KIT-negative gastrointestinal stromal tumors: proo... - ResearchGate

Medeiros et al Am J Surg Pathol • Volume 28, Number 7, July 2004
MATERIALS AND METHODS
The study group included 25 tumors that were typical of
GIST clinically and histologically, but were KIT negative by
immunohistochemistry in formalin-fixed, paraffin-embedded
tissue. Among 495 consecutive GISTs diagnosed between
1999 and 2002 at the Brigham and Women’s Hospital, 20 tumors
(4%) were KIT negative by immunohistochemistry. The
remaining 5 tumors were obtained from consultation files of
the Department of Pathology at Oregon Health & Science University.
Five of the 25 cases were included in a prior study. 9
Only surgical excision specimens were included to minimize
nonrepresentative sampling. Patients previously treated with
imatinib mesylate were excluded from the study to avoid treatment
effect as a cause of KIT negativity.
Clinical data, including age, gender, and tumor location,
were obtained in all cases. An average of five hematoxylineosin
archival slides per case was available for histopathologic
review. Paraffin-embedded, formalin-fixed tissue was used for
immunohistochemical analysis. Tissue sections were cut at 4
µm and incubated with the primary antibodies for 40 minutes at
room temperature. Immunohistochemistry was performed for
KIT (Dako Corporation, Carpinteria, CA; polyclonal A4502,
1:250) in all cases without epitope retrieval, as previously described.
12 Selected cases were also stained for CD34 (Dako
Corporation; clone Qbend10, 1:400), desmin (Dako Corporation;
D33, 1:500), S-100 (Dako Corporation; polyclonal,
1:3000), -smooth-muscle actin (Sigma, St. Louis, MO; clone
1A4, 1:20,000), and keratins (Dako Corporation; AE1/AE3;
1:200) with the Envison+ avidin-biotin peroxidase kit (Dako
Corporation) according to manufacturer’s specifications. KITpositive
GISTs were used as positive controls. Negative controls
consisted of substituting normal serum for the primary
antibody, which resulted in no staining of the tissues.
Cytogenetic analysis was performed in 4 cases according
to standard procedures. 7 Immunoblotting was performed
using total cell lysates from snap-frozen GIST specimens, as
described elsewhere. 8,17 Mutational analyses were performed
on DNA extracted from paraffin-embedded tumor tissue using
a combination of PCR amplification, denaturing high performance
liquid chromatography screening, and automated sequencing,
as described previously. 2,9,17
RESULTS
There were 17 males and 8 females (2:1). The median
age at diagnosis was 56 years (range 29–79 years). Most tumors
originated in the stomach (N = 14, 56%), followed by
omentum/mesentery (N = 5) and small bowel (N = 1). In 5
cases (20%), there was an intraabdominal mass, but no distinct
primary site could be identified at the time of presentation,
suggesting possible peritoneal origin. Tumor size ranged from
4 to 38 cm (median, 8.5 cm) (Table 1).
All tumors exhibited classic histologic features for
GIST, being composed of cellular sheets, fascicles, or nests of
cells that lacked significant nuclear pleomorphism. Tumor cell
cytoplasm was eosinophilic and slightly fibrillary with illdefined
cytoplasmic borders, producing a somewhat syncytial
appearance. Nuclei were spindled, ovoid, or rounded and had a
uniform appearance with evenly distributed chromatin. The
majority of cases showed epithelioid cell morphology (N = 13,
52%) (Fig. 1). Eight tumors were of mixed (epithelioid and
spindle) cell type (32%) and four were composed of spindle
cells only (16%). Mitoses ranged from 1 to 52 per 50 high
power fields (median 8) (Table 1). All cases were classified as
intermediate or high risk for aggressive behavior based on tumor
size and mitotic count. 5 All tumors completely lacked KIT
staining by immunohistochemistry (Fig. 1). Complementary
immunostains were performed in 23 cases to exclude other tumors
in the differential diagnosis. Eleven and 10 cases were
positive for CD34 and smooth muscle actin, respectively. Focal
S-100 protein positivity was detected in one tumor. All
cases that were evaluated for desmin and keratin were negative
for these markers.
Cytogenetic analysis revealed noncomplex karyotypes
and a typical loss of chromosome 14 in all four cases evaluated.
One tumor also showed deletion of chromosome 22
(Table 2). Mutational analysis revealed KIT mutations in 4
cases (16%) and PDGFRA mutations in 18 cases (72%) (Table
1). In only 3 cases, no KIT or PDGFRA mutations were identified.
Most PDGFRA mutations involved exon 18 (15 of 18,
83%), including 11 GISTs with missense mutations leading
to a substitution of valine (N = 9) or tyrosine (N = 2) for aspartic
acid 842 (D842V and D842Y, respectively). The D842Y
mutation is a novel mutation, whereas the D842V mutation
has been reported previously. 9 The remaining 4 GISTs with
PDGFRA exon 18 mutations had in-frame deletions. Two
GISTs had PDGFRA mutations in exon 12 (N = 2), encoding
the PDGFRA juxtamembrane region, and one GIST had a previously
undescribed point mutation in PDGFRA exon 14
(N659K). Four GISTs had KIT mutations involving either
exon 11 (N = 3) or exon 9 (N = 1) (Table 1). No tumor had
mutations of both PDGFRA and KIT or more than one mutation
in either of these genes. Immunoblotting, performed in 3
cases with available snap-frozen tissue, confirmed absence of
KIT protein expression (Fig. 2). Two of these GISTs expressed
phosphorylated and total PDGFRA strongly and had PDGFRA
oncogenic mutations (Fig. 2; Table 1). The third case expressed
neither KIT nor PDGFRA but had a KIT exon 11 mutation
(Fig. 2; Table 1).
DISCUSSION
GIST is a mesenchymal neoplasm that exhibits morphologic
and immunophenotypic features similar to the interstitial
cells of Cajal, which are pacemaker cells regulating gastrointestinal
peristalsis. 13 A characteristic feature of GISTs, similar
to the Cajal cells, is expression of the protein tyrosine kinase
KIT, which is readily detected by immunohistochemistry and
890 © 2004 Lippincott Williams & Wilkins