Characterization of patients with normal karyotype or no result after banding cytogenetics
As shown in Fig. 1, normal karyotype and cases without result after banding cytogenetics are approximately 55.8% of all patients. MLPA identifies copy number changes in 24 (16.7%,24/144 ) of 144 MDS patients. Among these 24 patients, 10 patients showed chromosome banding analysis failed. For patients with normal karyotype, 10.6% (14/132) were identified with CNVs. Characteristics of 144 patients were showed in Table 1.There were 86 males and 58 females in our study. According to the classification of WHO 2016 version, the most common subtype is MDS-MLD. We calculated the Revised International Prognostic Scoring System scores, confirming 8 patients were very high risk, 28 patients with high risk, 48 patients with intermediate risk, 50 patients with low risk༌and 10 patients with very low risk disease.
Table 1
Characteristics MDS Patients in our study (n = 144)
|
Total n (%)
|
Normal karyotype and MLPA+
|
Normal karyotype and MLPA-
|
Failed karyotype
|
N. of patients
|
144 (100%)
|
14
|
118
|
12
|
Median age (years) (range)
|
53 (15–83)
|
47.5(34–81)
|
57(15–83)
|
47.5(32–67)
|
Sex
|
Male
|
86
|
13
|
66
|
7
|
Median Hgb (g/L)
|
76
|
72
|
81
|
79
|
Median ANC (× 109/L)
|
1.2
|
1.0
|
1.1
|
0.9
|
Median platelet count(× 109/L)
|
72
|
89
|
76
|
69
|
WHO 2016
|
MDS-SLD
|
1(0.7%)
|
0
|
1
|
0
|
MDS-RS
|
6 (4.2%)
|
1
|
4
|
1
|
MDS-MLD
|
68 (47.2%)
|
6
|
56
|
6
|
MDS-EB-1
|
33 (22.9%)
|
4
|
27
|
2
|
MDS-EB-2
|
29 (20.1%)
|
3
|
25
|
1
|
MDS-U
|
7 (4.9%)
|
0
|
5
|
2
|
IPSS-R risk category
|
Very low
|
10 (7.0%)
|
2
|
8
|
0
|
Low
|
50(34.7%)
|
6
|
42
|
2
|
Intermediate
|
48 (33.3%)
|
1
|
46
|
1
|
High
|
28(19.4%)
|
4
|
16
|
8
|
Very high
|
8 (5.6%)
|
1
|
6
|
1
|
Abnormalities detected by MLPA in 14 MDS patients with normal karyotype
Among 24 patients, 14 patients showed normal karyotype, which were showed in Table 2. According to cytomorphology, the cohort comprised the following MDS subtypes: MDS-RS (n = 1), MDS-MLD (n = 6), MDS-EB-1 (n = 4), and MDS-EB-2 (n = 3). The most common CNV was − 17p (P53-4b, TP53-1).
Table 2
Copy number changes identified by MLPA in 14 MDS patients with normal karyotype
Case
|
Age
|
Gender
|
Diagnosis
|
Karyotype based on MLPA
|
Assumed karyotype according to MLPA
|
Patient 1
|
69
|
Male
|
MDS-EB-1
|
11q(4): KMT2A-4,KMT2A-36,TIRAP-3,ETS1-10
|
+ 11q
|
Patient 2
|
72
|
Male
|
MDS-RS
|
11q(4): KMT2A-4 ,KMT2A-36,TIRAP-3,ETS1-10
|
+ 11q
|
Patient 3
|
50
|
Male
|
MDS-MLD
|
20q(1): ASXL1-4
|
-20q
|
Patient 4
|
63
|
Male
|
MDS-EB-2
|
8q(3): NCOA2-5,MYC-3,PTK2-33 17p(2): TP53-4b,TP53-1
|
+ 8q -17p
|
Patient 9
|
39
|
Male
|
MDS-MLD
|
17p(2): TP53-4b,TP53-1
|
-17p
|
Patient 10
|
36
|
Male
|
MDS-MLD
|
17p(3): TP53-10,TP53-4b,TP53-1
|
-17p
|
Patient 11
|
46
|
Male
|
MDS-EB-2
|
17p(2): TP53-4b,TP53-1
|
-17p
|
Patient 12
|
51
|
Male
|
MDS-MLD
|
11q(1): KMT2A-4(+) 17q(1): NF1-17 SUZ12-12(+)
|
-17p
|
Patient 13
|
42
|
Male
|
MDS-EB-1
|
17q(1): NF1-17
|
-17q
|
Patient 14
|
34
|
Female
|
MDS-EB-2
|
19p(1):SMARCA4-25 19q(1): PRPF31-14
|
-19
|
Patient 15
|
32
|
Male
|
MDS-MLD
|
20q(2):MMP9-9,ZMYND8-14
|
-20q
|
Patient 16
|
57
|
Male
|
MDS-EB-1
|
20q(3): ASXL1-4,SRC-6,ZMYND8-14
|
-20q
|
Patient 19
|
47
|
Female
|
MDS-MLD
|
5q(3):APC-18,EGR1-1,EGR1-2
|
-5q
|
Patient 24
|
61
|
Female
|
MDS-EB-1
|
7q(4): CDK6-8 SAMD9L-5,MLL5-4,MET-13
|
-7q
|
Chromosome 8 abnormality was positive in 1 case (7.1%,1/14),only showed with 8q amplification. Chromosome 5 abnormality was positive in 1case (7.1%,1/14). Chromosome 7 abnormality was positive in 1case (7.1%༌1/14). Chromosome 20 abnormalities were positive in 3 cases (21.3%༌3/14). Chromosome17 abnormalities includingboth17p and 17q deletions were positive in 6cases (42.9%༌6/14), 5 patients for 17p deletion and 1 patient for 17q deletion. Chromosome11 abnormalities were positive in 2 cases (14.2%༌2/14) and both showed 11q amplifications. One patient was showed chromosome19 abnormalities including both 19p and 19q deletions. All detected aberrations are summarized in Table 2.
Abnormalities detected by MLPA in 10 MDS patients without result after banding cytogenetics
Among 24 patients, 10 patients without result after banding cytogenetics were showed in Table 3. The cohort involved the following MDS subtypes: MDS-RS (n = 1), MDS –MLD (n = 6), MDS-EB-1 (n = 2), MDS-EB-2 (n = 1) and MDS-U(n = 2).The most common CNVs were − 7 and + 8.
Table 3
Copy number changes identified by MLPA in 10 MDS patients with failed chromosome banding analysis.
Case
|
Age
|
Gender
|
Diagnosis
|
Karyotype based on MLPA
|
Assumed karyotype according to MLPA
|
Patient 5
|
81
|
Male
|
MDS-EB-1
|
8p(1): FGFR1-2 8q(4): NCOA2-5,RUNX1T1-8, MYC-3, PTK2-33
|
+ 8
|
Patient 6
|
49
|
Male
|
MDS-MLD
|
8p(1): FGFR1-2 8q(4): NCOA2-5,RUNX1T1-8,MYC-3, PTK2-33
|
+ 8
|
Patient 7
|
50
|
Male
|
MDS-EB-1
|
8p(1): FGFR1-2 8q(4): NCOA2-5, RUNX1T1-8,MYC-3,PTK2-33
|
+ 8
|
Patient 8
|
42
|
Male
|
MDS-MLD
|
8p(1): FGFR1-2 8q(4): NCOA2-5, RUNX1T1-8,MYC-3,PTK2-33 11q(1): KMT2A-4
|
+ 8 +11q
|
Patient 17
|
39
|
Female
|
MDS-U
|
5q(3): EGR1-1,EGR1-2,RPS14-3 17p(3):TP53-10,TP53-4b,TP53-1 19p(1):SMARCA4-25 19q(1): PRPF31-14
|
-5q -17p -19
|
Patient 18
|
48
|
Male
|
MDS-MLD
|
5q(4):EGR1-1,MIR145-1,SPARC-7,SPARC-1
|
-5q
|
Patient 20
|
37
|
Female
|
MDS-U
|
7p(1): IKZF1-30
7q(7):CDK6-8,SAMD9L-5,EPO-4,MLL5-4 ,MET-13,EZH2-20,EZH2-13
|
-7
|
Patient 21
|
41
|
Male
|
MDS-MLD
|
7p(1): IKZF1-30
7q(7): CDK6-8,SAMD9L-5,EPO-4,MLL5-4 ,MET-13,EZH2-20,EZH2-13
|
-7
|
Patient 22
|
41
|
Male
|
MDS-EB-2
|
7q(3): CDK6-8,MLL5-4,MET-13
|
-7q
|
Patient 23
|
67
|
Male
|
MDS-RS
|
7q(5): CDK6-8,MLL5-4,MET-13,EZH2-20,EZH2-13
|
-7q
|
Chromosome 8 abnormalities including both 8p and 8q amplifications were positive in 4 cases (40%, 1/10). Chromosome 5 abnormalities were positive in 2cases (20%, 2/10). Chromosome 7 abnormalities including both 7q deletion and 7p deletion were positive in 4 cases (40%, 4/10), two patients included both 7p and 7q deletions. Chromosome 20 abnormalities were not detected. Chromosome17 abnormality was positive in 1 cases(10%, 1/10). Chromosome11 abnormality was positive in 1 cases (10%, 1/10).One patient showed chromosome 19 abnormalities including both 19p and 19q deletions. All detected aberrations are summarized in Table 3.
Comparison Of Mlpa Assay And Fish
To evaluate the performance of MLPA as a candidate diagnostic method for the identification of CNVs in MDS patients, five abnormalities, including − 5/-5q, -7/-7q, + 8, -20q and 17p- were detected by both FISH and MLPA. FISH results of 144 cases were studied retrospectively and compared with MLPA results directly. Among 144 MDS patients, 137 results were concordant, and the whole consistency was 95.1%. The genetic lesions determined by FISH and MLPA were listed in Table 4. Using MLPA analysis, clonal cytogenetic abnormalities were detected in 24 MDS patients with normal and undetectable karyotype, and19/24 (79.2%) of those patients were reclassified into a higher-risk IPSS-R prognostic category. All the additional detected aberrations by MLPA are summarized in Table 4.
Table 4
Genetic lesions determined by FISH and MLPA
Case
|
Diagnosis
|
IPSS-R risk
|
karyotype
|
FISH
|
MLPA
|
IPSS-R risk group by MLPA
|
Patient 5
|
MDS-EB-1
|
Very high
|
failed
|
+ 8
|
+ 8
|
Very high
|
Patient 6
|
MDS-MLD
|
High
|
failed
|
+ 8
|
+ 8
|
Very high
|
Patient 7
|
MDS-EB-1
|
High
|
failed
|
+ 8
|
+ 8
|
Very high
|
Patient 8
|
MDS-MLD
|
High
|
failed
|
+ 8
|
+ 8 +11q
|
Very high
|
Patient 18
|
MDS-MLD
|
High
|
failed
|
-5q
|
-5q
|
High
|
Patient 20
|
MDS-U
|
Intermediate
|
failed
|
-7
|
-7
|
Very high
|
Patient 22
|
MDS-EB-2
|
High
|
failed
|
-7q
|
-7q
|
Very high
|
Patient 23
|
MDS-RS
|
Low
|
failed
|
-7q
|
-7q
|
Very high
|
Patient 17
|
MDS-U
|
Intermediate
|
failed
|
-5q
|
-5q -17p -19
|
Very high
|
Patient 4
|
MDS-EB-2
|
Very high
|
normal
|
+ 8 -17p
|
+ 8 -17p
|
Very high
|
Patient 9
|
MDS-MLD
|
Low
|
normal
|
-17p
|
-17p
|
High
|
Patient 10
|
MDS-MLD
|
Low
|
normal
|
-17p
|
-17p
|
High
|
Patient 15
|
MDS-MLD
|
Low
|
normal
|
-20q
|
-20q
|
Low
|
Patient 16
|
MDS-EB-1
|
Low
|
normal
|
-20q
|
-20q
|
Intermediate
|
Patient 24
|
MDS-EB-1
|
High
|
normal
|
-7q
|
-7q
|
Very high
|
Patient 21
|
MDS-MLD
|
High
|
failed
|
Negative*
|
-7
|
Very high
|
Patient 1
|
MDS-EB-1
|
High
|
normal
|
Negative
|
+ 11q
|
High
|
Patient 2
|
MDS-RS
|
High
|
normal
|
Negative
|
+ 11q
|
High
|
Patient 3
|
MDS-MLD
|
Very low
|
normal
|
Negative
|
-20q
|
Low
|
Patient 11
|
MDS-EB-2
|
Low
|
normal
|
Negative
|
-17p
|
High
|
Patient 12
|
MDS-MLD
|
Very low
|
normal
|
Negative
|
-17p
|
Low
|
Patient 13
|
MDS-EB-1
|
Intermediate
|
normal
|
Negative
|
-17q
|
Very high
|
Patient 14
|
MDS-EB-2
|
Low
|
normal
|
Negative
|
-19
|
Intermediate
|
Patient 19
|
MDS-MLD
|
High
|
normal
|
Negative
|
-5q
|
Very high
|
*Negative just for − 5/-5q, -7/-7q, + 8, -20q and 17p-. |
Survival Analysis
We performed survival analysis and compared the outcome of patients with normal and cases without result after banding cytogenetics, which were also confirmed by MLPA (n = 120) versus patients with aberrant karyotype as determined by MLPA (n = 24). We observed a significant difference in survival (median OS: undefined vs 27 months, p = 0.0071, Fig. 2). We performed a survival analysis of normal karyotypes and cases without result after banding cytogenetics, respectively. Data were shown in Supplement Fig. 1.
In addition, we compared the outcome of patients with normal karyotype (n = 132) to patients without result after banding cytogenetics (n = 12), Our result showed a significant difference in survival (median OS: undefined vs 26 months, p = 0.0059, Fig. 3), indicating patients without result after banding cytogenetics had worse survival.
In our study, we also explored the impact of cytogenetic aberrations detected by MLPA on OS of lower-risk and higher-risk patients (defined according to IPSS-R) with a normal or without result after banding cytogenetics via R-banding test. Lower-risk IPSS-R group included very low risk patients, low risk patients and intermediate patients with score ≤ 3.5. Higher-risk IPSS-R group included intermediate patients with score > 3.5, high risk, and very high risk patients. For lower-risk IPSS-R patients (73/144), there were no differences in OS (P = 0.5207; Fig. 4A). For higher-risk IPSS-R patients(71/144), OS was significantly shorter in the higher-risk patients(n = 19) with cytogenetic aberrations detected using MLPA compared with other higher-risk patients(n = 52) (median OS: 21 vs. undefined months, P = 0.0281; Fig. 4B).