Molecular and genomic characterisation of a panel of human anal cancer cell lines.

Anal cancer is a rare disease that has doubled in incidence over the last four decades. Current treatment and survival of patients with this disease has not changed substantially over this period of time, due, in part, to a paucity of preclinical models to assess new therapeutic options. To address this hiatus, we set-out to establish, validate and characterise a panel of human anal squamous cell carcinoma (ASCC) cell lines by employing an explant technique using fresh human ASCC tumour tissue. The panel of five human ASCC cell lines were validated to confirm their origin, squamous features and tumourigenicity, followed by molecular and genomic (whole-exome sequencing) characterisation. This panel recapitulates the genetic and molecular characteristics previously described in ASCC including phosphoinositide-3-kinase (PI3K) mutations in three of the human papillomavirus (HPV) positive lines and TP53 mutations in the HPV negative line. The cell lines demonstrate the ability to form tumouroids and retain their tumourigenic potential upon xenotransplantation, with varied inducible expression of major histocompatibility complex class I (MHC class I) and Programmed cell death ligand 1 (PD-L1). We observed differential responses to standard chemotherapy, radiotherapy and a PI3K specific molecular targeted agent in vitro, which correlated with the clinical response of the patient tumours from which they were derived. We anticipate this novel panel of human ASCC cell lines will form a valuable resource for future studies into the biology and therapeutics of this rare disease.

Cutaneous adnexal carcinosarcoma: Immunohistochemical and molecular evidence of epithelial mesenchymal transition.

Cutaneous carcinosarcomas are rare biphenotypic tumors that simultaneously show epithelial and mesenchymal differentiation. The most common carcinomatous components in skin carcinosarcomas are basal cell carcinoma and squamous cell carcinoma; adnexal carcinomas are rarely encountered. We report a case of an adnexal carcinoma with ductal and squamous differentiation and spindle cell component, which is interpreted as carcinosarcoma. Loss of immunohistochemical expression of E-cadherin and ß-catenin detected in the sarcomatous component suggested epithelial mesenchymal transition (EMT). RNA sequencing analysis identified several gene mutations and alterations such as translocations and upregulations/downregulations, either shared by the two components of the tumor or differentially present in the carcinoma or the sarcoma parts. Thus, mutations in genes, such as TP53, were found in both components of the tumor while mutations in PDGFRA and RB1 (a pathogenic missense mutation) were exclusively present in the sarcomatous areas, further supporting EMT. EMT is a dynamic process by which tumors acquire mesenchymal phenotype while simultaneously losing epithelial properties. Although the pathways involved in EMT have been extensively studied, this phenomenon still needs to be investigated in cutaneous tumors of adnexal origin for a better understanding of their pathogenesis. These molecular changes may represent promising targets for personalized therapies.

The RBP1-CKAP4 axis activates oncogenic autophagy and promotes cancer progression in oral squamous cell carcinoma.

Retinol-binding protein 1 (RBP1) is involved in several physiological functions, including the regulation of the metabolism and retinol transport. Studies have shown that it plays an important role in the pathogenesis of several types of cancer. However, the role of RBP1 and its correlation with autophagy in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. In this study, RBP1 was identified as the most significantly upregulated DEPs with a >2-fold change in OSCC samples when compared to normal tissues through iTRAQ-based proteomics analysis coupled with 2D LC-MS/MS. RBP1 overexpression was significantly associated with malignant phenotypes (differentiation, TNM stage, and lymphatic metastasis) of OSCC. In vitro experiments demonstrated that RBP1 was significantly increased in OSCC tissues and cell lines compared with control group. RBP1 overexpression promoted cell growth, migration, and invasion of OSCC cells. Silencing of RBP1 suppressed tumor formation in xenografted mice. We further demonstrated that the RBP1-CKAP4 axis was a critical regulator of the autophagic machinery in OSCC, inactivation of autophagy rescued the RBP1-CKAP4-mediated malignant biological behaviors of OSCC cells. Overall, a mechanistic link was provided by RBP1-CKAP4 between primary oncogenic features and the induction of autophagy, which may provide a potential therapeutic target that warrants further investigation for treatment of OSCC.

Automated oral squamous cell carcinoma identification using shape, texture and color features of whole image strips.

Despite profound knowledge of the incidence of oral cancers and a large body of research beyond it, it continues to beat diagnosis and treatment management. Post physical observation by clinicians, a biopsy is a gold standard for accurate detection of any abnormalities. Towards the application of artificial intelligence as an aid to diagnosis, automated cell nuclei segmentation is the most essential step for the recognition of the cancer cells. In this study, we have extracted the shape, texture and color features from the histopathological images collected indigenously from regional hospitals. A dataset of 42 whole slide slices was used to automatically segment and generate a cell level dataset of 720 nuclei. Next, different classifiers were applied for classification purposes. 99.4 % accuracy using Decision Tree Classifier, 100 % accuracy using both SVM and Logistic regression and 100 % accuracy using SVM, Logistic regression and Linear Discriminant were acquired for shape, textural and color features respectively. The in-depth analysis showed SVM and Linear Discriminant classifier gave the best result for texture and color features respectively. The achieved result can be effectively converted to software as an assistant diagnostic tool.

PET/MRI and genetic intrapatient heterogeneity in head and neck cancers.

PURPOSE: The relation between functional imaging and intrapatient genetic heterogeneity remains poorly understood. The aim of our study was to investigate spatial sampling and functional imaging by FDG-PET/MRI to describe intrapatient tumour heterogeneity. METHODS: Six patients with oropharyngeal cancer were included in this pilot study. Two tumour samples per patient were taken and sequenced by next-generation sequencing covering 327 genes relevant in head and neck cancer. Corresponding regions were delineated on pretherapeutic FDG-PET/MRI images to extract apparent diffusion coefficients and standardized uptake values. RESULTS: Samples were collected within the primary tumour (n = 3), within the primary tumour and the involved lymph node (n = 2) as well as within two independent primary tumours (n = 1). Genetic heterogeneity of the primary tumours was limited and most driver gene mutations were found ubiquitously. Slightly increasing heterogeneity was found between primary tumours and lymph node metastases. One private predicted driver mutation within a primary tumour and one in a lymph node were found. However, the two independent primary tumours did not show any shared mutations in spite of a clinically suspected field cancerosis. No conclusive correlation between genetic heterogeneity and heterogeneity of PET/MRI-derived parameters was observed. CONCLUSION: Our limited data suggest that single sampling might be sufficient in some patients with oropharyngeal cancer. However, few driver mutations might be missed and, if feasible, spatial sampling should be considered. In two independent primary tumours, both lesions should be sequenced. Our data with a limited number of patients do not support the concept that multiparametric PET/MRI features are useful to guide biopsies for genetic tumour characterization.

CircCDR1as upregulates autophagy under hypoxia to promote tumor cell survival via AKT/ERK½/mTOR signaling pathways in oral squamous cell carcinomas.

Autophagy, as an important non-selective degradation mechanism, could promote tumor initiation and progression by maintaining cellular homeostasis and the cell metabolism as well as cell viability. CircCDR1as has been shown to function as an oncogene in cancer progression, however, it remains largely unknown as to how autophagy is regulated by circCDR1as in oral squamous cell carcinoma (OSCC). In this study, we validated the functional roles of circCDR1as in regulation of autophagy in OSCC cells and further investigated how circCDR1as contributed to cell survival via up-regulating autophagy under a hypoxic microenvironment by using combination of human tissue model, in vitro cell experiments and in vivo mice model. We found that hypoxia promoted the expression level of circCDR1as in OSCC cells and elevated autophagy. In addition, circCDR1as further increased hypoxia-mediated autophagy by targeting multiple key regulators of autophagy. We revealed that circCDR1as enhanced autophagy in OSCC cells via inhibition of rapamycin (mTOR) activity and upregulation of AKT and ERK½ pathways. Overexpression of circCDR1as enhanced OSCC cells viability, endoplasmic reticulum (ER) stress, and inhibited cell apoptosis under a hypoxic microenvironment. Moreover, circCDR1as promoted autophagy in OSCC cells by sponging miR-671-5p. Collectively, these results revealed that high expression of circCDR1as enhanced the viability of OSCC cells under a hypoxic microenvironment by promoting autophagy, suggesting a novel treatment strategy involving circCDR1as and the inhibition of autophagy in OSCC cells.

Carcinoma cuniculatum in the tongue of a patient with oral lichen planus: Unusual presentation.

Carcinoma cuniculatum (CC) is a rare variant of squamous cell carcinoma (SCC). Only 27 cases have been published in English. A 50-year-old male, who presented a white nodule with erythematous areas, localized in the lateral border of the tongue with 2 months of duration. The patient presents oral lichen planus lesions on the tongue, commissure, and buccal mucosa. The microscopy evaluation of the nodular lesion of the tongue revealed a malignant epithelial neoplasia characterized by cuniculatum architecture, similar in appearance to "rabbit burrows" and the final diagnosis was of CC. The management of CC needs cooperation between surgeons and pathologists to establish a correct diagnosis and treatment. CC is a rare entity and must be recognized by oral pathologist so that it is not misdiagnosed as verrucous carcinoma or oral SCC (OSCC). Regarding prognosis, CC must be evaluated and distinguished from other variants of OSCC.

CerS6 regulates cisplatin resistance in oral squamous cell carcinoma by altering mitochondrial fission and autophagy.

Chemoresistance remains a challenge in the effective treatment of solid tumors, including oral squamous cell carcinoma (OSCC). Mitochondrial dynamics and autophagy have recently been implicated in the chemoresistance of cancer cells. The neutralization of ceramide is also associated with multidrug resistance, and ceramide synthase 6 (CerS6) is known to induce apoptosis. However, whether CerS6 regulates chemoresistance in OSCC is not clearly understood. Therefore, we investigated the role of CerS6 in the susceptibility of OSCC cells to cisplatin. In this study, we observed that cisplatin-resistant OSCC cells process lower levels of fission-state mitochondria and cell apoptosis than cisplatin-sensitive cells, and autophagy was activated in cisplatin-resistant OSCC cells. We found lower CerS6 expression in cisplatin-resistant OSCC cells. Overexpression of CerS6 with lentivirus-encoded CerS6 complementary DNA in cisplatin-resistant OSCC cells increased cisplatin sensitivity. Overexpression of CerS6 enhanced mitochondrial fission and apoptosis and attenuated cisplatin-induced autophagy in cisplatin-resistant OSCC cells. Further investigation indicated that CerS6 might function through altering calpain expression to enhance cisplatin sensitivity. Cisplatin-resistant OSCC cells xenografted onto a nude mouse model confirmed that CerS6 enhanced cisplatin chemotherapy sensitivity to reduce tumor volume. These data indicate that CerS6 could mediate an effective response to cisplatin in chemoresistant OSCC.

Interactive Visual Exploration of 3D Mass Spectrometry Imaging Data Using Hierarchical Stochastic Neighbor Embedding Reveals Spatiomolecular Structures at Full Data Resolution.

Technological advances in mass spectrometry imaging (MSI) have contributed to growing interest in 3D MSI. However, the large size of 3D MSI data sets has made their efficient analysis and visualization and the identification of informative molecular patterns computationally challenging. Hierarchical stochastic neighbor embedding (HSNE), a nonlinear dimensionality reduction technique that aims at finding hierarchical and multiscale representations of large data sets, is a recent development that enables the analysis of millions of data points, with manageable time and memory complexities. We demonstrate that HSNE can be used to analyze large 3D MSI data sets at full mass spectral and spatial resolution. To benchmark the technique as well as demonstrate its broad applicability, we have analyzed a number of publicly available 3D MSI data sets, recorded from various biological systems and spanning different mass-spectrometry ionization techniques. We demonstrate that HSNE is able to rapidly identify regions of interest within these large high-dimensionality data sets as well as aid the identification of molecular ions that characterize these regions of interest; furthermore, through clearly separating measurement artifacts, the HSNE analysis exhibits a degree of robustness to measurement batch effects, spatially correlated noise, and mass spectral misalignment.

A nomogram for predicting the risk of neck node metastasis in pathologically node-negative oral cavity carcinoma.

OBJECTIVE: To generate a nomogram for predicting the risk of neck node metastasis in pathologically node-negative patients using a combination of variables comprising of protein expression, ultrastructural alterations and clinicopathological parameters. MATERIALS AND METHODS: Surgically removed oral tumours (n = 103) were analysed for the expression of desmosomal and hemidesmosomal assembly proteins by immunohistochemistry and ultrastructural alterations by transmission electron microscopy (TEM). Protein expression, ultrastructural alterations and clinicopathological variables were used to construct nomogram from the training set in 75 patients. Clinical utility of the nomogram was validated in a discrete set of 28 patients. RESULTS: Univariate and multivariate analyses were performed on the training set, and obtained significant variables comprising of integrin ß4 expression (p = .027), number of hemidesmosomes (p = .027)/desmosomes (p = .046), tumour differentiation grade (p = .033) and tumour thickness (p = .024) were used for construction of the nomogram. The area under the curve was calculated for both training 0.821 (95% CI 0.725-0.918) and validation sets 0.880 (95% CI 0.743-1.000). The nomogram demonstrated a predictive accuracy of 73.3% and 78.6% with the sensitivity of 81.4% and 83.3% in the training and validation sets, respectively. CONCLUSIONS: The nomogram constructed on postsurgical tumour samples will be a value addition to histopathology for the detection of neck node metastasis in pathologically node-negative patients.

Molecular imaging of biological systems with a clickable dye in the broad 800- to 1,700-nm near-infrared window.

Fluorescence imaging multiplicity of biological systems is an area of intense focus, currently limited to fluorescence channels in the visible and first near-infrared (NIR-I; ∼700-900 nm) spectral regions. The development of conjugatable fluorophores with longer wavelength emission is highly desired to afford more targeting channels, reduce background autofluorescence, and achieve deeper tissue imaging depths. We have developed NIR-II (1,000-1,700 nm) molecular imaging agents with a bright NIR-II fluorophore through high-efficiency click chemistry to specific molecular antibodies. Relying on buoyant density differences during density gradient ultracentrifugation separations, highly pure NIR-II fluorophore-antibody conjugates emitting ∼1,100 nm were obtained for use as molecular-specific NIR-II probes. This facilitated 3D staining of ∼170-µm histological brain tissues sections on a home-built confocal microscope, demonstrating multicolor molecular imaging across both the NIR-I and NIR-II windows (800-1,700 nm).

Does pTis exist in HPV-driven tonsillar carcinomas? An ultrastructural review and examination of two cases.

Many tonsillar tumors present clinically as cervical nodal metastases and the primary tumor is often difficult to find. HPV-driven tonsillar carcinoma begins in the reticulated crypt epithelium, possibly through viral integration. The basement membrane is not complete in the reticulated crypt epithelium, which may enhance the immune function. We examined the reticulated crypt epithelium in a normal case and five neoplastic tonsils with cervical metastasis as the presenting symptom to further investigate whether tonsil carcinoma in-situ exists. Our results suggest that in-situ carcinoma may need to be excluded from the future staging for human papilloma virus associated tonsillar tumors.

Combinatorial effects of geopropolis produced by Melipona fasciculata Smith with anticancer drugs against human laryngeal epidermoid carcinoma (HEp-2) cells.

The identification of natural products exerting a combined effect with therapeutic agents could be an alternative for cancer treatment, reducing the concentration of the drugs and side effects. Geopropolis (Geo) is produced by some stingless bees from a mixture of vegetable resins, gland secretions of the bees and soil. It has been used popularly as an antiseptic agent and to treat respiratory diseases and dermatosis. To determine whether Geo enhances the anticancer effect of carboplatin, methotrexate and doxorubicin (DOX), human laryngeal epidermoid carcinoma (HEp-2) cells were treated with Geo alone or in combination with each drug. Cell growth, cytotoxicity and apoptosis were evaluated using 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release, and flow cytometry. Scratch assay was used to analyze cell migration and transmission electron microscopy to observe morphologic alterations. The influence of Geo on drug resistance was also investigated assessing P-glycoprotein (P-gp) action. Geo inhibited cell proliferation and migration. The combination Geo+DOX led to the highest cytotoxic activity and induced apoptosis, leading to loss of membrane integrity. Geo had no effect on P-gp-mediated efflux of DOX. Data indicate that Geo combined with DOX could be a potential clinical chemotherapeutic approach for laryngeal cancer treatment.

Epidemiological changes in the histological subtypes of 35,018 non-small-cell lung cancer cases in Brazil.

OBJECTIVES: Regarding the fatality rates stemming from various existing forms of cancers worldwide, lung cancer (LC) is ranked as the main cause of death amongst those who suffer from cancer. Although the epidemiological, clinical, and histological profile of patients with this type of cancer is largely unknown, Brazil has made tremendous efforts to generate data for supporting healthcare policies concerning lung cancer. Taking these factors into account, this study aims to analyse the epidemiological, clinical, and histological profiles of patients with non-small-cell lung cancer (NSCLC) in Brazil. MATERIAL AND METHODS: For this study, a cross-sectional epidemiological study was conducted to nationally analyse patient's data within the cancer hospital registries found in the National Cancer Institute (INCA) and the São Paulo Cancer Foundation (FOSP) between 2000 and 2011. RESULTS: A total of 35,018 patients diagnosed with NSCLC in Brazil between 2000 and 2011 were analysed. The analysis demonstrated the occurrence of an epidemiological shift, related to the most prevalent histological type of NSCLC in the study population from 2003. The shift resulted in a higher percentage of adenocarcinoma (43.3%) over squamous cell carcinoma (36.5%). Additionally, there was a significant increase in both the number of cases of LC in women and in the rates of patients diagnosed with metastatic disease. CONCLUSION: The use of filtered cigarettes since the 60's and the increase in the number of LC cases in women, were one of the causes for the switch in the histological profile of NSCLC in Brazil. Consequently, adenocarcinoma is now the predominant type of cancer detected. Late diagnosis is a hallmark sign.

[Physical Properties of Squamous Cell Carcinoma Cells using Atomic Force Microscopy]. / Bestimmung elastischer Eigenschaften von Plattenepithelkarzinomzellen mittels Rasterkraftmikroskopie.

INTRODUCTION: Malignant and benign cells differ according to their elasticity. An atomic force microscope is a useful tool for measuring these mechanical cell properties. If cells of different dignity show different resonance behavior, due to their different elasticity, a selective ablation of specific tissue types by ultrasound would be possible. The goal is a highly selective ablation of tumor tissue without damaging healthy tissue. MATERIALS AND METHODS: We performed elasticity measurements of tumor cells (UD-01 cell line) with an atomic force microscope. In a further step, an ultrasound applicator has been positioned and the morphological changes of the cells during the treatment were documented. RESULTS: Different elasticities on the squamous cells were measured, depending on the location. Below a defined maximum amplitude the morphological cell changes were caused solely by ultrasonic excitation. SUMMARY: The atomic force microscope is suitable for the determination of the individual cell elasticity. The data collected could be the basis for treatment modalities that lead to a very selective damage for malignant cells.

Cytomorphologic findings of malignant mesothelioma in FNA biopsies and touch preps of core biopsies.

BACKGROUND: Given the lack of recent literature regarding the aspiration cytology of immunohistochemically confirmed malignant mesothelioma (MM), we were interested in reviewing the experience of our institution and establishing useful morphologic criteria. METHODS: Seventeen aspiration and touch preparation specimens with a diagnosis of MM obtained between 2002-2013 were reviewed along with 20 cases of adenocarcinoma and 16 cases of squamous cell carcinoma. The utility of a number of morphologic features was evaluated. RESULTS: In most cases of MM, a consistent pattern emerged. Aspirates and touch preps were cellular with irregularly shaped 2 and 3 dimensional clusters. The individual cells were predominantly angulated and had dense cytoplasm with eccentric nuclei. In every case, a minority of tumor cells contained prominent microvacuoles. The chromatin pattern tended to be fine with small nucleoli. While most cases were cytologically monotonous, five cases displayed striking pleomorphism and three cases contained occasional large atypical cells. Two cases contained metachromatic background material. Features which were most useful in discriminating MM from adenocarcinoma were angulated cell shape(P = 0.0002), dense cytoplasm(P = 0.0001), and cytoplasmic microvacuoles(P = 0.0001). In our material, cases of squamous cell carcinoma were often difficult to distinguish from MM. Useful discriminatory features present in squamous cell carcinoma included ink dot nuclei(P = 0.0003), a "dirty" cystic, necrotic background (P = 0.0027) and tumor balls with peripheral spindling(P = 0.0041). CONCLUSION: Most cases of MM have a consistent appearance in core biopsy touch preps and FNAs. Distinguishing MM from adenocarcinoma and squamous cell carcinoma can be facilitated by evaluating a few key morphologic features.

Absolute CT perfusion parameter values after the neoadjuvant chemoradiotherapy of the squamous cell esophageal carcinoma correlate with the histopathologic tumor regression grade.

PURPOSE: To analyze value of the computed tomography (CT) perfusion imaging in response evaluation of the esophageal carcinoma to neoadjuvant chemoradiotherapy (nCRT) using the histopathology as reference standard. METHODS: Forty patients with the squamous cell esophageal carcinoma were re-evaluated after the nCRT by CT examination, which included low-dose CT perfusion study that was analyzed using the deconvolution-based CT perfusion software (Perfusion 3.0, GE). Histopathologic assessment of tumor regression grade (TRG) according to Mandard's criteria served as reference standard of response evaluation. Statistical analysis was performed using Spearman's rank correlation coefficient (r(S)) and Kruskal-Wallis's test. RESULTS: The perfusion CT parameter values, measured after the nCRT in the segment of the esophagus that had been affected by neoplasm prior to therapy, significantly correlated with the TRG: blood flow (BF) (r(S)=0.851; p<0.001), blood volume (BV) (r(S)=0.732; p<0.001) and mean transit time (MTT) (r(S)=-0.386; p=0.014). Median values of BF and BV significantly differed among TRG 1-4 groups (p<0.001), while maximal esophageal wall thickness did not (p=0.102). Median BF and BV were gradually rose and MTT decreased as TRG increased, from 21.4 ml/min/100 g (BF), 1.6 ml/100 g (BV) and 8.6 s (MTT) in TRG 1 group, to 37.3 ml/min/100 g, 3.5 ml/100 g and 7.5 s in TRG 2 group, 81.4 ml/min/100 g, 4.1 ml/100 g and 3.8 s in TRG 3 group, and 121.1 ml/min/100 g, 4.9 ml/100 g and 3.7 s in TRG 4 group. In all 15 patients who achieved complete histopathologic regression (TRG 1), BF was <30.0 ml/min/100 g. CONCLUSIONS: CT perfusion could improve the accuracy in response evaluation of the esophageal carcinoma to nCRT.