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Met expression is an independent prognostic risk factor in patients with oesophageal adenocarcinoma

Met expression is an independent prognostic risk factor in patients with oesophageal adenocarcinoma Molecular Diagnostics British Journal of Cancer (2008) 98, 1102 – 1108 & 2008 Cancer Research UK All rights reserved 0007 – 0920/08 $30.00 www.bjcancer.com Met expression is an independent prognostic risk factor in patients with oesophageal adenocarcinoma ,1,2 1 3 2 1,4 JB Tuynman , SM Lagarde , FJW ten Kate , DJ Richel and JJB van Lanschot 1 2 Department of Surgery, Academic Medical Centre at the University of Amsterdam, Amsterdam, The Netherlands; Department of Medical Oncology, Academic Medical Centre at the University of Amsterdam, Amsterdam, The Netherlands; Department of Pathology, Academic Medical Centre at the University of Amsterdam, Amsterdam, The Netherlands; Department of Surgery, Erasmus Medical Centre, Rotterdam, The Netherlands Oesophageal adenocarcinoma is an aggressive malignancy with propensity for early lymphatic and haematogenous dissemination. Since conventional TNM staging does not provide accurate prognostic information, novel molecular prognostic markers and potential therapeutic targets are subject of intense research. The aim of the present study was to study the prognostic significance of Met, the hepatic growth factor (HGF) receptor and a possible target for therapy in comparison to cyclooxygenase-2 (COX-2). Tumour sections from 145 consecutive patients undergoing intentionally curative surgery for oesophageal adenocarcinoma were immunohistochemically analysed for Met and COX-2 expression. Clinicopathological data were prospectively collected for all patients. Patients with high Met expression had significantly reduced overall and disease-specific 5-year survival rates (Pp0.001 and Pp0.001, respectively) and were more likely to develop distant metastases (P¼ 0.002) and local recurrences (P¼ 0.004) compared to patients with low Met expression. High COX-2 expression tended to be correlated with poor long-term survival but this did not reach statistical significance. Expression of Met was recognised as a significant and independent prognostic factor by stage-specific analysis and multivariate analysis (relative risk¼ 2.3; 95% CI¼ 1.3–4.1). These findings support the importance of Met in oesophageal adenocarcinoma and support the concept of Met tyrosine kinase inhibition as (neo-) adjuvant treatment. British Journal of Cancer (2008) 98, 1102–1108. doi:10.1038/sj.bjc.6604251 www.bjcancer.com Published online 18 March 2008 & 2008 Cancer Research UK Keywords: oesophageal adenocarcinoma; Met; HGF receptor; COX-2; prognosis Oesophageal adenocarcinoma (OA) is a highly aggressive malig- and haematogenous dissemination are still poorly understood nancy with early lymphatic and haematogenous dissemination. (Lagarde et al, 2006). Identification of growth factor receptors with The incidence of OA is increasing rapidly in the Western World tyrosine kinase activity, highly expressed in advanced cancer, has (Enzinger and Mayer, 2003). Despite advances in diagnosis and been shown to provide both prognostic information and potential treatment of the disease, even after potentially curative surgery the molecular targets for (neo-) adjuvant therapy (Krause and Van overall 5-year survival rate rarely exceeds 35% (Hulscher et al, Etten, 2005). A promising development in cancer therapy is the 2002; Cunningham et al, 2006). Subgroup analysis in early-stage combination of surgery with potent selective growth factor tumours have shown good survival rates although even early-stage receptor inhibitors as (neo-) adjuvant therapy resulting in tumours show early lymphatic dissemination. improved overall and disease-specific survival (Verweij et al, For adenocarcinoma of the oesophagus the most important 2004; Krause and Van Etten, 2005; Gold and Dematteo, 2006; conventional prognostic factors are summarised in the pTNM Motzer et al, 2007; Smith et al, 2007). Therapeutic usage of small stage of the oesophagus. Also other pathological aspects such as molecules selectively inhibiting c-KIT, a growth factor receptor extracapsular lymph node involvement and (relative) number of present in gastrointestinal stromal cell tumours (GIST), has positive nodes have prognostic impact. However, these conven- resulted in remarkable responses and has enhanced prognosis tional prognostic factors have limited accuracy (Lagarde et al, for patients with GIST to a great extent (Gold and Dematteo, 2006). 2006). Therefore, molecular prognostic markers, which can serve Other examples of targeted (neo-) adjuvant therapy are the as targets for therapy are subject of intense research. For OA only inhibition of vascular endothelial growth factor receptor (VEGFr) few molecular prognostic factors have been identified and in patients with advanced colorectal cancer, the inhibition of molecular events responsible for the development of lymphatic HER2-Neu, an epidermal growth factor receptor in patients with breast cancer and the inhibition of both VEGFr and platelet- derived growth factor receptor (PDGFr) in patients with renal cell *Correspondence: Dr JB Tuynman, Department of Surgery, Academic carcinoma (Hurwitz et al, 2004; Verweij et al, 2004; Geyer et al, Medical Centre, PO Box 22660, Meibergdreef 9, Amsterdam 1100DD, 2006; Motzer et al, 2007). The Netherlands; E-mail: J.B.Tuynman@amc.uva.nl Growth factor receptors have been identified in OA and some Received 28 August 2007; revised 7 January 2008; accepted 15 January show higher expression in later stages of cancer development 2008; published online 18 March 2008 (Lagarde et al, 2006; Vallbohmer et al, 2006). However, the Met expression identifies poor survival in oesophageal cancer JB Tuynman et al prognostic significance of growth factors expressed in OA has only histologically proven Barrett’s segment (defined by the presence been investigated in relatively small patient cohorts and no of goblet cells). Patients with an adenocarcinoma of the cardia or significance in multivariate analysis was demonstrated so far. The gastro-oesophageal junction without a clear Barrett’s segment were only independent molecular prognostic factor demonstrated for excluded (n¼ 161). This careful selection of patients has been OA is cyclooxygenase-2 (COX-2) expression as published by our described in our previous report (Buskens et al, 2002). Archival group (Buskens et al, 2002; Lagarde et al, 2006). Recently, we have materials of the remaining 145 patients were re-evaluated to obtain reported a clinical study in which neo-adjuvant selective COX-2 the sample with deepest invasion of each tumour. inhibition downregulates Met expression in conjunction with COX-2 expression in patients with OA (Tuynman et al, 2005). Met Surgical tissue specimens is the hepatocyte growth factor (HGF) receptor and is identified in OA (Tuynman et al, 2005; Anderson et al, 2006). Overexpression of All 145 patients were treated with subtotal oesophagectomy and Met and/or its ligands has been shown to contribute to progression resection of the lesser curvature of the stomach. Between April and dissemination of several malignancies including lung, colo- 1994 and February 2000, 96 patients (66%) were randomly rectal, gastric, breast, prostate, thyroid, pancreas, and oesophageal assigned to either transhiatal or transthoracic oesophagectomy as cancer (Hu et al, 2001; Saeki et al, 2002; Christensen et al, 2003; part of a randomized trial comparing both techniques (Hulscher Murai et al, 2004; Anderson et al, 2006). In experimental models, et al, 2002). In the remaining 49 patients, a standard transhiatal activation of Met (endogenously by mutations in its tyrosine procedure was performed. In 95 patients (65.5%), resection was kinase domain, or exogenously by HGF and prostaglandins performed by a transhiatal approach without thoracotomy. produced by COX-2) causes decreased apoptosis and enhanced Lymphadenectomy comprised en bloc removal of all lymphatic proliferation, angiogenesis, and invasion (Shinomiya et al, 2004; tissue in the lower posterior mediastinum, along the cardia and the Herrera et al, 2005; Boccaccio and Comoglio, 2006). Thus, COX-2 lesser curvature of the stomach. Fifty patients (34.5%) underwent and Met seem functionally connected. In cancer development, oesophagectomy through a right-sided thoracotomy followed by a COX-2 is present in early stages of dysplasia, initiating cancer laparotomy in combination with two-field lymph node dissection. growth and progression whereas Met is an important key regulator This procedure included an abdominal lymphadenectomy as of molecular processes in later stages of cancer development and described plus the removal of lymph nodes along the common progression (Tuynman et al, 2004). Small molecules selectively hepatic artery, the splenic artery, and the coeliac trunk as well as inhibiting Met have been shown to inhibit dissemination and an extended lymph node dissection in the chest (i.e., including the cancer growth both in vitro as in animal studies (Christensen et al, right paratracheal, infra-aortic arch, and subcarinal lymph nodes). 2003; Kim et al, 2003; Hov et al, 2004; Herrera et al, 2005; Martens et al, 2006; Watson et al, 2006). Consequently, inhibition of Met as Immunohistochemistry (neo-) adjuvant therapy for OA seems a promising strategy. A relation between Met expression and stage of disease has been Of all patients 5-mm thick sections of paraffin and formaldehyde- described previously (Anderson et al, 2006). However, the fixed tissue of the resection specimens were cut. For immuno- potential value of Met expression in OA as an independent histochemical staining, sections were incubated overnight at 371C prognosticator calculated by multivariate analysis has not yet been and subsequently deparaffinised in xylene, rehydrated, and treated addressed in a large consecutive cohort. Therefore, the aim of the with 3% H O in methanol for 10 min to block endogenous 2 2 present study was to characterize further the prognostic signifi- peroxidase activity. All specimens were subjected to heat-induced cance of Met expression in a large consecutive cohort of patients antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for with OA. 10 min at 951C. To block aspecific binding the slides were incubated with Tris-buffered saline (TBS) supplemented with 5% goat serum. Sections were incubated with the primary antibodies anti-Met c-Met (3D4; Zymed, San Francisco, CA, USA) (1 : 100), PATIENTS AND METHODS and anti-human COX-2 (160112; Cayman Chemical Co., Ann Arbor, MI, USA) (1 : 200) diluted in TBS with 1% bovine serum Patients albumin overnight at 41C. For the Met staining the sections were A consecutive series of 306 patients who underwent potentially incubated after washing steps with anti-mouse/rabbit-peroxidase curative oesophagectomy at the Department of Surgery of the polymer for 30 min at room temperature (Powervision; Immuno- Academic Medical Centre at the University of Amsterdam, The vision Inc., Daly City, CA, USA). Diaminobenzidine chromogen Netherlands for adenocarcinoma of the mid/distal oesophagus (Sigma, St Louis, MO, USA) was used for visualisation. For the between January 1993 and December 2000 was selected. Pre- COX-2 staining the sections were treated with biotinylated horse operative workup included endoscopy with histological biopsy, anti-mouse immunoglobulin (1 : 200; Vector Laboratories Inc., external ultrasonography of the abdomen and neck, CT scan of the Burlingame, CA, USA) and avidin–biotin peroxidase complex abdomen and chest, radiography of the chest, oesophageal (Vectastain ABComplex; Vector Laboratories). After these steps for endosonography, and indirect laryngoscopy. Lymph node metas- Met and COX-2 staining the sections were counterstained with tases at the coeliac trunk were a contraindication for resection only haematoxylin and embedded. Specificity of the antibodies was when considered non-resectable (i.e., larger than 2 cm in diameter) confirmed by controls using irrelevant immunoglobulins instead and confirmed by cytological puncture. Patients did not receive of primary antibodies. Colon cancer tissue was included as a additional (neo-) adjuvant chemotherapy and/or radiotherapy. positive control. Clinicopathological data from all operated patients were perma- nently prospectively collected. Follow-up was complete for all Scoring patients and extended until July 2006, ensuring a minimal potential follow-up of 5.5 years. Recurrence of disease was diagnosed on Met and COX-2 immunohistochemical staining were scored clinical grounds. However, whenever a relapse was suspected, semiquantitatively using a four-step scale as used and validated radiologic, endoscopic, or histological confirmation was sought. in previous reports (Buskens et al, 2002; Tuynman et al, 2005). The Recurrent disease was classified as locoregional (occurring in the following scoring criteria of tumour cells were agreed upon before upper abdomen or mediastinum) or distant (including cervical the analysis: (0) no staining or equal to background; (1) weak recurrences). All pathology reports were reviewed to identify those diffuse cytoplasmic staining (may contain stronger intensity in less patients in whom the adenocarcinoma had developed in a than 10% of cancer cells); (2) moderate granular cytoplasmic & 2008 Cancer Research UK British Journal of Cancer (2008) 98(6), 1102 – 1108 Molecular Diagnostics Molecular Diagnostics Met expression identifies poor survival in oesophageal cancer JB Tuynman et al Table 1 Correlation of clinicopathological findings and Met expression Patient Low Met High Met characteristics expression expression (n¼ 145) Overall (N¼ 67) (N¼ 78) P-value Median age (range) 67 (35 – 85) 67 (35 – 83) 68 (44 – 85) 0.493 Str Sex Male (%) 120 (83%) 63 (94%) 57 (73%) 0.435 Tumour characteristics T stage 0.017 T1 44 (30%) 29 (43%) 15 (19%) Tu T2 18 (12%) 8 (12%) 10 (13%) T3 83 (57%) 30 (45%) 53 (68%) N stage p0.001 N0 65 (45%) 38 (57%) 27 (35%) N1 80 (55%) 29 (43%) 51 (65%) M stage 0.086 M0 122 (84%) 58 (87%) 64 (82%) M1a 23 (16%) 9 (13%) 14 (18%) Differentiation grade 0.078 Good 11 (8%) 5 (7%) 6 (8%) Moderate 56 (39%) 35 (52%) 21 (27%) Poor 78 (54%) 27 (40%) 51 (65%) Overall 5-year 35% 57% 16% p0.001 survival Disease-specific 5- 48% 66% 33% p0.001 Str year survival T1, tumour limited to (sub)mucosa; T2, tumour infiltrates muscularis propia; T3, tumour infiltrates adventitia layer; as determined in the pathological resection specimens. N0, no tumour positive locoregional lymph nodes; N1, locoregional lymph node metastasis. M0, no distant metastasis, M1a, metastasis in coeliac lymph Tu nodes. Figure 1 Representive samples of immunohistochemical staining of Met causes. The Cox proportional hazards regression model was used (A) and COX-2 (B) (Str¼ stroma, T¼ tumour). to identify prognostic factors. To identify independent prognostic factors multivariate Cox regression analysis was carried out. Variables with multiple categories were recoded into dichotomous staining in 10–90% of cancer cells; (3) over 90% of tumour cells variables by combining categories with a comparable prognosis stained with strong intensity (Figure 1). (differentiation grade, good vs moderate and poor (poor); tumour T stage, stage 1 and 2 vs 3; Met expression, no or weak staining The analysis of all tissue sections was performed independently by three different investigators (JBT, SML, and FJWTK) without (low) vs moderate to strong staining (high); COX-2 expression, no patient identification parameters to correct for observer accuracy. or weak staining (low) vs moderate to strong staining (high). The semiquantitative scoring by the investigators had a low observer variation; 92% of the specimens were categorized identically. In cases of disagreement (n¼ 6 for COX-2 expression RESULTS and N¼ 13 for Met expression) consensus was reached after re- evaluation by the investigators using a multiheaded microscope. A total of 145 consecutive patients with OA were included for Data regarding COX-2 staining intensity are equal as previously immunohistochemical analysis. Of these patients 120 were men described and used for the present analysis. Areas of diffuse (83%) and 25 were women (17%) with a median age of 67 years haemorrhage or necrosis were neglected. (range¼ 35–85) (Table 1). The majority of patients (N¼ 83, 57%) had a T3 tumour and 80 patients (55%) had positive lymph nodes. The overall 5-year survival in the included group was 35% and the Statistics disease-specific 5-year survival was 48%. Two patients (1.4%) died Statistical calculations were performed using SPSS version 14.0 within 90 days due to post-operative complications (myocardial (Statistical Package for the Social Sciences, Chicago, IL, USA). The and respiratory failure in one patient and cerebrovascular event in association between demographic and clinicopathological features another one patient). and protein expression was analysed using Student’s t-test High Met staining (as opposed to low Met staining) was (continuous data) and w -test (categorical data). Overall and observed in 78 cases (54%). Of these 78 patients, 28 cases were disease-specific 5-year survival rates were estimated according to scored as strong Met expression and 50 as moderate Met the Kaplan–Meier method and compared between groups using expression. In 67 patients (46%) Met expression was classified as the log-rank test. Overall survival was calculated using deaths since low; 56 patients had weak Met expression and 11 patients had no time of surgery irrespective of cause. For disease-specific survival or equal to background staining of Met. Met expression was all non-disease-related deaths were excluded including in-hospital mainly localised in neoplastic cells (Figure 1A and B) but was also death within 90 days of surgery, since we assumed that these weakly identified in non-neoplastic epithelial cells (both squamous patients had died because of comorbidity and surgery-related and columnar epithelium) and in stromal cells. Interobserver British Journal of Cancer (2008) 98(6), 1102 – 1108 & 2008 Cancer Research UK Met expression identifies poor survival in oesophageal cancer JB Tuynman et al 1.0 1.0 P0.001 P0.001 0.8 0.8 Met low Met low 0.6 0.6 0.4 0.4 Met high Met high 0.2 0.2 0.0 0.0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Overall survival in months Disease-specific survival in months 1.0 1.0 P=0.238 P=0.180 0.8 0.8 COX-2 low 0.6 COX-2 low 0.6 COX-2 high COX-2 high 0.4 0.4 0.2 0.2 0.0 0.0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Overall survival in months Disease-specific survival in months Figure 2 Kaplan –Meier survival curves of 145 patients with adenocarcinoma of the oesophagus. Patients with high Met expression had a significantly worse overall 5-year survival. (A)(Pp0.001) and disease-specific 5-year survival (Pp0.001) (B) as compared to patients with low Met expression. Overall 5-year survival and disease-specific 5-year survival tended to be worse in patients with high COX-2 expression (C and D, respectively) as compared to patients with low COX-2 expression but this did not reach statistical significance (P¼ 0.180 and P¼ 0.238, respectively). variation was 8% for Met expression. All specimens that were Table 2 Results of univariate analysis of clinical, pathological and discrepant (n¼ 13) were re-evaluated and the consensus score was immunohistochemical parameters related to disease-specific 5-year survival used for further analysis. Results of COX-2 expression have been Odds ratio described previously in this cohort of patients. Briefly, COX-2 (confidence interval) P-value expression was negative to weak in 21% (COX-2 low) and moderate to strong in 79% (COX-2 high) of the carcinomas Patient sex (male vs female) 1.2 (0.7 – 2.3) 0.435 (Buskens et al, 2002; Tuynman et al, 2005). High Met expression was observed more often in patients with Patient ASA classification 1.0 (0.5 – 2.0) 0.974 higher T stage (P¼ 0.003), in patients with positive lymph nodes (0 and 1 vs 2or3) (Pp0.001) and a poor differentiation grade (P¼ 0.003) (Table 1). Met expression was not correlated with COX-2 expression Patient age (70 and higher vs 1.1 (0.5 – 2.2) 0.283 lower than 70 years) (P¼ 0.839). During 5-year follow-up, 92 patients died: 17 patients died of Tumour T stage (3 vs 1 and 2) 4.1 (2.4 – 7.2) 0.001 unrelated causes and 75 patients died of recurrent disease. Of these patients, 23 had locoregional recurrences, 39 patients had Tumour N stage (1 vs 0) 4.9 (2.8 – 8.6) 0.000 haematogenous recurrences and 13 patients had both locoregional tumour M1a stage (1 vs 0) 3.7 (2.2 – 6.5) 0.035 and haematogenous recurrences. After a complete follow-up, overall 5-year survival was Differentiation grade 2.2 (1.3 – 3.6) 0.015 significantly lower in patients with high Met expression as (moderate and poor vs good) compared to patients with low Met expression; 16 vs 57% Met expression (high vs low) 3.5 (2.0 – 5.9) 0.000 (Pp0.001). Furthermore, disease specific 5-year survival was significantly lower in patients with high Met expression as COX-2 expression (high vs low) 1.4 (0.8 – 2.6) 0.234 compared to patients with low Met expression; 33 vs 66% (Pp0.001). Patients with high Met expression were more likely to develop distant metastases (P¼ 0.002) as well as local recurrences (P¼ 0.004). Patients with high COX-2 expression (Figure 2). Univariate analysis revealed that T stage, N stage, M1a tended to have a poor overall and disease-specific 5-year survival stage, differentiation grade, and Met expression were all significant as compared to patients with low COX-2 expression but in contrast prognostic indicators for disease-specific 5-year survival (Table 2). to previous reports this did not reach statistical significance Multivariate analysis of these variables demonstrated that T3 stage & 2008 Cancer Research UK British Journal of Cancer (2008) 98(6), 1102 – 1108 Probability of overall survival Probability of overall survival Probability of disease-specific survival Probability of disease-specific survival Molecular Diagnostics Molecular Diagnostics Met expression identifies poor survival in oesophageal cancer JB Tuynman et al (relative risk (RR)¼ 1.9, (95% confidence interval (95% CI¼ 1.0– high Met expression as compared to patients with low Met 3.5)), (P¼ 0.035)), lymph node involvement (RR¼ 2.8, (95% expression (P¼ 0.007 and Pp0.001, respectively) (Figure 3). CI¼ 1.5–5.3), (P¼ 0.001)) and high Met expression (RR¼ 2.3, Patients with stage 3 disease and high Met expression tended to (95% CI¼ 1.3–4.1), (P¼ 0.004)) were independent prognostic have poor overall 5-year survival as compared to patients with low factors (Table 3). Met expression but no statistical significance was reached Subgroup analysis in patients with Stages 1 and 2 OA revealed (P¼ 0.064). In contrast to patients with stage 1 and 2, in patients that overall 5-year survival was significantly lower in patients with with stage 4 OA the Met expression level did not discriminate poor vs better overall 5-year survival. Table 3 Results of multivariate analysis of pathological and immunohis- tochemical parameters related to disease-specific 5-year survival according DISCUSSION to the Cox regression model This study provides evidence that Met expression level (as detected Relative risk by immunohistochemical analysis) is an independent prognostic (confidence interval) P-value factor in OA. Overall 5-year survival after potentially curative resection is significantly worse in patients with tumours expressing T stage (3 vs 1 and 2) 1.9 (1.0 – 3.5) 0.035 high Met levels compared to low Met levels. In literature, lymphatic dissemination as identified on histo- N stage (1 vs 0) 2.8 (1.5 – 5.3) p0.001 pathological examination is the single most important prognostic M1a stage (1a vs 0) 1.8 (0.9 – 3.4) 0.056 factor in patients with oesophageal cancer (Lagarde et al, 2006). Also in the present study, lymph node involvement is a strong Tumour differentiation grade 1.6 (0.9 – 2.7) 0.077 independent prognostic factor next to T stage and Met expression (moderate and poor vs good) level. Since Met expression was correlated stage of disease the subgroup analysis revealed that especially in stage 1 and 2 OA Met Met expression (high vs low) 2.3 (1.3 – 4.1) 0.004 expression is a significant and valuable prognostic factor. In stage Stage 1 Stage 2 P=0.007 1.0 P0.001 1.0 Met low 0.8 0.8 0.6 0.6 Met low Met high 0.4 0.4 0.2 0.2 Met high 0.0 0.0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Overall survival in months Overall survival in months Stage 4 Stage 3 1.0 1.0 P =0.061 P=0.915 0.8 0.8 0.6 0.6 0.4 0.4 Met low 0.2 0.2 Met low Met high 0.0 0.0 Met high 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Overall survival in months Overall survival in months Figure 3 Stage specific Kaplan – Meier survival curves for high vs low Met expression. Patients with stage 1 (T1, N0, and M0) (A) and stage 2 (T2, 3, N0, M0 or T1, N1, M0) (B) and high Met expression had a significantly worse overall 5-year survival as compared to patients with stage 1 or 2 with low Met expression (P¼ 0.007 and Pp0.001, respectively). Patients with stage 3 (T3, N1, M0 or T4, N0, 1, M1a) (C) with high Met expression had a worse overall 5- year survival as compared to patients with stage 3 with low Met expression however, this did not reach statistical significance (P¼ 0.061). High or low Met expression in patients with stage 4 OA did not change the 5-year overall survival (P¼ 0.915) (D). British Journal of Cancer (2008) 98(6), 1102 – 1108 & 2008 Cancer Research UK Probability of overall survival Probability of overall survival Probability of overall survival Probability of overall survival Met expression identifies poor survival in oesophageal cancer JB Tuynman et al 4 disease, Met expression level did not discriminate poor vs good enhanced in inflammation and has been shown to be involved in 5-year overall survival indicating that in advanced stage OA other early progression of oesophageal metaplasia and dysplasia into factors determine survival. Since Met expression appears to be an (adeno-) carcinoma (Morris et al, 2001; Buskens et al, 2002; important independent prognosticator and especially, this might Abdalla et al, 2004; Ling et al, 2007). Increased COX-2 expression offer an attractive opportunity for targeted therapy. Selective causes activation of several cancer-related genes including the HGF inhibitors of Met have recently become available and successful receptor Met (Boon et al, 2004; Han et al, 2006). Vice versa COX-2 inhibition of tumour progression, stromal and endothelial adhe- inhibition causes downregulation of cancer-related genes including sion and dissemination has been reported both in vitro and in Met as it has been published previously by our group (Tuynman animal studies. Targeted therapy of growth factor receptors has et al, 2005). In comparison to COX-2, Met is involved later in the been shown clinically effective in other cancer types such as process of cancer development and has been shown vital in cancer chronic myelogenous leukaemia, gastrointestinal stromal tumours, progression (Boccaccio and Comoglio, 2006). The proto-oncogene HER-2/NEU overexpressing breast cancer, colorectal cancer and Met, also known as the scatter factor, has been shown particularly non-small cell lung cancer (Verweij et al, 2004; Krause and Van important in morphogenic differentiation and organisation of Etten, 2005; Gold and Dematteo, 2006; Motzer et al, 2007; Smith three-dimensional tubular structures as well as in cell growth and et al, 2007). loss of cellular adhesion causing migration (dissemination) of cells A possible limitation of the present study is the semiquantitative (Boccaccio and Comoglio, 2006). Since OA is known for its evaluation of immunohistochemistry. The rational to semiquanti- propensity to early lymphatic and haematogenous dissemination, tatively score Met and COX-2 immunohistochemically was to the strong prognostic significance of high Met expression for both compare results from earlier reports. These scoring methods have overall and disease-specific 5-year survival can explain this clinical been used and validated in our previous reports (Buskens et al, behaviour at least partly. These results suggest that employment of 2002; Tuynman et al, 2005). A significant advantage of immuno- new therapeutic agents targeting Met might be of value as (neo-) histochemistry is the cellular morphology, which helps to correct adjuvant therapy in patients with OA, especially if Met expression for false-positive staining (blood vessels, stromal expression etc.). is high. Future studies using microarray gene expression technique can In conclusion, our data indicate that high Met expression is a help to validate results obtained in this patient cohort. significant independent indicator of poor long-term survival in Surprisingly, COX-2 expression was not a significant prognostic patients after potentially curative resection of OA. Targeting this factor in this study. The same cohort of patients was employed for receptor by a selective Met kinase inhibitor is an attractive (neo-) the current analysis of Met expression as reported on earlier for adjuvant treatment option that should be tested especially in COX-2 expression (Buskens et al, 2002). In this study, a minimal patients with high tumoral Met expression. follow-up of 60 months was available whereas in the previous study the median follow-up was only 27 months. Although survival in patients with high COX-2 expression tended to be poorer than that in patients with low COX-2 expression and this did not reach ACKNOWLEDGEMENTS statistical significance. Theoretically, the difference between COX- 2 expression and Met expression as prognostic indicators can We thank CJ Buskens for the previous work regarding COX-2 probably be explained by their function. The COX-2 enzyme is expression in oesophageal adenocarcinoma. REFERENCES Abdalla SI, Lao-Sirieix P, Novelli MR, Lovat LB, Sanderson IR, Fitzgerald Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, RC (2004) Gastrin-induced cyclooxygenase-2 expression in Barrett’s Jagiello-Gruszfeld A, Crown J, Chan A, Kaufman B, Skarlos D, Campone carcinogenesis. Clin Cancer Res 10: 4784–4792 M, Davidson N, Berger M, Oliva C, Rubin SD, Stein S, Cameron D (2006) Anderson MR, Harrison R, Atherfold PA, Campbell MJ, Darnton SJ, Lapatinib plus capecitabine for HER2-positive advanced breast cancer. 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Int J Oncol 25: 831–840 949–955 British Journal of Cancer (2008) 98(6), 1102 – 1108 & 2008 Cancer Research UK http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

Met expression is an independent prognostic risk factor in patients with oesophageal adenocarcinoma

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Springer Journals
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Copyright © 2008 by The Author(s)
Subject
Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
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0007-0920
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1532-1827
DOI
10.1038/sj.bjc.6604251
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Abstract

Molecular Diagnostics British Journal of Cancer (2008) 98, 1102 – 1108 & 2008 Cancer Research UK All rights reserved 0007 – 0920/08 $30.00 www.bjcancer.com Met expression is an independent prognostic risk factor in patients with oesophageal adenocarcinoma ,1,2 1 3 2 1,4 JB Tuynman , SM Lagarde , FJW ten Kate , DJ Richel and JJB van Lanschot 1 2 Department of Surgery, Academic Medical Centre at the University of Amsterdam, Amsterdam, The Netherlands; Department of Medical Oncology, Academic Medical Centre at the University of Amsterdam, Amsterdam, The Netherlands; Department of Pathology, Academic Medical Centre at the University of Amsterdam, Amsterdam, The Netherlands; Department of Surgery, Erasmus Medical Centre, Rotterdam, The Netherlands Oesophageal adenocarcinoma is an aggressive malignancy with propensity for early lymphatic and haematogenous dissemination. Since conventional TNM staging does not provide accurate prognostic information, novel molecular prognostic markers and potential therapeutic targets are subject of intense research. The aim of the present study was to study the prognostic significance of Met, the hepatic growth factor (HGF) receptor and a possible target for therapy in comparison to cyclooxygenase-2 (COX-2). Tumour sections from 145 consecutive patients undergoing intentionally curative surgery for oesophageal adenocarcinoma were immunohistochemically analysed for Met and COX-2 expression. Clinicopathological data were prospectively collected for all patients. Patients with high Met expression had significantly reduced overall and disease-specific 5-year survival rates (Pp0.001 and Pp0.001, respectively) and were more likely to develop distant metastases (P¼ 0.002) and local recurrences (P¼ 0.004) compared to patients with low Met expression. High COX-2 expression tended to be correlated with poor long-term survival but this did not reach statistical significance. Expression of Met was recognised as a significant and independent prognostic factor by stage-specific analysis and multivariate analysis (relative risk¼ 2.3; 95% CI¼ 1.3–4.1). These findings support the importance of Met in oesophageal adenocarcinoma and support the concept of Met tyrosine kinase inhibition as (neo-) adjuvant treatment. British Journal of Cancer (2008) 98, 1102–1108. doi:10.1038/sj.bjc.6604251 www.bjcancer.com Published online 18 March 2008 & 2008 Cancer Research UK Keywords: oesophageal adenocarcinoma; Met; HGF receptor; COX-2; prognosis Oesophageal adenocarcinoma (OA) is a highly aggressive malig- and haematogenous dissemination are still poorly understood nancy with early lymphatic and haematogenous dissemination. (Lagarde et al, 2006). Identification of growth factor receptors with The incidence of OA is increasing rapidly in the Western World tyrosine kinase activity, highly expressed in advanced cancer, has (Enzinger and Mayer, 2003). Despite advances in diagnosis and been shown to provide both prognostic information and potential treatment of the disease, even after potentially curative surgery the molecular targets for (neo-) adjuvant therapy (Krause and Van overall 5-year survival rate rarely exceeds 35% (Hulscher et al, Etten, 2005). A promising development in cancer therapy is the 2002; Cunningham et al, 2006). Subgroup analysis in early-stage combination of surgery with potent selective growth factor tumours have shown good survival rates although even early-stage receptor inhibitors as (neo-) adjuvant therapy resulting in tumours show early lymphatic dissemination. improved overall and disease-specific survival (Verweij et al, For adenocarcinoma of the oesophagus the most important 2004; Krause and Van Etten, 2005; Gold and Dematteo, 2006; conventional prognostic factors are summarised in the pTNM Motzer et al, 2007; Smith et al, 2007). Therapeutic usage of small stage of the oesophagus. Also other pathological aspects such as molecules selectively inhibiting c-KIT, a growth factor receptor extracapsular lymph node involvement and (relative) number of present in gastrointestinal stromal cell tumours (GIST), has positive nodes have prognostic impact. However, these conven- resulted in remarkable responses and has enhanced prognosis tional prognostic factors have limited accuracy (Lagarde et al, for patients with GIST to a great extent (Gold and Dematteo, 2006). 2006). Therefore, molecular prognostic markers, which can serve Other examples of targeted (neo-) adjuvant therapy are the as targets for therapy are subject of intense research. For OA only inhibition of vascular endothelial growth factor receptor (VEGFr) few molecular prognostic factors have been identified and in patients with advanced colorectal cancer, the inhibition of molecular events responsible for the development of lymphatic HER2-Neu, an epidermal growth factor receptor in patients with breast cancer and the inhibition of both VEGFr and platelet- derived growth factor receptor (PDGFr) in patients with renal cell *Correspondence: Dr JB Tuynman, Department of Surgery, Academic carcinoma (Hurwitz et al, 2004; Verweij et al, 2004; Geyer et al, Medical Centre, PO Box 22660, Meibergdreef 9, Amsterdam 1100DD, 2006; Motzer et al, 2007). The Netherlands; E-mail: J.B.Tuynman@amc.uva.nl Growth factor receptors have been identified in OA and some Received 28 August 2007; revised 7 January 2008; accepted 15 January show higher expression in later stages of cancer development 2008; published online 18 March 2008 (Lagarde et al, 2006; Vallbohmer et al, 2006). However, the Met expression identifies poor survival in oesophageal cancer JB Tuynman et al prognostic significance of growth factors expressed in OA has only histologically proven Barrett’s segment (defined by the presence been investigated in relatively small patient cohorts and no of goblet cells). Patients with an adenocarcinoma of the cardia or significance in multivariate analysis was demonstrated so far. The gastro-oesophageal junction without a clear Barrett’s segment were only independent molecular prognostic factor demonstrated for excluded (n¼ 161). This careful selection of patients has been OA is cyclooxygenase-2 (COX-2) expression as published by our described in our previous report (Buskens et al, 2002). Archival group (Buskens et al, 2002; Lagarde et al, 2006). Recently, we have materials of the remaining 145 patients were re-evaluated to obtain reported a clinical study in which neo-adjuvant selective COX-2 the sample with deepest invasion of each tumour. inhibition downregulates Met expression in conjunction with COX-2 expression in patients with OA (Tuynman et al, 2005). Met Surgical tissue specimens is the hepatocyte growth factor (HGF) receptor and is identified in OA (Tuynman et al, 2005; Anderson et al, 2006). Overexpression of All 145 patients were treated with subtotal oesophagectomy and Met and/or its ligands has been shown to contribute to progression resection of the lesser curvature of the stomach. Between April and dissemination of several malignancies including lung, colo- 1994 and February 2000, 96 patients (66%) were randomly rectal, gastric, breast, prostate, thyroid, pancreas, and oesophageal assigned to either transhiatal or transthoracic oesophagectomy as cancer (Hu et al, 2001; Saeki et al, 2002; Christensen et al, 2003; part of a randomized trial comparing both techniques (Hulscher Murai et al, 2004; Anderson et al, 2006). In experimental models, et al, 2002). In the remaining 49 patients, a standard transhiatal activation of Met (endogenously by mutations in its tyrosine procedure was performed. In 95 patients (65.5%), resection was kinase domain, or exogenously by HGF and prostaglandins performed by a transhiatal approach without thoracotomy. produced by COX-2) causes decreased apoptosis and enhanced Lymphadenectomy comprised en bloc removal of all lymphatic proliferation, angiogenesis, and invasion (Shinomiya et al, 2004; tissue in the lower posterior mediastinum, along the cardia and the Herrera et al, 2005; Boccaccio and Comoglio, 2006). Thus, COX-2 lesser curvature of the stomach. Fifty patients (34.5%) underwent and Met seem functionally connected. In cancer development, oesophagectomy through a right-sided thoracotomy followed by a COX-2 is present in early stages of dysplasia, initiating cancer laparotomy in combination with two-field lymph node dissection. growth and progression whereas Met is an important key regulator This procedure included an abdominal lymphadenectomy as of molecular processes in later stages of cancer development and described plus the removal of lymph nodes along the common progression (Tuynman et al, 2004). Small molecules selectively hepatic artery, the splenic artery, and the coeliac trunk as well as inhibiting Met have been shown to inhibit dissemination and an extended lymph node dissection in the chest (i.e., including the cancer growth both in vitro as in animal studies (Christensen et al, right paratracheal, infra-aortic arch, and subcarinal lymph nodes). 2003; Kim et al, 2003; Hov et al, 2004; Herrera et al, 2005; Martens et al, 2006; Watson et al, 2006). Consequently, inhibition of Met as Immunohistochemistry (neo-) adjuvant therapy for OA seems a promising strategy. A relation between Met expression and stage of disease has been Of all patients 5-mm thick sections of paraffin and formaldehyde- described previously (Anderson et al, 2006). However, the fixed tissue of the resection specimens were cut. For immuno- potential value of Met expression in OA as an independent histochemical staining, sections were incubated overnight at 371C prognosticator calculated by multivariate analysis has not yet been and subsequently deparaffinised in xylene, rehydrated, and treated addressed in a large consecutive cohort. Therefore, the aim of the with 3% H O in methanol for 10 min to block endogenous 2 2 present study was to characterize further the prognostic signifi- peroxidase activity. All specimens were subjected to heat-induced cance of Met expression in a large consecutive cohort of patients antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for with OA. 10 min at 951C. To block aspecific binding the slides were incubated with Tris-buffered saline (TBS) supplemented with 5% goat serum. Sections were incubated with the primary antibodies anti-Met c-Met (3D4; Zymed, San Francisco, CA, USA) (1 : 100), PATIENTS AND METHODS and anti-human COX-2 (160112; Cayman Chemical Co., Ann Arbor, MI, USA) (1 : 200) diluted in TBS with 1% bovine serum Patients albumin overnight at 41C. For the Met staining the sections were A consecutive series of 306 patients who underwent potentially incubated after washing steps with anti-mouse/rabbit-peroxidase curative oesophagectomy at the Department of Surgery of the polymer for 30 min at room temperature (Powervision; Immuno- Academic Medical Centre at the University of Amsterdam, The vision Inc., Daly City, CA, USA). Diaminobenzidine chromogen Netherlands for adenocarcinoma of the mid/distal oesophagus (Sigma, St Louis, MO, USA) was used for visualisation. For the between January 1993 and December 2000 was selected. Pre- COX-2 staining the sections were treated with biotinylated horse operative workup included endoscopy with histological biopsy, anti-mouse immunoglobulin (1 : 200; Vector Laboratories Inc., external ultrasonography of the abdomen and neck, CT scan of the Burlingame, CA, USA) and avidin–biotin peroxidase complex abdomen and chest, radiography of the chest, oesophageal (Vectastain ABComplex; Vector Laboratories). After these steps for endosonography, and indirect laryngoscopy. Lymph node metas- Met and COX-2 staining the sections were counterstained with tases at the coeliac trunk were a contraindication for resection only haematoxylin and embedded. Specificity of the antibodies was when considered non-resectable (i.e., larger than 2 cm in diameter) confirmed by controls using irrelevant immunoglobulins instead and confirmed by cytological puncture. Patients did not receive of primary antibodies. Colon cancer tissue was included as a additional (neo-) adjuvant chemotherapy and/or radiotherapy. positive control. Clinicopathological data from all operated patients were perma- nently prospectively collected. Follow-up was complete for all Scoring patients and extended until July 2006, ensuring a minimal potential follow-up of 5.5 years. Recurrence of disease was diagnosed on Met and COX-2 immunohistochemical staining were scored clinical grounds. However, whenever a relapse was suspected, semiquantitatively using a four-step scale as used and validated radiologic, endoscopic, or histological confirmation was sought. in previous reports (Buskens et al, 2002; Tuynman et al, 2005). The Recurrent disease was classified as locoregional (occurring in the following scoring criteria of tumour cells were agreed upon before upper abdomen or mediastinum) or distant (including cervical the analysis: (0) no staining or equal to background; (1) weak recurrences). All pathology reports were reviewed to identify those diffuse cytoplasmic staining (may contain stronger intensity in less patients in whom the adenocarcinoma had developed in a than 10% of cancer cells); (2) moderate granular cytoplasmic & 2008 Cancer Research UK British Journal of Cancer (2008) 98(6), 1102 – 1108 Molecular Diagnostics Molecular Diagnostics Met expression identifies poor survival in oesophageal cancer JB Tuynman et al Table 1 Correlation of clinicopathological findings and Met expression Patient Low Met High Met characteristics expression expression (n¼ 145) Overall (N¼ 67) (N¼ 78) P-value Median age (range) 67 (35 – 85) 67 (35 – 83) 68 (44 – 85) 0.493 Str Sex Male (%) 120 (83%) 63 (94%) 57 (73%) 0.435 Tumour characteristics T stage 0.017 T1 44 (30%) 29 (43%) 15 (19%) Tu T2 18 (12%) 8 (12%) 10 (13%) T3 83 (57%) 30 (45%) 53 (68%) N stage p0.001 N0 65 (45%) 38 (57%) 27 (35%) N1 80 (55%) 29 (43%) 51 (65%) M stage 0.086 M0 122 (84%) 58 (87%) 64 (82%) M1a 23 (16%) 9 (13%) 14 (18%) Differentiation grade 0.078 Good 11 (8%) 5 (7%) 6 (8%) Moderate 56 (39%) 35 (52%) 21 (27%) Poor 78 (54%) 27 (40%) 51 (65%) Overall 5-year 35% 57% 16% p0.001 survival Disease-specific 5- 48% 66% 33% p0.001 Str year survival T1, tumour limited to (sub)mucosa; T2, tumour infiltrates muscularis propia; T3, tumour infiltrates adventitia layer; as determined in the pathological resection specimens. N0, no tumour positive locoregional lymph nodes; N1, locoregional lymph node metastasis. M0, no distant metastasis, M1a, metastasis in coeliac lymph Tu nodes. Figure 1 Representive samples of immunohistochemical staining of Met causes. The Cox proportional hazards regression model was used (A) and COX-2 (B) (Str¼ stroma, T¼ tumour). to identify prognostic factors. To identify independent prognostic factors multivariate Cox regression analysis was carried out. Variables with multiple categories were recoded into dichotomous staining in 10–90% of cancer cells; (3) over 90% of tumour cells variables by combining categories with a comparable prognosis stained with strong intensity (Figure 1). (differentiation grade, good vs moderate and poor (poor); tumour T stage, stage 1 and 2 vs 3; Met expression, no or weak staining The analysis of all tissue sections was performed independently by three different investigators (JBT, SML, and FJWTK) without (low) vs moderate to strong staining (high); COX-2 expression, no patient identification parameters to correct for observer accuracy. or weak staining (low) vs moderate to strong staining (high). The semiquantitative scoring by the investigators had a low observer variation; 92% of the specimens were categorized identically. In cases of disagreement (n¼ 6 for COX-2 expression RESULTS and N¼ 13 for Met expression) consensus was reached after re- evaluation by the investigators using a multiheaded microscope. A total of 145 consecutive patients with OA were included for Data regarding COX-2 staining intensity are equal as previously immunohistochemical analysis. Of these patients 120 were men described and used for the present analysis. Areas of diffuse (83%) and 25 were women (17%) with a median age of 67 years haemorrhage or necrosis were neglected. (range¼ 35–85) (Table 1). The majority of patients (N¼ 83, 57%) had a T3 tumour and 80 patients (55%) had positive lymph nodes. The overall 5-year survival in the included group was 35% and the Statistics disease-specific 5-year survival was 48%. Two patients (1.4%) died Statistical calculations were performed using SPSS version 14.0 within 90 days due to post-operative complications (myocardial (Statistical Package for the Social Sciences, Chicago, IL, USA). The and respiratory failure in one patient and cerebrovascular event in association between demographic and clinicopathological features another one patient). and protein expression was analysed using Student’s t-test High Met staining (as opposed to low Met staining) was (continuous data) and w -test (categorical data). Overall and observed in 78 cases (54%). Of these 78 patients, 28 cases were disease-specific 5-year survival rates were estimated according to scored as strong Met expression and 50 as moderate Met the Kaplan–Meier method and compared between groups using expression. In 67 patients (46%) Met expression was classified as the log-rank test. Overall survival was calculated using deaths since low; 56 patients had weak Met expression and 11 patients had no time of surgery irrespective of cause. For disease-specific survival or equal to background staining of Met. Met expression was all non-disease-related deaths were excluded including in-hospital mainly localised in neoplastic cells (Figure 1A and B) but was also death within 90 days of surgery, since we assumed that these weakly identified in non-neoplastic epithelial cells (both squamous patients had died because of comorbidity and surgery-related and columnar epithelium) and in stromal cells. Interobserver British Journal of Cancer (2008) 98(6), 1102 – 1108 & 2008 Cancer Research UK Met expression identifies poor survival in oesophageal cancer JB Tuynman et al 1.0 1.0 P0.001 P0.001 0.8 0.8 Met low Met low 0.6 0.6 0.4 0.4 Met high Met high 0.2 0.2 0.0 0.0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Overall survival in months Disease-specific survival in months 1.0 1.0 P=0.238 P=0.180 0.8 0.8 COX-2 low 0.6 COX-2 low 0.6 COX-2 high COX-2 high 0.4 0.4 0.2 0.2 0.0 0.0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Overall survival in months Disease-specific survival in months Figure 2 Kaplan –Meier survival curves of 145 patients with adenocarcinoma of the oesophagus. Patients with high Met expression had a significantly worse overall 5-year survival. (A)(Pp0.001) and disease-specific 5-year survival (Pp0.001) (B) as compared to patients with low Met expression. Overall 5-year survival and disease-specific 5-year survival tended to be worse in patients with high COX-2 expression (C and D, respectively) as compared to patients with low COX-2 expression but this did not reach statistical significance (P¼ 0.180 and P¼ 0.238, respectively). variation was 8% for Met expression. All specimens that were Table 2 Results of univariate analysis of clinical, pathological and discrepant (n¼ 13) were re-evaluated and the consensus score was immunohistochemical parameters related to disease-specific 5-year survival used for further analysis. Results of COX-2 expression have been Odds ratio described previously in this cohort of patients. Briefly, COX-2 (confidence interval) P-value expression was negative to weak in 21% (COX-2 low) and moderate to strong in 79% (COX-2 high) of the carcinomas Patient sex (male vs female) 1.2 (0.7 – 2.3) 0.435 (Buskens et al, 2002; Tuynman et al, 2005). High Met expression was observed more often in patients with Patient ASA classification 1.0 (0.5 – 2.0) 0.974 higher T stage (P¼ 0.003), in patients with positive lymph nodes (0 and 1 vs 2or3) (Pp0.001) and a poor differentiation grade (P¼ 0.003) (Table 1). Met expression was not correlated with COX-2 expression Patient age (70 and higher vs 1.1 (0.5 – 2.2) 0.283 lower than 70 years) (P¼ 0.839). During 5-year follow-up, 92 patients died: 17 patients died of Tumour T stage (3 vs 1 and 2) 4.1 (2.4 – 7.2) 0.001 unrelated causes and 75 patients died of recurrent disease. Of these patients, 23 had locoregional recurrences, 39 patients had Tumour N stage (1 vs 0) 4.9 (2.8 – 8.6) 0.000 haematogenous recurrences and 13 patients had both locoregional tumour M1a stage (1 vs 0) 3.7 (2.2 – 6.5) 0.035 and haematogenous recurrences. After a complete follow-up, overall 5-year survival was Differentiation grade 2.2 (1.3 – 3.6) 0.015 significantly lower in patients with high Met expression as (moderate and poor vs good) compared to patients with low Met expression; 16 vs 57% Met expression (high vs low) 3.5 (2.0 – 5.9) 0.000 (Pp0.001). Furthermore, disease specific 5-year survival was significantly lower in patients with high Met expression as COX-2 expression (high vs low) 1.4 (0.8 – 2.6) 0.234 compared to patients with low Met expression; 33 vs 66% (Pp0.001). Patients with high Met expression were more likely to develop distant metastases (P¼ 0.002) as well as local recurrences (P¼ 0.004). Patients with high COX-2 expression (Figure 2). Univariate analysis revealed that T stage, N stage, M1a tended to have a poor overall and disease-specific 5-year survival stage, differentiation grade, and Met expression were all significant as compared to patients with low COX-2 expression but in contrast prognostic indicators for disease-specific 5-year survival (Table 2). to previous reports this did not reach statistical significance Multivariate analysis of these variables demonstrated that T3 stage & 2008 Cancer Research UK British Journal of Cancer (2008) 98(6), 1102 – 1108 Probability of overall survival Probability of overall survival Probability of disease-specific survival Probability of disease-specific survival Molecular Diagnostics Molecular Diagnostics Met expression identifies poor survival in oesophageal cancer JB Tuynman et al (relative risk (RR)¼ 1.9, (95% confidence interval (95% CI¼ 1.0– high Met expression as compared to patients with low Met 3.5)), (P¼ 0.035)), lymph node involvement (RR¼ 2.8, (95% expression (P¼ 0.007 and Pp0.001, respectively) (Figure 3). CI¼ 1.5–5.3), (P¼ 0.001)) and high Met expression (RR¼ 2.3, Patients with stage 3 disease and high Met expression tended to (95% CI¼ 1.3–4.1), (P¼ 0.004)) were independent prognostic have poor overall 5-year survival as compared to patients with low factors (Table 3). Met expression but no statistical significance was reached Subgroup analysis in patients with Stages 1 and 2 OA revealed (P¼ 0.064). In contrast to patients with stage 1 and 2, in patients that overall 5-year survival was significantly lower in patients with with stage 4 OA the Met expression level did not discriminate poor vs better overall 5-year survival. Table 3 Results of multivariate analysis of pathological and immunohis- tochemical parameters related to disease-specific 5-year survival according DISCUSSION to the Cox regression model This study provides evidence that Met expression level (as detected Relative risk by immunohistochemical analysis) is an independent prognostic (confidence interval) P-value factor in OA. Overall 5-year survival after potentially curative resection is significantly worse in patients with tumours expressing T stage (3 vs 1 and 2) 1.9 (1.0 – 3.5) 0.035 high Met levels compared to low Met levels. In literature, lymphatic dissemination as identified on histo- N stage (1 vs 0) 2.8 (1.5 – 5.3) p0.001 pathological examination is the single most important prognostic M1a stage (1a vs 0) 1.8 (0.9 – 3.4) 0.056 factor in patients with oesophageal cancer (Lagarde et al, 2006). Also in the present study, lymph node involvement is a strong Tumour differentiation grade 1.6 (0.9 – 2.7) 0.077 independent prognostic factor next to T stage and Met expression (moderate and poor vs good) level. Since Met expression was correlated stage of disease the subgroup analysis revealed that especially in stage 1 and 2 OA Met Met expression (high vs low) 2.3 (1.3 – 4.1) 0.004 expression is a significant and valuable prognostic factor. In stage Stage 1 Stage 2 P=0.007 1.0 P0.001 1.0 Met low 0.8 0.8 0.6 0.6 Met low Met high 0.4 0.4 0.2 0.2 Met high 0.0 0.0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Overall survival in months Overall survival in months Stage 4 Stage 3 1.0 1.0 P =0.061 P=0.915 0.8 0.8 0.6 0.6 0.4 0.4 Met low 0.2 0.2 Met low Met high 0.0 0.0 Met high 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Overall survival in months Overall survival in months Figure 3 Stage specific Kaplan – Meier survival curves for high vs low Met expression. Patients with stage 1 (T1, N0, and M0) (A) and stage 2 (T2, 3, N0, M0 or T1, N1, M0) (B) and high Met expression had a significantly worse overall 5-year survival as compared to patients with stage 1 or 2 with low Met expression (P¼ 0.007 and Pp0.001, respectively). Patients with stage 3 (T3, N1, M0 or T4, N0, 1, M1a) (C) with high Met expression had a worse overall 5- year survival as compared to patients with stage 3 with low Met expression however, this did not reach statistical significance (P¼ 0.061). High or low Met expression in patients with stage 4 OA did not change the 5-year overall survival (P¼ 0.915) (D). British Journal of Cancer (2008) 98(6), 1102 – 1108 & 2008 Cancer Research UK Probability of overall survival Probability of overall survival Probability of overall survival Probability of overall survival Met expression identifies poor survival in oesophageal cancer JB Tuynman et al 4 disease, Met expression level did not discriminate poor vs good enhanced in inflammation and has been shown to be involved in 5-year overall survival indicating that in advanced stage OA other early progression of oesophageal metaplasia and dysplasia into factors determine survival. Since Met expression appears to be an (adeno-) carcinoma (Morris et al, 2001; Buskens et al, 2002; important independent prognosticator and especially, this might Abdalla et al, 2004; Ling et al, 2007). Increased COX-2 expression offer an attractive opportunity for targeted therapy. Selective causes activation of several cancer-related genes including the HGF inhibitors of Met have recently become available and successful receptor Met (Boon et al, 2004; Han et al, 2006). Vice versa COX-2 inhibition of tumour progression, stromal and endothelial adhe- inhibition causes downregulation of cancer-related genes including sion and dissemination has been reported both in vitro and in Met as it has been published previously by our group (Tuynman animal studies. Targeted therapy of growth factor receptors has et al, 2005). In comparison to COX-2, Met is involved later in the been shown clinically effective in other cancer types such as process of cancer development and has been shown vital in cancer chronic myelogenous leukaemia, gastrointestinal stromal tumours, progression (Boccaccio and Comoglio, 2006). The proto-oncogene HER-2/NEU overexpressing breast cancer, colorectal cancer and Met, also known as the scatter factor, has been shown particularly non-small cell lung cancer (Verweij et al, 2004; Krause and Van important in morphogenic differentiation and organisation of Etten, 2005; Gold and Dematteo, 2006; Motzer et al, 2007; Smith three-dimensional tubular structures as well as in cell growth and et al, 2007). loss of cellular adhesion causing migration (dissemination) of cells A possible limitation of the present study is the semiquantitative (Boccaccio and Comoglio, 2006). Since OA is known for its evaluation of immunohistochemistry. The rational to semiquanti- propensity to early lymphatic and haematogenous dissemination, tatively score Met and COX-2 immunohistochemically was to the strong prognostic significance of high Met expression for both compare results from earlier reports. These scoring methods have overall and disease-specific 5-year survival can explain this clinical been used and validated in our previous reports (Buskens et al, behaviour at least partly. These results suggest that employment of 2002; Tuynman et al, 2005). A significant advantage of immuno- new therapeutic agents targeting Met might be of value as (neo-) histochemistry is the cellular morphology, which helps to correct adjuvant therapy in patients with OA, especially if Met expression for false-positive staining (blood vessels, stromal expression etc.). is high. Future studies using microarray gene expression technique can In conclusion, our data indicate that high Met expression is a help to validate results obtained in this patient cohort. significant independent indicator of poor long-term survival in Surprisingly, COX-2 expression was not a significant prognostic patients after potentially curative resection of OA. Targeting this factor in this study. The same cohort of patients was employed for receptor by a selective Met kinase inhibitor is an attractive (neo-) the current analysis of Met expression as reported on earlier for adjuvant treatment option that should be tested especially in COX-2 expression (Buskens et al, 2002). In this study, a minimal patients with high tumoral Met expression. follow-up of 60 months was available whereas in the previous study the median follow-up was only 27 months. 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Int J Oncol 25: 831–840 949–955 British Journal of Cancer (2008) 98(6), 1102 – 1108 & 2008 Cancer Research UK

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British Journal of CancerSpringer Journals

Published: Mar 18, 2008

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