Ana Alarcão

Promoter hypermethylation of DNA repair genes MLH1 and MSH2 in adenocarcinomas and squamous cell carcinomas of the lung

Five years survival of lung cancer is 16%, significantly lower than in prostate (99.9%), breast (88.5%) and colon (64.1%) carcinomas. When diagnosed in the surgical stage it increases to 50% but this group only comprises 14-16% of the cases. DNA methylation has emerged as a potential cancer-specific biomarker. Hypermethylation of CpG islands located in the promoter regions of tumour suppressor genes is now firmly established as an important mechanism for gene inactivation. This retrospective study included 40 squamous cell carcinomas and 40 adenocarcinomas in various surgical TNM stages to define methylation profile and possible silencing of DNA repair genes - MLH1 and MSH2 - using Methylation-Specific PCR and protein expression by immunohistochemistry in tumoural tissue, preneoplastic lesions and respiratory epithelium with normal histological features. The protein expression of MLH1 and MSH2 genes, in the available preneoplastic lesions and in normal cylindrical respiratory epithelium appeared reduced. The frequency of promoter hypermethylation found on these DNA repair genes was elevated, with a higher prevalence of methylation of MLH1 gene in 72% of squamous cell carcinoma. The differences are not so obvious for MSH2 promoter hypermethylation. No correlation was found among the status of methylation, the protein expression and the clinicopathological characteristics. With a larger study, a better characterization of the hypermethylation status of neoplastic and preneoplastic lesions in small biopsies would be achieved, inherent to tumour histology, heterogeneity and preservation, and finally differences in the study population to elucidate other possible mechanisms of altered expression of the hMLH1 and hMSH.

Deficient Production of Reactive Oxygen Species Leads to Severe Chronic DSS-Induced Colitis in Ncf1/p47phox-Mutant Mice

Background Colitis is a common clinical complication in chronic granulomatous disease (CGD), a primary immunodeficiency caused by impaired oxidative burst. Existing experimental data from NADPH-oxidase knockout mice propose contradictory roles for the involvement of reactive oxygen species in colitis chronicity and severity. Since genetically controlled mice with a point-mutation in the Ncf1 gene are susceptible to chronic inflammation and autoimmunity, we tested whether they presented increased predisposition to develop chronic colitis. Methods Colitis was induced in Ncf1-mutant and wild-type mice by a 1st 7-days cycle of dextran sulfate sodium (DSS), intercalated by a 7-days resting period followed by a 2nd 7-days DSS-cycle. Cytokines were quantified locally in the colon inflammatory infiltrates and in the serum. Leukocyte infiltration and morphological alterations of the colon mucosa were assessed by immunohistochemistry. Results Clinical scores demonstrated a more severe colitis in Ncf1-mutant mice than controls, with no recovery during the resting period and a severe chronic colitis after the 2nd cycle, confirmed by histopathology and presence of infiltrating neutrophils, macrophages, plasmocytes and lymphocytes in the colon. Severe colitis was mediated by increased local expression of cytokines (IL-6, IL-10, TNF-α, IFN-γ and IL-17A) and phosphorylation of Leucine-rich repeat kinase 2 (LRRK2). Serological cytokine titers of those inflammatory cytokines were more elevated in Ncf1-mutant than control mice, and were accompanied by systemic changes in functional subsets of monocytes, CD4+T and B cells. Conclusion This suggests that an ineffective oxidative burst leads to severe chronic colitis through local accumulation of peroxynitrites, pro-inflammatory cytokines and lymphocytes and systemic immune deregulation similar to CGD.

EGFR/erB-1, HER2/erB-2, CK7, LP34, Ki67 and P53 expression in preneoplastic lesions of bronchial epithelium: an immunohistochemical and genetic study

A prognostic interpretation of preneoplastic lesions would have impact in bronchial carcinoma early diagnosis and through the study of Erb-B family receptors as they have an important role in lung carcinogenesis. The existence of drugs as tyrosine kinase inhibitors stressed the importance of studying gene alterations for selected chemoprevention schemes and characterization of carcinogenesis. Bronchial preneoplastic lesions were characterized by immunohistochemistry using the antibodies LP34 (high weigh molecular cytokeratin), CK7, chromogranin A, Ki67, p53, C-erbB-2 and EGFR. HER2 and EGFR gene copy number was also evaluated by fluorescent in situ hybridization in those lesions. The expected results defined the origin cell for basal cell hyperplasia and squamous metaplasia as adaptative lesions and dysplasia. By known experiences and published data, beyond the stem cell, the spectral evolution of bronchial preneoplastic lesions was demonstrated by characterizing basal cells (LP34) and their neoplastic potentiality. Dysplasias showed a higher expression of EGFR, Ki67 and p53 with a stepwise increase with the gravity of the respective grading. C-erbB-2 immunohistochemical overexpression was a rare event in preneoplastic lesions. Polysomy was the main mechanism for EGFR and HER2/neu higher gene copy number and together with increased proliferation index (Ki67) will account to preview bronchial carcinogenesis.

Bronchial-pulmonary adenocarcinoma subtyping relates with different molecular pathways

Lung cancer is one of the most common cancers in the world with a high mortality rate. We analyzed 45 surgical samples of the adenocarcinoma, 13 with lymph node metastasis. APC, BCL2, chromogranin A, CK 5/6/18 (LP34), CK20, CK7, cyclin D1, EGFR, ERCC1, HER2, Ki67, LRP, MRP, P53, RB and TTF1 expressions were evaluated by immunohistochemistry (IHC). Higher Ki67, APC, ERCC1 expressions and lower TTF1 expression were identified in advanced stages (IIA and IIIA) of adenocarcinomas, which reflect a more aggressive, less differentiated, possibly a non-TRU adenocarcinoma. Acinar, micropapillary and BA/lepidic adenocarcinoma patterns were the most similar patterns and papillary was the most different pattern followed by solid pattern, according to expression of these markers. Different adenocarcinoma patterns are engaged with different molecular pathways for carcinogenesis, based on the differences of expression. Acinar, BA/lepidic and micropapillary showed higher TTF1 expression (type TRU), and papillary and solid patterns revealed less TTF1 expression, exhibiting a non-TRU/bronchial phenotype. Solid pattern revealed lower HER2 and higher EGFR and ERCC1 (this compared to papillary) expression; papillary higher HER2 and lower ERCC1 expressions; micropapillary higher RB expression; and acinar lower ERCC1 and higher EGFR expressions. Ciclin D1 seems to have more importance in acinar and BA/lepidic patterns than in micropapillary. ERCC1 protein expression in micropapillary, solid and BA/lepidic patterns may indicate DNA repair activation. Inhibition of apoptosis could be explained by BCL2 overexpression, present in all adenocarcinoma patterns. MRP-1 and LRP were overexpressed in all patterns, which may have implications for drug resistance. Further studies are needed to interpret these data regarding to therapy response in advanced staged bronchial-pulmonary carcinomas.

Prevalence and characteristics of Epstein–Barr virus-associated gastric carcinomas in Portugal

BackgroundGastric cancer (GC) is one of the most common malignant tumors of the digestive tract and is the third leading cause of cancer death worldwide. Epstein–Barr virus (EBV) has been associated with approximately 10% of the total cases of gastric carcinomas. No previous study has analyzed the prevalence of EBV infection in gastric cancer of the Portuguese population.MethodsIn the present study, we have analyzed 82 gastric carcinoma cases and 33 healthy individuals (control group) from Coimbra region for the presence of EBV by polymerase chain reaction (PCR) and by in situ hybridization (ISH) for EBV-encoded small RNAs (EBERs). The status of H. pylori infection was assessed by serology and by PCR.ResultsEBV was detected by PCR in 90.2% of stomach cancer cases, whereas EBERs were detected in 11%. In our series, EBV-associated gastric carcinoma (EBVaGC) were significantly associated with gender and the majority of them presented lymph node metastasis. These cases were generally graded in more advanced pTNM stages and, non-surprisingly, showed worse survival. H. pylori infection was detected in 62.2% of the gastric cancers and 64.7% of these patients were CagA+. On the other hand, the H. pylori prevalence was higher in the EBV-negative gastric carcinomas (64.4%) than in those carcinoma cases with EBV+ (44.4%).ConclusionsThe present study shows that prevalence of EBVaGC among Portuguese population is in accordance with the worldwide prevalence. EBV infection seems to be associated to poorer prognostic and no relation to H. pylori infection has been found. Conversely, the presence of H. pylori seems to have a favourable impact on patient’s survival. Our results emphasize that geographic variation can contribute with new epidemiological data on the association of EBV with gastric cancer.

Association Between IL-4 and IL-6 Expression Variants and Gastric Cancer Among Portuguese Population

Introduction Multiple studies have reported strong associations between Helicobacter pylori (Hp) inflammation and gastric cancer (GC) development. Altered expressions of pro/anti-inflammatory cytokines have a crucial role in Hp and GC proliferation. Although there are many studies related to cytokines polymorphisms involvement in GC risk, the role of Interleukin-4 (IL-4) and Interleukin-6 (IL-6) in gastric inflammation process is not yet clarified. Aim This study aimed to investigate the impact of common IL-4 and IL-6 polymorphisms in GC development risk among Portuguese population. Methods A total of 100 GC biopsies (50 with intestinal type, IGC, 50 with diffuse type, DGC) and 50 chronic gastritis cases, used as control group, were included in this case-control study. IL-4 and IL-6 common polymorphisms were genotyped by PCR-SSP, using commercially available kits. Results IL-4 low producer genotypes, IL-4-590TT (OR = 6.7; 95% CI 1.4–32.4) and IL-4-1098GG (OR = 4.4; 95% CI 1.7–16.9) were found associated with IGC and DGC, respectively. We also verified that IL-4 TTT haplotype was linked with both IGC (OR = 5.8; 95% CI 2.3–14.4) and DGC (OR = 2.3; 95% CI 1.0–5.5) groups. Concerning IL-6 results, IL-6-174CG genotype showed a higher prevalence among IGC cases (OR = 7.3; 95% CI 2.7–20.3), and IL-6-174CC (OR = 3.8; 95% CI 1.7–8.7) showed upper prevalence within DGC subjects. Finally, IL-6-174/nt565CG haplotype showed a significant association with both IGC (OR = 7.3; 95% CI 2.7–20.3) and DGC (OR = 7.9; 95% CI 4.2–14.9). Conclusion IL-6 and IL-4 expression variants seem to have an important role in GC risk mechanisms. This study provides preliminary evidence that IL-4 and IL-6 polymorphisms, although not directly linked to the disease, may be useful tools in the study of this multifactorial disease.

Lung adenocarcinoma: Sustained subtyping with immunohistochemistry and EGFR, HER2 and KRAS mutational status

Pulmonary adenocarcinomas are still in the process of achieving morphological, immunohistochemical and genetic standardization. The ATS/ERS/IASLC proposed classification for lung adenocarcinomas supports the value of the identification of histological patterns, specifically in biopsies. Thirty pulmonary adenocarcinomas were subjected to immunohistochemical study (CK7, CK5, 6, 18, CK20, TTF1, CD56, HER2, EGFR and Ki-67), FISH and PCR followed by sequencing and fragment analysis for EGFR, HER2 and KRAS. Solid pattern showed lower TTF1 and higher Ki-67 expression. TTF1 expression was higher in non-mucinous lepidic and micropapillary patterns when compared to acinar and solid and acinar, solid and mucinous respectively. Higher Ki67 expression was present in lepidic and solid patterns compared to mucinous. EGFR membranous staining had increasing expression from non-mucinous lepidic/BA pattern to solid pattern and micropapillary until acinar pattern. EGFR mutations, mainly in exon 19, were more frequent in females, together with non-smoking status, while KRAS exon 2 mutations were statistically more frequent in males, especially in solid pattern. FISH EGFR copy was correlated gross, with mutations. HER2 copy number was raised in female tumours without mutations, in all cases. Although EGFR and KRAS mutations are generally considered mutually exclusive, in rare cases they can coexist as it happened in one of this series, and was represented in acinar pattern with rates of 42.9% and 17.9%, respectively. EGFR mutations were more frequent in lepidic/BA and acinar patterns. Some cases showed different EGFR mutations. The differences identified between the adenocarcinoma patterns reinforce the need to carefully identify the patterns present, with implications in diagnosis and in pathogenic understanding. EGFR and KRAS mutational status can be determined in biopsies representing bronchial pulmonary carcinomas because when a mutation is present it is generally present in all the histological patterns.

Is Preterm Labor Influenced by the Maternal-Fetal Interface?

ABSTRACT Preterm labor (PTL) accounts for almost 11% of deliveries, and is a major cause of neonatal morbidity and mortality. T regulatory (Treg) cells may prevent fetal rejection by the maternal immune system under the influence of progesterone. Case control study was conducted to determine Treg cells, IL-10, TGF-β, and membrane progesterone receptorα (mPRα) in the maternal–fetal interface (placenta), including eight pregnant women with threatened PTL (study group) and 16 normal-delivery women (control group). Comparing study group versus control, mean gestational age of delivery differed significantly (p = 0.02), as did endothelial hyperplasia in the upper half (p = 0.035) and the lower half (p = 0.005) of the placenta. Besides, there was higher expression of mPRα and IL-10 in all layers, while Foxp3 expression occurred equally and only in the decidua. TGF-β expression was similar in both groups. Preterm group placentas showed higher endothelial hyperplasia in both upper and lower halves of the placenta.

Evaluation of HER2 by automated FISH and IHC in gastric carcinoma biopsies.

Purpose The use of trastuzumab (Herceptin) to target HER2 has been applied in breast carcinoma and gastric carcinoma (GC). Previous studies have tested trastuzumabs effectiveness by assessing HER2 expression or HER2 amplification by means of immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). In this work we aimed to evaluate automated FISH and IHC technologies for HER2 detection in GC biopsies to be used in routine pathology practice. Methods The study used an Oracle HER2 IHC System and an LSI HER2/CEP17 Dual Probe on an automated Bond™ system (Leica Microsystems). One hundred GC biopsies were evaluated including 44 intestinal type, 38 diffuse type and 18 indeterminate type according to Laurens classification. Results The overall concordance rate between the automated FISH and IHC methods was 94% (κ = 0.87), as 6 samples were scored as equivocal (4 in IHC and 2 in FISH). Moreover, HER2 positivity was significantly different between the 3 types of GC (p<0.05), being more frequent in intestinal-type GC (23%) than in the other 2 histological types (5% and 0%). Finally, the HER2/CEP17 FISH ratio was significantly different (p<0.01) between disomic and polysomic samples, being higher in polysomic samples (mean 1.633 ± 0.509) than in disomic samples (mean 1.231 ± 0.675). Conclusions Automated HER2 testing of GC biopsies using the Leica Bond system was useful and efficient. This method allowed us to improve normal routine procedures, minimizing time and costs as well as handling and observation errors.

Senescent bronchial fibroblasts induced to senescence by Cr(VI) promote epithelial–mesenchymal transition when co-cultured with bronchial epithelial cells in the presence of Cr(VI)

Cellular senescence is a physiological process that serves as a powerful barrier for tumorigenesis. However, senescent cells can be deleterious for the tissue microenvironment. Such is the case of senescent fibroblasts that release several pro-tumorigenic factors that promote malignant transformation in the nearby epithelial cells. Occupational exposure to hexavalent chromium [Cr(VI)] compounds is a cause of respiratory cancers. Although Cr(VI) is known to induce senescence in human foreskin fibroblasts, the role of senescent fibroblasts in the Cr(VI)-induced malignant transformation of human bronchial epithelial cells was never assessed. Thus, to study the evolutionary dynamics generated by the interaction between human bronchial epithelial cells and senescent bronchial fibroblasts, the non-tumorigenic human bronchial epithelial BEAS-2B cells were co-cultured with Cr(VI)-induced senescent human bronchial fibroblasts for 4 weeks. Under the pressure of 0.5 µM Cr(VI), senescent fibroblasts promoted the acquisition of mesenchymal features on BEAS-2B cells, e.g. the fusiform shape and increased Vimentin expression, consistent with the occurrence of an epithelial-mesenchymal transition-like process. Features of transformed cells including larger nuclei, as well as nuclei with heterogeneous size, were also observed. Altogether the results obtained demonstrate that besides acting over the epithelium, Cr(VI) also affects bronchial fibroblasts driving them senescent. As a consequence, a paracrine communication loop is established with the above-placed epithelium prompting the epithelial cells for malignant transformation and thus facilitating the initial steps of tumorigenesis.