Morphologic, immunophenotypic, and molecular genetic comparison study in patients with clonal cytopenia of undetermined significance, myelodysplastic syndrome, and acute myeloid leukemia with myelodysplasia-related changes: A single institution experience

Abstract

Introduction:

Next-generation sequencing (NGS) analysis showed clonal cytopenia of undetermined significance (CCUS) as an immediate precursor to myelodysplastic syndrome (MDS).

Methods:

We evaluated and compared morphologic, multiparametric flow cytometry (MFC), and molecular genetic findings in patients with CCUS (n = 37), MDS (n = 75), and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC, n = 24).

Results:

CCUS patients showed variable MFC abnormalities including >2% CD34+ myeloblasts (5.8%), altered antigen expression on myeloblasts, monocytes, and granulocytes (1.2, 1.5, and 0.2/case), abnormal maturation of myeloblasts (45.8%), decreased hematogones (17.6%), and decreased side scatter (SSC) of granulocytes (11.4%). CCUS patients with high-risk mutations showed significantly more MFC abnormalities. However, CCUS patients with >20% variant allelic fraction (VAF) did not show more MFC aberrations than the rest of the group. MDS patients showed significantly more MFC abnormalities compared with CCUS patients (p = 7.8E-05 – 0.047). Low-grade MDS patients showed significantly fewer MFC abnormalities compared with high-grade MDS or AML-MRC patients (p = 1.89E-05– 0.04). AML-MRC patients showed significantly elevated blast counts, more antigen aberrations, decreased hematogones, and decreased SSC of granulocytes compared with CCUS patients (p = 2.0E-05– 0.01). CCUS patients carried predominantly TET2/DNMT3A/ASXL1 mutations. They harbored fewer mutations in gene coding splicing factors compared with MDS patients (p = .0001–. 02) and fewer mutations in tumor suppressor and transcription factor genes compared with AML-MRC patients (p = .0006– .02).

Conclusions:

CCUS is an immediate precursor to low-g rade MDS. The progression from CCUS to MDS to AML-MRC is a stepwise process that requires acquisition of mutations in splicing, transcription factor, and tumor suppressor genes with accumulations of additional MFC abnormalities.

1 |. INTRODUCTION

Myelodysplastic syndromes (MDSs) are clonal hematopoietic neoplasms characterized by inefficient hematopoiesis, morphologic dysplasia, progressive cytopenia, and transformation to acute myeloid leukemia (AML).¹ MDS remains a diagnostic challenge as these clinical features of MDS are not specific and the diagnostic boundaries between MDS and its related conditions can be hard to define.² With the advent of NGS technology, these molecular studies can provide new insight to clarify the diagnostic ambiguity between MDS and its mimics and potentially identify precursors.^2,3

Increased utilization of NGS-based gene panel testing for unexplained cytopenia helps to detect genomic mutations in patients without morphologic features of MDS. Therefore, new entities that have defined precursor states of MDS have emerged.^2–4 Clonal hematopoiesis of indeterminate potential (CHIP) is a condition that carries similar somatic mutation(s) as MDS without cytopenia. Clonal cytopenia of undetermined significance (CCUS) describes a condition of cytopenic patients that carry similar somatic mutation(s) as MDS patients without MDS-defining features of dysplasia. Both CHIP and CCUS are regarded as a premalignant status that can progress to MDS, AML, and other hematologic neoplasms.

Limited number of multiparametric flow cytometry (MFC) studies have been performed in CCUS patients.^5–7 In our current study, we have evaluated morphologic changes, MFC abnormalities, and genomic alterations in CCUS patients and compared these findings in patients with MDS and AML with myelodysplasia-related changes (AML-MRC).

2 |. MATERIALS AND METHODS

2.1 |. Case selection

We retrospectively identified 159 patients via keyword search in our electronic medical record system from 2017–2021. These selected cases included patients with CCUS (n = 37), MDS with single lineage dysplasia with or without ring sideroblasts (MDS-SLD, n = 28), MDS with multilineage dysplasia with or without ring sideroblasts (MDS-MLD, n = 21), MDS with excess blasts (MDS-EB, n = 26), and AML-MRC (n = 24). All AML cases had a prior history of MDS or chronic myelomonocytic leukemia (CMML). We also included 23 patients with history of myeloid neoplasms status post chemotherapy and allogenic stem cell transplant and carrying 100% donor cells as negative control for MFC baseline evaluation on myeloblasts.

Peripheral blood (PB) cytopenias were defined as platelets below 150 × 10⁹/L, neutrophils below 1.8 × 10⁹/L, and hemoglobin below 12.0 g/dl or 13.5 g/dl for women and men, respectively. CCUS patients were included based on persistent cytopenia for 6 months and harboring at least one somatic mutation of pathogenic, likely pathogenic or variant of uncertain significance (VUS) (using accepted variant classification methods) with variant allelic fraction (VAF) of at least 4%. Patients without persistent cytopenia were excluded.

All MDS and AML diagnoses were made according to 2016 World Health Organization (WHO) classification.⁸ Clinical, morphologic, immunophenotypic, molecular, and cytogenetic data were reviewed. Perl’s iron stain was evaluated in each case. Morphologic dysplasia and blast count were evaluated by four hematopathologists (DZ, SK, JW, and WC). This retrospective study was approved by the University of Kansas Medical Center Institutional Review Board (IRB).

2.2 |. Multiparametric flow cytometric study

Eight-color MFC was performed on PB (n = 1) or bone marrow (BM, n = 155) specimens using BD FACSCanto II instruments (BD Biosciences) according to the manufacturer’s instructions. For initial diagnosis of AML, an acute leukemia panel was performed with the following antibody combinations: (1) CD13/CD15/CD33/ CD34/CD45/CD56/HLA-DR, (2) CD11b/CD11c/CD14/CD34/CD64/CD45/CD117, (3) CD19/CD20/CD5/CD10/CD38/κ/λ/CD45, (4) CD1a/CD2/CD3/CD4/CD5/CD7/CD8/CD45, and (5) CyMPO/ cyCD3/CD19/cyCD22/cyCD79/CD34/CD45/nTDT. If MDS was suspected or treated AML or MDS was evaluated, an MRD panel was performed with the following antibody combinations: (1) CD7/ CD33/CD11b/CD34/CD13/CD45/CD38/CD19, (2) HLA-DR/ CD117/CD4/CD34/CD123/CD45/CD38/CD19, (3) HLA-DR/CD56/ CD36/CD34/CD64/CD45/CD14/CD19, and (4) CD2/CD22/CD5/ CD34/CD15/CD45/CD38/CD19. Antibody clones and manufacturer’s instructions for application were listed in Table S1. MRD panel was used for evaluation of MDS due to its higher detection sensitivity for neoplastic myeloblasts. Data analysis was performed using FCS Express 5 software (De Novo Software, Pasadena, CA).

A total of 20,000 cell events (range and median of viable events: 15 176– 17 236 and 15 366) were acquired for acute leukemia panel at the initial diagnosis. Major cell populations were defined by CD45/SSC (side-scatter) characteristics. All populations were further refined by forward-and side-scatter gates to exclude debris and doublets. A CD45 dim “blast” gate was drawn. A positive flow cytometry result was defined as identification of a population of CD34⁺ or CD117⁺ myeloblasts or CD64⁺/CD14⁺ monocytoid cells with aberrant myeloid or lymphoid antigen expression within the “blast” gate, usually comprising more than 20% of gated cells.

A total of 500 000 cell events were routinely acquired for MRD assessment with a range of 4623 to 500 000 and a median of 500 000 cell events. CD45 dim, monocyte, granulocyte and lymphocyte gates were drawn on a traditional CD45/SSC display (Figure S1A). Then a broad CD45 dim “blast” gate including monocytes and a CD45 dim blast gate was drawn, followed by back-gating to identify the CD34⁺ population (Figure S1B,C), CD117⁺ cells, and CD64⁺ monocytoid cells (data not shown) on CD45/SSC plot. Among the CD34⁺ cells, CD19 is used as an exclusion gate on most plots, to separate out normal type 1 hematogones from the CD45 dim gate (Figure S1D). In cases of myeloblasts with aberrant CD19 expression, this CD19 gate will be moved aside to display all blasts on all of the analysis plots. CD34⁺ myeloblasts, CD34⁻ populations in the CD45 dim⁺ cells, as well as monocytic and granulocytic populations were separately evaluated, following the previously published analytic strategy.^9–12

MFC data were analyzed based on five quantifiable parameters. They included three parameters of Ogata score (<2% CD34+ myeloblasts, SSC ratio of granulocytes versus lymphocytes >6, and type 1 hematogones >5% of CD34⁺ cells), number of aberrant antigens expressed on myeloblasts, monocytes and granulocytes, and evaluation of myeloid blast maturation by assessment of CD13, CD33, and CD38 expression.^9,11,13 MFC baseline parameters were evaluated in CD34⁺ myeloblasts with a range of 78–3368 and a median of 911 CD34⁺ myeloblasts in 23 negative control patients. Neoplastic blasts were defined as residing in a tight cluster of at least >20 cells and had at least two abnormalities. Aberrantly expressed antigen was defined as normalized median fluorescence intensity (MFI) and percentage of antigen expression on blasts, monocytes, or granulocytes deviated from the normal range established in our laboratory. The normal ranges of MFI and antigen expression level were defined as values between means ±2 standard deviations on myeloblasts, granulocytes, and monocytes previously established from 48 negative control patients who had either a negative staging BM for a newly diagnosed lymphoma or who had a history of myeloid neoplasm but had been successfully treated with hematopoietic stem cell transplantation and had been proven to be in remission by clinical, morphologic, chimerism-based molecular and cytogenetic means.

2.3 |. Next-generation sequencing method

Nucleic acid was purified from fresh BM or PB samples using QIAamp DNA Blood Mini Kit (Qiagen, Germantown, MD). Cellular material was lysed before binding to silica-based membrane spin columns, which were subjected to washing and elution. DNA was quantified using a NanoQuant Plate on an Infinite M200 Pro (Tecan, Männedorf, Switzerland), then 40 ng of input DNA underwent a multiplex PCR reaction targeting exons within 141 myeloid-related genes using the QIAseq Targeted DNA Human Myeloid Neoplasms Panel (Qiagen) as previously described.¹² Prepared libraries were subjected to next-generation sequencing on a NextSeq 500 instrument (Illumina, San Diego, CA) to generate FASTQ files. Reads were mapped to GRCh37 reference using the CLC Genomics Workbench (Qiagen) to generate Variant Call Files, which were processed using Clinical Insight-Interpret (Qiagen) to assess pathogenicity based on ACMG-AMP guidelines.¹⁴ Quality control metrics such as depth of coverage, variant allele frequency (VAF) and average quality scores for reported variants were evaluated individually for Pathogenic and Likely Pathogenic calls compared with VUS. Somatic mutation was defined as any mutation with a VAF of <40% or >60%. For any mutations with VAF between 40% and 60%, somatic variant curation was determined by database search on COSMIC and ClinVar sites.^15,16

We further classified these genes into functional groups according to those previously reported as follows: epigenetic modifier: TET2, DNMT3A, ASXL1, IDH1, IDH2, EZH1, EZH2, SETBP1, KDM6A; RNA spliceosome: SF3B1, SRSF2, ZRSF2, ZRSR2, ZRSR2, U2AF1, and U2AF2; signaling transduction: FLT3, KIT, NRAS, KRAS, PTPN11, CBL, CBLB, CBLC, JAK1, JAK2, and MPL; transcription factor: RUNX1, CEBPA, GATA1, GATA2, BCOR, BCORL1, NPM1, ETV6, PHF6; tumor suppressor: TP53, WT1; cohesion: STAG2, SMC1A; and others.¹⁷

2.4 |. Statistical analysis

Student t-test was performed to compare ages, levels of Hb, counts of neutrophil, platelet, and blast, and frequencies of aberrantly expressed MFC antigen and mutation between groups using Microsoft Excel software (Microsoft, Richmond, VA). Chi-squared test was performed to compare the frequencies of gender, cytopenia, MFC abnormality, and abnormal cytogenetics between groups using the online GraphPad calculator (GraphPad Software, La Jolla, CA). A p-value less than .05 was considered statistically significant.

3 |. RESULTS

3.1 |. Clinicopathologic characteristics of patients

The study included 78 male and 58 female patients with a mean age of 67 years (range, 21– 92 years). The characteristics including age, sex, complete blood count, BM blast count, dysplasia, MFC, cytogenetics, and NGS molecular findings were evaluated and compared. Results are summarized in Table 1.

TABLE 1.

Clinicopathologic characteristics and comparison of patients in different groups

	CCUS (n = 37)	MDS-SLD (n = 28)	MDS-MLD (n = 21)	MDS-EB (n = 26)	AML-MRC (n = 24)	CCUS versus MDS-SLD, p value	CCUS versus MDS-MLD, p value	CCUS versus MDS-EB, p value	CCUS versus AML-MRC, p value
Age	62 (22–86)	68 (21–90)	69 (23–90)	68 (39–92)	66(31–82)	.14	.13	.13	.37
Gender	F: 16; M: 21	F: 12; M: 16	F:8; M:13	F: 8; M:18	F:14; M:10	1.00	.78	.31	.43
Hemoglobin (mg/dl)	11.0 (6.6–17.3)	10.0 (6.9–15.5)	10.2 (6.6–13.9)	9.5(5.8–15.5)	9.2 (7–12.7)	.19	.26	.04	.005
Neutrophils (K/vl)	1.6(0.4–5.38)	2.2 (0.7–4.8)	2.5 (0.3–5.9)	1.4 (0.06–8.4)	0.9 (0–4.4)	.81	.44	.29	.03
Platelets (K/vl)	135.7(16–356)	155.6(10–410)	131.2 (24–390)	98.6(9–421)	37.8 (7–112)	.42	.86	.14	1.6E-08
Dysplastic lineage(s)
0	37	0	0	2	0	<.0001	<.0001	<.0001	<.0001
1	0	28	0	4	6	<.0001	1.0	.03	.002
2	0	0	12	13	5	1.0	<.0001	.0001	.007
3	0	0	9	7	13	1.0	<.0001	.001	<.0001
BM blasts (%) (range)	1.6 (0–9)	1.8 (0–4)	2.0 (1–4)	9.2 (5–18)	31.3 (20–57)	.68	.31	1.7E-10	1.1E-11
Flow cytometry
Blasts (%) (range)	0.6(0.01–3)	0.7 (0.03–2)	1.1 (0.1–5.2)	5.1 (0.4–16)	17.0 (1.2–59)	.63	.16	4.9E-06	2.0E-05
Abn marker numbers (range)	1.2 (0–4)	1.4 (0–5)	2.7 (0–7)	2.8 (0–6)	4.4 (2–8)	.47	.002	.0003	7.8E-05
Abnormal maturation	46% (11/24)	63.0% (17/27)	61.9% (13/21)	85.7% (22/25)	95% (19/20)	.40	.55	.005	.002
Decreased hematogone	17.6% (6/34)	51.8% (14/27)	90.5% (19/21)	84.6% (22/26)	87.5% (21/24)	.006	<.0001	<.0001	<.0001
Decreased SSC	11.4% (4/35)	3.7% (1/27)	47.6% (10/21)	26.9% (7/26)	41.7% (10/24)	.38	.004	.18	.01
Abn marker numbers on monocytes (range)	1.5 (0–6)	1.5 (0–7)	2.8 (0–6)	4.0 (1–7)	3.2 (0–9)	1.0	.02	.0002	.047
Abn marker numbers on granulocytes (range)	0.2 (0–2)	0.6 (0–2)	0.7 (0–2)	0.8 (0–2)	1.3 (0–3)	.08	.026	.007	8.7E-05
NGS
Pathogenic	0.44 (0–3)	0.86(0–3)	1.24(0–5)	1.35(0–4)	1.63 (0–5)	.07	.01	.001	.0003
Likely	0.44 (0–3)	0.29 (0–2)	0.91(0–6)	0.81(0–3)	0.92 (0–4)	.33	.18	.09	.06
VUS	2.8 (0–10)	1.60 (0–5)	1.90(0–5)	1.77(0–4)	2.8 (0–7)	.01	.07	.02	.96
Abnormal cytogenetics	13.5% (5/37)	32.1% (9/28)	42.9% (9/21)	57.7% (15/26)	70.8% (17/24)	.13	.02	.0003	.0001

Abbreviations: Abn, abnormal; AML-MRC, acute myeloid leukemia with myelodysplasia-related changes; BM, bone marrow; CCUS, clonal cytopenia of undetermined significance; MDS-EB, myelodysplastic syndrome with excess blasts; MDS-SLD, myelodysplastic syndrome with single linage dysplasia with or without ring sideroblasts; MDS-MLD, myelodysplastic syndrome with multilineage dysplasia with or without ring sideroblasts; NGS, next-generation sequencing; SSC, side scatter; VUS, variant of uncertain significance.

3.2 |. Clinical characteristic comparison between patients with clonal cytopenia of undetermined significance, myelodysplastic syndrome, and acute myeloid leukemia

There were no differences in age and sex between CCUS patients and others. CCUS patients were less anemic compared with MDS-EB and AML-MRC patients. There were no significant differences in absolute neutrophil count and platelet count between CCUS and MDS groups. AML-MRC patients were significantly more thrombocytopenic compared with CCUS patients.

3.3 |. Morphologic comparison in patients with clonal cytopenia of undetermined significance, myelodysplastic syndrome, and acute myeloid leukemia

CCUS patients did not meet the minimal WHO diagnostic criteria of MDS based on the degree of morphologic dysplasia. However, 9 (24.3%) and 3 (8.1%) CCUS patients showed single-and bi-lineage dysplasia in less than 10% of the cells, respectively. In contrast, MDS patients showed significant dysplasia including single-lineage dysplasia (42.6%; dyserythropoiesis ±ring sideroblasts: 17; dysmegakaryopoiesis: 15), bi-lineage dysplasia (33.3%; dysplasia of erythroid and megakaryocytic lineages: 11; dysplasia of erythroid and granulocytic lineages: 4; dysplasia of granulocytic and megakaryocytic lineages: 10), and trilineage dysplasia (21.3%, n = 16). Two MDS-EB patients did not show any dysplasia. Similarly, AML-MRC patients also showed significant dysplasia including single-lineage dysplasia (25%; dyserythropoiesis: 1; dysmegakaryopoiesis: 4; dysgranulopoiesis: 1), bi-lineage dysplasia (20.8%; dysplasia of erythroid and megakaryocytic lineages: 2; dysplasia of granulocytic and megakaryocytic lineages: 3), and tri-lineage dysplasia (54.1%, n = 13).

3.4 |. Flow cytometry comparison in patients with clonal cytopenia of undetermined significance, myelodysplastic syndrome, and acute myeloid leukemia

Like MDS patients, CCUS patients showed variable MFC abnormalities including >2% CD34⁺ myeloblasts (5.8%), aberrant antigen expression on myeloblasts, monocytes, and granulocytes (1.2, 1.5, and 0.2/case), abnormal maturation of myeloid blasts (45.8%), reduction of type I hematogones (17.6%), and decreased SSC of granulocytes (11.4%) (Table 1 and Figure 1). CCUS patients showed significantly less reduction of type I hematogones compared with MDS-SLD patients (p = .006). MFC detected more aberrancies as MDS progressed from low grade to high grade to AML when we compared patients with MDS-SLD to patients with high-grade MDS or AML-MRC (p = 1.89E-05–0 .04). CCUS patients demonstrated significantly lower frequencies in aberrant antigen expression on myeloblasts, monocytes, and granulocytes, abnormal maturation of myeloblasts, reduction of type I hematogones, and decreased SSC of granulocytes compared with high-grade MDS or AML patients (p = 7.8E-5 – 0.047, Table 1, Figure 1, and Figure S2).

FIGURE 1.

Flow cytometry abnormalities present in control, clonal cytopenia of undetermined significance (CCUS), myelodysplastic syndrome (MDS), and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) patients. (A) Normal side scatter (SSC) of granulocytes in control patient and reduced SSC of granulocytes (indicated by blue arrow) in CCUS, MDS, and AML-MRC patients. (B) Normal (NL) level of type I hematogones in control patient and reduction of type I hematogones (indicated by red arrow) in CCUS, MDS, and AML-MRC patients. (C) Normal (NL) expression of lymphoid marker (CD7) on myeloblasts in control patient and overexpression of CD7 on myeloblasts (indicated by red arrow) in CCUS, MDS, and AML-MRC patients. (D) Normal (NL) expression of mature myeloid marker (CD11b) on myeloblasts in control and CCUS patients and overexpression of CD11b on myeloblasts (indicated by red arrow) in MDS and AML-MRC patients. (E) Normal (NL) expression of CD38 measured by median fluorescence intensity (MFI) on myeloblasts in control and CCUS patients and underexpression of CD38 on myeloblasts (indicated by red arrow) in MDS, and AML-MRC patients. (F) Normal (NL) expression of CD117 measured by MFI on myeloblasts in control and AML-MRC patients and overexpression of CD117 on myeloblasts (indicated by red arrow) in CCUS and MDS patients. (G– I) Normal maturation pattern (expression of CD13, CD33, and CD38 indicated by black arrow) of myeloblasts in control patient and abnormal maturation pattern of myeloblasts in CCUS, MDS, and AML-MRC patients. (J) Normal (NL) expression of lymphoid marker (CD56) on monocytes in control patient and overexpression of CD56 on monocytes (indicated by red arrow) in CCUS, MDS, and AML-MRC patients. (K) Normal (NL) expression of monocytic marker (CD36) on granulocytes in control patient and overexpression of CD36 on granulocytes (indicated by red arrow) in CCUS, MDS, and AML-MRC patients

The most frequent aberrancies expressed on myeloblasts in CCUS group were altered expression of CD117 (30%, n = 9, Figure S2A) and CD13 (25.9%, n = 7, Figure S2B) and overexpression of CD7 (18.5%, n = 5). The other abnormalities included underexpression of CD38 and HLA-DR (7.4%, n = 2) and overexpression of CD2 and CD11b (3.7%, n = 1). MDS and AML-MRC patients showed similar aberrancies. Interestingly, overexpression of CD56 on myeloblasts was not detected in any of CCUS patients but present at higher frequency in patients with MDS-EB and AML-MRC (p = .051 and .02). Overexpression of CD56 on myeloblasts was detected in 1 MDS-SLD (3.7%), 1 MDS-MLD (4.8%), 5 MDS-EB (19.2%), and 6 AML-MRC patients (25%). 38.5% (n = 5) of patients with CD56⁺ myeloblasts also harbored a TP53 mutation.

The most frequent aberrancies expressed on monocytes in CCUS group were underexpression of CD14 (21.8%, n = 7, Figure S2C), CD64 (18.8%, n = 6), CD11b (15.6%, n = 5), CD13 (12.5%, n = 4), and CD36 (8.3%, n = 3). The other abnormalities included overexpression of CD56 in two patients with SRSF2 mutation (6.3%), underexpression of CD33 (6.3%, n = 2), overexpression of CD15 and CD123 (3.2%, n = 1), and underexpression of HLA-DR (3.2%, n = 1). MDS and AML-MRC patients showed similar aberrancies. The frequency of HLA-D R underexpression was significantly increased in patients with MDS-MLD, MDS-EB, and AML-MRC compared with CCUS patients (p = .0001– .008). The frequency of CD36 underexpression was also increased in patients with MDS-MLD and MDS-EB compared with CCUS patients (p = .001 and .0001). CD56⁺ monocytes were present in 2 MDS-SLD, 2 MDS-MLD, 5 MDS-EB, and 3 AML-MRC patients. 28.6% (n = 4) and 21.4% (n = 3) of patients with CD56+ monocytes harbored a TP53 mutation and a SRSF2 mutation, respectively.

Underexpression of CD33 and overexpression of HLA-DR, CD36, and CD56 were evaluated on granulocytes. CCUS group showed overexpression of CD36 (Figure S2D) and underexpression of CD33 in 3 and 2 patients, respectively. MDS and AML-MRC patients showed similar aberrancies. Overexpression of HLA-DR on granulocytes was not detected in any of CCUS patients but present at higher frequency in patients with MDS-EB and AML-MRC (p = .001 and .0006). Overexpression of CD56 on granulocytes was present in 1 MDS-MLD, 4 MDS-EB, and 2 AML-MRC patients, but not detected in CCUS group. 42.8% (n = 3) of patients with CD56⁺ granulocytes harbored a TP53 mutation.

In our study, only MDS-EB and AML-MRC patients showed significantly more abnormal maturation of myeloblasts compared with CCUS patients (Table 1). Therefore, we assessed the baseline of MFC parameters on myeloblasts in 23 negative control patients. The results are summarized in Table S3. Negative control patients showed low level of MFC alterations including aberrant antigen expression (0.86/case), altered maturation pattern (17.4%), reduced type I hematogones (34.8%), and decreased SSC of granulocytes (4.3%). These alterations were not specific and did not meet the diagnostic criteria of neoplastic myeloblasts.

We then compared these MFC findings between negative control and other groups (Table S4). CCUS and MDS-SLD patients displayed significantly higher frequency of abnormal maturation of myeloblasts compared with negative control. MDS-SLD patients also showed significantly elevated blast count. In contrast, high-grade MDS and AML patients demonstrated significant differences in all evaluated MFC parameters. These findings suggest altered maturation pattern of myeloid blasts might be an early MFC detectable event in CCUS and MDS-SLD patients.

3.5 |. Flow cytometry comparison in CCUS patients based on high-risk mutation or variant allelic fraction

We also evaluated the impact of “high-risk” mutations on MFC alterations in CCUS patients. These mutations included ASXL1, SRSF2, GATA2, RUNX1, EZH2, TP53, ETV6, IDH2, and U2AF1 and were reported to associate with poor outcome in CCUS patients.⁵ We identified nine CCUS patients with high-risk mutations. These high-risk CCUS patients showed significantly lower blast count and higher frequencies in aberrant antigen expression and reduction of type I hematogones compared with low-risk CCUS patients (Table 2). They also displayed similar MFC abnormalities as MDS-SLD patients. In contrast, low-risk CCUS patients had significantly lower frequencies in aberrant antigen expression and reduction of type I hematogones compared with MDS-SLD patients.

TABLE 2.

Comparison of flow cytometry findings in CCUS with low-or high-risk mutations and MDS patients

Flow cytometry	Low-risk CCUS+ (n = 28)	High-risk CCUS (n = 9)	Low-risk vs. high-risk p-value	Low-risk vs. MDS-SLD p-value	High-risk vs. MDS-SLD p-value
Blasts (%)	0.78 (0.01– 3)	0.4 (0.1– 0.9)	.03	.4	.08
Abn marker numbers	0.65 (0– 2)	2.25 (1– 4)	.002	.03	.10
Abnormal maturation	38% (6/16)	75% (6/8)	.19	.12	.7
Decreased hematogone	8.0% (2/25)	44.4% (4/9)	.03	.0008	1.0
Decreased SSC	7.6% (2/26)	22.2% (2/9)	.26	.6	.1

Abbreviations: Abn, abnormal; CCUS, clonal cytopenia of undetermined significance; MDS-SLD, myelodysplastic syndrome with single linage dysplasia with or without ring sideroblasts; SSC, side scatter.

We then evaluated the impact of high level of VAF of >20% on MFC alterations in CCUS patients. We identified 14 CCUS patients with a VAF of >20%. These patients showed significantly lower blast count and no significantly statistical differences in other MFI findings compared with the rest of the patients (data not shown).

3.6 |. Genomic and cytogenetics comparison in patients with CCUS, MDS, and AML

As illustrated in Table 1, CCUS patients carried 0.44 pathogenic variants, 0.44 likely pathogenic variants, and 2.8 VUS per case. In comparison, MDS and AML patients carried 1.13 and 1.63 pathogenic variants, 0.64 and 0.92 likely pathogenic variants, and 1.74 and 2.8 VUS per case, respectively. CCUS patients displayed significantly lower frequency of pathogenic variants compared with patients with MDS-MLD, MDS-EB, and AML-MRC (Table 1, p = .0003– .01). CCUS patients also displayed higher frequency of VUS compared with MDS patients (p = .01– .07). CCUS patients displayed similar frequencies of likely pathogenic variants compared with MDS and AML patients. CCUS carried low frequency of pathogenic variant of mutation compared with MDS-SLD patients (p = .07). Lastly, CCUS patients also harbored significantly fewer cytogenetic abnormalities compared with MDS-MLD, MDS-EB, and AML-MRC patients. Five CCUS patients carried a karyotype of -Y chromosome (Table S2). The rest of CCUS patients showed normal diploid karyotype. There were no significant differences in cytogenetic abnormalities in both CCUS and MDS-SLD patients. Negative control patients had no cytogenetic abnormalities.

We next evaluated and compared mutational distribution based on the function of the proteins encoded by the effected genes in different groups of patients (Tables 3 and 4). TET2, DNMT3A, and ASXL1 mutations were the most common pathogenic variants, likely pathogenic variants, and VUSs in CCUS patients, which were consistent with age-related clonal hematopoiesis.¹⁸ Compared with MDS patients, CCUS patients harbored significantly lower frequency of deleterious mutations in splicing factors (p = .0001– .02). SF3B1 mutations were the most common splicing mutation in MDS-SLD and MDS-MLD patients. In contrast, SF3B1 mutations were not present in any CCUS patients. SRSF2 mutations were identified in 4 CCUS patients (Tables S2).

TABLE 3.

Somatic mutation distribution in different groups of patients

	CCUS (n = 37)			MDS-SLD (n = 28)			MDS-MLD (n = 21)			MDS-EB (n = 26)			AML-MRC (n = 24)
Mutation	Path	Likely	VUS	Path	Likely	VUS	Path	Likely	VUS	Path	Likely	VUS	Path	Likely	VUS
Epigenetic modifier TET2/DNMT3A/ASXL1	6	5	14	4	4	11	4	4	5	7	5	2	8	4	4
Other epigenetic modifiers IDH1/2/EZH1/SETBP1/KDM6A	1	0	2	2	0	0	2	0	0	2	1	1	5	0	2
Splicing mutation SF3B1/SRSF2/U2AF1/2	3	1	1	13	4	2	12	2	2	9	0	1	4	3	2
Signaling transduction FLT3/KIT/NRAS/KRAS/PTPN11/CBL/CBLB/C/MPL	4	0	10	0	0	2	3	0	1	4	0	2	5	1	2
Transcription factor RUNX1/CEBPA/GATA1/2/BCOR/BCORL1/NPM1/ETV6/PHF6	0	1	5	0	0	1	2	4	3	4	4	3	4	5	6
Tumor suppressor TP53/WT1	1	2	0	2	0	1	2	0	0	4	2	1	9	0	2
Cohesion STAG2/SMC1A	0	0	0	0	0	0	0	2	0	3	2	1	0	3	2
Others	1	4	6	2	0	29	3	4	26	2	4	33	1	4	41

Abbreviations: AML-MRC, acute myeloid leukemia with myelodysplasia-related changes; CCUS, clonal cytopenia of undetermined significance; MDS-EB, myelodysplastic syndrome with excess blasts; MDS-MLD, myelodysplastic syndrome with multilineage dysplasia with or without ring sideroblasts; MDS-SLD, myelodysplastic syndrome with single linage dysplasia with or without ring sideroblasts; VUS, variant of uncertain significance.

TABLE 4.

Comparison of mutations in CCUS versus others

	CCUS vs. MDS-SLD			CCUS vs. MDS-MLD			CCUS vs. MDS-EB			CCUS vs AML-MRC
Mutation	Path	Likely	VUS	Path	Likely	VUS	Path	Likely	VUS	Path	Likely	VUS
Epigenetic modifier TET2/DNMT3A/ASXL1	1.00	1.00	1.00	1.0	0.71	0.38	0.35	0.73	0.008	0.13	0.73	0.09
Other epigenetic modifiers IDH1/2/EZH1/SETBP1/KDM6A	0.57	1.00	0.51	0.55	1.00	0.53	0.56	0.42	1.00	0.03	1.00	0.64
Splicing mutation SF3B1/SRSF2/U2AF1/2	0.0009	0.16	0.57	0.0001	0.55	0.55	0.02	1.0	1.00	0.42	0.29	0.56
Signaling transduction FLT3/KIT/NRAS/KRAS /PTPN11/CBL/CBLB/C/MPL	0.13	1.00	0.05	0.70	1.00	0.04	0.71	1.00	0.10	0.29	0.39	0.10
Transcription factor RUNX1/CEBPA/GATA1/2/BCOR/BCORL1/NPM1/ETV6/PHF6	1.00	1.00	0.22	0.12	0.05	1.00	0.03	0.15	1.00	0.02	0.03	0.32
Tumor suppressor TP53/WT1	0.57	0.51	0.13	0.13	1.00	0.40	0.15	1.00	0.73	0.0006	0.51	0.13
Cohesion STAG2/SMC1A	1.00	1.00	1.00	1.00	0.12	1.00	0.07	0.17	0.41	1.00	0.056	0.15

Abbreviations: AML-MRC, acute myeloid leukemia with myelodysplasia-related changes; CCUS, clonal cytopenia of undetermined significance; MDS-EB, myelodysplastic syndrome with excess blasts; MDS-MLD, myelodysplastic syndrome with multilineage dysplasia with or without ring sideroblasts; MDS-SLD, myelodysplastic syndrome with single linage dysplasia with or without ring sideroblasts; VUS, variant of uncertain significance.

Compared with MDS-EB and AML-MRC patients, CCUS patients harbored significantly lower frequency of deleterious mutations in transcription factors, that is, only 1 likely pathogenic variant in PHF6. CCUS patients also harbored a significantly lower frequency of mutations in tumor suppressors, particularly TP53 compared with AML-MRC patients. One pathogenic variant and 1 likely pathogenic variant of TP53 were identified in CCUS patients. Two out of 3 MDS-EB and 7 out of 9 AML-MRC patients also showed deletion or loss of chromosome 17 in addition to a TP53 mutation (Tables S2). MDS-SLD and MDS-MLD patients carried significantly lower frequency of mutations in tumor suppressors compared with AML-MRC patients (p = .009 and .038).

Because acquisition of splicing mutations leading to the development of morphologic dysplasia is the first step of progression from CCUS to MDS,¹ we further evaluated morphologic and MFI findings in these four CCUS patients carrying SRSF2 mutations. Interestingly, all four patients showed <10% dysplasia in one-or two-cell lineage. Furthermore, MFC studies showed aberrant antigen expression on myeloblasts (n = 3), abnormal maturation of myeloblasts (n = 3), reduction of type I hematogones (n = 3), decreased SSC of granulocytes (n = 2), and abnormal monocytes with CD56 expression (n = 2).

3.7 |. Evaluation of immunophenotypic, molecular genetics, and cytogenetic changes during disease progression

We then evaluated immunophenotypic, molecular genetics, and cytogenetic changes in 9 patients who had disease evolution (Table S5). One CCUS patient (#1) with mutations of SRSF2, TET2, NRAS, and PHF progressed to MDS-MLD within 50 months. While immunophenotypic and cytogenetic abnormalities remained unchanged, NRAS mutation was no longer present at the diagnosis of MDS. One MDS-SLD patient (#2) gained MFC aberrancies and pathogenic variant of NBN mutation during progression to MDS-MLD. One MDS-EB patient (#3) had a prior history of MDS-SLD and acquired additional MFC abnormalities and SRSF2 and ASXL1 mutations. Three AML-MRC patients (#4, #8, and #9) showed increased frequencies of MFC aberrancies, whereas one patient (#5) presented blasts with completely different immunophenotype compared with that in low-grade disease. One leukemic patient (#7) evolved from MDS-MLD to MDS-EB1 to AML. This patient had additional MFC aberrancies at onset of MDS-EB1 and sequential gain of RUNX1, BCOR, and CEBPA mutations during transformation from low-grade MDS to AML. All AML patients acquired additional somatic mutations including TP53 and 3 AML patients showed cytogenetic evolutions.

4 |. DISCUSSION

In our current study, flow cytometry analysis demonstrated progressive accumulation of abnormalities when we compared CCUS group with low-grade MDS, high-grade MDS, or AML-MRC group. The accumulation of MFC aberrancies were consistent with morphologic progression. CCUS patients showed significantly less reduction of type I hematogones compared with MDS-SLD patients. CCUS patients demonstrated significantly lower frequencies in altered antigen expression on myeloblasts, monocytes and granulocytes, reduction of type I hematogones, and decreased SSC of granulocytes compared with high-grade MDS or AML patients. Lastly, gain of additional MFC aberrancies occurred in six out of eight MDS patients during disease progression.

Both CCUS with sub-diagnostic dysplasia and CCUS without dysplasia patients were reported to display significantly fewer MFC abnormalities compared with MDS patients.⁶ Our current study was consistent with this finding. Dimopoulos et al showed that MFC had lower diagnostic sensitivity to differentiate patients with idiopathic cytopenia of undetermined significance from patients with CCUS.⁵ However, MFC could identify a group of patients with higher degree of morphologic dysplasia and higher mutation burden. Our study supported the positive correlation between the severity of morphologic dysplasia and the degree of flow cytometric abnormalities.

Nirmalanantham et al. demonstrated MFC abnormalities in CCUS patients with high-risk mutations were similar to low-grade MDS. In addition, they also showed the frequency of MFC abnormalities in CCUS with >20% VAF was in between low-grade MDS and control.⁷ Our current study showed similar findings in CCUS cases with high-risk mutations. However, CCUS patients with >20% VAF did not show significantly more MFC abnormalities compared with the rest of patients in our study, probably due to small sample size.

Normal myeloid blast maturation by assessment of CD13, CD33, and CD38 expression was reported.^9,11 In this study, we demonstrated that alteration of myeloblast maturation was an early abnormality detected by MFC when we compared CCUS and MDS-SLD patients with negative control patients. Reduction of type I hematogones occurred next when we compared CCUS group with MDS-SLD group. Indeed, preservation of CD34+ hematogones was reported in low-grade MDS.^7,19 We also showed reduction of type I hematogones only occurring in 51.8% of MDS-SLD patients while being present in 84.6%– 90.5% patients with high-grade MDS or AML-MRC (p < .05).

CD56+ myeloblasts were enriched in patients with MDS-EB and AML-MRC but not detected in any of CCUS patients in current study. Nirmalanantham et al. demonstrated that CD56+ blasts were more frequently present in MDS than in CCUS patients.⁷ Interestingly, close to one third of patients with CD56+ myeloblasts, monocytes or granulocytes carried a TP53 mutation. More studies to evaluate the relationship between CD56 expression and TP53 mutation are needed.

Our study showed CCUS patients carried significantly fewer somatic mutations in splicing factors and similar frequency of somatic mutations in TET2/DNMT3A/ASXL1 compared with MDS patients. These findings were consistent with previous observations that mutations in genes coding for splicing factors and epigenetic modulators were early events in the evolution of MDS.^1,20 A recent clinical, molecular, and prognostic comparison study from Mayo Clinic demonstrated low-grade MDS patients were likely to carry more SF3B1 mutation and similar mutation distribution in TET2, DNMT3A, ASXL1, and SRSF2 compared with CCUS patients.²¹ Our study showed similar findings. Jajosky et al. demonstrated CCUS patients with sub-diagnostic dysplasia were enriched with MDS-type of mutations compared with CCUS patients without dysplasia.⁶ All four cases of CCUS with mutation in splicing factors also showed <10% dysplasia in one-or two-cell lineage in our study.

Our study showed MDS-EB and AML-MRC patients harbored significantly more somatic mutations in transcription factors compared with CCUS patients. Previous studies showed mutations in genes coding for transcription factors such as RUNX1, GATA2, and CEBPA, played important roles in progression of MDS to AML.¹ AML-MRC patients carried significantly more somatic mutations in tumor suppressor genes such as TP53 in our study. Our study also showed MDS patients gained RUNX1, CEBPA, and TP53 mutations during disease progression. Furthermore, seven out of nine AML-MRC patients and two out of three MDS-EB patients with a TP53 mutation displayed deletion or loss of chromosome 17. These findings further supported the important role of biallelic inactivation of TP53 in leukemic progression.²²

The study presented is limited by lack of follow-up in most of CCUS patients. In addition, our flow cytometry panel did not include markers to evaluate erythroid dysplasia and granulocyte maturation pattern. Erythroid dysplasia was only evaluated by morphology. Nevertheless, our study supports the notion that CCUS is an immediate precursor to low-grade MDS. The authors from Mayo Clinic proposed to include CCUS-HighVAF (>20%), as an MDS subtype in the next edition of the WHO classification system due to significant clinical and molecular overlap between these two groups.²¹ All four CCUS cases with SRSF2 mutations in current study showed sub-diagnostic morphologic dysplasia and significant flow cytometric abnormalities including decreased SSC of granulocytes. One of these patients progressed to MDS within 50 months.

In summary, we demonstrated a stepwise acquisition of somatic mutations in gene coding for splicing factors, transcription factors, and tumor suppressors, which were responsible for progression from CCUS to MDS then ultimately AML transformation. This progressive acquisition of somatic mutations also paralleled morphologic progression in dysplasia and increased blast counts in MDS and leukemic transformation in AML-MRC as well as progressive accumulation of MFC abnormalities.

DATA AVAILABILITY STATEMENT

Data available in article supplementary material