The aim of the present study was to evaluate by immunohistochemistry the prognostic meaning of the tumor marker MET (hepatocyte growth factor) in patients submitted to surgical resection due to primary colorectal adenocarcinoma.
PATIENTS AND METHODS:
A retrospective study was carried out that included 286 consecutive patients with colorectal adenocarcinoma, submitted to surgical resection at Barretos Cancer Hospital, from 1993 to 2002. The histopathological expression of the MET tumor marker was evaluated using an anti-protein monoclonal antibody against MET by the streptavidin-biotin-peroxidase technique. The expression of the tumor marker was semi-quantitative, and the slide samples were independently analyzed by three pathologists unaware of patient clinical and histopathological data.
RESULTS:
The tumor marker expression was positive in 236 (79%) out of a total of 286 patients. This expression was statistically significantly different between stages I and IV (p=0.004), for overall survival (p=0.009), and for cancer-related mortality rates (p=0.022). However, no association between the tumor marker and recurrence (p=0.89) or disease-free interval (p=0.91) was observed.
CONCLUSION:
MET has shown significant expression at advanced stages of the disease, as well as for overall survival and cancer-related mortality rates demonstrating to be a valuable marker for poor prognosis in colorectal cancer patients

Logullo, Angela Flavio

Longatto Filho, Adhemar

Matos, Delcio

Neto, Ricardo Artigiani

Oliveira, Antônio Talvane Torres

Saad, Sarhan Sydney

Silva, Sandra Regina Morini da

Anticancer Research

The aim of the present study was to evaluate by immunohistochemistry the prognostic meaning of the tumor marker MET (hepatocyte growth factor) in patients submitted to surgical resection due to primary colorectal adenocarcinoma. Patients and Methods: A retrospective study was carried out that included 286 consecutive patients with colorectal adenocarcinoma, submitted to surgical resection at Barretos Cancer Hospital, from 1993 to 2002. The histopathological expression of the MET tumor marker was evaluated using an anti-protein monoclonal antibody against MET by the streptavidin-biotin-peroxidase technique. The expression of the tumor marker was semi-quantitative, and the slide samples were independently analyzed by three pathologists unaware of patient clinical and histopathological data. Results: The tumor marker expression was positive in 236 (79%) out of a total of 286 patients. This expression was statistically significantly different between stages I and IV (p=0.004), for overall survival (p=0.009), and for cancer-related mortality rates (p=0.022). However, no association between the tumor marker and recurrence (p=0.89) or disease-free interval (p=0.91) was observed. Conclusion: MET has shown significant expression at advanced stages of the disease, as well as for overall survival and cancer-related mortality rates demonstrating to be a valuable marker for poor prognosis in colorectal cancer patients

OLIVEIRA, Antonio Talvane Torres De

MATOS, Delcio

LOGULLO, Angela Flavia

SILVA, Sandra Regina Morini Da

ARTIGIANI NETO, Ricardo

LONGAT FILHO, Adhemar

SAAD, Sarhan Sydney

Biblioteca Digital da Produção Intelectual da Universidade de São Paulo (BDPI/USP)

MET Is Highly Expressed in Advanced Stages of Colorectal Cancer and Indicates Worse Prognosis and Mortality

Universidade do Minho: RepositoriUM

Abstract. The aim of the present study was to evaluate byimmunohistochemistry the prognostic meaning of the tumormarker MET (hepatocyte growth factor) in patients submittedto surgical resection due to primary colorectal adenocarcinoma.Patients and Methods: A retrospective study was carried outthat included 286 consecutive patients with colorectal adeno-carcinoma, submitted to surgical resection at Barretos CancerHospital, from 1993 to 2002. The histopathological expressionof the MET tumor marker was evaluated using an anti-proteinmonoclonal antibody against MET by the streptavidin-biotin-peroxidase technique. The expression of the tumor marker wassemi-quantitative, and the slide samples were independentlyanalyzed by three pathologists unaware of patient clinical andhistopathological data. Results: The tumor marker expressionwas positive in 236 (79%) out of a total of 286 patients. Thisexpression was statistically significantly different between stagesI and IV (p=0.004), for overall survival (p=0.009), and forcancer-related mortality rates (p=0.022). However, noassociation between the tumor marker and recurrence (p=0.89)or disease-free interval (p=0.91) was observed. Conclusion:MET has shown significant expression at advanced stages ofthe disease, as well as for overall survival and cancer-relatedmortality rates demonstrating to be a valuable marker for poorprognosis in colorectal cancer patients.Colorectal cancer is the most important cause of cancer deadamong gastrointestinal cancer patients (1). In the UnitedStates of America, 106,680 new colorectal cancer cases wereexpected for 2006 and 41,930 cases of rectal cancer (2).Similarly in Brazil, colorectal cancer is one of the five moreprevalent types of cancer among women and men, mainly inindividuals 50 to 70 years old (3).Several clinical, pathological and epidemiological factorscan influence the patient’s prognosis (4, 5) but the classificationof tumor stage is the most significant parameter used toevaluate clinical behavior. Consequently, the presence of lymphnode metastasis indicates that only 30% of patients survive forfive years, and for those with hepatic metastasis, lifeexpectancy is severely limited. However, tumor stage alone isfar from comprehensive in terms of prognosis which seriouslylimits its use for this purpose. Theoretically, patients staged I orII should demonstrate good prognosis; however, one third ofthese patients die in five years with distant metastasis or localrelapse (6). Conversely, there are patients with veryvoluminous tumoral masses involving contiguous tissues andorgans without lymph node invasion or distant metastasis, andbenevolent clinical behavior (7-10). Consequently, it is crucialto find one or more parameters informative for prognosis.Among a plethora of tumoral markers, the activity ofmesenchymal-epithelial transition factor, a proto-oncogenethat encodes a protein MET, known as MET hepatocytegrowth factor receptor (HGFR), is believed to be particularlyimportant in cancer prognosis. HGF is the unique ligandknown for MET which is involved in carcinogenesis ofseveral types of tumors, invasion, differentiation and tumoralangiogenesis (11-19). Perceptibly, the expression of METprotein is believed to be crucial to determine prognosis. It hasalready been demonstrated that MET can enhance colorectal4807Correspondence to: Antônio Talvane Torres de Oliveira, RuaAntenor Duarte Vilella, 1331, Bairro Dr. Paulo Prata, CEP 14784-400, Hospital de Câncer de Barretos, Fundação Pio XII, Barretos,SP, Brazil. e-mail: netto123@uol.com.brKey Words: Colorectal carcinoma, immunohistochemistry, MET,prognosis, survival, carcinogenesis.ANTICANCER RESEARCH 29: 4807-4812 (2009)MET Is Highly Expressed in Advanced Stages of ColorectalCancer and Indicates Worse Prognosis and MortalityANTÔNIO TALVANE TORRES DE OLIVEIRA1, DELCIO MATOS2, ANGELA FLÁVIA LOGULLO3, SANDRA REGINA MORINI DA SILVA4, RICARDO ARTIGIANI NETO3, ADHEMAR LONGATTO FILHO4,5 and SARHAN SYDNEY SAAD21Departments of Surgery and Pathology of Barretos Cancer Hospital, Pio XII Foundantion, Barretos, São Paulo;Departments of 2Gastroenterology and 3Pathology, School of Medicine, Federal University of São Paulo, São Paulo, Brazil;4Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 5Laboratory of Medical Investigation (LIM) 14, Department of Pathology, University of São Paulo School of Medicine, São Paulo, Brazil0250-7005/2009 $2.00+.40tumoral cell motility, facilitating invasion and metastasis (13).Interestingly, HGF is not detectable in normal hepatic cellsbut its expression is enhanced in hepatocytes neighboring themalignant colorectal tumor cells metastasized to the liver(20). High expression of MET in malignant transformation isalso associated with an augmented capacity for invasion andmetastasis (21). In fact, MET was reported to be expressed inmore than 50% of colorectal lesions from dysplastic adenomato invasive carcinoma, which suggests that MET influenceoccurs from the early stages of malignant disease and ishighly associated with advanced disease (22). Despite its thewell-documented role in the metastatic potential of colorectalcancer (23), MET did not demonstrate any independent valueas a prognostic factor (17). Indeed, the alleged participationof MET amplification in colorectal cancer development is notunequivocal (24), but the majority of investigations havereported data supporting the participation of MET incolorectal cancer. Increased MET expression is frequentlyassociated with concomitant augmentation of HGFexpression, which infers a paracrine effect that optimizes thecellular mass expansion (18). Moreover MET/HGF is moresignificantly expressed in Dukes’ C than Dukes’ B tumors.Both conditions are limited to the organ but differ in lymphnode status, which infers that MET/HGF expression canpredict metastasis (11).The aim of this work was to verify if HGFR was morehighly expressed or not in advanced stages of colorectal cancerand to correlate these findings with prognosis and mortality.Patients and MethodsAll 286 patients enrolled in this study were consecutively examinedand treated by surgery at Barretos Cancer Hospital, in São PauloState, Brazil, between 1993 and 2002 due to colorectaladenocarcinoma. Clinical and pathological data were retrospectivelyobtained from the files of the hospital medical archive. Cases withhistory of any previous cancer treatment were excluded. Generalinformation included size of the tumor, histological classification ofthe tumor, including type and degree of histopathologicaldifferentiation, invasion of colonic wall, lymph node invasion anddistant metastasis, time to recurrence, survival rates and cause ofdeath when death occurred.Immunohistochemistry reaction. Immunohistochemistry wasperformed according to the avidin–biotin–peroxidase complexprinciple (Dako Co., San Diego, CA, USA), using a primaryantibody raised against MET oncoprotein (HGTR), NCL-MET(Dako Co.) diluted at 1:1500. Briefly, deparaffinized and rehydrated sections were immersed inEDTA (pH 8.0), heated to 98˚C in a water-bath for 15 minutes andwashed in phosphate-buffered saline (PBS). Endogenousperoxidases were then inactivated using 3% hydrogen peroxide inmethanol for 10 minutes, followed by washing in PBS. Tissuesections were then incubated with blocking solution for 10 minutesand incubated at room temperature with the primary antibody for 2hours. Sections were then sequentially washed in PBS and incubatedwith biotinylated goat anti-polyvalent antibody for 10 minutes,streptavidin peroxidase for 10 minutes, and developed with 3,3’-diaminobenzidine (DAB+ Substrate System; Dako, Carpinteria, CA,USA) for 10 minutes. Tissue sections were counterstained withhematoxylin and permanently mounted.Immunohistochemical evaluation. The immunoreaction was scoredsemi-quantitatively according to Wielenga et al. (25): the reactionswere considered augmented when ≥50% of the malignant cells werepositively stained; (Figure 1) and diminished when the positive cellswere between >10% to <50% (Figure 2). Cases were considerednegative for MET when ≤10% of cells gave positive reactions(Figure 3).Evaluation of MET immunohistochemical expression wasperformed blindly by two independent observers and discordantresults were reassessed using in a double-head microscope and afinal score was agreed.Statistical analysis. Data were stored and analyzed using SPSSstatistical software (version 14.0; SPSS Inc., Chicago, IL, USA).The comparison of MET expression between tumor and normalcells, as well as the relationship between MET expressions and theclinicopathological parameters, were examined for statisticalsignificance using Pearson’s chi-square (χ2) test, using a thresholdfor significance of p-values <0.05. Survival curves were plottedusing the method of Kaplan and Meier and data compared using thelog-rank test.Ethics. The study was approved by the local Committees of Ethicson Research (No. 0436/04).ResultsA total of 286 patients were enrolled in the study, with meanage of 63 years (ranged from 28 to 93): 154 (50.3%) maleand 142 (49.7%) female. Adenocarcinoma without otherspecification was the predominant histopathological type with251 (88%) cases, followed by 24 (8%) mucinous type, 9 (3%)tubular type and 2 (1%) squamous adenocarcinoma. In 210cases (73%), the colonic wall had been invaded and the tumorreached the serosal membrane or adjacent structures (T4); in43 (15%), the tumor had invaded the subserosal zone (T3);in 28 (9%) the muscular propria, and in 5 (2%) cases thetumor was restricted to the submucosal (T1). There were 195(68%) cases exhibiting lymph node invasion and 91 (32%)cases without lymph node invasion. Distant metastases werenot observed in 217 patients (76%) at first examination and69 (24%) patients already showed signs of metastatic disease.TNM classification showed 40 (14%) stage I, 110 (39%)stage II; 67 (23%) stage III and 69 (24%) stage IV tumors. MET reaction was positive in 236 (79%) and negative in50 (21%) cases. The expression of MET according tumorstage is depicted in Table I. A statistically significantdifference (p=0.004) in MET expression between stages wasobserved, with stage IV associated with augmented and stageI with diminished MET expression. Table II shows thecomparison between MET expression and recurrence. StageANTICANCER RESEARCH 29: 4807-4812 (2009)4808IV patients and patients lost from follow-up were excludedfrom this analysis, but no significant difference was found.Figure 4a shows the Kaplan-Meier survival curves and bylog-rank test, no statistical difference was observed amongthe three groups (p=0.96), which implies that the disease-free interval is not associated with MET expression. Figure 4b shows the curve of general survival ratesaccording to MET expression. Interestingly, longer survivalrates (p=0.009) were observed for the MET diminishedgroup. The overall correlation of MET and mortality is givenin Table III, which shows that 49.3% of the patients withaugmented expression of MET were dead by the end of thisstudy, whilst the majority of the MET-negative group werenot (χ2=14.82; p=0.022).DiscussionWe sought to investigate MET expression as a possible tool toindicate better or worse prognosis in cases of colorectal cancer.As previously mentioned there is a progressive augmentation ofMET expression in the early stages of adenoma/carcinomatransformation, but this pattern is not useful for predictingDe Oliveira et al: MET and Colorectal Cancer4809Figure 1. Highly augmented MET expression (×100).Figure 4. Recurrence-free (a) and overall (b) survival according to METexpression. DE, Diminished expression; AE, augmented expression.Log-rank test a, p=0.96; b, p=0.009.Figure 2. Diminished MET expression (×100).Figure 3. Negative case, without MET expression (×100).recurrence or the disease-free interval. For this reason, TNMclassification is still believed to be a better option in evaluatingprognosis. However, we observed that MET was proportionallyless expressed in incipient carcinomas than in advanced stagesof colorectal cancer. Our data are in part corroborated by somedata already published where MET was indeed demonstrated tobe a good marker for predicting the metastatic potential ofcolorectal tumors (11, 26). Despite its utility in demonstratingtumor aggressiveness, there was no valid indication for anyassociation between MET expression and survival rates (27).Conversely, the data obtained herein significantly demonstratedthat the patients with diminished expression of MET had greatersurvival rates when compared with the group of patients withhigh expression of MET, which endorses the basis of our studythat highly expressed MET is more commonly related to worseprognosis; however, this assumption is not consensual (27).Moreover, we found that MET correlated to TNM stages with aperceptible and progressive increase of MET expression fromstage I to stage IV (22, 23, 26). Additionally, we opted forimmunohistochemical evaluation of MET because this methodis reproducible and easily applicable in routine of pathology. Despite controversies, we demonstrate that immunohisto-chemical expression of MET is useful in colorectal cancerprognostic evaluation.References1 Landis SH, Murray T, Bolden S and Wingo PA: Cancer statistics,1998. CA Cancer J Clin 48: 4-12, 1998.2 American Cancer Society. Cancer statistics 2006. Georgia:American Cancer Society, 2006. Available at: http://www.cancer.org/downloads/STT/Cancer_Statistics_2006_Presentation.ppt#256,1,Cancer Statistics 2006. Accessed 15 march 2006.3 Instituto Nacional do Câncer. Estimativas de câncer no Brasil2006. Rio de Janeiro: INCA, 2006. Available at: http://www.inca.gov.br/estimativa/2006/ Accessed 20 February 2006.4 Hermanek P, Wiebelt H, Staimmer D and Riedl S: Prognosticfactors of rectum carcinoma: experience of the GermanMulticentre study SGCRC. German Study Group ColorectalCarcinoma Tumori 81: 60-64, 1995.5 Benson AB 3rd. New approaches to assessing and treating early-stage colon and rectal cancers: cooperative group strategies forassessing optimal approaches in early-stage disease. Clin CancerRes 13: 6913s-20s, 2007.6 Frati L and Codacci-Pisanelli G: Chemotherapy and immuno-therapy in metastatic colorectal cancer. N Engl J Med 360: 2134-2135, 2009.7 Vieira RAC. Prognostic factors related to the patients submittedto colon cancer surgery. Master Thesis – Faculty of Medicine,São Paulo University,  1999.8 Zhao DB, Wu YK, Shao YF, Wang CF and Cai JQ: Prognosticfactors for 5-year survival after local excision of rectal cancer.World J Gastroenterol 15: 1242-1245, 2009.9 Ivanov K, Kolev N, Tonev A, Nikolova G, Krasnaliev I, SoftovaE and Tonchev A: Comparative analysis of prognosticsignificance of molecular markers of apoptosis with clinicalstage and tumor differentiation in patients with colorectalcancer: a single institute experience. Hepatogastroenterol 56:94-98, 2009.10 Golfinopoulos V, Salanti G, Pavlidis N and Ioannidis JP: Survivaland disease-progression benefits with treatment regimens foradvanced colorectal cancer: a meta-analysis. Lancet Oncol 8:898-911, 2007.ANTICANCER RESEARCH 29: 4807-4812 (2009)4810Table I. MET expression distributed according to clinical stage ofcolorectal cancer.Expression of METAugmented Diminished Negative TotalStage No. % No. % No. % No. %I 12 8.5 22 23.4 6 12.0 40 14.0II 50 35.2 41 43.6 19 38.0 110 38.5III 35 24.6 19 20.2 13 26.0 67 23.4IV 45 31.7 12 12.8 12 24.0 69 24.1Total 142 100.0 94 100.0 50 100.0 286 100.0p=0.004Table II. MET immunohistochemical expression according to therecurrence of colorectal cancer.Expression of METAugmented Diminished Negative TotalRecurrence No. % No. % No. % No. %Absent 82  84.6 72  87.8 33  86.8 187  86.2Present 15  15.4 10  12.2 5 13.2 30 13.8Total 97 100.0 82  100.0 38 100.0 217 100.0p=0.89Table III. MET immunohistochemical expression according to mortalitycaused by colorectal cancer.Expression of METMortality Augmented Diminished NegativeNo.  % No.   % No.  %No 72  50.7 68   72.3 31  62.0Yes 70  49.3 26   27.7 19  38.0Total 142  100.0 94  100.0 50 100.0p=0.003 11 Fazekas K, Csuka O, Köves I, Rásó E and Tímár J:Experimental and clinicopathologic studies on the function ofthe HGF receptor in human colon cancer metastasis. Clin ExpMetastasis 18: 639-649, 2000.12 Herynk MH, Tsan R, Radinsky R and Gallick GE: Activation ofc-MET in colorectal carcinoma cells leads to constitutiveassossiation of tyrosine-phosphorylated beta-catenin. Clin ExpMetastasis 20: 291-300, 2003.13 Jiang WG, Lloyds D, Puntis MC, Nakamura T and Hallett MB:Regulation of spreading and growth of colon cancer cells byhepatocyte growth factor. Clin Exp Metastasis 11: 235-242,1993.14 Kataoka H, Hamasuna R, Itoh H, Kitamura N and Koono M:Activation of hepatocyte growth factor/scatter factor incolorectal carcinoma. Cancer Res 60: 6148-6150, 2000.15 Kermorgant S, Aparicio T, Dessirier V, Lewin MJ and Lehy T:Hepatocyte growth factor induces colonic cancer cellinvasiveness via enhanced motility and protease overproduction:evidence for Pl3 kinase and PKC involvement. Carcinogenesis22: 1035-1042, 2001.16 Nabeshima K, Shimao Y, Inoue T, Itoh H, Kataoka H and KoonoM: Hepatocyte growth factor/scatter factor induces not onlyscattering but also cohort migration of human colorectal-adenocarcinoma cells. Int J Cancer 78: 750-759, 1998.17 Hiscox SE, Hallett MB, Puntis MC, Nakamura T and Jiang WG:Expression of the HGF/SF receptor, c-MET, and its ligand inhuman colorectal cancers. Cancer Invest 15: 513-521, 1997.18 Otte JM, Schmitz F, Kiehne K, Stechele HU, Banasiewicz T,Krokowicz P, Nakamura T, Fölsch UR and Herzig K: Functionalexpression of HGF and its receptor in human colorectal cancer.Digestion 61: 237-246, 2000.19 Tsarfaty I, Resau JH, Rulong S, Keydar I, Faletto DL and VandeWoude GF: The met proto-oncogene receptor and lumenformation. Science 257: 1258-1261, 1992.20 Yoshinaga Y, Matsuno Y, Fujita S, Nakamura T, Kikuchi M,Shimosato Y and Hirohashi S: Immunohistochemical detectionof hepatocyte growth factor/scatter factor in human cancerousand inflammatory lesions of various organs. Jpn J Cancer Res84: 1150-1158, 1993.21 Baldus SE, Kort EJ, Schirmacher P, Dienes HP and Resau JH:Quantification of MET and hepatocyte growth factor/scatterfactor expression in colorectal adenomas, carcinomas and non-neoplastic epithelia by quantitative laser scanning microscopy.Int J Oncol 31: 199-204, 2007.22 Zeng ZS, Weiser MR, Kuntz E, Chen CT, Khan SA, Forslund A,Nash GM, Gimbel M, Yamaguchi Y, Culliford AT 4th, D’AlessioM, Barany F and Paty PB: c-Met gene amplification isassociated with advanced stage colorectal cancer and livermetastases. Cancer Lett 265: 258-269, 2008.23 Fujita S and Sugano K: Expression of c-MET proto-oncogene inprimary colorectal cancer and liver metastases. Jpn J Clin Oncol27: 378-383, 1997.24 Umeki K, Shiota G and Kawasaki H: Clinical signifiance of c-MET oncogene alterations in human colorectal cancer. Oncology56: 314-321, 1999.25 Wielenga VJ, van der Voort R, Taher TE, Smit L, Beuling EA,van Krimpen C, Spaargaren M and Pals ST: Expression of c-METand heparan-sulfate proteoglycan forms of CD44 in colorrectalcancer. Am J Pathol 157: 563-573, 2000. 26 Takeuchi H, Bilchik A, Saha S, Turner R, Wiese D, Tanaka M,Kuo C, Wang HJ and Hoon DS: c-MET expression level inprimary colon cancer: a predictor of tumor invasion and lymphnode metastases. Clin Cancer Res 9: 1480-1488, 2003.27 Resnick MB, Routhier J, Konkin T, Sabo E and Pricolo VE:Epidermal growth factor receptor, c-MET, β-catenin, and p53expression as prognostic indicators in stage II colon cancer: Atissue microarray study. Clin Cancer Res 10: 3069-3075, 2004.Received June 4, 2009Revised September 14, 2009Accepted September 22, 2009De Oliveira et al: MET and Colorectal Cancer4811

MET Is highly expressed in advanced stages of colorectal cancer and indicates worse prognosis and mortality

RCAAP - Repositório Científico de Acesso Aberto de Portugal

Universidade de São Paulo

http://repositorium.sdum.uminho.pt/bitstream/1822/30089/1/oliveira%20att_%20anticancer%20res%202009.pdf

MET Is highly expressed in advanced stages of colorectal cancer and indicates worse prognosis and mortality

Abstract

Similar works

Full text

Available Versions

Biblioteca Digital da Produção Intelectual da Universidade de São Paulo (BDPI/USP)

Universidade do Minho: RepositoriUM

Universidade do Minho: RepositoriUM

RCAAP - Repositório Científico de Acesso Aberto de Portugal

Universidade de São Paulo