Maria Reis Silva

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.

Evaluation of dentin formed in autogenous tooth transplantation in the dog: a comparison between one- and two-stage surgical techniques

This study was designed to compare the thickness of dentin formed associated with autogenous tooth transplantation in dogs, using either one- or two-stage surgical techniques. The study consisted of three Beagles, older than 5 months, in which six incisors and six premolars were transplanted to mechanically prepared recipient sockets. One group was transplanted using a one-stage method to recipient beds prepared immediately before transplantation. The second groups of teeth were transplanted using a two-stage method in which the recipient beds were prepared and left to heal for 7 days before transplantation. Dogs were injected with xylenol orange, calcein and oxytetracycline at 2 days before, 3 and 9 weeks after transplantation, respectively, for vital staining. Clinical examinations were carried out every week, and the animals were euthanized 9 weeks later. The jaws were resected, fixed in formaldehyde and embedded in resin. Undemineralized sections were cut and examined by fluorescent microscopy. The thickness of dentin formed in the third week after transplantation and 9 weeks was evaluated by undertaking histomorphometric analysis and analysed using the Mann-Whitney U test (P = 0.05). All the transplanted teeth in both groups survived, and the dentin was formed. No statistically significant difference was found in the thickness of dentin formed in the third week and formed in the third to ninth week between the treatment groups (P = 0.999 and P = 0.998, respectively). This study demonstrated that there was no difference between the two surgical techniques in terms of the thickness of dentin formed in transplanted teeth.

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.

MicroRNA-21 in lung cancer: overexpression in metastasis of pulmonary adenocarcinomas and squamous cell carcinomas

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level. Expression profiling has identified miRNA signatures in cancers that associate with diagnosis, staging, progression, prognosis, and response to treatment. MiRNAs are ideal biomarkers in FFPE-tissue because, unlike mRNA, miRNA integrity is affected very little by formalin fixation. Previous studies have shown that miR-21 overexpression correlated with poor prognosis in NSCLC patients. In this study we investigated the expression of mir-21 in primary carcinoma and metastasis and near non-tumor parenchyma. FFPE samples from surgical specimens and biopsies of 7 pulmonary adenocarcinomas and 5 squamous cell carcinomas and respective metastasis together with normal lung tissue from the same case; these areas were separated by laser-capture microdissection prior to miRNA analysis. The expression level of miR-21 by qRT-PCR was significantly higher in tumor tissues than in adjacent normal tissues (p = 0.005). The overexpression in the metastasis samples compared to adjacent normal tissue was almost satistically significant (p = 0.051). MiR-21 was overexpressed in tumor tissues relative to adjacent non-tumor tissues. We found an increase in miR-21 expression in primary carcinoma and metastasis in pulmonary adenocarcinomas when compared with miR-21 lower expression in squamous cell carcinoma. Despite the small sample studied, further investigation may indicate therapeutic and prognostic relevance of this determination, previous studies suggest that miR-21 and has a role in tumor growth, invasion and metastasis by targeting multiple suppressor genes [1]. Therefore, suppression of miR-21 may provide a novel approach for the treatment of advanced cancers through regulation of tumour suppressor genes.

KRAS and EGFR mutations coexisting in lung adenocarcinoma

Lung adenocarcinoma represents about 42% and 28% of NSCLC in women and men. Adenocarcinomas incidence is still rising being the most frequent type of NSCLC diagnosed in USA. Both EGFR and KRAS gene mutations can contribute to the development of NSCLC, namely adenocarcinomas. EGFR and KRAS mutations are considered by some authors as mutually exclusive explained by the fact that KRAS-MAPK pathway is one of the downstream signalling pathways of EGFR. Lung cancers with KRAS mutations are resistant to EGFR tyrosine kinase inhibitors. Sections of the adenocarcinoma of the lung, formalin-fixed paraffin-embedded tissues (FFPE), were selected to analyze mutations in EGFR exons 19 and 21 and KRAS - codons 12 and 13 by DNA extraction for polymerase chain reaction (PCR). Exon 19 was studied by fragment analysis and exon 21, codons 12 and 13 were studied by direct sequencing. The analysis of FISH results was done by Cappuzzo’s score to EGFR gene. Determination of EGFR protein expression was done by immunohistochemistry (IHC) (Zymed Laboratories). The authors present a rare case with synchronous EGFR and KRAS mutations. The patient is a 77 years old, male with a central 3cm mixed adenocarcinoma. The tumor showed EGFR protein overexpression identified by IHC and chromosome 7 high polyssomy by FISH. The authors call attention to the fact that although EGFR and KRAS mutations are almost always mutually exclusive in some cases they may coexist in the same neoplasia.

EGFR/erb-1, HER2/erb-2, CK7, LP34, Ki67 and p53 expression in preneoplastic lesions of bronchial epithelium

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 tirosine kinase inhibitors (TKis) 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 (FISH) 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.