Material and Method: Eighty consecutive resected cases were genotyped for KIT exons 11, 9, 13, 17 and PDGFRA exons 18, 14, 12 and correlated with histomorphology by nonparametric tests.
Results: Forty-seven cases (58.8%) were in the high-risk group. Males had higher rates of KIT exon 11 and PDGFRA exon 18 mutations than females (p=0.03). KIT and PDGFRA mutation frequencies were lower (58.8%) than western data showing KIT exon 11 mutation in 63.8%, KIT exon 9 mutation in 19% and PDGFRA exon 18 mutation in 17% of the cases. Extragastrointestinal stromal tumours (n=6) showed 100% mutation. KIT exon 11 deletion was associated with gastric location (60%) (p=0.04), spindle cells (63.3%), and high-risk stratification (66.6%) (p=0.01) while KIT exon 9 mutation was common in small intestine (66.7%) (p=0.04), in higher risk groups (66.7%) (p=0.01) and 75% of codon 502-503 duplications (p=0.03). PDGFRA 18 mutation was common in males (p=0.03), in gastric location (62.5%) (p=0.04), in cases showing mild to moderate atypia (62.5%) (p=0.01) and lower risk stratification (62.5%) (p=0.01). KIT/PDGFRA mutations were significantly associated with gender (p=0.03), location (p=0.04), nuclear atypia (p=0.01) and risk stratification (p=0.01).
Conclusion: Morphological features and anatomic location may be useful in deciding molecular testing strategy, particularly in resource-limited settings, when a plethora of targetable mutations are present. An algorithm may be derived for genotyping with KIT exon 11 and PDGFRA exon 18 heading the list of targetable mutations. This approach may reduce financial burden on patients as well as workload on hospital staff.
Molecular Analysis
DNA extraction was done from formalin-fixed paraffinembedded
tissue using the QiaAmp FFPE kit. Polymerase
chain reaction (PCR) was performed for KIT (exons 11, 9,
13, 17) and PDGFRA (exons 18, 14, 12). Briefly, each PCR
reaction was done in 25 μl volume using 250 ng of DNA on
the ABI SimplyAmpTM thermal cycler followed by Sanger
sequencing on the ABI 3130 Genetic Analyzer.
Statistical Analysis
Categorical variables were correlated using the Chi square
test and Fishers exact test where appropriate. Recurrencefree
survival was analysed using Kaplan-Meier log-rank
analysis. SPSS version 16 was used. A p value of < 0.05
was considered significant. The study was approved by the
institutes ethics committee.
Histopathology
The majority of the gastric (89.5%), small intestinal
(90.9%) and EGIST cases (83.3%) showed moderate to
high cellularity with colonic GISTs having conspicuously
low cellularity (66.7%), (p=0.15). The spindle cell type
(n=52, 65%) was predominantly present in all locations,
followed by mixed (n=20, 25%) and epithelioid cell type
(n=8, 10%). Multinucleated tumour giant cells were seen
scattered in 2 cases. The most common pattern was that
of fascicles (n=63, 78.7%) followed by palisading (10 cases,
12.5%) and storiform (8 cases, 10%) (p=0.15). Secondary
changes included cystic degeneration and skenoid fibres in
11 cases each (13.7%), of which 10 (12.5%) were present
in the small intestine, hyalinization (8 cases, 10%), myxoid
degeneration (7 cases, 8.7%), paranuclear vacuolization (3 cases, 3.7%), calcification (1 case, 1.2%), congested vessels
(4 cases, 5%) and significant haemorrhage (6 case, 7.5%).
The extent of tumour infiltration was limited to the submucosa in one case and muscularis propria in 9 cases. Exophytic GISTs (n=15) involving the serosa alone were seen in 5 and serosa to muscularis propria in 10 cases. Twenty-nine cases (36.2%) had entire thickness involvement with ulceration of overlying mucosa.
Overall CD117 and DOG-1 immunoreactivity were 93.8% (n=75) and 91.3%, respectively, with high concordance between the two markers; excluding five cases (6.25%) with the CD117 (+)/DOG1(-); three (4%) cases with CD117(-)/DOG1(+) and two cases (2.5%) with CD117 (-)/DOG1(-) immune profile. The latter two cases were also KIT/PDGFRA wild type and the diagnosis was based on characteristic histomorphology, anatomical association with the gastrointestinal tract, presence of CD34 and absence of diffuse and strong expression of other immunohistochemical markers. The detailed gross, histological and immunohistochemical features are mentioned in Table I.
Table I: Gross, histological and immunohistochemical features of GIST at different locations.
Mutational Analysis
Overall KIT and PDGFRA mutations were present in 47
cases (58.8%).
KIT exon 11 mutations were seen in 30 (63.8%) cases with simple deletions in 17 (56.7%), point mutations in 10 (33.3%) and complex mutations (deletions and duplication/ insertions) in 3 (10%) cases. Codon 557-558 deletions (Figure 1A) were found in 15 cases (50%).
KIT exon 9 mutations were found in 9 cases (19%) which showed duplications Tyr502-503Asp in 4 cases and point mutations in 5 cases. These mutations were common in the small intestine (66.7%) followed by EGIST (22.2%) and stomach (11.1%). The spectrum of mutations in KIT exon 11 and 9 are summarized in Table II.
Table II: Spectrum of KIT mutations with location and immunohistochemistry.
PDGFRA exon 18 mutations were present in 8 cases (17%), commonly located in the stomach (62.5%), and had epithelioid/mixed morphology. D842V mutation was seen in 1 case whereas D842E was seen in 5 cases (Figure 1B). The spectrum of PDGFRA exon 18 mutations are summarised in Table III. No mutations were found in PDGFRA exons 12 and 14.
Table III: Spectrum of PDGFRA exon 18 mutations with locations and immunohistochemistry.
Genotype and Clinicopathological Features
Males had higher KIT exon 11 and PDGFRA mutations
than females (p=0.03). GISTs with PDGFRA exon 18
mutations had smaller tumour size than cases with KIT exon 9 and exon 11 mutations (p=0.01). Nearly 67%
of cases with KIT exon 11 mutations were present in
the stomach that included 12 simple deletions, 7 point
mutations and one complex mutation (Table III). Overall
66.7% of KIT exon 9 mutations were found in the small
intestine (p=0.04) of which codon 502-503 duplication was
present in 75% of the small intestine (p=0.03). PDGFRA
exon 18 mutations were mostly present in the stomach (62.5%) and all D842 point mutations were found in the
gastric location as well (p=0.04). All colonic GISTs were
wild type whereas all EGISTs were mutant (KIT exon 11
deletion in 2 cases, KIT exon 11 point mutation in one case,
KIT exon 9 duplication/point mutation and PDGFRA exon
18 point mutation in one case each). The correlation of the
GIST genotype with clinicopathological variables and risk
stratification is shown in Table IV.
Table IV: Correlation of histopathological variables with mutation.
Genotype and Histomorphology
KIT exon 11 and 9 mutations were predominantly associated
with spindle cell morphology (Figure 2A) compared to
PDGFRA mutations that had epithelioid (Figure 2B) or
mixed phenotype (p=0.4). Mild to moderate nuclear atypia
was significantly associated with KIT exon 11 (100%) and
exon 9 mutations (100%) whereas 37.5% cases with PDGFRA
exon 18 mutation and 15% of wild type GISTs showed severe
atypia including plasmacytoid cells and binucleate tumour
giant cells (p=0.01) (Figure 2C).
A diffuse sheet-like pattern was seen in 10 cases; 6 of which harbored KIT exon 11 deletions and 4 were wild type. All cases with substitutions and insertions had fascicles and/ or a palisaded pattern. Paranuclear vacuolization was commonly observed in cases with KIT exon 9 mutations (Figure 2D). It may be assumed that cases with both KIT exon11 deletions and wild type have an aggressive course with high cellularity and hence have a diffuse sheet-like pattern.
Genotype and Survival
Follow-up was available in 73 patients with median followup
of 27 and mean follow-up of 34.2 months ± 26.7 (range:
1-101 months). Recurrence was observed in 12 patients
(9 males, 3 females) within 6 to 34 months post-surgery
in 9 high risk, 2 intermediate risk and 1 low malignant
risk cases. Six recurrent cases (50%) showed KIT exon
11 mutation with 4 cases (33.4%) having codon 557-558
deletion and two (16.7%) having point mutations. Median
recurrence free survival (RFS) in patients with wild type
GIST was higher than in patients with mutated GIST (79
months vs. 67 months, respectively; p=0.9) (Figure 3A).
The 5-year RFS in patients with KIT and PDGFRA wild
type was also higher (87% vs.48%) than in mutant GIST
(p=0.6). The median RFS in patients with codon 557-558
deletion was 67 months compared to 84 months in patients
with all other missense mutations (p=0.6) (Figure 3B).
The 5-year RFS was 42% in patients with codon 557-558
deletion and 53% in other missense mutations (p=0.7) of KIT 11. The codon 557-558 deletion also showed decreased
median RFS of 68 months (5-year survival: 42%) compared
to wild type GISTs with RFS of 79 months (5-year survival:
92%) (p=0.4) (Figure 3C).
In the present study, tumour size ranged from 1.5-30 cm with a mean of 10.3 cm, with larger tumours in large intestinal (mean: 17 cm) and extraintestinal location (mean: 12.9 cm) compared to the stomach and small intestine (mean-9.8 cm). In the series by Miettinen et al., the median size of gastric GISTs was 6 cm [9] and several European series have reported a median size of 5-5.6 cm (2-10 cm) [10,11]. On the contrary, median tumour size from other Indian series varies from 10.6 to 10.9 cm [12,13], similar to our series which could be related to later admission to the hospital at an advanced symptomatic stage.
Mixed and epithelioid cell types are usually encountered in a gastric location [14]. Interestingly, in the present study 50% of our EGISTs also showed a mixed cell type and this has not been observed in the EGIST of other series [9,10].
High concordance (87.5%) was observed between CD117 and DOG1 immunoreactivity; however, four of five (80%) CD117 negative cases stained for DOG1. Foo et al. have reported one third of CD117 negative cases to be positive for DOG1 [15].
KIT/PDGFRA mutation frequency was lower in the present study compared to western data. This may be explained by ethnical variation which is well known for lung and colorectal cancers [16]. Although genotyping studies from India and other Asian countries are fewer, the series from Taiwan and China also showed similar lower rate of mutations in KIT and PDGFRA genes [17-19]. In our cases, KIT exon 11 and PDGFRA exon 18 mutations were commonly seen in the stomach whereas KIT exon 9 mutations were seen in the small intestine, similar to other studies [20,21]. However, Daniel et al. showed KIT 11 deletions to have different locations having a wide spectrum of morphology [22]. A peculiar finding noted in this study was the presence of 100% mutation in EGISTs.
Male gender had higher rates of both KIT11 and PDGFRA mutations as well as wild genotype as compared to females (p=0.03), indicating that all kinds of GIST genotype is possible in males [20]. Lv et al. showed that all mutations were more common and associated with poor RFS in males [23]. PDGFRA mutation showed a significant correlation with male gender in the present study (p=0.02), similar to Daniels et al.; however, KIT 9 mutation was equally present among both sexes [22]. PDGFRA exon 18 mutations had smaller tumour size than cases with KIT exon 9 and 11 mutations (p=0.01), similar to the series of Wozniak et al. [20].
Agaimy et al. and Daum et al. noted that epithelioid or mixed cell morphology, presence of mast cells, multinucleated giant cells and myxoid stroma were more frequently associated with PDGFRA mutation [7,24]. We also observed similar findings where 5 of 8 PDGFRA mutated cases had epithelioid morphology and two cases had plasmacytoid cells and multinucleated cells. Unfortunately, SDH, KRAS and BRAF mutations could not be performed in KIT-PDGFRA wild type cases due to financial constraints, which is one of the major limitations of this study.
Morphological predictors of prognosis or RFS were mitotic activity (p=0.009), tumour necrosis (p=0.04), and cellular atypia (p=0.03).
Suggested Algorithm for Molecular Testing in GIST
Based on Morphological Features
Considering all histomorphological or genotypical features
observed in the present study, we can argue that certain
morphological features can be associated with a particular
genotype. Therefore, we can suggest that the sequence of
testing can be modified especially for patients/centres
where financial affordability for multiple investigations is
a concern (Figure 4). As most of the GIST mutations are
mutually exclusive, the order of mutation testing can be
devised for each set of morphological features. Tumours in
the gastric location with spindle cell morphology and high
mitosis may be tested for KIT exon 11 while gastric tumours
with low mitosis, epithelioid morphology and low cellular
atypia may be tested initially for PDGFRA exon 18 in the
first phase, or if feasible, gastric GISTs should be first tested
for KIT exon 11 followed by PDGFRA exon 18. If both are
wild type then one may proceed with KIT exon 9 testing.
About 20-40% of double negative GISTs are positive for
SDH mutations and another 15% of triple negative (KIT,
PDGFRA, SDH negative) cases harbour mutations in
RAS/BRAF [25,26], which may be checked if the above
mutations are negative. SDH immunohistochemistry can
be done either following or in conjunction with KIT exon
11 and 9 mutation testing. Later, the BRAF, KIT exons 13,
17 and PDGFRA exon 12 mutations may be tested, which
account for <5% of all mutations. Most SDH deficient
GISTs have characteristic dumb bell/lobulated shape with
thick fibrous bands, epithelioid morphology and frequent
lymph node metastasis; SDH testing may precede KIT exon
11 mutation testing for suspected cases. It should be noted
that this suggested algorithm is suited mostly for resource
limited situations, and does not disagree with studying
multiple mutations if laboratory resources and the patients
status permits.
Figure 4: The figure shows suggested algorithm for molecular testing in GIST.
In conclusion, this study is dedicated to the present association of morphological features with mutations occurring in GIST. Genotyping GIST is becoming mandatory as it is recommended to predict dosage and response to imatinib therapy. The only matter of concern is its availability and affordability for the individual patient. Since GISTs harbor multiple mutations, next generation sequencing (NGS) is the most suitable technology today to study mutations. However, the biggest limitations with NGS are the cost and the necessary expertise which is not available in all centres. Therefore, most of the laboratories have to depend on the gold standard Sanger sequencing and it is always prudent to prioritize the mutation testing especially in settings with limited financial means. We suggest testing initially for KIT exon 11 and/or PDGFR exon 18 in gastric GISTs depending on spindle cell and epithelioid or mixed cell morphology respectively, followed by KIT exon 13, 17 and PDGFRA exon12.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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