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.

Expression of autophagy-related proteins Beclin-1 and LC3A and proliferation marker Ki-67 in calculous and acalculous human gallbladder epithelium.

BACKGROUND: Autophagy is an inducible intracellular process that has been studied mostly in cancer and less in inflammatory diseases. To establish the relation between cholecystitis (calculous and acalculous) and autophagy, we studied the expressions of immunohistochemical markers Beclin-1, LC3A, and Ki-67 in gallbladder epithelium and their significance in the induction of autophagy. METHODS: Adult human gallbladder tissues were obtained from 100 patients (45 male, 55 female) who underwent cholecystectomy. According to the findings, the patients were divided into two groups: group A (calculous gallbladder: 24 male, 46 female; mean age 52.6 ± 16.0 years) and group B (acalculous gallbladder: 21 male, nine female; mean age 65.3 ± 12.4 years). The expressions of immunohistochemical markers Beclin-1, LC3A, and Ki-67 in gallbladder epithelium were studied using immunohistochemistry techniques. RESULTS: Beclin-1 expression was correlated with LC3A expression in group A with increased Beclin-1 expression promoting LC3A expression (p =0.0001). In group B, the LC3A expression did not follow Beclin-1 expression (p =0.09). The mean percentage of Beclin-1 expression in group A patients was 23.8 % compared to group B patients, where the corresponding percentage was only 17.3 %. Corresponding mean percent expressions of LC3A in groups A and B were 38.9 % and 50.7 %, respectively. The expression of Ki-67 was higher in group A patients compared to group B patients. The mean percentage of Ki-67 expression in group A patients was 3.75 %, whereas, in group B patients, it was only 0.5 % (statistically significantly different; p =0.0003). CONCLUSION: In the epithelium of calculous cholecystitis, overexpression of LC3A is related to Beclin-1 overexpression, which reinforces the view that Beclin-1 promotes autophagy in stone cholecystitis. HIPPOKRATIA 2019, 23(2): 64-69.

Brenner tumour of the ovary--an incidental histological finding.

Brenner tumours of the ovary are uncommon neoplasms and mostly benign. There is general agreement that Brenner tumors are derived from the surface epithelium of the ovary or the pelvic mesothelium through transitional cell metaplasia. It is essential to categorise these tumours as benign, borderline or malignant type as the biologic behaviour and choice of surgery differs in all of the three categories. The authors report a case of Brenner tumour that had only a single area with a beginning indistinct stroma vessel invasion. However the presence of characteristic epithelial nests, fibromatous stroma, and marked cytological metaplasia without atypia provided important clues to the correct diagnosis--proof of a benign tumour.

Presentation of a patient with in situ amelanotic melanoma of the vulva.

Amelanotic malignant melanoma of the vulva is extremely rare. The authors describe here a case of amelanotic malignant melanoma of the vulva, occurring in a 71-year-old woman without any clinical symptoms. The woman had a small nodular lesion in the left labia majora. Local excision was performed. Histological examination revealed an in situ malignant melanoma without any evidence of invasive disease. All suspicious lesions in the vulva region, even if there are no clinical symptoms, should be biopsied, and if an in-situ melanoma is identified, partial or total vulvectomy should be considered.

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.

Immunohistochemical studies of wound healing after monopolar electrocautery and ultrasound submucosal inferior nasal turbinate reduction in sheep.

BACKGROUND: Extracellular matrix (ECM) proteins such as fibronectin and collagen III, enzymes such as matrix metalloproteinases and macrophages have been demonstrated to intervene in nasal and paranasal sinuses wound healing. AIM OF THE STUDY: To compare concentration of ECM proteins, enzymes and the recruitment of macrophages during wound repair after monopolar electrocautery in contrast with ultrasound submucosal surgical tissue reduction of inferior nasal turbinate (INT) tested in sheep. MATERIALS AND METHODS: Prospective controlled study in sheep. Immunostaining for collagen III, fibronectin, CD68 and matrix metalloproteinase-9 (MMP9) was applied in tissue specimens of INT mucosa after monopolar electrocoagulation (MEC) and ultrasound tissue reduction (UTR). Twelve INTs were studied 1, 3 and 8 weeks post-operatively in each interventional group (MEC and UTR) and 5 INTs were studied in animals of the control group (without surgery). The immunoreactivity was quantitatively graded between 0% to 100% immunoreactivity by a blinded senior pathologist. RESULTS: At the end of the study period collagen III, fibronectin and MMP9 were increased in both groups compared to the levels of the control group. When compared to control group, CD68 immunoreactivity was found higher in MEC group but not in UTR group. Fibronectin subepithelial immunoreactivity exhibited a substantial negative correlation with mucosal epithelial cell necrosis, a substantial positive correlation with fibrosis in MEC-treated specimens and a significant positive correlation with sinusoid engorgement in UTR-treated specimens. Collagen III tissue immunoreactivity showed a particularly significant negative correlation with sinusoid engorgement in MEC-treated specimens. CONCLUSION: Correlation of fibronectin and collagen III immunoreactivity to histopathologic findings suggests different ECM repair processes between MEC and UTR turbinate tissue reduction. The use of CD68 and MMP9 provides additional clues to the mode of actions of these techniques and to the molecular and cellular events of the nasal mucosa wound healing process.

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.

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.

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.

Intestinal-type metaplasia in the original squamous epithelium of the cervix.

There have been a number of reports on cervical carcinomas, both invasive and intraepithelial (CIN III), indicating the presence of intracellular mucins in the absence of glandular differentiation. Yet, the expression of such cells in the normal/original squamous epithelium of the cervix remains unexplored. We investigated the presence of mucin-distended goblet cells at this site, after examining retrospectively normal cervices from 250 hysterectomy specimens. Goblet cells were detected in 3.2% (8/250) of the cervices examined using haematoxylin and eosin stained sections and confirmed by mucin histochemistry: alcian blue (AB) pH 2.5, periodic acid-Schiff reaction with and without diastase digestion (PAS-d, PAS) and the combined AB/PAS. Additional sections were stained with Diazo and Masson-Fontana for argentaffin granules and Grimelius for argyrophil cells, but were all negative and no other cell types were identified. It is believed that this incomplete type of intestinal metaplasia is an acquired change in the cervix, derived from multi-potential stem cells of Müllerian duct origin.

Myxoid leiomyosarcoma of the uterus: a diagnostic challenge.

Myxoid leiomyosarcoma is an extremely rare variant of leiomyosarcoma, masquerading almost to perfection as a benign lesion. For, indeed, the tumor lacks the defining features of high mitotic activity, cellular atypia or necrosis, and the microscopic picture is dominated by abundant myxoid stroma containing sparse spindle cells. We report here such a case occurring in the uterus and discuss the differential diagnosis. The relevant literature is reviewed.

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.

Prognostic and predictive factors of invasive ductal breast carcinomas.

PURPOSE: To investigate the significance of certain immunohistochemical markers, namely estrogen (ER) and progesterone receptors (PgR), c-erbB-2 oncogene, p53 tumor suppressor gene and E-cadherin adhesion molecule, in invasive ductal breast carcinomas. METHODS: A series of 102 primary breast carcinomas of the ductal type and a standard immunohistochemical technique was used to detect the aforementioned biological markers. The findings were related to various clinical and pathological tumor characteristics, including lymph node metastases. RESULTS: ER and E-cadherin were expressed more commonly in tumors of low histological grade and small number (< or =3) of metastatic lymph nodes, whereas c-erbB-2 and the p53 gene were usually expressed in breast tumors of high histological grade and increased number (>3) of metastatic lymph nodes. PgR, on the other hand, was detected frequently in patients with early menarche and metastases in <3 lymph nodes, but this tendency was not statistically significant. CONCLUSION: The use of these biomarkers, preferably in combination, may provide additional prognostic and therapeutic information which may be proved useful in planning breast cancer treatment.

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.

Port-site metastasis of extrahepatic bile duct carcinoma after laparoscopic cholecystectomy without evidence of a primary tumour.

A 75-year-old man suffering from symptomatic cholelithiasis underwent laparoscopic cholecystectomy using the four-port technique. No malignancy was observed in the resected gall-bladder and the patient exhibited a good postoperative course. Eleven months postoperatively he presented with two subcutaneous tumours: one at the port-site on the right anterior axillary line (at the position of the vacuum drain) and the other at the subumbilical port-site. The patient underwent an incisional biopsy, which revealed metastatic adenocarcinomas of the primary extrahepatic duct, with no evidence of a primary tumour or other distant metastasis. The patient underwent wide excision of the subcutaneous tumours. Six months later he again presented with subcutaneous tumours at the same positions. Magnetic resonance imaging of the abdomen revealed only the subcutaneous tumours. The patient again underwent wide excision of the subcutaneous tumours, followed by radiotherapy. At a 21-month follow-up the patient was symptom-free. Magnetic resonance imaging of the abdomen and magnetic resonance cholangiopancreatography results were normal, and there was no evidence of other metastasis. Four months later the patient died from metastatic disease of the abdomen.