Overexpression of P73 As a Tissue Marker for High-Risk Gastritis

Published OnlineFirst June 8, 2010; DOI: 10.1158/1078-0432.CCR-09-2491 Published OnlineFirst on June 8, 2010 as 10.1158/1078-0432.CCR-09-2491

Clinical Imaging, Diagnosis, Prognosis Cancer Research Overexpression of p73 as a Tissue Marker for High-Risk Gastritis

Gonzalo Carrasco1, Jose Diaz2, Jose R. Valbuena1, Paulina Ibanez1, Paz Rodriguez1, Gabriela Araya1, Carolina Rodriguez1, Javiera Torres1, Ignacio Duarte1, Edmundo Aravena3, Fernando Mena4, Carlos Barrientos3, and Alejandro H. Corvalan1,2

Abstract Purpose: Histologic assessment of high-risk gastritis for the development of gastric cancer is not well defined. The identification of tissue markers together with the integration of histologic features will be required for this assessment. Experimental Design: Matched tumor/nontumor adjacent mucosa (NTAM) of 91 early gastric cancer and 148 chronic gastritis cases were evaluated for histologic characteristics (atrophy, intestinal metaplasia, chronic inflammation, polymorphonuclear infiltration, and Helicobacter pylori) by the Sydney System. At- rophy risk assessment was also evaluated by the Operative Link on Gastritis Assessment (OLGA) staging system. Eight tissue markers (BRCA1, HSP90, STAT1, FHIT, EGFR, p73, p53, p16INK4a) and EBV were also evaluated by tissue microarray/immunohistochemistry/in situ hybridization platform. Data were analyzed by contingency tables (2 × 2) using Fisher's exact two-tailed test (P < 0.001) and integrated by Significance Analysis of Microarrays (SAM) and clustering analysis. Results: Histologically, NTAM have severe intestinal metaplasia/chronic inflammation and severe atrophy assessed by Sydney and OLGA staging systems. H. pylori infection was similar in both groups, and EBV was found only in 5.5% of the tumor samples. Overexpression of p73 was higher in NTAM (50.5%) than in chronic gastritis (10.8%; P < 0.0001). Integration of histologic features and tissue markers showed that overexpression of p73, severe atrophy, and OLGA stage 4 were the most relevant features in NTAM. Clustering analysis correctly assigned NTAM and control cases (P < 0.0001). Conclusions: Overexpression of p73 should be considered for the assessment of high-risk chronic gastritis. SAM allows the integration of histology and tissue markers for this assessment. Clin Cancer Res; 16(12); 3253–9. ©2010 AACR.

Gastric cancer is the second leading cause of cancer- gastric cancer is causally linked to Helicobacter pylori and related deaths and the fourth most common malignancy consensus conference recommended population-based worldwide (1). The incidence of gastric cancer shows con- screening and treatment in highly prevalent areas (3), siderable geographic variation. The East Asia, Eastern\ screening tests for the early detection of gastric cancer Europe, and Latin American regions still remain highly are needed. We recently found that DNA methylation of prevalent areas, whereas mortality from gastric cancer is Reprimo was present in 90% (pair tumor and plasma sam- declining globally (2). In spite of convincing evidence that ples) of gastric cancer cases but only in 10% of plasma from healthy donors (4). These findings support the idea Authors' Affiliations: 1Department of Anatomic Pathology and that DNA methylation of Reprimo could be a potential 2Laboratory of Molecular Pathology and Epidemiology-Department of noninvasive marker for the early detection of gastric Hematology-Oncology, Pontificia Universidad Católica de Chile, and 3Instituto Chileno Japones de Enfermedades Digestivas, Hospital cancer. However, early detection of this malignancy also Clinico San Borja Arriaran, Santiago, Chile; and 4Department of requires histologic assessment of premalignant conditions. Pathology, Hospital Max Peralta, Cartago, Costa Rica In spite of that the histologic features of noninvasive neo- Note: Current address for Gonzalo Carrasco: Department of Anatomic plasia (formerly dysplasia) are well defined (5), whereas Pathology, Fundacion Hospital Parroquial de San Bernardo, Santiago, Chile. criteria for risk assessments of premalignant conditions Current address for Carlos Barrientos: Department of Gastroenterology, such as chronic gastritis are not (6, 7). In this scenario Fundacion Arturo Lopez Perez, Santiago, Chile. tissue markers, such as overexpressed proteins, have re- Corresponding Author: Alejandro H. Corvalan, Laboratory of Molecular cently been explored as candidates for risk assessment Pathology and Epidemiology, Department of Hematology-Oncology, Pontificia Universidad Católica de Chile-School of Medicine, 391 Marco- (8). However, integration with the histologic features of leta St., Santiago 8330074, Chile. Phone: 02-3548289; Fax: 56-2- premalignant conditions will be required to assess the 3548289; E-mail: [email protected]. potential clinical impact of these tissue markers. In this doi: 10.1158/1078-0432.CCR-09-2491 study, we have identified the overexpression of p73, intes- ©2010 American Association for Cancer Research. tinal metaplasia, and severe atrophy assessed by the

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Histologic variables Translational Relevance Paraffin blocks were cut and stained with H&E and Giemsa stains for histologic evaluation and H. pylori Recently, we found that DNA methylation of detection. Early gastric cancer cases were classified by Reprimo could be a potential noninvasive marker for histologic type (intestinal or diffuse) according to Lauren the early detection of gastric cancer (Clin Cancer Res. classification (11) and location. Chronic inflammation, 2008;14:6264-9). However, early detection also polymorphonuclear activity, atrophic gastritis, intestinal requires risk assessment of premalignant conditions metaplasia, and the presence of H. pylori were evaluated at tissue level. Histologic criteria for risk assessment in nontumor adjacent mucosa (NTAM) from early gastric are not well defined. Tissue markers, such as over- cancer and chronic gastritis controls according to the Up- expressed proteins, have been recently explored as dated Sydney System (10). For atrophy risk assessment, we candidates for high-risk gastritis. Here, we have identi- also applied the new histological staging system, OLGA, fied that among eight tissue markers (BRCA1, HSP90, proposed by Rugge et al. (12–14). The evaluation was STAT1, FHIT, EGFR, p73, p53, and p16INK4a), p73 made by two double-blinded independent pathologists was overexpressed in tumor and nontumor adjacent who were unaware of clinical data and histologic diagnoses. mucosa at a similar level in early gastric cancer cases in comparison with chronic gastritis cases Tissue microarray construction P ( < 0.0001). Integration of histologic features and tis- Tissue microarrays (TMA) were done by using a Manual sue markers showed that overexpression of p73 and Tissue Array II instrument (Beecher Instruments) as previ- Operative Link on Gastritis Assessment (OLGA) stage ously described (15). In brief, paraffin blocks from 91 4 were the most relevant features in nontumor adja- paired tumor/NTAM and from 148 endoscopic gastric bi- cent mucosa. Our data suggest that overexpression of opsies were selected, cut, and stained with H&E for the p73 might have a role in the assessment of high-risk gas- best histologic area. After whole-section glass slide evalu- tritis for gastric cancer development. ation, tissue area was selected for placement into the TMA by a circling on the glass slide and was identified in the corresponding paraffin block. A 1-mm stylet in the inner diameter was used to take two cylindrical core biopsies Sydney and Operative Link on Gastritis Assessment from each tissue block (donor block), with subsequent (OLGA) staging systems as being associated with high-risk arraying into a new recipient paraffin block. In this way, chronic gastritis. all paired tumor and NTAM from early gastric cancer and endoscopic gastric biopsies from control cases were held in seven recipient blocks. An adequate case was de- Materials and Methods fined as a tumor occupying >10% of the core area. Each case was in duplicate to avoid the loss of tissue during Clinical samples the cutting of the block. Slides (4 μm) were cut from each Between 1984 and 2005, 91 surgically resected early tissue array block, deparaffinized, and dehydrated for H&E gastric cancer cases from the Department of Pathology– and immunohistochemical staining. Pontificia Universidad Católica de Chile (PUC) and the Instituto Chileno Japones de Enfermedades Digestivas– Immunohistochemistry and in situ hybridization Hospital Clinico San Borja Arriaran (ICHJED-HCSBA), Eight biomarkers were selected based on genes whose Santiago, Chile, and from Hospital Max Peralta-Cartago, alterations in gastric carcinoma play a significant role in Costa Rica were collected. All cases were surgically resected the promotion of chronic gastritis (STAT1; ref. 16), gastric by total gastrectomy and were classified as early gastric epithelial cell response to H. pylori infection (p73; ref. 17), cancer in accordance with the depth of invasion as pro- prognosis (FHIT, p16INK4a; refs. 18–21), early onset posed by the Japanese Research Society for Gastric Cancer (BRCA1; ref. 15), and emerging molecular target for ther- (9). A total of 148 cases with history of symptomatic chron- apy (HSP90, EGFR; refs. 22, 23). In addition, the tumor ic gastritis were used as controls. These control cases were suppressor gene p53 was also included because it is the obtained from PUC, and mucosal biopsies were sampled most common genetic abnormality in gastric cancer according to the Updated Sydney System protocol of biopsy (24). Immunohistochemistry (IHC) was done on 4-μm- sampling of the stomach (10). Gender distribution (male/ thick section TMA blocks as previously described (15). female) was 63(69.2%)/28(30.8%) for early gastric cancer Antibodies used in this study were STAT-1 (clone Rbx, Ab- cases and 87(58.8%)/61(41.2%) for chronic gastritis con- cam), p73 protein α (clone 24, Novocastra), FHIT (clone trols The average age was 58 years (range, 26-89 years) for SMP472, LabVision), p16 (clone 6H12, Novocastra), early gastric cancer cases and 54 years (range, 25-80 years) BRCA1 (clone SG-11, Zymed), HSP90 (clone JPBZ4, Vision for controls, with no differences according to gender (data Biosystems/Novocastra), EGFR (clone 31G7, Zymed), and not shown). The Institutional Review Boards of PUC, p53 (clone BP53-12-1, Biogenex). The results of the ICHJED-HCSBA, and Hospital Max Peralta approved this immunostaining were considered to be positive if ≥10% study, and all patients gave informed consent. of the cells were stained in the nucleus (BRCA1 and

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Table 1. Histologic characteristic of NTAM from early gastric cancer cases and chronic gastritis controls

Histologic characteristics NTAM (n = 91) Controls (n = 148) P n (%) n (%)

Chronic inflammation Negative 9 (9.9) 61 (41.2) <0.0001* Mild 27 (29.7) 66 (44.6) Moderate 25 (27.5) 16 (10.8) Severe 30 (33) 5 (3.4) Polymorphonuclear activity Negative 47 (51.6) 81 (54.7) 0.06 Mild 31 (34.1) 54 (36.5) Moderate 9 (9.9) 8 (5.4) Severe 4 (4.4) 3 (2) Atrophy Negative 32 (35.2) 125 (84.5) <0.001* Mild 17 (18.7) 18 (12.2) Moderate 16 (17.6) 3 (2) Severe 26 (28.6) 0 (0) Intestinal metaplasia Negative 20 (22) 142 (95.9) <0.001* Mild 26 (28.6) 3 (2) Moderate 20 (22) 3 (2) Severe 25 (27.5) 0 (0) H. pylori Positive 51 (56) 100 (67.5) 0.049 Negative 40 (44) 43 (29)

*Fisher's exact test, two-sided P values for P.

p53), cytoplasm (HSP90, STAT1, and FHIT), and nuclear/ in the antigen retrieval method (Pepsin, 10 minutes at cytoplasm (p16, p73) or cell membrane (EGFR). Immuno- 37°C). In situ hybridization analysis for EBV was done histochemical staining against H. pylori (rabbit polyclonal; using flourescein-labeled peptide nucleic acid probe spe- clone CH-20 429, Novocastra) was also done using a cific for EBER-1 and the DAKO hybridization kit according similar protocol as described with a slight modification to the manufacturer's instructions. Evaluation was made by

Table 2. Tissue marker expression in tumor and NTAM from early gastric cancer cases and superficial gastritis mucosa from control cases

Marker Tumor (n = 91) NTAM (n = 91) Controls (n = 148) P* P† Positive (%) Positive (%) Positive (%)

BRCA-1 61 (67) 60 (66) 73 (49.3) 0.123 0.004 HSP-90 88 (97) 90 (99) 141 (95.3) 0.249 0.103 STAT-1 87 (95.6) 88 (97) 135 (91.2) 0.278 0.057 FHIT 83 (91) 82 (90) 123 (83.1) 0.194 0.05 EGFR 26 (28.5) 20 (21) 11 (7.4) 0.08 0.002 p73 38 (41.7) 46 (50.5) 16 (10.8) 0.058 0.000001‡ p16 10 (11) 3 (3.3) 0 (0) 0.031 0.054 p53 20 (22) 2 (2.2) 0 (0) 0.000021‡ 0.1439

*Tumor versus NTAM. †NTAM versus control. ‡Fisher's exact test, two-sided P values for P.

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two double-blinded independent observers who were un- an unsupervised hierarchical clustering analysis (HCA) to aware of clinical data and histologic diagnoses. explore the reliability of self-classification of early gastric cancer/NTAM and control cases, using the previous inte- Statistical analysis and integration approaches grated histologic and tissue marker analysis. HCA is anoth- Histologic and tissue marker data were evaluated by er genomic method previously used in TMA/IHC studies contingency (2 × 2) table using Fisher's exact test for cate- including gastric cancer (27–29). Both SAM and clustering gorical variables to determine differences between tumor analysis were done by using MultiExperiment Viewer 4.4 and NTAM from early gastric cancer cases and control software (http://www.tm4.org/mev/; refs. 30–32). cases. All of the values were two-tailed, and statistical significance was defined as P < 0.001. Integration of histo- Results logic and tissue markers was done by Serial Analysis for Microarray (SAM), a multiple testing approach method Histologic characteristics of NTAM from early gastric that has been extensively applied in genomic research cancer and chronic gastritis control cases (25). Although SAM was originally designed for continu- According to Lauren classification, 72 early gastric ous variables, here we adapted it to dichotomous variables cancers were intestinal type and 19 were diffused type. (0 or 1), so we were able to combine histologic and tissue Table 1 details the histologic characteristics of NTAM marker variables. Data found by SAM were confirmed by and controls cases. NTAM has moderate to severe atrophy, logistic regression, a multivariate method for analysis of intestinal metaplasia, and chronic inflammation in binominal-distributed variables (26). Finally, we carried out comparison with controls. These differences were highly

Fig. 1. A, representative examples of positive and negative immunostaining for eight tissue markers and EBER-1 expression by in situ hybridization (×100) in tumor and NTAM from early gastric cancer cases and chronic gastritis controls. B, high magnification (×400) of (a) BRCA1- and (b) p73-positive controls, (c) p73-positive NTAM, (d) FHIT-negative control, (e) FHIT-negative NTAM, (f) p16-positive NTAM, (g) p16-positive tumor, and (h) positive EBV staining. No cases of chronic gastritis cases were positive for p16, p53, and EBER by in situ hybridization.

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Fig. 2. SAM of NTAM and chronic gastritis controls. NTAM group is significantly characterized by the overexpression of p73, OLGA stages 3 to 4, and severe atrophy (ATR-3), intestinal metaplasia (IM-3), and chronic inflammation (CI-3) according to the Sydney System. Control group cases were significantly characterized by lack of intestinal metaplasia (IM-0), atrophy (ATR-0), and chronic inflammation (CI-0). False discovery rate = 0. significant (P < 0.001). Polymorphonuclear activity was ciated with location or tumor histology (data not shown). predominantly negative or mild in both groups (85.7% ver- Sensitivity and specificity for association between p73 protein sus 91.2%, respectively). Twenty (58.8%) of 34 NTAM but expression and early gastric cancer were 74.2% and 74.6%, re- only 1 (1.3%) of 75 chronic gastritis cases were OLGA stages spectively. EBV detection by EBER1 expression was found only 3to4(P < 0.001). No differences were found according to in tumor tissue from 5 (5.5%) of early gastric cancer cases. Lauren histologic classification and/or tumor location. The prevalence of H. pylori infection was similar in both groups. Integration of histologic variables and tissue markers To integrate histologic variables and tissue markers we Tissue marker expression in tumor and NTAM from applied SAM, a popular method for expression profile early gastric cancer and chronic gastritis control cases analysis that can be adapted to other types of experimental Seven array blocks containing 660 cores and representing data such as survival time or tumor stage (25). As seen in 91 early gastric cancers (91 paired tumors and NTAMs) and Fig. 2, SAM shows, in a graphical representation, that 148 chronic gastritis control cases were built and tested by the chronic gastritis control group is characterized only IHC for eight tissue markers. A total of 646 (98%) cores by histologic features such as the lack of intestinal meta- were correctly evaluated by H&E examination. Missing cores plasia, atrophy, and chronic inflammation. However, were usually caused by exhaustion of tissue material. Table 2 NTAM is characterized by overexpression of p73 and shows a comparison of tissue marker expression among OLGA stages 3 and 4 intermingled with severe atrophy, tumor and NTAM from early gastric cancer cases and chronic intestinal metaplasia, and chronic inflammation accord- gastritis control cases. Figure 1 shows representative examples ing to the Sydney System. These data were confirmed by of positive and negative immunostainings of tumor/NTAM logistic regression analysis (data not shown). and control cases. In early gastric cancers a similar tissue marker profile was found between tumor and NTAM with Self-classification of cases by clustering analysis the exception of p53, which was significantly overexpressed Next, to explore the possibility that this integrated in tumors (P < 0.001). The comparison of tissue marker profile approach was also able to self-classify NTAM and chronic between NTAM and chronic gastritis control samples shows gastritis, we carried out an unsupervised clustering analy- significant differences in p73 protein expression (50.5% sis. This widely used method in cDNA microarray analysis versus 10.8%). These differences were highly significant (P < has also been used in TMA/IHC in several tumors includ- 0.0001). Comparisons of tissue marker profiles were not asso- ing gastric cancer (27, 28). Thus, as shown in Fig. 3, this

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Fig. 3. Unsupervised multivariate hierarchical clustering analysis (Euclidean distance and complete linkage) of 91 NTAM and 148 chronic gastritis cases were based on integration of histology and tissue marker expression. Two groups were formed: group A composed of 75 NTAM and 21 chronic gastritis controls, and group B composed of 127 gastritis and 16 NTAM. CI, chronic inflammation; ACT, polymorphonuclear activity; ATR, atrophy; IM, intestinal metaplasia; 0, negative; 1, mild; 2, moderate; 3, severe.

analysis produced a dendrogram that successfully self- Sydney System and the new OLGA staging system for atro- classified NTAM versus chronic gastritis cases (P < 0.0001). phy risk assessment. Furthermore, this novel approach con- firms that overexpression of p73 might become the most significant feature that identifies high-risk chronic gastritis Discussion in cases of severe atrophy. These data can be obtained by logistic regression, a multivariate method of analysis of In this study we have found differences in histologic fea- binominal-distributed variables (26). One of the main advan- tures such as atrophy, intestinal metaplasia, and chronic tagesofSAM,however,isthatitgivesasequenceofalltested inflammation between NTAM and chronic gastritis by variables and graphical representation showing the strength of the Sydney System. The new OLGA staging system for at- variables from each group. Similarly, unsupervised HCA rophy risk assessment successfully assigned stages 3 and 4 was also able to self-classify NTAM and control cases. to NTAM cases. In summary, our findings have shown that integration of Although differences in expression of p73 have been histologic and tissue marker variables is possible. The over- described between tumor and chronic gastritis (33, 34), expression of p73, being present mostly in NTAM from early the most important and novel finding in this study is gastric cancer, but rarely in chronic gastritis controls, suggests that the expression of p73 in NTAM is significantly higher a potential role of p73 as a tissue marker of high-risk gastri- than that in chronic gastritis. Because atrophy is considered tis for development of gastric cancer. Finally, the goal of sys- a preneoplastic field for gastric carcinoma development, tems pathology is to present the data in an integrated way, overexpression of p73 could be useful for risk assessment which might be useful in switching from an interpretive in cases of severe atrophy as shown by the OLGA staging and subjective morphologically oriented approach to a more system. Besides gastric carcinoma, overexpression of p73 objective, evidence-based tissue marker approach (39–41). has been described in other neoplasm such as prostate, lung, bladder, and renal carcinoma. In these malignancies, Disclosure of Potential Conflicts of Interest however, it has to be shown at tissue level, as we have shown here, that such overexpression occurs in a preneo- No potential conflicts of interest were disclosed. plastic lesion at the same frequency as that in tumors. There- Acknowledgments fore, p73 overexpression might emerge as a tissue marker not only for gastric carcinoma but also for other malignan- We thank Dr. Claudia Bustamante and Dr. Ivan Gallegos for H. pylori cies. We have also found differences in accumulation of p53 evaluation, Sabina L. Magedson for tissue microarray construction and in tumor versus NTAM as has been described previously immunohistochemistry experiments, and Camila Malta for premium editing and proofreading of the manuscript. (35, 36). This finding supports the concept that inactiva- tion of p53 occurs at early stages of gastric carcinoma. Grant Support Having found that p73 is a potential marker for risk assessment of chronic gastritis and that the new OLGA Grants-in-Aid for Fondo Nacional de Ciencia y Tecnologia (FONDECYT) staging system successfully assigned stages 3 and 4 to #1080563 to A.H. Corvalan from the Government of Chile. The costs of publication of this article were defrayed in part by the NTAM cases, we attempted to integrate histologic features payment of page charges. This article must therefore be hereby marked and tissue markers by using a systems pathology approach advertisement in accordance with 18 U.S.C. Section 1734 solely to (37–41). To this end we applied SAM, a multiple testing indicate this fact. approach that has been used mostly in genomic research Received 10/26/2009; revised 03/15/2010; accepted 04/09/2010; (37). Therefore, SAM shows the admixture of the classical published OnlineFirst 06/08/2010.

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Overexpression of p73 as a Tissue Marker for High-Risk Gastritis

Gonzalo Carrasco, Jose Diaz, Jose R. Valbuena, et al.

Clin Cancer Res Published OnlineFirst June 8, 2010.

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