bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression Pattern of a Homeotic Gene, HOXA5, in Normal Breast and in Breast Tumors
Authors: Henderson Gregory S. , van Diest Paul J. , Burger Horst , Russo Jose , Raman Venu
Published: January 2021

Introduction: Homeotic (HOX) gene products are now known to be functionally associated with breast cancer biogenesis. Recent evidence has indicated that HOXA5 regulates both p53 and progesterone receptor expression levels in breast cancer cells. In addition, HOXA5 has been shown to interact and regulate the activity of another protein referred to as Twist. As homeotic genes play a pivotal role in development, we sought to decipher the expression pattern in both normal breast tissues and in breast carcinomas. Methods: RT‐PCR and immunohistochemistry were performed, to assay the levels of HOXA5 expression, on a panel of normal breast tissue and its corresponding primary breast tumors. Results and Conclusions: We show that HOXA5 expression was maintained at stable levels at different reproductive stages of a woman′s life, except during lactation. This evidence indicates that HOXA5 may play a role in maintaining the differentiated state within the breast epithelium. However, nearly 70% of all breast carcinomas had decreased HOXA5 protein levels as compared to normal breast tissues. In addition, we demonstrate that HOXA5 protein expression levels in breast carcinomas inversely co‐relates with Epidermal Growth Factor Receptor (EGFR) expression. Furthermore, we found that the survival rate amongst the different low levels of HOXA5 expressing breast tumors was not significant, indicative of an early tumorigenesis process in the absence of innate levels of HOXA5 in normal breast cells.
Transcription Factor E2F‐1 Is Upregulated in Human Gastric Cancer Tissues and Its Overexpression Suppresses Gastric Tumor Cell Proliferation
Authors: Xiao Qiang , Li Lei , Xie Yubo , Tan Ning , Wang Changqing , Xu Junyi , Xia Keyi , Gardner Kevin , Li Qingdi Q.
Published: January 2021

The E2F family members play a critical role in cell cycle regulation and other biological processes in the cell. To better understand the involvement of E2F‐1 in the development and progression of gastric tumors, we investigated the mutation and expression of E2F‐1 in human gastric cancer tissues and the effect of E2F‐1 overexpression on the proliferation of gastric carcinoma cells. In this study, 80 pairs of gastric cancer specimens and paratumor tissues from different patients and 40 stomach mucosa specimens from healthy individuals were examined. PCR‐SSCP analysis demonstrated that mutations were not detected in any of the gastric cancer and normal tissue specimens. In addition, the results of an immunohistochemistry assay revealed higher expression rates of E2F‐1 (P < 0.01) in gastric cancer tissues (72.5%) than in paratumor tissues (30.0%) of the same individuals and stomach mucosa from healthy individuals (22.5%). However, no correlation was observed between the E2F‐1 levels and patients’ clinical features, such as sex, age, histological types, lymph node metastasis, and clinical stages (P >0.05). Finally, the influence of E2F‐1 overexpression on the growth of human gastric carcinoma MKN‐45 cells in vitro was assessed by measuring colony formation, cell survival, and cell cycle progression. Our data clearly showed that cell growth and proliferation were significantly inhibited in MKN‐45 tumor cells transfected with the expression vector encoding E2F‐1 in comparison with nontransfected cells or cells transfected with empty vector. These findings suggest that E2F‐1, a stable and conservative gene during the oncogenesis and progression of stomach cancers, may potentially serve as a biomarker for clinical diagnosis of gastric carcinomas and as a target for the development of novel therapeutic interventions to treat this disease.
TNFα–308G/A Polymorphism as a Risk Factor for HPV Associated Cervical Cancer in Indian Population
Authors: Kohaar Indu , Thakur Nisha , Salhan Sudha , Batra Swaraj , Singh Veena , Sharma Anita , Sodhani Pushpa , Das B. C. , Sarkar Debi P. , Bharadwaj Mausumi
Published: January 2021

Background: Investigation of the potential association of single nucleotide polymorphisms (SNPs) at –308 G/A and –238 G/A of Tumor necrosis factor α (TNFα) with susceptibility to HPV‐16 associated cervical cancer in Indian women. Methods: The study included 165 histologically confirmed cases with 45 precancer and 120 cancer patients and an equal number (165) of healthy controls with normal cervical cytology. PCR‐RFLP was employed to analyze TNFα promoter polymorphisms, which were confirmed by direct sequencing. Both patients and controls were screened for Human Papillomavirus (HPV) infection. Results: The frequency of –308 A allele in TNFα was significantly higher in cases compared with control subjects (21% in cases vs. 9% in controls; p < 0.01), with an odds ratio of 2.7 (95% CI = 1.41–5.15). Also, women carrying A allele for this locus presented 3 times increased susceptibility to HPV 16 infection as evident from carrier genotype distribution between HPV positive cases and control subjects (24% in HPV positive cases vs. 9% in controls; p < 0.01; OR = 3.1; 95% CI = 1.60–6.03). No such association was found for TNFα–238 (G/A) polymorphism with the risk of development of cervical cancer. Conclusion: It suggests that SNP at –308 (G/A) of TNFα promoter may represent an increased risk for HPV infection and development of cervical cancer in Indian women.
Hypoxia and Angiogenesis in Endometrioid Endometrial Carcinogenesis
Authors: Horrée Nicole , van Diest Paul J. , van der Groep Petra , Sie-Go Daisy M. D. S. , Heintz A. Peter M.
Published: January 2021

Background: Hypoxia‐inducible factor 1α (HIF‐1α) plays an essential role in the adaptive response of cells to hypoxia, triggering biologic events associated with aggressive tumor behavior. Methods: Expression of HIF‐1α and proteins in the HIF‐1α pathway (Glut‐1, CAIX, VEGF) in paraffin‐embedded specimens of normal (n = 17), premalignant (n = 17) and endometrioid endometrial carcinoma (n = 39) was explored by immunohistochemistry, in relation to microvessel density (MVD). Results: HIF‐1α overexpression was absent in inactive endometrium but present in hyperplasia (61%) and carcinoma (87%), with increasing expression in a perinecrotic fashion pointing to underlying hypoxia. No membranous expression of Glut‐1 and CAIX was noticed in inactive endometrium, in contrast with expression in hyperplasia (Glut‐1 0%, CAIX 61%, only focal and diffuse) and carcinoma (Glut‐1 94.6%, CAIX 92%, both mostly perinecrotically). Diffuse HIF‐1α was accompanied by activation of downstream targets. VEGF was significantly higher expressed in hyperplasias and carcinomas compared to inactive endometrium. MVD was higher in hyperplasias and carcinomas than in normal endometrium (p < 0.001). Conclusion: HIF‐1α and its downstream genes are increasingly expressed from normal through premalignant to endometrioid adenocarcinoma of the endometrium, paralleled by activation of its downstream genes and increased angiogenesis. This underlines the potential importance of hypoxia and its key regulator HIF‐1α in endometrial carcinogenesis.
Predictive Value of Tumor Load in Breast Cancer Sentinel Lymph Nodes for Second Echelon Lymph Node Metastases
Authors: van Deurzen C. H. M. , van Hillegersberg R. , Hobbelink M. G. G. , Seldenrijk C. A. , Koelemij R. , van Diest P. J.
Published: January 2021

Background: The need for routine axillary lymph node dissection (ALND) in patients with invasive breast cancer and low‐volume sentinel node (SN) involvement is questionable. Accurate prediction of second echelon lymph node involvement could identify those patients most likely to benefit from ALND.

Methods: A consecutive series of 317 patients with invasive breast cancer and a tumor positive axillary SN followed by ALND was reviewed. Clinicopathologic features of the primary tumor and the SN were assessed as possible predictors of second echelon lymph node involvement.

Results: Second echelon metastases were found in 116/317 cases (36.6%). Frequency of second echelon lymph node involvement in patients with isolated tumor cells (ITC, N = 23), micro‐ (N = 101) and macrometastases (N = 193) was 13%, 20% and 48%, respectively (p < 0.001). Based on the area % of SN occupied by tumor no subgroup of patients could be selected with less than 20% second echelon lymph node involvement. However, none of the patients with SN ITC or micrometastases and a primary tumor size ≤1 cm (N = 12, 3.8%) had second echelon lymph node involvement.

Conclusions: Accurately measured SN tumor load predicts second echelon lymph node involvement. However, even in patients with ITC, the second echelon lymph nodes are involved in 13% justifying ALND.
Aberrant Regulation of Messenger RNA 3′‐Untranslated Region in Human Cancer
Authors: López de Silanes Isabel , Paz Quesada María , Esteller Manel
Published: January 2021

The messenger RNA 3′‐untranslated region (3′UTR) is emerging as critically important in regulating gene expression at posttranscriptional levels. The 3′UTR governs gene expression via orchestrated interactions between mRNA structural components (cis‐elements) and specific trans‐acting factors (RNA‐binding proteins and non‐coding RNAs). Alterations in any of these components can lead to disease. Here, we review the mutations in 3′UTR regulatory sequences as well as the aberrant levels, subcellular localization, and posttranslational modifications of trans‐acting factors that can promote or enhance the malignant phenotype of cancer cells. A thorough understanding of these alterations and their impact upon 3′UTR‐directed posttranscriptional gene regulation will uncover promising new targets for therapeutic intervention.
Expression of Integrin Alpha10 Is Induced in Malignant Melanoma
Authors: Wenke Ann-Kathrin , Kjellman Christian , Lundgren-Akerlund Evy , Uhlmann Christian , Haass Nikolas K. , Herlyn Meenhard , Bosserhoff Anja K.
Published: January 2021

Recently, integrin alpha10 was described as a collagen type II‐binding integrin expressed mainly in chondrocytes. However, by array studies we detected integrin alpha10 also to be upregulated in malignant melanoma compared to primary melanocytes. Subsequent analysis of melanoma cell lines and melanoma tumor samples confirmed this finding. Further, we demonstrated that expression of integrin alpha10 is controlled by AP‐2 and Ets‐1, two transcription factors known to be involved in melanoma development and progression. To investigate the functional relevance of integrin alpha10, expression was downregulated via stable antisense transfection. Proliferation assays and colony forming assays revealed no differences comparing antisense integrin alpha10 cell clones with control and wild type melanoma cells, respectively. However, antisense integrin alpha10 cell clones and Mel Im cells treated with an inhibitory antibody against integrin alpha10 showed a reduced migratory potential.

In summary, these data indicate that AP‐2 and Ets‐1 regulated expression of integrin alpha10 plays a role in migration of malignant melanoma cells.
Deletions of N33, STK11 and TP53 Are Involved in the Development of Lymph Node Metastasis in Larynx and Pharynx Carcinomas
Authors: Guervós Marta Alonso , Marcos César Álvarez , Hermsen Mario , Nuño Andrés Sampedro , Suárez Carlos , Llorente José Luis
Published: January 2021

Background: Lymph node metastasis is the mayor cause of mortality in patients with head and neck squamous cell carcinomas (45%). The genetic changes underlying metastasis are still largely unknown and genetic markers to predict lymph node positivity still need to be found. The aim of this study was to search such markers by using Multiplex Ligation‐dependent Probe Amplification (MLPA), a semi‐quantitative PCR technique to detect gene copy number alterations. Methods: Thirty‐seven genes were analysed by MLPA in 34 larynx and 22 pharynx carcinomas. Results: Losses of CDKN2A (9p21) and MLH1 (3p22) and gains of CCND1, EMS1 (both at 11q13), RECQL4 and PTP4A3 (both at 8q24) were the most frequent aberrations in both larynx and pharynx carcinomas. Amplifications were detected at EMS1, CCND1 and ERBB2 (17q21). A correlation between loss of N33 (8p22) and poor survival was found (p=0.02). Gain of EMS1 had the same relation with survival but not significant (p=0.08). Lymph node positive tumors presented a specific pattern of genetic alterations, with losses of N33, STK11 (19p13) and TP53 (17p13), the latter especially in larynx tumors. Conclusion: We propose that these 3 genes might play a role in the development of metastasis in larynx and pharynx squamous cell carcinomas.
Neurotensin is a Versatile Modulator of In Vitro Human Pancreatic Ductal Adenocarcinoma Cell (PDAC) Migration
Authors: Mijatovic Tatjana , Gailly Philippe , Mathieu Véronique , De Nève Nancy , Yeaton Paul , Kiss Robert , Decaestecker Christine
Published: January 2021

Background: While the neurotensin (NT) roles in pancreatic cancer growth are well documented, its effects on pancreatic cancer cell migration have not been described. Methods: The NT‐induced effects on the migration process of human pancreatic ductal adenocarcinoma cells (PDACs) were characterized by means of various assays including computer‐assisted video‐microscopy, fluorescence microscopy, ELISA‐based, small GTPase pull‐down and phosphorylation assays. Results: The NT‐induced modifications on in vitro PDACs migration largely depended on the extra‐cellular matrix environment and cell propensity to migrate collectively or individually. While NT significantly reduced the level of migration of collectively migrating PDACs on vitronectin, it significantly increased the level of individually migrating PDACs. These effects were mainly mediated through the sortilin/NTR3 receptor. Neurotensin both induced altered expression of αV and β5 integrin subunits in PDACs cultured on vitronectin resulting in modified adhesion abilities, and caused modifications to the organization of the actin cytoskeleton through the NT‐mediated activation of small Rho GTPases. While the NT effects on individually migrating PDACs were mediated at least through the EGFR/ERK signaling pathways, those on collectively migrating PDACs appeared highly dependent on the PI 3‐kinase pathway. Conclusion: This study strongly suggests the involvement of neurotensin in the modulation of human PDAC migration.
Biomarkers for Risk Stratification of Neoplastic Progression in Barrett Esophagus
Authors: Kerkhof Marjon , Kusters Johannes G. , van Dekken Herman , Kuipers Ernst J. , Siersema Peter D.
Published: January 2021

Barrett esophagus (BE) is caused by chronic gastroesophageal reflux and predisposes to the development of esophageal adenocarcinoma through different grades of dysplasia. Only a subset of BE patients will finally develop esophageal adenocarcinoma. The majority will therefore not benefit from an endoscopic surveillance program, based on the histological identification of dysplasia. Several studies have been performed to find additional biomarkers that can be used to detect the subgroup of patients with an increased risk of developing malignancy in BE. In this review, we will summarize the most promising tissue biomarkers, i.e. proliferation/cell cycle proteins, tumor suppressor genes, adhesion molecules, DNA ploidy status and inflammation associated markers, that can be used for risk stratification in BE, and discuss their respective clinical application.
EGF‐R is Expressed and AP‐1 and NF‐κ:B Are Activated in Stromal Myofibroblasts Surrounding Colon Adenocarcinomas Paralleling Expression of COX‐2 and VEGF
Authors: Konstantinopoulos Panagiotis A. , Vandoros Gerasimos P. , Karamouzis Michalis V. , Gkermpesi Maria , Sotiropoulou-Bonikou Georgia , Papavassiliou Athanasios G.
Published: January 2021

Background: COX‐2 and VEGF are important triggers of colon cancer growth, metastasis and angiogenesis. Cox‐2 promoter contains transcriptional regulatory elements for AP‐1 and NF‐κ:B transcription factors whilst vegf is a known AP‐1 downstream target gene. We investigated whether stromal myofibroblasts surrounding colon adenocarcinomas express COX‐2 and VEGF and whether activation of AP‐1 and NF‐κ:B, as well as expression of EGF‐R parallel expression of COX‐2 and VEGF in these cells. Methods: Immunohistochemical methodology was performed on archival sections from 40 patients with colon adenocarcinomas. We evaluated c‐FOS, p‐c‐JUN (phosphorylated c‐JUN), p‐Iκ:B‐α (phosphorylated Iκ:B‐α), EGF‐R, COX‐2, NF‐κ:B and VEGF expression in stromal myofibroblasts surrounding colon adenocarcinomas. Double immunostaining with a‐smooth muscle actin and each antibody was done to verify the expression of these molecules in stromal myofibroblasts. Results: VEGF, p‐Iκ:B‐α, NF‐κ:B, c‐FOS, p‐c‐JUN, EGF‐R and COX‐2 were expressed in stromal myofibroblasts surrounding colon adenocarcinomas in the majority of cases. EGF‐R, p‐Iκ:B‐α, NF‐κ:B, c‐FOS and p‐c‐JUN correlated positively with COX‐2 and VEGF expression. Conclusion: Stromal myofibroblasts surrounding colon adenocarcinomas are an important source of VEGF and COX‐2 production, while AP‐1 and NF‐κ:B transcription factors are activated and EGF‐R is expressed in these cells and associated with COX‐2 and VEGF production.
Molecular Alterations of KIT Oncogene in Gliomas
Authors: Gomes Ana L. , Reis-Filho Jorge S. , Lopes José M. , Martinho Olga , Lambros Maryou B. K. , Martins Albino , Schmitt Fernando , Pardal Fernando , Reis Rui M.
Published: January 2021

Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK), is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117) immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17) and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH) and quantitative real‐time PCR (qRT‐PCR) were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179) of cases, namely in 25% (1/4) of pilocytic astrocytomas, 25% (5/20) of diffuse astrocytomas, 20% (1/5) of anaplastic astrocytomas, 19.5% (15/77) of glioblastomas and one third (3/9) of anaplastic oligoastrocytomas. Only 5.7% (2/35) of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0–22.2% of cases, being more frequent in high‐grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT‐PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24) of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti‐KIT RTK inhibitors.
Estrogen Receptor Beta Displays Cell Cycle-Dependent Expression and Regulates the G1 Phase through a Non-Genomic Mechanism in Prostate Carcinoma Cells
Authors: Hurtado Antoni , Pinós Tomàs , Barbosa-Desongles Anna , López-Avilés Sandra , Barquinero Jordi , Petriz Jordi , Santamaria-Martínez Albert , Morote Joan , de Torres Inés , Bellmunt Joaquim , Reventós Jaume , Munell Francina
Published: January 2021

Background: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERβ) remain elusive.

Methods: We have analyzed the levels of ERβ1 and ERβ2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERβ1 in the human prostate cancer LNCaP cell line.

Results: Both ERβ1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERβ2 levels decreased during the S phase and increased in the G2/M phase. ERβ1 protein was detected in both the nuclear and non-nuclear fractions, and ERβ2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERβ was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFκB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERβ1 or ERβ1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERβ1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1–ERβ1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested.

Conclusions: Our results demonstrate that, in LNCaP prostate cancer cells, both ERβ isoforms are differentially expressed during the cell cycle and that ERβ regulates the G1 phase by a non-genomic mechanism.
Tumour-Associated Transcripts and EGFR Deletion Variants in Colorectal Cancer in Primary Tumour, Metastases and Circulating Tumour Cells
Authors: Lankiewicz Silke , Rother Eva , Zimmermann Silke , Hollmann Christiane , Korangy Firouzeh , Greten Tim F.
Published: January 2021

The clinical relevance of circulating tumour cells (CTC) in peripheral blood of patients with colorectal cancer (CRC) has been described as an independent prognostic factor useful to monitor drug effects and clinical status. The aim of the present study was to compare the epidermal growth factor receptor (EGFR) status of primary tumour, related metastases and CTC of patients with CRC. Therefore, in addition to EGFR, the tumour-associated transcripts gastrointestinal tumour-associated antigen 733-2 (GA733-2) and carcinoembryonic antigen (CEA) were analyzed in a multiplex RT-PCR to characterize CTC. 55% patients were positive for CTC. EGFR expression was detected in 18% of these patients. EGFR was expressed more frequently in metastatic and primary tumour tissues as revealed by immunohistochemistry. Besides, detailed expression profiling of EGFR variants in various colorectal and glioma cell lines has been performed to generate positive controls, resulting in the discovery of two new transcript deletion variations (cEX12_15del, cEX12_14del) located on the extracellular domain of the EGFR.
Differential Proteomic Analysis of Nuclear Matrix in Muscle‐Invasive Bladder Cancer: Potential to Improve Diagnosis and Prognosis
Authors: Barboro Paola , Rubagotti Alessandra , Orecchia Paola , Spina Bruno , Truini Mauro , Repaci Erica , Carmignani Giorgio , Romagnoli Andrea , Introini Carlo , Boccardo Francesco , Carnemolla Barbara , Balbi Cecilia
Published: January 2021

Introduction: Although several molecular markers for bladder cancer have been identified, at present little information on prognostic biomarkers is available in the literature. Prognostication of this tumor is largely based on clinicopathological characteristics. Our aim was to identify nuclear matrix (NM) proteins that might serve to better characterize the phenotype of the invasive bladder cancer and to investigate their diagnostic and prognostic roles.

Methods: NM proteins expressed in normal (n=3) or non‐tumoral (n=9) tissue specimens and muscle‐invasive bladder cancer (n=21) specimens were analyzed by two dimensional (2D) gel electrophoresis. PDQuest image analysis software was used to generate a comparative NM proteome analysis. Selected spots were characterized by liquid chromatography coupled to tandem mass spectrometry and Western blot.

Results: We detected over 800 protein spots in each 2D map and 43 spots were identified. 30 proteins were differentially expressed by bladder tumor cells; among these, 19 proteins were detected in bladder tumoral tissues but not in normal and non‐tumoral tissues and seven proteins correlated with tumor stage. One protein (p54nrb) was strongly correlated with vascular invasions and appeared to be also significantly (P <0.0001) associated with a decreased probability of survival.

Conclusion: Important alterations in NM proteins occur in muscle‐invasive bladder cancer. The differentially expressed proteins include biomarkers potentially useful for disease diagnosis, progression and prognosis. Our findings beyond improving the understanding of the biology of bladder cancer, could help to stratify patients into different prognostic subgroups and to select those who might be better candidate to multimodal therapeutic approaches.
Protein Profiling of B‐Cell Lymphomas Using Tissue Biopsies: A Potential Tool for Small Samples in Pathology
Authors: Jansen Corine , Hebeda Konnie M. , Linkels Marianne , Grefte Johanna M. M. , Raemaekers John M. M. , van Krieken Johan H. J. M. , Groenen Patricia J. T. A.
Published: January 2021

Non‐Hodgkin’s lymphoma comprises many related but distinct diseases and diagnosis and classification is complex. Protein profiling of lymphoma biopsies may be of potential value for use in this lymphoma classification and the discovery of novel markers. In this study, we have optimized a method for SELDI‐TOF MS based protein profiling of frozen tissue sections, without dissection of tumour cells. First we have compared chip surfaces and lysis buffers. Also, we have determined the minimal input using laser dissection microscopy. Subsequently, we have analyzed and compared protein profiles of diffuse large B‐cell lymphoma (n=8), follicular lymphoma (n=8) and mantle cell lymphoma (n=8). Benign, reactive lymph nodes (n=14) were used as a reference group.

CM10 chip surface in combination with urea lysis buffer and an input of approximately 50,000 lymphocytes allowed the detection of many differential peaks. Identification of the diffuse large B‐cell lymphoma cases was reliably made in the supervised classification. Unsupervised clustering showed segregation into a benign/indolent cluster predominantly formed by benign, reactive lymph nodes and follicular lymphoma cases and into a more aggressive cluster formed by diffuse large B‐cell lymphoma and mantle cell lymphoma cases. In conclusion, our protocol enables protein profiling of protein lysates derived from small histological samples and the subsequent detection of many differentially expressed proteins, without the need of tumour cell dissection. These results support further evaluation of protein profiling of small lymphoma biopsies as an additional tool in pathology.
Fas-Induced Apoptosis of Renal Cell Carcinoma is Mediated by Apoptosis Signal-Regulating Kinase 1 via Mitochondrial Damage-Dependent Caspase-8 Activation
Authors: Hassan Mohamed , Feyen Oliver , Grinstein Edgar
Published: January 2021

Renal cell carcinoma (RCC) is a prototype of a chemo refractory tumour. It remains the most lethal of the common urologic cancers and is highly resistant to conventional therapy. Here, we confirmed the efficiency of anti-Fas monoclonal antibody (CH11) as alternative therapeutic approach for the treatment of RCC and investigated the molecular mechanism(s), whereby CH11 induces apoptosis of RCC cells. The present study shows an essential role for apoptosis signal-regulating kinase 1 (ASK1), together with both c-jun-N-terminal kinase (JNK) and p38 pathways, and caspase-8 in this process. Furthermore, CH11-dependent induction of the ASK1–JNK/p38 pathways was found to activate the transcription factors AP-1 and ATF-2, and FADD-caspase-8-Bid signalling, resulting in the translocation of both Bax and Bak proteins, and subsequently mitochondrial dysregulation that is characterized by the loss of mitochondrial membrane potential (ΔΨm), cytochrome c release and cleavage of caspase-9, caspase-3 and PARP. Thus, the described molecular mechanisms of CH11-induced apoptosis suggest the reliability of Fas activation as an alternative therapeutic approach for the treatment of patients with advanced renal cell carcinoma.
Genetic Classification of Oral and Oropharyngeal Carcinomas Identifies Subgroups with a Different Prognosis
Authors: Smeets Serge J. , Brakenhoff Ruud H. , Ylstrab Bauke , van Wieringen Wessel N. , van de Wiel Mark A. , Leemans C. René , Braakhuis Boudewijn J. M.
Published: January 2021

The common risk factors for oral and oropharyngeal cancer are tobacco smoking and alcohol consumption, and recently the human papillomavirus (HPV) was shown to be involved in a subgroup. HPV-positive and -negative carcinomas can be distinguished on basis of their genetic profiles. Aim of this study was to investigate patterns of chromosomal aberrations of HPV-negative oral and oropharyngeal squamous cell carcinomas (OOSCC) in order to improve stratification of patients regarding outcome. Thirty-nine OOSCCs were classified on basis of their genetic pattern determined by array comparative genomic hybridization (aCGH). Resulting groups were related to patient and tumor characteristics using the Fisher’s exact test and in addition to survival with the Kaplan–Meier and log rank tests. Classification distinguished three groups, one characterized by hardly any chromosomal aberration (N = 8) and another by a relatively high level (N = 26), and one with a very high level (N = 5) of chromosomal aberrations. This classification was significantly (p = 0.003) associated with survival, with the best survival in the genetically ‘silent’ group and the worst survival in the most aberrant group. The silent profile was significantly (p < 0.05) associated with wild-type TP53, an absence of alcohol consumption and a female gender. These carcinomas were negative for microsatellite instability. This classification of OOSCC was confirmed in an independent set of 89 oral carcinomas. In conclusion, the discovery of these new classes of oral and oropharyngeal cancer with unique genetic and clinical characteristics has important consequences for future basic and clinical studies.
Cyclooxygenase-2 Mediated Regulation of E-Cadherin Occurs in Conventional But Not Early-Onset Gastric Cancer Cell Lines
Authors: Sitarz R. , Leguit R. J. , de Leng W. W. J. , Morsink F. H. M. , Polkowski W. P. , Maciejewski R. , Offerhaus G. J. A. , Milne A. N.
Published: January 2021

Background: COX-2 and E-cadherin, involved in invasion and metastasis, are molecules critical for gastric carcinogenesis. A relationship between them is documented in non-small cell lung and prostate cancer. We present novel evidence of a relationship between COX-2 and E-cadherin expression in gastric cancer.

Methods: Using qPCR and Western blots analysis on celecoxib and PGE2 treated and untreated gastric cancer cell lines derived from tumours of the intestinal type (MKN45, MKN28, AGS3, MKN7) and immunohistochemistry of 178 gastric cancers on tissue microarrays (TMA), we examined the COX-2/E-cadherin relationship.

Results: Down-regulation of COX-2 by celecoxib led to up-regulation of E-cadherin mRNA and protein levels in conventional gastric cancer cell lines, whereas expression was down regulated in the early-onset gastric cancer (EOGC) cell line. Immunohistochemistry on TMAs of 178 gastric cancers showed no correlation between COX-2 and E-cadherin expression in the conventional or early gastric cancer groups.

Conclusions: The results suggest that COX-2 has an impact on transcriptional regulation of E-cadherin in gastric cancer and our findings further highlight the intriguing nature of EOGCs which appear to have a molecular phenotype distinct from conventional gastric cancer. In addition, our findings also suggest that reduction of COX-2 using nonsteroidal anti-inflammatory drugs in gastric cancer chemoprevention may only be relevant for older patients.
Absence of Granzyme B Positive Tumour-Infiltrating Lymphocytes in Primary Melanoma Excisional Biopsies is Strongly Associated with the Presence of Sentinel Lymph Node Metastasis
Authors: van Houdt I. S. , Sluijter B. J. R. , van Leeuwen P. A. M. , Moesbergen L. M. , Hooijberg E. , Meijer C. J. L. M. , de Gruijl T. D. , Oudejans J. J. , Boven E.
Published: January 2021

Background: Sentinel Lymph Node (SLN) status is strongly related to clinical outcome in melanoma patients. In this study we investigated the possible association between the presence of activated and/or suppressive Tumour Infiltrating Lymphocytes (TILs) and SLN status in clinically stage I/II melanoma patients.

Methods: Diagnostic primary melanoma samples from 20 patients with a sentinel lymph node metastasis were compared to melanoma samples from 20 patients with a negative sentinel lymph node, who were matched for gender, age and Breslow thickness. Presence of activated Granzyme B positive (GrB+) TILs, presence of suppressive (FoxP3+) TILs and MHC class I antigen expression on tumour cells were analysed by immunohistochemistry.

Results: FoxP3 and MHC-I expression had no direct bearing on the presence of melanoma metastases in the SLN. Whereas the presence of activated GrB+ TILs in the primary melanoma had no predictive value for SLN status either, their absence was strongly associated with the presence of metastasis in the SLN (p=0.001). While both GrB+ and FoxP3+ TILs could be detected in SLN metastases, a majority did not display MHC-I expression.

Conclusions: These data support a role for cytotoxic T cells in the prevention of early metastasis of melanoma to the draining lymph nodes.
Potential Biomarkers of Colorectal Adenoma–Dysplasia–Carcinoma Progression: mRNA Expression Profiling and In Situ Protein Detection on TMAs Reveal 15 Sequentially Upregulated and 2 Downregulated Genes
Authors: Galamb Orsolya , Sipos Ferenc , Spisák Sándor , Galamb Barnabás , Krenács Tibor , Valcz Gábor , Tulassay Zsolt , Molnár Béla
Published: January 2021

Background: As most colorectal cancers (CRC) develop from villous adenomas, studying alterations in gene expression profiles across the colorectal adenoma–dysplasia–carcinoma sequence may yield potential biomarkers of disease progression.

Methods: Total RNA was extracted, amplified, and biotinylated from colonic biopsies of 15 patients with CRC, 15 with villous adenoma and 8 normal controls. Gene expression profiles were evaluated using HGU133Plus2.0 microarrays and disease progression associated data were validated with RT-PCR. The potential biomarkers were also tested at the protein level using tissue microarray samples of 103 independent and 16 overlapping patients.

Results: 17 genes were validated to show sequentially altered expression at mRNA level through the normal–adenoma–dysplasia–carcinoma progression. Prostaglandin-D2 receptor (PTGDR) and amnionless homolog (AMN) genes revealed gradually decreasing expression while the rest of 15 genes including osteonectin, osteopontin, collagen IV–alpha 1, biglycan, matrix GLAprotein, and von Willebrand factor demonstrated progressively increasing expression. Similar trends of expression were confirmed at protein level for PTGDR, AMN, osteopontin and osteonectin.

Conclusion: Downregulated AMN and PTGDR and upregulated osteopontin and osteonectin were found as potential biomarkers of colorectal carcinogenesis and disease progression to be utilized for prospective biopsy screening both at mRNA and protein levels. Gene alterations identified here may also add to our understanding of CRC progression.
Oncogenic K-Ras Activates p38 to Maintain Colorectal Cancer Cell Proliferation during MEK Inhibition
Authors: van Houdt Winan J. , de Bruijn Menno T. , Emmink Benjamin L. , Raats Danielle , Hoogwater Frederik J. H. , Rinkes Inne H. M. Borel , Kranenburg Onno
Published: January 2021

Background: Colon carcinomas frequently contain activating mutations in the K-ras proto-oncogene. K-ras itself is a poor drug target and drug development efforts have mostly focused on components of the classical Ras-activated MEK/ERK pathway. Here we have studied whether endogenous oncogenic K-ras affects the dependency of colorectal tumor cells on MEK/ERK signaling.

Methods: K-ras mutant colorectal tumor cell lines C26, HCT116 and L169 were used. K-ras or components of the MEK/ERK and p38 pathway were suppressed by RNA interference (RNAi). MEK was inhibited by U0126. p38 was inhibited by SB203850.

Results: MEK inhibition, or suppression of MEK1/2 or ERK1/2 by RNA interference, reduced the proliferation rate of all colorectal cancer cell lines. However, cell proliferation returned to normal after two weeks of chronic inhibition, despite the continued suppression of MEK or ERK. In contrast, K-ras-suppressed tumor cells entered an irreversible senescent-like state following ERK pathway inhibition. MEK inhibition or ERK1/2 suppression caused activation of p38α in a K-ras-dependent manner. Inhibition or suppression of p38α prevented the recovery of K-ras mutant tumor cells during prolonged MEK inhibition.

Conclusions: Oncogenic K-ras activates p38α to maintain cell proliferation during MEK inhibition. MEK-targeting therapeutics can create an acquired tumor cell dependency on p38α.
Gene Promoter Methylation Patterns throughout the Process of Cervical Carcinogenesis
Authors: Yang Nan , Nijhuis Esther R. , Volders Haukeline H. , Eijsink Jasper J. H. , Lendvai Ágnes , Zhang Bo , Hollema Harry , Schuuring Ed , Wisman G. Bea A. , van der Zeea Ate G. J.
Published: January 2021

Objectives: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL).

Methods: QMSP was performed in normal cervix, low-grade (L)SIL, high-grade (H)SIL, adenocarcinomas and squamous cell cervical cancers, and in corresponding cervical scrapings.

Results: Only CCNA1 was never methylated in normal cervices and rarely in LSILs. All other genes showed methylation in normal cervices, with CALCA, SPARC and RAR-β2 at high levels. Methylation frequency of 6 genes (DAPK, APC, TFPI2, SPARC, CCNA1 and CADM1) increased with severity of the underlying cervical lesion. DAPK showed the highest increase in methylation frequency between LSIL and HSIL (10% vs. 40%, p < 0.05), while CCNA1 and TFPI2 were most prominently methylated in cervical cancers compared to HSILs (25% vs. 52%, p < 0.05, 30% vs. 58%, p < 0.05). CADM1 methylation in cervical cancers was related to depth of invasion (p < 0.05) and lymph vascular space involvement (p < 0.01), suggesting a role in invasive potential of cervical cancers. Methylation ratios in scrapings reflected methylation status of the underlying lesions (p < 0.05).

Conclusion: Methylation of previously reported cervical cancer specific genes frequently occurs in normal epithelium. However, frequency of methylation increases during cervical carcinogenesis, with CCNA1 and DAPK as the best markers to distinguish normal/LSIL from HSIL/cancer lesions.
Pre T-Cell Receptor Alpha (pTα) Expression Patterns and Functional Analysis in Human T-Cell Lymphoblastic Leukemia
Authors: Ivanyi Philipp , Morgan Michael , Piao Wenji , Ukena Sya N. , Steube Klaus , Ganser Arnold , Franzke Anke
Published: January 2021

Background: The pTα/preTCR regulates the β-selection, a crucial T-cell developmental checkpoint, providing a most potent survival advantage to thymocytes mediated by the src-kinase p56Lck.

Methods: To define the relevance of pTα in human T-cell lymphoblastic leukemia (T-ALL), we analyzed in T-ALL cell lines (n=14) pTα and p56Lck mRNA and protein expression as also the tyrosine-phosphorylation. The p56Lck specific src-protein-tyrosine kinase inhibitor (PTK-I) PP1 was used in growth inhibition assays. IC50 value determination, cell cycle- and apoptosis analyses were performed in T-ALL-, non-T-ALL- and murine transgenic cell lines.

Results: pTα expression patterns were markedly different in T-ALL cell lines as compared to those reported for normal lymphoid counterparts. PP1 induced in 6/11 T-ALL cell lines a survival disadvantage resulting from a cell cycle arrest in the G1/0 phase in thymic lymphoblastic cells and apoptosis induction in the immature cell line HSB-2, respectively. PP1 sensitive cell lines expressed the target protein p56Lck and showed a corresponding P-Tyr signal.

Conclusions: Sensitivity of thymic T-ALLs to PP1 clearly underlines the impact of pTα mediated proliferation in this leukemic sub-type. In addition, p56Lck represents also independently of pTα a promising therapeutical target for the src-kinase inhibitors in neoplastic lymphoid diseases.
A Virtual Microscopy System to Scan, Evaluate and Archive Biomarker Enhanced Cervical Cytology Slides
Authors: Grabe Niels , Lahrmann Bernd , Pommerencke Thora , von Knebel Doeberitz Magnus , Reuschenbach Miriam , Wentzensen Nicolas
Published: January 2021

Background: Although cytological screening for cervical precancers has led to a reduction of cervical cancer incidence worldwide it is a subjective and variable method with low single-test sensitivity. New biomarkers like p16 that specifically highlight abnormal cervical cells can improve cytology performance. Virtual microscopy offers an ideal platform for assisted evaluation and archiving of biomarker-stained slides.

Methods: We first performed a quantitative analysis of p16-stained slides digitized with the Hamamatsu NDP slide scanner. From the results an automated algorithm was created to reliably detect cells, nuclei and p16-stained cells. The algorithm's performance was evaluated on two complete slides and tiles from 52 independent slides (11,628, 4094 and 25,619 cells/clusters, respectively).

Results: We achieved excellent performance to discriminate unstained cells from nuclei and biomarker-stained cells. The automated algorithm showed a high overall and positive agreement (99.0–99.7% and 70.9–83.4%, respectively) with the gold standard and had a very high sensitivity (89.1–100.0%) and specificity (98.9–100.0%) to detect biomarker-stained cells.

Conclusions: We implemented a virtual microscopy system allowing highly efficient automated prescreening and archiving of biomarker-stained slides. Based on the initial results, we will evaluate the performance of our system in large epidemiologic studies against disease endpoints.
Relevance of Germline Mutation Screening in Both Familial and Sporadic Head and Neck Paraganglioma for Early Diagnosis and Clinical Management
Authors: Hermsen Mario A. , Sevilla María A. , Llorente José Luis , Weiss Marjan M. , Grimbergen Anneliese , Allonca Eva , Garcia-Inclán Cristina , Balbín Milagros , Suárez Carlos
Published: January 2021

Background: Head and neck paraganglioma (PGL) are benign tumors that can cause important direct or surgery induced morbidity. Almost all familial and 11–29% of sporadic PGL are caused by inactivating germline mutations in succinate dehydrogenase (SDH) genes. Our aim was to screen for such mutations and to evaluate clinical parameters as predictors of germline mutation.

Methods: Seventy-four PGL patients were analyzed for germline mutations and large deletions in SDH genes, VHL and RET. Results were correlated to clinical characteristics including gender, age, tumor localization and multifocality. The surgical approach was evaluated in terms of tumor origin, sequelae and subsequent evolution.

Results: Mutations in SDHB and SDHD were identified in equal proportion in 13/13 (100%) of familial and in 15/61 (25%) of sporadic cases. Familiarity, age ≤50 years and male gender were predictors of any germline mutation, while multifocality and carotid/vagal localization were indicative of SDHD mutation in particular.

Conclusions: In contrast to other series, this cohort of Spanish patients showed many SDHB mutations. Sporadic cases with germline mutation are frequent and underline the importance of mutational screening of all PGL patients, allowing the identification of relatives at risk and the early diagnosis of the disease, reducing or avoiding morbidity.
Three-Dimensional Fourth-Order Time-Fractional Parabolic Partial Differential Equations and Their Analytical Solution
Authors: Mussa Yesuf Obsie , Gizaw Ademe Kebede , Negassa Ayana Deressa
Published: January 2021

In this study, the fractional reduced differential transform method (FRDTM) is employed to solve three-dimensional fourth-order time-fractional parabolic partial differential equations with variable coefficients. The fractional derivative used in this study is in the Caputo sense. A few important lemmas which are essential to solve the problems using the proposed method are proved. The novelty of this method is that it uses appropriate initial conditions and finds the solution to the problems without any discretization, linearization, perturbation, or any restrictive assumptions. Two numerical examples are considered in order to validate the efficiency and reliability of the method. Furthermore, the FRDTM solution when α = 1 is compared with other analytical methods available in the existing literature. Computational results are shown in tables and graphs. The obtained results revealed that the method is capable and simple to solve fractional partial differential equations. The software used for the calculations in this study is Mathematica 7.
General Complex-Valued Overlap Functions
Authors: Chen Ying , Bi Lvqing , Hu Bo , Dai Songsong
Published: January 2021

Overlap function is a special type of aggregation function which measures the degree of overlapping between different classes. Recently, complex fuzzy sets have been successfully applied in many applications. In this paper, we extend the concept of overlap functions to the complex-valued setting. We introduce the notions of complex-valued overlap, complex-valued 0-overlap, complex-valued 1-overlap, and general complex-valued overlap functions, which can be regarded as the generalizations of the concepts of overlap, 0-overlap, 1-overlap, and general overlap functions, respectively. We study some properties of these complex-valued overlap functions and their construction methods.
Investigation on Methods of Determining the Grouting Quality of Embedded Rock Bolts Using High Frequency Guided Waves
Authors: Li Peng , Zhang Changsuo , Yang Guanlin
Published: January 2021

Two experiments, with differing equipment setups, were used to test rock bolts, with differing structures and grouting qualities, using low frequency (20–200 kHz) and high frequency (700 kHz‐3 MHz) guided waves to determine the effect of grouting quality on the propagating velocity of the guided waves. The results indicate that grouting quality has a significant effect on the velocity at which waves of low frequencies propagate through embedded rock bolts. As guided wave frequency increases, the sensitivity of the propagating velocity of guided waves to grouting quality decreases. Furthermore, the influence of grouting quality on propagating velocity becomes negligible once the frequency of the guided wave is greater than or equal to 1.0 MHz. An investigation was conducted to ascertain the feasibility of utilizing high frequency guided waves to determine the grouting quality of embedded rock bolts. Moreover, this study discusses a method of evaluating the grouting quality of embedded rock bolts using the peak ratios and average amplitude ratios of the high frequency guided waves. Through an analysis of the results of the abovementioned method, it was discovered that the optimal guided wave frequency is 2.65 MHz for the evaluation of 20 mm fully‐embedded rock bolts because waves with this frequency have the largest average amplitude ratios.