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Polycythaemia vera (pv), essential thrombocythaemia and primary myelofibrosis

 

MPNs (myelo-proliferative neoplasms) are chronic haemopathies affecting the myeloid lineage. The initial anomaly is an acquired clonal anomaly of the haematopoietic stem cell which leads to proliferation of myeloid precursors of one or more lineages with complete differentiation and maturation of the cells.

The classification, according to the affected lineage, distinguishes chronic myeloid leukaemia (CML), polycythaemia vera (PV), essential thrombocythaemia (ET), primary myelofibrosis (PMF) and other rarer MPNs: chronic neutrophilic leukaemia, chronic eosinophilic leukaemia, mastocytosis, non-classifiable MPNs.

Setting aside CML and chronic neutrophilic leukaemia, we look more in detail here at PV, ET and PMF.

1- POLYCYTHAEMIA VERA (PV):

A polycythaemia is an increase in haematocrit and haemoglobin concentration.

1.1- How to deal with polycythaemia?

First, there is the possibility of a “false” polycythaemia:

  • from reduction in plasma volume (dehydration, burns, etc.),
  • from an anomaly in the red blood cells (RBC) (thalassemia),

Then, secondary “true” polycythaemia should also be ruled out:

  • most often linked to chronic tissue hypoxia: smoking (HbCO > 3% in blood gases), COPD, respiratory failure, sleep apnoea, being at high altitudes, etc.)
  • or hypersecretion of erythropoietin: in the context of a renal tumour or stenosis of the renal arteries (measure serum erythropoietin),
  • in rare cases, to certain specific types of Hb: Hyperaffine Hb for O2 which mimics hypoxia (request a haemoglobin electrophoresis).

Primary polycythaemia vera has no identifiable reaction-related cause explaining the increase in RBC production. This excessive production of RBC by the marrow is malignant, independent of EPO.

1.2- In which contexts should polycythaemia vera be suspected?

Polycythaemia vera is often discovered fortuitously in a CBC or in the context of non-specific symptoms: erythrosis of the face (skin and mucous membranes) and palms, splenomegaly (frequent), sometimes hepatomegaly, neurological signs (headaches, dizziness, paresthesias, tingling, ringing in the ears, etc.), erythromelalgia (toe pain), thrombosis, gout attacks.

After eliminating false polycythaemia and secondary polycythaemia, polycythaemia vera is diagnosed in adults, with a median age of 65 years, slightly more often in men than in women (R = 1.3). The incidence is 2 to 3 new cases / 100,000 inhabitants / year.

1.3- Which criteria confirm the diagnosis of PV?

The 2017 WHO diagnostic criteria are M1 + M2 + M3 or M1 + M3 + m if Hb > 18.5 g/dL (or Hct> 55.5%) in men or if Hb > 16.5 g /dL (Hct> 49.5%) in women.

Diagnostic criteria for PV:
Major criteria (M)
M1: Haemoglobin (Hb), haematocrit (Hct)Man
Hb > 16.5 g/dl or
Hct > 49% or
increased blood mass (> 25% above expected normal value)
Woman
Hb > 16 g/dl or
Hct > 48% or
increased blood mass (> 25% above expected normal value)
M2: bone marrow biopsy (“BMB”)M2: bone marrow biopsy (“BMB”)
M3: mutation JAK2V617F (exon 14) or exon 12
Minor criteria (m)
Serum EPONormal or subnormal serum assay

1.4- Which biological analyses should be ordered and what to expect?

Initially a CBC-platelets, an assay of serum erythropoietin, then a myelogram (or a bone marrow biopsy BMB) with bone marrow cytological examination, performance of a bone marrow karyotype and molecular biology tests screening for JAK2 V617F mutations (exons 14 and 12), then CALR and MPL or an “MPN diagnostic” NGS panel.

For any diagnosis and/or establishment of the prognosis of a haematological malignancy, the values for cellular haematology, histology (osteomedullary biopsy), cytogenetics and molecular biology must be compared.

In polycythaemia vera, the results are:

  • CBC-platelets: increase of the 3 lines, especially the red line. Erythrocyte constants (MCH, MCHC, MCV) are normal and there are no RBC morphological abnormalities. WBCs are often between 10 G/L and 20 G/L (blood count rich in neutrophils, some granular precursors). Platelets are usually around 400-500 G/L;
  • The myelogram is generally of little diagnostic use. The BMB, if performed, shows very rich marrow with global hyperplasia (predominant erythroblast lineage, assessment of fibrosis).
  • The bone marrow karyotype should be performed: it may be normal or show non-specific clonal abnormalities: +8, +9, del(9p), del(13q), del(20q), dup(1q), etc.
  • Molecular biological analysis: essential, it has revolutionised the diagnosis of polycythaemia vera. The analysis can be performed on blood or bone marrow.
    • Mutation JAK2 V617F in exon 14 of JAK2: positive in 96% of cases (often mutated allelic fraction > 50%);
    • Mutation JAK2 exon 12: 2% of cases;
    • Absence of mutation CALR and MPL.

In less than 2% of cases, no mutation is observed in the gene JAK2.

Contribution of NGS in PV diagnosis

Screening for mutation JAK2 V617F is conventionally carried out by selective PCR (with or without quantification).

The NGS approach means a complete analysis of the JAK2 gene can be performed with a single technique with, in particular, the analysis of exons 12 and 14.

At the prognostic level, some additional abnormalities detected in NGS are of interest, in particular the SRFS2 mutation associated with an unfavourable evolution (towards primary myelofibrosis or acute leukaemia, see below).

The contribution of the NGS panels is presented in schematic form in Fig. 1.

 

Prescription advice for a suspicion of MPN or a MPN follow-up

1.5- What is the course of the disease and how is it treated?

Polycythaemia vera is a chronic condition with a median survival of around 15 years. The immediate risks are thrombotic (due to hyperviscosity) or haemorrhagic, and the occurrence of gout attacks.

The disease progresses to primary myelofibrosis associated with bone marrow failure in 10% of cases at 10 years, or to acute myeloid leukaemia in 1 to 2% of cases (with poor prognosis) in the long term.

Treatment is based on the practice of bloodletting associated with anti-coagulation treatment in emergencies.

The first line basic treatment is with hydroxyurea (Hydrea®) and second line treatment is with pipobroman (Vercyte®) or ruxolitinib (Jakavi®). Interferon α can be used as an alternative.

2- ESSENTIAL THROMBOCYTHEMIA (ET)

2.1- Which contexts are suggestive of essential thrombocythemia?

The disease with an incidence of 1.5 to 2.5 cases/100,000 inhabitants/year, occurs in adults aged 50-60 or younger (30 years, especially in women). In 2/3 of the cases, it is asymptomatic (fortuitous discovery in the CBC in context of significant thrombocytosis) and in 1/3 of the cases, it is manifested by haemorrhagic or thrombotic signs (sometimes microthrombi causing headaches, ischaemia of the extremities, etc.); splenomegaly may be observed, rather moderate.

2.2- What criteria confirm the diagnosis of ET?

The 2017 WHO criteria specify that the diagnosis of ET is made on the basis of 4 major criteria or the first 3 major criteria and a minor criterion.

MAJOR criteria

  • Platelet count > 450 G/L (confirmed 1 month apart);
  • BMB showing proliferation mainly of the megakaryocyte lineage with an increased number of large mature megakaryocytes with hyperlobed nuclei. No significant increase or hiatus in granulocyte or erythroblast lineage and absent or rare increase in reticulin fibres;
  • Exclusion of Phi+ CML, PV, PMF, MDS or other myeloid neoplasia;
  • Presence of a JAK2, CALR or MPL mutation.

MINOR criterion

Presence of a clonal marker or absence of a sign suggesting reactive thrombocytosis.

2.3- Which biological analyses should be ordered and what to expect?

Initially a CBC-platelets (repeated after a 1 month interval), then a myelogram (or a BMB) with bone marrow cytological examination, performance of a bone marrow karyotype and molecular biology tests screening for mutations JAK2 V617F (exons 14 and 12), CALR and MPL or an “MPN diagnostic” NGS panel.

The results expected for ET are:

  • In the CBC, platelet count > 450 G/L (verified in 2 tests 1 month apart), or > 1000 G/L in half of the cases (in the absence of another cause of thrombocytosis: inflammatory syndrome, iron deficiency). On the blood smear, look for giant platelets or fragments of megakaryocytes.
  • Myelograms are important in ET because they support other differential diagnoses such as CML. The marrow is of normal or increased richness, with an increase in the number of megakaryocytes and sometimes some giant-sized elements with a hypersegmented nucleus, without quantitative or qualitative anomaly of the other lines.
  • In the BMB, bone marrow cellularity is normal or increased with rare adipocytes. Megakaryocytes are numerous (enlarged in size and multilobed nucleus with a “deer antler” appearance), scattered or sometimes grouped in small clumps. Above all, there is no fibrosis.
  • Cytogenetics: the bone marrow karyotype should be performed to:
    • rule out a translocation t(9;22)(q34;q11) which would signal a CML with thrombocytemic presentation;
    • rule out a 5q- syndrome (EGR1 deletion);
    • identify non-specific clonal anomalies, found in 5 to 10% of cases (+8, +9, del(20q)).
  • Molecular biology plays an essential role

Screening for mutations has revolutionised the diagnosis of ET. Indeed, this diagnosis is confirmed by molecular biology which makes it possible to find:

    • the JAK2 V617F mutation (exon 14) in 50 to 60% cases (often the mutated allelic fraction is less than 50%) or a mutation in exon 12 of JAK2 (< 5% of cases) – note, an association between mutated JAK2 and thromboses;
    • a mutation in the gene CALR, in 25% of cases. The type of CALR mutation must be specified because there is a prognostic value (CALR type 1: higher risk of transformation into MF; type 2 associated with more marked thrombocytosis, without increased thrombotic risk and with a better prognosis);
    • a MPL mutation in 5% of cases.

Fifteen percent of ETs show no JAK2/CALR/MPL mutations (triple negative).

2.4- What are the differential diagnoses?

  • Reactive thrombocytoses (post-surgery, infection, inflammation, iron deficiency, haemolysis, etc.);
  • Thrombocytoses associated with haematological malignancy
    • thrombocytemic form of CML (platelets rarely > 1000 G/L; prescribe laboratory tests for CML);
    • PV (platelets rarely > 600 G/L; JAK2+);
    • PMF (platelets sometimes > 1000 G/L; fibrosis in BMB);
    • MDS with thrombocytoses: Sd 5q- (platelets rarely > 600 G/L; cytogenetic test);
    • borderline form of MPN/MDS with sideroblasts and thrombocytosis (WHO entity 2017 – platelets sometimes > 1000 G/L, gene SF3B1 mutated)).

Specialised clinical biology analyses are essential to differentiate these entities.

2.5- Contribution of NGS

NGS analysis is particularly useful in the diagnosis of ET because it allows both analysis of the JAK2 “full exon”/CALR/MPL trio in a single step (faster diagnosis, detection sensitivity for CALR and MPL) and makes it possible to rule out the WHO entity MDS/MPN-with ringed sideroblasts-thrombocytosis, characterised by a mutation in the SF3B1 gene (possibly associated with a JAK2 mutation), or to identify one or more other mutations which would be proof of clonality to confirm the diagnosis. Note that around 15% of ETs are triple-negative and the value of NGS in these cases is significant.

In addition, NGS may be of prognostic value in ET: the presence of mutations in the spliceosome genes (SF3B1, SRSF2 and U2AF1) is associated with a poor prognosis and mutations in the TP53 gene are predictive of acutisation to AML. Recent bibliographic data suggest the beneficial effect of ASXL1, RUNX1 and EZH2 gene mutations on the risk of arterial thrombosis.

2.6- What is the course of the disease and how is it treated?

Regardless of the number of platelets, there is a risk of thrombosis; the risk of bleeding is correlated to the number of platelets (+++ if > 1,500 G/L).

The risk of progression from ET to primary myelofibrosis with myeloid metaplasia is 10% at 10 years. The risk of progression to MDS/AML is 2% to 5% at 10 years. This risk increases beyond the age of 60, when the platelet count is greater than 1500 G/L and in the presence of co-morbidities.

The treatment is based on a combination of aspirin and/or Hydrea® in the elderly with a history of thrombosis (otherwise, aspirin alone) or IFN alpha, before the age of 40.

3- PRIMARY MYELOFIBROSIS (PMF)

3.1- Which contexts are suggestive of primary myelofibrosis?

It is a rare disease (1 case/100,000 inhabitants/year), typically affecting adults aged 60.

At an early stage, the disease can be discovered fortuitously in connection with an anomaly in the CBC. Most often, there is significant splenomegaly (in more than 90% of cases), hepatomegaly (in 50% of cases) and varying clinical symptoms related to cytopaenias: anaemia, bleeding (25%), infections (more rarely). In 30% of cases, there are general symptoms: weight loss, fatigue, fever, night sweats, bone pain, etc.

Myelofibrosis begins with a prefibrotic then fibrotic stage and can finally transform into AML.

The decision on whether to perform an allograft requires the availability of robust prognostic markers.

3.2- Which criteria confirm the diagnosis of MFP?

The WHO in 2017 defined the diagnostic criteria of prefibrotic or early stage (pre-PMF) and those for fibrotic stage (PMF).

3.3- Which biological tests should be prescribed?

Initially a CBC-platelets (repeated after a 1 month interval), then a myelogram, or preferably a BMB, with bone marrow cytological examination, a blood karyotype and molecular biology tests screening for JAK2 V617F (exons 14 and 12), CALR and MPL mutations or an “MPN diagnostic” NGS panel. These tests are essential to confirm the diagnosis and establish the prognostic factors that will inform treatment of the condition.

In PMF:

  • the CBC is variable (non-specific). It usually shows:
    • normochromic normocytic anaemia (normal or slightly increased reticulocytes) due to decreased haematopoiesis, splenic sequestration, or even haemolysis;
    • variable leukocytosis: increased WBC (> 100 G/L, especially with neutrophils) in more than 50% of cases, normal in 25% of cases and decreased (leukopaenia) in 25% of cases.
    • the platelets are also variable: > 400 G/L in 30% of cases, normal in 55% of cases or < 100 G/L in 15% of cases.
    • On the blood smear, there are many non-specific abnormalities:
      • moderate myelemia (5-15%) with possibly rare blasts;
      • erythroblasts (2-20%), the presence of which is linked to extramedullary erythropoiesis;
      • significant RBC abnormalities: anisocytosis, dacryocytes (fibrosis), basophil pitting +/- Cabot rings;
      • platelet abnormalities: hypo- or degranulated macroplatelets, micromegakaryocytes or free nuclei of circulating megakaryocytes.
  • The myelogram (not very informative) is difficult to aspirate (fibrosis), often poor quality. It shows the presence of dacryocytes, erythroblasts (more or less in clumps), micromegakaryocytes and large megakaryocytes with large nuclei.
  • The BMB is essential: it identifies the megakaryocytic abnormalities and makes it possible to evaluate the fibrosis of reticulin (silver staining) and collagen (trichrome). It also provides a basis for differential diagnosis with ET and the other MPNs (overall survival and different therapies).
  • Cytogenetics

The karyotype must be performed on peripheral blood (and not on marrow because, due to fibrosis, there are many culture failures), to rule out CML (translocation t(9;22)(q34,q11)) and screen for non-specific clonal abnormalities with prognostic value (found in 30% of cases):

    • normal karyotype / +9 / del(13q) / del(20q): fairly good prognosis;
    • inv(3) / -7 / i(17q) / +21 / +19 / del(12p) / del(11q) / complex karyotype: poor prognosis.
  • Molecular biology plays an essential role
    • the JAK2 V617F mutation (exon 14) is found in 50 to 60% of cases;
    • a mutation on the gene CALR, in 30% of cases. The best prognosis for CALR mutations in PMF appears to be restricted to type 1 mutations (or type 1-like);
    • a MPL mutation, in 5 to 10% of cases.

In the absence of JAK2/CALR/MPLmutations (10% of cases), it is recommended to perform an NGS panel to look for other mutations (ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2, SF3B1, etc.).

3.4- What is the course of the disease?

Myelofibrosis begins with a prefibrotic then fibrotic stage and can finally transform into AML.

The course is more or less rapid from the pre-fibrotic stage to the fibrotic stage with a decrease in haemoglobin (Hb) and platelets, an increase in splenomegaly, LDH, erythromyelaemia and the presence of blasts.

Complications are mainly infectious and haemorrhagic; they can also be linked to hepatosplenomegaly or transfusions (haemochromatosis).

In the accelerated phase, blood blasts increase (5% of cases) or the disease turns into AML (10-15% of cases). It is advisable to look for any alteration of the general state or a worsening of the CBC.

Note: 5 to 15% of ET cases and 30% of PV evolve into a PMF after 10-15 years.

3.5- What are the differential diagnoses?

  • With other MPNs
    • ET if there is thrombocytosis, but there is little or no splenomegaly and no fibrosis in the ET.
    • CML if there is hyperleukocytosis and myelaemia, but there are no erythroblasts or dacryocytes in CML during which, on the other hand, the BCR-ABL transcript and basophilia are found.
    • PV if the Hb is high, but it is higher in the PV and the splenomegaly is less.
  • With other causes of myelofibrosis
    • Hairy cell leukaemia: no blasts or myelaemia, but hairy cells present
    • Other lymphomas
    • Sometimes with acute leukopenic leukaemia
    • Or MDS with fibrosis, but absence of splenomegaly and presence of dysmyelopoiesis.
  • Other pathologies with bone abnormalities
    • Metastases, metabolic bone changes.

3.6- How does NGS contribute to PMF diagnosis?

The diagnostic interest of NGS for the diagnosis of PMFs is to screen in a single step for the mutations of the JAK2/CALR/MPL trio and to search, if negative, for other mutations in order to characterise the triple negatives.

On the prognostic level, an NGS panel can help the clinician decided between an allograft and simple clinical-biological monitoring by calculating MIPSS70+ scores (status of genes CALR type 1/1-like, ASXL1, SRSF2, EZH2, IDH1 and IDH2) or GIPSS (status of genes CALR type1/1-like, ASXL1, SRSF2 and U2AF1).

Other genes are also of prognostic interest in PMF (TP53, CBL, NRAS or KRAS).

Finally, mutations in the RAS and CBL genes may be predictive of resistance to JAK2 inhibitors.

3.7- What is the treatment?

In the absence of a negative risk factor, the rule is to forego treatment, with regular clinical and biological monitoring.

In the presence of negative prognostic factors, an allograft may be proposed, in particular for young patients. If the allograft is not possible, the therapeutic alternatives are the prescription of Hydrea® or JAK2 inhibitors, and sometimes splenectomy.