A systematic classification of megakaryocytic dysplasia and its impact on prognosis for patients with myelodysplastic syndromes

The study was approved by the ethics committees of the institute of hematology, Chinese Academy Of Medical Sciences (CAMS) and Peking Union Medical College (PUMC) according to guidelines of the declaration of Helsinki. In this retrospective analysis, the study cohort included 422 consecutive new-diagnosed subjects that were seen at the Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences from January, 2000 to April, 2014. 8 subsequently received a haematopoietic cell transplant, 14, decitabine, 45, other chemotherapy and the remainder cyclosporine or thalidomide and best supportive care. Cases were re-reviewed by two blinded pathologists (W Cui and W Cai) and classified using the 2008 WHO criteria [2]. Subjects with suspected therapy-related MDS were excluded as the clinical course was typically progressive and treatment with conventional therapy was usually associated with a poor prognosis [19]. Furthermore, there was no Down Syndrome patient in the cohort. Follow-up data were available for 370 subjects (88 %). Date of last follow-up was December 15, 2014 or date of last contact. Median follow-up was 22 months (range 1–180 months). Subjects with lower-risk MDS fall into the international prognostic scoring system-revised (IPSS-R) categories of very low-, low-, and intermediate-risk groups and those with higher-risk MDS into the high- and very high-risk groups [20].

Bone marrow smears from diagnosis were reviewed using an avidin–biotin-complex method (ABC; CD41 immune staining) by the experts who were blinded for patients’ diagnoses, cytopenias and cytogenetic status in cytology. The preparation of bone marrow smear was a relatively uniform procedure. The marrow area on every smear was approximate to 1.5 × 3.0 cm with proper and relatively uniform thickness. ≥30 megakaryocytes were evaluated and the frequency of morphologic abnormalities was recorded. The presence of nuclear hypolobation, single or multiple separate small round nuclei were considered as main characteristics of dys-megakaryocytopoiesis.

Statistical analyses were performed using SPSS 19.0 software or SAS software. The best discriminator threshold was detected using the minimal P value approach (a method aimed at minimizing the identification of rare classes of subjects) and considering survival (log-rank statistic) as the dependent variable [21, 22]. The functional form of the covariate under study was also evaluated using Martingale residual analysis [23].

Numerical variables were summarized by median and range. Categorical variables were described with count and relative frequency (%) of subjects in each category. Comparison of numerical variables between groups was carried out using a non-parametric approach (Mann–Whitney test). Comparison of the distribution of categorical variables in different groups was performed with χ2 test (unordered categorical variable) or the non-parametric approach (ordinal categorical variable).

Median survival was estimated using the Kaplan–Meier method and compared using the log-rank test. Cox proportional hazard regression model was used for multivariate analyses. P values were two-tailed and statistical significance was set as the level of P < 0.05.

Median age at diagnosis was 50 years (range 16–83 years). 286 subjects (68 %) were male. Distribution of WHO subtypes, IPSS-R cytogenetic category and IPSS-R classification are indicated in Table 1.

Megakaryocyte dysplasia was detected in 374 subjects (89 %). Median frequency of dysplastic megakaryocytes was 14 % (range 0–100 %). Patients without megakaryocytic dysplasia included RA (17 %), RARS (15 %), RCMD (42 %), RAEB-1 (17 %), RAEB-2 (6 %), MDS-U (4 %). Dysplastic megakaryocytes were assigned to 7 categories: (1) micro-megakaryocytes (<12 µm); (2) micro-megakaryocytes (12–40 µm) with 1 nucleus; (3) micro-megakaryocytes (12–40 µm) with 2 nuclei; (4) micro-megakaryocytes (12–40 um) with multiple (more than 2) nuclei; (5) dysplastic megakaryocytes (≥40 µm) with 1 nucleus; (6) dysplastic megakaryocytes (≥40 µm) with 2 nuclei; and (7) dysplastic megakaryocytes (≥40 µm) with multiple (more than 2) nuclei (Fig. 1). The most frequent dysplastic megakaryocytes were micro-megakaryocytes (12–40 µm) with 1 nucleus and dysplastic megakaryocytes (≥40 µm) with 1 nucleus, with median frequency of 28 % (0–91 %) and 29 % (0–78 %), respectively. Distribution of each type is shown in Additional file 1: Figure S1.

To analyze the prognostic impact of dys-megakaryopoiesis, we identified the cutoff point for the two prognostic classes with the greatest differences according to the smallest P value for micro-megakaryocytes at 25 % as well as mono-nucleated dys-megakaryopoiesis at 30 %. Subjects without megakaryocytic dysplasia were all grouped to micro-megakaryocytes <25 % and mono-nucleated dys-megakaryopoiesis <30 %.

Clinical and laboratory variables in subjects with micro-megakaryocytes <25 and ≥25 % are compared in Additional file 1: Table S1. A similar comparison between subjects with mono-nucleated dys-megakaryopoiesis <30 and ≥30 % is outlined in Additional file 1: Table S2.

Increased micro-megakaryocytes and mono-nucleated dys-megakaryopoiesis were significantly associated with lower levels of platelet count (P < 0.001 and P < 0.001) and higher levels of bone marrow blasts (P < 0.001 and P < 0.001). Distributions of WHO 2008 subtypes (P = 0.001 and P < 0.001), IPSS-R cytogenetic category (P = 0.002 and P = 0.001) and IPSS-R risk cohorts (P < 0.001 and P < 0.001) were also significantly different. There was no significant difference in age, gender, hemoglobin concentration and blood neutrophil counts at diagnosis between the two groups. In addition, levels of micro-megakaryocytes and dysplastic mono-nucleated megakaryocytes were significantly associated with abnormal karyotype (P = 0.026 and P = 0.014), complex karyotype (CK) (P = 0.034 and P = 0.022) and chromosome 7 aberrations (P = 0.004 and P = 0.003), but not with monosomal karyotype (MK) or del(5q) (Additional file 1: Table S3).

In univariate analyses, subjects with micro-megakaryocytes ≥25 % had poorer survival (median, 19 months [95 % CI 14–23 months]) than those with micro-megakaryocytes <25 % (46 months [28–64 months], P < 0.001; Fig. 2a). Similarly, patients with dysplastic mono-nucleated megakaryocytes ≥30 % demonstrated poorer survival as compared to those with dysplastic mono-nucleated megakaryocytes <30 % (18 months [14–23 months] vs. 49 months [31–68 months], P < 0.001; Fig. 2b). Other significant predictors of survival in univariate analyses were male gender (P = 0.009), age ≥60 years (P < 0.001), hemoglobin concentration <80 g/L (P = 0.001), neutrophils <0.8 × 10E + 9/L (P = 0.001), platelets <50 × 10E + 9/L (P = 0.002), bone marrow blasts >10 % (P < 0.001), IPSS-R cytogenetic category (P < 0.001) and IPSS-R score (P < 0.001).

We performed a multivariate Cox regression model including gender, age, micro-megakaryocytes and IPSS-R. Male gender (hazard ratio [HR] = 1.5; 95 % CI 1.0–2.0; P = 0.029), age ≥60 years (HR = 1.5; [1.1–2.0]; P = 0.016), micro-megakaryocytes ≥25 % (HR = 1.6 [1.1–2.2]; P = 0.010) and IPSS-R score (P < 0.001) were significantly associated with survival (Table 2). In a similar analysis including gender, age, dysplastic mono-nucleated megakaryocytes and IPSS-R, only male gender (HR = 1.5; 95 % CI 1.0–2.0; P = 0.028), age ≥60 years (HR = 1.5; 95 % CI 1.1–2.0; P = 0.018), dysplastic mono-nucleated megakaryocytes ≥30 % (HR = 1.533; [1.1–2.2]; P = 0.014) and IPSS-R (P < 0.001) remained in the final model (Table 3).

In the IPSS-R lower-risk subjects, those with micro-megakaryocytes ≥25 % had a poorer survival than patients with micro-megakaryocytes <25 % (P < 0.001; Fig. 3a). Similarly, there was a significant difference in survival between patients with mono-nucleated dys-megakaryopoiesis <30 and ≥30 % (P < 0.001; Fig. 3b). This association was not significant in subjects in the IPSS-R higher-risk cohort. Consequently, we performed stratified multivariate analyses to further evaluate prognostic implications of megakaryocytic dysplasia in IPSS-R lower-risk subjects only. In the model of cell size including age, gender, IPSS-R and micro-megakaryocytes, only age, IPSS-R and micro-megakaryocytes were significantly correlated with survival (Table 4). In a similar analysis considering dysplastic mono-nucleated megakaryocytes, only age, IPSS-R and dysplastic mono-nucleated megakaryocytes were significantly correlated with survival (Table 5).