Clinicopathologic and Prognostic Significance of Overexpression of Her-2/neu and p53 Oncoproteins in Gastric Carcinoma Using Tissue Microarray

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1 Journal of the Egytian Nat. Cancer Inst., Vol. 19,. 2, June: , 2007 Clinicoathologic and Prognostic Significance of Overexression of Her-2/neu and 53 Oncoroteins in Gastric Carcinoma Using Tissue Microarray HODA M. ISMAIL, M.D. 1 ; MANAR MONEER, M.D. 2 ; MANAL EL-BARADIE, M.D. 3 ; OLA KHORSHID, M.D. 4 and AHMED TOUNY, M.D. 5 The Deartments of Pathology 1, Biostatistics and Eidemiology 2, Radiation Oncology & Nuclear Medicine 3, Medical Oncology 4 and Surgical Oncology 5, National Cancer Institute, Cairo University. ABSTRACT Background: The aim of the study was to verify the frequency of the immunohistochemical overexression of her-2/neu and 53 in gastric carcinoma and their relation to the other clinico-athological features and the imact on survival rates. Patients and Methods: A total of 93 atients of gastric carcinoma, who had a otential curative surgery in the eriod from and with reresentative araffin blocks, and sufficient follow-u data were included in this study. They were arrayed and evaluated for rotein marker overexression using tissue microarray (TMA). Patients, tumor and treatment characteristics were collected from the atients files. The ossible rognostic significance of 53 and her-2/neu over exression and different clinico-athological features on survival rates were exlored. Results: Twenty four (25.8%) cases were her-2/neu and 53 ositive. ne of the examined clinico-athologic factors had a significant relation to her-2/neu overexression. 53 was overexressed in intestinal tye, 14/34 (41.2%), more than in diffuse tye, 10/59 (16.9%), (= 0.01). There was no relation between the overexression of 53 and her-2/neu. The median survival eriod was 17.7 months. The survival rates at 12 months were 64.2%, 52.2%, 55.6% and 45.0% for overall (OS), local control (LC), metastasis free survival (MFS) and disease free survival (DFS) rates, resectively. Patients with advanced stages had a significantly lower OS and MFS. Age above 57 years was associated with significantly lower OS, LC, MFS and DFS. Patients who received radiotheray had significantly higher OS, LC, MFS and DFS. ne of the survival rates had been affected by the overexression of 53, or her-2/neu. Conclusion: Although, this study failed to show any rognostic effect of 53 and her-2/neu on survival rates, Corresondence: Dr Hoda M. Ismail, 17 El Mathaf El Zeraie St., Hodanci@hotmail.com we may suggest that 53 overexression may lay a role in the athogenesis of intestinal gastric adenocarcinoma. It could also demonstrate the significantly imroved survival rates with adjuvant chemoradiation. Also, TMA is a useful technique for raid identification of rotein exression rofiles using minimal samles from archived tissues. Key Words: Her-2/neu 53 Gastric carcinoma Tissue microarray Prognostic factors. INTRODUCTION According to the Deartment of Pathological Registry at the National Cancer Institute (NCI), Cairo University for years , 2.1% of the total malignancies were gastric tumors, 68% of them were adenocarcinoma [1]. According to the annual statistical reort for the year 2005 in the NCI, gastric cancer reresented 1.7% of the total malignancies in that year, 85.5% of these cases were confirmed athologically [2]. Among the rognostic factors now available for gastric cancer, the most imortant is the TNM stage. However, the rognosis varies among atients of the same stage and hence it is necessary to investigate other rognostic factors. Treatment and other tumor biologyrelated factors could be of unexlored significance. Much is being done in the field of microarray studies, gene rofiling and the develoment of molecular markers as a means of roviding rognostic information and new theraeutic targets and agents to treat gastric cancer atients, although many of these markers have not yet been translated into clinical ractice. The her-2/neu gene is located on chromosome 17 at q21 and encodes a 185-kDa cell- 147

2 148 surface glycorotein [3]. Gene amlification or overexression of her-2/neu and its relation to rognosis has been reorted in several tumors [4]. Several studies mentioned a number of genetic alterations in gastric carcinoma including amlification of her-2/neu gene or overexression of its rotein [5-7]. 53 is a DNA binding cell cycle-regulating transcrition factor and articiates in the balance between cell survival and death. It is also located on chromosome 17. Losses, deletions, and mutations of 53 have been determined in the athogenesis of a wide range of tumors including the stomach [8,9]. Kononen et al. [10] develoed tissue microarray (TMA) techniques that simultaneously roduce comrehensive rotein exression rofiles for numerous tumors. TMA aears to be articularly useful for immunohistochemical characterization of tumors [11-13]. The basic treatment for gastric carcinoma is radical surgery with extensive lymh node dissection. Having high incidence of locally advanced stage, adjuvant treatment arose to be of significant to imrove control and survival rates [14]. However, both the utilization of adjuvant theray and the different regimens used vary greatly among countries and institutions. Since 2001, adjuvant treatment was imlemented in atients with gastric carcinoma at NCI, Cairo University. In this study, the overexression of her-2/neu and 53 roteins was investigated in gastric carcinoma and the association of their overexression, histoathological features and their influence on survival rates was evaluated. PATIENTS AND METHODS This is a retrosective clinico-athological study of surgically treated gastric carcinoma atients at NCI, Cairo University, during the eriod from 2001 to A total of 93 evaluable atients who had otentially curative resection surgery without neo-adjuvant theray, with sufficient follow-u data as well as the availability of araffin blocks with reresentative tissue were eligible to be included in this study. All atients were subjected to full diagnostic and metastatic worku reoeratively; including full laboratory investigation, chest radiograhy, Clinicoathologic & Prognostic Significance uer gastrointestinal endoscoy, biosy in addition to C.T abdomen and elvis. Treatment rotocols: Surgery: After excluding metastatic and unfit atients, an initial surgical exloration was done. In the last 5 years initial laaroscoic exloration together with laaroscoic intraoerative ultrasound were alied for most of the cases osted for surgery. The extent of gastric resection was according to the site and extent of the tumor. Patients having distal tumors were subjected to distal gastrectomy, atients with roximal tumors were subjected to a total gastrectomy in most of the cases and those having limited disease were subjected to a roximal oesohagogastrectomy or total gastrectomy. Total gastrectomy was erformed in large tumors involving the body of the stomach and linitis lastica. Reconstruction after distal gastrectomy was by a gastrojejuenostomy and jejunojejenostomy. Oesohagogastrostomy was erformed after roximal gastrectomy and Roux-en Y anastomoses was the rocedure of choice after total gastrectomy with few cases having ouch reconstruction. The extent of lymh node dissection was variable. Patients were ket on intravenous fluids for the first 3-4 days and fluid diet was introduced on return of intestinal function. Radiation theray: Postoerative adjuvant treatment was indicated for gastric tumors with ositive lymh nodes, extension to serosa or ositive margin. A 6 MV linear accelerator was used. An isocentric technique was adoted at SAD of 100 cm. All atients were treated in the suine osition. Conventional simulator-based 2 antero-osterior fields, with or without cone down technique, are the standard of care in the radiotheray deartment. More localized 2D/3D CT multile fields are used but not in standard level. Irradiation was given in a dose of 45 Gy/4.5 weeks, 1.8-2Gy/fraction. Treatment was given 5 days/ week. Chemotheray: Postoerative adjuvant treatment was indicated for gastric tumors with ositive lymh nodes, extension to serosa or ositive margin. The adjuvant treatment consisted of one cycle of Mayo regimen (425 mg of fluorouracil/m 2 / day, lus 20 mg of leucovorin/m 2 /day, for five

3 Hoda M. Ismail, et al. days) followed by modified doses of bolus fluorouracil (400mg/m 2/day) and leucovorin 20mg/m2/day on the first four and the last three days of radiotheray. One month after the comletion of radiotheray, two five-days cycles of fluorouracil (425 mg/m2/day) lus leucovorin (20 mg/m2/day) were given one month aart. Pathological evaluation: The histological tye of the tumors was determined according to Lauren s criteria [15] and the WHO classification [16]. The tumors were classified according to (TNM) staging [17]. TMA Construction: A ma of receiver block was reared with coordinates for each samle to correctly identify the tumor. Under a microscoe, areas of interest that were non-necrotic and rich in tumoral glands were marked on the whole hematoxylin and eosin stained (H&E) section of each donor s block. TMAs were constructed using a manual tissue arrayer (Beacher Instruments Inc.). The system consists of thin-walled stainless-steel needles with an inner diameter of about 2 mm and a stiletto for transferring and emtying needle contents. Secimens were retrieved from selected regions of donor tissue and recisely arrayed in a new reciient block. One core er donor block for each case samling a 600µm diameter core was extracted and using 0.8 mm edge-to-edge sacing was arrayed in 3 reciient blocks. H&E sections were cut from each block to identify tumor tissue as well as ositively charged sections for immunohistochemistry staining. Immunohistochemical staining: Slides were dearaffinized, rehydrated, incubated for 15 min with 3% hydrogen eroxidase to inhibit endogenous eroxidase activity and then heated in 0.01 mol/l citrate buffer (H6.0) in a microwave oven for 5 min at 100ºC for antigen retrieval. After cooling down, the sections were incubated for 15 min in a blocking solution containing 10% normal goat serum in PBS. A ready-to-use antibody against 53 (N1518 Dako) was used. Her-2/neu was stained using Herce Test Kit (K5204) according to the manufacturer s instructions (DAKO). Detection was by Biotin-Stretavidin Amlified (B-SA) system with diaminobenzidine chromogen as in routine rotocol. Meyer s hematoxylin was used for counterstaining. The slides were then dehydrated, cleared and mounted. 149 Evaluation of scores: The DAKO Herce Test Protocol system [18] was used to grade the degree of membrane staining. The stain was scored as negative (0) when no membrane staining was observed, or when membrane staining was observed in less than 10% of the tumor cells; weak ositive (1+) if weak focal membrane staining was seen in more than 10% of the tumor cells; intermediate (2+) if weak to moderate, comlete membrane staining was seen in more than 10% of the tumor cells; and strongly ositive (3+) if intense comlete membrane staining was seen in more than 10% of the tumor cells (Fig. 1). Scores 0 and 1 were considered negative, while, scores 2 and 3 were considered ositive. Sections were scored as ositive for 53 when more than 10% of tumor cells dislayed nuclear immunostaining (Fig. 2) [19]. Fig. (1): Tissue microarray section of adenocarcinoma grade I with ositive nuclear immunostaining for 53 (x250). Fig. (2): Tissue microarray section of adenocarcinoma grade II with ositive membranous immunostaining for her-2/neu (x400).

4 150 Clinicoathologic & Prognostic Significance Statistical analysis: SPSS ackage for Windows, version 15 (SPSS Inc., Chicago, Illinois, USA) was used. Chi-square test (Fisher's exact test) was used to detect association between qualitative variables. Survival analysis was erformed using the Kalan-Meier (roduct limit) method. Comarison between survival curves was done using the log-rank test. Over all survival (OS) was calculated from the date of diagnosis to the date of last follow-u, while the local control rate (LC) and metastasis free survival (MFS) and disease free survival (DFS) were calculated from date of surgery till date of relase (local, distant or both) that was confirmed clinically and/or radiologically using CT of the abdomen and elvis. Multivariate analysis was done on the significant variables affecting survival using Cox regression: forward likelihood ratio method. A was considered significant [20]. RESULTS The atients, tumor and treatment characteristics are resented in Table (1). The age of the studied oulation ranged between 19 and 81 years with a mean of 56.4±11.8 years. The male gender constituted 61.3% (57 atients) of them. Thirty two tumors (34.4%) were located roximally, 35 distally and 26 tumors diffusely involved the stomach. The average size of tumors was 7.0±3.2 cm ranging from 2.5 to 22 cm. According to the Lauren classification, there were 34 cases (36.6%) of the intestinal tye and 59 cases (63.4%) of the diffuse tye. Histoathologically, there was 1 (1.1%) case well differentiated, 30 (32.3%) moderately differentiated, 26 (27.9%) oorly differentiated adenocarcino ma, 24 (25.8%) signet and 12 (12.9%) mucinous adenocarcinoma. Two thirds of the cases (62 cases) were of grade 3. Only 2 cases had early tumor invasion limited to the mucosa or submucosa. dal metastasis was resent in 66 (70.9%) atients. Almost half (45) of the cases were of stage III. Staging was inalicable in 3 cases. Nine atients (9.7%) were left with ositive margin ost-surgery. Surgery: Curative surgery was done for all atients excet one who had artial gastrectomy as a alliative rocedure. Total gastrectomy was done in 31 cases, subtotal gastrectomy in 51 cases and roximal oesohagogastrectomy in 11 cases. D1 lymh node dissection was done in 66 cases and D2 dissection was done in 27 cases. The mean number of retrieved lymh nodes was 13.1±8.4 (range 2-40) in all cases. The mean number of ositive lymh nodes was 5.3±7 (range 0-40). Oerative mortality was 19.3%. Oerative morbidity was 34%; the commonest surgical morbidity was anastomotic leakage and sesis. R 0 (curative resection with no residual) was achieved in 84 cases, nine atients (9.6%) had R 1 resection (microscoic residual with ositive resection margin). Radiation theray: Only 21 atients (22.6%) received radiotheray, 20 of them (95.2%) on adjuvant bases. Radiotheray dose had a mean of 4389±446cGy (range from 30-50Gy). Cases received dose more than 46Gy had cone down technique with or without 3D lanning. Adjuvant chemotheray: Similarly, 32 (34.4%) atients received ostoerative chemotheray. Two atients received chemotheray on alliative bases since they were stage IV. Overall the treatment was well tolerated. The main encountered side effects were hematological and gastrointestinal toxicities. Grade 2 gastrointestinal toxicities were encountered in 17 (53%) mainly in the form of oral mucositis and diarrhea. Grade 3 diarrhea occurred in only 1 atient. Grade 3 thrombocytoenia was encountered in only 1 atient. Immunohistochemical results: 53 was overexressed in 25.8% of the cases, being more in intestinal than diffuse tye. The difference was statistically significant, = There was no correlation with the overexression of 53 and gender, tumor grade, stage, or lymh node involvement. Similarly there was no significant relation with the age of atients or tumor site (Table 2). Her-2/neu was overexressed in 25.8% of the cases. It was overexressed more in smaller tumors, =0.038, but there was no relation between overexression of her-2/neu and any of the other clinico-athologic arameters examined in this study (Table 2). It was also exressed more in distally located tumors, diffuse tye, high grade tumors, node-ositive cases and in late stages, but this difference was not statistically significant. Also, there was no correlation with 53 overexression.

5 Hoda M. Ismail, et al. 151 Survival and attern of treatment failure: The median follow-u eriod was 6.7 months (range ). At the end of the follow-u, 39 atients (41.9%) were dead, 4 (4.3%) had disease and 50 (53.8%) were free of disease. Twenty two atients had treatment failure, 6 had isolated local recurrence, 6 had isolated metastasis and 10 had both local recurrence and metastasis. OS rate at the end of follow-u eriod was 22.7%, with a median survival of 17.7 months (Fig. 3). Table (3) shows the survival rates at 12 months with 64.2%, 52.2%, 55.6% and 45.0% for OS, LC, MFS, DFS rates, resectively. As exected, atients with higher stages and those with lymh node involvement had a significantly lower survival; OS and MFS. Age above 57 years was associated with significantly lower OS, LC, MFS and DFS. Patients who received radiotheray had significantly higher OS, LC, MFS and DFS. However, adding chemotheray imroved OS and MFS but did not affect LC or DFS rates. ne of the survival rates had been affected by the overexression of 53, nor her-2/neu. Table (4) shows the results of multivariate analysis of the significant factors that affects survival. Radiotheray and lymh node ositivity significantly affected DFS, MFS and LC. OS was also significantly affected by radiotheray in addition to tumor stage. Percent surviving Time (months) Uer 95% CI Lower 95% CI Fig. (3): Overall survival with the 95% confidence interval curves. 96 Table (1): Characteristics of studied gastric carcinoma atients, tumor and treatment. Characteristic Age: Mean ± SD Sex (M:F ratio): Male Female Tumor site: Proximal Distal Tumor size: Mean ± SD Median (range) Histological tye: Adenocarcinoma Signet Mucinous Pathological subtye: Intestinal Grade: I-II (Low) III (High) Stage* (n=90): I II III IV LN stage* (n=90): N0 N1 N2 N3 Surgical margin* (n=73): Her-2/neu: 53: Treatment received surgery: Curative Palliative Surgical oeration: Partial/subtotal G** Total G Radiotheray: Chemotheray: Median follow-u (Range) n = 93 Number (%) 56.4± :1 57 (61.3%) 36 (38.7%) 32 (34.4%) 35 (37.6%) 26 (28.0%) 7.0± ( ) 57 (61.3%) 24 (25.8%) 12 (12.9) 59 (63.4%) 34 (36.6%) 31 (33.3%) 62 (66.7%) 7 (7.3%) 29 (32.2%) 45 (50.0%) 9 (10.0%) 24 (26.7%) 40 (44.4%) 20 (22.2%) 6 (6.7%) 9 (9.7%) 64 (68.8%) 24 (25.8%) 69 (74.2%) 24 (25.8%) 69 (74.2%) 92 (98.9%) 1 (1.1%) 62 (66.7%) 31 (33.3%) 21 (22.6%) 72 (77.4%) 32 (34.4%) 61 (65.6%) 6.7 ( ) * Cases with no data were excluded. ** Gastrectomy.

6 152 Clinicoathologic & Prognostic Significance Table (2): Relation between overexression of tumors markers and different clinico-athological criteria in studied gastric carcinoma atients. Her-2/neu +ve -ve +ve 53 -ve Age: 57 years > 57 years 13 (26.0%) 11 (25.6%) 37 (74.0%) 32 (74.4%) (28.0%) 10 (23.3%) 36 (72.0%) 33 (76.7%) Tumor site: Proximal Distal 9 (28.1%) 10 (28.6%) 5 (19.2%) 23 (71.9%) 25 (71.4%) 21 (80.8%) (28.1%) 9 (25.7%) 6 (23.1%) 23 (71.9%) 26 (74.3%) 20 (76.9%) Tumor size: < 6 cm 6 cm 13 (38.2%) 11 (18.6%) 21 (61.8%) 48 (81.4%) (26.5%) 15 (25.4%) 25 (73.5%) 44 (74.6%) Histological cell tye: Adenocarcinoma Signet Mucinous 17 (29.8%) 4 (16.7%) 3 (25.0%) 40 (70.2%) 20 (83.3%) 9 (75.0%) (33.3%) 2 (8.3%) 3 (25.0%) 38 (66.7%) 22 (91.7%) 9 (75.0%) Pathological tye: Intestinal 15 (25.4%) 9 (26.5%) 44 (74.6%) 25 (73.5%) (16.9%) 14 (41.2%) 49 (83.1%) 20 (58.8%) Grade: Low High 9 (29.0%) 15 (24.2%) 22 (71.0%) 47 (75.8%) (35.5%) 13 (21.0%) 20 (64.5%) 49 (79.0%) Stage* (n=90): I & II III & IV 8 (22.2%) 16 (29.6%) 28 (77.8%) 38 (70.4%) (16.7%) 18 (33.3%) 30 (83.3%) 36 (66.7%) LN stage* (n=90): 18 (27.3%) 6 (25.0%) 48 (72.7%) 18 (75.0%) (30.3%) 4 (16.7%) 46 (69.7%) 20 (83.3%) SM* (n=73): 3 (33.3%) 13 (20.3%) 6 (66.7%) 51 (79.7%) (11.1%) 16 (25.0%) 8 (88.9%) 48 (75.0%) RTH: 6 (28.6%) 18 (25.0%) 15 (71.4%) 54 (75.0%) (23.8%) 19 (26.4%) 16 (76.2%) 53 (73.6%) CTH: 6 (18.8%) 18 (29.5%) 26 (81.3%) 43 (70.5%) (34.4%) 13 (21.3%) 21 (65.6%) 48 (78.7%) : 6 (25.0%) 18 (26.1%) 18 (75.0%) 51 (73.9%) *Cases with no data were excluded. LN: Lymh nodes. SM: Safety margin. RTH: Radiotheray. CTH: Chemotheray.

7 Hoda M. Ismail, et al. 153 Table (3): Survival rates at 12 months of studied gastric carcinoma atients. N OS LC MFS DFS Whole grou % 52.2% 55.6% 45.0% Age: 57 years > 57 years % 51.5% % 38.0% % 37.3% % 32.7% Sex: Male Female % 69.9% % 62.4% % 68.4% % 59.9% Tumor site: Proximal Distal % 68.4% 62.5% % 55.9% 48.3% % 59.8% 54.4% % 53.1% 44.2% Tumor size: < 6 cm 6 cm % 62.0% % 48.8% % 53.4% % 39.6% Pathological tye: Intestinal % 60.1% % 46.2% % 58.2% % 44.9% Grade: Low High % 60.4% % 49.0% % 58.3% % 44.6% : % 59.6% % 52.5% % 58.0% % 48.5% Her-2/neu: +ve -ve % 51.3% % 52.8% % 42.6% Stage (n=90): I & II III & IV % 53.5% % 36.9% % 40.9% % 30.5% LN stage (n=90): % 54.6% % 39.0% % 42.8% % 33.7% RTH: % 54.1% % 43.8% % 50.5% % 39.1% CTH: % 52.1% % 52.7% % 53.8% % 45.5% LN: Lymh nodes. RTH: Radiotheray. CTH: Chemotheray. OS: Overall survival. LC: Local recurrence rate. MFS: Metastasis free survival. DFS: Disease free survival.

8 154 Table (4): Multivariate analysis for factors significantly affecting survival of studied gastric carcinoma atients. OS: Stage RTH LC: LN ositivity RTH MFS: LN ositivity RTH DFS: LN ositivity RTH OS LC MFS DFS LN B SE OR : Overall survival. : Local recurrence rate. : Metastasis free survival. : Disease free survival. : Lymh nodes DISCUSSION % CI for OR RTH: Radiotheray. B : Regression coefficient. SE : Standard error. OR : Odds ratio. CI : Confidence interval. Excellent correlation has been reorted between data obtained from TMA and conventional blocks in a variety of tumor tyes. Also it has been found that samling with otimal cores was sufficient to accurately detect clinicoathological correlations [21,22]. 53 ositivity was detected in 25.8% of our gastric carcinoma cases. In this study, we found that intestinal tye cancer had more ositive 53 exression than the diffuse tye. Some studies have shown similar results [8,23] but others could not find such difference [24]. Generally, intestinal-tye carcinomas originate from the atrohic gastritis or intestinal metalasia of gastric eithelium, the latter being considered as an imortant recancerous lesion [25]. -tye carcinomas are derived from the stem cells of gastric gland neck. Although a recent study indicated that all gastric eithelial cells originated from the stem cells of bone marrow [26]. 53 alterations have been reorted to aear secifically at the stage of dyslasia during tumor genesis of the intestinal gastric cancer [27,28] and they also have been reorted to occur in intestinal metalasia [29]. Thus, we may suggest that 53 alterations have a crucial and early role in gastric carcinogenesis of intestinal tye, likely acting at the transition ste between intestinal metalasia and dyslasia. Clinicoathologic & Prognostic Significance There are conflicting results in studies of the rognostic significance of 53. In Western countries and Korea, 53 overexression had a oor rognostic effect [8,30,31], whereas in Jaan ositive 53 was not related to rognosis in gastric carcinoma atients [32,33]. In this study, 53 had no relation to rognosis. This may be attributed to our late stage tumors. Immunohistochemical membranous staining incidence of her-2/neu rotein has been reorted to be 10-45% in gastric carcinomas [6,7,34,35]. This is consistent with the ositive rate of 25.8% which was achieved for her-2/neu immunoreactivity in this study. We found that her-2/neu was significantly overexressed in smaller tumors. This may reflect its role in carcinogenesis at a rather early ste and thus may justify testing for its overexression in gastric biosies, being one of the risk factors. Some researchers reorted a significant relation with larger tumors [36], while others did not find any relation [8]. There are conflicting results in studies of her-2/neu regarding the relationshi with rognosis in gastric carcinoma. Some found that its overexression had a good rognostic significance [37], whereas others stated that its overexression was related to oor rognosis [38-40]. In our study, her-2/neu overexression was not related to rognosis of gastric carcinoma. Similar relation was reorted by several researchers [8,32,41]. In this study, a relatively small ercentage of her-2/neu ositive cases was observed. In general, low incidence of an event can substantially attenuate the statistical ower of a study and can easily lead to false negative results; a fact which could otentially be one reason why some studies on her-2/neu in gastric cancer cases failed to determine a significant rognostic imact. Many factors may account for the variable rates of 53 and her-2/neu immunoreactivity, as well as the conflicting reorts suggesting that they are associated with adverse clinical outcomes, in some studies but not others. These factors include small study samle sizes, tissue fixation, rocessing, choice of rimary antibodies, detection systems, methods of antigen retrieval, as well as the subjective scoring system. Most of the atients who resented to the NCI were in a relatively advanced stage. In this study, survival rates were worse in older age and advanced stage. Park and col-

9 Hoda M. Ismail, et al. 155 leagues [36] reorted similar relation, while Jang et al. [42] found that only the stage affected the survival. Using chemotheray alone on an adjuvant base has generally been disaointing. Desite the marginal benefit shown by several metaanalyses, the majority of individual studies of adjuvant chemotheray in gastric cancer showed negative results. Several factors may contribute to these outcomes. Many trials have included relatively small numbers of atients and were, therefore, underowered to detect clinically significant survival differences between treatment and control arms. Moreover, the chemotheray regimens used in many of these adjuvant studies were subotimal, as demonstrated by their low resonse rates and a short duration of resonse in advanced gastric cancer. Therefore, lack of efficacy in the adjuvant setting is not unexected. During the last 4 decades, many trials addressed adjuvant chemoradiation to imrove treatment outcome of gastric cancer atients [14,43-45]. Adjuvant chemoradiotheray has a sound rationale in gastric cancer to imrove locoregional control, which is a valid objective given the high relase rates after surgery alone. The combination of radiation for local control and chemotheray to control systemic micrometastases and radiosensitization makes combined chemoradiotheray a logical ostoerative adjuvant treatment for gastric cancer [46]. However, none of the early trials could draw a definitive survival benefit until the Gastrointestinal Cancer Intergrou Trial (INT 0116) was initiated in 1991 [47]. This trial was a landmark study in the adjuvant treatment of gastric cancer. In this trial, good erformance status atients with stage IB-1V M0 gastric adenocarcinoma who had en-bloc resection and negative surgical margins were randomized after their oeration to either an observation arm or to adjuvant chemoradiotheray. This consisted of 5-FU/ leucovorin followed by 4500cGy radiation accomanied by 5-FU/LV and followed by a further two cycles of the same chemotheray regimen. In considering the survival outcome of the trial, it should be noted that 54% of atients included had a less than D1 dissection (involving incomlete removal of N1 nodes), while 36% had a D1 dissection. Only in 10% of atients was the D2 rocedure used. Based on data noted above, it is therefore likely that 60%-70% of atients had residual cancer in nodes not removed during surgery. The outcome of the trial was excetionally ositive. Comared with atients in the observation-only arm, those treated with chemoradiotheray exerienced significantly longer rogression-free survival (median, 30 Vs 19 months; =0.0001) and significantly longer overall survival (35 Vs 28 months; =0.01). The latter data are based on a median followu of 58.8 months. Furthermore, the imroved survival following chemoradiotheray in resected gastric cancer is achieved with accetable toxicity [14]. In conclusion, this study failed to show any rognostic effect of 53 and her-2/neu on survival rates. However, it could demonstrate the significantly imroved survival rates with adjuvant chemoradiation. Thus, we stress on the imortance of adjuvant chemoradiotheray as a standard of care management. TMA is a useful technique for raid identification of rotein exression rofiles using minimal samles from archived tissues and we recommend the alication of TMA technique for its economic imortance and reliable quick throughut. Also, almost all of our atients sought treatment in a relatively late stage, so early detection rotocols for gastric malignancy should be adoted to establish the diagnosis at an early stage. This may hel in erforming further studies to establish the role of 53 in the athogenesis of intestinal tye adenocarcinoma and to investigate more accurately the redictive and rognostic significance of 53 and her-2/neu. REFERENCES 1- Mokhtar N, Gouda I, Adel I. Cancer Pathology Registry and Time Trend Analysis. National Cancer Institute. 2007, El Attar IA, Ali Eldin NH, Moneer MM, El Basmy AA, Belal D, Aref NA, et al. Annual statistical reort 2005, Eidemilogy & biostatistics Deartment, National Cancer Institute, Cairo University. 2006, ww.nci. cu.edu.eg. 3- Yamamoto T, Ikawa S, Akiyama T, Semba K, mura N, Miyojima N, et al. Similarity of rotein encoded by the human c-erbb-2 gene to eidermal growth factor recetor. Nature. 1986, 319: Wright C, Agnus B, Nicholson S, Sainsbury JR, Cairns J, Gullick WJ, et al. Exression of c-erbb-2 oncorotein; a rognostic indicator in human breast cancer. Cancer Res. 1989, 49: Lemoine NR, Jain S, Silvestre F, Loes C, Hughes CM, McLelland E, et al. Amlification and overex-

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