Luís Pedro Afonso

Association of ERBB2 gene status with histopathological parameters and disease-specific survival in gastric carcinoma patients.

The clinical significance of ERBB2 amplification/overexpression in gastric cancer remains unclear. In this study, we evaluated the ERBB2 status in 463 gastric carcinomas using immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH), and compared the findings with histopathological characteristics and with disease-specific survival. ERBB2 overexpression (2+ and 3+) and amplification (ratio ERBB2/CEP17⩾2) were found in 43 (9.3%) and 38 (8.2%) gastric carcinomas, respectively. Perfect IHC/FISH correlation was found for the 19 cases scored as 0 (all negative by FISH), and also for the 25 cases scored as 3+ (all positive by FISH). One out of six carcinomas scored as 1+ and 12 out of 18 carcinomas scored as 2+ were positive by FISH. ERBB2 amplification was associated with gastric carcinomas of intestinal type (P=0.007) and with an expansive growth pattern (P=0.021). ERBB2 amplification was detected in both histological components of two mixed carcinomas, indicating a common clonal origin. A statistically significant association was found between ERBB2 amplification and worse survival in patients with expansive gastric carcinomas (P=0.011). We conclude that ERBB2 status may have clinical significance in subsets of gastric cancer patients, and that further studies are warranted to evaluate whether patients whose gastric carcinomas present ERBB2 amplification/overexpression may benefit from therapy targeting this surface receptor.

Increased Expression of Toll-like Receptors (TLR) 2, 4 and 5 in Gastric Dysplasia

TLRs are important innate immunity receptors. Even though TLR2, 4 and 5 appear to be important for Helicobacter pylori (HP) recognition, their role in the evolution of gastritis to more advanced lesions is still unknown. To compare the expression of TLR2, 4 and 5 in normal gastric mucosa, HP+ gastritis, intestinal metaplasia, dysplasia and adenocarcinoma. Immunohistochemistry for TLR2, 4 and 5 was performed with anti-TLR2-TLR4-TLR5 antibodies in 117 histological samples of normal gastric mucosa (n = 22), HP+ gastritis (n = 20), intestinal metaplasia (n = 33), dysplasia (mucosectomy specimens, n = 20) and intestinal type adenocarcinoma (surgery specimens,n = 22); quantification of expression was performed independently by two pathologists taking into account the percentage of positive epithelial cells and the degree of expression (zero to three score). A statistically significant trend for progressive increase of TLRs expression from normal mucosa to gastric dysplasia was found (mean expression: normal mucosa 0.1; gastritis 1.0; metaplasia 2.2; dysplasia 2.8, p < 0.01). All dysplasia samples presented more than 90% positive epithelial cells with strong expression (2.8;95%CI2.7–3). There was less TLRs expression in carcinomas (TLR2:1.0; TLR4:2.0 and TLR5:1.2, p < 0.05) when compared with dysplasia, with TLR4 being more expressed than TLR2 and 5 in these lesions (p = 0.03). A score of all markers’ expression of eight leads to a low (4%) false positive rate in patients with precancerous conditions. Progression of gastric lesions associated with gastric carcinogenesis is associated with increased TLRs expression. Gastric dysplasia presents a high level of TLRs expression, suggesting that these receptors may play a role in adenocarcinoma development.

MSI phenotype and MMR alterations in familial and sporadic gastric cancer.

Microsatellite instability (MSI) is a major pathway involved in gastric carcinogenesis occurring in 20% of gastric cancer (GC). However, it is not clear whether MSI phenotype preferentially occurs in the sporadic or familial GC, when stringent inclusion criteria are used. The aim of this study was to compare the frequency of MSI and hypermethylation of MLH1 promoter in a large series of familial GC patients (non‐HNPCC and non‐CDH1‐related) and sporadic cases. Additionally, we analysed the immunoexpression of MMR proteins in a fraction of cases. Overall, the frequency of familial GC was 7.1%, and the frequency of hereditary tumours was 4.6%. MSI phenotype and MLH1 hypermethylation frequencies were not statistical different between familial and sporadic GC settings. Further, the MSI phenotype was not associated with any clinico‐pathological features studied in the familial GC setting, whereas in the sporadic setting, it was associated with older age, female gender and intestinal histotype. Using our stringent Amsterdam‐based clinical criteria to select familial GC (number of cases, age of onset), we verified that sporadic and familial cases differed in gender but shared histopathological features. We verified that the frequency of MSI was similar in familial and sporadic GC settings, demonstrating that this molecular phenotype is not a hallmark of familial GC in contrast to what is verified in HNPCC. Moreover, we observed that the frequency of MLH1 hypermethylation is similar in sporadic and familial cases suggesting that in both settings MSI is not associated to MMR genetic alterations but in contrast to epigenetic deregulation.

Helicobacter pylori Induces Increased Expression of Toll‐Like Receptors and Decreased Toll‐Interacting Protein in Gastric Mucosa that Persists Throughout Gastric Carcinogenesis

Toll‐like receptors (TLR) are essential for Helicobacter pylori (HP) recognition. Their role in the progression of gastric lesions leading to cancer is not established.

Long-term follow-up after endoscopic resection of gastric superficial neoplastic lesions in Portugal

BACKGROUND AND STUDY AIMS Although endoscopic resection for the treatment of gastric superficial neoplastic lesions is an established first-line treatment in Eastern countries, its role has yet to be considered in Western guidelines, mostly due to a lack of long-term studies. The aim of this study was to describe long-term outcomes for endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) in the treatment of gastric neoplasias in Portugal. PATIENTS AND METHODS This was a single-center, retrospective, cohort study between March 2003 and April 2013. A total of 162 consecutive patients with 195 gastric superficial neoplasias underwent EMR (n = 54) or ESD (n = 141) and were followed up for a median of 3.2 years. RESULTS Resection was feasible in 97 %, with en bloc and R0 resection rates of 85 % (94 % ESD vs. 61 % EMR; P = 0.001) and 81 % (91 % ESD vs. 54 % EMR; P < 0.001), respectively. The recurrence rate was 7 %, and recurrence was associated with Rx/R1 resection irrespective of resection technique (OR 5.8; 95 % confidence interval 3.9 - 8.8). The long-term curative resection rate was 86 % after one procedure and 91 % after two procedures. Adverse events were observed in 13 % of cases: 8 % bleeding and 2 % of perforations (EMR = ESD). Surgery was performed in 7 %: 6 % after noncurative endoscopic resection and 1 % due to complications. Metachronous lesion detection rate was 1 % - 1.5 % per patient year. Cancer-specific survival rate was 100 % at follow-up. CONCLUSIONS For the first time in a Western country, results are reported to be similar to those in Eastern countries. Endoscopic resection, particularly ESD, is a highly effective treatment for gastric superficial lesions, without compromising cancer survival. Endoscopic resection should also be considered as first-line treatment for gastric neoplasias in Western countries.

Increased hepatic expression of TLR2 and TLR4 in the hepatic inflammation-fibrosis-carcinoma sequence:

We evaluated expression of TLR2, TLR4 and proinflammatory genes [NF-κB, TNF-α, cyclooxygenase-2 (COX-2)] in liver samples of patients in different stages of liver disease. Fifteen patients with unexplained transaminases elevation (reference group), 22 with viral chronic hepatitis (hepatitis group), 14 with virus-induced severe fibrosis/cirrhosis (cirrhosis group) and 10 with hepatocarcinoma (hepatocarcinoma group) were consecutively included in the study. Quantification of TLR2, TLR4, NF-κB, TNF-α and COX-2 mRNA was done by real-time RT-PCR and TLR2 and TLR4 protein expression was evaluated by immunohistochemistry. Compared with reference, TLR2 and TLR4 mRNA was increased in hepatitis (TLR2: 2.66 ± 0.69; TLR4: 3.11 ± 0.79; P < 0.05) and cirrhosis (TLR2: 2.14 ± 0.5; TLR4: 1.74 ± 0.27; P < 0.05) and decreased in hepatocarcinoma (TLR2: 0.48 ± 0.15; TLR4: 0.54 ± 0.10; P < 0.05). This associated with increased TNF-α and COX-2 mRNA in hepatitis (TNF-α: 3.24 ± 0.79; COX-2: 2.47 ± 0.36; P < 0.05) and cirrhosis (TNF-α: 1.73 ± 0.28; COX-2: 1.8 ± 0.35, P < 0.05), whereas NF-κB mRNA was increased in hepatitis (2.42 ± 0.31; P < 0.05) and unchanged in cirrhosis (1.34 ± 0.17; P = 0.3). Hepatocarcinoma presented increased COX-2 mRNA (1.63 ± 0.15; P < 0.05) and maintained (at decreased levels) mRNA of NF-κB (0.52 ± 0.12) and TNF-α (0.52 ± 0.12; P < 0.05, all genes). Immunohistochemistry confirmed increased expression of TLR2 and TLR4 in hepatitis and cirrhosis and maintained expression in hepatocarcinoma. Upregulation of TLR2, TLR4 and their proinflammatory mediators is associated with virus-induced hepatic IFC sequence.

Probing the O-glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery

Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO “SimpleCell” (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer.

Colorectal carcinomas with microsatellite instability display a different pattern of target gene mutations according to large bowel site of origin

BackgroundOnly a few studies have addressed the molecular pathways specifically involved in carcinogenesis of the distal colon and rectum. We aimed to identify potential differences among genetic alterations in distal colon and rectal carcinomas as compared to cancers arising elsewhere in the large bowel.MethodsConstitutional and tumor DNA from a test series of 37 patients with rectal and 25 patients with sigmoid carcinomas, previously analyzed for microsatellite instability (MSI), was studied for BAX, IGF2R, TGFBR2, MSH3, and MSH6 microsatellite sequence alterations, BRAF and KRAS mutations, and MLH1 promoter methylation. The findings were then compared with those of an independent validation series consisting of 36 MSI-H carcinomas with origin from each of the large bowel regions. Immunohistochemical and germline mutation analyses of the mismatch repair system were performed when appropriate.ResultsIn the test series, IGFR2 and BAX mutations were present in one and two out of the six distal MSI-H carcinomas, respectively, and no mutations were detected in TGFBR2, MSH3, and MSH6. We confirmed these findings in the validation series, with TGFBR2 and MSH3 microsatellite mutations occurring less frequently in MSI-H rectal and sigmoid carcinomas than in MSI-H colon carcinomas elsewhere (P = 0.00005 and P = 0.0000005, respectively, when considering all MSI-carcinomas of both series). No MLH1 promoter methylation was observed in the MSI-H rectal and sigmoid carcinomas of both series, as compared to 53% found in MSI-H carcinomas from other locations (P = 0.004). KRAS and BRAF mutational frequencies were 19% and 43% in proximal carcinomas and 25% and 17% in rectal/sigmoid carcinomas, respectively.ConclusionThe mechanism and the pattern of genetic changes driving MSI-H carcinogenesis in distal colon and rectum appears to differ from that occurring elsewhere in the colon and further investigation is warranted both in patients with sporadic or hereditary disease.

Mitochondrial genome alterations in rectal and sigmoid carcinomas.

The scarce studies on the molecular pathways involved in the pathogenesis of rectal cancer indicate that these may vary, at least in part, from those relevant for colon cancer. Mitochondrial DNA alterations have been described in several human cancers. We aimed to study D310, ND1 and ND5 microsatellite sequence alterations and nuclear microsatellite instability in a series of 38 rectal carcinomas as compared to a series of 25 sigmoid carcinomas. D310 sequence alterations were observed in 34.3% and 37.5% of rectal and sigmoid carcinomas, respectively, whereas ND1 mutations were present in 2.6% in RC and ND5 mutations were detected in 5.3% and 8% of rectal and sigmoid carcinomas, respectively. A trend toward an association between nuclear and mitochondrial microsatellite instability was observed in sigmoid but not in rectal cancers. In conclusion, mitochondrial genome alterations are common in both rectal and sigmoid carcinomas and may contribute to their pathogenesis.

Association between environmental factors and CDX2 expression in gastric cancer patients.

Intestinal differentiation, mediated by CDX2, may occur both in intestinal and in diffuse gastric carcinomas. In an attempt to ascertain the environmental determinants of the intestinal differentiation pathway, we aimed to compare the exposure to environmental factors in gastric cancer cases according to the CDX2 expression status. We evaluated 270 patients undergoing gastrectomy due to gastric adenocarcinoma. Cases were classified according to tumour location within the stomach, Laurén’s histological type and CDX2 expression. Participants completed a comprehensive structured questionnaire on sociodemographic and behavioural characteristics and provided a blood sample for assessment of Helicobacter pylori infection status. Odds ratios with 95% confidence intervals were computed by unconditional logistic regression to compare cancer cases according to histological type and CDX2 expression status. Approximately 80% of the patients expressed CDX2, regardless of the histological type and location of the tumour. No statistically significant or consistent associations between sociodemographic or environmental exposures and CDX2 expression status were observed, except for a decreased risk of CDX2 expression in those with higher coffee consumption for tumours of the intestinal type (≥1 vs. <1 cup/day: odds ratio =0.36, 95% confidence interval: 0.13–0.97). In conclusion, this is the first study to use CDX2 expression as a finer marker of intestinal differentiation to distinguish aetiologically distinct subgroups of gastric cancer, adding new evidence to the previous research on the determinants of cancer of Laurén’s intestinal and diffuse types.