Effectiveness of Hyperthermia as Monotherapy and Adjuvant Therapy Approaches Against an In Vitro Model of Colorectal Carcinoma.

BACKGROUND/AIM: Despite improvement in current therapies, the 5-year overall survival rate of colorectal carcinoma is still low especially in its metastatic form. On the other hand, hyperthermia has been utilized as a cancer treatment approach to improve overall therapeutic efficacy. In the present study, we have aimed to develop an optimized hyperthermic protocol against an in vitro model of human colon carcinoma, as a single and/or adjuvant treatment approach. MATERIALS AND METHODS: We have utilized an in vitro model of human colorectal carcinoma consisting of colorectal carcinoma (HT29, CaCo2) and normal colon epithelial (CCD841CoN) cell lines. Cells were exposed to 45°C, over 120 min, in the presence or absence of chemotherapeutic (5-Fluorouracil, Capecitabine) and targeted (Bevacizumab, Cetuximab) drugs. Cell viability levels were determined by the Alamar-blue assay while determination of cell death, reactive oxygen species (ROS) production, mitochondrial membrane depolarization (ΔΨµ) levels and cell cycle progression were performed by flow cytometry. RESULTS: CaCo2 and HT29 cells showed a differential response towards i) cell viability, ii) cell death, iii) ROS and ΔΨµ levels as well as iv) cell cycle distribution, in the presence of hyperthermia alone (monotherapy) or in combination with the above-mentioned drugs (adjuvant therapy). Finally, normal colon epithelial (CCD841CoN) cells remained minimally affected. CONCLUSION: We have developed an optimized experimental hyperthermic protocol, as a promising monotherapy and/or adjuvant therapy approach, with the capacity to potentiate chemotherapeutic as well as targeted drug-induced cytotoxicity against a model of colorectal carcinoma, to a variable degree.

Endometrial Stromal Expression of ER, PR, and B-Catenin Toward Differentiating Hyperplasia Diagnoses.

Background. The interpretation of histopathological changes of endometrial hyperplasia with or without atypia can be challenging. We aim to investigate the role of specific immunohistochemical markers in the endometrial stroma to classify endometrial hyperplasia in difficult cases. Methods and Results. We retrospectively reviewed and reclassified (WHO 2014): 47 specimens with endometrial hyperplasia without atypia, 33 with atypical hyperplasia (AH), and 13 endometrioid adenocarcinomas. We performed IHC for B-catenin, E-cadherin, p16, estrogen receptors and progesterone receptors, and B-cell lymphoma 2 (BCL2). Percentage of positive stromal cells was calculated. B-catenin was equally expressed in the stroma of both hyperplasia and AH (mean 60%, 50%; P = .17) and was absent from adenocarcinoma (0%, hyperplasia vs adenocarcinoma; P < .0001, AH vs adenocarcinoma; P < .0001). E-cadherin was not expressed in the stroma of any lesion, while p16 expression levels were not statistically different (hyperplasia vs AH; P = .46, hyperplasia vs adenocarcinoma; P = .22, AH vs adenocarcinoma; P = .48). Estrogen and progesterone were highly identified in stromal cells of hyperplasia (80%) and diminished in AH (respectively, at 30% and 60%, hyperplasia vs AH; P < .0001), and in adenocarcinoma (0% and 40%, respectively). Finally, BCL2 was not differentially expressed (hyperplasia vs AH; P = .33, hyperplasia vs adenocarcinoma; P = .17, AH vs adenocarcinoma; P = .36). Conclusion. Estrogen and progesterone were strongly expressed in stroma exclusively of hyperplasia, while B-catenin was particularly expressed in hyperplasia and AH. Use of these markers can be useful in the differential diagnosis of hyperplasia from AH, and AH from adenocarcinoma in challenging cases.

Expression of Delta Like Ligand 4 (DLL4) in endometrial carcinomas and tumor vasculature.

PURPOSE: Delta like ligand 4 (DLL4) is a transmembrane ligand of the Notch Signalling pathway, that regulates blood vessel sprouting and maturation. We investigated the expression of DLL4 in endometrial cancer. METHODS: DLL4 was assessed in the plasma (with ELISA) and tissues (with immunohistochemistry) 33 patients with endometrial cancer, treated with radical hysterectomy for stage I endometroid carcinoma. The angiogenic activity (AA) of endometrial cancer was quantified by assessing the CD31+ microvessel density (MVD) in the invading tumor front. Vascular maturation index (VMI), defined as the percentage of CD31+ microvessels expressing DLL4, was calculated as the ratio of the CD31+ MVD to the DLL4+ MVD. RESULTS: The angiogenic activity was directly related with the histological grade (p=0.01). The VMI ranged from 0.1 to 0.7 (median 0.34). The concentration of DLL4 in the plasma ranged from 55-81pg/ml (mean 62.8) before, and dropped to 55-62 (mean 58.2) after hysterectomy (p<0.05). DLL4 was also expressed by cancer cells in 17/33 cases. No correlation between DLL4-related parameters with histopathological variables was noted. CONCLUSION: This pilot study shows that DLL4 is overexpressed in endometrial cancer cells, vasculature and is also elevated in the plasma of a fraction of patients before surgery. The percentage of DLL4+ vessels in the penetrating sample ranged from 10-70%, indicating a large difference in the quality of angiogenesis produced between the endometrial tumors of the same histological type and differentiation.

P16 detection in benign, precursor epithelial lesions and carcinomas of head and neck.

BACKGROUND: Human papillomavirus (HPV) had recently been implicated in the pathogenesis of Head and Neck SCCs. The biological role of HPV in benign and pre-cancerous lesions is far less studied. p16 is a widely accepted marker to detect immonohistochemically the presence of HPV. METHODS: We evaluated, immunohistochemically, expression of p16 in 212 specimens: glottis, supraglottis, oropharynx, nasal/paranasal, with various diagnoses: hyperplasia, polyp/nodule, keratosis, papilloma, inverted papilloma, dysplasia, cancer (SCC). Analysis was completed according to location and disease. RESULTS: Hyperplasias/polyps were all negative for p16. A small percentage of papillomas was p16+ regardless of their location (12.5 %), the majority of inverted papillomas were p16+ (78.6 %) and statistically significant (p < 0.04). In carcinomas, 18/59 were p16+ (30.5 %): nasal/paranasal SCCs had a significantly higher percentage of p16+ cancer cells compared to glottis (p = 0.009), while tumours of the supraglottis/oropharynx had an intermediate score for p16+ cells (p = 0.07). Dysplasias were p16+ in 9/64 (14 %) regardless of grading (p = 0.03 compared to carcinomas). CONCLUSION: p16 was highly detected in inverted papillomas and in certain anatomic sites; however, it failed to be traced in benign lesions and only rarely encountered in dysplasias.