Introduction
The cytomorphological enumeration of blast percentages in bone marrow (BM) smears is a critical parameter for classification of myelodysplastic syndromes (MDSs) [
1]. The blast percentage is also an important prognostic marker as reflected by the high intrinsic prognostic power of the WHO classification [
2]. Blast percentage cut-offs at 5%, 10%, 20%, and 30% are included as prognostic factors in the International Prognostic Scoring System (IPSS) [
3]. The further splitting of the lower blast range into two separate groups (0–2% vs > 2–< 5%) in the revised IPSS (R-IPSS) provided groups with very low risk versus low-risk features [
4]. Indeed, patients with ≤ 2% BM blasts had lower risk of disease progression into acute myeloid leukemia (AML) and a more favorable outcome [
4].
The International Working Group on Morphology of MDS proposed a consensus for the definition and enumeration of BM blasts, widely implemented in clinical practice [
5]. Although the reliability of blast cell count with respect to the 5% threshold has been demonstrated [
6‐
8], the reproducibility of counts within the lower blast range has been questioned. Font et al. found low agreement among cytologists when analyzing MDS patients with ≤ 2% BM blast counts [
6]. Therefore, even when good-quality BM smears are available, the reproducible identification and quantification of blast cells might be a challenge. Moreover, blast counts in smears may not be representative due to hemodilution or low cellularity of smears that are often seen in hypoplastic MDS or in MDS with significant (≥ grade 2 WHO) fibrosis [
9‐
12]. In addition, CD34+ cell clusters as identified by immunohistochemistry (IHC) that were reported as independent prognostic marker for progression to AML may be seen even in MDS without blast excess by cytomorphology (CM) [
13,
14]. In follow-up samples from MDS patients under treatment, BM topography may be severely altered with variation in cellularity and residual focal increase of blasts. Therefore, according to European Leukemia Network (ELN) guidelines and the revised WHO classification of MDS, the blast percentage by conventional CM should always be correlated to BM histology including IHC staining for CD34 [
15,
16].
Multiparameter flow cytometry (FCM) is an important complementary tool for the analysis of the blast compartment in MDS. Previous studies have shown a strong correlation between FCM and CM with regard to blast enumeration [
17‐
19]. Among other criteria, enumeration of CD34+ cells by FCM has been evaluated, but it proved to be considerably lower than morphological blast cell counts in a significant number of cases [
20‐
24]. However, it has been shown that a 2% cut-off for CD34+ cells by FCM reached higher prognostic significance than 2% blasts by CM [
25]. A recent multicenter quality assurance study stressed the need for a standardized approach for the enumeration of CD34+ myeloid blast cell count by FCM [
26]. A series of publications from the International MDS Flow Cytometry Working and the ELN group provided guidelines for FCM application in the diagnostic work-up of MDS [
23,
27‐
31]. However, as it has been stated in the revised WHO 2017, FCM results cannot replace the morphological differential count in the MDS subclassification [
1,
32].
A number of previous studies compared the percentages of blasts by CM to the assessment of the blast compartment by FCM. In this study, we assessed the percentage of CD34+ blasts by IHC in BM biopsies from MDS patients and compared the results to the blast counts obtained by conventional CM and, in a subset of patients, also to the enumeration of CD34+ myeloid blast cells by FCM. By using three different methods in a simultaneous setting, we demonstrate that BM histology with CD34 IHC identifies a significant number of patients with higher blast counts than found by CM and FCM. Importantly, diagnostically relevant differences in blast percentages were more often detected in trephine biopsies as compared to clots from aspirates, strongly supporting the use of the former at initial investigation for correct disease classification.
Discussion
Enumeration of blast cells in the BM of MDS patients is a key parameter for correct assignment to MDS subgroups, as well as for the differential diagnosis between MDS and AML. In addition, it is used as a single independent prognostic marker in currently available prognostic risk scores [
4,
35]. In the updated WHO guidelines [
1], despite inaccuracies inherent in manual differential counting, conventional CM is still considered the gold standard to determine blast counts.
Previous studies have shown a generally good correlation between CM and FCM for BM blast enumeration [
17,
19], but it has been shown that FCM may underestimate or overestimate blast cell counts in individual cases. The percentage of CD34+ myeloid cells by FCM has been tested as a substitute for a CM count; however, blasts in MDS may not express CD34 [
36,
37]. Other studies have shown that the CD34
high and/or CD117
+HLA-DR
+ phenotype of total events showed the highest degree of correlation and agreement with the morphological assessment of blast counts [
19]. However, BM samples taken in a simultaneous setting for cytomorphology and FCM analysis, respectively, may differ in terms of cellularity and blood contamination. FCM has not been accepted as a complementary tool for BM blast count in routine clinical practice but provides valuable information on blast immunophenotype and maturation patterns of various BM cell lineages. In our series, we observed good overall concordance between CM and FCM with respect to critical blast thresholds (88% of cases). In four of 53 MDS with excess blasts (≥ 5% by CM), the percentage of CD34+ myeloid blasts by FCM was ≤ 1% indicating that blast cells lacked this marker.
According to recommendations from the ELN and the revised WHO 2017 diagnostic guidelines, BM trephines should be performed in all patients with suspected MDS for whom BM examination is indicated [
15,
16]. Immunohistochemical analysis with CD34 is especially useful for assessing blast percentage in MDS with fibrosis or a hypocellular bone marrow, in which blast percentages are often underestimated. Moreover, it allows the enumeration of CD34+ blast cells and identification of CD34+ cell clusters, which were found to have an independent impact on both overall survival and leukemia-free survival [
13]. Previous studies in MDS and AML have shown that CD34 IHC increases diagnostic accuracy [
38]. Dunphy et al. found that IHC detected higher CD34+ blast counts as compared to the blast count obtained in smears, which resulted in a change of the initial classification [
38]. It has also been illustrated that during follow-up of treated AML patients with CD34+ myeloid blast cells at initial diagnosis, IHC detected higher blast counts (> 5%) as compared to CM (< 5%) in a number of cases [
39]. Other studies have proven high overall concordance for positive and negative results for CD34 by IHC in comparison to either CM or FCM [
40,
41]. Correlation with FCM may particularly be helpful in MDS cases with micromegakaryocytic hyperplasia and strong aberrant CD34 expression. We demonstrate here a high interobserver correlation for the blinded morphological assessment of CD34+ blasts cells in BM biopsies and good overall concordance between IHC and FCM with respect to critical blast thresholds. However, BM histology detected higher blast percentages by IHC as compared to CM in 17% of MDS cases, which would allocate patients to a higher subcategory and IPSS risk group. Discrepant cases were seen in both MDS-MLD and MDS-EB subtypes and associated with shorter overall survival and poor-risk cytogenetics. In seven of 21 AML patients, final diagnosis was established on the basis of histology and IHC, while cytomorphology of the smears did not fulfill the criteria for leukemia diagnosis. Importantly, discordant cases were more frequently detected in BM trephines as compared to clots from aspirates. Differences in blast percentages between CM and BM histology may partly be explained by uneven distribution of CD34+ blast cells and the presence of CD34+ clusters, which are more easily detected in a trephine biopsy and more frequently seen in high-risk MDS.
The automated assessment of CD34 in BM biopsies was found problematic due to technical issues and positive staining in different cellular and stromal compartments. In addition, the distribution and frequency of CD34-positive blast cells can vary within samples which may require an individual approach for the automated count including the selection of adequate annotation areas and the definition of negative and positive scores (e.g., size of blast cells).
In summary, our findings illustrate that CM does not establish correct blast counts in 100% of cases and should be used with the awareness that BM histology may reveal higher blast percentages, particularly in MDS with high-risk features. Accordingly, as the most reasonable and reliable approach to diagnosing and classifying MDS, we propose the combination of all three methods as gold standard for the assessment of marrow blasts. This should be applied in a simultaneous setting and as part of an integrative diagnostic approach for correct assignment of patients to specific risk categories and MDS subgroups. In cases with increased blast percentages in BM histology, final MDS classification should be based on the higher blast count even if the percentage of blasts by CM is below the 5% threshold. The CM blast count cannot be replaced by FCM, but if FCM finds significant higher counts in the BM aspirate, control sampling may be considered. To our knowledge, there are no larger published studies who have analyzed the percentage of blast cells in BM samples from MDS patients by all three methods (CM, FCM, and BM histology together with IHC) in a simultaneous setting. Additional studies to define standards for the enumeration of CD34 cells in BM samples are currently ongoing by members of the European Bone Marrow Working Group [
42]. The findings of our study will be followed up in a larger, population-based cohort of MDS patients at initial diagnosis and under treatment with correlation to clinical data and survival.
Acknowledgments
The authors thank the staff at the Department of Clinical Pathology, Hematopathology Laboratory, Karolinska University Hospital, Solna, for preparation of bone marrow samples for morphological and flow cytometric analysis.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.