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Myeloproliferative neoplasms (MPN) are chronic clonal haemopathies affecting the myeloid lineage. Myelodysplastic syndromes (MDS) are acquired clonal haemopathies in haematopoietic stem cells, resulting in ineffective hematopoiesis.

The WHO 2017 classification has a separate category named ‘myelodysplastic/myeloproliferative neoplasms (MDS/MPN)’ that includes chronic myelomonocytic leukaemia (CMML) and its juvenile form (JMML), atypical chronic myeloid leukaemia (aCML), chronic neutrophilic leukaemia (CNL), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) and unclassifiable mixed MDS/MPN syndromes. Here, we will discuss CMML, atypical CML and MDS/MPN with ring sideroblasts and thrombocytosis.

1. CHRONIC MYELOMONOCYTIC LEUKAEMIA

1.1. WHAT ARE THE DIAGNOSTIC CRITERIA FOR CMML?

In 2017, the World Health Organization defined new diagnostic criteria for chronic myelomonocytic leukaemia (CMML), within the category of myeloproliferative neoplasia (MPN) and myelodysplastic syndrome (MDS) overlap forms (1).

  1. Persistent absolute (> 1 G/L) and relative (≥ 10% white blood cells) blood monocytosis; no impact on bone marrow monocytosis.
  2. Absence of WHO criteria for chronic myeloid leukaemia (CML), polycythaemia vera (PV), essential thrombocythaemia (ET), primary myelofibrosis (PMF).
  3. If eosinophilia: absence of PDGFRA, PDGFRB or FGFR1 rearrangements; absence of PCM1-JAK2 fusion transcript.
  4. < 20% blasts in the bone marrow and blood (blasts + myeloblasts + monoblasts + promonocytes).
  5. At least 1 of the following criteria:
  • dysplasia of at least 1 lineage; if dysplasia is absent or minimal, the other criteria must be present and a cytogenetic abnormality or acquired clonal genetic abnormality must be present in the haematopoietic cells.
  • monocytosis present for at least 3 months and absence of other causes of monocytosis (infection / inflammation / cancer).

CLASSIFICATION OF CMML (See Table 1).
Table 1: Classification of CMML

BloodBone marrow
CMML-0< 2 % blastsAND< 5% blasts
CMML-12-4% blastsAND5-9% blasts and absence of Auer rods
CMML-25-19% blastsAND/OR10-19% blasts or presence of Auer rods, regardless of the number of blasts

The WHO also distinguishes 2 subtypes:
– leukocytosis < 13 G/L (around 60% of patients), also called myelodysplastic CMML.
– leukocytosis ≥ 13 G/L, or myeloproliferative CMML.

1.2. WHICH BIOLOGICAL EXAMINATIONS SHOULD BE PRESCRIBED FOR CMML

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

As such, it is recommended to request a CBC-platelet count as the first line analysis (to be confirmed in a second sample 3 months later). If the monocytosis is persistent, phenotyping of the blood monocytes (cannot be performed on bone marrow) can be carried out alongside the biological work-up to exclude other causes of peripheral monocytosis. Finally, if this work-up points towards CMML, a myelogram (or bone marrow biopsy – BMB) can be carried out, accompanied by a bone marrow karyotype and molecular biological examinations or a NGS panel on the blood or bone marrow

DS71-INTGB: NGS Panel – Chronic Myelomonocytic Leukemia (CMML) 

EXPECTED RESULTS

HAEMOGRAM TEST – BONE MARROW BIOPSY
Leukocytosis is variable, between 3 and 100 G/L with two subtypes, according to whether it is < 13 G/L (myelodysplastic) or ≥ 13 G/L (myeloproliferative).

What distinguishes CMML from other ‘overlap forms’ in adults is blood monocytosis, both absolute, above 1 G/L) and relative, above 10% of leukocytes, and myelaemia of under 10% (above this, diagnosis of aCML).

The bone marrow biopsy is rich in 75% of cases, with granulocytic and monocytic hyperplasia and dysmyelopoiesis.

The values for differential diagnosis with other MDS/MPN overlap forms are shown in Table 2.

Table 2: Factors for differential diagnosis of CMML

CriterionCMML-0CMML-1CMML-2aCMLCNLUnclassifiable MPN/MDSMDS/MPN-RS-T
Monocytes> 1 G/L (> 10%)< 10%< 1G/L  
Neutrophils    ≥ 80%  
Myelaemia< 10%≥ 10%< 10%  
WBC (x109/L)   ≥ 13≥ 25≥ 13 
Platelets (109/L)     ≥ 450≥ 450
Basophils   < 2%< 1%  
Circulating blasts*< 2%2-4%5-19%< 20%< 1% < 1%
Bone marrow blasts*< 5%5-9%10-19%< 20%< 5% < 5%
Sideroblasts ≥ 15%rare (> 5% of cases)NoNo Yes
Dysplasia**VariableDysGNoYes 
t(9;22)/BCR-ABL1

No

 

PDGFRA/BNo

aCML: atypical chronic myeloid leukaemia; CNL: chronic neutrophilic leukaemia; MDS/MPN-RS-T: MDS/MPN with ring sideroblasts and thrombocytosis; DysG: dysgranulopoiesis.
* The blasts count includes blasts, myeloblasts, monoblasts and promonocytes.
** The dysplasia criterion can be replaced by a cytogenetic or acquired genetic abnormality.

MONOCYTE PHENOTYPING USING FLOW CYTOMETRY (FC)

Although its results are not included in the WHO classification criteria, flow cytometry now has recognised diagnostic value for CMML according to the ELN criteria (2).

The diagnostic approach for CMML using FC is based on identification of monocyte sub-populations in peripheral blood according to the expression profile of the CD14 and CD16 markers.
Thus, a relative accumulation of ‘classical’ monocytes MO1 (CD14+, CD16-) of ≥ 94% of total monocytes, points towards CMML with a high degree of sensitivity and specificity (3). These findings can then be confirmed with a bone marrow biopsy and to pursue the diagnostic and prognostic work-up. Inversely, a decrease in the proportion of classical monocytes in peripheral blood points towards a cause other than monocytosis and in this case no bone marrow biopsy need be performed.

However, it is sometimes difficult to distinguish CMML, which can be associated with inflammation, from autoimmune or inflammation disorders accompanying reactive monocytosis. In this context, FC provides considerable support through specially developed panels. Thus, for an associated inflammatory syndrome that could interfere in the interpretation of the immunophenotype profile, the identification of a relative decrease in the percentage of non-standard monocytes expressing the slan marker, ≤ 1.7% points towards the diagnosis of CMML (4).

FC has now become an extremely useful examination in patients presenting with unexplained monocytosis.

CYTOGENETICS TO CONFIRM THE DIAGNOSIS, RULE OUT CML AND ESTABLISH THE PROGNOSIS OF CMML

A bone marrow karyotype can be used to rule out a t(9;22)(q34;q11) translation, that would suggest CML.
If eosinophilia is present, when combined with a FISH analysis, it can be used to rule out PDFGRA / PDFGRB / FGFR1 rearrangements and PCM1-JAK2 fusion.

Finally, it can be used to identify clonal abnormalities (in under 30% of cases) associated with a high risk (trisomy 8, ano chr 7 or complex karyotype), a low risk (normal karyotype or isolated Y loss) or an intermediate risk (other abnormalities).
CMML now has its own cytogenetic classifications: the European classification (favoured in France) is useful for identifying trisomy 8 as an abnormality with a poor prognosis. As such, it is now recommended to include this karyotyope criterion, which is now used as the basis (among other things) for the CPPS (CMML Prognostic Score System) (5), the simplest to use in clinical practice of the ‘modern’ scores validated for CMML (GFM, Mayo, CPPS-mol).

This score is based on the values for bone marrow blasts, leukocytosis, the existence or not of red blood cell transfusion dependence and molecular data (status of ASXL1, NRAS, RUNX1 and SETBP1 genes); it is divided into four groups, with median survival ranging from 5 to 72 months (6).

MOLECULAR ANALYSIS FOR DIAGNOSTICS, PROGNOSTICS AND THERANOSTICS

Screening for specific mutations is offered by Eurofins Biomnis using next-generation sequencing (NGS) for a panel of 19 target genes:
ASXL1/CBL/DNMT3A/EZH2/FLT3/IDH1/IDH2/JAK2/KRAS/NPM1/NRAS/RUNX1/SETBP1/SF3B1/SRSF2/TET2/TP53/U2AF1/ZRSR2

This panel can be performed on blood or bone marrow. This panel is a diagnostic, prognostic and theranostic tool.
– According to the WHO in 2017 (1), the analysis of the genes TET2, SRSF2, ASXL1 and SETBP1 can help in the diagnosis of CMML (criterion 5 of the WHO 2017 in combination with criteria 1 to 4), in particular in the absence of contributing cytogenetic data. In practice, the identification of TET2 and SRSF2 mutations is highly suggestive of CMML.
It should be noted that in the presence of a mutation in the NPM1 and/or FLT3 genes, diagnosis should be reconsidered between CMML-2 and LAM-M4 (re-examine the percentage of blasts in the bone marrow, below or above 20%).

– This panel can also aid in prognosis and in therapeutic decision-making.
According to ELN 2018 (2) recommendations, a panel with a minimum of 4 genes is mandatory (genes at high risk of unfavourable evolution: ASXL1, NRAS, RUNX1, SETBP1).

– The theranostic impact is also addressed with this panel, with respect to IDH1, IDH2, NPM1 and FLT3 mutations, which while rare, are also potential targets for therapy.

DS71-INTGB: NGS Panel – Chronic Myelomonocytic Leukemia (CMML) 

2. ATYPICAL CHRONIC MYELOID LEUKAEMIA BCR-ABL1 NEGATIVE

2-1 WHEN SHOULD ATYPICAL CHRONIC MYELOID LEUKAEMIA BCR-ABL1 NEGATIVE BE SUSPECTED?

This condition, which is very rare, affects patients over 65 years old. Clinically, it presents with symptoms linked to anaemia and/or thrombocytopaenia, splenomegaly, sometimes associated with general signs. It is primarily a differential diagnosis for an MPN or MDS.

It will be considered in cases with the appearance of clinical signs and/or identification of abnormalities in the CBC (see criteria below).

The 2017 WHO diagnostic criteria are:

  1. Blood leukocytosis ≥ 13 G/L (rarely over 100 G/L), with neutrophilic polyneucleosis and myelaemia ≥ 10% of leukocytes
  2. Marked dysgranulopoiesis (pseudo Pelger-Huët hyposegmentation, giant granulocytes, degranulation) sometimes with excessive or abnormal condensation of the chromatin
  3. No notable basophilic leukocytosis (in general < 2%)
  4. No or limited monocytosis (< 10% of leukocytes)
  5. Hypercellular bone marrow with marked granulocytic and monocytic hyperplasia and dysmyelopoiesis, with or without dysplasia of the other lineages
  6. Under 20% blasts in the blood and bone marrow
  7. Absence of PDGFRA, PDGFRB, FGFR1, or PCM1-JAK2 rearrangement
  8. Absence of criteria suggestive of CML with BCR-ABL1 fusion transcript, PV, ET or PMF.

Note:

  • Cases of accelerated phase MPN and in particular post-polycythaemia vera or post-essential thrombocythemia primary myelofibrosis may present with neutrophilic polyneucleosis and mimic aCML. However, the prior history and/or presence of a JAK2, CALR or MPL mutation normally rule out diagnosis of aCML.
  • Moreover, diagnosis of aCML is confirmed by the presence of SETBP1 and/or ETNK1 mutations.
  • The existence of a CSF3R mutation is rare in aCML and should point to a chronic neutrophilic leukaemia.

2-2- WHICH BIOLOGICAL EXAMINATIONS SHOULD BE PRESCRIBED AND WHICH WHAT RESULT ARE EXPECTED FOR ATYPICAL CML NEGATIVE BCR ABL1?

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

A CBC-platelets count should thus be requested initially, then phenotyping of blood monocytes in the event of peripheral monocytosis, a myelogram (or a bone marrow biopsy – BMB) with bone marrow cytological examination and molecular biology tests.

HAEMOGRAM TEST – BONE MARROW BIOPSY

Leukocytosis remains ≥ 13 G/L (median of 24 to 96 G/L), and, in certain cases, may exceed 300 G/L. The percentage of blood blasts is typically < 5% and always < 20%. The other characteristics are classification values (see above, § 2.1).

As in the blood, the percentage of bone marrow blasts is always < 20%.

A morphological variant of aCML, the abnormal chromatin condensation syndrome, is characterised by the presence in the blood and bone marrow of a high percentage of neutrophils and precursors (myelaemia) with accentuated condensation of the nuclear chromatin.

CYTOGENETIC AND MOLECULAR ABNORMALITIES

The karyotype did not show any specific cytogenetic abnormality, but allowed CML to be ruled out by the absence of t(9;22)(q34;q11) translocation.

Role of NGS: value of the MPN diagnostic panel (7 genes)

Atypical CMML BCR-ABL negative has a heterogeneous molecular profile. The mutations in the SETBP1 and ETNK1 genes seem to be suitable candidates to help diagnose aCML. Several other mutations have been reported in aCMLs.
The NGS panel may help with prognosis: if a SETBP1 is observed, it is suggestive of a poor prognosis.

3. MYELODYSPLASTIC/MYELOPROLIFERATIVE NEOPLASM SYNDROMES WITH RING SIDEROBLASTS AND THROMBOCYTOSIS (MDS/MPN -RS-T)

3.1- WHEN SHOULD MYELODYSPLASTIC/MYELOPROLIFERATIVE NEOPLASM SYNDROMES WITH RING SIDEROBLASTS AND THROMBOCYTOSIS (MDS/MPN-RS-T) BE SUSPECTED?

This syndrome affects adults, with a median age of 74 years at diagnosis. The clinical symptoms are similar to those of ET, with haemorrhagic or thrombotic signs (sometimes microthrombi causing headaches, ischaemia of the extremities, etc.) and, occasionally, splenomegaly, which is somewhat moderate.

It is primarily a differential diagnosis for an MPN or MDS.

It will be suspected in the presence of clinical signs and/or the detection of abnormalities on the CBC (see criteria below), in particularthrombocytosis (platelets ≥ 450 G/L) and the presence of ring sideroblasts (RS).

Sideroblasts are erythroblasts containing iron granules that appear as Prussian blue granules in the cytoplasm after Perls cytochemical staining. The presence of ring sideroblasts (grains forming a ring around the nucleus) is closely linked with a mutation of the SF3B1 gene.

As such, the WHO 2017 classification for MDS/MPN gives the following diagnostic criteria for MDS/MPN-RS-T:

  1. Anaemia with erythroid lineage dysplasia, whether or not associated with multilineage dysplasia; ≥ 15% RS in bone marrow, <1% blasts in blood and <5% in bone marrow.
  2. Persistent thrombocytosis (platelets ≥ 450 G/L).
  3. Presence of a SF3B1 mutation or, in the absence of a SF3B1 mutation, absence of a recent history of chemotherapy or growth factor treatment that could cause dysplastic elements suggestive of MDS/MPN.
  4. Absence of BCR-ABL1 fusion gene, PDGFRA, PDGFRB or FGFR1 or PCM1-JAK2 abnormality; nor t(3;3)(q21.3;q26.2), inv(3)(q21.3q26.2) or del(5q).
  5. Absence of history of MPN, MDS (except an MDS/MPN-RS-T) or other MDS/MPN overlap form.

Note:

  • ≥ 15% of RS in the bone marrow is an essential criterion, even with the presence of a SF3B1 mutation
  • Diagnosis of MDS/MPN-RS-T is strongly suggested by the presence of a SF3B1 mutation associated with a JAK2, CALR or MPL mutation.

3.2- WHICH BIOLOGICAL TESTS SHOULD BE PRESCRIBED AND WHAT ARE THE EXPECTED RESULTS FOR MDS/MPN-RS-T?

HAEMOGRAM TEST – BONE MARROW BIOPSY

The characteristics of this syndrome appear in the WHO diagnostic criteria.

For the haemogram, the anaemia is normo- or macrocytic. Anisocytosis in the red blood cell and platelet lineages may be observed. Persistent thrombocytosis ≥ 450 G/l is noted and there are under 1% blasts.

The bone marrow biopsy reveals less than 15% ring sideroblasts (required diagnostic criterion, even in the presence of a SF3B1 mutation). At least a few giant and hyperlobed megakaryocytes, similar to those seen in ET, with or without dysgranulopoiesis, and less than 5% blasts are found.

CYTOGENETIC AND MOLECULAR ABNORMALITIES

The karyotype is often normal.

The molecular analysis identifies a mutation of the SF3B1 gene in 90% of cases and a mutation of the JAK2 gene in 60% of cases (V617F in particular). Other mutations are also frequently identified, on TET2 (23.3%), DNMT3A (16.7%) and ASXL1 (14.3%).

On the other hand, the CALR gene is rarely mutated (under 10% of cases).

There is no BCR-ABL1 fusion transcript, nor PDGFRA, PDGFRB or FGFR1 or PCM1-JAK2 abnormality; nor t(3;3)(q21;q26), inv(3)(q21q26) or del(5q)

Some factors confirming the diagnosis:

  • Patients with a SF3B1 mutation have more ring sideroblasts (60% versus 40% in the absence of a mutation)
  • Patients with a JAK2 mutation have higher leukocyte and platelet counts: 10 versus 7 G/L and 800 versus 600 G/L respectively.
  • Myeloproliferative forms with platelet count above 600 G/L, moderate anaemia and, often, associated mild neutrophilic polynucleosis feature JAK2 mutation in 60% of cases; myelodysplastic forms, with platelet counts between 450 and 600 G/L, normoleukocytic and with anaemia of variable intensity, feature JAK2 mutation in only 10% of cases.

Role of NGS: value of the MPN diagnostic panel (7 genes)

MDS/MPN-RS-T are characterised by a SF3B1 mutation, but this mutation is often associated with other mutations found in

MPN such as ET, PV or PMF; thus the NGS panel is an important aid for differential diagnosis.

Finally, it is also useful for prognosis as, if it identifies a ASXL1 mutation associated with STBP1, the prognosis becomes poor.

DS69-INTGB: NGS Panel – Myeloproliferative neoplasms (MPN)

DS71-INTGB: NGS Panel – Chronic Myelomonocytic Leukemia (CMML)