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Loss of P16 Protein Defines High-Risk Patients with Gastrointestinal Stromal Tumors: a Tissue Microarray Study

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Loss of P16 Protein Defines High-Risk Patients with Gastrointestinal Stromal Tumors: a Tissue Microarray Study 638 Vol. 11, 638–645, January 15, 2005 Clinical Cancer Research Loss of p16 Protein Defines High-Risk Patients with Gastrointestinal Stromal Tumors: A Tissue Microarray Study Regine Schneider-Stock,1 Carsten Boltze,4 expected frequency [i.e., marker combinations: p16 positive, Jerzy Lasota,5 Brigitte Peters,2 Chris L. Corless,6 no metastases, and death of disease and p16 loss, metastases, and still alive]. The ‘‘type’’ whose observed frequency was Petra Ruemmele,8 Luigi Terracciano,10 3 11 significantly higher than the expected frequency consisted of Matthias Pross, Luigi Insabato, the following marker pattern: p16 loss, necrosis, and death of 11 9 Dolores Di Vizio, Igor Iesalnieks, disease (P < 0.001). In the multivariate Cox regression Stefan Dirnhofer,10 Arndt Hartmann,8 analysis, p16 loss, necrosis, and metastases each had Michel Heinrich,7 Markku Miettinen,5 independent prognostic value. P16 loss is a common molecular Albert Roessner,1 and Luigi Tornillo10 abnormality in GISTs and might be used in routine diagnosis to identify patients with high-risk tumors. Departments of 1Pathology, 2Biometrics, and 3General Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany; 4Department of Pathology, University of Rostock, Rostock, Germany; 5Department of Soft Tissue Pathology, Armed Forces Institute, INTRODUCTION Washington, District of Columbia; 6Department of Pathology and Gastrointestinal stromal tumors (GIST) are the most 7 Division of Hematology/Oncology, Oregon Health and Science common mesenchymal tumors of the gastrointestinal tract. University Cancer Institute and Portland Virginia Medical Center, Portland, Oregon; Departments of 8Pathology and 9General Surgery, They are characterized by the expression of the KIT (CD117, University of Regensburg, Regensburg, Germany; 10Institute of stem cell factor receptor) protein (1, 2). The mechanism Pathology, University of Basel, Basel, Switzerland; and 11Department responsible for constitutive activation of KIT protein in most of Biomorphological Sciences, Section of Pathology, University GISTs is a gain-of-function mutation in the KIT gene (3). ‘‘Federico II,’’ Naples, Italy Subsets of 5% to 7% of GISTs have activating mutations in the closely related PDGFRA gene (4). Interestingly, KIT and ABSTRACT PDGFRA mutation status in GISTs is predictive of clinical response to the tyrosine kinase inhibitor imatinib (5). Despite clearly defined histologic criteria, the prediction GISTs have a wide clinical spectrum that ranges from of tumor behavior for patients with gastrointestinal stromal benign to malignant behavior. Small tumors (V5 cm) that tumors (GIST) still poses a challenge to pathologists. show low mitotic frequency (V5 mitoses per 50 high-power Therefore, searching for alternative markers that allow for field) usually pursue a benign clinical course; however, a better prognostic evaluation is an important task. To small subset of mitotically inactive tumors do subsequently determine the practicability of immunohistochemical staining metastasize (1). Moreover, gastric tumors seem to behave less for p16 in clinical cases, we examined p16 protein expression aggressively than small intestinal tumors of similar size and in a group of 284 GISTs, a subset of which had long-term mitotic activity (6). Additional adverse prognostic factors, follow-up (median, 45 months; range, 1-204 months). P16 tested in large series of GISTs, include aneuploidy in DNA protein expression was ascertained on tissue microarrays as flow cytometry (7) as well as the presence of tumor necrosis well as on standard sections. Survival analyses were carried (8). No consensus has been reached regarding the prognostic out in 157 patients. P16 loss was found in 50% of GISTs, there value of a high Ki-67 score (1). being no correlation with age, sex, histologic subtype, signs of There are several indications that molecular alterations necrosis, or metastases. Patients having p16-negative tumors may predict the clinical outcome in GISTs, such as telomerase had a worse prognosis than those with p16-positive tumors activity (9, 10), DNA copy number changes (11, 12), KIT (P = 0.012) with a 2.3-fold relative increased risk of dying of mutation type (13, 14), hypermethylation of the E-cadherin disease. P16 loss identified a subgroup of gastric tumors with promoter (15), and expression of a set of six genes: CCNB1, a worse prognosis (P = 0.03). The multivariate configural CENP-F, FAK , HMG2, TSG101, and ezrin (16). Abnormal- frequency analysis identified two ‘‘antitypes,’’ whose observed ities in cell cycle regulators have been related to proliferation frequency was found to be significantly lower than the and progression of cancer cells. In a recent study, we reported on the prognostic significance of p16 gene alterations in 39 GISTs 17. The p16 tumor suppressor gene at 9p21 inhibits cell cycling by arresting cells in G1-S phase of the cell cycle Received 7/9/04; revised 9/2/04; accepted 9/17/04. (18). P16 operates through inhibition of cyclin-dependent The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked kinase 4, which in turn inhibits phosphorylation of pRb to advertisement in accordance with 18 U.S.C. Section 1734 solely to prevent cells from progressing into S phase (19). P16 loss has indicate this fact. been reported to predict poor clinical outcome in several Requests for reprints: Regine Schneider-Stock, Department of human tumors (20–24). Pathology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany. Phone: 49-391-6715060; Fax: 49-391- To confirm our previously reported data in a larger group 6715060; E-mail: [email protected]. of GISTs and to evaluate the reliability of immunohistochemical D2005 American Association for Cancer Research. p16 staining in the clinical setting, we determined p16 protein Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Clinical Cancer Research 639 expression in a group of 284 GISTs using tissue microarrays Table 2 Criteria for the definition of malignancy risk in GISTs (6) (TMA) and sections of conventionally cut tumor blocks. Risk scale Size (cm) Mitotic index Very low risk <2 <5 per 50 high-power field MATERIALS AND METHODS Low risk 2-5 <5 per 50 high-power field Clinical and Morphologic Features Intermediate risk <5 6-10 per 50 high-power field Patients. An overview of the clinical and morphologic data 5-10 <5 per 50 high-power field High risk >5 >5 per 50 high-power field is given in Table 1. We analyzed 284 primary GISTs from 284 >10 Any mitotic rate patients. The tumors were collected from the institutes of Any size >10 per 50 high-power field pathology at the University of Basel (Basel, Switzerland), University ‘‘Federico II’’ (Naples, Italy), Oregon Health and Science University (Portland, OR), University of Regensburg (Regensburg, Germany), and Armed Forces Institute of Pathology activity and tumor size (Table 2), GISTs were morphologically (Washington, DC). The study was approved by the investigator’s classified as very low risk, low-risk, intermediate-risk, or high- institutional review board. Our study included 160 men (56.3%) risk tumors. According to this histologic classification, we and 124 women (43.7%), their ages ranging from 9 to 91 years, investigated 28 very low risk, 55 low-risk, 43 intermediate-risk, with a mean (SE) of 60.9 (15.1) years. The primary tumors and 158 high-risk GISTs. (localization was known for 262 tumors) originated in the stomach Construction of TMA. For TMA construction, we used (n = 159), small intestine (n = 73), colon (n = 9), mesentery (n = a H&E-stained slide from each block to define representative 7), esophagus (n = 7), and rectum (n = 7). Tumors (data were tumor regions. Tissue cylinders with a diameter of 0.6 mm were available for 190 GIST) were histologically classified as punched from the tumor areas of each block and brought into predominantly spindled (n = 82), epithelioid (n = 49), or mixed a recipient paraffin block using a precision instrument (Beecher spindled-epithelioid (n = 59). Tumor size ranged from 0.4 to 45 cm Instruments, Sun Prairie, WI; ref. 25). Three different TMAs in greatest dimension, with a mean (SE) of 8.3 (6.6) cm. The were constructed, each containing tumors of the above- occurrence of necrosis was recorded in 100 cases: 38 cases (38%) mentioned institutes. To overcome the problem of tissue showed at least some tumor necrosis. Status of metastases was microheterogeneity and to increase the number of evaluable recorded for 195 patients, of whom 49 (25.1%) had metastases. cases, we replicated the TMAs by punching the donor tissue Morphologic Classification. The diagnosis of GISTs was blocks at least thrice. Thus, the analysis of p16 expression based on previously published criteria (6). Based on mitotic represents the mean of the staining of three different slides containing a single core per slide. Immunohistochemistry Table 1 Association between p16 loss and various clinicopathologic factors in GISTs KIT Protein. All GISTs were immunohistochemically positive for the KIT protein (antibody CD117, DAKO Corp., p16 loss, Carpinteria, CA). Factors n n (%) P P16 Protein. P16 immunohistochemistry was done as Morphologic classification described previously by Schneider-Stock et al. (17). Briefly, Very low risk 28 14 (50) 0.421 Low risk 55 22 (40) a monoclonal mouse antibody to p16 (1:100 dilution, Quartett, Intermediate risk 43 23 (53.5) Berlin, Germany) and antigen retrieval using microwave heating High risk 158 83 (52.5) [thrice for 10 minutes; 10 mmol/L citrate buffer (pH 6.0)] were Sex used after inhibition of endogeneous peroxidase activity. The Male 160 85 (53.1) 0.282 primary antibody was incubated for 1 hour at 37jC. The slides Female 124 57 (46) Age (mean, t test) were subsequently incubated with a 1:10 dilution of normal p16 loss 142 62.4 y 0.087 swine serum (Vector, Burlingame, CA).
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