Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children. (C) 2007 Wiley-Liss, Inc.

HLA-G polymorphisms in women with squamous intraepithelial lesions harboring human papillomavirus

Human papillomavirus (HPV) infection is etiologically associated with low-(LSIL) and high-grade squamous intraepithelial lesions (HSIL) and with cervical cancer. The progression or regression of the lesions may depend, among other factors, on the host heritable immune response. Because human leukocyte antigen (HLA)-G molecules are involved in the modulation of innate and adaptive immune responses, and because no previous studies have evaluated HLA-G polymorphism in patients with SIL, we conducted a study to assess the association between HLA-G polymorphisms and cervical lesions harboring HPV infection. Cervico-vaginal scrapings and blood samples were collected from 125 women with SIL (68 LSIL and 57 HSIL) and from 94 healthy women without HPV infection and cytological abnormalities. HPV type and HLA-G polymorphisms in exons 2, 3 and 8 (14 bp insertion/deletion) were evaluated by PCR methodology, and digested with restriction endonucleases. The Genepop software and the EM and PHASE algorithms were used for statistical analysis. A significant protective association was observed between the presence of the G*0103 allele and SIL and between the G0101/G0104 genotype and HSIL in the group of patients compared to control. The presence of the G0104/+14 bp and G0104/-14 bp haplotypes conferred susceptibility to SIL compared to control. In addition, patients possessing the G0104/+14 bp haplotype and harboring HPV-16 and -18 co-infections were particularly associated with HSIL. These findings suggest that HLA-G polymorphisms may be associated with HPV infection and SIL, consequently representing a profile of predisposition to cervical cancer. Modern Pathology (2009) 22, 1075-1082; doi: 10.1038/modpathol.2009.67; published online 1 May 2009

Classical and non-classical HLA molecules and p16(INK4a) expression in precursors lesions and invasive cervical cancer

Objectives: Viruses and turnout cells may regulate the expression of HLA molecules on the cell surface to escape immune system surveillance. Absence of classical HLA class I molecules may impair the action of specific cytotoxic cells, whereas non-classical HLA class I molecules may regulate innate and adaptive immune cells. We assess here the possible associations between classical/non-classical class I HLA and p16(INK4a) molecule expression in cervical biopsies of women infected with HPV, stratified according to grade of the lesion and HPV type. Study design: Cervical biopsies (N = 74) presenting cervical intraepithelial neoplasia grade 1 (CIN1) (n = 31), CIN2-3 (n = 19), and invasive cancer (n = 14) were evaluated alongside 10 normal cervical specimens. Results: HLA-A/B/C/G staining was observed in the early stages of HPV infection. A significant association was detected between HLA-A/B/C staining and HPV16/18 infection (OR = 0.12, 95%CI: 0.0163-0.7899; p = 0.04). HLA-E expression increased with the progression of the lesion (chi(2)-test for trend = 4.01; p = 0.05), and a significant association was found between HLA-E staining and HPV16/18 infection (OR = 11.25, 95%CI: 2.324-54.465; p = 0.003). Irrespective of the grade of the lesion, HLA-A/B/C staining and p16(INK4a) presented a good concordance (Kappa: 0.67). Conclusions: HLA-E overexpression seemed to be associated with invasive cancer and HPV16/18 infection. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Abnormal cell-cycle expression of the proteins p27, mdm2 and cathepsin B in oral squamous-cell carcinoma infected with human papillomavirus

Since the role of human papillomavirus (HPV) infection in oral carcinogenesis is still unclear, the purpose of this study was to verify the association between the expression of p27, mdm2 and cathepsin B and by HPV-related oral lesions. Fifty-five oral biopsies were studied and HPV detection and typing (6/11, 16, 18, 31 and 33) were performed using polymerase chain reaction techniques. The distribution p27, mdm2 and cathepsin B was determined by immunohistochemistry. Twenty-one (38%) out of the 55 oral lesions tested positive for HPV, of which 6(33%) were HPV 6/11, 1 (5%) was HPV 16,14 (72%) were HPV 18 and none was HPV 33/31. Among the 55 biopsies, immunopostivity for p27, mdm2 and cathepsin B was observed in 17 (30.9%), 37 (67.2%) and 37 (67.2%), respectively. Among 21 HPV-positive oral lesions, immunopostivity of mdm2, p27 and cathepsin B was found, respectively, in 6 (33%) out of 18 benign lesions (BL), 4(22%) out of 18 potential malignant epithelial lesions (PMEL) and 11(57.9%) out of 19 malignant lesions (ML). High-risk HPV types may be associated with oral carcinoma, by cell-cycle control dysregulation, contributing to oral carcinogenesis and the overexpression of mdm2, p27 and cathepsin B. (C) 2009 Elsevier GmbH. All rights reserved.

Cancer/Testis Antigen Expression on Mesenchymal Stem Cells Isolated from Different Tissues

Background/Aims: The expression of cancer/testis antigens (CTAs) on additional normal tissues or stem cells may restrict their use as cancer targets. The objective of the present study was to evaluate the mRNA levels of some CTAs in a variety of tissues. Materials and Methods: mRNA of pericytes, fibroblasts and mesenchymal stem cells (MSCs) derived from adult and fetal tissues, human umbilical vein endothelial cells, MSC-derived adipocytes, selected normal tissues and control cancer cell lines (CLs) were extracted and quantitative polymerase chain reaction was performed for MAGED1, PRAME, CTAG1B, MAGEA3 and MAGEA4. Results: MAGED1 was expressed in all normal tissues and cells evaluated. CTAG1B was expressed at levels comparable to control CLs on MSCs derived from arterial, fetal skin, adipose tissue and saphenous vein, heart, brain and skin tissues. MAGEA4 was detected only in fibroblasts and differentiated adipocytes from MSCs, at levels comparable to the control CLs. Conclusion: The potential use of CTAs in immunotherapy should take into account the potential off-target effects on MSCs.

BUBR1 expression in benign oral lesions and squamous cell carcinomas: Correlation with human papillomavirus

Oral squamous Cell carcinoma (OSCC) is the most common head and neck cancer. Only in Brazil, the estimate is for 14,160 new cases in 2009. HPV is associated with increasing risk of oral cancer, but its role in carcinogenesis is still controversial. BUBR1, all important protein in the mitotic spindle assembly checkpoint (SAC), has been associated with some virus-encoded proteins and cancer. The aim of the present study was to evaluate the expression of BUBR1 in non-malignant oral lesions and OSCC with and without metastasis associated with HPV infection. We performed immunohistochemistry for BUBR1 in 70 OSCC biopsies divided into three groups (in situ tumors, invasive tumors without metastasis and invasive tumors with metastasis) with their respective lymph nodes from samples with metastasis and in 16 non-malignant oral lesions. PCR was performed in order to detect HPV DNA. Significantly higher BUBR1 expression associated with shorter survival (p=0.0479) was observed in malignant lesions. There was also it significant correlation (r=1.000) with BUBR1 expression in lesions with metastasis and their lymph nodes. Ninety percent of OSCC and 100% of benign lesions were HPV positive. HPV16 and HVP18 were present in 13 and 24% of HPV-positive OSCC samples, respectively. HPV was more prevalent (76%) in samples with a high BUBR1 expression and the absence of viral DNA had no influence oil BUBR1 expression. These findings suggest that HPV could be associated with overexpression of BUBR1 in OSCC. but not in benign oral lesions.

Basal levels of DNA damage detected by micronuclei and comet assays in untreated breast cancer patients and healthy women

Breast cancer is the second most frequent type of cancer worldwide and is the most common malignant disease among women. Risk factors for breast cancer include early menarche, late menopause, hormonal therapies, exposure to environmental pollutants, smoking and alcohol use. However, increased or prolonged exposure to estrogen is the most important risk factor. It has been suggested that accumulation of DNA damage may contribute to breast carcinogenesis. Epidemiological studies suggest that cytogenetic biomarkers such as micronuclei in peripheral blood lymphocytes may predict cancer risk because they indicate genomic instability in target tissues. The objective of the present study was to evaluate the frequencies of micronuclei and the extent of DNA damage detected by comet assay in peripheral blood lymphocytes of untreated breast cancer patients and healthy women. The study was conducted using peripheral blood lymphocytes from 45 women diagnosed for Ductal ""in situ"" or invasive breast carcinoma and 85 healthy control women. Micronuclei and comet assays were performed to detect spontaneous DNA damage. The results showed that micronuclei frequencies and tail intensity, detected by comet assay, were significantly higher in the breast cancer group than in controls. The levels of DNA damage were similar in smokers and non-smokers, and aging did not influence the frequencies of micronuclei or tail intensity values observed in either group. In conclusion, the present work demonstrates higher levels of DNA damage in untreated breast cancer patients than in healthy women.

Determination of Cyclophosphamide Enantiomers in Plasma by LC-MS/MS: Application to Pharmacokinetics in Breast Cancer and Lupus Nephritis Patients

This article describes the enantioseleclive analysis of cyclophosphamide (CPA) in human plasma using LC-MS/MS. CPA enantiomers were extracted from plasma using a mixture of ethyl acetate and chloroform (75:25, v/v). The enantiomers were separated on a Chiralcel(R) OD-R column, with the mobile phase consisting of a mixture of acetonitrile and water (75:25, v/v) plus 0.2% formic acid. The protonaled ions and their respective product ions were monitored using two functions, 261 > 141 for CPA enantiomers and 189 > 104 for the internal standard (antipyrine). Recovery rates were higher than 95% and the quantification limit was 2.5-ng/ml plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of intra- and interassay precision and accuracy were less than 10%. The method was applied for the investigation of the enantioselective pharmacokinetics of CPA in a lupus nephritis patient treated with 1 g CPA infused over 2 h and in a breast cancer patient treated with 0.9 g infused over 1 h. No stereoselectivity in the pharmacokinetic parameters was observed for either patient. Clearance values of 2.63 and 2.93 l/h and of 3.36 and 3.61 l/h for (-)-(S) and (+)-(R)-CPA were obtained for the breast cancer and lupus nephritis patient., respectively. Chirality 21:383-389, 2009. (C) 2008 Wiley-Liss, Inc.

HIGH EXPRESSION OF CANCER TESTIS ANTIGENS MAGE-A, MAGE-C1/CT7, MAGE-C2/CT10, NY-ESO-1, AND GAGE IN ADVANCED SQUAMOUS CELL CARCINOMA OF THE LARYNX

Background. Despite diagnostic and therapeutic advances in head and neck cancer, the 5-year survival of patients with laryngeal cancer has not improved in the last 30 years. Several recent studies indicate that specific targets for immunotherapeutic approaches can be useful in the control of cancer. There is considerable interest in the expression of cancer testis antigens in human cancers since they may serve as the basis for an immunologic approach to therapy. Methods. We evaluated by immunohistochemical analysis the expression of cancer testis antigens MAGE-A4 (57B), MAGE-C1 (CT7-33), MAGE-A1 (MA454), MAGE-A3 (M3H67), MAGE-C2 (CT10.5), NY-ESO-1 (E978), and GAGE (GAGE) in squamous cell carcinoma (SCC) of the larynx. Results. A total of 63 cases (57 men and 6 women) of laryngeal SCC were available for this study. The findings were correlated with the clinical course and laboratory data. Expression of at least 1 cancer testis antigen was detected in 42 of 63 of the laryngeal SCCs (67%). In 34 of 42 of the positive cases (81%) there was simultaneous expression of >= 2 cancer testis antigens. There was significant correlation between antigen expression and advanced tumor stage (stage III/IV) in cases with reactivity to only 1 antibody (p = .01) as well as in the cases with reactivity to >= 2 primary antibodies (>= 2 mAbs, p = .04). There was no association between survival and expression of any of the analyzed antigens. Conclusions. We find a high incidence of cancer testis antigen expression in SCCs of the larynx, which was correlated with advanced clinical stage. Our data indicate that cancer testis antigens could be valuable vaccine targets in laryngeal tumors, especially in those with a worse prognosis. (C) 2010 Wiley Periodicals, Inc. Head Neck 33: 702-707, 2011

Expression of genes related to apoptosis, cell cycle and signaling pathways are independent of TP53 status in urinary bladder cancer cells

Urinary bladder cancer is the fourth most common malignancy in the Western world. Transitional cell carcinoma (TCC) is the most common subtype, accounting for about 90% of all bladder cancers. The TP53 gene plays an essential role in the regulation of the cell cycle and apoptosis and therefore contributes to cellular transformation and malignancy; however, little is known about the differential gene expression patterns in human tumors that present with the wild-type or mutated TP53 gene. Therefore, because gene profiling can provide new insights into the molecular biology of bladder cancer, the present study aimed to compare the molecular profiles of bladder cancer cell lines with different TP53 alleles, including the wild type (RT4) and two mutants (5637, with mutations in codons 280 and 72; and T24, a TP53 allele encoding an in-frame deletion of tyrosine 126). Unsupervised hierarchical clustering and gene networks were constructed based on data generated by cDNA microarrays using mRNA from the three cell lines. Differentially expressed genes related to the cell cycle, cell division, cell death, and cell proliferation were observed in the three cell lines. However, the cDNA microarray data did not cluster cell lines based on their TP53 allele. The gene profiles of the RT4 cells were more similar to those of T24 than to those of the 5637 cells. While the deregulation of both the cell cycle and the apoptotic pathways was particularly related to TCC, these alterations were not associated with the TP53 status.

Cancer stem cell immunophenotypes in oral squamous cell carcinoma

Background: The presence of cancer stem cell (CSC) antigens can be evidenced in some human tumors by phenotypic analysis through immunostaining. This study aims to identify a putative CSC immunophenotype in oral squamous cell carcinoma (OSCC) and determine its influence on prognosis. Methods: The following data were retrieved from 157 patents: age, gender, primary anatomic site, smoking and alcohol intake, recurrence, metastases, histologic classification, treatment, disease-free survival (DFS), and overall survival (OS). An immunohistochemical study for CD44 and CD24 was performed in a tissue microarray of 157 paraffin blocks of OSCCs. Results: In univariate analysis, the immunostaining pattern showed significant influences in relation to OS for alcohol intake and treatment, as well as for the CD44+ and CD44-/CD24- immunophenotypes. The multivariate test confirmed these associations. Conclusions: Based on our results, the CD44 immunostaining and the absence of immunoexpression of these two investigated markers can be used in combination with other clinicopathologic information to improve the assessment of prognosis in OSCC.

Laser Phototherapy as Topical Prophylaxis Against Head and Neck Cancer Radiotherapy-Induced Oral Mucositis: Comparison Between Low and High/Low Power Lasers

Background and Objective: Oral mucositis is a dose-limiting and painful side effect of radiotherapy (RT) and/or chemotherapy in cancer patients. The purpose of the present study was to analyze the effect of different protocols of laser phototherapy (LPT) on the grade of mucositis and degree of pain in patients under RT. Patients and Methods: Thirty-nine patients were divided into three groups: G1, where the irradiations were done three times a week using low power laser; G2, where combined high and low power lasers were used three time a week; and G3, where patients received low power laser irradiation once a week. The low power LPT was done using an InGaAlP laser (660 nm/40 mW/6 J cm(-2)/0.24 J per point). In the combined protocol, the high power LPT was done using a GaAlAs laser (808 nm, 1 W/cm(2)). Oral mucositis was assessed at each LPT session in accordance to the oral-mucositis scale of the National Institute of the Cancer-Common Toxicity criteria (NIC-CTC). The patient self-assessed pain was measured by means of the visual analogue scale. Results: All protocols of LPT led to the maintenance of oral mucositis scores in the same levels until the last RT session. Moreover, LPT three times a week also maintained the pain levels. However, the patients submitted to the once a week LPT had significant pain increase; and the association of low/high LPT led to increased healing time. Conclusions: These findings are desired when dealing with oncologic patients under RT avoiding unplanned radiation treatment breaks and additional hospital costs. Lasers Surg.Med. 41:264-270,2009. (C) 2009Wiley-Liss, Inc.

Insights from Studies with Oral Cleft Genes Suggest Associations between WNT-pathway Genes and Risk of Oral Cancer

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of the malignant neoplasms that arise in the mucosa of the upper aerodigestive tract. Recent studies of cleft lip/palate have shown the association of genes involved in cancer. WNT pathway genes have been associated with several types of cancer and recently with cleft lip/palate. To investigate if genes associated with cleft lip/palate were also associated with oral cancer, we genotyped 188 individuals with OSCC and 225 control individuals for markers in AXIN2, AXIN1, GSK3 beta, WNT3A, WNT5A, WNT8A, WNT11, WNT3, and WNT9B. Statistical analysis was performed with PLINK 1.06 software to test for differences in allele frequencies of each polymorphism between cases and controls. We found association of SNPs in GSK3B (p = 0.0008) and WNT11 (p = 0.03) with OSCC. We also found overtransmission of GSK3B haplotypes in OSCC cases. Expression analyses showed up-regulation of WNT3A, GSK3B, and AXIN1 and down-regulation of WNT11 in OSCC in comparison with control tissues (P < 0.001). Additional studies should focus on the identification of potentially functional variants in these genes as contributors to human clefting and oral cancer.

GP5+/6+ SYBR Green methodology for simultaneous screening and quantification of human papillomavirus

Background: Detection and quantification of human papillomavirus (HPV) may help in predicting the evolution of HPV infection and progression of associated lesions. Objectives: We propose a novel protocol using consensus primers GP5+/6+ in a SYBR Green quantitative real-time (Q-RT) polymerase chain reaction (PCR). The strategy permits screening for HPV infection and viral load quantification simultaneously. Study design: DNA from 153 archived cervical samples, previously tested for HPV detection by GP5+/6+ PCR and typed by EIA-RLB (enzyme immunoassay-reverse line blot) or sequence analysis, was analysed using SYBR Green Q-RT PCR. Melting temperature assay (T(m)) and cycle threshold (C(t)) were used to evaluate HPV positivity and viral load. The T(m) in the range of 77-82 degrees C was considered to be positive for HPV-DNA. HPV results generated through GP5+/6+ conventional PCR were considered the gold standard against which sensitivity and specificity of our assay were measured. Results: Out of 104 HPV positive samples, 100 (96.2%) were also determined as positive by SYBR Green Q-RT PCR; of the 49 HPV-negative samples, all were determined as negative. There was an excellent positivity agreement (K = 0.94) between the SYBR Green Q-RT and the previous methods employed. The specificity and sensitivity were 100% and 96.2%, respectively. Comparison of SYBR Green Q-RT and TaqMan oligo-probe technologies gave an excellent concordance (pc = 0.95) which validated the proposed strategy. Conclusions: We propose a sensitive and easy-to-perform technique for HPV screening and viral load quantification simultaneously. (C) 2009 Elsevier B.V. All rights reserved.

Interrelationship between Epstein-Barr virus infection in gastric carcinomas and the expression of apoptosis-associated proteins

Aims: Epstein-Barr virus (EBV) and its associated proteins may be protective against the occurrence of apoptosis that would normally inhibit cancer development and progression. Alternatively, the viral infection may cause altered or mutated expression of oncogenes or tumour suppressor genes that are necessary for tumour development. an action that may also involve apoptosis, In this study, a relationship was sought between occurrence of EBV infection, expression of apoptosis-associated proteins (tumour suppressor gene p53 and oncogenes c-myc and bcl-2) and levels of cell death (apoptosis or necrosis) in 119 cases of gastric carcinoma. Methods and results: The EBV status of the gastric carcinomas (using the EBV-encoded small RNA I (EBER-1) and in-situ hybridization), stage and grade of tumour and sex of patients were compared for bcl-2, p53 and c-myc expression patterns. EBER-1 was detected in approximately 20% of cases studied. There was no significant correlation between levels of cell death in the tumour tissue and EBV status. In the protein analyses, development and progression of gastric carcinoma, with or without EBV infection. was independent of bcl-2 expression. However, in gastric cancers with EBV infection, p53 overexpression was inhibited and c-myc expression was increased in early stage cancers, in comparison with decreased c-myc expression in late stage cancers. Conclusions: The p53 and c-myc expression patterns indicate that EBV-infected gastric carcinomas are less likely to have a natural regression via apoptosis at an early stage and explain, in part, the resistance to treatment of late stage of gastric cancers.