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

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


KIT-Negative Gastrointestinal Stromal Tumors

Proof of Concept and Therapeutic Implications

Fabiola Medeiros, MD,* Christopher L. Corless, MD,† Anette Duensing, MD,*

Jason L. Hornick, MD, PhD,* Andre M. Oliveira, MD,* Michael C. Heinrich, MD,‡

Jonathan A. Fletcher, MD,*§ and Christopher D. M. Fletcher, MD, FRCPath*

Abstract: The diagnosis of gastrointestinal stromal tumor (GIST) is

currently based on morphologic features and immunohistochemical

demonstration of KIT (CD117). However, some tumors (in our estimation

approximately 4%) have clinicopathologic features of GIST

but do not express KIT. To determine if these lesions are truly GISTs,

we evaluated 25 tumors with clinical and histologic features typical of

GIST, but with negative KIT immunohistochemistry, for KIT and

PDGFRA mutations using DNA extracted from paraffin-embedded

tissue. Most tumors originated in the stomach (N = 14) or

omentum/mesentery (N = 5). The neoplasms were composed of epithelioid

cells (13 cases), admixed epithelioid and spindle cells (8

cases), or spindle cells (4 cases). Absence of KIT expression was confirmed

by immunoblotting in 5 cases. Tumor karyotypes performed in

4 cases were noncomplex with monosomy 14 or 14q deletion, typical

of GIST. Mutational analysis revealed PDGFRA and KIT mutations

in 18 and 4 tumors, respectively, whereas 3 tumors did not have apparent

KIT or PDGFRA mutations. The PDGFRA mutations primarily

involved exon 18 (N = 15) and included 11 tumors with missense

mutation in codon 842 (PDGFRA D842V or D842Y). In conclusion,

a small subset of GISTs with otherwise typical clinicopathologic and

cytogenetic features do not express detectable KIT protein. When

compared with KIT-positive GISTs, these KIT-negative GISTs are

more likely to have epithelioid cell morphology, contain PDGFRA

oncogenic mutations, and arise in the omentum/peritoneal surface.

Notably, some KIT-negative GISTs contain imatinib-sensitive KIT or

PDGFRA mutations; therefore, patients with KIT-negative GISTs

should not, a priori, be denied imatinib therapy.

Key Words: gastrointestinal stromal tumor, KIT, PDGFRA

(Am J Surg Pathol 2004;28:889–894)

From the *Department of Pathology, Brigham and Women’s Hospital & Harvard

Medical School, Boston, MA; Departments of †Pathology and

‡Medicine, Oregon Health & Science University Cancer Institute and

Portland VA Medical Center, Portland, OR; and §Departments of Pediatric

and Medical Oncology, Dana-Farber Cancer Institute, Boston, MA.

Drs. Medeiros and Corless contributed equally to this publication.

The study was funded in part by a VA Merit Review Grant to Dr. Heinrich.

Reprints: Christopher D. M. Fletcher, MD, FRCPath, Department of Pathology,

Brigham and Women’s Hospital, 75 Francis Street, Boston, MA

02115 (e-mail:

Copyright © 2004 by Lippincott Williams & Wilkins

Gastrointestinal stromal tumor (GIST) is the most common

mesenchymal tumor of the digestive tract and shows lineage

differentiation similar to the interstitial cell of Cajal. 13

Until relatively recently, most GISTs were classified as visceral

leiomyoma or leiomyosarcoma, reflecting the histologic

similarities between these two types of neoplasms. However,

in most cases, GISTs are now distinguished readily from true

smooth muscle tumors based on results of immunohistochemical

staining for KIT (CD117 antigen) and desmin. 5 Most

GISTs have oncogenic KIT mutations that engender constitutive

activation of this receptor tyrosine kinase, resulting in increased

cell proliferation and survival, 17 and such mutations

appear to be early (and even initiating) events in the pathogenesis

of many GISTs.

The central and essential role of KIT in GIST pathogenesis

is underscored by the therapeutic success of the KIT inhibitor

imatinib (Gleevec in the United States, Glivec in the

rest of the world; Novartis, Basel, Switzerland). 4,21 Most patients

with metastatic GIST show a major clinical response after

treatment with single-agent imatinib. 4 KIT positivity by

immunohistochemistry has been considered the gold standard

for GIST diagnosis and, at least for the first clinical trials, this

has been an eligibility criterion for imatinib therapy. 5 However,

in our experience, a subset of tumors that are typical for

GIST both clinically and histologically lack apparent KIT expression,

having low to undetectable KIT protein expression

by immunohistochemistry and Western blot evaluations.

Whether these tumors are indeed GISTs and whether they may

respond to imatinib therapy have been controversial. Recently,

we identified platelet-derived growth factor receptor alpha

(PDGFRA) mutations as an alternative oncogenic mechanism

in a small group of GISTs lacking KIT mutations 9 and this has

been confirmed by others. 11 Because imatinib can also bind

and inhibit PDGFRA, 9,16 responses to this drug have been observed

in some, but not all, patients with PDGFRA-mutant tumors.

8 In this study, we characterize a series of 25 KITnegative

GISTs, which reveal an interesting spectrum of

pathologic and molecular features of potential clinical and

therapeutic relevance.

Am J Surg Pathol • Volume 28, Number 7, July 2004 889

Medeiros et al Am J Surg Pathol • Volume 28, Number 7, July 2004


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


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).


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

Am J Surg Pathol • Volume 28, Number 7, July 2004

KIT-Negative GISTs

TABLE 1. Clinicopathologic Features and Mutational Analysis in 25 KIT-Negative GISTs



Age (yr)/





Cell Type


50 HPF KIT Mutations PDGFRA Mutations

1 76/M Small bowel 15 Epithelioid 20 WT exon 9, 11, 13, 17 Exon 18 delDIMH842-845

2 70/F Stomach 8 Mixed

Medeiros et al Am J Surg Pathol • Volume 28, Number 7, July 2004

FIGURE 1. Epithelioid GIST (A) with negative KIT immunohistochemistry

(B). Note the positive mast cells.

myosarcoma, melanoma, schwannoma, and carcinoma. Notably,

the cytogenetic profiles obtained in four tumors were classic

for GIST. Each of the karyotypes was noncomplex and

featured loss of material from the long arm of chromosome 14,

which is the most frequent cytogenetic aberration in GIST. 10

TABLE 2. Cytogenetic Profiles of Four KIT-Negative GISTs



Cytogenetic Profile

1 47,XY,add(1)(p11),add(1)(p13),+8,del(14)(q22),−19,r(19)


2 43–44,X,−X,i(8)(q10),add(11)(p15),−14,−15,−21,+1–2rings

3 45,XX,add(1)(p32),del(14)(q22p32),−22

5 46,XY,der(1)t(1;13)(p11;q11),der(3)t(1;3)(p32,q11),


FIGURE 2. Expression of phosphorylated and total receptor

tyrosine kinases in KIT-positive and KIT-negative GISTs. KITpositive

GISTs, both with KIT exon 11 mutations, are in lanes

1 to 2. KIT-negative GISTs include two with PDGFRA mutations

(lanes 3–4) and one with KIT exon 11 mutation (lane 5). Phosphorylated

and total KIT are expressed strongly in the KITpositive/KIT-mutant

control GISTs, whereas phosphorylated

and total PDGFRA are expressed strongly in the KITnegative/PDGFRA-mutant

GISTs (case nos. 1 and 23). KIT and

PDGFRA are not demonstrably expressed in the KITnegative/KIT-mutant

GIST (case no. 4). The PI3-K stain provides

evidence for approximate equivalency in loading of intracellular


By contrast, leiomyosarcomas, and particularly those of higher

histologic grade, typically have extremely complex karyotypes.

Most GISTs are characterized by oncogenic mutations

of KIT and constitutive activation of the KIT receptor tyrosine

kinase, which seems to drive tumor formation. However, a

small subset of GISTs has been identified that lacks detectable

KIT mutations. 17,20 Recently, activating mutations in the related

receptor tyrosine kinase PDGFRA were reported in 35%

to 67% of GISTs lacking KIT mutations. 9,11 We evaluated

PDGFRA and KIT mutations in the 25 cases in our series and

found that the majority exhibited mutations of the PDGFRA

gene, most frequently involving exon 18. All pure epithelioid

tumors in this series harbored PDGFRA mutations. However,

we also identified four tumors with KIT mutations despite the

892 © 2004 Lippincott Williams & Wilkins

Am J Surg Pathol • Volume 28, Number 7, July 2004

KIT-Negative GISTs

consistent negativity of these tumors for KIT by both immunohistochemistry

and immunoblotting. No tumors had mutations

involving KIT and PDGFRA concomitantly, corroborating

previous evidence that these are mutually exclusive transforming

equivalents in GIST oncogenesis. 9 Three tumors

otherwise indistinguishable from the others in this study had

no detectable KIT or PDGFRA mutations, suggesting that

some KIT-negative GISTs arise through alternative oncogenic


The biologic basis for the absence of KIT expression, in

the subgroup of GISTs identified in our study, is unclear. Most

of the tumors contained PDGFRA mutations, and as discussed

above, such mutations appear to be alternate, or “either/or”

transforming mechanisms, compared with the more common

KIT oncogenic mutations in GISTs. In PDGFRA-mutant tumors,

KIT expression may simply be superfluous and therefore

down-regulated, as mutant PDGFRA appears to act as an

oncogenic substitute for KIT in these cases. It would have been

of interest, if feasible, to assess the PDGFRA-mutant cases immunohistochemically

for evidence of PDGFRA protein expression.

However, multiple trials using a variety of currently

available antibodies (in both our Boston and Portland laboratories)

have failed to identify any commercial antibody that

yields meaningful and reproducible results with a clean background.

Thus, we do not think that these presently available

antibodies have clinical utility. More puzzling are the four tumors

containing KIT gene mutations but in which KIT protein

expression was not demonstrated. It is counterintuitive that the

neoplastic GIST cells would select for a genomic KIT mutation

in the absence of the functional protein product of that gene.

One possibility is that these tumors originated through a KITactivated

pathway and then later became independent as a result

of additional secondary mutations. 6 Another possibility is

that these discordant genomic and protein findings could be

related to technical factors. For example, deletion of the KIT

C-terminus, containing the epitope against which the DAKO

KIT antibody was raised, could lead to false-negative immunostaining

results. On the other hand, C-terminal truncation

cannot account for the negative KIT immunohistochemistry in

all KIT-negative GISTs, because we corroborated absence of

KIT protein expression, using antibodies to a kinase domain

epitope, by immunoblotting in three PDGFRA-mutant or KITmutant

cases with available frozen tissue. Finally, it is conceivable

that routine immunohistochemistry is insufficiently sensitive

to detect the lowest biologically relevant levels of KIT

expression in all tumors. We recently reported a case of a KITmutant

GIST in which KIT expression was virtually absent,

excluding the patient from a clinical trial. The tumor became

PET negative and showed shrinkage on CT scan when the patient

was treated with imatinib off protocol. 1 Based on this anecdotal

example, it is possible that other KIT-mutant GISTs

that lack KIT expression, such as the 4 cases in the present

series, are imatinib sensitive.

In summary, we report that a small subset of GISTs lack

KIT expression but have otherwise typical clinical, histopathologic,

and cytogenetic features. Most of these tumors have at

least partly or totally epithelioid cytomorphology and harbor

PDGFRA mutations, but some examples have KIT mutations

despite the absence of demonstrable KIT protein expression.

Given that most KIT-negative GISTs contain PDGFRA or KIT

mutations, one might expect that patients with such tumors

could potentially respond to therapeutic PDGFRA and KIT inhibition

with imatinib. 8 Even acknowledging that the commonest

PDGFRA D842V mutation is intrinsically imatinib resistant,

8,11 approximately 30% of the PDGFRA mutations

identified are known to be potentially imatinib sensitive, 8 providing

a greater hope of treatment response than with any other

currently approved therapy. Therefore, it is important for pathologists

and oncologists to be aware that, in the context of

otherwise typical morphology, a GIST diagnosis should not be

precluded on the basis of negative immunohistochemical

staining for KIT and that such patients should not a priori be

denied imatinib therapy. Where testing is available, the finding

of KIT or PDGFRA mutations not only may help to confirm or

enable the diagnosis of a KIT-negative GIST, but can also provide

important prognostic information for patients in whom

imatinib therapy is being considered. 8


The authors thank Troy Bainridge, Laura McGreevey,

Andrea Haley, Ajia Town, Maureen Thyne, and Catherine

Quigley for excellent technical assistance.


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894 © 2004 Lippincott Williams & Wilkins

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