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July 2014 - Test your knowledge in the quiz!

What is the cause of the observed leukocytosis, anaemia and thrombocytopenia? Vitamin B12 or folate deficiency
Acute myeloid leukaemia (AML) under chemotherapy
Bacterial sepsis
Chronic myeloid leukaemia (CML)

Online version of this month`s case:

THE CORRECT ANSWER TO JULY’S QUIZ IS:

 

„Acute myeloid leukaemia (AML) under chemotherapy: AML with abnormal marrow eosinophils”

Scattergrams and microscopy

Patient history: a 54-year old man was admitted to the hospital with a high fever.

The WDF scattergram showed a leukocytosis with an extended monocyte population and the presence of immature granulocytes (IG). Without a reflex analysis in the WPC channel, the high-fluorescence cells would have triggered the flag ‘Blasts/Abn Lympho?’.
The WNR scattergram showed the presence of nucleated red blood cells (NRBC).
The SSC-FSC scattergram of the WPC channel showed the presence of blasts.
Thrombocytopenia with a slight increase of the relative IPF (IPF%) and a normal absolute IPF count (IPF#) could be inferred from the PLT-F scattergram.
Peripheral blood smear
Bone marrow smear

Table

Interpretation and Differential Diagnosis

 The answer can be inferred from…

          unusually high fluorescence intensity of eosinophils (WDF scattergram)

  • The presence of blast cells in the peripheral blood and bone marrow smears

 

Case history

This 54-year old man was admitted to the hospital with a high fever. A complete blood count with leukocyte differential (CBC-Diff) was ordered to investigate his condition.

Case results

The blood count revealed anaemia with severe thrombocytopenia and combined with a low immature platelet count (IPF#) this indicated ineffective erythropoiesis and thrombopoiesis. Furthermore, the WDF scattergram contained an increased cell population with high fluorescence intensity above the monocytes, which hinted at the presence of blasts. This triggered a reflex measurement with the WPC channel, which confirmed the presence of blasts and triggered the appearance of the ‘Blasts?’ flag. In addition, erythroid blasts (2.1% NRBC) and large numbers of immature granulocytes (18.8% IG) were present in the peripheral blood. The combination of these findings (ineffective erythropoiesis and thrombopoiesis, the presence of blasts, NRBC and immature granulocytes) pointed to leukaemic bone marrow infiltration and suppression of normal haematopoiesis. Therefore, these observations made a neoplastic disorder, such as an acute leukaemia or chronic myeloid leukaemia (CML) likely. The morphology of the cells in the peripheral blood and bone marrow smears, which showed myeloblasts from type 1 to 3 and monocytic blasts confirmed the presence of blasts. In addition, the presence of abnormal eosinophil precursors with basophilic and eosinophilic granulation made an AML-M4eo most likely. The increased fluorescence intensity of eosinophils (immature abnormal eosinophils) supported the finding of abnormal cells in the peripheral blood and bone marrow smears. Haematopoietic suppression resulting from bone marrow infiltration can be conducive of an infection. Indeed, the increased NEUT-SFL, indicating activation of neutrophils, and the presence of immature granulocytes may point to an infection and this could explain the patient’s fever.

The following answers are incorrect for the described reasons

 

Vitamin B12 or folate deficiency

Humans cannot synthesize vitamin B12 (cobalamin) and folate so dietary intake is essential. While cobalamin deficiency takes years to develop because the body stores large amounts of cobalamin, folate deficiency can develop within four to five months of reduced intake.
Cobalamin and folate deficiencies impair DNA synthesis, which leads to reduced cell division and oval macrocytosis of red blood cells. This results in hyperchromic macrocytic anaemia, as seen here, but it is typically not associated with such a severe thrombocytopenia, leukocytosis or high concentrations of immature granulocytes. Furthermore, cobalamin and folate deficiencies can cause hypersegmentation of neutrophils, but the NEUT-SSC in the presented patient (155.1 channels) is within the normal range (140-160 channels). However, the most important indication of a neoplastic condition rather than a vitamin B12 or folate deficiency is of course the presence of blasts. The presence of hyperchromic macrocytic cells is possibly caused by chemotherapy or by a secondary underlying vitamin B12 or folate deficiency in this patient but this is not the primary diagnosis.

Bacterial sepsis

The presence of highly fluorescent neutrophils (high NE-SFL) and immature neutrophils (18.8% IG) in this patient may point to a bacterial sepsis. However, the presence of blasts and abnormal eosinophils make a bacterial infection unlikely as the main diagnosis. An infection secondary to bone marrow infiltration could be the cause of the observed fever and is more likely than a bacterial sepsis.

Chronic myeloid leukaemia (CML)

Patients with CML have< 20% blasts in the peripheral blood or bone marrow but no severe thrombocytopenia as observed in this case. Furthermore, in most CML cases WBC counts are above 50,000/µL and basophilia in the peripheral blood and bone marrow is often observed. These two features were both absent in the presented patient. The total amount of blasts in the bone marrow was 30% and the presence of chromosomal abnormality inv(16) confirmed the diagnosis of AML-M4eo.

Underlying Disease

Acute leukaemia

Acute leukaemia usually presents suddenly with fever, pallor and purpura, indicating bone marrow failure. The blood count typically shows granulocytopenia, anaemia and thrombocytopenia. A marked increase of proliferating cells is frequently observed in the peripheral blood but aleukaemic forms are not uncommon. The cause of acute leukaemia remains enigmatic but certain factors have been implicated, such as radiation, chemicals and drugs, genetic factors and viruses. Increasing knowledge of the biology and molecular genetics of acute leukaemia has led to the introduction of therapeutic approaches that have transformed the inevitably fatal illness of three decades ago into high long-term survival rates after intensive treatment, particularly in children. Acute leukaemia is an uncommon disease but it may occur at any age. There is a peak in early childhood and a progressive rise in incidence with age in the elderly. Acute lymphoblastic leukaemia (ALL) is the most common acute leukaemia in childhood but acute myeloid leukaemia (AML) accounts for 75% of all adult cases. Clinical symptoms resulting from organ infiltration by leukaemic cells (generalised lymphadenopathy, splenomegaly and hepatomegaly, commonly observed in ALL) are not characteristic of AML but bone pain and bone tenderness are common manifestations.

 

Acute myeloid leukaemia

AML is extraordinarily heterogeneous, reflecting the complexities of myeloid cell differentiation. AML is believed to be triggered by a multistep process: changes in genes encoding transcription factors impair myeloid differentiation while a second genetic transformation promotes proliferation or survival of transformed cells. The resulting clone of haematopoietic cells undergoes massive cell division and fails to undergo apoptosis. The results from the haematology analyser may show circulating leukaemic blasts but the final diagnosis must be established by means of cell morphology, cytochemistry, flow cytometry, immunohistochemistry / immunofluorescence, cytogenetics and molecular genetics. However, results from the haematology analyser offer important guidance for the selection of further diagnostic tests.

 

The French-American-British (FAB) classification is generally accepted as useful and reproducible for AML (1). The 8 FAB subtypes of AML are defined by the percentage of blast cells, the presence of cytochemical myeloperoxidase (MPO), the major cell types present defined by morphology and esterase cytochemistry, and the immunophenotype.

Table 1: FAB subtypes of acute myeloid leukaemia

  • AML-M0: MPO negative; blast cells not distinctive; blasts express myeloid associated markers CD13 or CD33; lymphoid markers negative,
  • AML-M1: Marrow contains at least 90% medium to large blasts; few granules, Auer rods or vacuoles; > 3% blasts MPO positive,
  • AML-M2: >10% of bone marrow cells are differentiated at or beyond the promyelocytes stage, blasts 30-90%; Auer rods; 8;21 chromosomal translocation in 18% of patients,
  • AML-M3: Usually hypergranular or microgranular promyelocytes can be recognised; ‘faggot cells’; MPO strongly positive; associated with 15;17 chromosome translocation,
  • AML-M4: Both myeloid and monocytic differentiation; marrow blasts > 30%, many showing monocytic features; > 5% monoblasts, promonocytes, monocytes in peripheral blood, which show non-specific esterase (NSE) positivity (combined with the esterase procedure, cells containing both NSE and granulocyte enzyme chloroacetate esterase can be demonstrated); an eosinophilic variant can be recognised (AML-M4eo),
  • AML-M5: MPO is negative and NSE strongly positive; serum and urine lysozyme levels are much increased (AML-M5a: Large monoblasts with abundant basophilic cytoplasm (> 80%); may contain vacuoles and/or azurophilic granules – AML-M5b: Mostly promonocytes and abnormal monocytes),
  • AML-M6: Erythroid precursors are > 50% and blasts > 30%; intense Periodic acid-Schiff (PAS) block positivity with or without ringed sideroblasts,
  • AML-M7: Blast morphology polymorphic; MPO negative; variable PAS and esterase positivity; diagnosis depends on 1) demonstration of platelet peroxidase by ultracytochemistry or 2) immunophenotyping to identify CD41 or CD61 glycoproteins on the blast cell surface.

In 2001, the World Health Organization (WHO) published a new classification for haematopoietic and lymphoblastic neoplasms (2), which has been revised in 2008 (3). A fundamental principle of this new classification is that all available information, including genetic, immunophenotypic, biological, and clinical features should be used to define specific disease entities when possible.

Table 2: WHO classification of AML

AML with recurrent genetic abnormalities:

  • AML with t(8;21)(q22;q22); RUNX1-RUNX1T1
  • AML with inv(16)(p13.1;q22) or t(16;16)(p13.1;q22); CBFB-MYH11
  • Acute promyelocytic leukaemia with t(15;17)(q22;q12); PML-RARA
  • AML with t(9;11)(p22;q23); MLLT3-MLL
  • AML with t(6;9)(p23;q34); DEK-NUP214
  • AML with inv(3)(q21;q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1
  • AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1
  • AML with mutated NPM1
  • AML with mutated CEBPA

 
AML with myelodysplasia-related changes

Therapy-related myeloid neoplasms
Acute myeloid leukaemia, not otherwise specified:

  • AML with minimal differentiation
  • AML without maturation
  • AML with maturation
  • Acute myelomonocytic leukaemia
  • Acute monoblastic and monocytic leukaemia
  • Acute erythroid leukaemia
  • Acute megakaryoblastic leukaemia
  • Acute basophilic leukaemia
  • Acute panmyelosis with myelofibrosis

 

Myeloid sarcoma

Myeloid proliferations related to Down syndrome:

  • Transient abnormal myelopoiesis
  • Myeloid leukaemia associated with Down syndrome

Blastic plasmacytoid dendritic cell neoplasm

As with previous classifications, the importance of the blast cell count in defining myeloid diseases and predicting prognosis is recognised by the WHO, which recommends 1) determination of the blast percentage, 2) assessment of the degree of maturation, and 3) assessment of the degree of dysplasia in the neoplastic cells. When possible a 200-cell differential leukocyte count should be performed on the peripheral blood and a 500-cell differential leukocyte count should be performed on marrow aspirate smears stained by Romanowsky methods. The blast percentage should be correlated with an estimate of the blast cell count from the marrow histology preparation. Erythroid blasts are not included in the blast cell count except in the rare instance of a ‘pure’ erythroid leukaemia. The percentage of CD34-positive cells is not a substitute for a blast cell count: although CD34-positive cells are generally blasts cells, not all blast cells are CD34-positive. The percentage blasts relates to all nucleated marrow cells, with the exception of acute erythroid leukaemia. In addition, cytochemistry and immunophenotyping must provide evidence that the neoplastic cells belong to one or more myeloid lineage, i.e. granulocytic, monocytic, erythroid or megakaryocytic lineages. Since the WHO classification includes genetic subgroups, cytogenetic studies must be undertaken where possible, for the initial diagnosis and during the course of the disease to detect any genetic evolution.

The two most important features that are new in the WHO classification are 1) reduction in the blast threshold for diagnosis of AML from 30% to 20% and 2) grouping into clinical and biological subgroups (Table 2). Patients that do not satisfy any of these subgroups or for which no cytogenetic data is available are designated as AML, not otherwise specified. Patients with recurring cytogenetic abnormalities are considered to have AML regardless of the blast percentage.

While the FAB classification recognised the morphological diversity of AML, it did not always reflect the genetic or clinical diversity of the disease. Genetic abnormalities may predict the biological properties and prognosis more consistently than morphology. A further subdivision now considered to be important relates to certain biological features. The behaviour of AML evolving from myelodysplastic syndromes (MDS) or possessing features similar to those of MDS differs very significantly from AML arising de novo. The former (more common in the elderly) is associated with multilineage dysplasia, poor-risk cytogenetic abnormalities and poor response to therapy while the latter (more common in children and young adults) does not show multilineage dysplasia, is associated with low-risk cytogenetics and responds well to the appropriate therapy.

AML with inv(16)(p13.1;q22) or t(16;16)(p13.1;q22) / AML-M4eo

AML with abnormal marrow eosinophils (WHO: AML with inv(16)(p13.1;q22) or t(16;16)(p13.1;q22); FAB: AML-M4eo) often shows differentiation of monocytes and granulocytes. Eosinophil counts in the bone marrow are typically increased but counts in the peripheral blood are usually normal, though occasionally abnormal eosinophils are present, as in the presented case. The percentage blasts in the bone marrow is often close to the 20% threshold but cases with inv(16)(p13.1;q22) or t(16;16)(p13.1;q22) abnormalities should also be diagnosed as AML when the percentage blasts is lower than 20%. Myeloid sarcomas, extramedullary tumour masses containing myeloid blasts, may be present at diagnosis or during relapse. AML-M4eo is primarily found in paediatric patients but it may also occur in older patients and makes up 5-8% of all AML cases. It is associated with a better prognosis compared to other forms of AML but survival and treatment effectiveness decrease in older patients and in patients with c-KIT mutations.

Literature

Literature acute myeloid leukaemia (AML)

 

  1. Bennett JM, Catovsky D, Daniel MT, et al (1985): Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Intern Med 103 (4): 620-625
  2. Brunning RD, Matutes E, Harris NL, et al (2001): Acute Myeloid Leukaemias. In: Jaffe ES, Harris NL, Stein H, et al (Editors): World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. 3th Edition. Lyon, France. International Agency for Research on Cancer (IARC) Press: 75-107
  3. Arber DA, Brunning RD, Le Beau MM, et al (2008): Acute Myeloid Leukaemia and Related Precursor Neoplasms. In: Swerdlow SH, Campo E, Harris NL, et al (Editors): World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th Edition. Lyon, France. International Agency for Research on Cancer (IARC) Press: 109-147

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