Critical Issues About the Diagnosis of Myeloproliferative Neoplasms: World Health Organization Classification (General Issues in the Management of MPNs)

Polycythaemia Vera

Polycythaemia vera (PV) is a primary bone marrow disorder where excessive numbers of red blood cells are produced. A somatic gain-of-function mutation of the Janus Kinase (JAK) 2 gene is present in most cases. The disease may present in a latent form where there is only a borderline erythrocytosis, the classical overt polycythaemia form and finally may progress to a spent or post-polycythaemic myelofibrotic disorder (post-PV MF).

The World Health Organization (WHO) has now defined diagnostic criteria for PV (Thiele et al. 2008a).

The major criteria are

1. Haemoglobin >18.5 g/dl in men, 16.5 g/dl in women or other evidence of increased red cell volume. (This is further defined as haemoglobin or haematocrit >99th percentile of method specific reference range for age, sex, altitude of residence or haemoglobin >17 g/dl if associated with a documented and sustained increase of at least 2 g/dl from an individual’s baseline value that cannot be attributed to correction of iron deficiency, or elevated red cell mass >25% above mean normal predicted value).

2. Presence of JAK2V617F or other functionally similar mutation such as JAK2 exon 12 mutation.

The minor criteria are

1. Bone marrow biopsy showing hypercellularity for age with tri-lineage growth (panmyelosis) with prominent erythroid, granulocytic and megakaryocytic proliferation


2. Serum erythropoietin (EPO) level below the reference range for normal

3. Endogenous erythroid colony (EEC) formation in vitro

Diagnosis requires the presence of both major criteria and one minor criterion or the presence of the first major criterion and two of the minor criteria.

There are a number of issues with each of these criteria which must be considered when the criteria are being used to make the diagnosis in routine practice.

Haemoglobin

Haemoglobin levels for the diagnosis have been defined with important caveats. Haemoglobins are defined as above the 99th percentile for the measurement used or a sustained increase from baseline not due to iron deficiency or other ‘evidence of increased red cell mass’. This attempts to include a variety of situations where the haemoglobin is significantly increased. However, there are a number of issues with this which may be of importance.

The true measurement of an erythrocytosis is an increased red cell mass. This does not always equate to an increased haemoglobin or haemat-ocrit as shown by the study of Johansson where in both males and females, a raised haemoglobin was not always consistent with an absolute eryth-rocytosis by red cell mass measurement, and conversely, haemoglobins below the cut-off level could be present with an absolute erythrocytosis (Johansson et al. 2005).

Iron deficiency is also excluded, and this means that the patient presenting with an obvious iron deficiency will need to be considered carefully. Judicious and carefully monitored administration of iron may be indicated, but it may also be correct from a therapeutic point of view to withhold iron and in such a patient the haemoglobin would not make the diagnostic criteria.

Trephine in polycythaemia vera showing tri-lineage hypercellularity.

Fig. 4.1 Trephine in polycythaemia vera showing tri-lineage hypercellularity.

JAK2 Mutations

In 2005, the JAK2 V617F mutation was described in the majority of patients who were then classified with PV (Baxter et al. 2005; James et al. 2005; Kralovics et al. 2005; Levine et al. 2005). This is a gain-of-function acquired mutation in a bone marrow clone which leads to a constitutively active JAK2 protein. In a further group of patients with erythrocytosis, different mutations in exon 12 of the JAK2 gene were discovered (Scott et al. 2007; Percy et al. 2007). These mutations can be detected in the peripheral blood with increasingly sensitive techniques, and it is possible to detect the presence of small clones (Chen et al. 2007).

This criterion which has completely altered the new WHO diagnostic criteria for PV is a crucial major criterion and in clinical practice is increasingly an initial diagnostic test for investigating and confirming a diagnosis of a patient with raised haemoglobin.

Bone Marrow Biopsy

The bone marrow biopsy showing defined tri-lineage hypercellularity is a minor criterion (Fig. 4.1). Much of the work in coming to the histopathological interpretations has been done in retrospective and unblinded settings (Hussein et al. 2007) . There are also patients with congenital erythrocytosis/polycythaemia with mutations in the von Hippel-Lindau gene who have had their bone marrow interpreted as consistent with PV (Gordeuk et al. 2005), so the discriminatory power of the described bone marrow changes may not be sufficient. Widespread prospective use may be difficult, requiring very specific expertise, and this haematopathology experience may not be widely available. It may also be difficult to persuade patients and justify the expense of the widespread use of bone marrow biopsy to add to the diagnostic pathways in those who fulfil other criteria using less invasive tests.

Erythropoietin Level

The EPO level in a patient can be below the normal range, normal or elevated. A below normal levels suggests a primary bone marrow problem with production of increased red cells and is usually seen in cases of PV and as such is a useful discriminating test. However, the assay is only of limited availability, and the result may be influenced by other factors such as venesection and secondary causes of erythrocytosis such as smoking.

Endogenous Erythroid Colonies

The growth of EECs from the peripheral blood demonstrating in a patient with PV that erythroid colonies grow in the absence of added EPO is a skilled technique, requires 2 weeks of culture for each sample, and is only available in a very few centres. It can be standardised and quality controlled between centres, but this requires considerable technical effort (Dobo et al. 2004). Therefore, while this is a useful research tool and can be useful in those with the skills, it is unlikely to be used in any widespread fashion. The addition of this test as a minor criterion does not seem to add anything to the diagnostic process but merely acknowledges an old test (Prchal and Axelrad 1974) which was of use in the past before more practical, easy to perform and generally applicable tests became available.

Diagnostic Issues

The commentary above elucidates some of the issues with the individual tests which are required to make a diagnosis of PV in the WHO criteria, but the criteria require both major and one minor criterion or the first major criterion plus two minor criteria to make the diagnosis. Is this necessary and sufficient for this diagnosis?

If both major criteria are present does the addition of a minor criterion add anything? It can be argued that it does not. A patient with both major criteria present has been shown to have a clonal erythrocytosis and as such a PV-type disorder. The addition of minor criteria complements this diagnosis and acknowledges tests which have been used to discriminate in cases in the past before the discovery of the acquired clonal markers, but they do not actually add anything from the point of view of confirming the diagnosis. In clinical practice, there will be increasing reluctance to do invasive tests or those which are difficult to carry out if they do not add to the diagnostic process.

The other alternative set of criteria, the first major criterion and two minor criteria, is present to give a set of criteria for the diagnosis of PV in a case where no mutation in JAK2 can be detected. With the discovery of exon 12 mutations (Scott et al. 2007) and increasingly sensitive techniques, this situation is becoming vanishingly rare, and it could not be argued that if a JAK2 clone has not been discovered then it is necessary to look with a more sensitive technique. It must be questioned if there is truly such an entity as JAK2 mutation negative PV.

Latent Polycythaemia Vera

The WHO authors allude to the situation which is referred to as latent PV or a pre-polycythaemia phase. This is the situation where a patient is discovered to have a JAK2 mutation, a subnormal EPO level or typical EECs but does not fulfil the PV criteria. It is to be expected that these patients will go onto develop PV. One cannot set diagnostic criteria as by definition the issue is that they do not fulfil the necessary criteria, but these individuals require ongoing observation.

Post-Polycythaemia Vera Myelofibrosis

Patients with PV often develop an end-stage disease where they have anaemia and other cytope-nias, extramedullary haematopoiesis, ineffective haematopoiesis, increasing splenomegaly and bone marrow fibrosis. This has been known by many different terms in the literature, and the criteria for describing this phase have been confusing. The WHO set defined criteria for this condition which are set out as follows:

Diagnostic criteria for post-polycythaemic myelofibrosis Required criteria

1. Documentation of a previous diagnosis of WHO-defined PV

2. Bone marrow fibrosis grade 2-3 (on a 3 scale) or grade 3-4 (on a 0-4 scale)

Additional criteria (two are required)

1. Anaemia or sustained loss of either phlebotomy (in the absence of cytoreductive therapy) or cytoreductive treatment requirement for eryth-rocytosis. (anaemia defined as a haemoglobin below the reference range for the appropriate age, sex, gender and altitude considerations)

2. Leukoerythroblastic peripheral blood picture

3. Increasing splenomegaly defined as either an increase in palpable splenomegaly of >5 cm from baseline (distance from the left costal margin) or the appearance of newly palpable splenomegaly

4. Development of >1 of 3 constitutional symptoms: >10% weight loss in 6 months, night sweats, unexplained fever (>37.5°C)

These criteria are very helpful in categorising patients who fallen into this group with this constellation of signs and symptoms.

Primary Myelofibrosis

The revised WHO criteria for primary myelofi-brosis (PMF) are divided into major and minor criteria (Thiele et al. 2008b) ; To make the diagnosis, all three major criteria must be present and two minor criteria: Major criteria

1. Presence of megakaryocyte proliferation and atypia (small to large megakaryocytes with an aberrant nuclear/cytoplasmic ratio and hyper-chromatic, bulbous or irregularly folded nuclei and dense clustering), usually accompanied by reticulin and/or collagen fibrosis, or in the absence of significant reticulin fibro-sis, the megakaryocyte changes must be accompanied by an increased bone marrow cellularity characterised by granulocyte proliferation and often decreased erythropoiesis (i.e. prefibrotic cellular-phase disease).

2. Not meeting WHO criteria for polycythaemia vera, BCR-ABL 1+ chronic myelogenous leukaemia, myelodysplastic syndrome or other myeloid neoplasms.

3. Demonstration of JAK2 V617F or other clonal marker (e.g. MPL W515K/L) or in the absence of a clonal marker, no evidence that the bone marrow fibrosis or other changes are secondary to infection, autoimmune disorder or other chronic inflammatory condition, hairy cell leukaemia or other lymphoid neoplasm, meta-static malignancy or other toxic (chronic) myelopathies.

Minor criteria

1. Leukoerythroblastosis

2. Increase in serum lactate dehydrogenase level (which can be borderline or marked)

3. Anaemia

4. Splenomegaly

Histopathology

The histopathological features of myelofibrosis are clearly defined and included in the criteria. These are megakaryocyte proliferation and atypia and described further as small to large megakaryocytes with an aberrant nuclear/cytoplasmic ratio and hyperchromatic, bulbous or irregularly folded nuclei and dense clustering. It is stated that these megakaryocyte changes are usually accompanied by reticulin and/or collagen fibrosis. The grading of fibrosis must be done carefully and reproduc-ibly, and there are agreed scoring systems for this (Thiele et al. 2005). Figure 4.2 demonstrates some of these changes.

Trephine in primary myelofibrosis showing dense clustering, hyperchromatic, bulbous and irregularly folded nuclei.

Fig. 4.2 Trephine in primary myelofibrosis showing dense clustering, hyperchromatic, bulbous and irregularly folded nuclei.

Prefibrotic Myelofibrosis

The previous version WHO classification distinguished ‘prefibrotic’ myelofibrosis from ‘fibrotic’ myelofibrosis. This revision incorporates the described entity within major criterion 1. Prefibrotic myelofibrosis is defined as a cellular phase of the disorder where there is no fibrosis seen but mega-karyocyte proliferation and atypia accompanied by an increased bone marrow cellularity characterised by granulocyte proliferation and often decreased erythropoiesis. These changes have been defined in published literature mainly originating from the Cologne group in multiple retrospective analyses of an archive of trephine biopsies (Thiele et al. 1996, 1999, 2011; Thiele and Kvasnicka 2003a, b). It is claimed that the entity of prefibrotic myelofi-brosis is distinct from essential thrombocythaemia (ET) and can and must be distinguished by the his-topathological changes. However, several studies have now found that the histopathological differences are subjective and not reproducible (Wilkins et al. 2008; Brousseau et al. 2010), so there is considerable doubt about the existence of an entity of prefibrotic myelofibrosis.

The diagnostic criteria require all three major criteria plus two of the minor criteria to make the diagnosis so the presence of the so-called prefibrotic changes alone would not be sufficient without at least two of leukoerythroblastosis, raised lactate dehydrogenase, anaemia or splenomegaly. It is hoped that there would be some other features to support making a diagnosis of PMF. Nevertheless, the WHO continues to discriminate a category of prefibrotic myelofibrosis which cannot be identified reproducibility and is not of diagnostic or therapeutic benefit but could be dangerous for patients. If myelofibrosis is considered a more serious disorder with a worse prognosis, patients may be advised to have higher risk procedures like bone marrow transplant if a fibrotic process is considered to be the main pathology.

Myelofibrosis: An Accelerated Phase of Polycythaemia Vera or Essential Thrombocythaemia

The WHO discusses how myelofibrosis is characterised over time by increasing reticulin, collagen fibrosis and osteosclerosis. The disorder transforms to an acute leukaemia with increasing percentages of blasts present. PV and ET also transform to myelofibrosis over time and are indistinguishable from PMF. The evolution of the diseases over time is poorly understood. However, considering all the disorders, it may be logical to consider myelofibrosis as an accelerated phase of the myeloproliferative disorders with PV and ET as the chronic phases and all ultimately ending in a leukaemia transformation (Campbell and Green 2005).

Essential Thrombocythaemia

The WHO has defined a set of criteria which are required for the diagnosis of ET (Thiele et al. 2008c). To make the diagnosis, all four criteria must be fulfilled:

1. Sustained platelet count >450×109/L (sustained during the work-up process)

2. Bone marrow biopsy specimen showing proliferation mainly of the megakaryocytic lineage with increased numbers of enlarged, mature megakaryocytes. No significant increase or left shift of neutrophil granulopoiesis or erythropoiesis

3. Not meeting WHO criteria for polycythaemia vera, primary myelofibrosis, BCR-ABL1 positive chronic myelogenous leukaemia or myel-odysplastic syndrome of other myeloid neoplasm

4. Demonstration of JAK2 V617F of other clonal marker, or in the absence of JAK2 V617F, no evidence for reactive thrombocytosis

In criterion 3, there are defined criteria for the excluded diagnoses. To exclude PV, there is a requirement for failure of iron replacement therapy to increase haemoglobin level to the PV range in the presence of decreased serum ferritin. PV diagnosis is based on haemoglobin and hae-matocrit. Red cell mass measurement is not required. Exclusion of PMF requires the absence of the relevant reticulum fibrosis, collagen fibro-sis, peripheral blood leukoerythroblastosis or markedly hypercellular marrow accompanied by megakaryocyte morphology that is typical for PMF including small to large megakaryocytes with an aberrant nuclear/cytoplasmic ratio and hyperchromatic, bulbous or irregularly folded nuclei and dense clustering. Exclusion of chronic myeloid leukaemia (CML) requires the absence of BCR-ABL1. Exclusion of myelodysplastic syndrome requires absence of dyserythropoiesis and dysgranulopoiesis.

A reactive thrombocytosis must also be excluded, and the causes of this which have to be eliminated include iron deficiency, splenectomy, surgery, infection, inflammation, connective tissue disease, and metastatic cancer and lymphop-roliferative disorders. It is noted that a condition associated with a reactive thrombocytosis may not exclude the possibility of ET if the first three criteria are met.

Platelet Count

Compared to the previous set of WHO criteria for ET, these criteria require a lower platelet count of 450 x 107L rather than the higher limit of 600 x 107L. This will include all individuals with a platelet count above the 95th percentile for normal counts and means that the other diagnostic criteria need to be considered. However, it does seem logical as there is no longer a group with sustained platelet counts above the upper limit but below the 600 χ 109/L limit who did not fit the previous criteria.

 Trephine in essential thrombocythaemia with megakaryocytes clustering with large forms which have abundant mature cytoplasm and deeply lobulated nuclei.

Fig. 4.3 Trephine in essential thrombocythaemia with megakaryocytes clustering with large forms which have abundant mature cytoplasm and deeply lobulated nuclei.

Haematopathology

Haematopathological changes in ET and the distinction from prefibrotic myelofibrosis have been described by pathologists in large retrospective series with clearly defined cases (Thiele et al. 1996, 1999, 2011; Thiele and Kvasnicka 2003a, b). Changes include proliferation of megakaryocytes with large forms which have abundant mature cytoplasm and hyperlobulated or deeply lobulated (staghorn-like) nuclei (Fig. 4.3). Megakaryocytes may be clustered or dispersed throughout the marrow. Proliferation of erythroid and granulocytic precursors is minor. Reticulin should be essentially normal, and any significant increase in reticulin fibrosis excludes the diagnosis. These various his-topathological changes are described by the WHO, but no guidance is given on the importance of the various features. In a study of a cohort of patients, three experienced haematopathologists assessed the morphologic features and overall diagnosis according to the described WHO criteria. There was substantial interobserver variability for overall diagnosis and individual cellular characteristics including megakaryocyte morphology. Analysis suggested that cellularity, megakaryocyte clustering and degree of fibrosis were the three underlying processes which describe the morphologic patterns in the bone marrow in ET. Reticulin grade was the major factor which all three haematopathologists used to assign WHO classification. The conclusions from this study were that even experienced haematopathologists need special training to distinguish subtypes of ET, and therefore the general use of the WHO criteria in routine practice is difficult, or the criteria are not sufficiently robust to describe subtypes of ET (Wilkins et al. 2008).

Recently, a large retrospective study looked at over 1,000 patients who had been classified as ET prior to 2002 using the Polycythemia Vera Study Group criteria. The bone marrow biopsies in conjunction with clinical and laboratory data were reviewed by the local experts and by the haemato-pathology expert who originally described prefi-brotic myelofibrosis. Using all this data, the experts reclassified 16% of those evaluable who were originally labelled ET as prefibrotic myelofi-brosis. Outcomes were then looked at, and those labelled prefibrotic myelofibrosis had worse outcomes. Age, leucocyte count anaemia and thrombosis history were independent risk factors for survival.This study shows that there may be factors within a group of ET patients which are associated with poorer outcomes, but it does not prove the existence of a distinct independent pathological entity which can be identified reproducibly by haematopathologists.

Diagnostic Issues

The major point to note is that with these criteria, ET remains a diagnosis of exclusion. In the presence of a raised platelet count, typical histological changes must be observed, but other myeloprolif-erative and reactive causes have to be excluded.

The WHO criteria rely heavily on histopathol-ogy. However, there will be patients in whom it will be very difficult to undertake a bone marrow trephine in order to complete a diagnostic process. A patient with a persistent markedly raised platelet count with a clonal marker has a strong indication of a diagnosis of ET, and the clinician and patient may be very reluctant to undertake a biopsy. This perhaps should always be undertaken in a younger patient of if there is any doubt, but in routine practice, there will be patients where it may not be justified.

The WHO criteria distinguish between PV and JAK2 V617F positive ET. There may be overlap between these disorders as iron levels, sex EPO levels and other factors may influence the clinical presentation (Campbell et al. 2005) . The two disorders may represent a biological continuum rather than distinct entities. The long tradition of defining by splitting up the disorders may not be accurate, and in the future, the preference may be to consider the disorders on a spectrum rather than as separate. The therapeutic relevance of the distinction is not yet clear.

MPN in Children

Myeloproliferative disorders are very rare in children. The applicability of the revised WHO criteria to cases of MPN in children may not be appropriate. This is demonstrated in a series of 45 unselected children, 13 with PV and 32 with ET. Applying WHO criteria to this group, 1 familial erythrocytosis and all 12 familial thrombocytosis would have been classified as PV or ET according to the WHO criteria. The JAK V617F mutation was also present in a smaller number of sporadic cases of PV than would be expected in an adult series (Teofilli et al. 2007). When considering children, criteria need to exclude familial forms and consider that pathogenetic lesions found in adults are only present in a minority of children.

Conclusion

The revised WHO criteria for MPNs are useful in categorising and defining the different disorders. The criteria continue to emphasise differences in bone marrow morphology between disorders, and these differences have been developed mainly in small groups of patients and in retrospective settings. The criteria need to be developed and validated further so that they can be generally applicable.

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