Epithelial dysplasia of the stomach with gastric immunophenotype shows features of biological aggressiveness

2013/2014 Pedro Fernando Magalhães Valente Epithelial dysplasia of the stomach with gastric immunophenotype shows features of biological aggressiveness março, 2014 Mestrado Integrado em Medicina Área: Anatomia Patológica Trabalho efetuado sob a Orientação de: Doutora Maria de Fátima Machado Henriques Carneiro Trabalho organizado de acordo com as normas da revista: Gastric Cancer Pedro Fernando Magalhães Valente Epithelial dysplasia of the stomach with gastric immunophenotype shows features of biological aggressiveness março, 2014 Agradecimentos Esta dissertação que marca o fim de um ciclo da minha formação académica não teria sido possível sem a colaboração e apoio de muitas pessoas às quais não poderia deixar de exprimir a minha mais sincera gratidão: À Professora Doutora Fátima Carneiro, que me abriu a porta do Serviço de Anatomia Patológica e amavelmente aceitou orientar este projecto de investigação, tornando-o uma experiência pedagógica e pessoal verdadeiramente enriquecedora. Ao Serviço de Anatomia Patológica, em especial aos técnicos Bárbara Gomes, Helena Pereira, Armando Castro, Armindo Pereira e Manuel Moutinho. Ao Dr. Franclim Ribeiro por toda a formação e disponibilidade paciente. A todos que, de alguma forma, contribuíram para esta dissertação, nomeadamente a Dr.ª Irene Gullo, Dr.ª Helena Baldaia, Dr. Francisco Baldaque-Silva e Dr.ª Joanne Lopes. À Mónica, que me motivou para a realização conjunta deste projecto e pela construção partilhada de um futuro em comum. Aos meus pais, a quem devo tudo o que sou. A todos os meus, pelas longas horas de ausência. 1 Original article Epithelial dysplasia of the stomach with gastric immunophenotype shows features of biological aggressiveness Pedro Valente1*, Mónica Garrido1*, Irene Gullo1,2, Helena Baldaia3, Margarida Marques3, Francisco Baldaque-Silva3,4, Joanne Lopes3, Fátima Carneiro1,3,5 1 Faculty of Medicine of the University of Porto, Porto, Portugal 2 Faculty of Medicine and Surgery, Genova, Italy 3 Centro Hospitalar de São João, Porto, Portugal 4 Department of Gastroenterology and Hepatology, Karolinska University Hospital, Stockholm, Sweden 5 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal *contributed equally Figures: 2 Tables: 5 Short title: Aggressiveness of gastric-type dysplasia Word Count: 2870 Potential competing interest: The authors have no competing interest to declare. Correspondence to: Fátima Carneiro, M.D., Ph.D. Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP) Rua Dr. Roberto Frias S/N, 4200-465 Porto, Portugal Tel.: +351 225570700 Fax: +351 225570799 Email: [email protected] 2 ABSTRACT Background: Gastric dysplasia is classified as adenomatous/type I (intestinal phenotype) and foveolar or pyloric/type II (gastric phenotype) according to morphological (architectural and cytological) features. The immunophenotypic classification of dysplasia, based on the expression of mucins, CD10 and CDX2, recognizes the following immunophenotypes: intestinal (MUC2, CD10 and CDX2); gastric (MUC5AC and/or MUC6, absent of CD10 and absent or low expression of CDX2); hybrid (gastric and intestinal markers) and null. Methods: Sixty-six cases of non-polypoid epithelial dysplasia of the stomach were classified according to morphological features (histotype and grade) and immunophenotype. Immunohistochemical staining was performed with antibodies against MUC2, MUC5AC, MUC6, CD10, CDX2, chromogranin, synaptophysin, Ki-67 and TP53. HER2 alterations were analysed by immunohistochemistry and silver-enhanced in situ hybridization (SISH). Results: By conventional histology, dysplasia was classified as adenomatous/intestinal (n=42; 64%) and foveolar or pyloric/gastric (n=24; 36%) and graded as low-grade (n=37; 56%) and high-grade (n=29; 44%). Immunophenotypic classification showed intestinal (n=22; 33.3%), gastric (n=25; 37.9%), hybrid (n=17; 25.8%) or null (n=2; 3.0%) phenotypes. In 20 cases a coexistent intramucosal carcinoma was identified. The intestinal immunophenotype was shown to be significantly associated with low-grade dysplasia (p=0.001), high expression of CDX2 (p=0.015), TP53 (p=0.034), synaptophysin (p=0.003) and chromogranin (p<0.0001); the gastric immunophenotype was significantly associated with high-grade dysplasia (p=0.001), high Ki-67 proliferative index (p=0.05) and coexistence of 3 intramucosal carcinoma (p=0.013). HER2 amplification was observed in 3 cases, typed as gastric or hybrid. Conclusions: Epithelial non-polypoid dysplasia of the stomach with gastric immunophenotype shows features of biological aggressiveness and may represent the putative precursor lesion in a pathway of gastric carcinogenesis originated de novo from the native gastric mucosa, leading to gastric type adenocarcinoma. MINI ABSTRACT Epithelial dysplasia of the stomach encompasses two major immunophenotypes, intestinal and gastric, the latter significantly associated with features of biological aggressiveness: high-grade, high proliferative index and coexistent carcinoma. Key words: Gastric carcinogenesis; dysplasia; HER2; immunophenotype; mucins. 10 histology as gastric (35.3%) and intestinal (64.7%) and all cases of the intestinal immunophenotype displayed features of the adenomatous/intestinal histotype (p<0.0001). Table 5 shows the relationship between the presence of the coexistent intramucosal adenocarcinoma and the features of dysplasia (grade, histo and immunophenotypes). In 20 of 66 cases (30.3%), there was a coexistent carcinoma at the periphery of the dysplastic lesions, the latter displaying the following features: high-grade (75.0%; p=0.001); gastric histotype (60.0%; p=0.024); gastric immunophenotype (65.0%, 20.0% and 15.0% for gastric, hybrid and intestinal immunophenotypes, respectively; p=0.013). Gastric dysplasia at the periphery of invasive carcinoma, when compared with gastric dysplasia in the absence of invasive carcinoma, displayed significantly lower frequency of expression of synaptophysin and chromogranin (30.0% and 40.0%; p=0.006 and p=0.025, respectively). DISCUSSION Gastric carcinogenesis is a complex process, still requiring the elucidation of putative distinct pathways. According to the so-called Correa model (4), gastric carcinogenesis is a multistep and multifactorial process that, in many cases, appears to involve a progression from normal mucosa, through chronic atrophic gastritis and intestinal metaplasia, to dysplasia and invasive carcinoma. However, evidence from literature points to the possibility of the existence of alternative pathways in which intestinal metaplasia may not play a role. Evidence stems mainly from the study of tiny early gastric carcinomas arising in non-metaplastic mucosa, as described by Japanese authors (14, 15) as well as 11 the studies of the expression of markers of gastric differentiation in dysplasia and gastric adenocarcinoma (7, 10, 16, 17). The latter demonstrate that both types of lesions may express, predominantly or exclusively, markers of gastric differentiation, raising the possibility of an origin in native gastric mucosa, rather than in intestinal metaplastic lesions. It remains to be elucidated the role of spasmolytic polypeptide-expressing metaplasia (SPEM) in the pathogenesis of the lesions with gastric immunophenotype. Other evidences stem from hereditary gastric cancer models (Hereditary Diffuse Gastric Cancer and Gastric Adenocarcinoma and Proximal Polyposis of the Stomach – HDGC and GAPPS) in which gastric carcinoma, diffuse/poorly cohesive type and intestinal/tubular types, respectively, originate in non-metaplastic gastric epithelium (fundic gland polyps in the case of GAPPS) (18, 19). Our study provides additional evidence in favour of de novo neoplastic transformation from native gastric mucosa (37.9% of the dysplastic lesions displayed “pure” gastric immunophenotype). Another relevant issue is the risk of malignant transformation of the different types of gastric dysplasia. Our results show that within the group of cases immunophenotyped as gastric, the majority were classified as high-grade dysplasia (68.0%; p=0.001). At variance, within cases immunophenotyped as intestinal, low-grade dysplasia was the most frequent (86.4%; p=0.001). These findings are in keeping with those recently reported by Nishimura et al (14), but differ from the results reported by Abraham et al (20), the latter showing that intestinal-type adenomas were more likely than gastric-type adenomas to display high-grade dysplasia and adenocarcinoma in the polyps. A major difference from this stud concerns the fact that while the series studied by 12 Abraham et al (20) was constituted by polypoid adenomas, our series is constituted by non-polypoid dysplasia. HER2 amplification was observed in three cases, immunophenotyped as gastric or hybrid. These findings show that HER2 amplification may be an early event in gastric carcinogenesis as observed by Fassan et al (11). The results herein obtained in dysplasia with gastric immunophenotype (higher frequency of high-grade lesions, expression and amplification of HER2) suggest that this type of dysplasia may be an important player in gastric carcinogenesis. The high frequency of cases with high proliferative index (Ki-67) in gastric and hybrid immunophenotypes (84.0% and 94.1%, respectively) when compared with the intestinal immunophenotype (63.6%; p=0.05) is in keeping with the features of aggressiveness identified in dysplastic lesions with gastric differentiation. At variance with other studies (21, 22) we have not found a significant difference in the Ki-67 proliferation index according to the grade of dysplasia. In this study, we observed that the expression of CDX2 is correlated with the intestinal immunophenotype (100% of the cases), in keeping with data previously reported (9). In accordance with Park et al (9), a decreased expression of CDX2 was observed in cases with gastric immunophenotype (80% of the cases; decreased intensity of immunoreactivity). However, there is controversy in the literature regarding the expression of CDX2 in gastric dysplasia, probably reflecting the lack of sub-typing of dysplasia in the different studies (9, 23, 24). 13 In some cases with intestinal immunophenotype small nests of neuroendocrine cells were observed, qualifying for neuroendocrine hyperplasia as reported in the literature (25, 26). It is likely that adenomatous/intestinal dysplasia and neuroendocrine hyperplasia both arise in the setting of chronic atrophic gastritis, as previously suggested in neuroendocrine hyperplasia within gastric hyperplastic polyps (27). However, further studies are needed to elucidate the biological meaning of this event. In the present study we observed that higher expression of TP53 significantly correlated with the intestinal immunophenotype (p=0.034) and was also more frequently observed in high-grade dysplasia, though this association was not significant (p=0.070 – data not shown). In previous studies, it was observed an increased frequency of TP53 overexpression along the progress of gastric carcinogenesis. However, in these studies the immunohistochemical sub-typing of gastric dysplasia was not performed (28). Kushima et al (29) showed that the frequency of TP53 expression was significantly higher in intestinal-type adenomas than in gastric-type adenomas, in keeping with the present study, and was higher in high-grade dysplasia than in low-grade dysplasia, leading to the suggestion that TP53 alterations occur earlier in the carcinogenetic sequence along intestinal rather than gastric differentiation pathway (29). Summing up, our results point to the existence of two major types of nonpolypoid dysplasia in the stomach. The gastric immunophenotype is significantly associated with high-grade dysplasia (p=0.001), high proliferative index (Ki-67) (p=0.050) and coexistence of intramucosal adenocarcinoma (p=0.013). The intestinal immunophenotype was shown to be significantly associated with low- 14 grade dysplasia (p=0.001), overexpression of TP53 (p=0.034) and neuroendocrine markers (p=0.003 for synaptophysin and p<0.0001 for chromogranin). Recently, gene expression profiling using mRNA consensus clustering has revealed three distinct gastric cancer subtypes – mesenchymal, proliferative and metabolic (30). The metabolic subtype is characterized by the expression of genes normally expressed in gastric mucosa, involved in metabolic processes and digestion, and the expression of trefoil peptides (30) that are co-expressed in normal mucosa of the stomach with gastric mucins. These data are in keeping with the results of our previous studies showing the expression of trefoil peptides (and gastric mucins) in a subset of dysplastic and adenocarcinomatous lesions of the stomach (7, 16, 17), supporting the existence of a pathway of gastric carcinogenesis with gastric differentiation. In face of the evidence we collected and that from the literature, we feel tempted to suggest that non-polypoid epithelial dysplasia of the stomach with gastric immunophenotype may represent the putative precursor lesion in a pathway of gastric carcinogenesis originated de novo from the native gastric mucosa, leading to a subset of glandular gastric carcinomas with gastric differentiation. ACKNOWLEDGMENTS The authors want to thank the technical support of Bárbara Gomes, Armando Castro, Armindo Pereira and Manuel Moutinho (Centro Hospitalar de São João), Bárbara Sousa (IPATIMUP) and Franclim Ribeiro (Roche Diagnostics Portugal). This study was partly supported by a grant from the Portuguese 15 affiliate of Hoffmann–La Roche AG (Roche Pharmaceuticals & Roche Diagnostics - Portugal). 16 TABLES Table 1. Clinicopathological features of the series of cases. Age (y, mean ± SD) 65.95 ± 10.93 Sex Male 34 Female 32 Tumour size (cm, mean ± SD) 2.51 ± 2.23 Location Body/fundus 20 (31.3%) Antrum/pylorus 44 (68.7%)  Missing data for location (2 cases). 17 Table 2. Primary antibodies and immunohistochemistry conditions used in this study. Antibody Clone Antigen Retrieval Conditions Dilution Incubation time (min) at 37ºC Localization Source CDX2 EPR2764Y 64 minutes at 96ºC Prediluted 28 Nuclear Cell Marque, USA MUC2 Ccp58 52 minutes at 96ºC 1:100 36 Cytoplasmatic Novocastra, UK MUC5AC MRQ-19 36 minutes at 96ºC Prediluted 24 Cytoplasmatic Cell Marque, USA MUC6 MRQ-20 36 minutes at 95ºC Prediluted 28 Cytoplasmatic Cell Marque, USA CD10 SP67 64 minutes at 95ºC Prediluted 40 Membrane (Brush border) Ventana, USA Chromogranin NS55 52 minutes at 96ºC 1:300 36 Cytoplasmatic Invitrogen, USA Synaptophysin SP11 36 minutes at 95ºC 1:150 32 Cytoplasmatic Neomarkers, USA Ki-67 SP6 36 minutes at 95ºC 1:400 32 Nuclear Neomarkers, USA HER2 4B5 36 minutes at 95ºC Prediluted 12 Membrane Ventana, USA TP53 318-6-11 52 minutes at 96ºC 1:200 32 Nuclear DAKO, Denmark  Antigen retrieval performed with CC1 (Tris/borate/EDTA buffer with pH 8.4 - Ventana Medical Systems, catalogue number 950-124). 18 Table 3. Expression of the different markers according to the three immunophenotypes of gastric dysplasia. Immunophenotype p-value Gastric Hybrid Intestinal MUC2 <5% 25 (100%) 10 (58.8%) 14 (63.6%) ≥5% 0 7 (41.2%) 8 (36.4%) .002 MUC5AC <5% 3 (12.0%) 8 (47.1%) 22 (100%) ≥5% 22 (88.0%) 9 (52.9%) 0 .000 MUC6 <5% 2 (8.7%) 1 (5.9%) 22 (100%) ≥5% 21 (91.3%) 16 (94.1%) 0 .000 CD10 <5% 24 (96.0%) 3 (17.6%) 2 (9.1%) ≥5% 1 (4.0%) 14 (82.4%) 20 (90.9%) .000 CDX2 <25% 5 (20.0%) 0 0 ≥25% 20 (80.0%) 17 (100%) 22 (100%) .015 Ki-67 <50% 4 (16.0%) 1 (5.9%) 8 (36.4%) ≥50% 21 (84.0%) 16 (94.1%) 14 (63.6%) .050 TP53 <50% 21 (84.0%) 8 (47.1%) 13 (59.1%) ≥50% 4 (16.0%) 9 (52.9%) 9 (52.9%) .034 HER2 0, 1+ 18 (72.0%) 13 (76.5%) 22 (100%) 2+, 3+ 7 (28.0%) 4 (23.5%) 0 .029 Synaptophysin <5% 17 (68.0%) 7 (41.2%) 4 (18.2%) ≥5% 8 (32.0%) 10 (58.8%) 18 (81.8%) .003 Chromogranin <5% 18 (72.0%) 5 (29.4%) 1 (4.8%) ≥5% 7 (28.0%) 12 (70.6%) 20 (95.2%) .000  Missing data for MUC6 (2 cases) and chromogranin (1 case). 19 Table 4. Relation between immunophenotype and the histotype and grade of dysplasia. Immunophenotype p-value Gastric Hybrid Intestinal Histotype Gastric 18 (72.0%) 6 (35.3%) 0 Intestinal 7 (28.0%) 11 (64.7%) 22 (100%) .000 Grade Low-Grade 8 (32.0%) 9 (52.9%) 19 (86.4%) High-Grade 17 (68.0%) 8 (47.1%) 3 (13.6%) .001 26 sequences of p53 alteration in stomach carcinogenesis. Virchows Arch. 1996;428(4-5):223-7. 30. Lei Z, Tan IB, Das K, Deng N, Zouridis H, Pattison S, et al. Identification of molecular subtypes of gastric cancer with different responses to PI3-kinase inhibitors and 5-fluorouracil. Gastroenterology. 2013;145(3):554-65. 27 Fig. 1 28 Fig. 2 ANEXO NORMAS DE PUBLICAÇÃO “GASTRIC CANCER” Gastric Cancer - Instructions to Authors Revised February 1, 2014 Gastric Cancer, a joint official journal of the international Gastric Cancer Association and the Japanese Gastric Cancer Association, publishes significant studies related to stomach neoplasms. 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