as that reported by others at high concentrations of EGF. However, Sigismund and colleagues found that the cholesterol- interfering drug filipin can inhibit endocytosis of EGFR after stimulation with medium and high concentrations of EGF . We did not observe any effect of filipin on HB-EGF- and BTC- induced endocytosis (Figure 6). Thus, the same mechanisms do not appear to be involved. We were not able to positively identify an alternative mechanism of EGFR internalization, despite inhibiting a range of known mediators ofinternalization (Figure 4+6–7, Figures S1, S2, S3). Although macropinocytosis is a known pathway for EGFR internalization [11–13] we saw no effect on the uptake of EGFR after ligand-stimulation in our cells (Figure 4). Similarly we found no effect of inhibiting caveolin1 (Figure 7), RhoA, Arf6 or flotillin1/2 (Figures S1, S2, S3) on EGFR internalizationafter stimulation withdifferentligands. This could indicate the involvement of an internalization pathway that has yet to be characterized. Also, long-term inhibition of endocytosis, such Figure 6. EGFR internalizationafter filipin treatment. A: Cells were incubated with or without 1 mg/ml filipin for 1 hour, and then treated with 10 nM ligand for 15 minutes at 37uC. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing filipin to control treatment for each ligand was performed using two-way ANOVA with Bonferroni posttest. ns = non significant. B: Cells were incubated with or without 1 mg/ml filipin for 1 hour, and then allowed to bind fluorescently labeled cholera toxin on ice for 30 minutes. The cholera toxin solution was removed and the cells were allowed to internalize the cholera toxin for 1 hour at 37uC. After uptake the cells were washed and cholera toxin uptake was determined using flow cytometry. Statistical analysis comparing filipin to control treatment was performed using t-test. *** = p,0.001. C: Test of EGFR levels. Cells were lysed in RIPA buffer and resolved by SDS-PAGE and western blotting. Actin is used as a loading control.
Epidermalgrowthfactorreceptor (EGFR), member ofthe human epidermalgrowthfactorreceptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation ofthe EGFR signal- ing has been found to be associated withthe development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activationof additional bypass sig- naling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, themechanisms underpinning drug-resis- tance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling ofmechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI- resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization ofthe erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overex- pression and/or MET gene amplification and MET receptoractivation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI- resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptoractivation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.
Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing ofthe NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermalgrowthfactorreceptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by themechanismsof tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activationof signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials ofthe covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC.
detected output highly sensitive to any affinity changes. The measurements in this paper were conducted at room temperature, and no signs ofinternalization were detected for the U343 cell line during a 24 hour timeframe (data not shown) implying that internalization can be neglected in all our experiments. Thus, gefitinib binding to the intracellular part ofthe EGF receptor can apparently change the extracellular binding properties of EGFR. If this effect is due to conformational changes ofthereceptor or intracellular processes is yet to be determined, although the increased effect of gefitinib in starved A431 and SKOV3 cells indicate that the condition ofthe cell is also important.
Staphylococcus (S.) aureus is an important pathogen causing various infections including those ofthe skin. Keratinocytes are able to sense invading S. aureus and to initiate a fast defense reaction by the rapid release of innate defense mediators such as antimicrobial peptides and cytokines. There is increasing evidence that the cytokines IL-1alpha and IL- 1beta, which both signal through the IL-1 receptor, play an important role in cutaneous defense against S. aureus. The aim of this study was to gain more insight into the underlying mechanisms leading to the S. aureus-induced IL-1alpha and IL-1beta expression in kerati- nocytes. Infection of human primary keratinocytes with S. aureus led to the induction of gene expression and protein secretion of IL-1alpha and IL-1beta. Full S. aureus-induced IL- 1 protein release required the inflammasome components caspase-1 and ASC (apoptosis- associated speck-like protein containing a CARD) whereas gene induction of IL-1alpha and IL-beta by S. aureus was not dependent on caspase-1 and ASC. Since patients receiving anti-cancer therapy by inhibition oftheepidermalgrowthfactorreceptor (EGFR) often suffer from skin infections caused by S. aureus we additionally evaluated whether the EGFR path- way may be involved in the IL-1alpha and IL-1beta induction by S. aureus. Inactivation ofthe EGFR with a blocking antibody decreased the S. aureus-mediated IL-1alpha and IL- 1beta induction in primary keratinocytes. Moreover, the use of siRNA experiments revealed that ADAM17 (A Disintegrin and A Metalloprotease 17), a metalloproteinase known to medi- ate the shedding and release of EGFR ligands, was required for full induction of IL-1alpha and IL-1beta in keratinocytes infected with S. aureus. A failure of keratinocytes to ade- quately upregulate IL-1alpha and IL-1beta may promote S. aureus skin infections.
In the normal condition, the network signaling is initiated by binding ofepidermalgrowthfactor (EGF) to EGFR, followed by the dimerization and subsequent mutual trans-phosphorylation on several tyrosine residues of EGFR; although EGFR family has other three members including ErbB2, ErbB3, and ErbB4 in addition to EGFR (ErbB1) which can form different dimmers (either homo- or hetero-dimers) [30,35], just EGFR and EGFR- EGFR homo-dimers are considered in this model for simplifica- tion. These phospho-tyrosine residues act as docking sites that enable receptors to recruit adaptor proteins Shc and Grb2 , which can then recruit the guanosine nucleotide exchange factor SOS. SOS promotes the replacement of GDP by GTP in Ras, thereby activating Ras . The activated Ras subsequently results in theactivationof protein kinase Raf ; since the direct activator of Raf kinase is not known yet, we assume that Raf phosphorylation is caused directly by a Ras-GTP molecule in this model. The activated Raf finally activates the mitogen-activated protein kinase kinase1/2 (MEK) and ERK in a cascade way . The activated ERK can phosphorylate the upstream protein SOS, hence causing the dissociation of Grb2-SOS from thereceptor complex, which forms a negative feedback loop . In addition, Grb2 in cytomembrane can also recruit Gab1, which causes the phosphorylation of Gab1 and the consequent recruitment of PI3K . In cytomembrane, PI3K transforms phosphatidylinositol 4,5- bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3) that induces theactivationof AKT through cooperating with 3-phosphoinositide dependent protein kinase-1 (PDK1) . In this process, phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) can specifically dephosphorylate PIP3 and AKT respectively . Phosphorylated Raf, MEK and ERK can be dephosphorylated by their specific phosphatases . P38 that is thought as an important pro-apoptotic effector can
EGFR is becoming an important therapeutic target, with some anti-EGFR drugs already being used in clinical practice and several novel EGFR inhibitors under development (38). Among the two major approaches, using EGFR-TKIs and mAbs, the former seems to be more suitable for gliomas due to their low molecular weight potentially being better at overcoming the blood _ brain barrier (39). In NSCLC, the presence of activating mutations in the EGFR kinase domain was associated with selective EGFR-TKI sensitivity, allowing the selection of patients with a higher probability of clinical response to gefitinib and erlotinib (11). However, these mutations have never been found in glioma cell lines (11), or in glioma patients (11, 40, 41). Recently, Lee and colleagues reported EGFR activation in GBM due to missense mutations in the EGFR extracellular domain (42). They reported that transformed cells withthe EGFR ectodomain mutations had increased sensitivity to erlotinib, however studying DNA samples from a previous clinical trial, these authors were unable to associate EGFR ectodomain mutations with clinical responses to EGFR TKI inhibitors (42).
In this study, most of RC and DC cases and all KOT cases showed high positivity for p63 protein in all cell layers. Lo Muzio et al. (2) observed that the positivity for p63 in RC and DC was restricted to basal and parabasal layers ofthe cystic epithelial lining, whereas in KOT the positivity was more evident in the intermediate and superficial layers. These results are similar to the present study and may probably be associated with a disturbance in cellular cycle control, leading to a higher intrinsic growth potential, which could explain the more infiltrative and aggressive growth and higher rates of recurrence observed in KOT. Interestingly, in RC, the epithelium adjacent to regions with severe inflammatory reaction presented diminished p63 expression. This finding suggests that the alterations caused by the inflammatory cells in the epithelial lining contributed to decrease the expression of that protein.
Because lymphocytes play an important role in tumor eradication and macrophages are associated with tumor progression[12, 13], we presumed that patients with higher lymphocyte- to-monocyte ratio (LMR) might have better prognosis in EGFR-mutant NSCLC patients receiving first-line EGFR-TKIs. The LMR was found to be a prognostic factor in hematological cancer [14, 15] and in several types of solid tumors. [16–18] In addition, elevated LMR was found to be an independent prognostic factor in patents with early-stage lung cancer after com- plete resection and in patients with advanced-stage lung cancer who were undergoing plat- inum-based chemotherapies. However, to the best of our knowledge, the prognostic significance of baseline and trend of LMR in EGFR-mutant NSCLC patients receiving first-line EGFR-TKIs has not been established. We conducted a retrospective analysis to investigate the influence of baseline and trend of LMR on PFS and OS.
Gene expression analysis was performed in untreated pMSCs and pMSCs treated with 1 or 10 μg/mL LL-37 for 2 days. Cells were recovered and submitted to ribo- nucleic acid (RNA) extraction using the RNEasy Mini Kkit, as indicated by the manufacturer (Qiagen, Valencia, CA, USA). Briefly, after collection, 3 × 10 4 pMSCs were disrupted in RLT buffer, ethanol was added and samples were transferred to the kitʼs spin column. Total RNA was retained in the column while contaminants were washed away using buffer RW1 and buffer RPE. Then, total RNA was eluted. RNA amount and quality were determined using NanoDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and analyzed by a Bioanalyzer (Agilent Genomics, Santa Clara, CA, USA). Samples with RIN 8 or higher were used for comple- mentary DNA (cDNA) production. Total RNA was reverse transcribed using the High Capacity cDNA Arch- ive Kit, and real-time PCR was performed using TaqMan probes and MasterMix (Applied BioSystems, Foster City, CA, USA), following manufacturer’s instructions.
This research was approved by the Institutional Ethics Committee withthe following reference number: CAAE - 37156714.6.0000.5149. From January 2008 to December 2015, we selected patients undergoing surgical treatment and histological diagnosis of cholangiocarcinoma. We collected data regarding gender, age, previous history, history of smoking or alcoholism, presence of gallstones, family history for neoplasia, histological type, tumor staging and survival after treatment. The laboratory tests analyzed were bilirubins and Ca 19-9. The classification ofthe anatomical location ofthe tumors and the staging were based on the description ofthe radiological studies (Computed Tomography, Magnetic Resonance and/ or Magnetic Resonance Cholangiopancreatography) associated withthe findings ofthe surgical description and anatomopathological study. We revised the hematoxylin and eosin (HE) stained slides to classify tumors as polypoid and non-polypoid. Classification and staging followed the TNM pattern ofthe American Joint Cancer Committee/Union for International Cancer Control - 7th edition 20,21 .
promote signalling independently of G proteins. For instance, theactivationof ERK1/2 by angiotensin II has been reported as dual; one initial and rapid stage (after 2 minutes) driven by Gαq and a later and more sustained activation (after 10 minutes) driven by arrestins afterreceptorinternalization . Since ERK5 phosphorylation by Gq-coupled GPCR occurs at a relatively late stage afterreceptoractivation (usually peaking around 15/30 minutes ) it was tempting to suggest an involvement of β-arrestin through mechanisms dependent on receptor phosphorylation and internalization. In the same lines, neutrophin-induced ERK5 activation in neurons has been shown to require theinternalizationofthe p62/PKCζ complex . However, our results clearly show that activationof ERK5 by muscarinic M1/M3 receptors does not seem to involve receptorinternalization, which is one ofthemechanisms for β- arrestin to initiate signalling. The possibility remained that a β- arrestin-mediated event occurred at the plasma membrane. However, we found that phosphorylation-deficient muscarinic Figure 4. β-arrestins are not involved in ERK5 activation by Gq-coupled muscarinic receptors. (A) CHO cells stably expressing wild-type muscarinic M3 receptor (CHO-M3 cells) were transfected with cDNAs encoding for HA-ERK5 and either pcDNA3, Gαq, β-arrestin1-Flag or β-arrestin2-GFP. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with carbachol (10µM) for the indicated times. ERK5 phosphorylation was assessed as in Figure 2. Data (mean +/- SEM of 3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions (*p<0.05, **p<0.005, two-tailed T-test). Gαq, β-arrestin1-Flag and β-arrestin2-GFP, and α-tubulin expression levels were assessed with specific antibodies. (B) NIH-3T3-M1 cells were transfected with siRNA oligonucleotides targeting β-arrestin2 or non- targeting scrambled oligonucleotides (100nM) as detailed in the Materials and Methods section. After 72h of incubation, cells were serum-starved for 5h and challenged with carbachol (10µM) for the indicated times. Endogenous ERK5 phosphorylation was assessed in cell lysates with a phospho-ERK5 specific antibody. Total ERK5 appears as a double band corresponding to basal (lower) and hyper-phosphorylated (upper) kinase. A representative blot for 3 independent experiments with similar results is shown. (C) β-arrestin2 expression levels were also determined and quantified to estimate the overall efficiency of protein silencing. Data (mean +/- SEM of 3 independent experiments) were normalised using α-tubulin as loading control and expressed as the relative difference (%) to β-arrestin2 protein levels in scrambled siRNA-treated cells.
The inability of standard therapeutic protocols to effectively treat MBC has prompted a search for other therapeutic options, including those targeting EGFR. Aberrant signaling through EGFR overexpression is associated with neoplastic cell proliferation, migration, stro- mal invasion, resistance to apoptosis, and angiogenesis . Bae et al  reported that MBC exhibited higher expression of EGFR compared to triple negative infiltrating ductal carcinoma. Reis-Filho et al  observed that 19 of 25 (76%) MBC cases exhibited EGFR expression. In our study, EGFR overexpression was identified in 57.8% of MBCs, and in 71.9% ofthe triple negative MBCs. Basal-like MBC lacking EGFR and KIT activating mutations may exhibit high EGFR copy numbers . Reis-Filho al  reported EGFR gene amplification in 37% ofthe MBCs with EGFR overexpression. We found that 29.8% (14/47) of MBCs demonstrated EGFR gene amplification. These results beg the question of whether MBC patients with EGFR overex- pression and/or gene amplification might benefit from EGFR tyrosine kinase inhibitor and EGFR monoclonal antibody (cetuximab) therapies. We noted that squamous cell carcinoma had a significantly higher proportion of EGFR overexpression (82.1%) compared to other sub- types (p = 0.002). Whether squamous cell carcinoma will respond more favorably to cetuximab remains a valid clinical question to be answered. It seems reasonable to recommend a routine assessment ofthe EGFR status in MBC and to further explore this therapeutic option.
20. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S, Campos D, Maoleekoonpiroj S, Smylie M, Martins R, van Kooten M, Dediu M, Findlay B, Tu D, Johnston D, Bezjak A, Clark G, Santabárbara P, Seymour L; National Cancer Institute of Canada Clinical Trials Group. Erlotinib in previously treated non–small-cell lung cancer. N Engl J Med. 2005;353(2):123-32. 21. Kim ES, Hirsh V, Mok T, Socinski MA, Gervais R, Wu YL, et al. Gefitinib versus
The tested steels have wide application as a construction material, meeting particular conditions of loading during exploitation. Steel S355NL is often used in building constructions and machine construction, operating, among others areas in mining, drilling and motor industry. Steel X5CrNi18-10 according to the standard PN-EN 10088, is ranked as resistant to corrosion. It is used in many industrial branches such as in food and chemical industry, in devices used in medicine or households. The type is widely used due to its chemical capacity in contact with many types of chemical compounds.
Com o desenvolvimento da biologia molecular e da descoberta de genes e produtos protéicos que regulam o crescimento e a progressão tumoral, o foco das pesqui- sas tem-se voltado para o desenvolvimento de agentes que possam interferir direta ou indiretamente no ciclo bio- lógico da célula neoplásica, e não somente detectá-lo mais precocemente. Isso representa enorme contribuição no tratamento do câncer de pulmão, pois estaríamos objeti- vando alvos moleculares ao invés de somente células já formadas. Neste campo, a descoberta mais importante talvez tenha sido a dos fatores de crescimento epidérmicos. O receptor do fator de crescimento epidérmico ( EGFR )
For the cell migration assay, BALB/MK cells were transformed withthe same volume of virus from PA317/LESN clones. Since the viral titer differed among the clones, the number of infected BALB/MK cells was also different. Consequently, cells incubated with a higher viral concentration supposedly had a greater level of EGF production and greater migration. However, PA317/LESN clone 9, which had a viral titer > 1 x 10 6 cfu/mL, grew less than clone 6, which had a lower titer (Figure 4). The most likely explanation for this discrepancy is that the viral DNA incorporated into the cells suffered mutations or rearrange- ments that resulted in a low level of EGF gene expression. This hypothesis was supported by the finding that the viral genome in PA317/LESN clone 9 was smaller than that ofthe other clones, as shown by Southern blotting after diges- tion with Sal I, which cleaves in both LTR regions (not shown). In addition, northern blotting revealed a strong band of EGF RNA only in clone 6 (not shown). The latter finding supports the idea that a rearrangement of DNA in clone 9 resulted in low expression of EGF. Clone 4 of PA317/LESN had a viral titer < 1 x 10 5 cfu/mL but caused
Structural and ligand based methods supported 2-O-caffeoyl tartaric acid, Emetine, Rosmaricine, and 2-O-feruloyl tartaric acid as potential EGFR inhibitors. Structurally, the TCM candidates were capable of forming H-bonds with key residues Asp855, Lys716, and Lys728 and matched hydrophobic regions ofthereceptor. Bioactivity ofthe candidates were evaluated using validated MLR, SVM, CoMFA, and CoMSIA models. All models indicated that the TCM candidates have good predicted bioactivity. Molecular simulation results further supported the high potential for the TCM candidates in drug development. IressaH, the drug currently used clinically, bound to the ERGF receptor through a single H-bond at Asp855. In comparison, multiple H-bonds formed at Asp855 and addi- tional H-bonds formed at Ala722 and Arg841 increase the stability of Emetine and Rosmaricine, respectively. The ability of carboxyl groups in 2-O-caffeoyl tartaric acid and 2-O- feruloyl tartaric acid to form multiple H-bond networks that directly blocked the ATP binding site was also a unique characteristic worthwhile of further investigation. Contour to hydrophobic regions ofthe TCM candidates within thereceptor site provides additional support for the stability ofthe protein-ligand complex. In summary, using different simulation and validation methods, we have identified four TCM compounds that may have potential as novel EGFR inhibitors. As the four TCM compounds have two distinctive types of binding locations and bond formation within the EGFR binding site, we suggest exploring the possibility of connecting Emetine/Rosmaricine with 2-O-caffeoyl tartaric acid/2-O-feruloyl tartaric acid through a spacer. The connec- tion could allow more of points of attachment, which in turn would contribute to more stable binding within the tyrosine kinase site.
homogenate which confirmed previous exposure to AA; the insert shows the dA-AAI adduct on the thin-layer chromatography plate after autoradiography. (b) Tissue expression of alpha-smooth muscle actine (α-SMA) was observed within vessels walls (▶) and by several interstitial cells (#) predominantly outside of fibrotic areas ($). Glomeruli were well-preserved glomeruli (!). (c) Several interstitial cells expressed receptorof transforming growthfactor beta (TGFβ) (CD105-endoglin: !) and (d) phosphorylated Smad2/3 (!) around fibrotic areas ($). (e) Besides podocytes marked expression of vascular endothelial growthfactor (VEGF) (!) was found in several atrophic tubular cells (§) typically around fibrotic areas ($). (f) Cells expressing platelet-derived growthfactorreceptor beta (PDGFRβ) (!) massively accumulated in the interstitial areas around atrophic tubules (§). Immunohistochemical analysis of human formalin-fixed paraffin-embedded kidney tissue sections (immunoperoxidase technic counterstained with hematoxylin) using anti-human antibodies recognizing: (b) alpha-SMA (mouse monoclonal, Dako, Heverlee, Beglium), (c) TGFβR (CD105, endoglin) (mouse monoclonal, Thermo Scientific, Fremont CA, USA), (d) phosphorylated Smad2/3 (rabbit polyclonal affinity purified, Santa-Cruz Biotechnology, Inc, Dallas, TX, USA), (e) VEGF (rabbit polyclonal, Thermo Scientific, Fremont CA, USA), (f) PDGFRβ (rabbit monoclonal, Cell Signaling Technology, Leiden, The Netherlands). Original magnification: b: x40; c, d and f: x100; e: x200.
resection ofthe lesion and conirmation ofthe primary site by immunohistochemistry (positive expression of cytokeratin 7 [CK7], epithelial membrane antigen [EMA], mammaglobin, gross cystic disease luid protein 15 [GCDFP-15) and trans-acting T-cell- speciic transcription factor [GATA-3]; and negative expression of cytokeratin 20 [CK20], intestinal transcription factor [CDX-2], villin, cancer antigen 125 [CA125], thyroid transcription factor 1 [TTF-1], napsin A, paired box transcription factor 8 [PAX8], renal cell carcinoma [RCC]-associated antigen, and carbohydrate antigen 19.9 [CA19.9]). Secondary CNS involvement was detected by computerized tomography and/or magnetic resonance during patients’ clinical follow-up. For immunohistochemistry, 3-µm- thick histological sections were obtained from each sample and placed on silanized slides. Each slide was deparafinized with xylol and hydrated with ethanol. Antigen retrieval was carried out using a microwave oven, in a 10 mM solution of citric acid, pH 6, for two nine-minute cycles, rated at 750 W. Endogenous peroxidase blocking was performed with 3% hydrogen peroxide (10 volumes). The primary antibody was diluted in 1% albumin solution and 0.1% sodium azide, in phosphate-buffered saline (PBS), incubated in a humid chamber, during 30 minutes at 37ºC, and kept under refrigeration at 4ºC during 18 hours. Biotinylated secondary antibody was employed in a humid chamber at 37ºC, during 30 minutes, as well as the incubation withthe streptavidin-biotin- peroxidase complex (Strep ABC). The used chromogenic substrate was diaminobenzidine 60 mg% in PBS, and Harris hematoxylin was used as the counterstain. The following antibodies were used (Dako Corporation or Novocastra): ER, PR, c-erb-B2, CK7, CK20, EMA, mammaglobin, GCDFP-15, GATA-3, CDX-2, villin, CA125, TTF-1, napsin A, CA125, PAX8, RCC and CA19.9.