leading to the development of newly synthesized and/or improved drug compounds capable of targeting the epigenome of malignant melanoma.

Tumor microenvironment, immune response and post-radiotherapy tumor clearance.

Radiotherapy is the treatment of choice for many cancer patients. Residual tumor leads to local recurrence after a period of an equilibrium created between proliferating, quiescent and dying cancer cells. The tumor microenvironment is a main obstacle for the efficacy of radiotherapy, as impaired blood flow leads to hypoxia, acidity and reduced accessibility of radiosensitizers. Eradication of remnant disease is an intractable clinical quest. After more than a century of research, anti-tumor immunity has gained a dominant position in oncology research and therapy. Immune cells play a significant role in the eradication of tumors during and after the completion of radiotherapy. The tumor equilibrium reached in the irradiated tumor may shift towards cancer cell eradication if the immune response is appropriately modulated. In the modern immunotherapy era, clinical trials are urged to standardize immunotherapy schemes that could be safely applied to improve clearance of the post-radiotherapy remnant disease.

Characterization of the "Autophagic Flux" in Prostate Cancer Tissue Biopsies by LC3A/LAMP2a Immunofluorescence and Confocal Microscopy.

A method is described for the immunostaining of human prostate cancer biopsies for the autophagic marker LC3 and the lysosomal protein LAMP2A. Using this combination we can provide some information on autophagic flux, and specific patterns of staining have been recognized for normal prostate tissue and prostatic carcinomas.

Lactate dehydrogenase 5 isoenzyme overexpression defines resistance of prostate cancer to radiotherapy.

BACKGROUND: Radiotherapy provides high-cure rates in prostate cancer. Despite its overall slow clinical growth, high proliferation rates documented in a subset of tumours relate to poor radiotherapy outcome. This study examines the role of anaerobic metabolism in prostate cancer growth and resistance to radiotherapy. METHODS: Biopsy samples from 83 patients with prostate cancer undergoing radical hypofractionated and accelerated radiotherapy were analysed for MIB1 proliferation index and for lactate dehydrogenase isoenzyme LDH5, a marker of tumour anaerobic metabolism. Ninety-five surgical samples were in parallel analysed. Correlation with histopathological variables, PSA and radiotherapy outcome was assessed. Dose-response experiments were performed in PC3 and DU145 cancer cell lines. RESULTS: High MIB1 index (noted in 25% of cases) was directly related to Gleason score (P<0.0001), T3-stage (P=0.0008) and PSA levels (P=0.03). High LDH5 (noted in 65% of cases) was directly related to MIB1 index (P<0.0001), Gleason score (P=0.02) and T3-stage (P=0.001). High Gleason score, MIB1, LDH5 and PSA levels were significantly related to poor BRFS (P=0.007, 0.01, 0.03 and 0.01, respectively). High Gleason score (P=0.04), LDH5 (P=0.01) and PSA levels (P=0.003) were significantly related to local recurrence. MIB1 and T-stage did not affect local control. Silencing of LDHA gene in both prostate cancer cell lines resulted in significant radiosensitisation. CONCLUSIONS: LDH5 overexpression is significantly linked to highly proliferating prostate carcinomas and with biochemical failure and local relapse following radiotherapy. Hypoxia and LDHA targeting agents may prove useful to overcome radioresistance in a subgroup of prostate carcinomas with anaerobic metabolic predilection.

Prospective neoadjuvant analysis of PET imaging and mechanisms of resistance to Trastuzumab shows role of HIF1 and autophagy.

BACKGROUND: Although Trastuzumab has improved survival of HER2+ breast cancer patients, resistance to the agent pre-exists or develops through the course of therapy. Here we show that a specific metabolism and autophagy-related cancer cell phenotype relates to resistance of HER2+ breast cancer to Trastuzumab and chemotherapy. METHODS: Twenty-eight patients with locally advanced primary breast cancer were prospectively scheduled to received one cycle of Trastuzumab followed by a new biopsy on day 21, followed by taxol/Trastuzumab chemotherapy for four cycles before surgery. FDG PET/CT scan was used to monitor tumour response. Tissue samples were immunohistochemically analysed for metabolism and autophagy markers. RESULTS: In pre-Trastuzumab biopsies, the LC3A+/HER2+ cell population was correlated with HIF1α expression (P=0.01), while GLUT1 and LC3B expression were correlated with Ki67 proliferation index (P=0.01 and P=0.01, respectively). FDG PET tumour dimensions before therapy were correlated with LC3B expression (P=0.005). Administration of Trastuzumab significantly reduced clinical and PET-detected tumour dimensions (P<0.01). An inverse association of tumour response with the percentage of cells expressing HIF1α at baseline was documented (P=0.01). Administration of Trastuzumab resulted in a decrease of the proliferation index (P=0.004), GLUT1 (P=0.04) and HER2 (P=0.01) expression. In contrast, the percentage of LC3A+/HER2+ cells was increased (P=0.01). High baseline HIF1α expression was the only parameter associated with poorer pathological response to preoperative chemotherapy (P=0.001). CONCLUSIONS: As the HER2+/LC3A+ phenotype, which often overexpresses HIF1α, is a major subpopulation increasing after therapy with Trastuzumab, LC3A- and HIF1α-targeting therapies should be investigated for the augmentation of anti-HER2 therapy efficacy.

Autophagy and hypoxia in colonic adenomas related to aggressive features.

AIM: The study investigated whether autophagic activity and hypoxia parallel the adenoma-carcinoma sequence. METHOD: The study comprised 120 tubular adenomas with high-grade dysplasia, including 22 with questionable evidence of invasion, 37 with definite stromal invasion and 29 with severely dysplastic adenoma, 10 traditional serrated adenomas and 22 classical tubular adenomas lacking aggressive features. The samples were stained immunohistochemically for autophagy (LC3A and Beclin-1) and hypoxia-inducible factor1-alpha (HIF1α) markers. RESULTS: LC3A was detected as diffuse cytoplasmic staining and as dense "stone-like" structures (SLS) within cytoplasmic vacuoles. Beclin-1 reactivity was purely cytoplasmic, whereas that of HIF1α was both cytoplasmic and nuclear. SLS counts in noninvasive, nontransformed areas of tubular adenomas were consistently low (median SLS = 0.5; 200× magnification), whereas a progressive increase was noted from areas of equivocal invasion (median SLS = 1.3; 200× magnification) and intramucosal carcinoma (median SLS = 1.4; 200× magnification) to unequivocal invasive foci (median SLS = 2.1; 200× magnification) (P < 0.0001). A similar association was shown for Beclin-1 and HIF1α expression (P < 0.05). Traditional serrated adenomas yielded low SLS counts and weak HIF1α reactivity, but high cytoplasmic LC3A and Beclin-1 expression (P < 0.01). CONCLUSION: A hypoxia-driven autophagy in adenomatous polyps, when particularly intense and localized, is commonly associated with early invasion or severely dysplastic adenoma.

Reduction of the radiation dose received by interventional cardiologists following training in radiation protection.

The University General Hospital of Alexandroupolis was established in 2003 to cover Eastern Macedonia and Thrace Districts of Northern Greece. The hospital has two interventional cardiology units and the occupational radiation exposure of the cardiologists was the highest of all specialties using ionising radiation. In order to aid in decreasing the radiation dose levels, a seminar was organised for all personnel working in interventional radiology field. After this, an important reduction of the radiation dose of the cardiologists was noted. Training in radiation protection is essential to reduce the radiation doses and consequently the deterministic and stochastic effects of ionising radiation of cardiologists working in interventional radiology.

Vascular density analysis in colorectal cancer patients treated with vatalanib (PTK787/ZK222584) in the randomised CONFIRM trials.

BACKGROUND: Pharmacological inhibitors of vascular endothelial growth factor (VEGF) receptors, like vatalanib, have been tested in randomised trials (CONFIRM (Colorectal Oral Novel therapy For the Inhibition of Angiogenesis and Retarding of Metastases) 1 and 2) in colorectal cancer showing activity in a subgroup of patients with high serum LDH expression. In the current study, we assessed the predictive role of vascular density (VD) in patients treated in the above trials. METHODS: Paraffin-embedded materials from 141 patients were analysed with immunohistochemistry for the expression of the CD31 (pan-endothelial cell marker) and of phosphorylated pVEGFR2/KDR on endothelial cells. The VD was correlated with response to therapy and with progression-free (PFS) and overall survival (OS). RESULTS: A significant association of pVEGFR2/KDR+ VD with poor response in the placebo group was noted (response rates (RRs) 15% (3/20) when high VD vs 52% (26/50) when low VD; P=0.006). The RR increased from 15 (3/20) to 50% (11/22) in tumours with high VD when vatalanib was added to chemotherapy (P=0.02). A significantly improved PFS was noted in patients with high pVEGFR2/KDR+ VD when treated with vatalanib (P=0.002). A similar effect was also noted in patients with high CD31+ VD (P=0.07). Overall survival was marginally improved (P=0.07). CONCLUSION: Assessment of the activated vessel density may allow the stratification of patients recruited in randomised trials with VEGFR-targeting anti-angiogenic agents, unmasking their therapeutic potential and enabling their introduction in the clinical practice for the benefit of specific patient subgroups, at the same time reducing the cost of therapy.

Radiation damage and radioprotectants: new concepts in the era of molecular medicine.

Exposure to ionising radiation results in mutagenesis and cell death, and the clinical manifestations depend on the dose and the involved body area. Reducing carcinogenesis in patients treated with radiotherapy, exposed to diagnostic radiation or who are in certain professional groups is mandatory. The prevention or treatment of early and late radiotherapy effects would improve quality of life and increase cancer curability by intensifying therapies. Experimental and clinical data have given rise to new concepts and a large pool of chemical and molecular agents that could be effective in the protection and treatment of radiation damage. To date, amifostine is the only drug recommended as an effective radioprotectant. This review identifies five distinct types of radiation damage (I, cellular depletion; II, reactive gene activation; III, tissue disorganisation; IV, stochastic effects; V, bystander effects) and classifies the radioprotective agents into five relevant categories (A, protectants against all types of radiation effects; B, death pathway modulators; C, blockers of inflammation, chemotaxis and autocrine/paracrine pathways; D, antimutagenic keepers of genomic integrity; E, agents that block bystander effects). The necessity of establishing and funding central committees that guide systematic clinical research into evaluating the novel agents revealed in the era of molecular medicine is stressed.

Cancer stem cell phenotype relates to radio-chemotherapy outcome in locally advanced squamous cell head-neck cancer.

BACKGROUND: Cancer stem cells (CSCs) tend to repopulate malignant tumours during radiotherapy and, therefore, prolongation of the overall treatment time may result in radiotherapy failure. Thus, an estimate of the number of CSCs in tumour biopsies may prove most useful in predicting resistance to radiotherapy and a guide for development therapies aimed to eradicate a cancer cell population with effects on radiotherapy-related cancer regrowth. METHODS: The CSC population was investigated semi-quantitatively in 74 locally advanced squamous cell head-neck cancers (HNSCC) from an equal number of patients, treated with accelerated platinum-based radiotherapy. A standard immunohistochemical technique and the CSC markers CD44, CD24, Oct4, integrin-ß1 and aldehyde dehydrogenase isoform 1A1 (ALDHA1) was used, in parallel with the proliferation marker MIB-1. The results were correlated with the site of the tumour, the MIB-1 index, the tumour grade and stage, and prognosis. RESULTS: The expression of CD44, CD24 and Oct4 were significantly associated with the MIB-1 proliferation index. In addition, the CD44 was linked with the better differentiated HNSCC. The CD44, Oct4 and integrin-ß1 were all associated with poor prognosis but, in a multivariate analysis, the integrin-ß1 had an independent statistical significance in terms of local relapse, distant metastases and overall survival. Interestingly, ALDH1 was associated with favourable prognosis. CONCLUSION: CSC markers are linked with poor radiotherapy outcome in HNSCC, with integrin-ß1 being the strongest and independent prognostic factor. Targeting CSC molecules with monoclonal antibodies or pharmaceutical agents may prove important for the treatment of HNSCC.

Therapeutic interventions targeting organ preservation in muscle-invasive bladder cancer: a review

Nowadays in modern oncology there is a tendency towards therapies that target organ preservation. Organ preservation protocols have become standard in the treatment of laryngeal carcinoma, oesophageal cancer, breast carcinoma and soft tissue sarcomas. The three-combined therapy consisting of a transurethral resection of the bladder tumour followed by concomitant chemoradiotherapy has been shown to be an attractive alternative for bladder preservation in selected patients with muscle-invasive bladder cancer. In order to evaluate the organ preservation approaches in muscle-invasive bladder cancer we have conducted a comprehensive literature review. Data reported from the studies have shown that bladder preservation therapy with a trimodality approach is safe and effective. Moreover, such an approach provides patients with the opportunity to maintain an intact and functional bladder with a survival rate similar to that of radical cystectomy (AU)

Long-term survival of a patient with multiple abdominal metastasis from endometrial carcinoma treated with multi-portal conformal re-irradiation and chemotherapy.

A patient with recurrent endometrial cancer with multiple abdominal and pelvic tumoral masses was treated with re-irradiation combined with liposomal doxorubicin and oxaliplatin. A multiple field conformal technique was used to deliver a highly accelerated and hypofractionated scheme (15 fractions of 3.5 Gy, within 19 days). Complete response was confirmed four months after therapy. Four years later a lung metastasis appeared and was again treated with a similar course of therapy, once again resulting in a complete response. It is suggested that in the era of modern image-guided radiotherapy patients with endometrial cancer who have relapsed within or outside the loco-regional area, should be carefully assessed for an eventual gross tumor eradication using high-dose localized radiotherapy, leaving as the only target of chemotherapy the microscopic undetectable disease.

Beclin 1 over- and underexpression in colorectal cancer: distinct patterns relate to prognosis and tumour hypoxia.

INTRODUCTION: Autophagy enables cells to recycle long-lived proteins or damaged organelles. Beclin 1, the mammalian orthologue of the yeast Apg6/Vps30 gene, functions as a scaffold for the formation of autophagosomes. MATERIALS AND METHOD: The immunohistochemical patterns of Beclin 1 expression and their prognostic relevance were studied in formalin-fixed tissues from 155 patients with colorectal adenocarcinoma treated with surgery alone. RESULTS: Using the weak homogeneous expression of Beclin 1 in normal colonic tissues as a basis for assessing tumours, the following grouping/staining patterns were recognised in colorectal carcinomas: a normal-like pattern in 62 of 155 (40%) cases, an underexpression pattern in 24 of 155 (15.5%) cases, extensive overexpression of Beclin 1 in 33 of 155 (21.3%) tumours and limited overexpression of the protein in 36 of 155 (23.2%) tumours. Extensive overexpression of Beclin 1 was significantly linked with overexpression of HIF1α and LDH5, as well as with high histological grade, vascular invasion and nodal involvement. Furthermore, patients with extensive over- or underexpression of Beclin 1 had a significantly poorer overall survival compared with the other two groups (P<0.0001). Beclin 1 had an independent prognostic relevance in multivariate analysis. CONCLUSIONS: Beclin 1 has an important role in growth and metastasis of colorectal cancer. Loss of Beclin 1 expression (allelic loss or microRNA regulatory activity, as suggested in the literature) defines poor prognosis presumably by promoting anti-apoptotic pathways, while overexpression of the protein, being linked with tumour hypoxia and acidity, also defines subgroups of tumours with aggressive clinical behaviour.

Angiogenesis and angiogenic factor expression in thyroid cancer.

PURPOSE: Increased expression of angiogenic factors and high vascular density characterize tumors with increased invasive and metastatic capability. Anti-vascular endothelial growth factor (VEGF) therapies have shown an important potentiation of chemotherapy and radiotherapy in experimental and clinical studies. The purpose of this study was to investigate whether it could be possible to identify a subgroup of thyroid cancer patients with high angiogenic activity. METHODS: Formalin-fixed paraffin-embedded tissues from 25 papillary and 18 follicular thyroid carcinomas were assessed immunohistochemically for angiogenic activity, i.e. vascular density (VD) and expression of VEGF and basic fibroblast growth factor (bFGF). RESULTS: VD was significantly higher in follicular tumors (p=0.05). Tumors > 4 cm had a significantly higher VD (p=0.001). High VEGF expression was significantly related to high VD (p=0.05). There was no association of bFGF with histological characteristics. CONCLUSIONS: Increased angiogenic activity is a common feature of thyroid carcinomas, particularly in follicular tumors and larger carcinomas. These results support the testing of anti-VEGF therapies in combination with radiotherapy and chemotherapy in advanced thyroid tumors.

Capecitabine chemotherapy for metastatic colorectal cancer. Chemical structure, combinations and efficacy.

PURPOSE: capecitabine is an oral fluoropyrimidine which had been developed as a pro-drug of fluorouracil (FU), and had shown improved tolerability and intratumor drug concentrations through its tumor-specific conversion to the active drug. Our purpose was to make a comprehensive literature review regarding capecitabine's efficacy in metastatic colorectal cancer. METHODS: we searched the Pubmed and Cochrane databases regarding all available information on capecitabine, focusing on its clinical effectiveness against metastatic colorectal cancer. Special attention was paid on trials that compared capecitabine with standard folinic acid (leucovorin/ LV)-modulated intravenous 5-fluorouracil (5-FU) bolus regimens in patients with metastatic colorectal cancer. Moreover, the efficacy of capecitabine alone or in several combinations with other active drugs such as irinotecan and oxaliplatin on metastatic colorectal cancer were also assessed. RESULTS: comparative trials showed that capecitabine is at least equivalent to standard 5-FU/LV combination regarding progression-free and overall survival, expressing at the same time a better tolerability profile with a much lower incidence of stomatitis. CONCLUSION: nowadays it is known that capecitabine can be combined with other active drugs such as irinotecan and oxaliplatin and the combination of oxaliplatin with capecitabine represents a new standard of care for metastatic colorectal cancer.

Cytoprotective activity of amifostine on cultured human lymphocytes exposed to irinotecan.

Irinotecan (camptothecin, CAM) is a topoisomerase-I inhibitor with a well established action in the chemotherapy of colorectal and ovarian cancer. Hematological and intestinal toxicity are commonly noted in patients treated with CAM. In this study, we examined the cytoprotective efficacy of amifostine (ethyol, ETH) against chromosomal damage induced by this drug on cultured peripheral human lymphocytes. Cultured lymphocytes were exposed to CAM (50 and 100 ng/ml of final concentrations) without or with ETH (in concentrations varying between 40 and 800 microg/ml of final culture volume). CAM's genotoxicity was quantified by counting the Sister Chromatid Exchange (SCEs) rate. The mitotic index (MI) and proliferation rate index (PRI) were also assessed. The SCE rate was increased following incubation with CAM, but the combined treatment of CAM with ETH significantly reduced the SCE formation, especially when ETH added at high concentrations. The MIs and PRIs remained also unaltered in cultures with CAM, but MIs were reduced with the combined treatment at high ETH concentrations. Clinical studies are required to assess the predicted benefits from ETH in patients receiving CAM.

Multi-channel magnetoencephalogram on Alzheimer disease patients.

Magnetoencephalogram (MEG) recordings of 8 patients with advanced Alzheimer Disease (AD) and 9 normal individuals were obtained with a 122-channel whole head biomagnetometer SQUID (Superconductive Quantum Interference Device) to record the minute magnetic fields generated by the brain. The obtained MEG signals were analyzed using linear signal analysis techniques such as Fourier Transform in order to get the frequency distribution of MEG values. The obtained frequencies from all MEG sensors located outside the scalp of each subject were stored for evaluation. From this evaluation it was concluded that in patients with AD the dominant frequencies were significantly lower compared to normal individuals.

Post-surgical irradiation causes cellular immune suppression in patients with breast cancer.

According to several studies, even the locoregional irradiation of patients with carcinoma can cause a severe and rather alarming cellular immune defect. We thus designed a prospective research in order to study the effect of post-operative irradiation on cellular immunity in patients suffering from breast cancer. In 35 patients with breast cancer who required post-operative irradiation, four blood samples were taken at indicated point times. Nineteen out of 35 patients received post-surgical chemotherapy before irradiation. The total lymphocytes as well as CD4 and CD8 subpopulations were measured by using flow cytometry analysis. The mean T-lymphocyte (Tol) count dropped from 1487.77 to 1227.91 (P = 0.0013) and the CD4+ count from 674.17 to 580.91 (P = 0.0189). The mean value of CD8+ dropped from 421.31 to 314.00 (P = 0.0003). Moreover, a statistically significant difference regarding the pattern of temporal change was observed between a group of patients that received irradiation only and a group that received radiation therapy (RT) with chemotherapy (P-values 0.0015, 0.01 and 0.092 for Tol, CD4+ and CD8+ respectively). The group of patients that received RT only presented a more rapid decrease of Tol concerning the decrease observed in the group that underwent chemotherapy and RT.

The efficacy of combined treatment with cetuximab (erbitux) and radiation therapy in patients with head and neck cancer.

Squamous cell carcinoma of the head and neck (SCCHN) region is among the most frequent human tumors due to the alcohol and tobacco abuse. Its management has evolved gradually from surgery as the mainstay of therapy to irradiation as the principal treatment. When radiation therapy is combined with chemotherapy, additional benefit is obtained. The value of chemoradiotherapy (CRT) is, however, counterbalanced by increased and often prohibitive toxicity, particularly among patients with coexisting medical conditions and decreased performance status. A member of the ErbB family of receptor tyrosine kinases known as the epidermal growth factor receptor (EGFR) is abnormally activated in epithelial cancers, including head and neck cancers. Overexpression of EGFR is a feature associated with poor clinical outcome. It is observed that radiation increases the expression of EGFR in cancer cells and the blockade of EGFR signaling sensitizes cells to the effects of radiation. The cytotoxic effects of radiation therapy in squamous cell carcinoma could be enhanced by cetuximab (erbitux), a monoclonal antibody against the ligand-binding domain of EGFR. The major studies that focus on the efficacy of adding cetuximab to radiotherapy in the treatment of patients with head and neck cancer and its impact in quality of life are reviewed in this study.

PDK-1 regulates lactate production in hypoxia and is associated with poor prognosis in head and neck squamous cancer.

Here we describe the expression and function of a HIF-1-regulated protein pyruvate dehydrogenase kinase-1 (PDK-1) in head and neck squamous cancer (HNSCC). Using RNAi to downregulate hypoxia-inducible PDK-1, we found that lactate and pyruvate excretion after 16-48 h of hypoxia was suppressed to normoxic levels. This indicates that PDK-1 plays an important role in maintaining glycolysis. Knockdown had no effect on proliferation or survival under hypoxia. The immunohistochemical expression of PDK-1 was assessed in 140 cases of HNSCC. PDK-1 expression was not expressed in normal tissues but was upregulated in HNSCC and found to be predominantly cytoplasmic with occasional strong focal nuclear expression. It was strongly related to poor outcome (P=0.005 split by median). These results indicate that HIF regulation of PDK-1 has a key role in maintaining lactate production in human cancer and that the investigation of PDK-1 inhibitors should be investigated for antitumour effects.