Antibodies and kits for KIT
GIST prognosis based on histology
William Check, PhD
In the beginning, there were smooth muscle tumors of the
gastrointestinal tract. And darkness reigned over the face of these tumors.
"Among the small number of pathologists who cared what they were," says Christopher
Corless, MD, PhD, of Oregon Health and Science University, "no one had any idea
how to classify them." During the 1970s and 1980s, these tumors were variously
called leiomyomas (benign), leiomyosarcomas (malignant), or Schwannomas.
Then, in 1998, light shined forth over this "mass of confusion," when Japanese and Swedish investigators independently identified expression of the cell surface receptor KIT (also known as CD117) in up to 95 percent of a subset of GI tumors. "This was a real breakthrough," says Dr. Corless, who is professor of pathology and director, OHSU Cancer Pathology Shared Resource. "It was the first marker indicating that some of these tumors were distinct." The darkness was rolled back and a new category of GI tumors appeared, called gastrointestinal stromal tumors, or GISTs. "Detection of KIT became the new gold standard in making a diagnosis [among abdominal tumors]," Dr. Corless says.
Not only was KIT expression recognized in GISTs, but mutations in the kit
gene were established as the initiating neoplastic events, says Christopher
Fletcher, MD, FRCPath, professor and director of surgical pathology at Brigham
and Women’s Hospital. Dr. Fletcher calls these findings "revolutionary."
"Prior to this discovery, the term GIST was used," he says, "but purely descriptively. It was a diagnostic wastebasket for many spindle cell tumors in the GI tract." Now the term GIST is specifically used for a subset of GI lesions that are almost always KIT-positive. "KIT provides us with an immunohistochemical marker that we can use to identify most of these tumors, which was a big coup for pathologists," Dr. Fletcher says.
Markku Miettinen, MD, chairman of the Department of the Soft Tissue Division and distinguished scientist at the Armed Forces Institute of Pathology, says, "We now know that almost all tumors previously considered gastric or intestinal smooth muscle tumors are actually GISTs."
Recognition of KIT expression in GISTs provided a huge benefit beyond classification. "Obviously what is most newsworthy is that KIT is a tyrosine kinase receptor," Dr. Fletcher says. "All of a sudden there was a way to treat [GISTs]." Imatinib (Gleevec, Novartis) had just been proved to be effective therapy for chronic myelogenous leukemia, or CML, which is also initiated by a runaway tyrosine kinase, in that case produced by a chromosome translocation. A researcher at Dana Farber Cancer Institute, David Tuveson, hypothesized that the KIT receptor was structurally and functionally similar to BCR/ABL in CML and would also be inhibited by imatinib, Dr. Fletcher says. Tuveson produced in vitro evidence for this idea, which was followed by successful clinical trials in patients with GISTs. "So we rapidly went from having a nice classification, which was good because we pathologists could diagnose GISTs more reproducibly and define them in a meaningful way, to the marvelous discovery that there was a way to inhibit this new mechanism in patients," Dr. Fletcher says.
"This is one of the fastest examples I know of a bench-to-bedside story," he adds. "It sets the paradigm for developing targeted therapeutics by specifically switching off the mechanism that drives tumor cells. Most cancer treatments smash up DNA. You hope you kill more tumor cells than normal cells. Now we are beginning to see targeted therapies."
Dr. Corless agrees that GISTs are "basically a model for what is coming." For example, the kinase inhibitor gefitinib (Iressa, AstraZeneca) is being developed for lung and colon cancer. "We have a lot better handle on the biology of those tumors than for many other cancers," he says. "The correlation between mutation testing and treatment is pointing to where a lot of other cancer therapies will be going in the future."
At the root of all this is immunohistochemistry, or IHC. Dr. Fletcher wrote
in an editorial: "As other new pharmacologic agents are discovered by screening
for small molecule inhibitors of oncoproteins, it is likely that there will
be a rapid increase in the immunohistochemical stains that pathologists will
be asked to perform to detect potential therapeutic targets" (Am J Clin
Pathol. 2003; 119: 325-327).
Says Dr. Miettinen: "Pathologists have become very aware of GISTs because they represent a new type of diagnosis that has a special clinical correlation. There is a receptor tyrosine kinase activation that can be countered with a specific inhibitor drug." He sees KIT IHC as the key to GIST diagnosis: "GIST is one of not so many tumors that are almost always KIT-positive."
Elizabeth Montgomery, MD, associate professor of pathology and director of clinical GI pathology at the Johns Hopkins Medical Institutions, says, "When confronted with a spindle cell tumor in the GI tract, the two main points are that stromal tumors should always be in the differential diagnosis and that CD117 IHC should virtually always be performed." Anatomic pathologists are now so aware of GISTs that they sometimes overdiagnose them, though, for the most part, she says, "they do an excellent job with GISTs."
Beyond the simple fact of doing IHC for KIT in suspected GISTs lie a number
of complex and challenging issues:
- How do mutant KIT oncoproteins induce tumors?
- How does imatinib kill KIT-positive tumors?
- What histological features tip off the pathologist that a tumor is a possible
- Is there anything to help choose among antibodies or assays for KIT?
- What should be done when the KIT IHC turns up negative?
- What non-GIST tumors can give positive KIT results?
- Are all KIT-positive tumors candidates for imatinib therapy?
- Is it possible to determine the likelihood of an individual GIST responding
- How does the pathologist predict malignant potential in a GIST?
Clear answers are available to some of these questions, while answers to
others are evolving.
In its wild-type state, kit is a cell surface receptor whose
natural ligand is stem cell growth factor. At the same time that the Japanese
scientists recognized KIT on the surface of GISTs, they found that mutations
in the gene coding for this receptor activate the kinase constitutively. Mutant
isoforms of KIT become autonomous—they phosphorylate tyrosine residues on
all types of signaling proteins that bind to or interact with them in the
absence of the growth factor. Mutant KIT receptors also phosphorylate each
other, called autophosphorylation. "Assessing KIT autophosphorylation is a
commonly used approach in assessing the effectiveness of inhibitors like imatinib,"
Dr. Corless says.
"Mutant KIT escapes normal regulation," Dr. Miettinin adds. It sends signals
into the interior of the cell along the signal transduction pathway that ultimately
leads to the nucleus to promote cell proliferation and inhibition of apoptosis.
Imatinib inhibits autophosphorylation of KIT. Dr. Corless says that several
groups, including his, proposed to Novartis to treat GIST with imatinib. "Their
response," he recalls, "was ’What the hell is a GIST?’ Outside pathology, few
people had heard of GISTs." Eventually, a patient in Finland was given imatinib
for metastatic GIST to the liver in a compassionate-use protocol. "The patient
responded beautifully," Dr. Corless says. "Clinical trials with imatinib subsequently
showed that about 80 percent of patients have a response or significant stabilization
of their disease" (Demetri GD, et al. N Engl J Med. 2002;347: 472-480).
Imatinib is now approved for treating advanced GISTs, which make up about one-third
of the approximately 5,000 new cases of GIST diagnosed each year in the United
"Inhibitor treatment is now given to patients who have metastatic or unresectable
GISTs," Dr. Miettinen says. "Neoadjuvant imatinib has also been used as a
precautionary measure immediately following surgery even without evidence
of metastasis, mostly in clinical trials."
Already a second kinase in hib it or, SU-11248 (Pfizer), is in phase three
trials for GIST patients who show imatinib resistance. About 65 percent of
such patients have had either partial response or stabilization on this new
agent, Dr. Corless says. "Based on mutation analysis, we suspect it might
do even better as a first-line drug for some patients," he proposes.
What we now know as GISTs were initially considered smooth muscle tumors
simply because they occur in the gastric or intestinal wall amidst smooth
muscle, Dr. Miettinen explains. "However, it has turned out that these tumors
are closely related to interstitial cells of Cajal [ICC], which are cellular
intermediates between nerves and smooth muscle cells," he says. GISTs either
arise from ICC or share a common stem cell with them. What is key to the GIST
story is that ICC express kit.
Even before staining for KIT, a pathologist will typically suspect a GIST
based on histology. "You always know," Dr. Montgomery says. GISTs have a characteristic
morphological pattern and histologic features somewhat akin to—but not identical
to—smooth muscle tumors. "When one is confronted with a tumor that looks almost,
but not quite, like a classic muscle tumor," she says, "one says, ’Aha! This
is a GI stromal tumor.’"
Dr. Miettinen says Cajal cells are normally dispersed around the myenteric
plexus and in the muscle layer of the GI tract. "Whenever you see these cells
in masses," he says, "it is either Cajal cell hyperplasia or a GIST." Another
clue is that, in the stomach, GIST is by far the most common mesenchymal tumor.
In the intestine the proportion of GISTs to other tumors is less striking,
but still the majority of mesenchymal tumors in the intestine are GISTs as
Staining by IHC for KIT provides confirmation for a specimen
suspected from histology of being a GIST. A number of antibodies are available
for this purpose. Most are analyte-specific reagents, or ASRs, but Ventana’s
Pathway c-KIT assay has just been approved by the Food and Drug Administration.
It is approved as an automated aid to diagnosis and as an aid for the selection
of GIST patients who may qualify for Gleevec therapy.
Thomas M. Grogan, MD, Ventana’s chief medical officer, says this automated
assay offers two advancements. First, he says, "the automation addresses the
complex assay conditions including tight control of temperature and buffer
pH to allow reliable results. Second, the rabbit monoclonal [clone 9.7] reduces
the false-positivity found in polyclonal assays." Finally, Dr. Grogan says,
their comparison of the Pathway c-KIT clone 9.7 rabbit monoclonal to mutational
analysis shows an 88 percent concordance when mutations to exons 9, 11, 13,
and 17 are examined. "Importantly, among the GIST cases that were c-KIT negative
by IHC [10 percent of GISTcases], many of these [56 percent] demonstrated
PDGFRA mutational change," he says.
DakoCytomation’s c-KIT pharmDx assay is at the FDA now awaiting approval.
Dr. Corless has worked with most available IHC methods. "I don’t have a position
to take on any particular antibody," he says. "All of them work if used correctly."
In his view, it is more important to titer the antibody, whether it is a prepackaged
kit or an ASR. "It is important that the titer is set so that there is reasonable
staining of GIST samples but no staining of smooth muscle elements or epithelial
elements in control sections," Dr. Corless says.
Dr. Fletcher notes that one of the major commercial antibodies has been demonstrated
to be less specific, though both antibodies tested were unreliable when used
with heat-induced epitope retrieval and at low (1:50) dilution (Lucas DR, et
al. Am J Clin Pathol. 2003;119: 339-345). "Most IHC is performed with
antigen retrieval," says Dr. Fletcher. "It turns out if you use antigen retrieval
with the most widely utilized anti-KIT antibody, which comes from Dako, that
gives you false-positives in many other tumor types."
Kenneth Bloom, MD, medical director of ChromaVision, says workup of GIST
involves more than one antibody. At the time of his interview with CAPTODAY,
he was senior medical director of USLabs, where he used a panel consisting
of antibodies to KIT, s100, CD34, PDGFRA, smooth muscle actin, desmin, and
beta-catenin in the workup of most cases submitted to rule out a GIST. "Even
if there is aberrant staining with one of the antibodies, that should not
necessarily confuse the diagnosis," he says. The diagnosis can be made in
most cases by the overall histologic pattern plus the immunophenotype.
Under optimal conditions, about 95 percent of GISTs stain positive for KIT.
(About 85 percent of GISTs have KIT mutations, Dr. Corless explains; in some
cases KIT appears to be activated by a mechanism other than mutation). Drs.
Fletcher and Corless and others have shown that many KIT-negative GISTs (four
to five percent of all GISTs) have mutations in another transmembrane signaling
protein, platelet-derived growth factor receptor alpha, or PDGFRA (Heinrich
MC, et al. Science. 2003;299: 708-710). These authors wrote: "Tumors
expressing KIT or PDGFRA oncoproteins were indistinguishable with respect to
activation of downstream signaling intermediates and cytogenetic changes associated
with tumor progression." Importantly, some PDGFRA-positive GISTs respond to
imatinib. "A small but clinically relevant fraction of GI tumors will not be
stained by antibodies to KIT but are still GISTs, and these patients deserve
a trial of imatinib," Dr. Corless says.
Dr. Montgomery agrees: "Pa thologists should be aware that [a small fraction]
of GI stromal tumors don’t express CD117, but a subset of those may still respond
to imatinib" (Medeiros F, et al. Am J Surg Pathol. 2004; 28: 889- 894).
As a result, she says, there is a role for mutation analysis for KIT and PDGFRA
in a subset of tumors. A small number of laboratories, including Dr. Corless’,
offer mutation analysis.
In the original clinical trials of imatinib in GISTs, KIT staining was an
absolute requirement. "Now that we have learned that a subset of GISTs are
KIT-negative," Dr. Fletcher says, "we will have to go back to relying more
on morphology, at least in the short term." Dr. Corless puts it more strongly.
"If any company tries to say that you have to use their reagent to demonstrate
GIST and prescribe imatinib, I would say that is [nonsense]," he says. "A
diagnosis should come from a pathologist, not an antibody."
Faced with a KIT-negative tumor that looks like a GIST by morphology, an
oncologist might say, "I’m going to give imatinib anyway and see what happens."
Dr. Fletcher proposes an alternative: Send the case to a specialized center
where they have seen very large numbers of GISTs over the past few years—as
opposed to a few cases a year—and see if they think it is a GIST.
Says Dr. Corless: "This tumor has evolved from being a new diagnostic entity
to a very complex family within the space of a few years. It is not fair or
appropriate at this time to say something should be KIT-positive or -negative
for diagnosis and treatment. We have to be a lot more sophisticated in our
approach to these tumors."
Pathologists also need to be sophisticated about KIT-positive non-GISTs.
Dr. Miettinen says a number of other tumor types show non-specific CD117 staining.
"The one that causes the most trouble," he says, "is mesenteric fibromatosis.
In my consult material, I have more than once received a specimen in which
the referring physician was looking for a reason to use imatinib treatment
and it has been desmoid."
Positive IHC for KIT can also occasionally be seen in mastocytosis, testicular
and ovarian seminoma, sinonasal T-cell lymphoma, rare examples of thymic carcinoma,
angiosarcoma, and Ewing sarcoma. All must be considered in the differential
diagnosis of a KIT-positive tumor.
Dr. Montgomery agrees that KIT-positive desmoids are the most problematic,
because they occur in the GI tract. "If you have a mass in the bowel wall,
it isn’t going to be a seminoma," she says. "It will be a desmoid."
It is critical for pathologists to be aware of non-GIST tumors that can give
positive KIT results. Oncologists often look for an excuse to use imatinib
because it is relatively nontoxic and because patients have been bombarded
by promotional material about it. "We get requests for KIT testing on everything
because clinicians want to throw imatinib at it," Dr. Montgomery says. She
calls such requests "frivolous and inappropriate." She and her colleagues
do the test, "then tell the doctors that the result may be meaningless," she
Dr. Fletcher has had similar experiences. "It was believed for a while that
any type of tumor that was KIT-positive would be a reasonable target for imatinib,"
he says. "So some oncologists were asking that almost any bad cancer—ovarian,
breast—especially if it was metastatic, be stained for KIT." But KIT expression
in non-GIST tumors doesn’t result from mutations in the kit gene, so
it doesn’t have the same biological meaning as in GISTs. "Imatinib has no therapeutic
effect on those other tumors," Dr. Fletcher says. "The key message is that unless
there has been initial work to demonstrate that a given type of tumor has kit
mutations, there is no basis for doing KIT immunostaining and trying imatinib
empirically." His concern: "That this approach can destroy the value of a good
drug by using it in tumors that were destined never to respond."
Some good has resulted from overtesting for KIT. "It has emerged in recent
months, though it is not well understood, that other tumors that do not have
kit mutations may respond to imatinib therapy," Dr. Fletcher says.
For example, in some cases, dermatofibrosarcomas have activated platelet-derived
growth factor receptor beta, which can be susceptible to imatinib.
Because the basic biology of GISTs has been so well elucidated,
molecular characterization can be used to predict the likelihood of an individual
tumor responding to imatinib. "Molecular characterization of mutations in the
kit gene correlates very well with response to imatinib," Dr. Fletcher says.
"Such a test was not available at all four to five years ago," he notes, "then
it was developed as a research tool." Because it is so good at predicting treatment
response, it has now been made available as a clinical test by Dr. Corless’
laboratory, he says, and other laboratories are setting it up.
This test, performed on paraffin-embedded tumor, is an analysis to determine
the presence and exon location of mutations in kit or pdgfra.
Dr. Corless says the correlation between the location of the mutation in the
kit or pdgfra genes and response to imatinib came out in phase two
trials. Tumors with mutations in exon 11 of the kit gene have the best
response to drug and are, fortunately, most common. Those with exon 9 mutations
do not respond as well overall. Interestingly, the small fraction of tumors
that are wild-type for both kinases, about 12 percent, have the worst response,
presumably because they have a different molecular pathway to the oncogenic
state. Pediatric GISTs are virtually all wild-type, Dr. Corless says, as are
GISTs associated with neurofibromatosis; however, imatinib response data are
not yet available for these subsets of patients.
Despite its value, mutational analysis for GISTs is only available in a few
centers and is not always understood by treating clinicians, according to
Dr. Fletcher. "When advances go so fast," he says, "obviously there will be
a lag time for people to get used to these new approaches."
In contrast to the high-tech nature of the test for predicting
response to imatinib, the criteria for estimating malignancy and predicting
recurrence are standard anatomic pathology measurements: tumor size and mitotic
index. Says Dr. Miettinen, "I believe that these two factors are important
enough to warrant inclusion in any pathology report on GISTs." In general,
he says, tumors with five or fewer mitoses per 50?? field and those that are
5 cm or less in diameter are clinically favorable. "However," he notes, "now
we know that gastric tumors of equal parameters tend to behave better than
intestinal ones. Even large tumors that have very low mitotic activity tend
to be more benign in the stomach." Dr. Miettinen estimates that less than
25 percent of gastric GISTs are malignant by clinical course, compared with
40 to 50 percent of intestinal GISTs. Gastric tumors make up 60 to 70 percent
Guidelines for prognosis are included in the publication from the 2001 National
Institutes of Health consensus meeting on GISTs (Fletcher CDM, et al. Hum
Pathol. 2002;33:459-465). "Those guidelines produce a framework for individual
cases but are not perfect," Dr. Fletcher cautions. He agrees that mitotic
index and tumor size are powerful predictors. But he adds, "The fact remains
that there are still tumors that don’t live by those rules. There are well-characterized
examples of very small tumors—some even less than 2 cm—that give rise to metastases,
and examples of tumors greater than 2 cm with very few mitoses that give rise
to metastases. These exceptions," he says, "remain a focus of great attention."
It may be helpful to identify upfront GISTs that have the potential for aggressive
behavior and possibly even consider neoadjuvant treatment with imatinib before
there is evidence of spread, in Dr. Fletcher’s view. "There are clinical trials
going on right now in which persons with primary tumors without evidence of
spread are randomized to surgery followed by imatinib versus surgery alone,"
he says. Selecting the most aggressive tumors for such testing would be desirable.
More accurate identification of potentially malignant GISTs could have financial
benefits as well. Imatinib costs $30,000 to $45,000 per year of treatment.
"Greater understanding of the biological behavior of GISTs would help to optimize
treatment and target it to the patient population most likely to benefit,"
Dr. Miettinen says.
He points out another curious feature about the course of GISTs. "Some low-grade
GISTs metastasize 10 to 15 years after surgery," he says. The longest history
in the AFIP files is a patient who is alive 10 years after total gastrectomy
for a recurrence that happened 30 years after the primary tumor. Dr. Miettinen
and his colleagues at the AFIP are conducting a followup study on gastric
and intestinal GISTs to understand their natural history and to help optimize
In theory, Dr. Montgomery says, patients should probably be followed forever
after treatment for GIST. An exception might be made for the smallest tumors.
"If a tumor is less than 1 centimeter and completely excised, we can probably
let those patients relax," she says.
Adherence to size and mitotic index for determining prognosis in GISTs is
not universal. "Henry Appelman has published morphologic criteria for gastric
GISTs that he believes allow separation of benign from malignant tumors,"
Dr. Montgomery says. (See "GISTprognosis based on histology.") "The trouble
is that every once in a while a tumor with benign features will metastasize.
Prediction based on morphology is not so easy for GISTs," she points out.
"For adenomas or carcinoma of the colon, we are quite sure which ones are
benign and malignant by looking through the microscope. For GISTs, we will
probably need extra markers."
Summing up these exciting findings, Dr. Corless says: "This
has been a fun story. We have discovered that imatinib is effective not only
for GISTs and CML, but also for dermatofibrosarcoma protuberans, hypereosinophilic
syndrome, and CMML [chronic monomyelocytic leukemia]." The common theme is
that all five of these tumors have activation of a kinase pathway that is
targeted by imatinib. Dr. Miettinen believes that the KIT story presages the
future of this field.
"Because there are at least 60 known receptor tyrosine kinases," he predicts,
"new connections between receptor mutations and disease will undoubtedly be
made in the near future."
Dr. Corless notes a recent example. "It was just published that EGFR [epidermal
growth factor receptor] mutations in non-small-cell lung cancer correlate with
response to gefitinib, which is a completely analogous situation," he says (Lynch
TJ, et al. N Engl J Med. 2004;350:2129-2139; Paez JG, et al. Science.
2004;304(5676):1497-1500. Published online April 29, 2004). As more discoveries
of this type are made, he predicts, "we will see increasing emphasis on the
use of mutation testing as a way of assigning patients into therapeutic categories."
William Check is a medical writer in Wilmette, Ill.