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Pathological characteristics of gastrointestinal stromal tumours with PDGFRA mutations
Pathology (October 2009) 41(6), pp. 544–554
ANATOMICAL PATHOLOGY
Pathological characteristics of gastrointestinal stromal tumours with PDGFRA mutations JI EUN KWON*{, HYUN JU KANG*{, SE HOON KIM*, YONG CHAN LEE{, WOO JIN HYUNGx, SUNG HOON NOHx, NAM KYU KIMx AND HOGUEN KIM*{
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*Department of Pathology, {Brain Korea 21 Project for Medical Science, {Department of Internal Medicine, and xDepartment of Surgery, Yonsei University College of Medicine, Seoul, Korea
Summary Aims: The aim of this study was to identify the characteristics of PDGFRA-mutated gastrointestinal stromal tumours (GISTs) in comparison with KIT-mutated GISTs. Methods: A total of 151 GISTs were examined for KIT and PDGFRA mutations, and clinical and histopathological parameters were evaluated and analysed statistically according to mutation status. Results: KIT and PDGFRA mutations were detected in 123 (81.5%) and 15 cases (9.9%), respectively. Clinically, all PDGFRA-mutated GISTs were located in the stomach with no recurrences and tumour-related deaths were noted. Pathological parameters associated with PDGFRA mutations were epithelioid and mixed type, low to moderate cellularity, moderate to severe intratumoural lymphocytic infiltration, prominent myxoid change, frequent rhabdoid cells and multinucleated giant cells, increased cellular pleomorphism, low mitotic count, and lower risk assessment (p 5 0.05). Compared with KIT-mutated GISTs, PDGFRA-mutated GISTs had unique histopathological patterns, intermingling of spindle and epithelioid cells, and/or a jigsaw puzzle-like arrangement of epithelioid cells. Conclusions: PDGFRA-mutated GISTs have distinctive histological patterns that are differentiated from KIT-mutated GISTs. The algorithmic approach by a combination of several distinguishing histological and immunohistochemical features between KIT- and PDGFRA-mutated GISTs might be helpful in predicting the mutated gene of each GIST on pathological examination. Key words: Gastrointestinal stromal tumour, PDGFRA mutation, mixed type, epithelioid type. Received 16 March, revised 27 October, accepted 30 October 2008
INTRODUCTION GISTs are the most common mesenchymal tumours of the gastrointestinal tract.1,2 GISTs are characterised by differentiation similar to that of the interstitial cells of Cajal (ICC) and expression of KIT (CD117, stem cell factor receptor).3–5 Gain-of-function mutations in KIT are the most common genetic events in GISTs, resulting in autophosphorylation
of the KIT receptor, which in turn activates downstream signals.6,7 In-frame deletion mutation of exon 11 encoding the juxtamembrane domain is the most frequent mutation; however, internal tandem duplication of Ala502-Tyr503 in exon 9 encoding extracellular domain is also common.7–9 Point mutations of exons 11, 13, and 17 have also been reported.8–10 Recent studies have demonstrated that gain-of-function mutations in PDGFRA, a receptor tyrosine kinase and member of the type III receptor tyrosine kinase family, are present in 35–67% of GISTs lacking mutated KIT. These results suggest another molecular pathogenesis of GIST.11,12 Mutations of PDGFRA frequently occur in exon 18, which is involved in the activation of the second tyrosine kinase domain, exon 12 of the juxtamembrane domain, and exon 14 of the first tyrosine kinase domain.13–16 It is well known that mutations of KIT and PDGFRA are mutually exclusive,11,12,17 and that both are related to several clinicopathological factors.18–22 Identification of mutations in either KIT or PDGFRA in any case of GIST is important in prognostication and treatment. PDGFRA-mutated GISTs usually show favourable clinical courses,15 and GISTs with a substitution of aspartic acid to valine in 842 codon (D842V), the most common PDGFRA mutation, do not respond to imatinib mesylate (Gleevec; Novartispharma, Switzerland), which is known to be effective in treatment for metastatic or recurring GISTs with many types of KIT mutations.14,23 Recently, PDGRFRA-mutated GISTs have been identified as having gastric preponderance, and being of epithelioid or mixed type.15,18–20 However, the cellular component of epithelioid or mixed type is not enough for the identification of PDGFRA-mutated GISTs on routine histological examination by pathologists because KIT-mutated GISTs of mixed or epithelioid cell type are occasionally found. It might be necessary to choose the possible mutated gene, KIT or PDGFRA, by histological examination to reduce the cost of mutational study of both genes. The aim of this study was to analyse multiple clinical and pathological parameters of GISTs according to mutations in KIT and PDGFRA and demonstrate the distinguishing histological features of PDGFRA- from KIT-mutated GISTs, making prediction of the type of mutation in GISTs possible.
Print ISSN 0031-3025/Online ISSN 1465-3931 # 2009 Royal College of Pathologists of Australasia DOI: 10.1080/00313020903071421
PDGFRA-MUTATED GISTS
MATERIALS AND METHODS Case and clinicopathological features A total of 151 GISTs were retrieved from surgically resected or endoscopically removed gastrointestinal mesenchymal tumours between October 1995 and October 2004 from the Department of Pathology, Yonsei University College of Medicine, Korea, by reviewing haematoxylin and eosin (H&E) stained slides and immunohistochemical stain results. Clinical data including patient age, sex, tumour size, location, and follow-up information were obtained in all cases from hospital records. Another 61 GISTs for validation study were collected from GISTs diagnosed between January 2006 and July 2008 at the Department of Pathology, Yonsei University College of Medicine. All patients gave informed consent and authorisation for the use of tissues for research was obtained from the Institutional Review Board.
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Histopathological parameters and risk assessment Several histopathological parameters were analysed by reviewing H&E slides of all cases (Fig. 1). Cell type was divided into spindle cell, epithelioid, and mixed, according to previously described cell morphology.24 Spindle and epithelioid cells were differentiated by calculating the ratio of long to short diameter of cells. Cells with a ratio of less than 3 were regarded as epithelioid type. Mixed type was defined as a mixture of spindle and epithelioid cells when each proportion exceeded more than 10%. Cellularity was divided into three groups of low, moderate, and high (Fig. 1A–C). Intratumoural lymphocytic infiltration was recorded as none to mild [100 or fewer lymphocytes per 10 high power fields (HPFs)], moderate (101–200 lymphocytes per 10 HPFs), or severe (more than 200 lymphocytes per 10 HPFs) (Fig. 1D,E). Nuclear pleomorphism was recorded as none, mild, or moderate (Fig. 1G,I). The presence or absence of fascicular or syncytial arrangement, nuclear palisading, necrosis, ulceration, mucosal invasion, perinuclear vacuolisation, skeinoid fibre, rhabdoid cells, and bi- or multinucleated cells was also assessed (Fig. 1J,K). Angiocentric lobule formation was recorded and arbitrarily defined by lobular structure composed of tumour cells surrounding blood vessels and occasionally demarcated by a cleft or clear space associated with liquefactive change (Fig. 1L). Hyalinisation was recorded as none to focal (510%) and diffuse (410%), microcystic change as none to focal (520%) and diffuse (420%), and myxoid change as none to focal (550%) and extensive (450%) (Fig. 1M–O). Mitoses were counted in 50 HPFs. Based on mitotic counts per 50 HPFs and tumour size, GISTs were classified into four risk groups as previously described by Fletcher et al.24
Immunohistochemical stain Three representative cores (1.0 mm) of the area of tumour were taken from the original paraffin blocks and tissue microarray blocks were made. Immunohistochemistry was performed using an avidin–biotin peroxidase complex system with diaminobenizidine (DAB) as the chromogen. Primary antibodies included c-kit (polyclonal rabbit antibody, 1:50 dilution; Dako, Denmark) and CD34 (monoclonal mouse antibody, 1:50 dilution; Dako). Immunohistochemical results were evaluated semiquantitively as follows:21 Strong positivity, more than 10% of tumour cells were moderately or strongly positive; weak positivity, less than 10% of tumour cells were moderately or strongly positive or more than 10% of tumour cells were weakly positive; and negative, less than 10% of tumour cells were weakly positive or there were no positive cells.
Mutational analysis For DNA extraction, GIST tissues were microdissected at a thickness of 10 mm from paraffin blocks or fresh frozen tissues composed totally of tumour area. Exons 9, 11, 13, and 17 of the KIT gene and exons 12 and 18 of the PDGFRA gene were evaluated for mutations using PCR amplification of genomic DNA and direct sequencing. Genomic DNA was extracted using the SDS-proteinase K and phenol-chloroform
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method. The primers used in this study to amplify KIT and PDGFRA are shown in Table 1. PCR conditions consisted of an initial denaturation at 958C for 15 min, followed by 30 cycles of 948C for 45 s, 558C for 45 s, and 728C for 45 s. The final elongation step was at 728C for 5 min. PCR products were separated on 6% polyacrylamide gels, followed by autoradiography for single-strand conformation polymorphism analysis. The amplified products were directly sequenced on a 3100 Genetic Analyser (Applied Biosystems, USA).
Statistical analysis Statistical analysis was performed using SPSS for Windows (version 12.0; SPSS Inc., USA). To make comparisons between KIT- and PDGFRA-mutated GISTs, clinical or histopathological parameters, risk assessment, and immunohistochemical results were evaluated using either chi-square test, Fisher’s exact test, or MannWhitney test. Results with p values of 50.05 were considered statistically significant.
RESULTS Mutational analysis of KIT and PDGFRA in 151 GISTs Among the 151 analysed GISTs, KIT mutations were found in 123 cases (81.5%) and PDGFRA mutations were found in 15 (9.9%). No mutations of either gene were found in the remaining 13 (9.3%) GISTs (Table 2). For GISTs with mutations in KIT, the most frequent mutations were found in exon 11 (109/123, 89%), followed by 10 cases with mutations in exon 9 and two cases of mutations in exons 13 and 17, respectively (Table 2). Among the 109 mutations in exon 11, a deletion was found in 60 cases, insertion in 26, and point mutations in 23. The most frequent type of deletion (10 cases) was a 6 bp deletion of codons 557–558, leading to loss of tryptophan and lysine amino acids. The most frequent type of point mutation was V559D, which was found in 12 cases. In the 15 PDGFRA mutations, 14 cases were found in exon 18 and one in exon 12. The most frequent type of PDGFRA mutation was a substitution of aspartic acid with valine in codon 842 (D842V), which was found in 13 cases. The remaining cases showed a 12 bp deletion in codon 842–846 in exon 18 and a 15 bp deletion of codons 556–571 in exon 12. The results of mutational analysis of all cases are listed in Table 3. Clinical characteristics of GISTs according to mutation type Clinically, PDGFRA-mutated GISTs more commonly occurred in males in contrast to KIT-mutated GISTs with equal prevalence in both sexes: KIT-mutated GISTs included 61 females and 62 males whereas PDGFRAmutated GISTs included four females and 11 males (p ¼ 0.108). All of the PDGFRA-mutated GISTs were present in the stomach whereas 89 (72.4%) of the 123 KIT-mutated GISTs were present in the stomach (p ¼ 0.022). Of the 13 GISTs lacking KIT or PDGFRA mutations, nine were present in the stomach and four in the small intestine. We evaluated the recurrence and metastasis of the 151 GISTs during a mean follow-up period of 40.9 months and found that 27 of the 123 (22%) KIT-mutated GISTs showed recurrence or metastasis while none of the 15 PDGFRA-mutated GISTs showed recurrence or metastasis
KWON et al.
Pathology (2009), 41(6), October
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FIG. 1 Histopathological parameters of GISTs evaluated in this study. Cellularity: (A) low, (B) medium, (C) high. Infiltration of intratumoural lymphocytes: (D) none to mild, (E) moderate, (F) severe. Nuclear pleomorphism: (G) none, (H) mild, (I) moderate. (J) Necrosis; (K) skeinoid fibre; (L) angiocentric lobule formation; (M) hyalinisation; (N) microcystic change; and (O) myxoid stroma (H&E).
at either the time of surgery or during follow up. Among the 27 GISTs with recurrence or metastasis, six cases had metastasised at the time of surgery and 21 developed
metastasis after curative surgery. The comparison of clinicopathological factors and follow-up results are summarised in Table 4.
PDGFRA-MUTATED GISTS
TABLE 1
Primer sequences used for PCR of KIT and PDGFRA genes
Gene
Primers
Primer sequences
KIT
hEx9F hEx9R hEx11F hEx11R hEx13F hEx13R hEx17F hEx17R
50 -TCCTAGAGTAAGCCAGGGCTT 50 -TGGTAGACAGAGCCTAAACATCC 50 -CCTTTGCTGATTGGTTTCGT 50 -AAACAAAGGAAGCCACTGGA 50 -GTTCCTGTATGGTACTGCATGCT 50 -CAGTTTATAATCTAGCATTGCC 50 -CATCATTCAAGGCGTACTTTTG 50 -TCACAGGAAACAATTTTTATCGAA
hEx12F hEx12R hEx18F hEx18R
50 -AAGCTCTGGTGCACTGGGACTT 50 -ATTGTAAAGTTGTGTGCAAGGGA 50 -TACAGATGGCTTGATCCTGAGT 50 -AGTGTGGGAGGATGAGCCTG
TABLE 3 GISTs
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Results of mutational analysis for KIT and PDGFRA in 151
Type of mutation
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PDGFRA
TABLE 2 Distribution of mutation type in 138 GISTs with KIT and PDGFRA mutations PDGFRA mutated (n ¼ 15)
KIT mutated (n ¼ 123) Mutation type Point mutation Deletion Insertion Total (%)
Exon 9
Exon 11
2
23
0 8 10 (8)
60 26 109 (89)
Exon 13 Exon 17 Exon 12
Exon 18
2
2
0
13
0 0 2 (2)
0 0 2 (2)
1 0 1 (7)
1 0 14 (93)
Comparison of histopathological parameters of GISTs according to KIT and PDGFRA mutations Pathologically, the 123 KIT-mutated GISTs were categorised as spindle cell type in 96 (78%) cases, epithelioid type in three (2.4%), and mixed type in 24 (19.5%). Of the 15 PDGFRA-mutated GISTs, two (13.3%) cases were categorised as epithelioid type and 13 (86.7%) as mixed type. Besides cell type, we found that many characteristic histological factors were related to the type of mutation of GISTs. Individual histological factors frequently present in KIT-mutated GISTs (Fig. 2) were of high cellularity (47% versus 7%, p ¼ 0.002), showed fascicle/syncytium formation (95% versus 27%, p 5 0.001) (Fig. 2A), nuclear palisading (76% versus 13%, p 5 0.001) (Fig. 2E), angiocentric lobule formation (29% versus 0%, p ¼ 0.011) (Fig. 2F), and high mitotic rate (12.1 versus 1.5, p 5 0.001). In PDGFRA-mutated GISTs (Fig. 3), nuclear pleomorphism (51% versus 93%, p ¼ 0.002), rhabdoid cell formation (11% versus 60%, p 5 0.001) (Fig. 3E), binucleated or multinucleated cell formation (63% versus 100%, p ¼ 0.003) (Fig. 3F), myxoid change (51% versus 93%, p 5 0.001), infiltrative border (19% versus 53%, p ¼ 0.006), and intratumoural lymphocyte infiltration (22% versus 67%, p ¼ 0.001) were more frequent (Table 5). There were no significant differences in the other histological factors between KIT- and PDGFRA-mutated GISTs. Comparing the risk assessment of GISTs according to the consensus of the NCI24 [high risk (45 cm and 45 mitotic index (MI)/50 HPF, or 410 cm with any mitotic count, or 410 MI/50 HPF with any size), intermediate risk (5–10 cm and 5 MI/50 HPF, or 5 cm and 6–10 MI/50
Exon 9 9 9 9 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 13 17 12 18 18
No. cases 6 2 1 1 15 12 10 4 4 4 4 3 3 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 13 1 13
KIT
PDGFRA
A502_Y503 ins F506_F508 ins G498S V457M T574_Y578 ins V559D W557_K558 del V560_L576 del D579 del V559_T574 del W557R V560_E561 del V560D Q556_V559 del Q556_K558 del P551_V569 del M552_Y553 del Y553_Q556 del Y553_E561 del L576_R586 ins W557_G564 del L576_P577 ins W557G K550_Y568 del K550_Q556 del L576P P573_L589 ins W557_Y558 del K550_W557 del V560E L556_D569 ins V555_W557 del E554_W557 del Q575_F591 ins V559_D572 del M552_K558 del K550_V555 del P577_H580 del M552_N572 del K558_I563 del Q575_D580 del T574_K581 del Y553_W557del Y578_D579 del D572_R588 ins W557_K558 ins Q556_N564 del K550_K558 del V560 del V559A K558_V559 del T574_R586 ins P551_M552 del I571_K581 ins P577-H580 ins T574-Y578 ins M552_E554 del P551_V555 del K550_V559 del K642E N822K Wild Wild Wild Wild
Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild S556_E571 del D842V D842_D846 del Wild
del, deletion; ins, insertion.
HPF), low risk (2–5 cm and 5 MI/ 50HPF), and very low risk (2 cm and 5 MI/50 HPF)], 85 of the 123 (69.1%) KIT-mutated GISTs were categorised in the
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Pathology (2009), 41(6), October
TABLE 4 Comparison of clinical parameters between GISTs with KIT and PDGFRA mutations
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Variable and category
KIT mutated (n ¼ 123)
PDGFRA mutated (n ¼ 15)
KIT wild and PDGFRA wild (n ¼ 13)
Total (n ¼ 151)
Age, years Mean (SD) 57 (12.2) 53 (10.0) 56 (13.0) 57 (12.0) Gender Male 62 (50.4%) 11 (73.3%) 4 (30.8%) 77 (51.0%) Female 61 (49.6%) 4 (26.7%) 9 (69.2%) 74 (49.0%) Location Oesophagus 1 (0.8%) 0 0 1 (0.7%) Stomach 89 (72.4%)* 15 (100%) 9 (69.2%) 113 (74.8%)* Small bowel 23 (18.7%) 0 4 (30.8%) 27 (17.9%) Large bowel 10 (8.1%) 0 0 10 (6.6%) Follow up, months Mean (SD) 41.3 (32.9) 33.7 (31.0) 45.6 (30.2) 40.9 (32.4) No. of recurrence/metastasis Mets 6 (4.9%) 0 1 (7.7%) 7 (4.6%) Recur 21 (17.1%) 0 1 (7.7%) 22 (14.6%) 5-year survival Event free 68.1% 100% 82.1% 72.1% Overall 82.2% 100% 92.3% 85.2% *Includes one GIST located in the omentum. Mets, metastasis at the time of surgery; Recur, recurrence or metastasis after curative resection; SD, standard deviation.
intermediate- and high-risk groups while five of the 15 (33%) PDGFRA-mutated GISTs were in the intermediateand high-risk groups (p ¼ 0.035) (Table 5). Recurrence and/ or metastasis were noted in 24 of 51 (47.1%) KIT-mutated GISTs that were regarded as the high-risk group, in three of 34 (8.8%) KIT-mutated GISTs regarded as the intermediate-risk group, and none in the low- and very low-risk groups as well as PDGFRA-mutated GISTs. Immunohistochemical result of GISTs Among the 123 KIT-mutated GISTs, 117 (95.1%) were strongly positive for KIT immunostaining (Fig. 2G). In contrast, four of the 15 PDGFRA-mutated GISTs showed strong positivity for KIT immunostaining, and six cases showed weak expression of KIT (Fig. 3G). Furthermore, four cases with PDGFRA mutations were negative for KIT immunostaining. Immunohistochemical results for CD34 showed that 101 of the 123 (82.1%) KITmutated GISTs were positive and 10 of the 15 (66.7%) PDFGRA-mutated GISTs were positive (Table 6). Histological characteristics of PDGFRA-mutated GISTs: a pathological algorithmic approach for prediction of KIT or PDGFRA mutation type We found that no PDGFRA-mutated GIST was composed purely of spindle cells with a fascicular or syncytial growth pattern, which was the most common histological feature of KIT-mutated GISTs of spindle cell type. Because all of the PDGFRA-mutated GISTs and 22% of KIT-mutated GISTs were of epithelioid or mixed cell type, we compared the pathological characteristics between PDGFRA- and KIT-mutated GISTs of epithelioid and mixed type. Of the 15 PDFGRA-mutated GISTs, two were composed of pure epithelioid cells. Both of these cases showed
characteristic epithelioid cells with distinct cellular borders similar to a jigsaw puzzle pattern (Fig. 3A); specifically, each tumour cell resembled pieces of a jigsaw puzzle and had abundant cytoplasm. In the 24 KIT-mutated GISTs of epithelioid or mixed type, three cases were composed of pure epithelioid cells. These tumours were arranged in small clusters or a solid sheet; however, they were more cellular, and the tumour cells exhibited less distinct cellular borders and low amounts of cytoplasm compared with the epithelioid cell type of PDGFRA-mutated GISTs (Fig. 2C,D). In PDGFRA-mutated GISTs of mixed type, all but one case exhibited a so-called ‘intermingled pattern’ of discohesively and singly scattered spindle and epithelioid cells arranged in a haphazard fashion without areas of fascicle formation or syncytial growth. The tumour cells and their nuclei frequently showed either a fusiform or stellate shape and indented appearance with occasional multinucleated and binucleated cells. Tumour cells revealed mild to moderate pleomorphism due to the composition of cells of variable sizes and morphology but no evidence of obvious atypia. These tumours had in common a prominent myxofibrillary intervening stroma that was sometimes indistinct from the fibrillary cytoplasm, and moderate to low cellular density was also noted (Fig. 3B–F). In the one case of GIST with PDGFRA-mutation in exon 12, a patchy area of fascicular arrangement in addition to both intermingled areas of cells of various shapes and jigsaw puzzle-like areas of epithelioid cells were noted. In contrast, among the 24 KIT-mutated GISTs of mixed type, only three cases had areas showing intermingled pattern while the remaining 21 exhibited an alternative pattern composed of areas of spindle cells with fascicle formation and syncytial growth and areas of epithelioid cells with solid or clustered patterns (Fig. 2B). We highlighted the clinicopathological and immunohistochemical characteristics of PDGFRA-mutated GISTs, comparing them with those of previous studies, in Table 7. Based on the distinct histological patterns and differential KIT immunoreactivity, we suggest an algorithmic diagnostic approach for predicting mutation status (Fig. 4). Verification of algorithm for predicting mutational status of GISTs To verify the adequacy of this algorithm, two pathologists independently examined another set of 61 GIST cases according to this algorithm and predicted the mutated gene, either KIT or PDGFRA. After completion of the pathological examination, we performed mutational analysis by the same method and compared revealed mutation to putative mutation. The results are listed in Table 8. Among the 50 KIT-mutated GISTs, only one case was predicted to have PDGFRA mutation by pathologist A. Sensitivity, specificity, positive predictive value, and negative predictive value for KIT-mutated GISTs were 98.0%, 54.5%, 90.7% and 75.7%, respectively. The accuracy of this algorithm predicting KIT-mutated GIST is 90.2%. With regard to PDGFRA-mutation, sensitivity, specificity, positive predictive value, and negative predictive value were 85.7%, 85.7%, 98.1%, and 98.1%, respectively. The accuracy of the algorithm predicting PDGFRA-mutated GIST was 96.7%. Differences between sensitivity and positive
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PDGFRA-MUTATED GISTS
FIG. 2 Histological features of KIT-mutated GISTs. (A) Spindle cell type GIST composed of uniform spindle cells showing a fascicular and/or syncytial arrangement and high cellularity (H&E). (B) Mixed type GIST showing an alternative pattern of spindle cell area (centre) and epithelioid cell area (right lower) (H&E). (C) Epithelioid GIST arranged in a solid sheet with less distinctive cellular borders and high cellularity (H&E). (D) Epithelioid GIST arranged in small clusters. (E) Nuclear palisading was associated with KIT-mutated GISTs (H&E). (F) Angiocentric lobules were also associated with KIT-mutated GISTs (arrow) (H&E). (G) Most of the KIT-mutated GISTs were strongly positive for KIT immunostaining (c-kit).
predictive value in predicting KIT-mutated GISTs and between accuracy for predicting KIT-mutated GISTs and for predicting PDGFRA-mutated GISTs were derived from the existence of GISTs lacking both KIT and PDGFRA mutation, which were interpreted to have KIT mutation by both pathologists. When we excluded these four GISTs lacking KIT and PDGFRA mutation, sensitivity, specificity, and accuracy for KIT mutation rose to 98.0%, 85.7%, and 96.5%, respectively.
DISCUSSION Gain of function mutations in KIT and PDGFRA have been proposed as causative genetic events in the tumorigenesis of GISTs.6,11,12 Mutations of these two genes are known to be mutually exclusive. In this study, we demonstrated the presence of several different clinical and pathological characteristics of GISTs according to KIT and PDGFRA mutation.
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FIG. 3 Histological features of PDGFRA-mutated GISTs. (A) Eithelioid type GIST showing a jigsaw puzzle-like pattern (H&E). (B–D) Mixed type GISTs showing an intermingled pattern. Spindle cells, stellate-shaped cells, and epithelioid/round cells with mild to moderate nuclear pleomorphism were singly and discohesively intermingled without fascicular or syncytial formation. These GISTs were less cellular and had prominent myxofibrillary stroma and a moderate abundance of intratumoural lymphocytes. Binulceated cells were frequently noted (H&E). (E) Rhabdoid cells were more frequently noted (H&E). (F) Multinucleated giant cells were occasionally found (H&E). (G) Weak positivity to KIT immunostaining (c-kit).
Our study confirmed several previously reported clinical and pathological characteristics of GISTs. Of the 151 GISTs analysed, 123 (81.5%) had mutations in KIT and 15 (9.9%) had mutations in PDGFRA. PDGFRA-mutated GISTs had gastric and male preponderance compared with KIT-mutated GISTs.15,18–20 We also confirmed that PDGFRA-mutated GISTs had a marked preponderance of epithelioid and mixed type: all of the 15 PDGFRAmutated GISTs were epithelioid and mixed type while only 27 of the 123 KIT-mutated GISTs were classified as
epithelioid and mixed type. Because all of the PDGFRAmutated GISTs and 20–30% of KIT-mutated GISTs were categorised as epithelioid and mixed type, the identity of the mutated gene could not be predicted in these subsets of GISTs. We found that the overall pattern was more important than constituent cell type itself for predicting the genotype of GISTs. We found that PDGFRA-mutated GISTs of epithelioid cell type were characterised as a sheet of epithelioid cells with distinct cell borders and abundant cytoplasm, occasionally resembling pieces of a jigsaw
PDGFRA-MUTATED GISTS
TABLE 5 Comparison of histopathological parameters and risk assessment between KIT- and PDGFRA-mutated GISTs Type of mutation
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Histological variables and category
KIT (n ¼ 123)
Cell type, n (%) Spindle 96 (78.0) Epithelioid or mixed 27 (22.0) Cellularity, n (%) Low to moderate 65 (52.8) High 58 (47.2) Fascicle/syncytium, n (%) Absent 6 (4.9) Present 117 (95.1) Intratumoral lymphocytes, n (%) None to mild 96 (78.0) Moderate to severe 27 (22.0) Nuclear pleomorphism, n (%) None 60 (48.8) Mild to moderate 63 (51.2) Myxoid change, n (%) None to focal 72 (58.5) Prominent 51 (41.5) Giant cell, n (%) Absent 45 (36.6) Present 78 (63.4) Rhabdoid cell, n (%) Absent 109 (85.6) Present 14 (11.4) Necrosis, n (%) Absent 72 (58.5) Present 51 (41.5) Ulceration, n (%) Absent 94 (76.4) Present 29 (23.6) Infiltrative border, n (%) Absent 100 (81.3) Present 23 (18.7) Mucosal invasion, n (%) Absent 99 (80.5) Present 24 (19.5) Perinuclear vacuolisation, n (%) Absent 34 (27.9) Present 88 (72.1) Microcystic change, n (%) None to focal 77 (62.6) Prominent 46 (37.4) Nuclear palisading, n (%) Absent 30 (24.4) Present 93 (75.6) Skeinoid fibre, n (%) Absent 115 (93.5) Present 8 (6.5) Hyalinisation, n (%) None to focal 98 (79.7) Prominent 25 (20.3) Angiocentric lobule, n (%) Absent 87 (70.7) Present 36 (29.3) Mitosis/50 HPF 0–5, n (%) 65 (52.8) 6–10, n (%) 22 (17.9) 410, n (%) 36 (29.3) Mean 12.2 + 18.7 Size, cm 0–5, n (%) 68 (55.3) 6–10, n (%) 39 (31.7) 410, n (%) 16 (13.0) Mean 6.1 + 4.9 Risk assessment, n (%) Very low 11 (8.9) Low 27 (22.0) Intermediate 34 (27.6) High 51 (41.5) *p 5 0.05.
PDGFRA (n ¼ 15)
p value
0 (0) 15 (100)
50.001*
14 (93.3) 1 (6.7)
0.002*
11 (73.3) 4 (26.7)
50.001*
5 (33.3) 10 (66.7)
0.001*
1 (6.7) 14 (93.3)
0.002*
1 (6.7) 14 (93.3)
50.001*
0 (0) 15 (100)
0.003*
6 (40.0) 9 (60.0)
50.001*
13 (86.7) 2 (13.3)
0.047*
14 (93.3) 1 (6.7)
0.191
7 (46.7) 8 (53.3)
0.006*
14 (93.3) 1 (6.7)
0.306
7 (46.7) 8 (53.3)
0.145
7 (46.7) 8 (53.3)
0.233
13 (86.7) 2 (13.3)
50.001*
15 (100) 0 (0)
0.598
15 (100) 0 (0)
0.073
15 (100) 0 (0)
0.011*
14 (93.3) 1 (6.7) 0 (0) 1.5 + 1.9
0.006*
10 (66.7) 3 (20.0) 2 (13.3) 5.6 + 5.4 3 7 3 2
(20.0) (46.7) (20.0) (13.3)
50.001* 0.685
0.287 0.035*
551
puzzle. In contrast, the three KIT-mutated GISTs of epithelioid type showed high cellularity, indistinct cellular borders, and low amounts of cytoplasm. In the GISTs of mixed type, most of the KIT-mutated GISTs showed an alternative arrangement of spindle and epithelioid cell areas. In contrast, PDGFRA-mutated GISTs showed an intermingled pattern of singly and discohesively scattered epithelioid, fusiform, stellate-shaped, and spindle cells. Fascicular arrangement of spindle cells was the hallmark of KIT-mutated GISTs whereas few areas of fascicle arrangement were evident in most of the PDGFRA-mutated GISTs. Other characteristic features of PDGFRA-mutated GISTs were low cellularity, more prominent myxofibrillary stroma, increased cellular pleomorphism, occasional rhabdoid and binucleated or multinucleated giant cells, and increased intratumoural lymphocytic infiltration. In a previous study of the pathological features of GISTs according to mutational status, Pauls et al.21 reported that all PDGFRA-mutated GISTs with epithelioid or mixed type have multinucleated giant cells, and this finding can be useful to predict the genotype of GISTs. In our cases, we found that 63.4% of the KIT-mutated GISTs also had at least one bi- or multinucleated giant cell, thus the presence of giant cells is by itself not sufficient evidence to discriminate between KIT- and PDGFRA-mutated GISTs. Most of the KIT-mutated GISTs showed strongly positive immunoreactivity to KIT; however, only 26.6% of the PDGFRA-mutated GISTs were strongly positive for KIT. These results are in accordance with previous reports that many KIT-negative GISTs contain PDGFRA mutation.13,25 Pathological diagnosis of PDGFRA-mutated GISTs may be difficult due to negative or weak immunoreactivity to KIT and/or CD34. Therefore, pathologists should become familiar with unique morphological findings of PDGFRAmutated GISTs, which we have described above in detail in order to assist with the diagnosis of PDGFRA-mutated GISTs. Based on distinguishing histological patterns and differential KIT immunoreactivity, we propose an algorithmic approach to predict the type of mutation of GISTs and verified it by using another validation set of GISTs. Sensitivity and specificity for predicting PDGFRA mutation of this algorithm are 85.7% and 95.1%. We expect that this algorithmic approach will be helpful for the initial selection of the gene for mutational analysis because mutations of KIT and PDGFRA in GISTs are known to be mutually exclusive. The clinical and histological parameters for risk assessment of GISTs consist of tumour size and mitotic count of tumour cells. When we classified our GISTs according to the risk assessment proposed by Fletcher et al.,24 69.1% of KITmutated GISTs were categorised into the intermediate- and high-risk groups while 33.3% of PDGFRA-mutated GISTs were also categorised in the intermediate- and high-risk groups. Because there was no significant size difference between KIT- and PDGFRA-mutated GISTs, the lower mitotic index in PDGFRA-mutated GISTs was related to the favourable group preponderance of PDGFRA-mutated GISTs. Among the evaluated histological factors, multinucleated giant cells were related to poor prognosis in KITmutated GISTs but not in PDGFRA-mutated GISTs. In KIT-mutated GISTs with recurrence or metastasis, most of the tumours contained multinucleated giant cells (p 5 0.001). In contrast, even though multinucleated giant
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TABLE 6
Pathology (2009), 41(6), October
Immunohistochemical expression of KIT and CD34 according to mutation status KIT immunoreactivity
CD34 immunoreactivity
Type of mutation
Negative
Weak
Strong
p value
Negative
Weak
Strong
p value
KIT PDGFRA
4 (3.3%) 5 (33.3%)
2 (1.6%) 6 (40.0%)
117 (95.1%) 4 (26.6%)
50.001
13 (10.6%) 2 (13.3%)
9 (7.3%) 3 (20.0%)
101 (82.1%) 10 (66.7%)
0.145
TABLE 7
Comparison of characteristics of PDGFRA-mutated GISTs between previous studies and ours
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Characteristics of PDGFRA-mutated GISTs Comparison category
Histological findings
Immunohistochemical findings
Clinical findings
Epithelioid cell or mixed phenotype15,18–22* Multinuclear giant cells21* Rhabdoid cell (less specific but helpful)22* Low mitotic activity15*
Weak immunoreactivity to KIT20,21
Gastric preponderance15,18,19 Favourable course15
Concordant findings
Not evaluated in this study Dot-like staining of PDGFRA21 New findings Low cellularity Intermingled pattern: discohesively scattered cells Jigsaw puzzle-like: distinct cell membrane Rare fascicular arrangement
Lower expression of CD34
Lower-risk group
*These histological features can be found in some KIT-mutated GISTs.
FIG. 4 Algorithm for predicting the mutational status of GISTs by pathological parameters.
cells were present in all 15 of the PDGFRA-mutated GISTs, no recurrence or metastasis was present. The clinical significance of the presence of multinucleated cells differs according to the type of mutated gene of the GIST.
Molecular classification of GISTs is important for accurate prognostication and predicting therapeutic response. Many recent studies have demonstrated that there is a strong correlation between the type of KIT mutation
PDGFRA-MUTATED GISTS
TABLE 8
Comparison of predicted mutation and type of mutation of 61 GISTs for the validation of the proposed algorithm of this study Predicted mutation
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Case no.
Histological type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Pure spindle Pure spindle Mixed Pure spindle Pure spindle Pure spindle Mixed Pure spindle Epithelioid Pure spindle Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Mixed Mixed Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Mixed Mixed Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Mixed Pure spindle Mixed Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Pure spindle Mixed Mixed Pure spindle Pure spindle
Type of mutation
Pathologist A
Pathologist B
K K K K K K K K K K P K K K K K K K P K K K K K K K K K K K K K K K K K P K P K K K K K K P K K K P K K K K K K K K P K K
K K K K K K K K K K P K K K K K K K P K K K K K K K K K K K K K K K K K P K K K K K K K K P K K K P K K K K K K K P K K K
KIT
PDGFRA
T574_Y578 ins W557_E561 del A502_Y503 ins D579del N564_N566 del W557_K558 del K642E L576_P577 del Q575_L576 del L576_P577 del Wild W557_K558 del Q556_W557 del V560D L576P Wild K642E V559_E561 del Wild V560D V559D Wild I571_Y578 del V560D Q556-L576 del Wild A502_Y503 ins V560del K558_V560 del I563-L576 del T574_P577 ins K558_E562 del V590E Q556_V560 del M552-V569 del V560 del Wild A502_Y503 ins V560_E561 del P551-E554 del W557-K558 del V559G W557_K558 del V559A Y553_E554 del Wild M552_I571 del W557_V559 del A502_Y503 ins Wild W557A V559D V559D V559G Wild V559D Y570_L576 del Wild Wild W557_K558 del N822K
Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild D842V Wild Wild Wild Wild Wild Wild Wild D842V Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild D842V Wild Wild Wild Wild Wild Wild Wild Wild D842V Wild Wild Wild D842V Wild Wild Wild Wild Wild Wild Wild D842V D842V Wild Wild
del, deletion; ins, insertion; K, KIT; P, PDGFRA.
and therapeutic response to imatinib mesylate (Gleevec; Novartispharma).23 The mutation of D842V of exon 18, the most common mutation in PDGFRA, has been reported to be resistant to imatinib mesylate treatment both in vivo and in vitro.14,23 In contrast, the V561D
mutation of exon 12 and deletion mutation of exon 18 has been reported to be partially responsive to imatinib mesylate treatment in approximately two-thirds of patients.23 These findings indicate that mutation analysis of PDGFRA is also important for predicting chemotherapeutic
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response. PDGFRA-mutated GISTs are known to have more favourable prognosis, and this was again confirmed in our study. When we compared the prognosis of our GISTs of epithelioid and mixed type, the most striking differences were found between the PDGFRA- and KIT-mutated GISTs. In the 27 KIT-mutated GISTs of epithelioid or mixed type, 16 cases showed recurrence and metastasis and nine patients died during the follow-up period. In contrast, no PDGFRA-mutated GISTs having epithelioid cell or mixed type showed recurrence, metastasis, or death during follow up despite the fact that 33% were categorised into the intermediate- or high-risk groups. The good prognosis of our PDGFRA-mutated GISTs might be related to the type of mutations, as no case of the V561D mutation, which had been reported to show bad prognosis,12 was present in our 15 PDGFRA-mutated GISTs. These findings indicate that mutational analysis is important for the prognostication of GISTs, especially epithelioid and mixed type. In summary, we demonstrated that PDGFRA-mutated GISTs have unique clinicopathological features compared with KIT-mutated GISTs, and overall histological pattern or an intermingled or jigsaw puzzle-like arrangement is the most important difference that distinguishes PDGFRAfrom KIT-mutated GISTs. We propose an algorithmic approach by combining the most distinguishing features of PDGFRA-mutated GISTs, and this approach might be useful for predicting either KIT or PDGFRA mutation on histological examination prior to mutational analysis. ACKNOWLEDGEMENTS Ji Eun Kwon and Hyun Ju Kang contributed equally to this work. This work was supported by Korea Health 21 R&D Project [Ministry of Health and Welfare (03-PJ10-PG6-GP01-0002)] the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R11-2000-082-02005-0). Address for correspondence: Professor H. Kim, Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemungu, CPO Box 8044, Seoul 120–752, Korea. E-mail: [email protected]
References 1. Miettinen M, Lasota J. Gastrointestinal stromal tumors—definition, clinical, histological, immunohistochemical, and molecular genetic features and differential diagnosis. Virchows Arch 2001; 438: 1–12. 2. Miettinen M, Lasota J. Gastrointestinal stromal tumors (GISTs): definition, occurrence, pathology, differential diagnosis and molecular genetics. Pol J Pathol 2003; 54: 3–24. 3. Robinson TL, Sircar K, Hewlett BR, et al. Gastrointestinal stromal tumors may originate from a subset of CD34-positive interstitial cells of Cajal. Am J Pathol 2000; 156: 1157–63. 4. Sircar K, Hewlett BR, Huizinga JD, et al. Interstitial cells of Cajal as precursors of gastrointestinal stromal tumors. Am J Surg Pathol 1999; 23: 377–89. 5. Kindblom LG, Remotti HE, Aldenborg F, et al. pacemaker cell tumor (GIPACT): gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal. Am J Pathol 1998; 152: 1259–69.
Pathology (2009), 41(6), October
6. Hirota S, Isozaki K, Moriyama Y, et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science. 1998; 279: 577–80. 7. Rubin BP, Singer S, Tsao C, et al. KIT activation is a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res 2001; 61: 8118– 21. 8. Lux ML, Rubin BP, Biase TL, et al. KIT extracellular and kinase domain mutations in gastrointestinal stromal tumors. Am J Pathol 2000; 156: 791–5. 9. Lasota J, Wozniak A, Sarlomo-Rikala M, et al. Mutations in exons 9 and 13 of KIT gene are rare events in gastrointestinal stromal tumors. A study of 200 cases. Am J Pathol 2000; 157 :1091–5. 10. Kinoshita K, Isozaki K, Hirota S, et al. c-kit gene mutation at exon 17 or 13 is very rare in sporadic gastrointestinal stromal tumors. J Gastroenterol Hepatol 2003; 18: 147–51. 11. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 2003; 299: 708– 10. 12. Hirota S, Ohashi A, Nishida T, et al. Gain-of-function mutations of platelet-derived growth factor receptor alpha gene in gastrointestinal stromal tumors. Gastroenterology 2003; 125: 660–7. 13. Medeiros F, Corless CL, Duensing A, et al. KIT-negative gastrointestinal stromal tumors: proof of concept and therapeutic implications. Am J Surg Pathol 2004; 28: 889–94. 14. Corless CL, Schroeder A, Griffith D, et al. PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib. J Clin Oncol 2005; 23: 5357–64. 15. Lasota J, Dansonka-Mieszkowska A, Sobin LH, et al. A great majority of GISTs with PDGFRA mutations represent gastric tumors of low or no malignant potential. Lab Invest 2004; 84: 874–83. 16. Lasota J, Stachura J, Miettinen M. GISTs with PDGFRA exon 14 mutations represent subset of clinically favourable gastric tumors with epithelioid morphology. Lab Invest 2006; 86: 94–100. 17. Kang HJ, Nam SW, Kim H, et al. Correlation of KIT and plateletderived growth factor receptor alpha mutations with gene activation and expression profiles in gastrointestinal stromal tumors. Oncogene 2005; 24: 1066–74. 18. Wardelmann E, Hrychyk A, Merkelbach-Bruse S, et al. Association of platelet-derived growth factor receptor alpha mutations with gastric primary site and epithelioid or mixed cell morphology in gastrointestinal stromal tumors. J Mol Diagn 2004; 6: 197–204. 19. Wasag B, Debiec-Rychter M, Pauwels P, et al. Differential expression of KIT/PDGFRA mutant isoforms in epithelioid and mixed variants of gastrointestinal stromal tumors depends predominantly on the tumor site. Mod Pathol 2004; 17: 889–94. 20. Penzel R, Aulmann S, Moock M, et al. The location of KIT and PDGFRA gene mutations in gastrointestinal stromal tumours is site and phenotype associated. J Clin Pathol 2005; 58: 634–9. 21. Pauls K, Merkelbach-Bruse S, Thal D, et al. PDGFRalpha- and c-kitmutated gastrointestinal stromal tumours (GISTs) are characterized by distinctive histological and immunohistochemical features. Histopathology 2005; 46: 166–75. 22. Daum O, Grossmann P, Vanecek T, et al. Diagnostic morphological features of PDGFRA-mutated gastrointestinal stromal tumors: molecular genetic and histologic analysis of 60 cases of gastric gastrointestinal stromal tumors. Ann Diagn Pathol 2007; 11: 27– 33. 23. Heinrich MC, Corless CL, Demetri GD, et al. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 2003; 21: 4342–9. 24. Fletcher CD, Berman JJ, Corless C, et al. Diagnosis of gastrointestinal stromal tumors: A consensus approach. Hum Pathol 2002; 33: 459– 65. 25. Isaac JC, Willmore C, Holden JA, et al. A c-kit-negative gastrointestinal stromal tumor with a platelet-derived growth factor receptor alpha mutation. Appl Immunohistochem Mol Morphol 2006; 14: 52–6.
ANATOMICAL PATHOLOGY
Pathological characteristics of gastrointestinal stromal tumours with PDGFRA mutations JI EUN KWON*{, HYUN JU KANG*{, SE HOON KIM*, YONG CHAN LEE{, WOO JIN HYUNGx, SUNG HOON NOHx, NAM KYU KIMx AND HOGUEN KIM*{
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*Department of Pathology, {Brain Korea 21 Project for Medical Science, {Department of Internal Medicine, and xDepartment of Surgery, Yonsei University College of Medicine, Seoul, Korea
Summary Aims: The aim of this study was to identify the characteristics of PDGFRA-mutated gastrointestinal stromal tumours (GISTs) in comparison with KIT-mutated GISTs. Methods: A total of 151 GISTs were examined for KIT and PDGFRA mutations, and clinical and histopathological parameters were evaluated and analysed statistically according to mutation status. Results: KIT and PDGFRA mutations were detected in 123 (81.5%) and 15 cases (9.9%), respectively. Clinically, all PDGFRA-mutated GISTs were located in the stomach with no recurrences and tumour-related deaths were noted. Pathological parameters associated with PDGFRA mutations were epithelioid and mixed type, low to moderate cellularity, moderate to severe intratumoural lymphocytic infiltration, prominent myxoid change, frequent rhabdoid cells and multinucleated giant cells, increased cellular pleomorphism, low mitotic count, and lower risk assessment (p 5 0.05). Compared with KIT-mutated GISTs, PDGFRA-mutated GISTs had unique histopathological patterns, intermingling of spindle and epithelioid cells, and/or a jigsaw puzzle-like arrangement of epithelioid cells. Conclusions: PDGFRA-mutated GISTs have distinctive histological patterns that are differentiated from KIT-mutated GISTs. The algorithmic approach by a combination of several distinguishing histological and immunohistochemical features between KIT- and PDGFRA-mutated GISTs might be helpful in predicting the mutated gene of each GIST on pathological examination. Key words: Gastrointestinal stromal tumour, PDGFRA mutation, mixed type, epithelioid type. Received 16 March, revised 27 October, accepted 30 October 2008
INTRODUCTION GISTs are the most common mesenchymal tumours of the gastrointestinal tract.1,2 GISTs are characterised by differentiation similar to that of the interstitial cells of Cajal (ICC) and expression of KIT (CD117, stem cell factor receptor).3–5 Gain-of-function mutations in KIT are the most common genetic events in GISTs, resulting in autophosphorylation
of the KIT receptor, which in turn activates downstream signals.6,7 In-frame deletion mutation of exon 11 encoding the juxtamembrane domain is the most frequent mutation; however, internal tandem duplication of Ala502-Tyr503 in exon 9 encoding extracellular domain is also common.7–9 Point mutations of exons 11, 13, and 17 have also been reported.8–10 Recent studies have demonstrated that gain-of-function mutations in PDGFRA, a receptor tyrosine kinase and member of the type III receptor tyrosine kinase family, are present in 35–67% of GISTs lacking mutated KIT. These results suggest another molecular pathogenesis of GIST.11,12 Mutations of PDGFRA frequently occur in exon 18, which is involved in the activation of the second tyrosine kinase domain, exon 12 of the juxtamembrane domain, and exon 14 of the first tyrosine kinase domain.13–16 It is well known that mutations of KIT and PDGFRA are mutually exclusive,11,12,17 and that both are related to several clinicopathological factors.18–22 Identification of mutations in either KIT or PDGFRA in any case of GIST is important in prognostication and treatment. PDGFRA-mutated GISTs usually show favourable clinical courses,15 and GISTs with a substitution of aspartic acid to valine in 842 codon (D842V), the most common PDGFRA mutation, do not respond to imatinib mesylate (Gleevec; Novartispharma, Switzerland), which is known to be effective in treatment for metastatic or recurring GISTs with many types of KIT mutations.14,23 Recently, PDGRFRA-mutated GISTs have been identified as having gastric preponderance, and being of epithelioid or mixed type.15,18–20 However, the cellular component of epithelioid or mixed type is not enough for the identification of PDGFRA-mutated GISTs on routine histological examination by pathologists because KIT-mutated GISTs of mixed or epithelioid cell type are occasionally found. It might be necessary to choose the possible mutated gene, KIT or PDGFRA, by histological examination to reduce the cost of mutational study of both genes. The aim of this study was to analyse multiple clinical and pathological parameters of GISTs according to mutations in KIT and PDGFRA and demonstrate the distinguishing histological features of PDGFRA- from KIT-mutated GISTs, making prediction of the type of mutation in GISTs possible.
Print ISSN 0031-3025/Online ISSN 1465-3931 # 2009 Royal College of Pathologists of Australasia DOI: 10.1080/00313020903071421
PDGFRA-MUTATED GISTS
MATERIALS AND METHODS Case and clinicopathological features A total of 151 GISTs were retrieved from surgically resected or endoscopically removed gastrointestinal mesenchymal tumours between October 1995 and October 2004 from the Department of Pathology, Yonsei University College of Medicine, Korea, by reviewing haematoxylin and eosin (H&E) stained slides and immunohistochemical stain results. Clinical data including patient age, sex, tumour size, location, and follow-up information were obtained in all cases from hospital records. Another 61 GISTs for validation study were collected from GISTs diagnosed between January 2006 and July 2008 at the Department of Pathology, Yonsei University College of Medicine. All patients gave informed consent and authorisation for the use of tissues for research was obtained from the Institutional Review Board.
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Histopathological parameters and risk assessment Several histopathological parameters were analysed by reviewing H&E slides of all cases (Fig. 1). Cell type was divided into spindle cell, epithelioid, and mixed, according to previously described cell morphology.24 Spindle and epithelioid cells were differentiated by calculating the ratio of long to short diameter of cells. Cells with a ratio of less than 3 were regarded as epithelioid type. Mixed type was defined as a mixture of spindle and epithelioid cells when each proportion exceeded more than 10%. Cellularity was divided into three groups of low, moderate, and high (Fig. 1A–C). Intratumoural lymphocytic infiltration was recorded as none to mild [100 or fewer lymphocytes per 10 high power fields (HPFs)], moderate (101–200 lymphocytes per 10 HPFs), or severe (more than 200 lymphocytes per 10 HPFs) (Fig. 1D,E). Nuclear pleomorphism was recorded as none, mild, or moderate (Fig. 1G,I). The presence or absence of fascicular or syncytial arrangement, nuclear palisading, necrosis, ulceration, mucosal invasion, perinuclear vacuolisation, skeinoid fibre, rhabdoid cells, and bi- or multinucleated cells was also assessed (Fig. 1J,K). Angiocentric lobule formation was recorded and arbitrarily defined by lobular structure composed of tumour cells surrounding blood vessels and occasionally demarcated by a cleft or clear space associated with liquefactive change (Fig. 1L). Hyalinisation was recorded as none to focal (510%) and diffuse (410%), microcystic change as none to focal (520%) and diffuse (420%), and myxoid change as none to focal (550%) and extensive (450%) (Fig. 1M–O). Mitoses were counted in 50 HPFs. Based on mitotic counts per 50 HPFs and tumour size, GISTs were classified into four risk groups as previously described by Fletcher et al.24
Immunohistochemical stain Three representative cores (1.0 mm) of the area of tumour were taken from the original paraffin blocks and tissue microarray blocks were made. Immunohistochemistry was performed using an avidin–biotin peroxidase complex system with diaminobenizidine (DAB) as the chromogen. Primary antibodies included c-kit (polyclonal rabbit antibody, 1:50 dilution; Dako, Denmark) and CD34 (monoclonal mouse antibody, 1:50 dilution; Dako). Immunohistochemical results were evaluated semiquantitively as follows:21 Strong positivity, more than 10% of tumour cells were moderately or strongly positive; weak positivity, less than 10% of tumour cells were moderately or strongly positive or more than 10% of tumour cells were weakly positive; and negative, less than 10% of tumour cells were weakly positive or there were no positive cells.
Mutational analysis For DNA extraction, GIST tissues were microdissected at a thickness of 10 mm from paraffin blocks or fresh frozen tissues composed totally of tumour area. Exons 9, 11, 13, and 17 of the KIT gene and exons 12 and 18 of the PDGFRA gene were evaluated for mutations using PCR amplification of genomic DNA and direct sequencing. Genomic DNA was extracted using the SDS-proteinase K and phenol-chloroform
545
method. The primers used in this study to amplify KIT and PDGFRA are shown in Table 1. PCR conditions consisted of an initial denaturation at 958C for 15 min, followed by 30 cycles of 948C for 45 s, 558C for 45 s, and 728C for 45 s. The final elongation step was at 728C for 5 min. PCR products were separated on 6% polyacrylamide gels, followed by autoradiography for single-strand conformation polymorphism analysis. The amplified products were directly sequenced on a 3100 Genetic Analyser (Applied Biosystems, USA).
Statistical analysis Statistical analysis was performed using SPSS for Windows (version 12.0; SPSS Inc., USA). To make comparisons between KIT- and PDGFRA-mutated GISTs, clinical or histopathological parameters, risk assessment, and immunohistochemical results were evaluated using either chi-square test, Fisher’s exact test, or MannWhitney test. Results with p values of 50.05 were considered statistically significant.
RESULTS Mutational analysis of KIT and PDGFRA in 151 GISTs Among the 151 analysed GISTs, KIT mutations were found in 123 cases (81.5%) and PDGFRA mutations were found in 15 (9.9%). No mutations of either gene were found in the remaining 13 (9.3%) GISTs (Table 2). For GISTs with mutations in KIT, the most frequent mutations were found in exon 11 (109/123, 89%), followed by 10 cases with mutations in exon 9 and two cases of mutations in exons 13 and 17, respectively (Table 2). Among the 109 mutations in exon 11, a deletion was found in 60 cases, insertion in 26, and point mutations in 23. The most frequent type of deletion (10 cases) was a 6 bp deletion of codons 557–558, leading to loss of tryptophan and lysine amino acids. The most frequent type of point mutation was V559D, which was found in 12 cases. In the 15 PDGFRA mutations, 14 cases were found in exon 18 and one in exon 12. The most frequent type of PDGFRA mutation was a substitution of aspartic acid with valine in codon 842 (D842V), which was found in 13 cases. The remaining cases showed a 12 bp deletion in codon 842–846 in exon 18 and a 15 bp deletion of codons 556–571 in exon 12. The results of mutational analysis of all cases are listed in Table 3. Clinical characteristics of GISTs according to mutation type Clinically, PDGFRA-mutated GISTs more commonly occurred in males in contrast to KIT-mutated GISTs with equal prevalence in both sexes: KIT-mutated GISTs included 61 females and 62 males whereas PDGFRAmutated GISTs included four females and 11 males (p ¼ 0.108). All of the PDGFRA-mutated GISTs were present in the stomach whereas 89 (72.4%) of the 123 KIT-mutated GISTs were present in the stomach (p ¼ 0.022). Of the 13 GISTs lacking KIT or PDGFRA mutations, nine were present in the stomach and four in the small intestine. We evaluated the recurrence and metastasis of the 151 GISTs during a mean follow-up period of 40.9 months and found that 27 of the 123 (22%) KIT-mutated GISTs showed recurrence or metastasis while none of the 15 PDGFRA-mutated GISTs showed recurrence or metastasis
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FIG. 1 Histopathological parameters of GISTs evaluated in this study. Cellularity: (A) low, (B) medium, (C) high. Infiltration of intratumoural lymphocytes: (D) none to mild, (E) moderate, (F) severe. Nuclear pleomorphism: (G) none, (H) mild, (I) moderate. (J) Necrosis; (K) skeinoid fibre; (L) angiocentric lobule formation; (M) hyalinisation; (N) microcystic change; and (O) myxoid stroma (H&E).
at either the time of surgery or during follow up. Among the 27 GISTs with recurrence or metastasis, six cases had metastasised at the time of surgery and 21 developed
metastasis after curative surgery. The comparison of clinicopathological factors and follow-up results are summarised in Table 4.
PDGFRA-MUTATED GISTS
TABLE 1
Primer sequences used for PCR of KIT and PDGFRA genes
Gene
Primers
Primer sequences
KIT
hEx9F hEx9R hEx11F hEx11R hEx13F hEx13R hEx17F hEx17R
50 -TCCTAGAGTAAGCCAGGGCTT 50 -TGGTAGACAGAGCCTAAACATCC 50 -CCTTTGCTGATTGGTTTCGT 50 -AAACAAAGGAAGCCACTGGA 50 -GTTCCTGTATGGTACTGCATGCT 50 -CAGTTTATAATCTAGCATTGCC 50 -CATCATTCAAGGCGTACTTTTG 50 -TCACAGGAAACAATTTTTATCGAA
hEx12F hEx12R hEx18F hEx18R
50 -AAGCTCTGGTGCACTGGGACTT 50 -ATTGTAAAGTTGTGTGCAAGGGA 50 -TACAGATGGCTTGATCCTGAGT 50 -AGTGTGGGAGGATGAGCCTG
TABLE 3 GISTs
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Results of mutational analysis for KIT and PDGFRA in 151
Type of mutation
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PDGFRA
TABLE 2 Distribution of mutation type in 138 GISTs with KIT and PDGFRA mutations PDGFRA mutated (n ¼ 15)
KIT mutated (n ¼ 123) Mutation type Point mutation Deletion Insertion Total (%)
Exon 9
Exon 11
2
23
0 8 10 (8)
60 26 109 (89)
Exon 13 Exon 17 Exon 12
Exon 18
2
2
0
13
0 0 2 (2)
0 0 2 (2)
1 0 1 (7)
1 0 14 (93)
Comparison of histopathological parameters of GISTs according to KIT and PDGFRA mutations Pathologically, the 123 KIT-mutated GISTs were categorised as spindle cell type in 96 (78%) cases, epithelioid type in three (2.4%), and mixed type in 24 (19.5%). Of the 15 PDGFRA-mutated GISTs, two (13.3%) cases were categorised as epithelioid type and 13 (86.7%) as mixed type. Besides cell type, we found that many characteristic histological factors were related to the type of mutation of GISTs. Individual histological factors frequently present in KIT-mutated GISTs (Fig. 2) were of high cellularity (47% versus 7%, p ¼ 0.002), showed fascicle/syncytium formation (95% versus 27%, p 5 0.001) (Fig. 2A), nuclear palisading (76% versus 13%, p 5 0.001) (Fig. 2E), angiocentric lobule formation (29% versus 0%, p ¼ 0.011) (Fig. 2F), and high mitotic rate (12.1 versus 1.5, p 5 0.001). In PDGFRA-mutated GISTs (Fig. 3), nuclear pleomorphism (51% versus 93%, p ¼ 0.002), rhabdoid cell formation (11% versus 60%, p 5 0.001) (Fig. 3E), binucleated or multinucleated cell formation (63% versus 100%, p ¼ 0.003) (Fig. 3F), myxoid change (51% versus 93%, p 5 0.001), infiltrative border (19% versus 53%, p ¼ 0.006), and intratumoural lymphocyte infiltration (22% versus 67%, p ¼ 0.001) were more frequent (Table 5). There were no significant differences in the other histological factors between KIT- and PDGFRA-mutated GISTs. Comparing the risk assessment of GISTs according to the consensus of the NCI24 [high risk (45 cm and 45 mitotic index (MI)/50 HPF, or 410 cm with any mitotic count, or 410 MI/50 HPF with any size), intermediate risk (5–10 cm and 5 MI/50 HPF, or 5 cm and 6–10 MI/50
Exon 9 9 9 9 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 13 17 12 18 18
No. cases 6 2 1 1 15 12 10 4 4 4 4 3 3 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 13 1 13
KIT
PDGFRA
A502_Y503 ins F506_F508 ins G498S V457M T574_Y578 ins V559D W557_K558 del V560_L576 del D579 del V559_T574 del W557R V560_E561 del V560D Q556_V559 del Q556_K558 del P551_V569 del M552_Y553 del Y553_Q556 del Y553_E561 del L576_R586 ins W557_G564 del L576_P577 ins W557G K550_Y568 del K550_Q556 del L576P P573_L589 ins W557_Y558 del K550_W557 del V560E L556_D569 ins V555_W557 del E554_W557 del Q575_F591 ins V559_D572 del M552_K558 del K550_V555 del P577_H580 del M552_N572 del K558_I563 del Q575_D580 del T574_K581 del Y553_W557del Y578_D579 del D572_R588 ins W557_K558 ins Q556_N564 del K550_K558 del V560 del V559A K558_V559 del T574_R586 ins P551_M552 del I571_K581 ins P577-H580 ins T574-Y578 ins M552_E554 del P551_V555 del K550_V559 del K642E N822K Wild Wild Wild Wild
Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild S556_E571 del D842V D842_D846 del Wild
del, deletion; ins, insertion.
HPF), low risk (2–5 cm and 5 MI/ 50HPF), and very low risk (2 cm and 5 MI/50 HPF)], 85 of the 123 (69.1%) KIT-mutated GISTs were categorised in the
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TABLE 4 Comparison of clinical parameters between GISTs with KIT and PDGFRA mutations
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Variable and category
KIT mutated (n ¼ 123)
PDGFRA mutated (n ¼ 15)
KIT wild and PDGFRA wild (n ¼ 13)
Total (n ¼ 151)
Age, years Mean (SD) 57 (12.2) 53 (10.0) 56 (13.0) 57 (12.0) Gender Male 62 (50.4%) 11 (73.3%) 4 (30.8%) 77 (51.0%) Female 61 (49.6%) 4 (26.7%) 9 (69.2%) 74 (49.0%) Location Oesophagus 1 (0.8%) 0 0 1 (0.7%) Stomach 89 (72.4%)* 15 (100%) 9 (69.2%) 113 (74.8%)* Small bowel 23 (18.7%) 0 4 (30.8%) 27 (17.9%) Large bowel 10 (8.1%) 0 0 10 (6.6%) Follow up, months Mean (SD) 41.3 (32.9) 33.7 (31.0) 45.6 (30.2) 40.9 (32.4) No. of recurrence/metastasis Mets 6 (4.9%) 0 1 (7.7%) 7 (4.6%) Recur 21 (17.1%) 0 1 (7.7%) 22 (14.6%) 5-year survival Event free 68.1% 100% 82.1% 72.1% Overall 82.2% 100% 92.3% 85.2% *Includes one GIST located in the omentum. Mets, metastasis at the time of surgery; Recur, recurrence or metastasis after curative resection; SD, standard deviation.
intermediate- and high-risk groups while five of the 15 (33%) PDGFRA-mutated GISTs were in the intermediateand high-risk groups (p ¼ 0.035) (Table 5). Recurrence and/ or metastasis were noted in 24 of 51 (47.1%) KIT-mutated GISTs that were regarded as the high-risk group, in three of 34 (8.8%) KIT-mutated GISTs regarded as the intermediate-risk group, and none in the low- and very low-risk groups as well as PDGFRA-mutated GISTs. Immunohistochemical result of GISTs Among the 123 KIT-mutated GISTs, 117 (95.1%) were strongly positive for KIT immunostaining (Fig. 2G). In contrast, four of the 15 PDGFRA-mutated GISTs showed strong positivity for KIT immunostaining, and six cases showed weak expression of KIT (Fig. 3G). Furthermore, four cases with PDGFRA mutations were negative for KIT immunostaining. Immunohistochemical results for CD34 showed that 101 of the 123 (82.1%) KITmutated GISTs were positive and 10 of the 15 (66.7%) PDFGRA-mutated GISTs were positive (Table 6). Histological characteristics of PDGFRA-mutated GISTs: a pathological algorithmic approach for prediction of KIT or PDGFRA mutation type We found that no PDGFRA-mutated GIST was composed purely of spindle cells with a fascicular or syncytial growth pattern, which was the most common histological feature of KIT-mutated GISTs of spindle cell type. Because all of the PDGFRA-mutated GISTs and 22% of KIT-mutated GISTs were of epithelioid or mixed cell type, we compared the pathological characteristics between PDGFRA- and KIT-mutated GISTs of epithelioid and mixed type. Of the 15 PDFGRA-mutated GISTs, two were composed of pure epithelioid cells. Both of these cases showed
characteristic epithelioid cells with distinct cellular borders similar to a jigsaw puzzle pattern (Fig. 3A); specifically, each tumour cell resembled pieces of a jigsaw puzzle and had abundant cytoplasm. In the 24 KIT-mutated GISTs of epithelioid or mixed type, three cases were composed of pure epithelioid cells. These tumours were arranged in small clusters or a solid sheet; however, they were more cellular, and the tumour cells exhibited less distinct cellular borders and low amounts of cytoplasm compared with the epithelioid cell type of PDGFRA-mutated GISTs (Fig. 2C,D). In PDGFRA-mutated GISTs of mixed type, all but one case exhibited a so-called ‘intermingled pattern’ of discohesively and singly scattered spindle and epithelioid cells arranged in a haphazard fashion without areas of fascicle formation or syncytial growth. The tumour cells and their nuclei frequently showed either a fusiform or stellate shape and indented appearance with occasional multinucleated and binucleated cells. Tumour cells revealed mild to moderate pleomorphism due to the composition of cells of variable sizes and morphology but no evidence of obvious atypia. These tumours had in common a prominent myxofibrillary intervening stroma that was sometimes indistinct from the fibrillary cytoplasm, and moderate to low cellular density was also noted (Fig. 3B–F). In the one case of GIST with PDGFRA-mutation in exon 12, a patchy area of fascicular arrangement in addition to both intermingled areas of cells of various shapes and jigsaw puzzle-like areas of epithelioid cells were noted. In contrast, among the 24 KIT-mutated GISTs of mixed type, only three cases had areas showing intermingled pattern while the remaining 21 exhibited an alternative pattern composed of areas of spindle cells with fascicle formation and syncytial growth and areas of epithelioid cells with solid or clustered patterns (Fig. 2B). We highlighted the clinicopathological and immunohistochemical characteristics of PDGFRA-mutated GISTs, comparing them with those of previous studies, in Table 7. Based on the distinct histological patterns and differential KIT immunoreactivity, we suggest an algorithmic diagnostic approach for predicting mutation status (Fig. 4). Verification of algorithm for predicting mutational status of GISTs To verify the adequacy of this algorithm, two pathologists independently examined another set of 61 GIST cases according to this algorithm and predicted the mutated gene, either KIT or PDGFRA. After completion of the pathological examination, we performed mutational analysis by the same method and compared revealed mutation to putative mutation. The results are listed in Table 8. Among the 50 KIT-mutated GISTs, only one case was predicted to have PDGFRA mutation by pathologist A. Sensitivity, specificity, positive predictive value, and negative predictive value for KIT-mutated GISTs were 98.0%, 54.5%, 90.7% and 75.7%, respectively. The accuracy of this algorithm predicting KIT-mutated GIST is 90.2%. With regard to PDGFRA-mutation, sensitivity, specificity, positive predictive value, and negative predictive value were 85.7%, 85.7%, 98.1%, and 98.1%, respectively. The accuracy of the algorithm predicting PDGFRA-mutated GIST was 96.7%. Differences between sensitivity and positive
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PDGFRA-MUTATED GISTS
FIG. 2 Histological features of KIT-mutated GISTs. (A) Spindle cell type GIST composed of uniform spindle cells showing a fascicular and/or syncytial arrangement and high cellularity (H&E). (B) Mixed type GIST showing an alternative pattern of spindle cell area (centre) and epithelioid cell area (right lower) (H&E). (C) Epithelioid GIST arranged in a solid sheet with less distinctive cellular borders and high cellularity (H&E). (D) Epithelioid GIST arranged in small clusters. (E) Nuclear palisading was associated with KIT-mutated GISTs (H&E). (F) Angiocentric lobules were also associated with KIT-mutated GISTs (arrow) (H&E). (G) Most of the KIT-mutated GISTs were strongly positive for KIT immunostaining (c-kit).
predictive value in predicting KIT-mutated GISTs and between accuracy for predicting KIT-mutated GISTs and for predicting PDGFRA-mutated GISTs were derived from the existence of GISTs lacking both KIT and PDGFRA mutation, which were interpreted to have KIT mutation by both pathologists. When we excluded these four GISTs lacking KIT and PDGFRA mutation, sensitivity, specificity, and accuracy for KIT mutation rose to 98.0%, 85.7%, and 96.5%, respectively.
DISCUSSION Gain of function mutations in KIT and PDGFRA have been proposed as causative genetic events in the tumorigenesis of GISTs.6,11,12 Mutations of these two genes are known to be mutually exclusive. In this study, we demonstrated the presence of several different clinical and pathological characteristics of GISTs according to KIT and PDGFRA mutation.
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FIG. 3 Histological features of PDGFRA-mutated GISTs. (A) Eithelioid type GIST showing a jigsaw puzzle-like pattern (H&E). (B–D) Mixed type GISTs showing an intermingled pattern. Spindle cells, stellate-shaped cells, and epithelioid/round cells with mild to moderate nuclear pleomorphism were singly and discohesively intermingled without fascicular or syncytial formation. These GISTs were less cellular and had prominent myxofibrillary stroma and a moderate abundance of intratumoural lymphocytes. Binulceated cells were frequently noted (H&E). (E) Rhabdoid cells were more frequently noted (H&E). (F) Multinucleated giant cells were occasionally found (H&E). (G) Weak positivity to KIT immunostaining (c-kit).
Our study confirmed several previously reported clinical and pathological characteristics of GISTs. Of the 151 GISTs analysed, 123 (81.5%) had mutations in KIT and 15 (9.9%) had mutations in PDGFRA. PDGFRA-mutated GISTs had gastric and male preponderance compared with KIT-mutated GISTs.15,18–20 We also confirmed that PDGFRA-mutated GISTs had a marked preponderance of epithelioid and mixed type: all of the 15 PDGFRAmutated GISTs were epithelioid and mixed type while only 27 of the 123 KIT-mutated GISTs were classified as
epithelioid and mixed type. Because all of the PDGFRAmutated GISTs and 20–30% of KIT-mutated GISTs were categorised as epithelioid and mixed type, the identity of the mutated gene could not be predicted in these subsets of GISTs. We found that the overall pattern was more important than constituent cell type itself for predicting the genotype of GISTs. We found that PDGFRA-mutated GISTs of epithelioid cell type were characterised as a sheet of epithelioid cells with distinct cell borders and abundant cytoplasm, occasionally resembling pieces of a jigsaw
PDGFRA-MUTATED GISTS
TABLE 5 Comparison of histopathological parameters and risk assessment between KIT- and PDGFRA-mutated GISTs Type of mutation
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Histological variables and category
KIT (n ¼ 123)
Cell type, n (%) Spindle 96 (78.0) Epithelioid or mixed 27 (22.0) Cellularity, n (%) Low to moderate 65 (52.8) High 58 (47.2) Fascicle/syncytium, n (%) Absent 6 (4.9) Present 117 (95.1) Intratumoral lymphocytes, n (%) None to mild 96 (78.0) Moderate to severe 27 (22.0) Nuclear pleomorphism, n (%) None 60 (48.8) Mild to moderate 63 (51.2) Myxoid change, n (%) None to focal 72 (58.5) Prominent 51 (41.5) Giant cell, n (%) Absent 45 (36.6) Present 78 (63.4) Rhabdoid cell, n (%) Absent 109 (85.6) Present 14 (11.4) Necrosis, n (%) Absent 72 (58.5) Present 51 (41.5) Ulceration, n (%) Absent 94 (76.4) Present 29 (23.6) Infiltrative border, n (%) Absent 100 (81.3) Present 23 (18.7) Mucosal invasion, n (%) Absent 99 (80.5) Present 24 (19.5) Perinuclear vacuolisation, n (%) Absent 34 (27.9) Present 88 (72.1) Microcystic change, n (%) None to focal 77 (62.6) Prominent 46 (37.4) Nuclear palisading, n (%) Absent 30 (24.4) Present 93 (75.6) Skeinoid fibre, n (%) Absent 115 (93.5) Present 8 (6.5) Hyalinisation, n (%) None to focal 98 (79.7) Prominent 25 (20.3) Angiocentric lobule, n (%) Absent 87 (70.7) Present 36 (29.3) Mitosis/50 HPF 0–5, n (%) 65 (52.8) 6–10, n (%) 22 (17.9) 410, n (%) 36 (29.3) Mean 12.2 + 18.7 Size, cm 0–5, n (%) 68 (55.3) 6–10, n (%) 39 (31.7) 410, n (%) 16 (13.0) Mean 6.1 + 4.9 Risk assessment, n (%) Very low 11 (8.9) Low 27 (22.0) Intermediate 34 (27.6) High 51 (41.5) *p 5 0.05.
PDGFRA (n ¼ 15)
p value
0 (0) 15 (100)
50.001*
14 (93.3) 1 (6.7)
0.002*
11 (73.3) 4 (26.7)
50.001*
5 (33.3) 10 (66.7)
0.001*
1 (6.7) 14 (93.3)
0.002*
1 (6.7) 14 (93.3)
50.001*
0 (0) 15 (100)
0.003*
6 (40.0) 9 (60.0)
50.001*
13 (86.7) 2 (13.3)
0.047*
14 (93.3) 1 (6.7)
0.191
7 (46.7) 8 (53.3)
0.006*
14 (93.3) 1 (6.7)
0.306
7 (46.7) 8 (53.3)
0.145
7 (46.7) 8 (53.3)
0.233
13 (86.7) 2 (13.3)
50.001*
15 (100) 0 (0)
0.598
15 (100) 0 (0)
0.073
15 (100) 0 (0)
0.011*
14 (93.3) 1 (6.7) 0 (0) 1.5 + 1.9
0.006*
10 (66.7) 3 (20.0) 2 (13.3) 5.6 + 5.4 3 7 3 2
(20.0) (46.7) (20.0) (13.3)
50.001* 0.685
0.287 0.035*
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puzzle. In contrast, the three KIT-mutated GISTs of epithelioid type showed high cellularity, indistinct cellular borders, and low amounts of cytoplasm. In the GISTs of mixed type, most of the KIT-mutated GISTs showed an alternative arrangement of spindle and epithelioid cell areas. In contrast, PDGFRA-mutated GISTs showed an intermingled pattern of singly and discohesively scattered epithelioid, fusiform, stellate-shaped, and spindle cells. Fascicular arrangement of spindle cells was the hallmark of KIT-mutated GISTs whereas few areas of fascicle arrangement were evident in most of the PDGFRA-mutated GISTs. Other characteristic features of PDGFRA-mutated GISTs were low cellularity, more prominent myxofibrillary stroma, increased cellular pleomorphism, occasional rhabdoid and binucleated or multinucleated giant cells, and increased intratumoural lymphocytic infiltration. In a previous study of the pathological features of GISTs according to mutational status, Pauls et al.21 reported that all PDGFRA-mutated GISTs with epithelioid or mixed type have multinucleated giant cells, and this finding can be useful to predict the genotype of GISTs. In our cases, we found that 63.4% of the KIT-mutated GISTs also had at least one bi- or multinucleated giant cell, thus the presence of giant cells is by itself not sufficient evidence to discriminate between KIT- and PDGFRA-mutated GISTs. Most of the KIT-mutated GISTs showed strongly positive immunoreactivity to KIT; however, only 26.6% of the PDGFRA-mutated GISTs were strongly positive for KIT. These results are in accordance with previous reports that many KIT-negative GISTs contain PDGFRA mutation.13,25 Pathological diagnosis of PDGFRA-mutated GISTs may be difficult due to negative or weak immunoreactivity to KIT and/or CD34. Therefore, pathologists should become familiar with unique morphological findings of PDGFRAmutated GISTs, which we have described above in detail in order to assist with the diagnosis of PDGFRA-mutated GISTs. Based on distinguishing histological patterns and differential KIT immunoreactivity, we propose an algorithmic approach to predict the type of mutation of GISTs and verified it by using another validation set of GISTs. Sensitivity and specificity for predicting PDGFRA mutation of this algorithm are 85.7% and 95.1%. We expect that this algorithmic approach will be helpful for the initial selection of the gene for mutational analysis because mutations of KIT and PDGFRA in GISTs are known to be mutually exclusive. The clinical and histological parameters for risk assessment of GISTs consist of tumour size and mitotic count of tumour cells. When we classified our GISTs according to the risk assessment proposed by Fletcher et al.,24 69.1% of KITmutated GISTs were categorised into the intermediate- and high-risk groups while 33.3% of PDGFRA-mutated GISTs were also categorised in the intermediate- and high-risk groups. Because there was no significant size difference between KIT- and PDGFRA-mutated GISTs, the lower mitotic index in PDGFRA-mutated GISTs was related to the favourable group preponderance of PDGFRA-mutated GISTs. Among the evaluated histological factors, multinucleated giant cells were related to poor prognosis in KITmutated GISTs but not in PDGFRA-mutated GISTs. In KIT-mutated GISTs with recurrence or metastasis, most of the tumours contained multinucleated giant cells (p 5 0.001). In contrast, even though multinucleated giant
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TABLE 6
Pathology (2009), 41(6), October
Immunohistochemical expression of KIT and CD34 according to mutation status KIT immunoreactivity
CD34 immunoreactivity
Type of mutation
Negative
Weak
Strong
p value
Negative
Weak
Strong
p value
KIT PDGFRA
4 (3.3%) 5 (33.3%)
2 (1.6%) 6 (40.0%)
117 (95.1%) 4 (26.6%)
50.001
13 (10.6%) 2 (13.3%)
9 (7.3%) 3 (20.0%)
101 (82.1%) 10 (66.7%)
0.145
TABLE 7
Comparison of characteristics of PDGFRA-mutated GISTs between previous studies and ours
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Characteristics of PDGFRA-mutated GISTs Comparison category
Histological findings
Immunohistochemical findings
Clinical findings
Epithelioid cell or mixed phenotype15,18–22* Multinuclear giant cells21* Rhabdoid cell (less specific but helpful)22* Low mitotic activity15*
Weak immunoreactivity to KIT20,21
Gastric preponderance15,18,19 Favourable course15
Concordant findings
Not evaluated in this study Dot-like staining of PDGFRA21 New findings Low cellularity Intermingled pattern: discohesively scattered cells Jigsaw puzzle-like: distinct cell membrane Rare fascicular arrangement
Lower expression of CD34
Lower-risk group
*These histological features can be found in some KIT-mutated GISTs.
FIG. 4 Algorithm for predicting the mutational status of GISTs by pathological parameters.
cells were present in all 15 of the PDGFRA-mutated GISTs, no recurrence or metastasis was present. The clinical significance of the presence of multinucleated cells differs according to the type of mutated gene of the GIST.
Molecular classification of GISTs is important for accurate prognostication and predicting therapeutic response. Many recent studies have demonstrated that there is a strong correlation between the type of KIT mutation
PDGFRA-MUTATED GISTS
TABLE 8
Comparison of predicted mutation and type of mutation of 61 GISTs for the validation of the proposed algorithm of this study Predicted mutation
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Case no.
Histological type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Pure spindle Pure spindle Mixed Pure spindle Pure spindle Pure spindle Mixed Pure spindle Epithelioid Pure spindle Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Mixed Mixed Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Mixed Mixed Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Mixed Pure spindle Mixed Mixed Pure spindle Pure spindle Pure spindle Pure spindle Pure spindle Mixed Pure spindle Mixed Mixed Pure spindle Pure spindle
Type of mutation
Pathologist A
Pathologist B
K K K K K K K K K K P K K K K K K K P K K K K K K K K K K K K K K K K K P K P K K K K K K P K K K P K K K K K K K K P K K
K K K K K K K K K K P K K K K K K K P K K K K K K K K K K K K K K K K K P K K K K K K K K P K K K P K K K K K K K P K K K
KIT
PDGFRA
T574_Y578 ins W557_E561 del A502_Y503 ins D579del N564_N566 del W557_K558 del K642E L576_P577 del Q575_L576 del L576_P577 del Wild W557_K558 del Q556_W557 del V560D L576P Wild K642E V559_E561 del Wild V560D V559D Wild I571_Y578 del V560D Q556-L576 del Wild A502_Y503 ins V560del K558_V560 del I563-L576 del T574_P577 ins K558_E562 del V590E Q556_V560 del M552-V569 del V560 del Wild A502_Y503 ins V560_E561 del P551-E554 del W557-K558 del V559G W557_K558 del V559A Y553_E554 del Wild M552_I571 del W557_V559 del A502_Y503 ins Wild W557A V559D V559D V559G Wild V559D Y570_L576 del Wild Wild W557_K558 del N822K
Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild D842V Wild Wild Wild Wild Wild Wild Wild D842V Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild D842V Wild Wild Wild Wild Wild Wild Wild Wild D842V Wild Wild Wild D842V Wild Wild Wild Wild Wild Wild Wild D842V D842V Wild Wild
del, deletion; ins, insertion; K, KIT; P, PDGFRA.
and therapeutic response to imatinib mesylate (Gleevec; Novartispharma).23 The mutation of D842V of exon 18, the most common mutation in PDGFRA, has been reported to be resistant to imatinib mesylate treatment both in vivo and in vitro.14,23 In contrast, the V561D
mutation of exon 12 and deletion mutation of exon 18 has been reported to be partially responsive to imatinib mesylate treatment in approximately two-thirds of patients.23 These findings indicate that mutation analysis of PDGFRA is also important for predicting chemotherapeutic
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response. PDGFRA-mutated GISTs are known to have more favourable prognosis, and this was again confirmed in our study. When we compared the prognosis of our GISTs of epithelioid and mixed type, the most striking differences were found between the PDGFRA- and KIT-mutated GISTs. In the 27 KIT-mutated GISTs of epithelioid or mixed type, 16 cases showed recurrence and metastasis and nine patients died during the follow-up period. In contrast, no PDGFRA-mutated GISTs having epithelioid cell or mixed type showed recurrence, metastasis, or death during follow up despite the fact that 33% were categorised into the intermediate- or high-risk groups. The good prognosis of our PDGFRA-mutated GISTs might be related to the type of mutations, as no case of the V561D mutation, which had been reported to show bad prognosis,12 was present in our 15 PDGFRA-mutated GISTs. These findings indicate that mutational analysis is important for the prognostication of GISTs, especially epithelioid and mixed type. In summary, we demonstrated that PDGFRA-mutated GISTs have unique clinicopathological features compared with KIT-mutated GISTs, and overall histological pattern or an intermingled or jigsaw puzzle-like arrangement is the most important difference that distinguishes PDGFRAfrom KIT-mutated GISTs. We propose an algorithmic approach by combining the most distinguishing features of PDGFRA-mutated GISTs, and this approach might be useful for predicting either KIT or PDGFRA mutation on histological examination prior to mutational analysis. ACKNOWLEDGEMENTS Ji Eun Kwon and Hyun Ju Kang contributed equally to this work. This work was supported by Korea Health 21 R&D Project [Ministry of Health and Welfare (03-PJ10-PG6-GP01-0002)] the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R11-2000-082-02005-0). Address for correspondence: Professor H. Kim, Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemungu, CPO Box 8044, Seoul 120–752, Korea. E-mail: [email protected]
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