Human Pathology (2012) xx, xxx–xxx
www.elsevier.com/locate/humpath
Original contribution
Use of potassium channel tetramerization domain-containing 12 as a biomarker for diagnosis and prognosis of gastrointestinal stromal tumor Tadashi Hasegawa MD, PhD a,⁎, Hiroko Asanuma PhD a , Jiro Ogino MD, PhD a , Yoshihiko Hirohashi MD, PhD b , Yasuhisa Shinomura MD, PhD c , Hiroyuki Iwaki MD, PhD d , Hironobu Kikuchi MD, PhD e , Tadashi Kondo MD, PhD f a
Department of Surgical Pathology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543, Japan Department of Pathology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556, Japan c Department of Internal Medicine, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543, Japan d Department of Pathology, Sunagawa City Medical Center, Sunagawa 073-0196, Japan e Department of Surgery, Sunagawa City Medical Center, Sunagawa 073-0196, Japan f Division of Pharmacoproteomics, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan b
Received 17 August 2012; revised 12 October 2012; accepted 17 October 2012
Keywords: KCTD12; Diagnosis; Prognosis; Immunohistochemistry; Gastrointestinal stromal tumor; GIST
Summary Previously, we showed that the expression of potassium channel tetramerization domaincontaining 12 (KCTD12), which was discovered by a proteomics approach, is associated with high-risk behavior of gastrointestinal stromal tumors (GISTs). Here, we examined the distribution and expression of this protein by immunostaining with a commercially available polyclonal KCTD12 antibody in GISTs (n = 64) and other types of malignancy (n = 168) to clarify its diagnostic and clinical significance. Diffuse KCTD12 immunoreactivity was found in most GISTs (52 cases; 81%). KCTD12 expression was observed primarily in vascular endothelial cells, Purkinje cells of the cerebellum, and some neurons scattered throughout the cerebral cortex. KCTD12 was absent from not only the interstitial cells of Cajal but also interstitial cells of Cajal hyperplasia that was encountered incidentally in colon diverticulitis. KCTD12 immunostaining was also seen in malignant peripheral nerve sheath tumors (2/10 cases; 20%), synovial sarcomas (2/10; 20%), solitary fibrous tumor (1/8; 13%), angiosarcoma (1/7; 14%), and colon adenocarcinoma (1/24; 4%). In survival analyses, the 5-year recurrence-free survival rate of patients without KCTD12 expression was only 16.7% compared with 95.6% in those with KCTD12 expression (P b .0001). Ki-67 and KCTD12 were significant predictors of recurrence-free survival, and KCTD12 expression provided additional information about recurrence-free survival after accounting for Ki-67 status. Overall, KCTD12 expression was specific for GISTs from neoplastic and nonneoplastic adult tissues other than brain and served as a predictor of GIST recurrence. These findings suggest that KCTD12 is a useful and reliable biomarker for both the diagnosis and prognosis of GIST. © 2012 Elsevier Inc. All rights reserved.
⁎ Corresponding author. Department of Surgical Pathology, Sapporo Medical University School of Medicine, South 1 West 16, Chuo-ku, Sapporo 0608543, Japan. E-mail address:
[email protected] (T. Hasegawa). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2012.10.013
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T. Hasegawa et al.
1. Introduction
2. Materials and methods
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and occur most frequently in the stomach, followed by the small intestine, colon, and extragastrointestinal sites such as the omentum and mesentery [1]. They emerge from the interstitial cells of Cajal (ICCs) and are characterized by the presence of gain-of-function mutations in c-kit (KIT) or platelet-derived growth factor receptor α (PDGFRA) [2,3]. These mutations result in the constitutive activation of signaling pathways downstream of receptor tyrosine kinases. The malignant potential of GISTs varies from virtually benign tumors to aggressive sarcomas. Some patients are at a significant risk for tumor recurrence and progression to metastatic disease, even after complete excision of their tumors. A few risk-stratification schemes are available for operable GISTs [4-7]; tumor size, mitosis count, and tumor site are considered established risk factors for recurrence. Imatinib is now being explored as an adjuvant therapy in patients with primary resectable GISTs because it increases the time to GIST recurrence [8,9], and patients with a high risk of recurrence have longer survival with 3 years of adjuvant imatinib therapy compared with those with 1 year of treatment [9]. Although imatinib is generally well tolerated, nearly all patients report some adverse effects. To minimize these adverse effects, biomarkers to assess the malignant potential of GISTs are required for the selection of patients who are most likely to benefit from adjuvant imatinib therapy in routine clinical practice. In our previous report, patients with immunohistochemical positive expression of potassium channel tetramerization domain-containing 12 (KCTD12), which was discovered by a proteomics approach, had better metastasis-free survival than did those not expressing KCTD12 [10]. KCTD12 was originally cloned as a gene that is highly expressed in the fetal cochlea and brain [11], whereas it was recently identified as a component of the GABAB receptor [12]. The prognostic value of KCTD12 was validated in a multiinstitutional series of GISTs by using a monoclonal antibody against KCTD12 that was developed in our laboratory [13,14]. However, this monoclonal antibody is not yet available for clinical application, and KCTD12 expression in normal and abnormal tissues and other types of malignancies remains to be clarified. For this reason, here we studied the immunohistochemical expression of KCTD12 in GISTs and a variety of other soft tissue tumors and carcinomas, some of which appear in the differential diagnosis of GIST, using a commercially available polyclonal antibody. We correlated the expression of KCTD12 with the clinicopathologic characteristics of 64 cases of GIST to establish the diagnostic and prognostic use of the KCTD12 antibody.
2.1. Patients and tumor samples The medical records of 64 patients with primary, resectable c-kit protein/KIT–positive GIST who were diagnosed and treated between 2000 and 2011 were retrieved from the pathology files of Sapporo Medical University Hospital and Sunagawa City Medical Center. All patients underwent resection with curative intent. In our institutions, administration of adjuvant or neoadjuvant imatinib has been considered for patients with a high-risk tumor, a tumor with a Ki-67 labeling index of 10% or greater, or a marginally resectable tumor since this therapy was approved in July 2009. Patients who received adjuvant/neoadjuvant imatinib were excluded from this analysis because adjuvant imatinib influences the natural course of the disease [8] and morphologic changes such as extensive hyalinization and hypocellularity and even the loss of KIT expression occur in tumor cells after imatinib treatment [15]. In addition, a total of 168 cases of soft tissue tumors and carcinomas diagnosed pathologically at Sapporo Medical University Hospital were selected for study (Table 1): 12 leiomyosarcomas, 6 schwannomas, 10 malignant peripheral nerve sheath tumors (MPNSTs), 6 desmoid-type fibromatoses, 8 solitary fibrous tumors, 7 myxofibrosarcomas, 6 dedifferentiated liposarcomas, 10 synovial sarcomas, 6 Ewing sarcomas, 8 malignant melanomas, 7 angiosarcomas, 10 cutaneous squamous cell carcinomas, 24 breast carcinomas, 24 pulmonary adenocarcinomas, and 24 colon adenocarcinomas. Nonneoplastic adult tissues from a variety of sites in the body were also analyzed. The study protocol
Table 1 Immunohistochemical expression of KCTD12 in 64 cases of GIST, 86 of soft tissue tumors, and 82 of carcinomas Histologic type
No. of cases
KCTD12-positive cases
GIST Leiomyosarcoma Schwannoma MPNST Desmoid-type fibromatosis Solitary fibrous tumor Myxofibrosarcoma Dedifferentiated liposarcoma Synovial sarcoma Ewing sarcoma Malignant melanoma Angiosarcoma Cutaneous squamous cell carcinoma Breast carcinoma Pulmonary adenocarcinoma Colon adenocarcinoma
64 12 6 10 6 8 7 6 10 6 8 7 10
52 (81%) 0 (0%) 0 (0%) 2 (20%) 0 (0%) 1 (13%) 0 (0%) 0 (0%) 2 (20%) 0 (0%) 0 (0%) 1 (14%) 0 (0%)
24 24 24
0 (0%) 0 (0%) 1 (4%)
KCTD12 expression in GISTs for the collection of tumor samples and clinical information was approved by the ethics committees of Sapporo Medical University School of Medicine and Sunagawa City Medical Center.
2.2. Immunohistochemistry The tumor slides from all cases, which were stained with hematoxylin and eosin, were reviewed, and representative sections from the formalin-fixed, paraffin-embedded tissue blocks were examined using automated immunohistochemistry systems at Sapporo Medical University Hospital. The sections were loaded into a PT Link module (Dako, Carpinteria, CA) and subjected to an antigen retrieval/dewaxing protocol with the Dako EnVision FLEX Target Retrieval Solution, high pH, and then transferred to the Dako Autostainer Link 48 instrument. Immunostaining was performed using the primary rabbit polyclonal KCTD12 antibody (1:200 dilution; Abgent, San Diego, CA) and the Dako Envision FLEX detection system. Immunoreactivity was assessed by 2 observers. As described previously [10,13], tumor cells were defined as positively stained if their staining intensity with the KCTD12 antibody was higher (moderate to intense) than that of vascular endothelial cells, which served as internal positive controls. Cases with more than 20% of tumor cells staining positively with the KCTD12 antibody were considered to be KCTD12 positive because most cases that were classified as positive showed a diffuse (N50% of tumor cells) staining pattern. In most cases, the difference was so obvious that the 2 reviewers had consistent results. In case of disagreement, the staining was reevaluated and a consensus was reached on the reactivity.
2.3. Western blotting Frozen GIST samples were homogenized and suspended in ice-cold radioimmunoprecipitation assay buffer for 20 minutes. After centrifugation at 15 000 g for 10 minutes, the lysates were mixed in 2× sample buffer and boiled for 5 minutes. Then, the lysates were separated on 12% sodium dodecyl sulfate (SDS)–polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membrane was probed with the primary polyclonal KCTD12 antibody (1:100 dilution) and a secondary peroxidase-labeled antibody, then visualized using an Odyssey Fc Imaging System (LI-COR, Lincoln, NE) for the detection of protein by enhanced chemiluminescence.
2.4. Statistical analysis Fisher exact test was used to analyze the correlation between the clinicopathologic characteristics and KCTD12 expression. Recurrence-free survival (RFS) was defined as the time interval between the date of surgery and the date of GIST recurrence or of last follow-up. Prognostic value was
3 examined for demographic data (age at diagnosis, ≤69 years versus N69 years; sex), primary tumor site (stomach versus nonstomach), tumor size (≤5 cm versus 5-10 cm versus N10 cm), mitotic count (≤5 versus 6-10 versus N10 per 50 highpower fields [HPFs]), modified National Institutes of Health (NIH) criteria (low versus intermediate versus high risk), Ki67 (MIB-1) labeling index (b10% versus ≥10%), and KCTD12 (positive versus negative). Survival analyses were performed using Kaplan-Meier methods, and survival between the groups was compared by stratified and unstratified log-rank tests. A stepwise Cox proportional hazards model was used for multivariate analysis of the factors associated with RFS. P values less than .05 were considered statistically significant.
3. Results 3.1. Expression of KCTD12 in GISTs, nonneoplastic adult tissues, and other soft tissue tumors and carcinomas The immunohistochemical results of the 64 cases of GIST, 86 cases of soft tissue tumors, and 82 cases of carcinomas are summarized in Table 1. KCTD12 was positive in 52 (81%) of the GIST cases; KCTD12 immunoreactivity was diffusely positive in the membrane and cytoplasm of tumor cells (Fig. 1). Of the 22 GISTs analyzed for the presence of c-kit and PDGFRA gene mutations, 14 of the 19 cases with c-kit mutations and 2 of the 3 cases with PDGFRA mutations were positive for KCTD12. There was no correlation between KCTD12 expression and c-kit or PDGFRA mutation status. In Western blotting analysis (Fig. 2), the polyclonal KCTD12 antibody reacted with the lysates of GIST tissues with KCTD12 immunoreactivity (sample 2) but did not react with the lysates of tumor tissues without KCTD12 immunoreactivity (sample 1). In normal adult tissues, KCTD12 immunoreactivity was only observed in the dendrites of Purkinje cells in the cerebellum (Fig. 1) and in scattered, small stellate-shaped neurons of the cerebral cortex, but not in the stomach, small intestine, colon, lung, heart, liver, kidney, adrenal gland, pancreas, spleen, testis, ovary, uterus, bladder, prostate, skin, tonsil, or bone marrow. Both usual ICCs of the gastrointestinal tract and diffuse ICC hyperplasia associated with colon diverticulitis, highlighted by KIT immunostaining, were negative for KCTD12 (Fig. 1). For the soft tissue tumor groups, 2 (20%) of the 10 MPNSTs were positive for KCTD12; the immunoreactivity was focal and patchy in spindle tumor cells (Fig. 1). Two (20%) of the 10 synovial sarcomas showed focal and patchy KCTD12 expression in their epithelial and spindle cell components. Diffuse KCTD12 immunoreactivity was present in 1 (13%) of the 8 solitary fibrous tumors, whereas 1 (14%) of the 7 angiosarcomas showed KCTD12 expression in the solid
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Fig. 1 KCTD12 immunoreactivity in GISTs, normal and abnormal tissues, and soft tissue tumors. A, Diffuse positive KCTD12 expression in the membrane and cytoplasm of a GIST with c-kit exon 11 mutations. B, Negative KCTD12 expression in another GIST with c-kit exon 11 mutations. C, KCTD12 expression in the dendrites of Purkinje cells in the cerebellum. A KIT-positive ICC hyperplasia associated with colon diverticulitis (D) was negative for KCTD12 (E). F, Focal and patchy KCTD12 immunoreactivity in an MPNST. A-F, Original magnification ×200.
component. For the carcinoma groups, focal and patchy KCTD12 reactivity was seen in 1 (4%) of the 24 colon adenocarcinomas, whereas cutaneous squamous cell carcinomas, breast carcinomas, and pulmonary adenocarcinomas were negative for KCTD12.
3.2. Association of KCTD12 expression with clinicopathologic characteristics and survival analyses in 64 GIST cases The clinicopathologic characteristics of the 64 GIST patients are shown in Table 2. The median age at the time of diagnosis was 69 years (range, 25-87 years). Thirty-six tumors were located in the stomach, 22 in the small intestine, 4 in the rectum, and 2 in the omentum. The median tumor size was 6 cm (range, 1-25 cm). No patient had a ruptured GIST in our series. The median RFS was 108 months (range, 2-134 months), and the 5-year RFS rate was 76% (95% confidence interval [CI], 63.2-88.8). GIST recurred after surgery in 16 (25%) of the 64 cases during the follow-up.
As shown in Table 2, the absence of KCTD12 expression was associated with large tumor size (P = .004), high mitotic count (P b .0001), modified NIH high-risk criteria (P = .001), and high Ki-67 labeling index (P b .0001). In the survival analyses, a nonstomach site (P = .0041), tumor size greater than 10 cm (P = .0008), mitotic count more than 10/50 HPFs (P b .0001), modified NIH high-risk category (P b .0001), a Ki-67 labeling index of 10% or more (P b .0001), and absence of KCTD12 expression (P b .0001) had an adverse impact on RFS. The 5-year RFS rate of patients without KCTD12 expression was only 16.7% (95% CI, 4.437.8) compared with 95.6% (95% CI, 89.6-100) in those with KCTD12 expression (Fig. 3). However, on multivariate analysis, Ki-67 (relative risk, 0.09; 95% CI, 0.02-0.45; P = .003) and KCTD12 (relative risk, 5.45; 95% CI, 1.57-18.93; P = .008) were shown to be highly correlated predictors, although the Cox model did not give valid results for either individual predictor and did not reveal which predictor was redundant with respect to the other. Thus, a stratified logrank test was performed and showed that the 5-year RFS rate was significantly higher in the KCTD12-positive group than
KCTD12 expression in GISTs
Fig. 2 Evaluation of the polyclonal KCTD12 antibody by Western blotting (A) and immunohistochemistry (B) in GISTs. A single band of 35.7 kd was detected in tumor tissues of sample 2 with KCTD12 expression. In contrast, no detectable reaction was observed in tumor tissues of sample 1 without KCTD12 expression. B, Original magnification ×200.
in the KCTD12-negative group within each Ki-67 category. In the group with Ki-67 labeling index less than 10%, the 5-year RFS rate was 97.4% (95% CI, 92.5-100) for KCTD12-positive patients and 50% (95% CI, 0-100) for KCTD12-negative patients (P = .0052). In the group with a Ki-67 labeling index of 10% or more, the 5-year RFS rate was 85.7% (95% CI, 59.8-100) for the KCTD12-positive patients and 10% (95% CI, 0-100) for the KCTD12-negative patients (P = .0157).
4. Discussion The KCTD12 gene, which was first cloned from a human fetal cochlea library [11], is encoded by a locus on chromosome 13q22.3. KCTD12 protein, also known as pfetin, C13orf2, and PFET1, is an auxiliary subunit of GABAB receptors that determines the pharmacology and kinetics of the receptor response [12]. Recent in situ hybridization and immunohistochemical analyses of adult mouse tissues revealed that KCTD12 transcripts were detected at high levels in the brain and at low levels in the intestine, colon, kidney, heart, testis, and bone marrow, and the distinct axonal or dendritic localization of KCTD12 protein was shown in neuronal populations [16]. In this study, we first evaluated KCTD12 immunohistochemical expression using a commercially available polyclonal KCTD12 antibody in large numbers of GISTs and in mesenchymal and epithelial tumors that could be listed in the differential diagnosis of GIST. KCTD12 expression was observed primarily in vascular endothelial cells, Purkinje cells of the cerebellum, and some
5 neurons scattered throughout the cerebral cortex, but not in the other tissues examined, which was in agreement with previous studies [10,16]. The specificity of this polyclonal antibody for KCTD12 was confirmed by Western blotting, and a positive correlation between Western blotting and immunohistochemistry was demonstrated using some GIST cases. We found KCTD12 immunoreactivity in most GISTs (52/64 cases; 81%), irrespective of c-kit or PDGFRA mutation status, in line with a previous series using a monoclonal antibody (up to 80%) [13,14]. In KCTD12-positive GIST cases, because the tumor cells were stained diffusely with the polyclonal antibody in the membrane and cytoplasm, the assessment of immunohistochemical staining could be made easily. GISTs are currently thought to be derived from or to differentiate similar to gastrointestinal pacemaker cells, namely, ICCs. ICCs are KIT positive and are located in relation to the myenteric plexus of Auerbach and in the septa between circular muscle lamellae. Interestingly, KCTD12 was absent in not only ICCs but also ICC hyperplasia that was encountered incidentally in colon diverticulitis, which could be mistaken for small microscopic GISTs and represents early neoplasia [17,18]. We also found a predominantly focal and patchy pattern of KCTD12 immunostaining in rare examples of non-GISTs, including MPNSTs (2/10 cases; 20%), synovial sarcomas (2/10; 20%), solitary fibrous tumor (1/8; 13%), angiosarcoma (1/7; 14%), and colon adenocarcinoma (1/24; 4%). In particular, nonneoplastic ICCs and ICC hyperplasia were KCTD12 negative, whereas aggressive GISTs were also KCTD12 negative. This paradoxical phenomenon may be explained by the findings that KCTD12 is overexpressed in GISTs with c-kit or PDGFRA mutations [19], and its expression is then down-regulated by additional molecular alterations associated with a high Ki-67 labeling index and high mitotic count [20]. KIT (CD117) immunoreactivity is seen in approximately 95% of GIST cases. The high sensitivity and specificity of KIT make it a useful marker to differentiate GIST from other mesenchymal tumors of the gastrointestinal tract [1]. Another common marker that is not as sensitive or specific for GIST is CD34, which is expressed in approximately 70% of these tumors [1]. Likewise, KIT and CD34 immunoreactivity was found in 64 (100%) and 56 (87.5%) of our 64 GIST cases, respectively. There was no correlation between KIT or CD34 expression and KCTD12 expression (data not shown). Meanwhile, GIST1 (DOG1), which is a transmembrane protein recently shown to be up-regulated in GISTs by gene expression profiling, was shown to successfully identify most KIT-positive GISTs and approximately one-third of KIT-negative GISTs, the latter mostly harboring PDGFRA mutations. The overall sensitivity of DOG1 staining in GISTs is nearly 95%; however, depending on which DOG1 antibody was used in different studies, DOG1 staining has also been reported in normal gastric epithelium, carcinomas, germ cell tumors, melanomas, and rarely in some mesenchymal tumors [21]. Protein kinase C-θ is expressed in virtually all GISTs and is a very specific marker in Western
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T. Hasegawa et al. Table 2 Correlation between KCTD12 expression and clinicopathologic characteristics, survival analyses, and multivariate analysis in 64 cases of GIST Characteristic
No. of cases
KCTD12 positive
Age at diagnosis (y) ≤69 N69 Sex Female Male Tumor site Stomach Nonstomach Tumor size (cm) ≤5 5-10 N10 Mitotic count (/50 HPFs) ≤5 6-10 N10 Modified NIH risk criteria Low Intermediate High Ki-67 labeling index (%) a b10 ≥10 KCTD12 b Positive Negative a b
Correlation, P negative
5-y RFS (%) (95% CI)
.757
Log-rank P value .3934
34 30
27 25
7 5
69.8 (50.9-88.7) 84.7 (70.4-99.0)
38 26
31 21
7 5
36 28
32 20
4 8
24 32 8
24 23 5
0 9 3
43 4 17
42 4 6
1 0 11
22 14 28
22 13 17
0 1 11
47 17
45 7
2 10
94.7 (87.4-100) 30.9 (4.3-57.4)
52 12
52 0
0 12
95.6 (89.6-100) 16.7 (4.4-37.8)
1.000
.767 79.3 (63.4-95.3) 71.9 (51.8-92.0)
.108
.0041 88.9 (76.2-100) 60.9 (39.8-82.0)
.004
.0008 100 71.5 (51.8-91.2) 37.5 (3.9-71.1)
b.0001
b.0001 94.5 (87.1-100) 100 32.4 (5.9-58.8) b.0001
.001 100 100 50.9 (29.7-72.1) b.0001
b.0001 b.0001
Multivariate analysis for RFS, P = .003, 0.09 (relative risk: 95% CI, 0.02-0.45). Multivariate analysis for RFS, P = .008, 5.45 (relative risk: 95% CI, 1.57-18.93).
blotting. Immunohistochemical staining for protein kinase C-θ has been reported in GISTs, but because the commercially available antibodies to this protein show high background staining and limited specificity, they are of
Fig. 3 RFS curves for 64 patients with primary, resectable GISTs according to KCTD12 expression.
limited diagnostic use [21]. Overall, the specific expression of KCTD12 in GISTs from neoplastic and nonneoplastic adult tissues other than brain suggests that the role of KCTD12 extends beyond GABAB receptors and functions in the development and progression of GIST, and KCTD12 represents a significant improvement over well-established immunohistochemical panels for GIST diagnosis. In survival analyses of the 64 resectable GIST cases, along with other prognostic factors such as tumor size, mitotic activity, anatomic location, risk stratification, and Ki67 labeling index, KCTD12 expression correlated significantly with the RFS of the patients. The modified NIH consensus criteria used in this study are based on four factors: tumor size, tumor site, mitotic count, and the presence of tumor rupture, instead of only the 2 factors that are used in the NIH consensus criteria (ie, size and mitotic count) [7]. In an analysis of pooled population-based cohorts of operable patients with GIST, the modified NIH classification had the best criteria to identify a single high-risk group for the consideration of adjuvant therapy [22]. Ki-67 is a more objective proliferation marker than mitotic count [23], and patients with a Ki-67 labeling index of 10% or more had a
KCTD12 expression in GISTs worse 5-year survival rate than 140 resectable gastric GISTs with an index less than 10% [24]. The current univariate logrank test indicates that, taken alone, Ki-67 and KCTD12 are significant predictors of RFS, and KCTD12 expression provides additional information about RFS after accounting for Ki-67 status. As described previously, typically, the all-or-none KCTD12 staining pattern within tumor cells allows for the easy identification of high-risk patients with GISTs, even for small biopsy specimens taken with endoscopic ultrasoundguided fine-needle aspiration [25,26]. In fact, we observed a perfect concordance of a “positive” or “negative” assessment of KCTD12 staining between preoperative endoscopic ultrasound-guided fine-needle aspiration biopsy and resected specimens in several GIST cases examined so far. Moreover, the absence of immunostaining for KCTD12 with a polyclonal antibody is associated with the early recurrence of GIST within 5 years after surgery, and such patients might benefit from adjuvant imatinib therapy. Nevertheless, we found that a small number of KCTD12-positive GISTs can recur later. Therefore, we now also routinely include information about risk stratification and proliferative activity assessed by Ki-67 when reporting on resected GISTs. In summary, the present results suggest that KCTD12 is a useful and reliable biomarker for the diagnosis and prognosis of GIST.
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