Marilena Vered

Immunohistochemical study of epidermal growth factor receptor in adenoid cystic carcinoma of salivary gland origin

Epidermal growth factor (EGF) and its receptor (EGFR) are involved in the development of salivary gland tumors. Recently, treatment modalities for EGFR inhibition have shown an enhanced clinical response in carcinomas of different locations. Adenoid cystic carcinoma (ACC) of salivary gland origin is a malignant tumor with a poor long‐term outcome. If salivary gland ACC does exhibit EGFR, then immunotherapy could have a major impact on improving its prognosis.

Cancer-associated fibroblasts and epithelial-mesenchymal transition in metastatic oral tongue squamous cell carcinoma

We examined cancer‐associated fibroblasts (CAFs) and a panel of immunohistochemical markers of epithelial‐mesenchymal transition (EMT) in 19 pair‐matched oral tongue squamous cell carcinoma (SCC) and metastatic tumors to regional lymph nodes (RLNs). α‐Smooth muscle actin (α‐SMA) was studied to identify CAFs. EMT was studied with syndecan‐1, Cadherin‐11, fibroblast‐specific protein (FSP)‐1, secreted protein acidic and rich in cysteine (SPARC) and Twist. Triple immunostaining in RLNs was used to highlight the carcinoma cells (E‐cadherin and Ki‐67) and their relationship to the CAFs (α‐SMA). We found that metastatic RLNs hosted CAFs similarly as in pair‐matched primary tumors. Expression of EMT markers is common in both primary and metastatic tumors. We demonstrate that metastatic carcinoma cells (Ki‐67 positive) downregulate E‐cadherin expression at the periphery of cancer islands, where they are in direct contact with CAFs. The supporting connective tissue microenvironment also commonly expresses syndecan‐1, Cadherin‐11, FSP‐1, and SPARC. In conclusion, CAFs are common to both primary and metastatic SCC. We hypothesize that CAFs not only promote tumor invasion but also facilitate metastases, either by cometastasizing and/or being recruited to lymph nodes. Evidence of EMT is common within primary tumors and metastatic SCC and may be further modulated by CAFs.

Tumor-host histopathologic variables, stromal myofibroblasts and risk score, are significantly associated with recurrent disease in tongue cancer

Margin status, a major prognostic parameter in oral cancer, was analyzed vis‐à‐vis the histopathologic parameters of risk scores and stromal myofibroblasts. Specimens of tongue carcinoma (n = 50) were submitted to a risk score assignment consisting of the worst pattern of invasion, lymphocytic infiltration, and perineural invasion. Frequency of stromal myofibroblasts (alpha‐smooth muscle actin stain) was assessed. A triple immunostaining assay with E‐cadherin, Ki‐67 and alpha‐smooth muscle actin was used to identify carcinoma cells undergoing epithelial–mesenchymal transition. Margins were considered ‘clean’ if the tumor was ≥5 mm away from them. Patients ≤60 years were considered as ‘young’. Kaplan–Meier survival analysis with univariate and Cox multivariate regression model with stepwise forward selection, and Fisher’s exact tests were used. Abundant myofibroblasts were found in 27 (54%) cases. Carcinoma cells devoid of E‐cadherin but amalgamated with the stromal myofibroblasts were identified in 18 (36%) cases. Local recurrence and overall survival were negatively influenced by abundance of stromal myofibroblasts (P = 0.004 and P = 0.008, respectively). High‐risk scores (P = 0.011), positive margins, and ‘young’ age (P = 0.027, each) had an unfavorable impact on recurrence. Multivariate analysis revealed that only abundance of stromal myofibroblasts had an independent adverse effect on local recurrence (hazard ratio [HR] 4.369; P = 0.014; 95% confidence interval [CI], 1.356–14.074). It seems that abundant stromal myofibroblasts (camouflaging some malignant cells) and high‐risk scores have an unfavorable impact on the risk of recurrence in particular in ‘young’ patients. Therefore, the treatment concept should be adjusted accordingly and target concomitantly the epithelial malignancy and its allied stroma. (Cancer Sci 2009)

Cancer-associated fibroblasts, a parameter of the tumor microenvironment, overcomes carcinoma-associated parameters in the prognosis of patients with mobile tongue cancer

Mobile tongue squamous cell carcinoma (MTSCC) is known for its strong propensity for regional metastasis and poor patient survival despite aggressive treatment, thus calling for new and reliable markers for predicting prognosis and guiding therapeutic management. Towards this end, three classes of markers were investigated: cancer-associated fibroblasts (CAFs; α-SMA positivity) as a representative of the tumor microenvironment, maspin (mammary serine protease inhibitor) as a tumor marker likely to be modulated by factors within the tumor microenvironment, and DNA content and Ki-67 labeling index as inbuilt tumor markers in 128 cases of MTSCC using immunohistochemistry and image cytometry. Of these markers, only CAF density was independently and relatively strongly associated with elevated mortality from MTSCC. The hazard ratio in the CAF-rich type of tumor microenvironment was 4.85 (95% CI 1.41-16.6, versus the CAF-poor) when adjusted by proportional hazards modeling for the center where the patient was managed, gender, tumor stage, presence of neck metastasis and age at diagnosis. CAF density was unrelated to non-MTSSC mortality. Given the strong association between increased CAF density and higher mortality in MTSCC, routine assessment of CAF density for disease course prognosis and inclusion as an integral part of treatment protocols are recommended.

Update from the 4th Edition of the World Health Organization Classification of Head and Neck Tumours: Odontogenic and Maxillofacial Bone Tumors

The 4th edition of the World Health Organization’s Classification of Head and Neck Tumours was published in January of 2017. This article provides a summary of the changes to Chapter 4 Tumours of the oral cavity and mobile tongue and Chapter 8 Odontogenic and maxillofacial bone tumours. Odontogenic cysts which were eliminated from the 3rd 2005 edition were included in the 4th edition as well as other unique allied conditons of the jaws. Many new tumors published since 2005 have been included in the 2017 classification.

The hypoxic tumor microenvironment regulates invasion of aggressive oral carcinoma cells

Invasion is an important hallmark of cancer involving interactions between the tumor microenvironment and the cancer cells. Hypoxia, low oxygen level, is related to increased invasion and metastasis in many cancers. The aim was to elucidate the effect of hypoxia on invasion of oral squamous cell carcinoma cells (OSCCs), and the applicability of a novel 3-dimentional myoma organotypic invasion model in hypoxia experiments. OSCC cell lines (primary oral carcinoma derived cells UT-SCC-43A, recurrent oral carcinoma cells UT-SCC-43B and aggressive tongue carcinoma cells HSC-3) were studied for their migration and invasion capabilities under normoxia, hypoxia, and in the presence a hypoxia-mimicker cobalt chloride. As expected, the recurrent UT-SCC-43B cells were significantly more aggressive than the primary tumor derived cells. In contrast to tongue carcinoma HSC-3 cells, they only mildly responded to hypoxia in the migration or invasion assays, indicating a cell line specific response of hypoxia on the invasive potential. The modification of the organotypic human tissue-derived matrix via the removal of various yet unidentified soluble factors by rinsing the tissue resulting in stripped matrix substantially changed the invasion pattern of HSC-3 cells and the outcomes of hypoxic treatments. Only in the stripped tissue hypoxia significantly increased invasion, whereas in native intact tissue the induced invasion was not observed. This demonstrates the importance of the soluble factors to the invasion pattern and to the hypoxia response. A metastasis and poor prognosis marker, hypoxia-regulated lysyl oxidase (LOX), was present in the myoma tissue, but could be removed by rinsing. The inhibition of LOX resulted in a decrease in invasion area, but only very mildly in invasion depth. Thus, it may have a role in the modulation of the invasion pattern. Another hypoxia-related poor prognosis marker carbonic anhydrase 9 (CAIX) was induced in HSC-3 cells both by the hypoxic exposure and interestingly in invading HSC-3 cells inside the tissue even in normoxic conditions. In conclusion, this suggests that the intact myoma organotypic model offers optimally hypoxic surroundings, thus being an excellent human tumor microenvironment mimicker.

Human Saliva-Derived Exosomes Comparing Methods of Isolation

ExoQuick-TCTM (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.

Age-related histomorphometric changes in labial salivary glands with special reference to the acinar component.

PURPOSE To evaluate histomorphometric age-related changes in labial salivary glands (LSG) of healthy subjects, with special reference to the mucous and seromucous acini. METHOD Investigation of age-related histomorphometric changes was conducted on 120 samples of LSG obtained from autopsy subjects free of salivary gland tumors/diseases. Samples were divided into young (< 30 years, n = 30), adult (30-60 years, n = 45) and old (> 60 years, n = 45) age groups. Measurements were performed on hematoxylin and eosin stained slides. Parenchymal components included acini and ducts. The acinar component was presently analyzed separately in respect to mucous acinar cells and seromucous acinar cells. Mucous acinar cells presented pale eosinophilic cytoplasm, while that of the seromucous was darker and basophilic. Stromal components included connective tissue, blood and lymphatic vessels, inflammatory infiltrate and adipose tissue. The mean volume fraction (V(v)) of all components in each age group was calculated. Statistical analysis was performed by one-way ANOVA and Tamhane tests. RESULTS The mean V(v) of the seromucous acinar cells decreased by 49.3% (p < 0.0001) and that of the mucous acinar cells decreased only by 28.5% (p = 0.0001). Changes in the ducts were statistically non-significant (p = 0.312). Of the stromal components, the mean V(v) of the adipose tissue showed the highest increase, 2768% (p < 0.0001) followed by the inflammatory infiltrate, 512% (p < 0.0001). CONCLUSIONS The marked decrease in the V(v) of seromucous acinar cells accompanied by a lower decrease in the V(v) of mucous acinar cells may explain age-related changes in LSG secretion. These findings could bear important implications regarding functional age-related changes of these glands.

Clear Cell Odontogenic Carcinoma and Clear Cell Ameloblastoma: A Single Clinicopathologic Entity? A New Case and Comparative Analysis of the Literature

BACKGROUND Clear cell odontogenic carcinoma (CCOC) is histologically characterized by solid sheets and nests of clear cells. Clear cell ameloblastoma (CCAM) is histologically characterized by an ameloblastomatous component intermixed with an extensive clear cell component. In all literature reviews, no separation has been made between the clinicopathologic features of CCOC and CCAM. PURPOSE We sought to review and analyze the clinicopathologic and radiologic features and the biologic behavior of CCOC and to compare them with those of CCAM to evaluate the possible separation between the 2 lesions. MATERIALS AND METHODS A MEDLINE search of the English-language literature was carried out for CCOC and CCAM. Cases were classified according to their histologic features. RESULTS A total of 27 cases of CCOC (26 from the literature and 1 new case) and 8 cases of CCAM were found. CCOC patients showed a male-to-female ratio of 1:2.4, with a mean age of 59 years. CCAM patients showed a male-to-female ratio of 1.7:1, with a mean age of 44 years. CCOC and CCAM were predominantly found in the mandible. Both CCOC and CCAM showed a high rate of recurrence (50% and 63%, respectively) and metastases (33% and 25%, respectively). Several patients with CCOC presented with metastases at time of diagnosis, whereas patients with CCAM usually developed metastases only after several recurrences. CONCLUSION Based on the relatively small number of cases in the literature on CCOC and CCAM, it is difficult to confidently separate the 2 lesions. Both lesions should be considered low-grade malignancies and could well represent a clinicopathologic continuum of a single disease entity rather than 2 separate lesions.

Nutraceuticals as new treatment approaches for oral cancer – I: Curcumin

Oral squamous cell carcinoma (OSCC) is a growing global public health problem for which standard therapeutic strategies have failed to contribute significantly to improve the survival rates that have remained around 50% over the past three decades. Therefore, there is a pressing need for new therapeutic strategies. Curcumin is a natural dietary compound with known anti-neoplastic activities, hence its classification as a nutraceutical agent. This review presents the current in vitro and in vivo studies in which curcumin has been examined for its anti-cancer potential in treating OSCC. Its mechanisms of action are also beginning to become unveiled. The available studies have been focusing on the impact of curcumin on epithelial malignant cells, but overlooking the components of the tumor microenvironment. Curcumin has been emerging as a promising therapeutic agent in oral cancer, either alone or in combination with standard therapeutic agents, and will probably become of practical use once its route of administration has overcome its poor bioavailability.