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With molecular MPN testing, think positive

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Karen Titus

March 2015—If molecular tests for myeloproliferative neoplasms ever decide to write their autobiography, they could easily do a riff on the business bestseller Getting to Yes.

For myeloproliferative neoplasms, morphologic and clinical findings should guide molecular analysis, which can often be a helpful way to clinch the diagnosis, says Dr. David Czuchlewski (left), of TriCore Reference Laboratories, with Mohammad Vasef, MD, TriCore’s director of molecular diagnostics.

For myeloproliferative neoplasms, morphologic and clinical findings should guide molecular analysis, which can often be a helpful way to clinch the diagnosis, says Dr. David Czuchlewski (left), of TriCore Reference Laboratories, with Mohammad Vasef, MD, TriCore’s director of molecular diagnostics.

Diagnosing and monitoring MPNs produce more than their fair share of negative results. But in many cases, that “no” news is not necessarily good news, or even any news at all. Maybe the mutation truly isn’t there—or maybe the negative result is a hint to use a different set of primers. Maybe a more sensitive test is in order. Maybe the patient has acquired a new, resistant mutation during treatment—or maybe noncompliance has become an issue.

Not all negative results are created equal when it comes to MPNs, in other words. “Negative results can be helpful to clinicians,” says Todd Kelley, MD, medical director, molecular hematopathology, ARUP Laboratories, Salt Lake City. But, he adds, they need to know how to interpret a negative result and make the next testing decision.

“It’s a complicated area,” says David Czuchlewski, MD, associate professor of pathology, University of New Mexico, and associate director, molecular diagnostics, TriCore Reference Laboratories, Albuquerque. “Even for hematopathologists and others well-versed in it.”

It’s a topic familiar to Dr. Czuch­lewski, who gave a talk on the subject at CAP ’14 last fall. The subject has become even more engrossing, he says, with the discovery, in late 2013, of two new, highly relevant diagnostic mutations, CSF3R and CALR (calreticulin). “It was a big year,” he says.

Far from upending everything, the new discoveries fit into the continuum of MPN molecular testing. If the first molecular test or two doesn’t turn up a mutation, press on, like Sherman to the sea. If an initial test for JAK2 V617F is negative, for example, it might be smart to look for a less common mutation, such as JAK2 exon 12. In BCR-ABL1-negative cases, CSF3R testing might provide an answer.

Nor are new mutations shutting the door on morphology. Despite its importance, molecular testing—not even next-generation sequencing—isn’t particularly special, says Ayalew Tefferi, MD, professor of medicine and hematology, Mayo Clinic, Rochester, Minn. “The tools are different, but there are always new tools,” says Dr. Tefferi. “It’s like children: When there are new toys, they want new toys. But the concept is the same; the concepts don’t change,” a surprisingly low-key view from someone Dr. Czuchlewski calls “probably the world expert on new molecular approaches to MPNs.”

In his CAP ’14 talk, Dr. Czuchlewski listed several diagnoses that could mean a laboratory is on the wrong path, and perhaps relying too heavily on its molecular GPS, so to speak:

  • Essential thrombocytopenia (ET) with JAK2 exon 12 mutation (i.e. not V617F).
  • Primary myelofibrosis (PMF) with JAK2 exon 12 mutation (i.e. not V617F).
  • Polycythemia vera (PV) with MPL mutation.
  • PV with CALR mutation (typically, only rare patients now reported).

In essence, molecular testing of MPNs involves two roads, but unlike Robert Frost’s traveler, pathologists can and need to take them both. One starts with clinical criteria, then moves through morphology and molecular testing; the other is the molecular testing itself.

One obvious fork in the road is the BCR-ABL1 fusion gene. If it’s present, it points to chronic myelogenous leukemia (though it’s not specific to the disease); PV, ET, and PMF, on the other hand, are BCR-ABL1-negative MPNs. The fusion most often arises from a (9;22) translocation. The resulting Philadelphia chromosome was the first cytogenetic abnormality identified in the setting of MPNs, Dr. Czuchlewski notes. “Our approach to the newer tests can be somewhat similar to how we’ve approached the 9;22 translocation. The new stuff isn’t replacing old testing and old information—it’s adding to it.” The result is a much more complete picture of the MPN molecular landscape, he says.

Dr. Czuchlewski presented three cases in his talk to help illustrate the point. The first involved a 79-year-old woman with persistent leukocytosis. One of the first questions to arise in such a case is: Could this be chronic myelogenous leukemia?

Certainly there are specific morphologic clues that can lead pathologists to suspect a CML diagnosis, though they were absent in this particular case. True, the patient had a very high white blood cell count (> 50 × 109/L), but it consisted predominantly of neutrophils and lacked a significant percentage of myelocytes. And there was no basophilia or eosinophilia.

Nevertheless, says Dr. Czuchlewski, in a case like this it’s important to assess for a BCR-ABL1 fusion. “It’s a diagnosis that simply can’t be missed, especially because the targeted therapy [imatinib] is so effective.” Moreover, there are cases of CML that have unusual phenotypes, including ones that are neutrophil-predominant. Think of it as a stack of Eames chairs at Mount Vernon—puzzling but attention getting.

The lab can choose from several arrows in its quiver: karyotyping, FISH, and RT-PCR.

Karyotyping is a good option, says Dr. Czuchlewski, though it can miss a small number of cases involving cryptic rearrangements—the fusion occurs, but in an unusual form that cannot be detected on conventional cytogenetic analysis. “You could miss a true BCR-ABL fusion,” he says. Another consideration: Karyotyping may be suboptimal in peripheral blood samples.

FISH is terrific for initial assessment of CML, Dr. Czuchlewski says. It’s fast; it can detect cryptic rearrangements that elude karyotyping; and it will provide positive results even in cases that involve variant breakpoints.

RT-PCR has high analytic sensitivity. But it has a blind spot of sorts, Dr. Czuchlewski notes. Different forms of the transcript occur; the major one is found in the cluster region between exon 13 and exon 14 (corresponding to the p210 fusion protein) and is the one that’s most associated with CML. In his lab, “We use an RT-PCR strategy to account for this heterogeneity at the breakpoints, essentially.” But this requires using different primer sets to pick up the various transcript forms. “Because if it’s a negative result, you want to make sure it’s covered all the different possibilities that you’re interested in,” Dr. Czuchlewski says. There are rare cases of CML, for example, that have an alternative breakpoint in the BCR-ABL1 gene, occurring after exon 19 (corresponding to the p230 fusion), which is called the micro-breakpoint.

Explains Dr. Kelley: “If FISH testing is positive for BCR-ABL1 but then a quantitative RT-PCR test against p210 is negative, that’s evidence that a rarer fusion form may be involved. But you have to know what you’re looking for, and make sure you order a test that can detect that.” There is indeed a needle in that haystack. You just have to know to look for it, and how.

In the case of the 79-year-old woman, there were two normal BCR signals on 22q11.2, and two normal ABL1 signals on 9q34. “So what does she have?” Dr. Czuchlewski asks. In this instance, it seemed reasonable to consider chronic neutrophilic leukemia (CNL), an MPN characterized by marked neutrophilia with minimal left shift.

Enter that 2013 breakthrough, CSF3R, which indeed identified CNL as the culprit here. For too many years, labs had no good molecular test for CNL. But two years ago, it emerged that the majority of these cases carry mutations in this gene (Maxson JE, et al. N Engl J Med. 2013;368:1781–1790). “This is very useful, because it gives us a clonal marker that allows us to clinch the diagnosis,” says Dr. Czuchlewski. There’s also some early suggestion—emphasis on early—that for some patients this may open the door to targeted therapy, especially with JAK2 inhibitors.

More controversial is whether the CSF3R mutation is seen in atypical CML. In some early reports, including the New England Journal of Medicine paper, researchers have said yes. As so often happens in medicine (and with gubernatorial budgets), subsequent commentators have said no.

Given the evolving thoughts on this matter, pathologists might want to watch out for silly CSF3R test ordering. Clinicians, understandably, will be enthusiastic about the test, but, Dr. Czuchlewski says, “What we’re talking about are cases where there’s significant neutrophilia. And the vast majority of those cases are going to be patients who have an infectious or inflammatory etiology.” Ideally, he says, this can be sorted out clinically without relying on mutation testing. “We don’t want a situation where the reflex would be, ‘This patient’s neutrophils are increased. Let’s get this mutational analysis to rule out a neoplasm’—when in fact all the morphologic and clinical clues indicate otherwise.”

Mayo’s Dr. Tefferi seconds that notion. Though he harbors no doubts that genetic information will become more influential, he, too, argues on behalf of morphology’s strengths, even as the molecular trail grows longer. “Morphology can tell us so much,” he says. “The problem is, other scientists and clinicians who are not trained as pathologists can’t appreciate that. They think it’s too subjective.” He barely holds back a sigh.

Beyond BCR-ABL1 testing, laboratories need to consider JAK2 mutation testing. The most common mutation in the classic BCR-ABL1-negative MPNs is JAK2 V617F.

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