Comparative Analysis of Four Histopathological Classification Systems to Discriminate Benign and Malignant Behaviour in Gastrointestinal Stromal Tumors D. VALLBÖHMER 1, H.E. MARCUS 1, S.E. BALDUS 2, J. BRABENDER 1, G. LURJE 1, U. DREBBER 3, R. METZGER 1, A.H. HÖLSCHER 1 and P.M. SCHNEIDER 1,4 1 Department of Visceral and Vascular Surgery and 3 Institute of Pathology, University of Cologne; 2 Institute of Pathology, Heinrich Heine University Düsseldorf, Germany; 4 Department of Visceral and Transplantation Surgery, University Hospital Zurich, Switzerland Abstract. The prognostic value of the four most common histopathological classification systems in gastrointestinal stromal tumors (GISTs) was evaluated retrospectively. Patients and Methods: Twenty-five consecutive patients with resected GIST and a follow-up of five years or more for surviving patients were included in this analysis. All the tumors were c-kit (CD117) positive and were additionally re-evaluated for the number of mitoses per 50 high-power fields (HPF) and Ki-67 proliferation index. The four most commonly applied histopathological classification systems of the WHO, Franquemont (modified by using the Ki-67 proliferation index), Fletcher and Miettinen were applied to each patient. Results: The survival of patient groups classified by Franquemont (p=0.03) and the WHO (p=0.031) were of prognostic relevance, while the grouping of patients by classifications according to both, Fletcher and Miettinen did not show a significant prognostic value. Conclusion: The classification systems of Franquemont (modified) or WHO appear to be advantageous for the evaluation of malignant potential and clinical outcome in patients with GISTs. Our data are merely hypothesis generating and should be validated in larger clinical studies. Gastrointestinal stromal tumors (GISTs) are rare but distinct mesenchymal tumors of the gastrointestinal tract (1, 2). In the past decades, these tumors have been a source of Correspondence to: Paul M. Schneider, MD, Professor of Surgery, Department of Visceral and Transplantation Surgery, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. Tel: +41 44 2553791, Fax: +41 44 2558941, e-mail: paul.schneider@ usz.ch Key Words: Gastrointestinal stromal tumors, classification systems, prognosis. confusion and controversy with regard to classification and differentiation. Historically, GISTs were thought to be of smooth muscle origin, mainly classified as leiomyomas, leiomyoblastomas, or leiomyosarcomas (3). However, the introduction of electron microscopy in the early 1970s and immunohistochemistry in the early 1980s has demonstrated that only a few of the tumors have evidence of smooth muscle origin and show rather an overlap of smooth muscles and neuronal features (2, 4). Based on the findings, suggesting these neoplasms to be distinct mesenchymal tumors, Mazur and Clark introduced the term gastrointestinal stromal tumor in 1983 (5). The first molecular marker for GIST was CD34, a myeloid progenitor cell antigen, that is stained positive in approximately 70%, which achieved more sensitive recognition in the diagnosis of these tumors (6). The recent discovery of the CD117 antigen, part of the KIT receptor tyrosine kinase, which is expressed in approximately 85% of GISTs, introduced the most recent definition of GISTs via cellular spindle cell, epithelioid or occasionally pleomorphic mesenchymal tumors of the gastrointestinal tract that express CD117, as detected using immunohistochemistry (4, 7). In more than 50% of the patients with GISTs the neoplasm is located in the stomach, while the small intestine (25%), the rectum (5%) and the esophagus (2%) are less common sites (2). The tumors are commonly between 2 and 30 cm in diameter and have a broad spectrum of clinical behaviour. While some GISTs remain stable over years, others progress rapidly to metastatic disease (2, 8). In fact, up to 50% of patients with this tumor entity have metastatic disease at the time of diagnosis. Frequently, GISTs are asymptomatic and discovered incidentally, but may cause mass-related symptoms, including dysphagia, pain or gastrointestinal bleeding. Apart from the undisputed adverse prognostic factors, including the presence of peritoneal or distant metastases, 0250-7005/2008 $2.00+.40 367
Figure 2. Localization of the GIST. To date, no published data are available comparing the histopathological classification systems by the WHO, Franquemont, Fletcher and Miettinen for their impact on predicting clinical outcome in patients with GISTs. Therefore, in this retrospective study the prognostic value of the most commonly used classification systems for this tumor type were evaluated. Patients and Methods Figure 1. Primary symptoms of study patients with GIST. prediction of clinical outcome in patients with GIST remains difficult. In recent years several studies have been performed to establish prognostic factors, predicting the biological behaviour of GISTs (4, 9, 10). Thereby, three key prognostic factors have been characterized tumor size, mitotic index and tumor site. Tumors that are small (<2-5 cm) and have a mitotic index <5 mitoses per 50 high-power fields (HPF) have a statistically better prognosis compared to big tumors with a high mitotic index (4, 9, 10). In addition, Emory et al. demonstrated a significant difference in site-specific survival in patients with GISTs, patients with primary gastric tumors had a significantly better prognosis than those with tumors located in the small bowel and rectum (11). Based on these findings, histopathological classification systems by the WHO, Franquemont, Fletcher and Miettinen have been established to predict clinical outcome of patients with GISTs by using the parameters of mitotic rate and tumor size (4, 9, 10, 12). Furthermore, the original classification system by Franquemont et al. characterized additional assessment criteria for GISTs, based on immunohistochemical findings. They showed a prognostic value of the proliferating cell nuclear antigen (PCNA) in patients with this tumor entity while other studies demonstrated Ki-67 labelling index to be a sensitive prognostic fatcor in GISTs (9, 13, 14). Clinicopathological variables. Between 1996 and 2002, 264 patients with mesenchymal tumors underwent surgical resection at the Department of Visceral and Vascular Surgery, University of Cologne, Germany. All the surviving patients had a minimum follow-up of 5 years. All the histopathological reports of these patients were reviewed to identify patients with GIST. Only those primarily diagnosed and treated at our University Hospital were included in this retrospective analysis. Fourteen of the study patients had primary symptoms, while 11 had no symptoms. In five of these patients the diagnosis of a GIST was made randomly during an operation, in one during an abdominal ultrasound and in another during an endoscopy (Figure 1). The localizations of the GIST are shown in Figure 2. Documented patient data included age, gender, symptoms, tumor localization, date and type of operation, tumor size, immunohistochemical variables, mitotic rate, adjuvant therapy, recurrence, distant metastases, date of death or last date of follow-up. In addition, all the tumors were reclassified according to the histopathological classification systems of the WHO, Franquemont, Fletcher and Miettinen, as described below. Adjuvant therapy with imatinib. In February 2002, the European Comission approved imatinib for use in the treatment of patients with unresectable or metastatic GISTs. Only one patient of our study group with recurrent disease received adjuvant therapy with imatinib, so that its prognostic influence was not assessable in this study. Histopathology and immunohistochemistry. Formalin-fixed, paraffinembedded tumor tissues from specimens of all the study patients were available for immunohistochemical analysis. To determine the mitotic rate (haematoxylin and eosin stained sections) and Ki-67- positive tumor cells, 50 HPF (magnification x400) of the tumour tissue of each patient were evaluated. The frequencies were recorded as the number of positive cells (n) per 50 HPF. 368
Vallböhmer et al: Classification Systems in GISTs Immunohistochemical staining was performed by applying monoclonal antibodies directed against Ki-67 (MIB-1; 1:150; DakoCytomation, Hamburg, Germany), c-kit (CD117; 1:100; DakoCytomation) and CD34 (Qbend10; 1:100; DakoCytomation). Microwave pre-treatment was performed (2x7 min in citrate buffer ph 6) before Ki-67 and CD34 staining. C-kit immunoreactivity was enhanced by autoclave pre-treatment. The paraffin-embedded tumor tissues from pre-therapeutic biopsies and resected specimens were cut into 2-Ìm sections and mounted onto Superfrost Plus slides (Menzel-Glaeser, Braunschweig, Germany). The tissue sections were deparaffinized according to routine histopathological procedures. Immunostaining was performed using a DakoCytomation Tech-Mate 500 plus immunostainer according to the manufacturer's instructions applying the following reagents: Chem Mate Buffer Kit (K5006), peroxidase/aminoethyl carbazole (AEC) detection kit (K5003), Chem Mate peroxidase blocking solution (S2023) and Chem Mate Hematoxylin (S2020), all from DakoCytomation. All the slides were analyzed by two staff pathologists (S.E.B. and U.D.) who were blinded for all clinical data. Classification systems. The following classification systems were used to determine the prognosis of each patient: i) WHO classification (12): benign GIST: <5 mitoses/50 HPF and tumor size <5 cm; malignant GIST: >5 mitoses/50 HPF and tumor size >5 cm and borderline GIST: a maximum of 4 mitoses/50 HPF when tumor size >5 cm. ii) Original and modified classification according to Franquemont (9): high risk : tumor size >5 cm and mitotic rate >2 mitoses/10 HPF or tumor size >5 cm or mitotic rate >2 mitoses/10 HPF in combination with PCNA index > 10%. low risk : tumor size <5 cm and mitotic rate <2 mitoses/10 HPF or tumor size <5 cm or mitotic rate <2 mitoses/10 HPF in combination with PCNA index <10%. Franquemont et al. in their original classification system used the proliferation marker PCNA (9). However, recent studies have shown that use of PCNA as a proliferation marker is not practicable (13). In addition, Wong et al. demonstrated in 108 patients with GIST that a Ki-67-proliferation-index 5% is significantly associated with a worse prognosis (14). Therefore, in this study the Ki-67 proliferation index with a cut-off value of 5% was used to classify patients instead of using the PCNA as a proliferation marker. For that reason we refer to this as modified classification of Franquemont. iii) Classification according to Fletcher (4): very low risk: tumor size <2 cm, <5 mitoses/50 HPF; low risk: tumor size 2-5cm, <5 mitoses/50 HPF; intermediate risk: tumor size <5 cm, 6-10 mitoses/50 HPF or tumor size 5-10 cm, <5 mitoses/50 HPF and high risk: tumor size >5 cm and >5 mitoses/50 HPF or tumor size >10cm independent of the mitotic rate or >10 mitoses/50 HPF independent of the tumor size. iv) Classification according to Miettinen (10): probably benign: intestinal tumors, tumor size 2 cm and 5 mitoses/50 HPF and gastric tumors, tumor size 5 cm and 5 mitoses/50 HPF; probably malignant: intestinal tumors, tumor size 5 cm or 5 mitoses/50 HPF and gastric tumors, tumor size 10 cm or 5 mitoses/50 HPF and uncertain or low malignant potential: intestinal tumors, tumor size 2-5 cm and 5 mitoses/50 HPF and gastric tumors, tumor size 5-10 cm and 5 mitoses/50 HPF. Statistical analysis. Kaplan-Meier plots were used to describe the survival distribution by the classification systems or other clinicopathological variables. The log-rank test was used to Table πa. Tumors of the study patients classified according to the WHO. Benign 13 52% Borderline 4 16% Malignant 8 32% Table πb. Tumors of the study patients classified according to Miettinen. Probably benign 10 40% Uncertain or low malignant potential 6 24% Probably malignant 9 36% Table πc. Tumors of the study patients classified according to Fletcher. Very low risk 4 16% Low risk 9 36% Intermediate risk 3 12% High risk 9 36% Table πd. Tumors of the study patients classified according to Franquemont (modified classification). Low risk 15 60% High risk 10 40% evaluate the prognostic significance. The level of significance was set to p<0.05. The p-values were given for two-sided testing. All the statistical tests were performed using the Software Package SPSS for Windows, version 11.0 (Chicago, IL, USA). Results Tumor size and mitotic rate. The median tumor size was 4.7 cm (range: 0.3-21 cm). The median mitotic rate was 3 mitoses/50hpf (range: 0-175 mitoses/50hpf). Immunohistochemical analysis. All 25 tumors were c-kit positive and 24 tumors were CD34 positive. The median Ki- 67 proliferation index was 2% (range 1-20%). In 19 (76%) patients the Ki-67 proliferation index was 5% and in 6 (24%) patients it was <5%. Classification systems. Table πa-d display the classification of all 25 tumors according to the WHO, Miettinen, Fletcher and modified Franquemont systems. 369
Figure 3. Probability of survival for the study patients according to histopathological classification systems of the WHO, Franquemont, Fletcher and Miettinen. A. WHO classification B. Miettinen classification C. Fletcher classification D. Modified Franquemont classification. Prognostic evaluation by histopathological classification systems. WHO classification. The median survival of the 3 patient groups classified according to the WHO (benign, borderline, malignant) has not been reached. No tumor-related deaths occurred in the benign and borderline subgroup while 3 GIST-related deaths occurred in the malignant subgroup. The median survival of the patients in the benign and borderline subgroup was significantly longer compared to the patients in the malignant group (p=0.031, log rank test, Figure 3A). Miettinen classification. The median survival of the 3 patient groups classified according to the Miettinen classification (probably benign, uncertain or low malignant potential and probably malignant) has not been reached. No tumor-related deaths occurred in the first two groups. Out of the 9 patients in the probably malignant group 3 patients died because of the tumor and 1 patient had a recurrence. The median survival only tended to be shorter in this group compared to the probably benign and uncertain or low malignant potential group (p=0.059, log rank test, Figure 3B). 370
Vallböhmer et al: Classification Systems in GISTs Fletcher classification. The median survival of the 3 patient groups classified according to the Fletcher classification (very low risk, low risk, intermediate risk and high risk) has not been reached. In the first 3 groups no tumor-related death occurred. In the high risk group 3 of the 9 patients died because of the GIST. The survival curves of the 4 groups did not show any significant difference (p=0.12, log rank test, Figure 3C). Modified Franquemont classification. The median survival of the 2 patient groups classified according to the modified Franquemont classification (low risk and high risk) has not been reached. No tumor-related death occurred in the low risk group while 3 of the 10 patients in the high risk group died because of their tumor. There was a significantly longer survival in the low risk compared to the high risk group (p=0.03, log rank test, Figure 3D). Multivariate analysis. A multivariate analysis was not performed because of the small study group and the low number of events. Discussion Previous studies of the clinicopathological and immunohistochemical factors in patients with GISTs determined that prognostic indicators included tumor size, mitotic index, tumor site, PCNA and Ki-67 labelling index (4, 9, 11, 13, 14). However, some studies have produced different findings, which was probably due to analyses of small and/or poorly defined study groups (14-16). In fact, previous analyses occasionally investigated GISTs, smooth muscle or neural tumors as one study group, included c-kit negative neoplasms in their investigations or combined untreated or surgically resected patients in their survival analysis at the same time (14). Therefore, only patients with c-kit positive tumors undergoing primary surgical resection were included in our study. The WHO and modified Franquemont classifications significantly separated the groups of patients with good or bad prognosis in the present study. Interestingly, the modified classification by Franquemont, is the only model that combines the three most significant prognostic factors the Ki-67 proliferation index, tumor size and mitotic index suggesting that this system might be advantageous. The classification of GISTs according to Fletcher and Miettinen did not show a significant prognostic value. The Fletcher classification is the only model separating patients with a probably good prognosis into a very low risk and a low risk group which was primarily done for the reason that even small GISTs can metastasize (4). Nevertheless, in our study this sub-classification was not effective because survival of the very low risk, low risk and even the intermediate risk groups were similar. Future studies are needed to proove if this more detailed system is of any advantage. The classification by Miettinen divides GISTs into gastric and intestinal type tumors (10). In fact, Emory et al. demonstrated that intestinal are more aggressive than gastric neoplasms (11). However, in our study merely a trend regarding prognosis between these subgroups was detected, which might reflect the low patient numbers. In GISTs the c-kit protooncogene encodes the KIT receptor tyrosine kinase. Recent studies have shown that about 80% of GISTs have mutations within c-kit, leading to constitutive activation of KIT (2, 17). Moreover, it has been shown that a KIT mutation in exon 11 is an independent adverse prognostic factor in patients with GISTs (18). While the targeted inhibitor of tyrosine kinase activity imatinib (Gleevec, Novartis) has demonstrated dramatic efficacy in the majority of patients with advanced GISTs, the evaluation of c-kit mutation might become very important, as GISTs with c-kit mutations involving regulatory domains respond better to imatinib, whereas those possessing mutations affecting enzymatic domains typically have a lesser response (17, 19). Moreover, a recent study by DeMatteo et al., analyzing the efficacy of imatinib in the adjuvant setting after complete resection of primary GIST, demonstrated a significantly increased recurrence free survival in patients receiving imatinib (20). In conclusion, this retrospective study suggests that the classification systems of Franquemont (modified) or WHO appear to be advantageous for the evaluation of malignant potential and clinical outcome in patients with GISTs. Our data are merely hypothesis generating and should be validated in larger clinical studies. 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