Baseline characteristics for patients with AA and different controls
15 AA patients, 11 h-MDS patients and 20 age and sex matched healthy controls were enrolled in this study. The detailed baseline characteristics were shown in Table 1.
All the AA patients were non-severe and transfusion dependent. They were six females and nine males with a median age of 44 (18–61) years. Before treatment, the average hemoglobin (HGB) level was 94±25 g/L, the average absolute neutrophil count (ANC) was 1.31±0.58×109/L, and the platelet count was 48±61×109/L. The average reticulocyte count was 62±20×109/L. Seven patients had elevated ferritin levels, with the average value 659±511 ug/L. Only one patient had +8 abnormal chromosomes and none of them had PNH clones. AA patients had normal liver function but two of them had mild kidney dysfunction (Chronic Kidney Disease Stage I–II) at the time of the diagnosis. The median duration of the CsA treatment was 9 months (6~15 months) and the CsA concentration was maintained at 100~200 ng/mL during the treatment period. The median follow-up time was 12 months (9~15 months). After the 6-month CsA treatment, 13.3% (2/15) achieved a complete response (CR), 44.4% (8/15) achieved a partial response (PR), and the overall response rate (ORR) was 66.7%. There was no difference in the baseline characteristics between patients with or without response. No PNH clone expansion or death occurred during the study period.
For h-MDS patients, they were five females and six males with a median age of 55 (44~73) years. The age was significantly higher in patients with h-MDS than AA (P=0.009). Five were MDS with single-lineage dysplasia, three were MDS with multilineage dysplasia, and three were MDS-Excess Blasts 1 (MDS-RAEB1), according to 2016 WHO criteria. The International Prognostic Scoring System (IPSS) was 0 in three patients, 0.5–1 in six patients and 1.5–2 in two MDS patients. Six MDS patients had cytopenia of more than one lineage. The absolute platelet count was higher than that in AA patients (P=0.037). Among the 11 MDS patients, one had three chromosome abnormalities, four had two abnormalities and the others had no chromosome abnormalities. None of them had abnormal chromosome 7. There was no significant difference in the ANCs, HGBs, reticulocytes, ferritin level and other biochemical indices between the AA and h-MDS patients at baseline. Nine IPSS low- and intermediate-1 (Low/Int-1) risk MDS patients were treated with CsA and androgen, while 2 intermediate-2 (Int-2) risk patients were treated with thalidomide and androgen. The median follow-up time was 12 months (9~15 months). Two MDS-RAEB1 patients experienced transformation into acute myelocytic leukemia (AML) at the end of our follow-up. No deaths occurred during the follow-up.
Table 1: The baseline characteristics of AA patients, MDS patients and the control
Basic characteristics
|
AA
|
MDS
|
Healthy control
|
P-value (AA vs. MDS)
|
Sex, N (%)
|
|
|
|
0.485
|
Female
|
6(40%)
|
5(45%)
|
8(40%)
|
|
Male
|
9(60%)
|
6(55%)
|
12(60%)
|
|
Age, years, median (range)
|
44(18-61)
|
55(44-73)
|
47(18-69)
|
0.009
|
Baseline blood cell count
|
|
|
|
|
ANC, ×109/L, mean±SD
|
1.31±0.58
|
1.36±0.91
|
3.12±1.22
|
0.875
|
HGB, g/L, mean±SD
|
94±25
|
83±26
|
135±20
|
0.302
|
PLT, ×109/L, mean±SD
|
48±61
|
104±64
|
185±82
|
0.037
|
Ret#, ×109/L, mean±SD
|
62±20
|
59±22
|
-
|
0.813
|
ALT, U/L, mean±SD
|
19±17
|
22±17
|
15±20
|
0.623
|
Cr, umol/L, mean±SD
|
70±26
|
84±22
|
62±20
|
0.160
|
Fer, ug/L, mean±SD
|
659±511
|
583±492
|
-
|
0.715
|
PNH, N(%)
|
0(0%)
|
0(0%)
|
-
|
-
|
Abnormal chromosome, N(%)
|
1(7%)
|
5(45%)
|
-
|
0.04
|
Abbreviations: ANC: absolute neutrophil count, HGB: hemoglobin, PLT: platelet, Ret: reticulocyte, ALT: alanine transaminase, Cr: creatinine, Fer: ferritin, PNH: paroxysmal nocturnal hemoglobinuria.
Baseline lipid metabolism profile for AA patients
The PCA score plot from the discovery set showed different trends between the AA patients and the healthy controls. The PLS-DA model further confirmed the differences between the two groups. The cross-validation plot indicated that this model was non-overfitting (Fig. 1). The differential metabolites, defined by a VIP > 1, P-value< 0.05, and ≥ 2 or ≤ 0.5, are listed in Table S1. Compared with the healthy controls, 106 lipid molecules were upregulated and 208 were downregulated in ESI+ mode, while 71 molecules were upregulated and 118 were downregulated in ESI- mode for patients with AA. The KEGG pathway analysis indicated that these lipid metabolites were involved mainly in the metabolism of arachidonic acid, retinol, glycerophospholipids, aminoacyl-tRNA biosynthesis and the biosynthesis of unsaturated fatty acids. The volcano plot and heatmap was then plotted to display the significant difference (Fig. 1). Phosphatidylcholine (0.17 FC), arachidonic acid (0.4 FC), prostaglandin G2 (0.6 FC), prostaglandin E2 (0.66 FC), prostaglandin J2 (0.31 FC), leukotriene B4 (0.31 FC) and 15(S)-HETE (0.39 FC) in the arachidonic acid pathway were significantly downregulated in AA patients compared with normal controls (P<0.05). All-trans-retinal (0.48 FC), dehydroretinaldehyde (0.29 FC) and tretinoin (0.47 FC) in retinol metabolism were also lower in AA patients (P<0.05).
To analyze the differences between AA and h-MDS patients, PCA, PLS-DA and cross validation was first explored to confirm the reliability (Fig. 2). Compared to h-MDS patients, AA patients had 42 lipid molecules upregulated and 165 downregulated in the ESI+ mode, while 40 were upregulated and 76 downregulated in the ESI- mode. The differences were then visualized using volcano plots and heatmaps (Fig. 2). The differentially expressed molecules are listed in Table S2. According to the pathway analysis, the changes focused on the metabolism of arachidonic acid, retinol, glycerophospholipids, and sphingolipid metabolism. Compared with patients with h-MDS, patients with AA showed decreased arachidonic acid pathway metabolites, including phosphatidylcholine (0.16 FC), arachidonic acid (0.18 FC), prostaglandin G2 (0.6 FC), prostaglandin E2 (0.59 FC), prostaglandin J2 (0.16 FC), leukotriene B4 (0.23 FC), 15(S)-HETE (0.13 FC) and the retinol metabolism-related metabolites (P<0.05). These differences were unique for AA and h-MDS because there were no differences in the above pathways between the patients with h-MDS and the healthy controls. On the other hand, h-MDS patients showed increased metabolism of proline (1.91 FC) and threonine (1.76 FC) and abnormal sphingolipid metabolism including increased ceramide (d18:1/16:0) (1.34 FC), sphingosine-1-phosphocholine (1.52 FC) and decreased SM (d18:0/20:2(11Z,14Z)) (0.47 FC) compared with the normal controls, which was normal in AA patients as well.
Change of lipid profiles after 6-month CsA treatment in patients with AA
The lipid profiles of patients with AA were measured again after the 6-month of standard CsA treatment and compared with the baseline values. The PCA and reliability tests were performed, and the results are shown in Fig 3. After the 6-month CsA treatment, 105 lipid molecules increased and 9 decreased in the ESI+ mode, while 41 increased and 14 decreased in the ESI- mode (Table S3). The KEGG pathway analysis revealed that the main changed metabolites were involved in the metabolism of arachidonic acid and retinol. In details, leukotriene B4 (0.49 FC) and 15(S)-HETE (0.49 FC), which were involved in arachidonic acid metabolism, and all-trans-retinal (0.4 FC) and protectin D1 (0.42 FC), which were involved in retinol metabolism, decreased after the 6-month of CsA treatment.
Molecules at baseline were also compared between the ten patients who achieved at least a partial response (PR+CR) and five patients who did not achieve a response (NR) after CsA treatment. Patients who achieved PR+CR had significant higher levels of baseline protectin D1 (p=0.011), leukotriene B4 (p=0.011), 15(S)-HETE (p=0.004) and all-trans-retinal (p=0.000) than those who had no response (Fig. 4). Further analysis revealed that the levels of protectin D1 at baseline was significantly higher in CR patients than in PR patients (p<0.05), while other molecules such as leukotriene B4, 15(S)-HETE, and all-trans-retinal tended to be higher in patients with CR than those with PR, but not significant. The baseline lipid molecules were also correlated with the rapidity of the CsA response: patients who achieved CR+PR within a 3-month period tended to have higher baseline expression of the above four metabolites than those who achieved CR+PR after 3~6 months of CsA treatment, although the differences were not significant.