Diagnostics

Aplastic anemia (AA)

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Method:
Anticoagulant:
Recommendation:

Method:

Cytomorphology
Anticoagulant:

EDTA
Recommendation:

obligatory

Method:

Immunophenotyping
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EDTA or Heparin
Recommendation:

facultative

Method:

Chromosome analysis
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Heparin
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obligatory

Method:

FISH
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EDTA or Heparin
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facultative

Method:

Molecular genetics
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EDTA or Heparin
Recommendation:

obligatory

Classification
Diagnostic methods
Prognosis
Therapy
Recommendation

Aplastic anemia describes rare and heterogeneous diseases leading to bone marrow insufficiency due to cytopenias. Based on the current guidelines and the current state of research, there are different diagnostic recommendations for patients with aplastic anemia. We have summarized the most important information on classification and diagnostic methods at MLL. In addition, we provide further links on prognosis and therapy in aplastic anemia, so that you can inform yourself in more detail.

Aplastic anemia: Classification

Acquired aplastic anemias (AA) are characterized by bi- or tricytopenias resulting from hypo- or aplasia in the bone marrow. Patients with aplastic anemia typically initially experience benign oligoclonal hematopoiesis due to a reduction in the stem cell pool resulting from immune-mediated pathogenesis, in which autoreactive T cells appear to play a major role. During the pathogenesis of aplastic anemia, the majority of bone marrow cells are replaced by adipocytes (Marsh et al. 2009, Ogawa 2016, Young 2018).

For the diagnosis of aplastic anemia, the exclusion of acquired cytopenias, as well as congenital syndromes with bone marrow insufficiency and hypoplastic myelodysplastic neoplasm (MDS) is essential (Marsh et al. 2003, Marsh et al. 2009, Killick et al. 2016). In all cases, diagnostics should include cytomorphology, histology, and cytogenetics from bone marrow.

Aplastic anemia can be classified into the following severity levels:

Table 1: Classification of aplastic anemia (two of three criteria must be met) (Onkopedia Guideline Aplastic Anemia 2022).

	Moderately severe / non-severe AA	Severe AA	Very severe AA
Neutrophil granulocytes	< 1.2 G/L	< 0.5 G/L	< 0.2 G/L
Thrombocytes	< 70 G/L	< 20 G/L	< 20 G/L
Reticulocytes	< 60 G/L	< 20 G/L	< 20 G/L

To diagnose very severe AA, < 0.2 G/L neutrophil granulocytes must be present and, in addition, hypocellular bone marrow must be present (cellularity < 25% or 25-50% with < 30% hematopoietic cells in the bone marrow) in severe and very severe AA, whereas evidence of hypocellular bone marrow is sufficient to diagnose moderately severe AA (Onkopedia Guideline Aplastic Anemia 2022).

If the classification is made according to presumed etiology, it results in the following distribution (Onkopedia Guideline Aplastic Anemia 2022):

Idiopathic (> 80%)

Triggered by medication (< 20%)

Post-infectious (< 5%)

Hereditary forms manifesting only in adulthood (5-15%)

Aplastic anemia: Diagnostics

Cytomorphology and histology are mandatory for reliable diagnosis and differentiation from acute leukemias and MDS. Essentially, a bone marrow biopsy (1.5 - 2 cm biopsy cylinder) and histological examinations must be performed for every bone marrow aspiration in which insufficient material has been obtained for a definite diagnosis or in the case of punctio sicca. This generally applies for aplastic anemias.

In aplastic anemia, immunophenotyping may have significance for differential diagnosis when cytomorphology and especially histology are inconclusive. Immunophenotyping is used, for example, to clarify the question of a PNH subclone (paroxysmal nocturnal hemoglobinuria).

Genetic alterations detected by sequencing and SNP array analyses are present in approximately 50% of patients with aplastic anemia (Yoshizato et al. 2015, Ogawa 2016). Cytogenetic aberrations have been described in 5-15% of adult patients with severe aplastic anemia (SAA), and these patients were generally younger than patients with normal karyotype (Gupta et al. 2006, Kim et al. 2010). The following cytogenetic aberrations are typical in aplastic anemia:

Trisomy 8
7q deletion
Monosomy 7
Trisomy 6
13q deletion

In addition, various other cytogenetic alterations are found, such as deletions in the long arm of chromosome 1, an isochromosome 17q, and gains (such as trisomy 15) or losses (such as monosomy 21) of whole chromosomes (Maciejewski & Selleri 2004, Kim et al. 2010, Kulasekararaj et al. 2014). Differential diagnosis should also exclude an "Inherited Bone Marrow Failure Syndrome" (Onkopedia guideline Aplastic Anemia 2022).

Often, the cytogenetic abnormalities are found only in subclones and can only be detected by FISH on interphase nuclei. Therefore, our FISH panel investigates the following aberrations commonly found in aplastic anemia:

13q14 deletion (DLEU)
17p13 deletion (TP53)
Trisomy 6 (6q21 /SEC63, 6q23 / MYB)
7q31 deletion or monosomy 7 (D7S486, cen7)
Trisomy 8 (cen8)

In aplastic anemia, mutations occur that are also observed in MDS, but with different frequencies. Most commonly, mutations are found in the genes BCOR, BCORL1, DNMT3A, PIGA and ASXL1 (Yoshizato et al. 2015, Gurnari et al. 2023). In addition, an association between the presence of a mutation in the genes ASXL1 or DNMT3A and an increased risk of transformation to MDS or AML, respectively, has been demonstrated (Kulasekararaj et al. 2014). Studies of the mutational landscape following clonal evolution have shown that mutations are enriched in the RUNX1 and ASXL1 genes, as well as in splicing factors, CBL, and SETBP1 (Groarke et al. 2022, Gurnari et al. 2023).

Aplastic anemia: Prognosis

The risk of leukemic transformation is significantly increased when cytogenetic alterations are detected in chromosome banding analysis (Fig. 1).

Fig. 1: Cumulative probability of leukemic transformation within 5 years in AA with normal versus aberrant karyotype (prepared according to Kim et al. 2010).

Depending on the type of cytogenetic alteration, response to immunosuppressive therapy (IST) and prognosis may be worse (-7, complex karyotype, 5q syndrome) or better (+8, -13q) (Maciejewski & Selleri 2004, Kim et al. 2010).

In addition to cytogenetic alterations, molecular aberrations such as mutations in the genes ASXL1 and DNMT3A may also increase the risk of developing MDS or AML (see molecular genetics) (Kulasekararaj et al. 2014). In contrast to mutations in BCOR, BCORL1, and PIGA, patients with mutations in DNMT3A, ASXL1, TP53, and RUNX1 show poorer response to IST and poorer overall or progression-free survival (Ogawa 2016).

Aplastic anemia: Therapy

Whether therapy or a watch-and-wait approach is indicated depends on the severity of the disease and its causes. In the case of non-severe or moderately severe aplastic anemia, a watch-and-wait approach is usually recommended initially (Onkopedia Guideline Aplastic Anemia 2022). When therapy is indicated, with the help of immunosuppressive therapies (IST), approximately 50% of patients with aplastic anemia achieve remission, with significantly higher response in patients with age ≤ 67 years (p=0.002), coexistence of a PNH clone (p=0.017), and normal karyotype (p=0.024) (Maciejewski & Selleri 2004, Kim et al. 2010). Patients with mutations of the genes PIGA, BCOR and BCORL1 also respond significantly better to IST (Yoshizato et al. 2015, Ogawa 2016). Due to the rarity of the disease, it should be discussed with an expert center before starting therapy whether participation in a clinical trial is possible. An overview of the therapy structure can be found in the Onkopedia Guideline Aplastic Anemia.

Aplastic anemia: Recommendation

The differentiation of aplastic anemia from hypoplastic MDS is important for prognostic assessment as well as for the choice of therapeutic strategies despite the partially overlapping symptoms (Fig. 2), but morphological analyses are challenging due to the poor cellularity of the bone marrow. Therefore, histologic examination on a sufficiently large bone marrow cylinder (at least 15 mm biopsy length) should necessarily be performed to establish the diagnosis. Indications of clonal evolution in the course of aplastic anemia are an increased blast percentage, hypercellular bone marrow in the presence of recurrent or persistent cytopenia, and the appearance of new cytogenetic or molecular genetic aberrations (Maciejewski & Selleri 2004, Afable et al. 2011).

Fig. 2: Overview of common symptoms in AA and hypoplastic MDS (modeled according to Mufti et al. 2018).

Other differential diagnoses include clonal cytopenia of undetermined significance (CCUS) and paroxysmal nocturnal hemoglobinuria (PNH).

Afable MG et al. SNP array-based karyotyping: differences and similarities between aplastic anemia and hypocellular myelodysplastic syndromes. Blood 2011;117(25):6876-6884.

Betensky M et al. Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia. Cancer Genet 2016;209(1-2):1-10.

Groarke EM et al. Predictors of clonal evolution and myeloid neoplasia following immunosuppressive therapy in severe aplastic anemia. Leukemia 2022;36(9):2328-2337.

Gupta V et al. Clinical relevance of cytogenetic abnormalities at diagnosis of acquired aplastic anaemia in adults. Br J Haematol 2006;134(1):95-99.

Gurnari C et al. Clinical and Molecular Determinants of Clonal Evolution in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria. J Clin Oncol 2023;41(1):132-142.

Killick SB et al. Guidelines for the diagnosis and management of adult aplastic anaemia. Br J Haematol 2016;172(2):187-207.

Kim SY et al. The characteristics and clinical outcome of adult patients with aplastic anemia and abnormal cytogenetics at diagnosis. Genes Chromosomes Cancer 2010;49(9):844-850.

Kulasekararaj AG et al. Somatic mutations identify a subgroup of aplastic anemia patients who progress to myelodysplastic syndrome. Blood 2014;124(17):2698-2704.

Maciejewski JP, Selleri C. Evolution of clonal cytogenetic abnormalities in aplastic anemia. Leuk Lymphoma 2004;45(3):433-440.

Marsh JC et al. Guidelines for the diagnosis and management of aplastic anaemia. Br J Haematol 2009;147(1):43-70.

Marsh JC et al. Guidelines for the diagnosis and management of acquired aplastic anaemia. Br J Haematol 2003;123(5):782-801.

Mufti GJ et al. Diagnostic algorithm for lower-risk myelodysplastic syndromes. Leukemia 2018;32(8):1679-1696.

Ogawa S. Clonal hematopoiesis in acquired aplastic anemia. Blood 2016;128(3):337-347.

Onkopedia Leitlinie “Aplastische Anämie” 2022; DGHO 2022.

Yoshizato T et al. Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia. N Engl J Med 2015;373(1):35-47.

Young NS. Aplastic Anemia. N Engl J Med 2018;379(17):1643-1656.

Status: July 2023

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Aplastic anemia (AA)

Aplastic anemia: Classification

Table 1: Classification of aplastic anemia (two of three criteria must be met) (Onkopedia Guideline Aplastic Anemia 2022).

Aplastic anemia: Diagnostics

Cytomorphology

Immunophenotyping

Chromosome analysis

Fluorescence in situ hybridisation (FISH)

Molecular genetics

Aplastic anemia: Prognosis

Aplastic anemia: Therapy

Aplastic anemia: Recommendation

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