Serial Determination of FLT3-ITD and NPM1 Mutations and Its Clinical Significance in Patients with MDS at Diagnosis and After Progression to AML with Myelodysplasia-related Changes

Jae-Woo Chung; Hyun-Sook Chi; Eun-Hye Lee; Seongsoo Jang; Eul-Ju Seo; Chan-Jeoung Park

doi:10.3343/lmo.2011.1.3.138

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Chung, Chi, Lee, Jang, Seo, and Park: Serial Determination of FLT3-ITD and NPM1 Mutations and Its Clinical Significance in Patients with MDS at Diagnosis and After Progression to AML with Myelodysplasia-related Changes

Original Article

Lab Med Online 2011;1(3):138-146.

Published online: 20 July 2011

DOI: https://doi.org/10.3343/lmo.2011.1.3.138

Serial Determination of FLT3-ITD and NPM1 Mutations and Its Clinical Significance in Patients with MDS at Diagnosis and After Progression to AML with Myelodysplasia-related Changes

Jae-Woo Chung, M.D, Hyun-Sook Chi, M.D, Eun-Hye Lee, M.D, Seongsoo Jang, M.D, Eul-Ju Seo, M.D, Chan-Jeoung Park, M.D

Department of Laboratory Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea

Corresponding author: Hyun-Sook Chi, M.D. Department of Laboratory Medicine, University of Ulsan College of Medicine, Asan Medical Center, Pungnap 2-dong, Songpa-gu, Seoul 138-736, Korea Tel: +82-2-3010-4502, Fax: +82-2-478-0884, E-mail: hschi@amc.seoul.kr

Received 18 November 2010 Revised 2 March 2011 Accepted 2 March 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Accumulation of genetic aberrations in MDS is closely associated with progression to AML. FLT3-ITD is commonly found in AML and less frequently in MDS. FLT3-ITD in MDS is associated with a high risk of transformation to AML. Recently, significant interaction of NPM1 and FLT3-ITD was described in AML. This study was conducted to investigate the incidence and prognostic role of FLT3-ITD and NPM1 mutations (NPM1^mt) on paired samples at diagnosis of MDS and AML.

Methods

Patients who were diagnosed as MDS transforming to AML were included. FLT3-ITD was detected by PCR, and NPM1^mt was confirmed by direct sequencing after screening for NPM by immunohistochemistry.

Results

AML developed in 12.0% (43/357) of MDS patients. FLT3-ITD was detected in none of MDS and 14.7% (5/34) of AML. NPM1^mt was detected in 2.4% (1/41) of MDS and 11.6% (5/43) of AML. One patient with type B NPM1^mt at MDS transformed to type A NPM1^mt at AML. FLT3-ITD positive AML showed a tendency of shorter survival and a significantly longer time to achieve complete remission than FLT3-ITD negative AML (P=0.007). Normal karyotype AML with FLT3-ITD showed shorter overall survival than that group of AML without FLT3-ITD (P=0.017).

Conclusions

MDS patients acquired FLT3-ITD during AML transformation, and FLT3-ITD positive AML, especially that with normal karyotype, predicted a poor outcome. NPM1^mt was identified in both MDS and AML. NPM1^mt was rarely found in MDS patients, and mostly was acquired after AML transformation. Clonal evolution of NPM1^mt subtype was found in one patient during acute transformation.

Keywords: MDS, AML, Fms-like tyrosine kinase 3 (FLT3), Nucleophosmin (NPM)

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Fig. 1.

Results of NPMc⁺ and NPM1 exon 12 type B mutation in a patient with AML M2 transformed from RAEB1. (A) Some dysplastic cells show cytoplasmic expression with blurring margination for NPM. (B) Type B mutation of NPM1 with CATG insertion (GenBank Accession No. AY740635) was confirmed by sequencing. Abbreviations: See Tables 1 and 3.

Fig. 2.

Kaplan-Meier survival curves. MDS patients with FLT3-ITD (N=5, straight line) or without FLT3-ITD (N=29, dotted line) at AML transformation. Abbreviations: See Table 1.

Table 1.

Characteristics of different subtypes of MDS patients according to WHO classification

Patients	All	RA	RCMD	RAEB-1	RAEB-2
Number	43	4	7	10	22
Median age (yr, range)	43.8 (1.7–79.4)	46.0 (30.5–60.5)	63.8 (5.9–65.8)	34.9 (1.7–65.4)	42.9 (3.2–79.4)
Sex (male/female)	26/17	3/1	6/1	6/4	11/11
WBC (/μL)	9.4±20.6	2.3±0.9	3.6±2.1	6.1±5.5	14.0±24.0
Hemoblobin (g/dL)	7.8±2.7	8.4±0.9	8.9±2.5	7.7±2.6	7.4±3.0
Platelet (/μL)	91.7±71.4	79.0±22.7	91.3±52.7	96.0±85.1	92.2±78.7
Blasts in PB (%)	2.4±3.5	0	0.1±0.4	2.1±1.4	3.8±4.3
Blasts in BM (%)	8.8±5.1	1.7±0.8	3.1± 1.4	7.2±1.1	12.7±3.7
Myeloid/erythroid	3.8±5.7	0.4±0.1	1.3±0.5	6.5±9.4	3.9±4.3
Dysmegakaryopoiesis (%)	74.4	0	85.7	80	77.3
Dysgranulopoiesis (%)	81.4	0	100	100	77.3
Dyserythropoiesis (%)	95.3	100	100	90	95.5
Cellularity (%)	70.6_±24.0	63.8_±14.9	61.4_±16.5	85.5_±18.5	67.9_±27.5
Time to AML (mon)	11.6_±16.4	3.5_±1.9	19.5_±20.0	15.5_±26.4	8.8_±8.7
Abnormal karyotype at MDS (%)	11/30 (36.7)	0/1 (0.0)	3/6 (50.0)	3/7 (42.9)	5/16 (31.3)
IPSS N (%)
Low	1 (3.3)	–	1 (16.7)	–	–
Intermediate-1	14 (46.7)	1 (100.0)	4 (66.7)	5 (71.4)	4 (25.0)
Intermediate-2	12 (40.0)	–	1 (16.7)	2 (28.6)	9 (56.3)
High	3 (10.0)	–	–	–	3 (18.8)

Abbreviations: RA, refractory anemia; RCMD, refractory cytopenia with multilneage dysplasia; RAEB, refractory anemia with excess blasts; WBC, white blood cell; PB, peripheral blood; BM, bone marrow; IPSS, international prognostic scoring system.

Table 2.

Results of chromosomal analysis in patients with paired samples at MDS and AML stages

Age	Sex	Initial diagnosis	Karyotype
Age	Sex	Initial diagnosis	MDS	AML
41	M	RA	Normal	Normal
66	M	RCMD	Normal	Normal
25	M	RAEB-1	Normal	Normal
48	M	RAEB-1	Normal	Normal
65	F	RAEB-1	Normal	Normal
3	F	RAEB-2	Normal	Normal
25	F	RAEB-2	Normal	Normal
29	M	RAEB-2	Normal	Normal
42	F	RAEB-2	Normal	Normal
54	M	RAEB-2	Normal	Normal
60	F	RAEB-2	Normal	Normal
35	M	RAEB-1	+8	Normal∗
51	F	RAEB-2	–7, +21	–7,+21
27	M	RAEB-1	del (9q)	del (9q)
15	M	RAEB-2	t (6;9)	t (6;9)
27	M	RAEB-2	t (6;11)	t (6;11)
44	F	RAEB-2	t (6;9)	t (6;9)
48	F	RAEB-1	i (17),+9,+13	i (17)
51	F	RAEB-2	+8,-X	+8
6	M	RCMD	Normal	del (7q)
55	M	RCMD	Normal	del (20q)
57	M	RAEB-2	Normal	add (16),-17
59	F	RAEB-2	Normal	+9, i (17)
46	F	RCMD	+8, der (20;21),+del (22q)	Complex
65	M	RCMD	+8	+8, +8
64	M	RCMD	Complex	NT
2	F	RAEB-1	Normal	NT
4	M	RAEB-2	Normal	NT
21	F	RAEB-2	Normal	NT
47	M	RAEB-2	Normal	NT
31	F	RA	NT	Normal
61	M	RA	NT	Normal
65	M	RCMD	NT	del (12q)
40	M	RAEB-2	NT	t (6;9)
42	F	RAEB-2	NT i	inv (3), del (5q)
43	M	RAEB-2	NT	Normal
79	M	RAEB-2	NT	Normal

^∗ In 1995, initial MDS had been diagnosed and hematopoietic stem cell transplanted. After 8 years, AML transformation was confirmed. Abbreviations: M, male; F, female; NT, not tested; Others, See Table 1.

Table 3.

Results of immunohistochemistry for NPM and sequencing for exon 12 of NPM1

Patients			At MDS		At AML
Age	Sex	Initial diagnosis	IHC	Sequencing	IHC	Sequencing
63	M	RA	NPMc⁺	Type B	NPMc⁺	Type A
60	F	RAEB1	NPMc^–	Wild	NPMc⁺	Type B
43	M	RAEB2	NPMc^–	Wild	NPMc⁺	Type B
57	M	RAEB2	NPMc⁺	Wild	NT	Type B
29	M	RAEB2	NPMc^–	Wild	NPMc⁺	Type B

Abbreviations: IHC, immunohistochemistry; NPM, nucleophosmin; NPMc

⁺ positive for cytoplasmic NPM; NPMc

^- negative for cytoplasmic NPM; Others, See Tables 1 and 2.

Table 4.

Characteristics of patients with FLT3-ITD at AML transformation

Age (yr)/ sex	Initial MDS subtype	Karyotype		IPSS	FLT3-ITD		NPM		Time to AML (mon)	Overall survival (mon)	Survival after AML (mon)
Age (yr)/ sex	Initial MDS subtype	MDS	AML	IPSS	MDS	AML	MDS	AML	Time to AML (mon)	Overall survival (mon)	Survival after AML (mon)
60/M	RA	NT	Normal	–	Negative	Positive	NPMc⁻	NPMc⁻	3.6	7.1	3.6
5/M	RCMD	NT	del(7q)	0.5	Negative	Positive	NPMc⁻	NPMc⁻	6.3	46.3	41.9
34/M	RAEB-1	NT	Complex	–	Negative	Positive	NPMc⁻	NPMc⁻	17.8	25.4	7.6
28/M	RAEB-2	Normal	Normal	2	Negative	Positive	NPMc⁻	NPMc⁻	2.2	6.1	3.9
14/M	RAEB-2	t(6;9)	t(6;9)	2.5	Negative	Positive	NPMc⁻	NPMc⁻	3.6	9.1	5.5

Abbreviations: FLT3-ITD, Fms-like tyrosine kinase 3 internal tandem duplication; mon, month; Others, See Table 1.

Table 5.

Relationship between FLT3-ITD results at AML transformation and clinical characteristics.

	FLT3-ITD at AML transformation		P value
	Positive (N=5)	Negative (N=29)	P value
Time to AML (mon)∗	3.6 (3.6–4.4)	9.2 (4.1–18.2)	0.232
N of CR achievement (%)	3 (60.0)	19 (65.5)	0.274^†
Time to CR achievement (mon)∗	4.7 (3.7–5.1)	1.2 (1.1–1.6)	0.007
Duration of CR (mon)∗^‡	2.7 (2.4–21.4)	5.7 (3.9–28.3)	0.573
Survival rate (%)	1/5 (20.0)	6/25 (24.0)	0.671^†
Overall survival (mon)∗	9.1 (7.1–25.4)	26.3 (13.1–48.5)	0.163
Survival after AML (mon)∗	5.5 (3.9–7.6)	9.4 (2.3–30.9)	0.888

^∗ Values represent median (25–75 percentile), P value calculated by Mann-Whitney U-test,

^† P value calculated by Fisher's exact test,

^‡ Values are defined by duration from CR to relapse or death. Abbreviations: CR, complete remission; others, See Table 1.

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