Subclonal patterns in follow-up of acute myeloid leukemia combining whole exome sequencing and ultrasensitive IBSAFE digital droplet analysis

Figures & data

Table 1. Clinical information at diagnosis, NGS at first relapse, MFC-MRD and MRD for NPM1 (ddPCR and qPCR) until the first relapse.

Patient	Age at diagnosis/ sex	Diagnosis (WHO)	% blasts at diagnosis (morphology/ MFC)	Karyotype/ FLT3 status at diagnosis	Genetic riskgroup	NPM1 status at diagnosis (VAF% by IBSAFE/ MRD% by qPCR for the type A mutation/ VAF% by NGS)	NPM1- mutations at follow-up before first relapse, by IBSAFE (days after diagnosis, VAF%)	NPM1- mutations at follow-up before first relapse, by qPCR for the type A mutation (days after diagnosis, MRD%)	NPM1 status at first relapse (VAF% by IBSAFE/MRD% by qPCR for the type A mutation/ VAF% by NGS)	MFC-MRD (days after diagnosis, % leukemic cells)	First relapse time point (days after diagnosis)/ SCT (days after diagnosis)	Remarks	Alive/† (days after diagnosis)
1	29/M	Acute myelo-blastic leukemia with maturation	58/60	normal/wt	favourable	wt	–	–	wt	N.D.	2054/auto (2160)		Alive
2	64/F	AML with mutated NPM1	70/73	+8/wt	intermediate	type A (29.3/89/26)	36: N. D. 73: N. D. 233: neg 352: neg 409: neg 498: neg	36: 0.015 73: 0.0057 233: neg 352: neg 409: neg 498: neg	wt (neg/neg/neg)	36: <0.1% 73: <0.1% 233: <0.1% 352: <0.1% 409: <0.1% 498: <0.1%	736/allo (911)	pre leukemic hematopoesis, relapse NPM1 wt	† (1051)
3	55/F	AML with mutated NPM1	60/60	normal/ITD	intermediate	type DD5 (57.9/ N. D./24)	17: 10.8 83: 0.08 122: 0.3	–	type DD5 (42.7/ N. D./31)	17: N. D. 83: <0.1% 122: pos 0.2%	161/no		† (220)
4	43/F	AML with mutated NPM1	30/26	normal/ITD	intermediate	type A (26.8/79.4/25)	30: 0.02 58: neg	30: 0.013 58: 0.0028	type A (32.3/57.0/28)	30: <0.1% 58: <0.1%	241/no		† (389)
5	41/M	AML with inv(16)	48/48	inv(16)/wt	favourable	wt	–	–	wt	80: <0.2%, 108: <0.1% 158: <0.1% 341: pos 0.8% 355: pos 0.8%	388/allo (453)	inv(16), c-kit neg, at relapse +22. Monitored with RT-qPCR for inv(16), molecular remission.	Alive
6	61/M	AML with MDS-related changes	21/21	complex/wt	adverse	wt	–	–	wt	17: 5% 32: <0.1% 40: <0.1% 90: <0.1% 109: <0.01% 136: <0.1%	189/no	hemophagocytosis at diagnosis	† (191)
7	60/F	AML with mutated NPM1	19/20	normal/wt	favourable	type A (31.6/60/31)	71: neg 119: neg 191: neg	71: pos <0.0024 119: pos <0.0024 191: 0.011	type A (9.5/12/3.7)	71: <0.1% 119: <0.1% 191: <0.1%	339/allo (438)		† (1067)
8	37/F	AML with t(3;3), MECOM+	82/86	complex/wt	adverse	wt	–	–	wt	93: pos 0.2-0.3% 133: <0.1%	179/allo (114)	t(3;3), MECOM present at diagnosis and at relapse.	† (251)
9	48/F	AML with mutated NPM1	60/57	normal/wt	favourable	type A (40.3/47.9/17)	29: 0.08 63: 0.006 102: 0.03 149: neg	29: 0.059 63: 0.021 102: 0.021 149: pos <0.0024	type A (40.3/33.2/26)	29: <0.5% 63: <0.1% 102: <0.1% 149: <0.1%	553/allo (647)		† (927)
10	71/F	AML with mutated NPM1	34/12	normal/ITD	intermediate	type D (37.8/ N. D./31)	63: 0.08	–	type D (43.6/ N. D./18)	63: <0.1%	145/no	pre leukemic hematopoiesis	† (304)
11	66/M	AML with mutated NPM1	92/80	normal/AKD	favourable	type A (47/62/33)	14: N. D.^a 28: 0.006 73: neg 176: neg	14: 13 28: 0.0045 73: neg 176: neg	–	14: <0.1% 28: <0.1% 73: <0.1% 176: <0.1%	no/no		Alive
12	47/F	AML with t(8;21)	33/16	t(8;21) + additional chromosome changes/wt	favourable	wt	–	–	–	68: <0.01% 216: <0.01% 375: <0.01% 551: <0.01% 720: <0.1%	no/no		Alive
13	71/M	Acute myelo-monocytic leukemia	70/77	trisomy 13/wt	adverse	wt	–	–	–	90: 0.03% 167: <0.1% 276: 0.03% 337: <0.1% 895: <0.1%	no/allo (189)		Alive
14	51/F	Acute myelo-blastic leukemia with minimal differentiation	90/90	normal/wt	intermediate	wt	–	–	–	75: <0.1% 139: <0.1% 237: <0.1%	no/allo (58)		Alive

WHO: World Health Organization classification; MFC: multicolor flow cytometry; FLT3: FMS-like tyrosine kinase 3; NPM1: nucleophosmin 1 gene; VAF: variant allele frequency; IBSAFE: in house digital droplet polymerase chain reaction; MRD%: %leukemic cells; qPCR: real-time quantitative polymerase chain reaction; NGS: next generation sequencing; MRD: measurable residual disease; SCT: stem cell transplantation; †: dead; M: male; F: female; AML: acute myeloid leukemia; inv: inversion; MDS: myelodysplastic syndrome; MECOM+: “MDS1 and EVI1 complex locus”; wt: wildtype; ITD: internal tandem duplication; AKD: activated kinase domain; N.D: not determined; neg: negative; pos: positive; auto: autologous stem cell transplantation; allo: allogeneic stem cell transplantation; RT-qPCR: reverse transcriptase PCR; molecular remission: morphological remission and two negative MRD-samples.

^aMutational status not determined due to insufficient DNA.

Figure 1. Performance of IBSAFE and comparison of IBSAFE and WES measured variant allele frequencies (VAFs) in diagnosis and relapse samples. (A) Dilution series for IBSAFE assay NPM1 type A for constructed samples with known VAFs at 10%, 1%, 0.1%, 0.01%, 0.001%, and 0%. (B) Scatterplot for agreement between the methods with Pearson’s coefficient of determination R²=0.77 and p-value <.0001, N = 144, (C) Bland Altman plot.

Table 2. Number of mutations at diagnosis and first relapse.

Patient	Number of genes known to be recurrently mutated in AML at diagnosis	Number of non-recurrently mutated genes in AML at diagnosis	Number of recurrently mutated genes at first relapse (lost/new)	Number of non-recurrently mutated genes at first relapse (lost/new)
1	5	9	5 (1 / 1)	10 (2 / 3)
2	6	24	5 (2 / 1)	12 (17 / 5)
3	2	15	2^a (0 / ND^a)	7 ^a (8 / ND ^a)
4	3	13	4 (1 / 2)	13 (6 / 6)
5	2	11	0^a (2 / ND^a)	8^a (3 / ND^a)
6	1	14	1 (0 / 0)	15 (1 / 2)
7	7	10	2 (5 / 0)	28 (4 / 22)
8	0	10	ND^b	ND^b
9	4	9	4 (0 / 0)	15 (0 / 6)
10	3	14	3 (0 / ND^a)	10 (4 / ND^a)
11	4	7	–	–
12	0	24	–	–
13	6	21	–	–
14	4	18	–	–

ND: not determined.

^aNGS analysis not possible to interpret for new mutations due to a high background noise.

^bNo NGS analysis performed on the relapse sample because germline variants could not be determined after allo-SCT before the relapse.

Figure 2. Monitoring leukemic mutations using ultrasensitive IBSAFE for ten relapsing AML patients (A) #4, (B) #9, (C) #6, (D) #8, (E) #7, (F) #5, (G) #1, (H) #3, (I) #10 and (J) #2. In each plot, the y-axis represents the detected variant allele frequency (VAF %) for each tracked mutation (key to the right; genes known to be recurrently mutated in AML in bold), and the x-axis indicates the days after diagnosis with therapies indicated by shading (chemotherapy) and clinical events indicated along the top of each plot. The inverted triangles indicate the flow cytometry MRD results, with the color-key indicated in the lower-right of plot (A). Dx: diagnosis; R: relapse; †: dead: SCT: stem cell transplantation; FU; follow-up; H: harvest; ND: not detected (VAF below lower effective limit of detection of 0.003% determined by input DNA quantity).

Figure 3. Monitoring leukemic mutations using ultrasensitive IBSAFE for four non-relapsing AML patients (A) #13, (B) #14, (C) #11 and (D) #12. In each plot, the y-axis represents the detected variant allele frequency (VAF %) for each tracked mutation (key to right; genes known to be recurrently mutated in AML in bold), and the x-axis indicates the days after diagnosis with therapies indicated by shading (chemotherapy) and clinical events indicated along the top of each plot. The inverted triangles indicate the flow cytometry MRD results, with the color-key indicated in the lower-right of plot (A). Dx: diagnosis; FU: follow-up; SCT: stem cell transplantation; ND: not detected (VAF below 0.003%).