May 2003
Cover Story
William Check, PhD
Our
progress in understanding chronic myeloid leukemia, or CML,
might be expressed by a paraphrase of the well-known tribute to
George Washington: First with a chromosomal abnormality, first with
an oncogene, and first with specific targeted molecular therapy.
“It was not chance that imatinib [Gleevec] was the first gene-targeted
molecular therapy to come to the clinic,” says Vesna Najfeld,
PhD, director of tumor cytogenetics and oncology-molecular testing
at Mt. Sinai School of Medicine, New York City. In 1960, when Nowell
and Hungerford discovered the Philadelphia chromosome in CML cells,
it was the first specific chromosomal abnormality implicated in
human cancer. In 1973 Rowley established the genetic basis of this
abnormality: The Philadelphia chromosome represents a balanced translocation
between chromosomes
9 and 22.
A decade later, only a few years after oncogenes had been accepted
as the basis of cancer, CML became one of the first cancers in which
an oncogene was demonstrated. Several laboratories showed that the
t(9;22) translocation produces a fusion of two genes, BCR and ABL,
leading to constitutive expression of the ABL tyrosine kinase activity,
which initiates the leukemogenic process. This set the stage for
development of imatinib,
a specific inhibitor of the ABL tyrosine kinase, which many oncologists
now consider to be first-line treatment for most, if not all, newly
diagnosed CML patients.
CML also epitomizes another fundamental process: collaboration between
laboratorians and clinicians. At every stage in the management of
CML, laboratory tests make an essential contribution. Conventional
cytogenetics and fluorescence in situ hybridization, or FISH, contribute
important information about diagnosis and prognosis. Both of these
tests, along with molecular methods, notably PCR, are important
in evaluating the impact of imatinib therapy, documenting remission,
and monitoring for recurrence. In the latest development, these
and other technologies are being used to detect imatinib-resistant
clones, which are appearing with increasing frequency with longer
followup, initially in patients treated in accelerated or advanced
stages, more recently in patients treated in the chronic phase.
“Given a burden of at least 1012 leukemic cells
at diagnosis, the likelihood of harboring a preexisting mutation
or developing one during therapy is reasonably high,” says
Neil Shah, MD, PhD, clinical instructor in the Division of Hematology/Oncology
of the David Geffen School of Medicine at the University of California,
Los Angeles. Although imatinib is in his view the best single agent
for CML, he and his colleagues are concerned that resistance will
be a growing problem. “We are seeing it more as time goes
on, even in patients treated in the chronic phase of disease. Our
belief is that ultimately it will probably occur in the majority
of cases,” he says. An emerging question is whether and when
clinical laboratories should institute assays for imatinib resistance.
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In the diagnosis of CML, laboratory methods confirm a clinical
suspicion, says William Finn, MD, director of hematopathology
at the University of Michigan Medical Center. “CML classically
has a distinct peripheral blood morphology, which leads us strongly
to suspect a diagnosis in the majority of cases,” Dr. Finn
says. “But the morphologic findings need to be confirmed by
other methods.”
Conventional cytogenetics is the mainstay of laboratory confirmation.
Also essential are FISH and reverse transcriptase (RT)-PCR. (Technical
issues make it preferable to measure cDNA reverse transcribed from
BCR-ABL mRNA, rather than genomic BCR-ABL DNA itself.) “I
would say in modern usage, for the sole purpose of diagnosis, these
two methods are equivalent,” Dr. Finn says. “FISH has
become more popular,” in his view. However, he notes, “As
molecular assays become easier, practice is evolving toward detecting
BCR-ABL fusion transcripts at the molecular level."
"PCR can be done with a very small amount of material and can
identify the specific type of BCR-ABL fusion by the size of the
mRNA product,” Dr. Finn continues. Different mRNAs are associated
with CML or acute lymphoblastic leukemia (ALL). But FISH also can
detect these different-length fusions. And FISH can see additional
abnormal duplications of the Philadelphia chromosome and trisomy
8, which have prognostic significance. Other genetic abnormalities
that portend poor prognosis are seen on karyotyping.
Karyotyping is also useful in following the progression of CML to
acute myeloid leukemia. By six to seven years, a “very substantial”
proportion of CML cases evolve to acute leukemia or blast crisis,
Dr. Finn says. Before this transformation, CML goes through an accelerated
phase, which can be detected by clinicopathologic correlation. “Clinical
signs of impending blast crisis include adenopathy and splenomegaly,”
Dr. Finn says. “Laboratory measurements are increased basophil
counts, an increase in the blast count, and additional cytogenetic
abnormalities.”
Rita Braziel, MD, director of hematopathology at the Oregon Health
Sciences University, describes her institution’s approach
to diagnosing and monitoring CML. “At initial diagnosis, we
do karyotyping,” she says. “Virtually all metaphases
are positive for fusion at this time, even if the patient is not
in blast crisis or accelerated phase.”
At the first followup, FISH is added to karyotyping. “We continue
to do both assays until the patient becomes cytogenetically negative,”
Dr. Braziel says. At that time some oncologists substitute quantitative
RT-PCR for cytogenetics. Cytogenetically negative samples typically
remain FISH- and PCR-positive, at least at a low level for a prolonged
time. Using single-fusion probes, FISH has a background cell false-positive
rate of five percent or higher. However, Dr. Braziel says, “With
the dual-fusion FISH probes, our cytogeneticists will sometimes
report down to a less than one percent sensitivity level.”
False-positive results are not a problem with the dual-fusion probes,
because that would require superimposing the BCR and
ABL signals from both normal alleles, which Dr. Braziel calls “virtually
impossible.”
LoAnn
Peterson, MD, director of hematopathology at Northwestern University
Medical School, agrees that the ability of conventional cytogenetics
to visualize additional chromosomal abnormalities warrants its use
as the initial confirmatory test. “FISH is rapid and RT-PCR
is very sensitive,” she says, “but neither tells you
about additional chromosomal abnormalities. If the clinician suspects
CML but you don’t see the BCR-ABL fusion by cytogenetics,
then you can go to FISH or RT-PCR. In a small percentage of cases
the Philadelphia chromosome can be masked.”
At Specialty Laboratories, a guideline for CML disease management
recommends diagnosis by hematopathology and FISH detection of the
BCR-ABL fusion. “At that point we recommend baseline quantitation
of BCR-ABL by RT-PCR,” says Jean Amos, PhD, Specialty’s
scientific director of molecular genetics. “FISH is really
the gold standard for diagnosis,” she says, “since some
healthy people are positive for BCR-ABL with PCR. We would expect
PCR to have a higher false-positive rate than FISH because PCR is
more sensitive.”
The most common platforms for quantitative RT-PCR are Taqman chemistry
from ABI and Roche’s LightCycler. “These two very different
technologies give similar results,” says Daniel Arber, MD,
director of the clinical hematology laboratories and associate director
of molecular pathology at Stanford University Medical Center. “There
are differences, but they haven’t been studied as well as
they should be.”
One of the biggest problems, he says, is how laboratories report
results of quantitative testing for BCR-ABL. “It is very hard
to compare results from one laboratory to another, which poses a
problem if patients move around—they might get a baseline
value at one hospital and followup at another. Unless we can correlate
results between laboratories,” Dr. Arber cautions, “our
results aren’t going to be very helpful in these situations.”
(See “Quantitative RT-PCR: how
well do labs do?”)
When using FISH, it is essential to understand probes. The standard
probe has a major drawback, says Dr. Najfeld—it has a false-positive
rate as high as 10 percent. “At remission, with this probe
we wouldn’t be able to distinguish residual disease from a
false-positive signal,” she says. “So it is not good
for monitoring.” She uses the BCR-ABL ES (extra signal) probe,
in which the ABL probe includes an additional gene called ASS and
is marked with red fluorochrome, reducing the false-positive rate
from two percent to 0.2 percent. “This gives us a 99 percent
chance of detecting residual disease post-therapy,” Dr. Najfeld
says.
FISH is as accurate on peripheral blood as on bone marrow, Dr. Najfeld
has shown. So blood is used in monitoring therapy, when testing
is done every three months, since drawing blood is easier on the
patient. For diagnosis, however, bone marrow is the tissue of choice,
because conventional cytogenetics can be used to look for additional
chromosomal abnormalities. At diagnosis, about 10 percent of patients
may have variants of the Philadelphia chromosome. Others have deletion
of chromosome 9 at the translocation breakpoint. “I think
we need to know that,” Dr. Najfeld says. These patients have
a worse prognosis, and the deletion furnishes a marker for followup.
Once a diagnosis of CML is established, a therapeutic dilemma
arises. Should the patient be treated immediately with imatinib?
“That standard is shifting right now under our feet,”
says Dr. Finn. Originally, treatment with hydroxyurea was geared
toward keeping the white blood cell count low and preventing the
hematologic sequelae of disease—clotting and bleeding. Such
treatment did not limit the natural tendency of CML to become acute.
“With improved treatment that has changed somewhat,”
Dr. Finn says. “Interferon is quite effective [for cytogenetic
remission], but we don’t know if it delays the acute phase.
“Because of the ease of treatment with imatinib and its efficacy,”
Dr. Finn continues, “some clinicians are now treating the
diagnosis rather than the condition.” Many patients remain
in remission by hematologic criteria on imatinib alone. “Will
they be cured?” Dr. Finn asks. “The jury is still out,”
he says, pending longer followup.
Before imatinib was introduced, the treatment of choice was interferon
plus cytarabine. But Michael Deininger, MD, PhD, assistant professor
of bone marrow transplantation/ leukemia at OHSU, cites a recent
trial showing that imatinib monotherapy significantly delays progression
to accelerated phase or blast crisis relative to interferon/cytarabine
in newly diagnosed patients in chronic phase (N Engl J Med.
2002;348:994-1004). At 18 months, 96.7 percent of patients treated
with imatinib were progression-free. Moreover, the rate of complete
cytogenetic responses with imatinib was significantly higher than
with interferon/cytarabine: 76 percent versus 14.5 percent. However,
Dr. Deininger notes, “Since followup was limited, we don’t
know if these responses will hold up long-term.”
Dr. Shah, too, interprets the data cautiously. “We surmise
that imatinib is going to be far superior with respect to survival,”
he says, “but it is still too early to draw that conclusion.”
An obvious next step is to combine imatinib with interferon or cytarabine
as initial therapy in newly diagnosed patients. In the United States
at this time only uncontrolled phase two studies of this strategy
are being conducted, Dr. Deininger says. Studies in Germany are
comparing imatinib combinations to imatinib monotherapy. “Available
data suggest that response may be achieved more quickly with imatinib
plus another drug,” Dr. Deininger says, “but we don’t
know whether responses will be achieved in more patients or whether
they will last longer.”
Dr. Peterson notes another dilemma: “We don’t know if
imatinib is curative,” she says, “but we do know that
bone marrow transplantation has curative potential.” If a
patient is a candidate for bone marrow transplantation, should the
clinician initiate imatinib or attempt BMT? “It is really
tempting to use imatinib,” Dr. Peterson says, “but we
don’t have long-term data.” Making the choice even more
difficult is the lessened toxicity of contemporary BMT. “I
would not want to be in that situation,” Dr. Peterson says.
Dr. Shah calls this “a
controversial issue.”
“If a patient is eligible and has an available donor,”
he says, “transplantation remains in most people’s minds
the recommendation of choice.” But many patients are reluctant
to proceed with bone marrow transplantation because of its morbidity
and are postponing it due to imatinib availability. “Because
we know that transplantation for CML is more effective when done
within one year of diagnosis,” Dr. Shah says, “we continue
to recommend early transplantation. How imatinib will change that
remains to be seen.”
In Dr. Shah’s experience, the majority of clinicians are now
treating first with imatinib. “In patients eligible for transplantation,
they might treat with imatinib while waiting for a donor.”
Whether imatinib alters the benefit of BMT, particularly if remission
is achieved, is an open question and the subject of ongoing clinical
trials.
In practice, the majority of patients are not straightforward candidates
for BMT because of age or lack of a suitable donor. As a result,
Dr. Deininger observes, “For the average patient with newly
diagnosed CML, imatinib is the first-line treatment of choice, and
most clinicians initiate it immediately upon diagnosis.” Unfortunately,
he says, once imatinib has been started, the patient may no longer
be eligible for inclusion in a clinical trial. Thus, centers should
be contacted immediately if a trial may be an option for the patient.
Bear in mind, too, he adds, that “imatinib-induced responses
in advanced disease are frequently not durable. In such patients,
BMT at the time of remission is the best option.”
Data show that, among patients treated with imatinib in
chronic
phase, about 90 percent get a hematologic response, Dr.
Amos says. In about half of these patients the Philadelphia chromosome
is no longer detectable. However, only 60 percent of patients in
advanced-stage CML respond. “Almost all of these patients
relapse despite continued therapy,” she says.
“Imatinib
is fantastic when it works,” Dr. Najfeld says. She draws an
impressionistic portrait of the drug’s varying efficacy by
contrasting two groups of patients she followed:
A
detailed picture of the response to imatinib was provided by a substudy
of chronic-phase CML patients resistant to or intolerant of interferon
enrolled in a phase three trial. Dr. Braziel participated in that
work (Blood. 2002;100:435-441). A “typical sequence,”
Dr. Braziel says, is normalization of the blood count within a few
weeks of starting imatinib while bone marrow remains consistent
with CML. Marrow normalizes morphologically by two to four months.
Says Dr. Peterson, whose laboratory conducted similar studies on
the patients enrolled at Northwestern, “All patients had a
reduction in bone marrow cellularity, and myeloid proliferation
decreased, which shows that imatinib was affecting the root process
that gave rise to abnormal cells.”
Cytogenetic and FISH analysis of bone marrow showed that most patients
had a decrease in the fraction of BCR-ABL—or Philadelphia-positive
cells, though typically marrow remained positive, often in a fairly
high percentage of cells. Eventually some values fell within the
normal range. “If we look with RT-PCR, which is more sensitive,”
Dr. Peterson says, “many were still positive for BCR-ABL.”
Patients who remained positive for BCR-ABL later progressed; some
developed accelerated phase or blast crisis. “So we don’t
know that imatinib can cure CML,” Dr. Peterson says. “That
is our hope. And we see such good responses that we think survival
will be lengthened. But we don’t yet know that clearly.”
However, she adds, this study was done in pretreated patients. Similar
studies in newly diagnosed patients
are ongoing.
Dr. Deininger’s experience is the same. “Even in patients
with complete cytogenetic response, as a rule we can detect residual
leukemia with RT-PCR. Only a tiny minority of patients treated with
imatinib monotherapy become PCR-negative,” he says. Among
newly diagnosed patients, this fraction is 3.3 percent.
Dr. Arber recently took part in a study seeking to determine whether
imatinib remissions will be durable. Investigators asked whether
residual BCR-ABL-positive hematopoietic progenitor cells were present
in patients who achieved complete cytogenetic response (less than
or equal to 6 percent BCR-ABL-positive cells by FISH). In CD34+
stem cells from the patients’ bone marrow grown in culture,
residual BCR-ABL-positive progenitor cells were detected by FISH
and RT-PCR in all 15 patients studied (Blood. 2003; Feb.
6 e-pub ahead of print). “Imatinib never completely wiped
out cells carrying the BCR-ABL fusion,” Dr. Arber concludes.
Clinical studies show that a significant number of people treated
with imatinib relapse. “This is probably the mechanism,”
he says.
A de facto algorithm is emerging for following imatinib-treated
patients in which cytogenetics and FISH on peripheral blood are
used initially. Data suggest that a 20 percent decrease in BCR-ABL-positive
cells after two months of therapy predicts cytogenetic remission,
Dr. Arber says. As blood becomes negative, cytogenetic and FISH
analysis of bone marrow begins. “When peripheral blood becomes
BCR-ABL-negative, that does not necessarily indicate that bone marrow
will be negative,” Dr. Najfeld says, since the burden of CML
is much higher in the marrow. As the number of BCR-ABL-positive
cells in marrow decreases, Dr. Finn says, people are moving toward
highly sensitive quantitative PCR methods.
Dr. Najfeld cautions that, during monitoring, the fraction of cells
positive for Philadelphia chromosome by cytogenetics may be much
lower than the fraction of BCR-ABL-positive cells by FISH. “We
feel these cases are important because they tell us that, in the
absence of treatment, cells with the Philadelphia chromosome have
a proliferative advantage,” she says. Imatinib interferes
with proliferation, so the number of positive metaphases is low.
Dr. Najfeld suggests using both cytogenetics and FISH for monitoring
if possible.
Once remission is achieved, monitoring for recurrence begins.
“In this situation we look for trends,” says Karen Mann,
MD, PhD, director of molecular hematopathology at Emory University
Medical Center. “If you are detecting one positive cell per
hundred thousand by PCR, you want to see the fraction of positive
cells rise for two or three consecutive measurements. When it increases
by tenfold, that is getting clinically significant.”
Monitoring for recurrence by FISH also requires a high level of
sensitivity.
“If you validate your probes, you can reliably pick up two
percent of BCR-ABL-positive cells,” Dr. Najfeld says. “We
take anything above one percent seriously.”
Dr. Braziel describes a potential pitfall in monitoring for recurrence.
As marrow becomes negative by cytogenetics and FISH, and PCR remains
positive only at a low level, many clinicians switch to following
patients with quantitative PCR on peripheral blood. A clue that
something atypical is occurring is the development of peripheral
blood abnormalities not usually associated with CML. “They
may be more myelodysplasia related,” Dr. Braziel says, “such
as cytopenia rather than basophilia or leukocytosis.” If unexpected
abnormalities persist, she suggests, “re-marrow the patient
and repeat the entire karyotype, as well as FISH, PCR, and morphology.”
“Recurrence” may be due to proliferation of cells other
than the original BCR-ABL-positive clone. Dr. Braziel cites a recent
study from OHSU in Leukemia (2003;17:481-487). “In
these patients imatinib is not effective,” she says. In fact,
imatinib may be unmasking a preexisting cytogenetic abnormality,
most commonly trisomy 8. This phenomenon is well-recognized in patients
treated with other drugs, such as interferon,
before imatinib therapy. So far, Dr. Braziel says, additional cytogenetic
abnormalities in Philadelphia chromosome-negative cells have not
been seen in the Oregon cohort among patients treated with imatinib
as initial therapy.
“This phenomenon was only recognized in the last nine months
as a result of imatinib therapy,” Dr. Najfeld says. Her laboratory
identified five of 300 patients treated with imatinib who developed
pancytopenia and trisomy 8 in Philadelphia-negative cells. None
of the patients developed clinical or hematologic signs of progression
to a more advanced phase of CML. She and several other groups reported
on this topic at the 2002 American Society of Hematology meeting.
These abnormalities may be induced by an effect of imatinib on the
marrow. “A more intriguing, though speculative, possibility,”
Dr. Najfeld says, “is that Philadelphia-negative, trisomy
8 cells are present in some patients with CML but are not detectable
when Philadelphia-positive cells populate the marrow. During therapy
with imatinib, which is specific for Philadelphia-positive cells,
they may be uncovered.” In this hypothesis, Philadelphia-negative,
trisomy 8 cells were not observed during treatment with non-specific
agents such as interferon or chemotherapy because they suppress
all malignant clones. “This may be the first clinical observation
in support of a multi-step pathogenesis of CML,” Dr. Najfeld
suggests, though she acknowledges that this idea is “speculative.”
Among advanced-phase patients
treated with imatinib in initial clinical trials, many relapsed.
Further work showed that these patients’ leukemic cells had
become resistant to the drug. Mercedes Gorre, a graduate student
working in the same group as Dr. Shah at UCLA, rapidly identified
the first BCR-ABL kinase domain mutation in six of nine such patients
(Science. 2001;293:876-880). Dr. Shah then studied 32 patients
whose cancers were resistant to imatinib upon relapse. He identified
BCR-ABL kinase domain mutations in 29 of them, including 10 of 11
patients in chronic phase (Cancer Cell. 2002;2:117-125).
“In
our laboratory, as well as in most laboratories that have published
using material derived from patients, the overwhelming majority
of acquired resistance to imatinib seems to be associated with emergence
of point mutations in the BCR-ABL kinase domain,” Dr. Shah
sums up. Mutations in nearly 20 codons have been found so far.
Gorre also explored phosphorylation downstream of the BCR-ABL kinase.
She found that downstream phosphorylation was elevated before therapy
and at the time of resistance, but not during imatinib response.
“These results suggest quite strongly that BCR-ABL signaling
is restored at the time of relapse,” Dr. Shah says. “A
kinase domain mutation is the most likely mechanism of that restoration.”
Dr. Deininger’s work also shows that the majority of patients
who relapse on imatinib have mutations in the BCR-ABL protein. However,
he says, “We think that some kinase domain mutations seen
in patients confer only limited or no resistance.” Distinguishing
such mutations could be important, since those cells may be sensitive
to an increased dose of imatinib (Blood. 2003; Feb. 6 e-pub
ahead of print. To be published June 1 in Blood. 2003;101[11]).
“It is likely that in some patients resistance will not be
mediated by BCR-ABL mutations,” Dr. Deininger adds. “Alternative
signaling pathways could conceivably be activated and produce the
resistance phenotype.” Data suggest that in some patients
in blast crisis the disease has become BCR-ABL-independent.
A sensitive PCR-based assay for imatinib resistance mutations using
in-house reagents and a Sequenom MALDI-TOF detection instrument
has been put in place at Specialty Laboratories. “Instead
of sequencing and looking for all genetic changes, we do targeted
mutation analysis,” Dr. Amos says. Specialty now offers a
test to detect one mutation shown to cause clinical resistance.
Two other mutations have been validated and will be implemented.
In the seven months since the assay came online, demand has been
light. “This test may be too esoteric for clinical practice,”
Dr. Amos acknowledges. “Clinicians may suspect resistance
based on other tests, such as reappearance of BCR-ABL positivity,
and discontinue imatinib on that basis.”
Which raises a question for molecular diagnostic laboratories: With
the increase in imatinib use, should they begin thinking about an
imatinib resistance assay?
“Pressure on clinical laboratories will exist,” says
Dr. Finn. “We will have to determine if and when we should
offer an assay for specific molecular mutations. I am not sure we
are there yet.” Ultimately, he says, laboratories will have
to adapt to emerging clinical needs to identify resistance to imatinib
by some method.
Dr. Deininger says research assays should go into standard practice
as soon as possible. “But there is still a lot of work to
do before resistance assays become routine tests,” he says.
“At a minimum, we need to know that they improve therapy.”
Dr. Shah and his colleagues believe that the best therapeutic response
to emerging resistance is to identify the most common mutations
and to develop new agents against them. “I think the data
show pretty convincingly that BCR-ABL remains a useful target for
the treatment of imatinib-resistant CML,” he says. “Given
at least short-term success of imatinib, there are other inhibitors
currently undergoing trials.” For example, Dr. Deininger and
his colleagues have identified a new compound that binds the ABL
kinase differently than imatinib, so that it inhibits some mutated
forms of ABL that are imatinib-resistant (Cancer Res. 2002;62:7149-7153).
Says Dr. Shah, “My hope is that we can identify two or three
other compounds that are tolerated well and have activity against
some of the more common mutated forms of ABL kinase.” Ideally,
he says, we will have a cocktail of inhibitors that we can use in
a manner similar to the cocktail for HIV. “Perhaps,”
Dr. Shah says, “combination targeted therapy for BCR-ABL will
substantially boost the overall survival of CML patients.”
William Check is a medical writer in Wilmette, Ill.