CTNNB1, AXIN1 and APC expression analysis of different medulloblastoma variants

OBJECTIVES: We investigated four components of the Wnt signaling pathway in medulloblastomas. Medulloblastoma is the most common type of malignant pediatric brain tumor, and the Wnt signaling pathway has been shown to be activated in this type of tumor. METHODS: Sixty-one medulloblastoma cases were analyzed for β-catenin gene (CTNNB1) mutations, β-catenin protein expression via immunostaining and Wnt signaling pathway-related gene expression. All data were correlated with histological subtypes and patient clinical information. RESULTS: CTNNB1 sequencing analysis revealed that 11 out of 61 medulloblastomas harbored missense mutations in residues 32, 33, 34 and 37, which are located in exon 3. These mutations alter the glycogen synthase kinase-3β phosphorylation sites, which participate in β-catenin degradation. No significant differences were observed between mutation status and histological medulloblastoma type, patient age and overall or progression-free survival times. Nuclear β-catenin accumulation, which was observed in 27.9% of the cases, was not associated with the histological type, CTNNB1 mutation status or tumor cell dissemination. The relative expression levels of genes that code for proteins involved in the Wnt signaling pathway (CTNNB1, APC, AXIN1 and WNT1) were also analyzed, but no significant correlations were found. In addition, large-cell variant medulloblastomas presented lower relative CTNNB1 expression as compared to the other tumor variants. CONCLUSIONS: A small subset of medulloblastomas carry CTNNB1 mutations with consequent nuclear accumulation of β-catenin. The Wnt signaling pathway plays a role in classic, desmoplastic and extensive nodularity medulloblastoma variants but not in large-cell medulloblastomas

Phenotypic and immunohistochemical characterization of sarcoglycanopathies

INTRODUCTION: Limb-girdle muscular dystrophy presents with heterogeneous clinical and molecular features. The primary characteristic of this disorder is proximal muscular weakness with variable age of onset, speed of progression, and intensity of symptoms. Sarcoglycanopathies, which are a subgroup of the limb-girdle muscular dystrophies, are caused by mutations in sarcoglycan genes. Mutations in these genes cause secondary deficiencies in other proteins, due to the instability of the dystrophin-glycoprotein complex. Therefore, determining the etiology of a given sarcoglycanopathy requires costly and occasionally inaccessible molecular methods. OBJECTIVE: The aim of this study was to identify phenotypic differences among limb-girdle muscular dystrophy patients who were grouped according to the immunohistochemical phenotypes for the four sarcoglycans. METHODS: To identify phenotypic differences among patients with different types of sarcoglycanopathies, a questionnaire was used and the muscle strength and range of motion of nine joints in 45 patients recruited from the Department of Neurology - HC-FMUSP (Clinics Hospital of the Faculty of Medicine of the University of São Paulo) were evaluated. The findings obtained from these analyses were compared with the results of the immunohistochemical findings. RESULTS: The patients were divided into the following groups based on the immunohistochemical findings: a-sarcoglycanopathies (16 patients), b-sarcoglycanopathies (1 patient), y-sarcoglycanopathies (5 patients), and nonsarcoglycanopathies (23 patients). The muscle strength analysis revealed significant differences for both upper and lower limb muscles, particularly the shoulder and hip muscles, as expected. No pattern of joint contractures was found among the four groups analyzed, even within the same family. However, a high frequency of tiptoe gait was observed in patients with a-sarcoglycanopathies, while calf pseudo-hypertrophy was most common in patients with non-sarcoglycanopathies. The a-sarcoglycanopathy patients presented with more severe muscle weakness than did y-sarcoglycanopathy patients. CONCLUSION: The clinical differences observed in this study, which were associated with the immunohistochemical findings, may help to prioritize the mutational investigation of sarcoglycan genes.CNP

Extracellular Matrix Proteome Remodeling in Human Glioblastoma and Medulloblastoma

Medulloblastomas (MBs) and glioblastomas (GBMs) are high-incidence central nervous system tumors. Different origin sites and changes in the tissue microenvironment have been associated with the onset and progression. Here, we describe differences between the extracellular matrix (ECM) signatures of these tumors. We compared the proteomic profiles of MB and GBM decellularized tumor samples between each other and their normal decellularized brain site counterparts. Our analysis revealed that 19, 28, and 11 ECM proteins were differentially expressed in MBs, GBMs, and in both MBs and GBMs, respectively. Next, we validated key findings by using a protein tissue array with 53 MB and 55 GBM cases and evaluated the clinical relevance of the identified differentially expressed proteins through their analysis on publicly available datasets, 763 MB samples from the GSE50161 and GSE85217 studies, and 115 GBM samples from RNAseq-TCGA. We report a shift toward a denser fibrillary ECM as well as a clear alteration in the glycoprotein signature, which influences the tumor pathophysiology. MS data have been submitted to the PRIDE repository, project accession: PXD023350

Expressão imunohistoquímica do p53 carcinoma hepatocelular de pacientes brasileiros

Hepatocellular carcinoma (HCC) is an important type of cancer etiologically related to some viruses, chemical carcinogens and other host or environmental factors associated to chronic liver injury in humans. The tumor suppressor gene p53 is mutated in highly variable levels (0-52%) of HCC in different countries. OBJECTIVE: The objective of the present study was to compare the frequency of aberrant immunohistochemical expression of p53 in HCC occurring in cirrhotic or in non-cirrhotic patients as well as in liver cell dysplasia and in adenomatous hyperplasia. We studied 84 patients with HCC or cirrhosis. RESULTS: We detected p53 altered immuno-expression in 58.3% of patients in Grade III-IV contrasting to 22.2% of patients in Grade I-II (p = 0.02). Nontumorous areas either in the vicinity of HCC or in the 30 purely cirrhotic cases showed no nuclear p53 altered expression, even in foci of dysplasia or adenomatous hyperplasia. No significant difference was found among cases related to HBV, HCV or alcohol. CONCLUSION: The high frequency of p53 immunoexpression in this population is closer to those reported in China and Africa, demanding further studies to explain the differences with European and North American reports.O carcinoma hepatocelular (CHC) é um importante tipo de câncer relacionado etiologicamente a alguns vírus, carcinógenos químicos e outros fatores ambientais que causam danos crônicos ao fígado em humanos. A freqüência de mutação do gene p53 em CHC é altamente heterogênea (0-52%) nos diversos países. OBJETIVO: O objetivo deste estudo foi determinar, imuno-histologicamente, a freqüência da expressão anômala de p53 em CHCs em pacientes cirróticos versus não-cirróticos, bem como em displasia hepática e hiperplasia adenomatosa. Para isso, foram estudados 84 pacientes com carcinoma hepatocelular ou cirrose. RESULTADOS: Foram detectadas expressões do p53 alterado em 58,3% dos pacientes com CHC graus III-IV, contrastando com os 22,2% dos pacientes com CHC graus I-II (p = 0,02). Áreas não tumorais, tanto nas proximidades do CHC como nos 30 casos de cirrose não mostraram expressão nuclear alterada do p53, mesmo nas displasias ou hiperplasias adenomatosas. Quando se considerou HBV, HCV ou alcoolismo nos casos estudados, não se encontrou diferença significativa. CONCLUSÃO: A elevada freqüência de imuno-expressão de p53 nesta população é próxima à relatada na China e África, tornando necessárias outras pesquisas para explicar as diferenças com os CHC estudados na Europa e na América do Norte

Immunocytochemical expression of p16 INK4A and Ki-67 in cytologically negative and equivocal pap smears positive for oncogenic human papillomavirus

This study was designed to analyze the cross-sectional comparison of the p16 INK4A and Ki-67 immunocytochemical expression in negative and equivocal (atyp-ical squamous cells of undetermined significance (ASC-US)) liquid-based cytology (LBC) samples testing positive for high-risk human papillomavirus (HPV) types with HC2 assay or polymerase-chain reaction (PCR). A series of 199 consecutive LBC speci-mens derived from the same number of women participating in the ongoing Latin American Screening Study at Leonor Mendes de Barros Hospital, São Paulo, were analyzed using immunocytochemistry for expression of p16 INK4A and Ki-67 in negative and equivocal LBC samples testing positive for high-risk HPV types with hybrid capture II test (HC2) or PCR. All patients with at least one test positive (cytology, PCR, and/or HC2) were followed each 6 months for 3 years. The follow-up procedure consisted of visual examination, colposcopic inspection, cytology, and HC2 assay. Among the neg-ative cytologic samples, 101 were HPV-positive and 55 HPV-negative. Of the HPV-pos-itive group, 59 of 101 cases (58.4%) were positive for both p16 and Ki67 immunostaining, and 17 of 101 (16.8%) were negative for both. The proportion of Ki-67-positivity increased almost in parallel with the increasing grade of p16-positivity (p = 0.0001 for linear trend). In the HPV-negative group, both markers were negative in 41 of 55 cases (74.5%), and no statistical relationship was observed between the two markers (Pearson, p = 0.595). HPV-positive ASC-US samples demonstrated a simulta-neous positive immunoreaction for p16 and Ki67 in 11 of 16 cases (68.7%), whereas 3 (18.7%) were concurrently negative. The relationship between the two markers was of borderline significance (Pearson, p = 0.053), but no linear relationship was found be-tween the graded p16 and Ki-67 expression (p = 0.065 for linear trend). In the HPV-negative ASC-US group, there was no statistical association between the graded p16 and Ki-67 positivity (Pearson, p = 0.281). After 36 months of follow-up of the ASC-US patients, 6 women still displayed ASC-US smear, of which 4 of 6 were HPV-positive and expressed both p16 and Ki-67 markers. Two of 43 ASC-US smears had high-grade squamous intraepithelial lesions diagnosed (4.6%), and 1 had low-grade squamous intraepithelial lesion (2.3%). All of those were positive for HPV, p16 and Ki-67. Patients with ASC-US diagnosis and positive high-risk HPV status and positive for p16 INK4A Ki67 should be carefully observed to exclude occurrence of a squamous intraepithelial lesion. The combination of these two markers can be a useful implement for manage-ment of women with equivocal cytology.European Commission (EC) - INCO-DEV Programme - Project #ICA4-CT-2001-10013

Lymphatic vessel density and VEGF-C expression are significantly different among benign and malignant thyroid lesions

Thyroid cancer is the most frequent endocrine neoplasia worldwide. The route for metastasis and loco-regional invasion preferentially occurs by lymphatic vessels. For this reason, the assessment of lymphatic vessel density (LVD) is supposed to represent both a prognostic parameter and also a potential therapeutic target. In order to evaluate the value of LVD in benign and malignant thyroid lesions, we analyzed 110 thyroidectomy specimens using D2-40, a specific marker for lymphatic vessels and vascular endothelial growth factor C (VEGF-C), the most potent molecule of lymphatic proliferation. LVD was significantly different between papillary and follicular carcinomas in total (p = 0.045) and peritumoral area (p = 0.042). Follicular adenoma and follicular carcinoma showed an important difference of intra- (p = 0.019) and peritumoral (p = 0.033) LVD. VEGF-C was more markedly expressed in malignancies than in benign lesions (p = 0.0001). Almost all cancers with high positive VEGF-C expression also exhibited increased peritumoral LVD (p = 0.049) when compared with the benign lesions. Indeed, the high peritumoral LVD of malignant thyroid lesions is an important finding for surgery planning and supports the practice of total thyroidectomy in malignant thyroid neoplasm's since the lymphatic peritumoral vessels definitely are an escape path for tumor cells

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Background\ud Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray.\ud \ud \ud Results\ud The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue.\ud \ud \ud Conclusions\ud The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.FAPESP doctoral fellowship n°. 05/50781-2CTC/CEPID/FAPESP [grant n. 1998/14247-6

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Abstract\ud \ud Background\ud Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray.\ud \ud \ud Results\ud The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue.\ud \ud \ud Conclusions\ud The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.FAPES

Combined use of immunohistochemical markers of basal and luminal subtypes in urothelial carcinoma of the bladder: Association with clinicopathological features and outcomes

OBJECTIVES: Whole genome expression profiles allow the stratification of bladder urothelial carcinoma into basal and luminal subtypes which differ in histological patterns and clinical behavior. Morpho-molecular studies have resulted in the discovery of immunohistochemical markers that might enable discrimination between these two major phenotypes of urothelial carcinoma. METHODS: We used two combinations of immunohistochemical markers, i.e., cytokeratin (CK) 5 with CK20 and CK5 with GATA3, to distinguish subtypes, and investigated their association with clinicopathological features, presence of histological variants, and outcomes. Upon searching for tumor heterogeneity, we compared the findings of primary tumors with their matched lymph node metastases. We collected data from 183 patients who underwent cystectomy for high-grade muscle-invasive urothelial carcinoma, and representative areas from the tumors and from 76 lymph node metastasis were organized in tissue microarrays. RESULTS: Basal immunohistochemical subtype (CK5 positive and CK20 negative, or CK5 positive and GATA3 negative) was associated with the squamous variant. The luminal immunohistochemical subtype (CK5 negative and CK20 positive, or CK5 negative and GATA3 positive) was associated with micropapillary and plasmacytoid variants. Remarkably, only moderate agreement was found between the immunohistochemical subtypes identified in bladder tumors and their lymph node metastasis. No significant difference in survival was observed when using either combination of the markers. CONCLUSION: This study demonstrates that these three routinely used immunohistochemical markers could be used to stratify urothelial carcinomas of the bladder into basal and luminal subtypes, which are associated with several differences in clinicopathological features