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The Classic Myeloproliferative Neoplasms

Optimizing Laboratory Testing for Hematologic Disorders Series


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Published: October 2011

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Dr. Hanson is presenting the second in our series “Optimizing Laboratory Testing for Hematologic Disorders.” This series will address guidelines for appropriate laboratory test utilization in hematologic disorders and represents the combined effort of the Divisions of Hematopathology and Laboratory Genetics. This presentation will discuss the appropriate utilization of the JAK2 and MPL gene tests for evaluating possible myeloproliferative neoplasms.

Presenter: Curtis A. Hanson, MD

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Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenterfor this program is Dr. Curtis Hanson, from the Division of Hematopathology at Mayo Clinic in Rochester, Minnesota. Dr. Hanson is presenting the second in our series "Optimizing Laboratory Testing for Hematologic Disorders." This series will address guidelines for appropriate laboratory test utilization in hematologic disorders and represents the combined effort of the Divisions of Hematopathology and Laboratory Genetics. This presentation will discuss the appropriate utilization of the JAK2 and MPL gene tests for evaluating possible myeloproliferative neoplasms. Thank you, Dr. Hanson.

Thank you, Sharon, for that introduction. As Sharon mentioned, my talk today is entitled Optimizing Laboratory Testing for Hematology Disorders Series, The Classic Myeloproliferative Neoplasms.

Optimizing Laboratory Testing for Hematologic Disorders Series

This is a continuation of a series of hot topics that will address the issue of appropriate laboratory test utilization in hematologic disorders. This is a combined effort of the Divisions of Hematopathology and Laboratory Genetics with the primary focus of identifying the correct algorithmic testing approach for various hematologic diseases and secondly to demonstrate how the laboratory can and should be engaged in reducing unnecessary testing.

Goals for Today's Presentation

Today, we will be talking about the classic myeloproliferative neoplasms or MPNs. Our goals today include understanding the laboratory approach for evaluating patients with a possible MPN. Second, to understand the appropriate utilization of JAK2 V617F and sequencing studies for JAK2 exon 12 and MPL in the MPNs. And, finally, to understand how a utilization scheme for molecular assays in the MPNs can be employed in a clinical laboratory and pathology setting.

Example of a Recent MPN Referral

I do want to start off and give you an example of a recent bone marrow report that I saw from a patient who had been studied outside of Mayo Clinic that shows why I believe test utilization is such an important issue. This patient had a straight-forward diagnosis of a MPN that was consistent with essential thrombocythemia. When looking at the accompanying bone marrow report, one saw that a whole plethora of assays had been done prior to coming to Mayo, as shown in this slide. But yet, only 4 of these were clinically necessary and warranted in the workup of this patient, while most of the assays were unnecessary. This, I think, points out as an example the magnitude of the issues that we can see within a laboratory hematology practice.

Why Do We Have Test Utilization Issues?

So why do we have test utilization issues? First of all, laboratories typically do not provide sufficient guidance on how to use assays in hematologic diseases. Instead, it is frequently just a listing of available tests. I think we all realize that also, as new technology emerge, there can be a lack of understanding or certainly a lack of effort put forth to understand how these tests should be utilized in the context of other existing assays. Laboratories frequently do not have processes to either review test requests or to sequentially add or delete tests after initial results are determined. Laboratory information systems don’t make it easy for clinicians to efficiently order assays and get information. And finally, laboratory reports do not always transmit the intended information and often come across as just lists of results instead of a correlation and diagnosis based on all the results for that episode of care.

Why Do We Have Test Utilization Issues?

Test utilization issues also come about because of the realities within today’s clinical practices. There are certainly varying levels of understanding how to use hematologic associated assays amongst clinicians, even hematologists, as today’s practice has gotten significantly more complex. Also realize that patients with particular hematologic diseases may see a hematologist or internist who may not have experience with that particular disease. Probably most important is that clinical knowledge at the time of test ordering is incomplete, and a clinician is compelled to order everything because it may be the only chance to get that information. Finally, we must realize that initial laboratory studies can help narrow the diagnostic choices and the specific testing needs. If laboratories don’t have a review and ordering process in place, clinicians have no choice but to order excess testing.

So What Should We Do?

So what should we do? I think it’s clearly appropriate for laboratories and pathologists to develop laboratory utilization guidelines and algorithms for ancillary studies in hematologic diseases, to have test review processes in place, and to be sure that reports contain appropriate content and clinical relevance, all with the goal of reducing unnecessary testing and performing appropriate testing.

WHO Classification

Today’s talk is about the classic myeloproliferative neoplasms. In the WHO classification, as shown on this slide, the chronic myeloid neoplasms include various subgroups and unique disease processes. Today, we will be talking about the classic myeloid neoplasms that are BCR/ABL1-negative and include polycythemia vera or PV, essential thrombocythemia or ET, and primary myelofibrosis or PMF.

Important Laboratory Assays in the MPNs

The diagnosis of a myeloproliferative neoplasm requires a multidisciplinary approach including the correlation of clinical findings, CBC and differential count, serum erythropoietin, and bone marrow morphology. Today’s standard practice of care also requires chromosome karyotype, BCR/ABL – whether done by RT-PCR or by FISH to exclude the diagnosis of chronic myelogenous leukemia, JAK2 V617F point mutation analysis, JAK2 exons 12 to 14 sequencing, and MPL exon 10 sequencing studies. The focus of today’s presentation will be on these 3 latter laboratory assays: JAK2 V617F and whether those studies should be done in peripheral blood or bone marrow, JAK2 exon 12 sequencing studies, and MPL exon 10 sequencing studies.

JAK2 V617F Background

As background, the JAK2 V617F plays a very important diagnostic role in the evaluation of the myeloproliferative neoplasms. If the mutation is present, it certainly confirms the presence of a myeloid disorder and strongly favors a myeloproliferative neoplasm over a myelodysplastic syndrome diagnosis. However, a positive JAK2 V617F mutation does not help distinguish between the various myeloproliferative neoplasms. As you can see in the right-hand column, the JAK2 V617F mutation is present in over 95% of cases of PV, approximately half of PMFs and ETs, and only rare cases of myelodysplastic syndrome or chronic myelomonocytic leukemia.

Conversely, if the JAK2 V617F mutation is absent, it’s usually not helpful diagnostically. However, the absence of the V617F mutation does argue against a diagnosis of PV or the end-stage of PV, postpolycythemic myelofibrosis. JAK2 V617F is absent in CML, in secondary erythrocytosis, reactive thrombocytosis and leukocytosis, solid tumors, and in lymphoid disorders.

Question #1 — JAK2 V617F

So our first question is: should JAK2 V617F mutation analysis be done on peripheral blood or bone marrow or both?

Microscopic Examination: Biopsy

We looked back at 267 Mayo Clinic patients in whom both peripheral blood and bone marrow studies for JAK2 V617F were done. As you can see, 98.5% of those patients had concordant results between peripheral blood and bone marrow with approximately one-half being positive and one-half being negative. Four out of the 267 showed discrepant results: 2 with a positive bone marrow and negative peripheral blood, and 2 the converse, having a positive peripheral blood and negative bone marrow.

JAK2 V617F at Mayo Clinic

Those negative discrepant cases all had very low mutation burdens just below the normal cutoff value and, conversely, the positive discrepant cases had a very low level of positivity just above the normal cutoff value, so basically all had very similar results. The bone marrow diagnoses in these 4 discrepant cases included 1 case of a myelodysplastic/myeloproliferative disorder that was unclassified and 3 cases in which the bone marrow showed no features of myeloid malignancy. Our findings confirmed that either a low-level positive or negative JAK2 result had no impact on the clinical management or outcome of these 4 patients.

Conclusion #1 — JAK2 V617F

So our first conclusion is that blood and marrow studies for the JAK2 V617F mutation will give similar diagnostic results. Thus, only 1 specimen, either peripheral blood or bone marrow, needs to be analyzed, but certainly not both. And finally, in data that I didn’t show, the mutation levels of the JAK2 V617F also appear to be comparable between blood and bone marrow.

JAK2 Exon 12 Sequencing Background

Now I would like to give you some background information regarding the JAK2 exon 12 sequencing assay. Approximately 5% of PVs will be JAK2 V617F-negative, but will have an identifiable novel mutation in exons 12 to 14 of the JAK2 gene that can be identified only by sequencing studies. Thus, it appears that virtually all cases of PV will have some type of a JAK2 mutation. JAK2 exon 12 to 14 mutations are either felt to be absent or extremely rare in patients with ET or PMF. At Mayo Clinic, we have never seen a bona fide ET or PMF with a JAK2 exon 12 mutation. Remember, sequencing studies will also identify the JAK2 V617F mutation, which is in exon 14. But sequencing is more expensive, takes longer to get results, ie, a slower turnaround time, and is certainly less sensitive than RT-PCR, having a detection level of approximately 10% – 20% versus PCR, which has a detection level of much less than 0.01%.

Question #2 — JAK2 Exon 12 Sequencing

So, our next question to pose today is which is a better assay for evaluating a patient’s JAK2 status? The JAK2 V617F mutation assay or the JAK2 exon 12 sequencing study and, as a follow-up question, when should JAK2 exon 12 sequencing studies be performed?

JAK2 Exon 12 Sequencing: When Not to Use

Well, it’s quite clear when we should not do JAK2 exon 12 sequencing. It should not be used as the routine JAK2 screening method in a possible myeloproliferative neoplasm for reasons that I pointed out in the previous slide. Because of the infrequency of exon 12 mutations, sequencing studies should not be done until the much more common JAK2 V617F results are available and, since JAK2 exon 12 mutations occur almost exclusively in PV, these studies should not be done unless the CBC and serum erythropoietin studies raise the possibility of a PV.

JAK2 Exon 12 Sequencing

When we looked back at 92 cases at Mayo Clinic that had JAK2 exon 12 sequencing studies done, we found that only 25% were appropriately requested – those that had a negative V617F and had a PV-like CBC with decreased serum erythropoietin. 75% of these 92 requests appeared to be inappropriately ordered since they had no JAK2 V617F testing, or had positive JAK2 V617F results, or had a bone marrow with no morphologic features of a myeloproliferative disorder, or a peripheral blood that had no PV-like CBC with normal serum erythropoietin values.

Conclusion #2 — JAK2 Exon 12 Sequencing

Thus, our second conclusion of the day: JAK2 V617F mutation assay is the preferred screening assay for JAK2 mutations. Second of all, JAK2 exon 12 sequencing will frequently be inappropriately ordered instead of the V617F as physicians are not aware of the differences between the various JAK2 assays. And finally, sequencing studies should be reserved for those patients who are negative for JAK2 V617F and have CBC and serum erythropoietin findings suggestive of polycythemia vera.

MPL Exon 10 Sequencing Studies: Background

The final topic today regards MPL exon 10 sequencing studies. As background, MPL exon 10 mutations are relatively infrequent and have been identified in approximately 10% of primary myelofibrosis and about 5% of ET patients, while rarely being identified in PV and never in CML. MPL mutations are only rarely present with JAK2 mutations and, in general, MPL mutations are not associated with significant clinical findings or outcomes that would affect the clinical management of those patients.

Question #3 — MPL Exon 10 Sequencing

Thus, our third question of today, should every patient who has a possible myeloproliferative neoplasm be sequenced for a MPL mutation, and when should MPL exon 10 sequencing studies be ordered?

MPL Exon 10 Sequencing: When to Use

MPL exon 10 sequencing is primarily useful in those borderline bone marrow cases in which the morphology cannot confidently distinguish between a benign and a myeloproliferative diagnosis. For example, a bone marrow where there is a question of a possible essential thrombocythemia. Frequently, these are cases with thrombocytosis, but without megakaryocyte clusters or only slight megakaryocyte atypia. A second common scenario, is it a possible primary myelofibrosis? The marrow may appear slightly fibrotic but without any megakaryocyte clusters and, again, at best, only slight megakaryocyte atypia. Another possible situation where MPL exon 10 sequencing studies may be useful in peripheral blood studies is if a bone marrow procedure is not feasible – but realizing that this is a low return assay in this situation.

MPL Exon 10 Sequencing: When Not to Use

There are 3 scenarios in which exon 10 sequencing should not be done. It’s certainly not useful in classic myeloproliferative neoplasms that can be diagnosed by bone marrow morphologic features. It’s not useful when JAK2 V617F studies are positive, and it’s not useful in patients who are likely to have PV on the basis of their CBC and serum erythropoietin findings.

MPL Exon 10 Sequencing

We looked back at 60 Mayo Clinic patients who had MPL exon 10 sequencing done and noted that only 22, or 37%, appeared to be appropriately ordered, ie, those who had negative V617F and had borderline bone marrow features as described on the previous slide. 38 were inappropriately ordered as they had no V617F testing done, had already been identified has having the V617F mutation, had diagnostic bone marrow features, ie, definite myeloproliferative neoplasm, or were normal bone marrows with no features of a myeloproliferative disorder.

Conclusion #3 — MPL Exon 10 Sequencing

Our third conclusion today: Only selective patients with possible MPNs should be sequenced for a MPL mutation. Physicians will frequently order MPL exon 10 sequencing when it is not needed in the evaluation of a patient with a possible myeloproliferative neoplasm. And finally, MPL exon 10 sequencing should be limited to patients with negative JAK2 studies and borderline bone marrow morphologic studies as we have talked about previously.

Roles of Other Assays in Classic MPN

What about other assays in the evaluation of the patient with a possible classic myeloproliferative neoplasm? Cytogenetic karyotype is considered as a standard of care in the workup of a patient with a MPN. And BCR-ABL1 studies, either by RT-PCR or by FISH, need to be done at diagnosis to exclude CML. However, FISH studies for myelodysplasia are not indicated and typically do not add anything beyond which can be obtained by cytogenetic karyotyping. Flow cytometric immunophenotyping is not indicated unless there is an increase in blasts or is suspicious for a concurrent diagnosis of malignant lymphoma and T- and B-cell gene rearrangement studies are also not indicated.


In summary, JAK2 V617F results are similar in peripheral blood and bone marrow. JAK2 exon 12 and MPL exon 10 sequencing studies frequently are not needed and should not be ordered without knowing results of the JAK2 V617F study. In particular, the JAK2 exon 12 sequencing studies should not be ordered unless the CBC and serum erythropoietin are suggestive of PV, and MPL exon 10 sequencing should not be ordered unless the bone marrow findings are suggestive, but not diagnostic, of a myeloproliferative neoplasm.

Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation

In the next 2 slides, I would like to introduce to you 2 algorithms that are available on the Mayo Medical Laboratories Web page that summarize our approach to the MPNs, whether in blood or bone marrow. In peripheral blood, if JAK2 V617F is positive, then understanding the CBC and serum erythropoietin values will determine whether or not you can make the diagnosis of PV by peripheral blood studies. If the JAK2 is negative or equivocal and PV is still possible on the basis of the CBC and serum erythropoietin, then JAK2 exon 12 sequencing should be performed. If, however, the CBC and serum erythropoietin do not support PV, then the next steps revert to whether or not there is clinical suspicion for a myeloproliferative neoplasm. That will determine the necessity of doing a bone marrow study or not.

Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation

In the evaluation of a bone marrow specimen, the initial review process centers on whether or not the bone marrow morphology is diagnostic of a myeloproliferative neoplasm or not or whether it has equivocal findings. After making the decision of whether or not the bone marrow represents a myeloproliferative neoplasm, then JAK2 V617F studies are the next critical step. If JAK2 is positive, this finding, together with the bone marrow studies confirms the diagnosis of a myeloproliferative neoplasm. If the JAK2 study is negative, then whether to proceed with JAK2 exon 12 sequencing is dependent on whether there is suspicion for PV or not on the basis of CBC and serum erythropoietin values. Finally, MPL exon 10 sequencing should only be for those cases with equivocal bone marrow morphology where there is suspicion for a myeloproliferative neoplasm and a negative JAK2 V617F assay.

Implementing a Utilization Approach

I want to conclude by bringing up what I think are 5 important points in trying to implement a utilization approach for the MPNs in the laboratory. Most importantly, there needs to be effective communication between the clinician, pathologist, and laboratory. This is essential if any kind of utilization screening is to work. It is also necessary to obtain pertinent information, CBC, erythropoietin, morphologic findings, JAK2 V617F, etc, so a sequential evaluation can occur. One needs to use a consistent and standard process. For example, following a mutually agreed upon algorithm. It’s appropriate and necessary to use your reports to communicate your process, decisions, and findings, and finally, auditing your results is necessary to assure that the process is giving you and your clinical colleague the desired outcome.


Thank you for the opportunity to speak to you today and hopefully I have been able to introduce you to the concept of how the laboratory can help control utilization of ancillary testing in the classic myeloproliferative neoplasms. I look forward in future Hot Topics to continue our discussion of the laboratory’s and pathologist’s roles in appropriate laboratory test utilization in hematologic disorders.