College of American Pathologists
Printable Version

At every stage, labs stay in step with CML

Quantitative RT-PCR: how well do labs do?
Acute myeloid leukemia and FLT3

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.

[Back to top]

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:

  • 20 patients who became BCR-ABL fusion-negative after 16 months to four years of therapy.
  • 15 patients who never achieved remission by cytogenetics or FISH after six months to three years of therapy.

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.