Diagnostics of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML)

Introduction

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are malignant diseases of the hematopoietic stem cell, characterized by differentiation and maturation disorders of hematopoiesis, cytopenia and clonal expansion of varying degrees. Various pathophysiological phenomena are observed in this context, such as chromosomal, molecular and epigenetic changes in the hematopoietic stem cell, as well as alterations to the bone marrow stroma.

Both groups of diseases are closely related pathophysiologically, and transitions from MDS to AML are frequent. The differentiation of MDS from AML is based, on the one hand, on cytogenetic or molecular-genetic markers that define AML and, on the other hand, on the assessment of the medullary and peripheral proportion of blasts. According to World Health Organization (WHO) recommendations, detecting ≥20% of blasts in the blood and/or bone marrow is a basic prerequisite for diagnosing AML. This number is arbitrary and cannot be taken dogmatically due to the imprecision of how the percentage of blasts is estimated. It is, instead, more of a pragmatic instruction. The detection of certain cytogenetic and molecular-cytogenetic markers defines some AML entities also independently of the blasts.

The following will explain in detail the diagnostic steps taken in connection with MDS and AML, and the WHO recommendations on the classification of MDS and AML will be explained and described in a tabular format.

MDS diagnostics

The diagnosis of an MDS is typically taken into account when trying to clarify a diagnosis of anemia and bi- or pancytopenia. Most patients have an isolated anemia, and only about 20% have pancytopenia. Initially, it is necessary to rule out, through suitable methods, a varied range of hematological and non-hematological differential diagnoses (Table 1). The most common MDS differential diagnosis is an iron level disorder in terms of “tumor – inflammatory anemia”. If the cause of the cytopenia cannot be identified, it is essential to do a thorough cytomorphological test of the blood and bone marrow to diagnose MDS. In around half of the cases of patients with MDS, the differential blood count shows signs of dysplasia, particularly of the granulocytes, and also, but less frequent, platelet anisometry [1]. In the bone marrow cytology, a number of dysplasia signs are mostly observed that, by definition, make up more than 10% of the nucleated cells of one, two or all three cell lines. None of the dysplasia signs is pathognomonic of MDS, because myeloproliferative neoplasms and AML, as well as other hematological and non-hematological conditions can show signs of dysplasia as well.

In addition to standard staining, iron staining and, ideally, a peroxidase reaction (POX) must be prepared in all cases. Ringed sideroblasts will become visible only with iron staining. They represent in many cases a clear criterion of dysplasia, and are required to diagnose ringed sideroblastic anemia. A classic indicator of dysplasia of granulopoiesis is a partial POX defect. PAS-positive erythroblasts are less common. The detection of a monocyte population is often difficult, which is why an esterase reaction is recommended, particularly in cases of suspected chronic myelomonocytic leukemia (CMML). The individual dysplasia criteria are listed in Table 2.

The main criteria for differentiating between various MDS types are the expressions of dysplasia signs (unilineage vs. multilineage), the medullary and peripheral proportions of blasts (<>5% and <>10%), as well as the detection of chromosomal changes, particularly the deletion of part of the long arm in chromosome 5 [2].

One distinguishes RCUD and RARS – entities for which the dysplasia signs are limited to one lineage, mostly erythropoiesis – from RCMD where two or mostly all three cell lines exhibit dysplastic characteristics. These patients have a median probability of survival of approximately three years, a significantly worse prognosis and a higher risk of leukemia than RCUD and RARS patients (median probability of survival of approx. 6 years). All of the aforementioned MDS types have medullary blasts of <5%. In the case of isolated del(5q) without blast proliferation, a diagnosis of MDS with del(5q) will be made. This entity comes with a good prognosis, but is also characterized by transitions to acute leukemia [3]. It is interesting to note that typical morphological characteristics are usually observed that lead attentive cytologists to think of a deletion of the long arm in chromosome 5. These characteristics are a) mononuclear megakaryocytes, b) a reduced, but usually only slightly dysplastic erythropoiesis, c) mild dysplasia signs of granulopoiesis, and d) increased lymphocytes in the bone marrow smear.

All cases of MDS require an analysis of the medullary blast proportion that must be as exact as possible, because a patient’s prognosis depends greatly on the proportion of blasts according to the revised version of the International Prognostic Scoring System [4]. The classification of blast categories (0–2%, 3–4%, 5–9%, 10–19%) demands precise morphology.

A diagnosis of RAEB I or II requires a relevant, malignant cell population with a blast proportion of 5–9% and/or 10–19% in the bone marrow. These patients have a high risk of developing a subsequent AML, and their median probability of survival is approximately 20 months in RAEB I and about one year in RAEB II. The current WHO classification of MDS [3] is shown in Table 3.

CMML, previously grouped with MDS, now makes up an entity within the group of mixed myelodysplastic-myeloproliferative neoplasms together with ringed sideroblastic anemia with thrombocytosis (RARS-T) [5]. CMML I and II as well as RARS-T also exhibit characteristics of myeloproliferative neoplasms that manifest themselves in an increase of monocytes in the blood (>1000/μL absolute) for CMML (I and II) and thrombocytes of >450,000/μL for RARS-T, as well as in the common detection of JAK2 and SF3B1mutations. There is also a further phenotype-genotype correlation for RARS-T, because it is mostly SF3B1 mutations that are found [6]. The classification of CMML into the types CMML I and II is analogous to the classification of RAEB I and II, that is, in accordance with the peripheral and medullary content of blasts, while promonocytes with CMML are classified as blasts.

The group of refractory anemia with excess blasts in transformation (RAEB-T), which was initiated with the first MDS classification, is now allocated to AML with multilineage dysplasia, because the new limit of medullary blasts to differentiate between MDS and AML is now at 20%.

It may also be useful diagnostically to do a histomorphological assessment of the bone marrow, which allows for a far better estimation of cellularity [7], which in turn may be especially relevant in differentiating from an aplastic syndrome, but may also detect bone marrow fibrosis [8], which can have a negative impact on the course of the disease. Ringed sideroblasts can be represented only in the smear, but not in a section slide. This is why the pathologist should be given some bone marrow smears as well.

A chromosome analysis of bone marrow cells is absolute essential, both for classification purposes and for a prognosis estimation [9]. At the time of diagnosis, around 60% of all MDS patients exhibit cytogenetic aberrations. Clonality testing may be diagnostically indicative in cases of mild dysplasia. The paramount significance of chromosomal findings in connection with MDS was most recently reflected in the revised version of the International Prognostic Scoring System (IPSS-R) [4], which is greatly dominated by cytogenetic findings. The key preparatory work with respect to cytogenetics was done under the leadership of the German-Austrian MDS Group [9].

Table 4 shows the frequency of different chromosomal findings and their allocation to risk groups. Ideally, 20–25 metaphases would be analyzed using banding cytogenetics so as to detect also smaller clones [10]. In a few cases of dry tap aspiration, the cytogenetic analysis may also be performed on cells of peripheral blood, although the analytical quality will be somewhat reduced. Fluorescence in situ hybridization (FISH) in the blood, however, is particularly successful with CD34+ selected cells. This method can also be used for monitoring treatment [11]. Immunophenotyping of bone marrow cells taken from MDS patients can generally trace and identify dysplasia as well as provide an estimation of the medullary blast percentage. But this requires an extended marker panel (ELN) in the hands of experienced personnel. This is why this method is not part of the standard repertoire of MDS diagnostics.

Testing for genetic mutations has already become part of clinical routine with respect to the pathogenesis and treatment decisions for AML. Recent years have also seen more and more molecular aberrations in MDS patients as well. Molecular analysis can already be used for diagnostic purposes where there are no cytogenetic aberrations or sufficient cytological criteria.

Several studies [12–14] have shown that molecular aberrations can be relevant to the prognosis, particularly mutations of the genes ASXL1, TP53, RUNX1 and EZH2. Molecular markers will soon be integrated with prognostic scoring systems, and likely become therapy-relevant and predictive in nature. More recent analytical methods, such as proteomics, are of great scientific interest, but not yet part of routine diagnostics [15–17].

Table 5 summarizes standard MDS diagnostics [18, 19].

The MDS Registry, based in Düsseldorf, captures diagnostic, clinical, prognostic and therapeutic data collected by many participating centers in order to promote joint scientific projects. A central biobank has also been established, which is part of the joint MDS-Cancer Aid project. It also offers bone marrow reference cytology. An interactive, web-based learning program with lectures and demonstrations of diagnostic steps and virtual microscopy is available at www.mdsdiagnosis.com free of charge.

Diagnosis of acute myeloid leukemia

Both the diagnosis and classification of AML are subject to the current 2008 WHO classification, like MDS (Table 6) [20].