Clinico-Pathological and Prognostic Significance of p53, Bcl-2 and Her-2/neu Protein Markers in Colorectal Cancer Using Tissue Microarray

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1 Journal of the Egytian Nat. Cancer Inst., Vol. 19, No. 1, March: 3-14, 2007 Clinico-Pathological and Prognostic Significance of 53, Bcl-2 and Her-2/neu Protein Markers in Colorectal Cancer Using Tissue Microarray HODA M. ISMAIL, M.D. 1 ; MANAL EL-BARADIE, M.D. 2 ; MANAR MONEER, M.D. 3 ; OLA KHORSHID, M.D. 4 and AHMED TOUNY M.D. 5 The Deartment of Pathology 1, Radiation Oncology & Nuclear Medicine 2, Biostatistics and Eidemiology 3, Medical Oncology 4 and Surgical Oncology 5, National Cancer Institute, Cairo University. ABSTRACT Background: The rognostic role of her-2/neu has been established in breast cancer but remains controversial in colorectal cancer (CRC). Widesread genetic mutations in colorectal carcinogenesis exist on chromosome 17. Her- 2/neu gene and the tumor suressor gene 53 are both located on this chromosome. Bcl-2 rotein rolongs survival of a variety of cells by blocking aotosis. The aim of this study is to evaluate the relationshi between the overexression of 53, bcl-2 and her-2/neu rotein markers and the clinico-athologic characteristics of CRC, and their influence on survival rates. Patients and Methods: One hundred and four cases of CRC had araffin blocks with reresentative tissue, and sufficient follow-u data. They were arrayed and evaluated for rotein marker exression using tissue microarray (TMA). Results: Ten (9.6%), 35 (33.7%) and 27 (26%) of the atients were her-2/neu, 53 and bcl-2 ositive, resectively. None of the examined clinico-athologic factors had a significant relation with her-2/neu overexression. Patients with bcl-2 had a significantly higher mean age (52.4±13.3years) comared to 45.4±14.4 years for bcl-2 negative atients, =0.03. Positive 53 was overexressed in 20/44 (45.5%), 6/17 (35%), 9/43 (21%) cases of the colon, recto-sigmoid, and rectal sites, resectively, =0.05. For the whole oulation, 53 overexression had a significantly lower disease-free survival (DFS). For atients with Dukes' stage B, overexression of 53 rotein had a significant reduced overall survival (OS) =0.04, metastasis free survival (MFS) =0.004, and DFS =0.01 rates. Exression of bcl-2 had a significantly better MFS =0.001, while her-2/neu overexression worsened the OS rate significantly, =0.04. Corresondence: Dr Hoda M. Ismail, 17 El Mathaf El Zeraie St., hodanci@hotmail.com Conclusion: This study recommends the alication of TMA technique for its economic imortance and reliable quick throughut. The results from this study also suggest that overexression of 53, bcl-2, and her-2/neu rotein markers aear to be useful in selecting a grou of CRC atients with a worse rognosis and constitute otential candidates for adjuvant theray. Key Words: Her-2/neu 53 Bcl-2 Colorectal cancer Tissue microarray Prognostic factors. INTRODUCTION Colorectal cancer (CRC) is the fifth most common cancer and ersists as a significant cause of cancer mortality in Egyt. It constitutes 4.34% of total malignancies [1]. Prognosis remains oor, desite the considerable number of cancer researches, and deends on the stage of cancer at the time of resentation. Colorectal carcinogenesis is comlex and robably matched by deendent genetic alterations. Recently, researchers have tried to identify biological markers in order to individualize chemotheray and redict clinical outcome and tumor sensitivity [2]. Her-2/neu oncogene is a member of the tyrosine kinase family similar to the eidermal growth factor recetor. It is located on chromosome 17q21 and encodes a 185kD transmembrane rotein. Her-2/neu activation initiates signal cascade athways that are essential for cell roliferation and differentiation [3]. Though the tyrosine kinase family recetors are found on normal cells, there is evidence that they are over-exressed in many tyes of tumors [4-6]. Clinically, her-2/neu amlification and/or overexression have been associated with oor rognosis in a number of tumor tyes such as breast and ovarian cancer [4]. Conflicting data exist 3

2 4 about the revalence of her-2/neu overexression in CRC, which ranges from 0 to 83%, as well as the relationshi between her-2/neu overexression and clinico-athologic features like Dukes classification and survival [7-12]. Trastuzumab, an anti-her-2/neu human monoclonal antibody, is now an FDA-aroved drug for the treatment of breast cancer, and this drug, lus ICM-225, ADAM-like rotease inhibitors, and EGF tyrosine inhibitors, are now in clinical trials for the treatment of CRC [13]. 53 is a DNA binding cell cycle-regulating transcrition factor and articiates in the balance between cell survival and death. Losses, deletions, and mutations of 53 have been determined in the athogenesis of a wide range of tumors including CRC [14,15]. The term aotosis, originally defined as ''falling off of leaves from trees'', was first used scientifically to describe energy-deendent cell death by Kerr et al. in 1972 [16]. The members of the bcl-2 family are a grou of crucial regulatory factors in aotosis. Aotosis can, not only maintain the body in well stable condition, but also lays an imortant role in regulating and controlling tumor occurrence, develoment and treatment [17]. It has been roved that occurrence of cancer is due to the loss of control of normal aotosis and the disturbance of balance between cell roliferation and aotosis [18,19]. Aotosis related genes such as bcl-2 family are divided into two categories: roaotosis genes and anti-aotosis genes. Bcl- 2 is an imortant aotosis reressor [20]. Tissue microarray (TMA) allows the analysis of a huge number of samles in a single rocedure using all tyes of in-situ analysis including immunohistochemistry, fluorescence-in-situ hybridization (FISH), and RNA in-situ hybridization. Thus, TMA technology has the otential to greatly facilitate the translation of basic research into clinical ractice [21]. In this study, the relationshis of the immunohistochemical overexressions of 53, bcl- 2 and her-2/neu in CRC secimens with clinicoathological arameters and their imact on survival rates were studied in CRC atients who had undergone otentially curative surgery, aiming at evaluating the relationshi between these rotein markers and the clinico-athologic characteristics of CRC, and their influence on survival rates. Clinico-Pathological & Prognostic Significance PATIENTS AND METHODS A retrosective clinico-athological study was carried out for the atients with CRC, surgically treated during the eriod from 1995 to A total of 104 evaluable cases (64 treated in the National Cancer Institute (NCI), Cairo University, and 40 treated in Tanta Cancer Centre) who had otentially curative resection surgery without any 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. Forty-six atients who did not fulfill study criteria were excluded. Pathological evaluation: Histological tyes and grading were reviewed and classified according to the World Health Organization classification criteria [22], and the athologic stage was determined according to Dukes' staging system [23]. Tissue microarray construction: Localization of a reresentative tumorous tissue on the donors' araffin block after reviewing a hematoxylin and eosin (Hx and E) section cut from the block was the first ste. A 600µm diameter core was extracted using a Beecher Instrument tissue extractor (at King Faisal Secialist Hosital and Research Centre, KSA) and these cores were re-embedded into a reciient araffin block. Four such reciient blocks were constructed containing 280 tissue cores, each arranged in 2 17x9 sectors [24]. A tae-based tissue transfer system (Instrumedics, Hachensack) was used. An adhesive tae was laced on the araffin block before cutting, the tae received with the 4 micron cut section adherent to it was laced on a secial slide with adhesivecoated surface. The section was then ultravioletly cross-linked to the slide before the tae was removed with a secial degreasing reagent included with the set. Hx and E sections were cut from each block to identify tumor tissue on the array slide (Fig. 1). Immunohistochemical staining: Slides were dearaffinized in xylene twice for 10 min, rehydrated through graded ethanol to distilled water, 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

3 Hoda M. Ismail, et al. 5 down to room temerature for 30 min, slides were arranged in Ventana auto-immunohistochemical stainer (Tucson, AZ, USA) according to the manufacturer s guidelines. Ready-to-use antibodies against bcl-2 (N1587 Dako) and 53 (N1518 Dako) were used. Her-2/neu was stained using Herce Test Kit (K5204) according to the manufacturer s instructions (DAKO). Ventana/View DAB detection kit for stretavidin horseradish eroxidase was used. Mayer s Hx was used for counterstaining. Evaluation of scores: The DAKO Herce test rotocol system [25] 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. Scores 0 and 1 were considered negative, while scores 2 and 3 were considered ositive. The results of bcl-2 were scored as follows [26]. The ercentage of tumor cells was graded as follows: 0: 0%; 1: 1-25%; 2: 26-50%; 3: more than 50% ositive cells. Immunostaining intensity was scored as follows: 0: none; 1: weak; 2: moderate; 3: intense. When a score of intensity multilied by ercentage of ositive cells was equal to or more than 1, the secimen was considered as immunoositive. Only the cytolasmic staining was evaluated and nuclear reaction was interreted to be nonsecific staining. Sections were scored as ositive for 53 when more than 10% of tumor cells dislayed nuclear immunostaining [27]. Treatment rotocol: Surgery: Colectomy, abdomino-erineal resection, osterior elvic excentration or low anterior resection, were erformed according to the site and extent of the tumor with a curative intention. Radiation theray: Postoerative adjuvant treatment was indicated for recto-sigmoid and rectal tumors with ositive regional lymh nodes or extension to eri-rectal fat. A 6 MV linear accelerator was used. An isocentric technique was adoted at SAD of 100 cm. All atients were treated in the rone osition with a full bladder to dislace the small bowel anteriorly and sueriorly and to reduce the ostero-anterior searation in obese atients. Careful CTlanning, to limit the dose to risk organs mainly the bladder and femoral heads, is the standard of care in the radiotheray deartment. Irradiation was given in a dose of 50Gy/5 weeks. All the atients were treated with 2Gy/fraction, treating 5 days/week. Chemotheray: Chemotheray, on adjuvant basis, was indicated for atients with athologically ositive lymh nodes, Dukes' C. As for Duke' B, it was indicated for those with oor rognostic factors such as resentation with signs of obstruction, signet-mucoid athology and/or bleeding er rectum. Mayo regimen (in the form of bolus intravenous fluorouracil 425mg/m 2 and leucovorin, 20mg/m 2 ) was given for 5 consecutive days every 28 days for 6 cycles. The treatment was commenced 4-6 weeks after surgery. For rectal carcinoma, chemotheray was given as a radio-sensitizer, with bolus intravenous fluorouracil 375mg/m 2 and leucovorin 20mg/m 2, both were administered during the first and last three days of ostoerative irradiation. Chemotheray (Mayo Clinic regimen for 6 cycles) was continued immediately after the end of irradiation if comlete blood icture and laboratory investigations were satisfactory. Statistical analysis: The atients had a median follow-u eriod of 18 ( ) months. All the survival rates were calculated from the date of surgery to the date of death from disease or last follow-u (the overall survival, OS), date of loco-regional relase (local control rate, LC), and date of metastasis (metastasis free survival, MFS). Disease-free survival (DFS) was calculated from the date of surgery to the date of relase (whether local, distant, or combined as confirmed clinically and/or radiologically using CT of the abdomen and elvis). For statistical analysis, Statistical Package for Social Sciences (SPSS) for Windows, version 15 (SPSS Inc., Chicago, Illinois, USA) was used. Numerical data were described in terms of means and medians for central tendency and standard deviation and range, minimum and maximum for disersion. For quantitative data, comarison between two grous was done using the student t-test or the Mann-Whitney test. Chi-square test

4 6 (Fisher's exact test) was used to comare qualitative variables. Survival analysis was erformed using the Kalan-Meier roduct limit method. Comarison between survival curves was done using the log-rank test was considered significant. RESULTS The atients, tumor and treatment characteristics are resented in Table (1). The age of the studied oulation ranged between 18 to 77 years with a mean of 47.4±14.4 years. The male gender constituted 43% (45 atients) of them. Intestinal tye adenocarcinoma constituted 75% (78 cases) with about 42% of the lesions found in the colon, 41% in the rectum and 16% in the rectosigmoid. About 60% were of grade I-II. More than 98% (102) of the atients underwent curative surgery. However, the majority of the atients resented with locally advanced disease (extra-colonic extension and/or ositive lymh nodes, 56 atients) or with intraoerative detection of liver metastasis (2 atients). Twenty eight atients (26.7%) received adjuvant ostoerative radiotheray and only one atient had alliative radiotheray for local recurrence. The remaining atients either were not indicated (73 atients, 69.5%), or assed the date of adjuvant radiation theray (4 atients, 3.8%) Sixty three atients (61.5%) received chemotheray, 57 (90.6%) of them on adjuvant basis, 6 (9.4%) for metastasis or local recurrence. The 53 was overexressed in 33.3% (35/104) (Fig. 2) of the tumors, bcl-2 in 26% (27/104) (Fig. 3) and her-2/neu in 9.5% (10/104) (Fig. 4). There was no relation between her-2/neu and any of the atients' or tumor characteristics. The only significant effect for 53 was found with the tumor site, where 45% (20/44) of the colonic tumors had ositive 53 comared to 25% (15/60) of the rectal and rectosigmoid lesions, (=0.05). The atients who had bcl-2 exressed in their tumor tissue were significantly older (52.4±13.3 years) comared to those with negative bcl-2 (45.4±14.4 years) (=0.03), (Table 2). There was a significant concordance (in 70.2% of the cases) between results of 53 and her-2/neu (=0.015). No relation was found between bcl-2 and 53 (=0.638) or her-2/neu and bcl-2 (=0.276). Clinico-Pathological & Prognostic Significance None of the examined markers had any relation with the OS or LC rates. DFS was significantly lower (60%) for 53 comared to negative 53 (83%), =0.01 (Fig. 5). However, there was an imrovement in the MFS for atients with 53, short of statistical significance, =0.09 (Table 3). For Dukes' stage B (Table 4), atients with 53 had a statistically lower OS (=0.04), MFS (=0.004), and DFS (=0.01) rates (Fig. 6). For this subgrou of atients, bcl-2 exression had 100% MFS rate comared to their counterart (89%), = Similarly, atients with her-2/neu had a better OS of 95% comared to 80% with her-2/neu, =0.04 (Fig. 7). Table (1): Patient, tumor and treatment characteristics. Sex ratio (M:F) Mean age (years) Tumor site: Colon Recto-sigmoid Rectum Tumor tye: Intestinal adenocarcinoma Mucoid Signet ring Tumor grade: I-II III Stage: A-B C-D Markers: 53 Bcl-2: Her-2/neu: Treatment received: surgery Curative alliative Radiotheray: Yes No Adjuvant chemotheray: Yes No Pattern of failure: No Loco-regional Metastasis Both Median follow-u (range) 45: 59 (0.8:1) 47.4± (42.3%) 17 (16.3%) 43 (41.3%) 78 (75.0%) 21 (20.2%) 5 (4.8%) 62 (59.6%) 42 (40.4%) 49 (47.1%) 55 (52.9%) 35 (33.7%) 69 (66.3%) 27 (26%) 77 (74%) 10 (9.6%) 94 (90.4%) 102 (98.1%) 2 (1.9%) 29 (27.9%)* 75 (72.1%)** 57 (54.7%) 47 (45.3%) * One atient treated with alliative intention. ** Four atients missed for date. Total no= 104 number (%) 62 (59.6%) 20 (19.2%) 19 (18.3%) 3 (2.9%) 18 ( ) months

5 Hoda M. Ismail, et al. 7 Table (2): Relationshi between clinico-athological arameters and 53, bcl-2 and her-2/neu immunohistochemical exression. 53 Bcl-2 Her-2 No=35 No=69 No=27 No=77 No=10 No=94 Age Mean ± SD Gender: Male Female Pathology: Adenocarcinoma Signet/mucoid Grade: I-II III Stage*: A-B C-D Positive LN.>3: Yes No Site: Colon Recto-sigmoid Rectum 48.4± ± ± ± ± ± ** 1.00*** 1.00*** 1.00*** 0.51*** 1.00*** * Dukes' staging system [23] ** Mann-Whitney test, *** Fisher's exact test, no - because of small number of cases within grous. Table (3): Relationshi between survival rates and 53, bcl-2 and her-2/neu immunohistochemical overexression. No OS (at 18 months) LCR (at 12 months) MFS (at 12 months) DFS (at 12 months) 53: % 85% % 86% % 93% % 83% 0.01 Bcl-2: % 82% % 83% % 86% % 73% 0.28 Her-2/neu: % 81% % 84% % 89% % 76% 0.39 Table (4): Relationshi between survival rates and 53, bcl-2 and her-2/neu immunohistochemical overexression for Dukes' stage B. No OS (at 18 months) LCR (at 12 months) MFS (at 12 months) DFS (at 12 months) 53: % 97% % 80% % 100% % 91% 0.01 Bcl-2: % 100% % 73% % 89% % 53% 0.12 Her-2/neu: % 95% % 75% % 83% % 64% 0.26

6 8 Clinico-Pathological & Prognostic Significance Fig. (1): Tissue microarray section (H & E x 25). Fig. (2): Tissue microarray section of adenocarcinoma grade II with ositive nuclear immunostaining for 53 (x 250). Fig. (3): Tissue microarray section of adenocarcinoma grade II with ositive cytoasmic immunostaining for bcl-2 (x 400). Fig. (4): Tissue microarray section of adenocarcinoma grade II with ositive membranous immunostaining for her-2/neu (x 400). Cum survival rortion grous censored censored Time (months) Fig. (5): Disease free survival curve for 53 overexression.

7 Hoda M. Ismail, et al. 9 Cum survival rortion Cum survival rortion Fig. (6): 53 overexression in stage Dukes' B: Time (months) Fig. (6-A): Overall survival Time (months) Fig. (6-B): Metastasis free survival. 53 grous censored censored grous censored censored Cum survival rortion Cum survival rortion bcl2 grous censored censored Time (months) Fig. (7-A): Metastasis free survival curve for stage Dukes' B for bcl-2 exression. Her2/neu-grou censored censored Time (months) Fig. (7-B): Overall survival curve for stage Dukes' B for her-2/neu overexression. Cum survival rortion Time (months) Fig. (6-C): Disease free survival. 53 grous censored censored DISCUSSION Studies indicate that tumor stage is the most imortant rognostic variable in CRC [28,29]. Desite this fact, considerable stage-indeendent variability in clinical outcome is observed that may be a consequence of altered rates of aotosis and cell roliferation. The her-2/neu rotein is intimately involved with normal cell roliferation and tissue growth, as it is extensively homologous and is related to the eidermal growth factor recetor. This henomenon has been most intensively studied in the context of breast carcinoma, in which

8 10 its amlification and overexression correlate with the overall course of disease and oor rognoses, and also constitute a redictive factor of oor resonse to chemotheray and endocrine theray [12]. However, when concerned with CRC, conflicting data exist about the revalence of her-2/neu overexression ranging from 0 to 83% [8-10,12,30]. The results of the current study reorted an overexression of her-2/neu in 9.1% of the cases. These considerable discreancies in the frequencies and distribution of her-2/neu overexression in CRC were robably due to differences in tissue-fixation methods, the antibodies used, the detection techniques and criteria for evaluating the results. A major cause of these discreancies seems to be the different criteria for evaluating the results. Studies that scored intra-cytolasm staining as ositive reorted larger frequencies, whereas studies that scored only membrane staining as ositive reorted lower or zero frequency [7,31,32]. In the resent study, there was no correlation between her-2/neu overexression and any of the atients' or tumor characteristics. While some authors reorted similar results to ours [11,33-35], others could demonstrate a significant correlation with tumor stage [9,31,36,37] and grade [9,31,38]. Our data did not show any significant effect of her-2/neu overexression on outcome. In this asect, the resent study agreed with other reorts [11,35,39]. On the contrary, a recent study [12] demonstrated that her-2/neu was indeendently related to ostoerative recurrence and survival of CRC and that the atients exhibiting her-2/neu overexression might constitute otential candidates for a new adjuvant theray. The same findings were resented by other studies as well [31,40]. In the resent study, 53 was overexressed in 33.7% of the cases. The reorted figures ranged from 11.8% [41] u to 76% [42-44] Bcl- 2 was exressed in 26% of our cases. Similar results were reorted [42,43]. Higher rates ranging from 46% to 82% were also reorted [41,44-46]. This may suggest that the athogenesis of our CRC cases may rogress through the athway characterized by the inactivation or mutation of the DNA mismatch reair rotein comlex and not through the multiste rocess involving a series of genetic changes involving roliferation and aototic athways. Clinico-Pathological & Prognostic Significance In the resent study, exression of bcl-2 rotein was associated with older age, while Zavrides et al. [47] reorted that only stage was correlated to the exression of bcl-2 rotein. The relationshi between bcl-2 exression and survival revealed variable results. Our cases revealed that the exression of bcl-2 had only a trend for a better MFS rate; that did not reach a significant level. However, others [45,47-49] showed that CRC exressing bcl-2 had a favorable rognosis. Bhatavdekar et al. [50] found that tumors ositive for bcl-2 had a worse rognosis, with a direct correlation between tumor grade and exression. These conflicting results may be related to the athway of bcl-2 acting as an antiaototic member of the bcl-2 family which could be modified according to the resence or absence of other members of the bcl- 2 family that may modulate the bcl-2 function. For the subgrou of our atients with Dukes B, there was a significantly higher MFS in cases exressing bcl-2. This result agreed with the ublished data by Meterissian and colleagues [46] who demonstrated that bcl-2 exression correlated with imroved survival, secifically in Dukes B colon carcinoma. Therefore, it can be used as a rognostic marker and can hel in the selection of a high-risk grou who would theoretically benefit from adjuvant theray. In this study, 53 had only a significant relation with the site of the tumor being significantly higher in the roximal colon comared to the distal rectosigmoid region. Others reorted that distal tumors had a higher frequency of allelic loss, 53 accumulation and aneuloidy [51]. This may require further studying of our cases on the genetic level. For the whole studied oulation, 53 overexression significantly worsen the DFS rate, with trend for a low MFS. In the Dukes' B grou of atients, the overexression of 53 was associated with lower OS, MFS, and DFS rates. Other researchers reorted a similar inverse relationshi between 53 and survival [43,52]. Yet, some others found that 53 had no rognostic otential for atients with CRC [41,53]. In the resent study, due to the small number of atients, we could not stratify them by stage and treatment grous versus marker exression. However, in view of the significant effect of 53 on survival rates, it might be useful to be

9 Hoda M. Ismail, et al. 11 further studied in rosective trials concerning adjuvant chemotheray in CRC. Identification of molecular markers redicting rognosis in Dukes B colon carcinoma can allow selection of atients for adjuvant theray. In the resent study, atients with 53 overexression, in Dukes B stage, had a significantly lower OS, MFS as well as DFS rates without any effect on LC rate. This could reflect the aramount imortance of adding chemotheray for this grou of atients. Lin et al. [54], examined whether 53, and bcl-2 (as well as other markers), could be used to redict the tumor resonse of rectal cancer to neoadjuvant theray and determine the OS and DFS rates of atients following neoadjuvant radio-chemotheray. They found increase of the rate of ositive identification of bcl-2 following neoadjuvant theray, and that 53 status was a good redictor for fair resonse of the atients in the reradiation biosy secimens. In a small randomized clinical trial the relation of neoadjuvant radiochemotheray on marker overexression (including 53) in the low rectal cancer, there was a significant dro of the level of 53 from 57.9% before neoadjuvant chemoradiation to 26.3% after surgery, while there was no significant change in the ositive rate of 53 in radiotheray alone grou. No single randomized clinical trial has demonstrated a survival benefit for adjuvant theray in atients with Stage II colon cancer. A ooled analysis of 7 studies demonstrated an equal 5- year OS in atients who received fluorouracilbased adjuvant theray and those who underwent surgery alone (80%) (=0.11) [55]. It has been roosed that adjuvant chemotheray may benefit atients with Stage II disease and T4 tumor stage, bowel erforation, or clinical bowel obstruction [56]. Although this hyothesis has not been validated in a rosective, randomized clinical trial, a retrosective subset analysis of 318 atients with Stage II disease enrolled in the ECOG INT-0035 study of fluorouracil and levamisole suggested a survival benefit for adjuvant theray in these atient subgrous [57]. It is to be noted that the benefit of chemotheray in atients with high-risk features, such as inadequate lymh node samling, lymhovascular or erineural invasion, oorly differentiated histology, microsatellite stability, and loss of heterozygosity at chromosome 18q, are also known to carry a higher risk of recurrence [58]. The most imortant advantage of the TMA technology include increased caacity, negligible damage caused to the original tissue blocks, the recise ositioning of tissue samles and the ossibility for automated construction and analysis. It is ossible to retrieve dozens of unch samles (each 0.6mm) from each donor block without significantly damaging it, thus enabling the generation of multile relicate array blocks. This technology enables extensive analysis of even small rimary tumor secimens for a larger number of analyses that may be of interest in future investigations. The question of whether minute tissue samles of otentially heterogeneous tumors are reresentative enough of their donor tumor to allow meaningful studies is a critical challenge to the tumor array concet. Some investigators have used core samles that are larger in diameter (>2-4mm). This does not substantially increase the information content of TMA analysis, since the likelihood of finding heterogeneity within such a small area is often quite low [59]. On the contrary, unching multile small cores from different regions catures the heterogeneity of the tumors more effectively and recent studies in fibroblastic tumors [60,61], rostate cancer, and bladder cancer have shown that the exression of Ki-67 by immunohistochemistry is highly correlated with the results in whole-slide rearations if trilicate or quadrulicate arrays were used, as done in this study. In conclusion, our results suggest that exression of 53, bcl-2, and her-2/neu rotein markers aear to be useful in selecting a grou of CRC atients with a worse rognosis and constitute otential candidates for adjuvant theray. Because of its beneficial economic asect, it is noteworthy to mention that TMA alication should be widely alied articularly in develoing countries. REFERENCES 1- Mokhtar N, Gouda I, Adel I. Cancer Pathology Registry and Time Trend Analysis. National Cancer Institute. 2007, Nanni O, Voli A, Frassinetti GL, De Paola F, Granato AM, Dubini A, et al. Role of biological markers in the clinical outcome of colon cancer. Br J Cancer. 2002, 88:

10 12 Clinico-Pathological & Prognostic Significance 3- Schlessinger J. Cell signaling by recetor tyrosine kinase. Cell. 2000, 103: Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: Correlation of relase and survival with amlification of HER- 2/neu oncogenes. Science. 1987, 234: Saranath D, Panchal RG, Nair R, Metha AR, Sanghavi VD, Deo MG. Amlification and over-exression of eidermal growth factor recetor gene in human oroharyngeal cancer. Eur J Cancer. 1992, 28B: Rusch V, Baselga J, Cordon-Cardo C, Orazem J, Zaman M, Hoda S, et al. Differential exression of the eidermal growth factor recetor and its ligand in rimary non-small cell lung cancers and adjacent benign lung. Cancer Res. 1993, 53: Nathanson DR, Culliford AT, Shia J, Chen B, D Alessio MD, Zeng Z, et al. HER 2/neu exression and gene amlification in colon cancer. Int J Cancer. 2003, 105: Kim JY, Lim SJ, Park K. Cyclooxygenase-2 and c- erbb-2 exression in colorectal carcinoma assessed using tissue microarray. Al Immunohistochem Mol Morhol. 2004, 12 (1): Tavangar SM, Shariftabrizi A, Soroush AR. Her 2/neu over-exression correlates with more advanced disease in Iranian colorectal cancer atients. Med Sci Monit. 2005, 11 (3): Demirbas S, Sucullu I, Yild irim S, Celen KT. Influence of the c-erbb-2, nm23, bcl-2 and 53 rotein markers on colorectal cancer. Turk J Gastroentrol. 2006, 17 (1): Schuell B, Gruenberger T, Scheithauer W, Zielinski1 Ch, Wrba F. HER 2/neu rotein exression in colorectal cancer. BMC Cancer. 2006, 6: Park DI, Kang MS, Oh SJ, Kim HJ, Cho YK, Sohn CL, et al. Her-2/neu overexression is an indeendent rognostic factor in colorectal cancer. Int J Colorectal Dis. 2007, 22 (5): Barnard J. Eidermal growth factor recetor blockade: An emerging theraeutic modality in gastroenterology. Gastroenterology. 2001, 120 (7): Thomas G. Advances in the genetics and molecular biology of colorectal tumors. Curr Oin Oncol. 1994, 6: Pereira H, Silva S, Juliao R, Garcia P, Peretua F. Prognostic markers for colorectal cancer. Exression of 53 and bcl2. World J Surg. 1997, 21: Kerr JF, Wyllie AH, Currie AR. Aotosis: A basic biological henomenon with wide-ranging imlications in tissue kinetics. Br J Cancer. 1972, 26: Heinke MY, Yao M, Chang D, Einstein R, Dos Remedios CG. Aotosis of ventricular and atrial myocytes from acing-induced canine heart failure. Cardiovasc Res. 2001, 49: Kanzler S, Galle PR. Aotosis and the liver. Semin Cancer Biol. 2000, 10: Park YN, Chae KJ, Kim YB, Park C, Theise N. Aotosis and roliferation in heatocarcinogenesis related to cirrhosis. Cancer. 2001, 92: Mao JD, Wu P, Xia XH, Hu JQ, Huang WB, Xu GQ. Correlation between exression of gastrin, somatostatin and cell aotosis regulation gene bcl-2/bax in large intestine carcinoma. World J Gastroenterol. 2005, 11 (5): Al Kuraya K, Simon R, Sauter G. Tissue microarrays for high-throughout molecular athology. Ann Saudi Med. 2004, 24 (3): Hamilton S, Aaltonen L (eds). WHO classification of tumors. Pathology and genetics of tumors of digestive system. Lyon, IARC Press. 2000, Dukes C. The classification of cancer of the rectum. J Pathol Bacteriol. 1932, 35: Schraml P, Kononen J, Bubendorf L, Moch H, Bissig H, Nocito A. Tissue microarrays for gene amlification surveys in many different tumor tyes. Clin Cancer Res. 1999, 5: Dako Herce Test for Immunoenzymatic Staining (ackage insert) DAKO A/S, Glastru, Denmark Krajewska M, Krajewski S, Estein JI, Shabaik A, Sauvageot J, Song K, et al. Immunohistochemical analysis of bcl-2, bax, bcl-x and mcl-1 exression in rostate cancers. Am J Pathol. 1996, 48: Abd El Hamid A. Survivin exression in colorectal adenocarcinoma using tissue microarray. J Egyt Nat Cancer Inst. 2005, 17 (1): Dowell SP, Hall PA. The 53 tumour suressor gene and tumour rognosis: Is there a relationshi? J Pathol. 1995, 177: Fielding LP, Pettigrew N. College of American Pathologists Conference XXVI on clinical relevance of rognostic markers in solid tumors. Reort of the Colorectal Cancer (Phila.) Working Grou. Arch Pathol Lab Med. 1995, 119: Ooi A, Takehana T, Li X, Suzuki S, Kunitomo K, Iino H, et al. Protein overexression and gene amlification of HER-2 and EGFR in colorectal cancers: An immunohistochemical and fluorescent in situ hybridization study. Mod Pathol. 2004, 17: Kaitanovic S, Radosevic S, Kaitanovic M, Andelinovic S, Ferencic Z, Tavassoli M, et al. The exression of 185 (HER-2/neu) correlates with the stage of disease and survival in colorectal cancer. Gastroenterol. 1997, 112: McKay JA, Loane JF, Ross VG, Ameyaw MM, Murray GI, Cassidy J, et al. C-erbB-2 is not a major factor in the develoment of colorectal cancer. Br J Cancer. 2002, 86: Hugh TJ, Dillon SA, Taylor BA, Pignatelli M, Poston GJ, Kinsella AR. Cadherin catenin exression in

11 Hoda M. Ismail, et al. 13 rimary colorectal cancer: A survival analysis. Br J Cancer. 1999, 80 (7): Karatzas G, Karaylannakis AJ, Syrigos KN, Chatzigianni E, Paanikolaou S, Riza F, Paanikolaou D. E-cadherin exression correlates with tumor differentiation in colorectal cancer. Heatogastroenterol. 1999, 46: Kountourakis P, Pavlakis K, Psyrri A, Rontogianni D, Xiros N, Patsouris E. Clinicoathologic significance of EGFR and Her-2/neu in colorectal adenocarcinomas. Cancer J. 2006, 12 (3): Mohri Y. Prognostic significance of E-cadherin exression in human colorectal cancer tissue. Jn J Surg. 1997, 27: Ikeguchi M, Taniguchi T, Makino M, Kaibara N. Reduced E-cadherin exression and enlargement of cancer nuclei strongly correlate with hematogenic metastasis in colorectal adenocarcinoma. Scand J Gastroenterol. 2000, 8: Ishida H, Sadahiro S, Suzuki T, Ishikawa K, Tajima T, Makuuchi H. C-erbB-2 rotein exression and clinicoathologic features in colorectal cancer. Oncol Re. 2000, 7: Jesus EC, Matos D, Artigiani R, Waitzberg AFL, Goldenberg A, Saad SS. Assessment of staging, rognosis and mortality of colorectal cancer by tumor markers: Recetor erbb-2 and cadherins. Acta Cirúrgica Brasileira. 2005, 20 (6): Osako T, Miyahara M, Uchino, Inomata M, Kitano S, Kobayashi M. Immunohistochemical study of c-erbb- 2 rotein in colorectal cancer and the correlation with atient s survival. Oncology. 1998, 55: Lustosa SAS, Logull A, Artigiani R, Saad SS, Goldenberg A, Matos D. Analysis of the correlation between 53 and bcl-2 exression with staging and rognosis of the colorectal adenocarcinoma. Acta Cirúrgica Brasileira. 2005, 20 (5): Giatromanolaki A, Stathooulos GP, Tsiobanou E, Paadimitriou C, Georgoulias V, Gatter KC, et al. Combined role of tumor angiogenesis, bcl-2, and 53 exression in the rognosis of atients with colorectal carcinoma. Cancer. 1999, 86 (8): Garrity MM, Burgart LJ, Mahoney MR, Windschitl HE, Salim M, James MW, et al. Prognostic of roliferation, aotosis, defective DNA mismatch reair and 53 overexression in atients with resected Dukes B2 or C colon cancer: A North Central Cancer Treatment Grou Study. J Clin Oncol. 2004, 22 (9): Qiu Ch Z, Wang Ch, Huang Z, Zhu Sh, Wu Y, Qiu JL. Relationshi between somatostatin recetor subtye exression and clinicoathology, Ki-67, Bcl-2 and 53 in colorectal cancer W J Gastentrol. 2006, 12 (3): Sinicroe FA, Hart J, Michelassi F, Lee JJ. Prognostic of bcl-2 oncorotein exression in stage II colon carcinoma. Clin Cancer Res. 1995, 1: Meterissian SH, Kontogiannea M, Al-Sowaidi M, Linjawi A, Halwani F, Jamison B, et al. Bcl-2 Is a Useful Prognostic Marker in Dukes B Colon Cancer. Ann Surg Oncol. 2001, 8 (6): Zavrides H, Zizi-Sermetzoglou A, Elemenoglou I, Paatheofanis I, Peros G, et al. Immunohistochemical exression of bcl-2 in UICC stage I and III colorectal carcinoma atients: Correlation with c-erbb-2, 53, ki-67, CD44, laminin and collagen IV in evaluating rognostic significance. Pol J Pathol. 2006, 57 (3): Ofner D, Riehemann K, Maier H, Riedmann B, Nehoda H, Totsch M, et al. Immunohistochemically detectable bcl-2 exression in colorectal carcinoma: Correlation with tumor stage and atient survival. Br J Cancer. 1995, 77: Baretton GB, Diebold J, Christoforis G, Vogt M, Muller C, Dofer K, et al. Aotosis and immunohistochemical bcl-2 exression in colorectal adenomas and carcinomas. Asects of carcinogenesis and rognostic significance. Cancer. 1996, 77: Bhatavdekar JM, Patel DD, Ghosh N, Chikhlikar PR, Trivedi TI, Suthar TP, et al. Coexression of bcl-2, c-myc and 53 oncoroteins as rognostic discriminants in atients with colorectal carcinoma. Dis Colon Rectum. 1997, 40: Kaiteijin E, Liefers GJ, Los LC. Mechanisms of oncogenesis in colon versus rectal cancer. J Pathol. 2001, 195: Allegra CJ, Parr AL, Wold LE. Investigation of the rognostic and redictive of thymidine synthase, 53 and ki-67 in atients with locally advanced colon cancer. J Clin Oncol. 2002, 20: Lyall MS, Dundas SR, Curran S, Murray GI. Profiling markers of rognosis in colorectal cancer. Clin Cancer Res. 2006, 12 (4): Lin LC, Lee HH, Hwang WS, Li CF, Huang CT, Que J, et al. P53 and 27 as redictors of clinical outcome for rectal-cancer atients receiving neoadjuvant theray. Surg Oncol. 2006, 15 (4): Gill S, Lorinzi CL, Sargent DJ, Thome SD, Alberts SR, Haller DG, et al. Pooled analysis of fluorouracilbased adjuvant theray for stage II and III colon cancer: Who benefits and by how much? J Clin Oncol. 2004, 22: Benson AB III, Schrag D, Somerfield MR. American Society of Clinical Oncology recommendations on adjuvant chemotheray for stage II colon cancer. J Clin Oncol. 2004, 22: Moertel CG, Fleming TR, Macdonald JS, Haller DG, Laurie JA, Tangen CM, et al. Intergrou study of fluorouracil lus levamisole as adjuvant theray for stage II/Dukes' B2 colon cancer. J Clin Oncol. 1995, 13: Wolin BM, Meyerhardt JA, Mamon HJ, Mayer RJ. Adjuvant Treatment of Colorectal Cancer. CA Cancer J Clin. 2007, 57:

12 14 Clinico-Pathological & Prognostic Significance 59- Kallioniemi OP, Wagner U, Kononen J, Sauter G. Tissue microarray technology for high-throughut molecular rofiling of cancer. Human Mol Gen. 2001, 10: Hoos A, Urist M, Stojadinovic A, Mastorides S, Dudas M, Leung D, et al. Validation of tissue microarrays for immunohistochemical rofiling of cancer secimens using the examle of human fibroblastic tumors. Am J Pathol. 2001, 158: Rubin M, Dunn R, Strawderman M, Pienta K. Tissue microarray samling strategy for rostate cancer biomarker analysis. Am J Surg Pathol. 2002, 26: