Targeted next generation sequencing and identification of risk factors in World Health Organization defined atypical chronic myeloid leukemia

Abstract

Atypical chronic myeloid leukemia (aCML) is an aggressive myeloid neoplasm with overlapping features of myelodysplastic syndromes (prominent granulocytic dysplasia) and myeloproliferative neoplasms (neutrophilic leukocytosis). We studied 25 molecularly-annotated and World Health Organization defined aCML patients; median age 70 years, 84% males. Cytogenetic abnormalities were seen in 36% and gene mutations in 100%. Mutational frequencies were, ASXL1 28%, TET2 16%, NRAS 16%, SETBP1 12%, RUNX1 12%, ETNK1 8% and PTPN11 4%. Fifteen patients (60%) had >1 mutation, while 9 (36%) had ≥3. The median overall survival (OS) was 10.8 months and at last follow up (median 11 months), 17 (68%) deaths and 2 (8%) leukemic transformations were documented. On univariate analysis, survival was adversely impacted by advanced age (p=0.02), low hemoglobin (p=0.01), red blood cell transfusion dependence (p=0.03), high white blood cell count (p=0.02), TET2 (p=0.03), NRAS (p=0.04), PTPN11 (p=0.02) mutations and the presence of ≥3 gene mutations (p=0.006); ASXL1, SETBP1, and ETNK1 mutations did not impact OS. In multivariable analysis, advanced age (p=0.003) [age >67: HR 10.1, 95% CI 1.3–119], low hemoglobin (p=0.008) [HB< 10gm/dl: HR 8.2, 95% CI 1.6–23.2] and TET2 mutations (p=0.01) [HR 8.8, 95% CI 1.6–47.7] retained prognostic significance. We then used age >67 years, hemoglobin <10 gm/dl and the presence of TET2 mutations (each counted as one risk factor) to create a hazard ratio weighted prognostic model; effectively stratifying patients into two risk categories, low (0–1 risk factor) and high (≥2 risk factors), with median OS of 18 and 7 months respectively.

Introduction

Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1 negative, clonal, hematopoietic stem cell disorder, with overlapping features of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN).[1] Atypical CML is a rare disease, with an estimated frequency of 1 to 2 cases for every 100 patients with BCR-ABL1 rearranged CML.[2] The 2008 and recently revised 2016 World Health Organization (WHO) classification recommendations for the diagnosis of aCML include; 1) peripheral blood (PB) leukocytosis due to increased numbers of neutrophils and their precursors comprising ≥10% of leukocytes, 2) dysgranulopoiesis, which may include abnormal chromatin clumping, 3) no or minimal absolute basophilia (<2% of leukocytes), 4) no or minimal absolute monocytosis (<10% of leukocytes), 5) hypercellular bone marrow (BM) with granulocytic proliferation and granulocytic dysplasia, with or without dysplasia in the erythroid or megakaryocytic lineages, 6) <20% blasts in the PB and BM and 7) not meeting diagnostic criteria for BCR-ABL1 rearranged CML, primary myelofibrosis (PMF), polycythemia vera (PV) or essential thrombocythemia (ET). [1, 3] In addition, great caution is suggested while making a diagnosis of aCML in a patient with a previous history of MPN, or in the presence of MPN like features in the BM, or in the presence of MPN-associated driver mutations such as JAK2, CALR and MPL. [3]

The median overall survival (OS) in patients with aCML is ~ 25 months (range, 14–30 months), with high leukemic transformation rates (~40%, within 18 months of diagnosis). [2, 4, 5] Reported prognostic factors impacting survival include; age >65 years, female gender, leukocyte count >50 × 10(9)/L, hemoglobin <10 gm/dl and the presence of circulating immature myeloid cells (IMC).[2, 5] Next-generation sequencing (NGS) technology has identified recurrent mutations involving ASXL1, TET2, SETBP1, ETNK1, NRAS, EZH2, and U2AF1 amongst others, in affected patients. [6–8] SETBP1 mutations are seen in ~25% of patients and are associated with leukocytosis and a shorter OS.[6, 7] TET2 (~40%) and ETNK1 (~8%) mutations have also been reported in aCML, with an indeterminate prognostic impact. [6, 8] We performed this study to i) identify prognostically-relevant mutations in aCML, ii) determine the prognostic impact of the number of concurrent mutations; and iii) identify clinical and laboratory parameters impacting OS and leukemia-free survival (LFS).

Materials and Methods

Twenty-five patients with atypical CML were included in the study. All patients had bone marrow biopsies and cytogenetic studies performed at diagnosis. Pathology slides including PB smears, BM iron stains, and BM reticulin stains were centrally re-reviewed in order to accurately quantify BM fibrosis and BM atypia in concordance with the 2008 and 2016 WHO classification recommendations.[1, 3] Twenty-nine gene targeted capture assays were carried out on BM DNA specimens of all 25 patients obtained at diagnosis for the following genes; TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, KRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, ETNK1 and SETBP1.

Paired-end indexed libraries were prepared from individual patient DNA in the Mayo Clinic Genomic Sequencing Core Laboratory using the NEBNext Ultra Library prep protocol on the Agilent Bravo liquid handler (NEB, Ipswich, MA/Agilent Technologies Inc, Santa Clara, CA). Capture libraries were assembled according to Nimblegen standard library protocol (Roche Nimblegen, Inc, Basel, Switzerland). The panel including the regions of 27 heme-related genes was selected for custom target capture using Agilent SureSelect Target Enrichment Kit. Capture libraries were pooled at equimolar concentrations and loaded onto paired end flow cells at concentrations of 7–8 pM to generate cluster densities of 600,000–800,000/mm² following Illumina’s standard protocol using the Illumina cBot and HiSeq Paired end cluster kit version 3, in batches of 48 samples per lane (Illumina Incorporated, San Diego, CA). The flow cells were sequenced as 101 × 2 paired end reads on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and HiSeq data collection version 2.0.12.0 software. Base-calling was performed using Illumina’s RTA version 1.17.21.3.

Genesifter® software was utilized (PerkinElmer, Danvers, Massachusetts) to analyze targeted sequence data. Reads from the sequencing in fastq format were aligned using Burrows-Wheeler Aligner (BWA) against the genomic reference sequence for Homo sapiens (Build 37.2; NCBI http://www.ncbi.nlm.nih.gov/). An additional alignment, post-processing set of tools was then used to do local realignment, duplicate marking, and score recalibration to generate a final genomic aligned set of reads. Nucleotide variants were called using the Genome Analysis Toolkit (GATK -Broad Institute, Cambridge, MA), which identified single nucleotide and small insertion/deletion events using default settings. Specific variants were deemed as mutations if they are associated with a heme malignancy (as identified by COSMIC database), or if they have not been associated with a dbSNP.

Based on prior observations in chronic myelomonocytic leukemia (CMML), only frame shift and non-sense ASXL1 mutations were considered pathogenic.[9, 10] For TET2, frame shift, non-sense, and missense mutations, along with insertions and deletions were considered pathogenic.[11] Previously annotated single nucleotide polymorphisms (http//www.hapmap.org) in all the aforementioned genes, were considered non-pathogenic.

All statistical analyses considered parameters obtained at time of referral to the Mayo Clinic, which in most instances coincided with time of BM biopsy. Differences in the distribution of continuous variables between categories were analyzed by either Mann-Whitney (for comparison of two groups) or Kruskal-Wallis (comparison of three or more groups) test. Patient groups with nominal variables were compared by chi-square test. Over-all survival was calculated from the date of first referral to date of death (uncensored) or last contact (censored). Leukemia-free survival was calculated from the date of first referral to date of leukemic transformation (uncensored) or death/last contact (censored). Overall and LFS curves were prepared by the Kaplan-Meier method and compared by the log-rank test. Cox proportional hazard regression model was used for multivariable analysis. P values less than 0.05 were considered significant. Receiver operating characteristic (ROC) curves were used to find statistically significant discriminant thresholds for age and hemoglobin values, so that they could be utilized to develop an aCML specific prognostic model. The Stat View (SAS Institute, Cary, NC, USA) and JMP Version10 (SAS Institute Inc., Cary, NC, USA) statistical packages were used for all calculations.

Results

Twenty-five patients with WHO defined atypical CML, with a median age of 70 years (range, 49–91), 84% males, seen at the Mayo Clinic from 1990–2015, were included in this study. Table 1 lists the clinical and laboratory details and subsequent events in these patients, stratified by their TET2 mutational status. At a median follow up of 11 months (range, 1–29), 17 (68%) deaths and 2 (8%) leukemic transformations were documented. Five (62%) of 8 patients alive were lost to follow up after a median of 7 months (range, 0–13), while 3 patients are being actively followed with a median follow up of 12 months (range, 6–13). All patients had BM dysgranulopoiesis, whereas dyserythropoiesis and dysmegakaryopoiesis were present in 16% (4/25) and 20% (5/25), respectively. Of 22 evaluable patients, BM reticulin fibrosis (WHO grades1–2 of 3) was seen in 14 (67%). Sixteen (64%) patients were red blood cell transfusion dependent at diagnosis, and 11(44%) had palpable splenomegaly, of which, 3 (12%) had massive splenomegaly (spleen size palpable >10 cm below the left costal margin).

Table 1.

Clinical and laboratory features in 25 patients with World Health Organization defined atypical chronic myeloid leukemia (aCML), stratified by the presence or absence of TET2 mutations.

Variable	All patients with aCML (n= 25)	TET2 mutated aCML patients (n=4)	TET2 wild type aCML patients (n=21)	P value
Age in years; median (range)	70 (49–91)	68 (64–76)	71 (49–91)	0.5
Males; n (%)	21 (84%)	4 (100%)	17 (81%)	0.3
Hemoglobin g/dL; median (range)	9.1 (6.3–14.9)	9.1 (8.7–12.3)	9.7 (6.3–14.9)	0.6
MCV femtoliter; median (range)	96 (82–111)	92 (86–103)	97 (82–111)	0.7
WBC × 109/L; median (range)	32 (8.3–192.7)	27.2 (14.4–21.8)	34.6 (8.3–192.7)	0.7
ANC × 109/L; median (range)	20.4 (0.4–153.2)	19.7 (12–77.9)	20.7 (0.4–153.2)	0.9
AMC × 109/L; median (range)	1.0 (0–5.4)	1.6 (0.1–4.8)	1.0 (0–5.4)	0.9
ALC × 109/L; median (range)	2.1 (0–7.7)	3.6 (0.8–6.1)	2.1 (0–7.7)	0.6
ABC × 109/L; median (range)	0.5 (0–2.2)	0.1 (0–1.2)	0.5 (0–2.2)	0.4
AEC × 109/L; median (range)	0.6 (0–5.7)	0.6 (0–1.2)	0.6 (0–5.7)	0.7
Platelets × 109/L; median (range)	95 (12–647)	120 (95–398)	81 (12–647)	0.2
Presence of circulating immature myeloid cells; n (%)	25 (100%)	4 (100%)	21 (100%)	0.9
PB blast %; median (range)	1 (0–12)	1 (1–2)	1 (0–12)	0.9
BM blast % ; median (range)	2 (0–15)	1.5 (0–4)	3 (0–15)	0.4
BM cellularity %	95 (80–100)	92 (90–100)	95 (80–100)	0.7
BM dyserythropoiesis; yes (%)	4 (16%)	1 (25%)	3 (14%)	0.6
BM dysmegakaryocytopoiesis; yes (%)	5 (20%)	1 (25 %)	4 (19%)	0.8
Red blood cell transfusion dependence	16 (64%)	3 (75%)	13 (62%)	0.6
Palpable splenomegaly; n (%)	13 (52%)	2 (50%)	11 (52%)	0.9
Palpable splenomegaly>10 cm below LCM; n (%)	8 (32%)	2 (50%)	6 (29%)	0.4
*Cytogenetic abnormalities; n (%)
Normal	14 (67%)	2 (50%)	12 (57%)
Trisomy 8	6 (27%)	2 (50%)	4 (21%)	0.6
Trisomy 9	1 (4%)	0 (0%)	1 (5%)
Trisomy 21	1 (4%)	0 (0%)	1 (5%)
Leukemic transformations; n (%)	2 (8%)	0 (0%)	2 (10%)	0.5
Deaths; n (%)	17 (68%)	3 (75%)	14 (67%)	0.7
Median follow up in months (range)	11 (1–29)	5 (1–6)	13 (1–29)	0.03

Key: aCML- Atypical Chronic Myeloid Leukemia, MCV- mean corpuscular volume, WBC- white blood cell count; ANC- absolute neutrophil count; AMC- absolute monocyte count; ALC- absolute lymphocyte count; ABC-absolute basophil count; AEC-absolute eosinophil count; PB- peripheral blood; BM- bone marrow; LCM- lower costal margin.

^*Cytogenetic studies were available for 23 patients with atypical chronic myeloid leukemia at diagnosis. In this study loss of chromosome Y was considered normal. One patient showed the following cytogenetic abnormality – 46,XY, inv(6)(p11.2q25)?c[30]- that was considered constitutional.

Cytogenetic information was available in 22 (88%) patients, with 8 (36%) displaying an abnormal karyotype. There were 6 (23%) patients with trisomy 8, one with trisomy 8 along with an associated structural abnormality (47, XY,+8[4]/47,idem,del (12)(p11.2) [16]) ; while there were one (5%) each, with trisomy 9 and trisomy 21, respectively. All patients tested negative for BCR-ABL1 by conventional metaphase cytogenetics and/or fluorescence in situ hybridization techniques. There was no statistically significant difference in the occurrence of cytogenetic abnormalities in the TET2 mutated and wild type aCML patients (p=0.6).

Mutational frequencies were 28% (7/25) for ASXL1, 16% (4/25) for TET2 and NRAS, 12% (3/25) for SETBP1, RUNX1, and SRSF2, 8% (2/25) for ETNK1, SF3B1, KRAS, EZH2, CSF3R, and JAK2V617F, and 4% (1/25) for IDH2, ZRSR2, PTPN11 and FLT3-TKD, respectively. No mutations were found in the following genes; Tp53, CALR, MPL, IDH1, NPM1, CEBPA, U2AF1, IKZF, SUZ12, BCOR, SH2B3 and CBL. ASXL1 was the most commonly mutated gene (28%), with 3 nonsense and 4 frame-shift mutations. The most common ASXL1 mutation was c.1888_1907del p.H630Gfs*21(43%), followed by c.1934dupG; p.G646WfsX12 (28%). TET2 mutations (n=4) were associated with advanced age (p=0.03), with 2 (50%) of 4 patients having two different TET2 mutations each. SRSF2 mutations were seen in 3 patients, with 2 demonstrating the c.284C>A; p.P95H change and 1 demonstrating the c.284_307del; p.P95_R102del deletion. Only 1 (33%) of three SRSF2 mutated patients had a concomitant TET2mutation; and in this patient the absolute monocyte count (AMC) was 0.2 × 10(9)/L, with no BM monocytosis and a negative BM dual esterase stain, thus ruling out a diagnosis of chronic myelomonocytic leukemia (CMML). There were 3 patients with SETBP1 mutations; all of whom demonstrated the c.2062G>A; p. D868N change. There were 2 patients with ETNK1 mutations, both of whom demonstrated the c.731A>G; p.N244S change. Both these patients had normal absolute eosinophil counts. There were two (8%) patients with the JAK2V617F mutation, with WHO grade 1 BM fibrosis, in whom a diagnosis of primary myelofibrosis was excluded. There were two (8%) patients with CSF3R mutations, both demonstrating the c.1853C>T T618I change. These patients had both, marked neutrophilia (absolute neutrophil count > 50 × 10(9)/L) and marked granulocytic dysplasia, meeting WHO criteria for a diagnosis of aCML. Bone marrow ring sideroblasts were not detected in the 2 SF3B1 mutated patients with aCML. TET2 mutated aCML patients were more likely to have concurrent mutations involving SH2B3, NRAS and PTPN11, in comparison to their wild type counterparts (table 1).

Treatment data was available on all 25 patients. Fifteen (60%) patients received hydroxyurea for the management of progressive myeloproliferation, while 4 (16%) received erythropoiesis stimulating agents (ESA) for anemia. One patient each received interferon alpha, thalidomide and lenalidomide as disease modifying agents. In all three cases, the respective agents were discontinued due to poor response and poor tolerability. Five (25%) patients were treated with hypomethylating agents (HMA) for refractory anemia and concomitant myeloproliferation; 4 (16%) with 5-azacitidine and 1 (4%) with decitabine. 5-Azacitidine was administered for a mean of 5 cycles (range, 1–10), with the best response being stable disease (40%), while one patient received 2 cycles of decitabine and had to discontinue the same due to progressive neutrophilia. No patients underwent allogeneic stem cell transplantation. Cause of death was known in 7 (41%) patients and included 2 (28%) deaths secondary to complications from leukemic transformation, 3 (43%) from complications related to sepsis and one each (14%) from subarachnoid hemorrhage and intracerebral hemorrhage, respectively. The remaining deaths occurred outside our institution and were attributed to the patients underlying disease; however the exact cause of death could not be ascertained.

i) Predictors of over-all survival

The median OS for the cohort was 10.8 months. In an univariate OS analysis which included the following; gender (p=0.7), AMC (p=0.6), absolute lymphocyte count (p=0.2), platelet count (p=0.2), peripheral blood (p=0.9) and BM blasts (p=0.3), circulating IMC (p=0.07), palpable splenomegaly (p=0.6), BM reticulin fibrosis (p=0.4), abnormal karyotype (p=0.4), and myeloid-relevant gene mutations (as listed in Table 2), advanced age (p=0.02), low hemoglobin (p=0.01), red blood cell transfusion dependence (p=0.03), high white blood cell count (WBC, p=0.02), high absolute neutrophil count (ANC, p=0.023), TET2 (p=0.03), NRAS (p=0.04), PTPN11 (p=0.02) mutations and the presence of ≥3 myeloid-relevant gene mutations (p=0.006), adversely impacted OS. Interestingly on univariate analysis ASXL1, SETBP1, SRSF2 and ETNK1 mutations did not impact OS. In multivariable analysis that included advanced age, low hemoglobin, high WBC, high ANC, red blood cell transfusion dependence, TET2, NRAS, PTPN11 mutations and ≥3 concurrent mutations; advanced age (p=0.003) [age >67: HR (hazard ratio) 10.1, 95% CI 1.3–119], low hemoglobin (p=0.008) [HB< 10gm/dl: HR 8.2, 95% CI 1.6–23.2] and TET2 mutations (p=0.01) [HR 8.8, 95% CI 1.6–47.7] retained independent prognostic significance. Statistically significant hemoglobin and age discriminants were determined using ROC curve analysis.

Table 2.

Next-Generation DNA Sequencing (NGS) analysis data in 25 patients with World Health Organization defined atypical chronic myeloid leukemia (aCML), stratified by the presence or absence of TET2 mutations.

Gene	All patients with aCML (n= 25)	TET2 mutated aCML patients (n=4)	TET2 wild type aCML patients (n=21)	P value
Next-generation sequencing analysis; n (%)
1. Epigenetic regulators
DNMT3A	0 (0%)	0 (0%)	0 (0%)	-
IDH1	0 (0%)	0 (0%)	0 (0%)	-
IDH2	1 (4%)	0 (0%)	1 (5%)	0.7
2. Chromatin regulation
ASXL1	7 (28%)	2 (50%)	5 (24%)	0.3
EZH2	2 (8%)	0 (0%)	2 (10%)	0.5
SUZ12	0 (0%)	0 (0%)	0 (0%)	-
3. Transcription factors
RUNX1	3 (12%)	0 (0%)	3 (14%)	0.4
4. Spliceosome components
SF3B1	2 (8%)	0 (0%)	2 (10%)	0.5
SRSF2	3 (12%)	1 (25%)	2 (10%)	0.4
U2AF1	0 (0%)	0 (0%)	0 (0%)	-
ZRSR2	1 (4%)	0 (0%)	1 (5%)	0.7
5. Cell signalling
JAK2V617F	2 (8%)	1 (25%)	1 (5%)	0.2
CALR	0 (0%)	0 (0%)	0 (0%)	-
MPL	0 (0%)	0 (0%)	0 (0%)	-
SH2B3	1 (4%)	1 (25%)	0 (0%)	0.02
CBL	0 (0%)	0 (0%)	0 (0%)	-
KRAS	2 (8%)	1 (25%)	1 (5%)	0.2
NRAS	4 (16%)	2 (50%)	2 (10%)	0.04
PTPN11	1 (4%)	1 (25%)	0 (0%)	0.02
CSF3R	2 (8%)	1 (25%)	1 (5%)	0.2
C-KIT	1 (4%)	0 (0%)	1 (5%)	0.7
FLT3TKD	1 (4%)	0 (0%)	1 (5%)	0.7
NPM1	0 (0%)	0 (0%)	0 (0%)	-
6. Tumor suppressor genes
Tp53	0 (0%)	0 (0%)	0 (0%)	-
7. Others
SETBP1	3 (12%)	1 (25%)	2 (10%)	0.5
ETNK1	2 (8%)	0 (0%)	2 (10%)	0.3
BCOR	0 (0%)	0 (0%)	0 (0%)	-

Key: aCML- atypical chronic myeloid leukemia, NGS- next-generation sequencing analysis, DNA- deoxyribonucleic acid.

Based on these results, we created a HR weighted prognostic model that effectively risk stratifies patients with aCML. Patients received 1 point each for age >67 years, hemoglobin <10 gm/dl and the presence of TET2 mutations. Stratification categorized patients into two risk categories; low (0–1 points), and high (≥2 points), with median OS of 18 and 7 months respectively (figure one).

Figure 1.

Over-all survival of 25 patients with World Health Organization (WHO) defined atypical chronic myeloid leukemia (aCML) stratified by the Mayo prognostic model for aCML.

ii) Leukemic transformation

There were 2 (2%) leukemic transformations documented. Given the low number, a formal LFS analysis could not be carried out. The first patient was an 83-year-old man who was diagnosed to have aCML with normal cytogenetics. TET2 and PTPN11 mutations were detected at diagnosis. He was treated with hydroxyurea and three months after diagnosis, transformed to acute myeloid leukemia (AML) with normal cytogenetics. Given his age and comorbidities he elected for supportive care measures. The second patient was a 73-year-old woman who was diagnosed to have aCML with normal cytogenetics, with ASXL1 and JAK2V617F mutations. She received ESA for anemia without response and within 6 months of diagnosis transformed to AML. She too elected for supportive care measures. Both patients had passed away at last follow up.

Discussion

Atypical CML is an uncommon BCR-ABL1 negative myeloid neoplasm with overlapping features of MDS and MPN.[3] Important features include the presence of neutrophilic leukocytosis (≥ 13 × 10 (9)/L) with left shift, prominent granulocytic dysplasia (immature granulocytes ≥10% of leukocytes) with or without multilineage dysplasia and <20% blasts in the PB and BM.[3] Most patients have hypercellular BM without significant monocytosis or basophilia (<2% PB basophils and <10% PB monocytes).[3] Myeloid neoplasms in the differential diagnosis include, chronic neutrophilic leukemia (CNL), an entity associated with marked neutrophilic leukocytosis with immature granulocytes comprising <10% of leukocytes and CMML, a MDS/MPN overlap syndrome characterized by absolute monocytosis (PB AMC >1 × 10(9)/L for >3 months) and BM dysplasia.[12] In concordance with prior studies, the median age at presentation in our study was 70 years, with a male preponderance (84%). Bone marrow dysgranulopoiesis was seen in all (100%) patients, while dyserythropoiesis and dysmegakaryopoiesis were present in 16% and 20%, respectively. The median survival was 10.8 months (similar to prior estimates of 14–30 months).[2, 5]

The molecular pathogenesis of aCML remains elusive, with no specific recurrent genomic or karyotypic association. The frequency of karyotypic abnormalities reported in literature varies between 20–80%, with trisomy 8 and del(20q) being common.[2, 4, 5] In our series 36% had an abnormal karyotype; 75% with trisomy 8 and 5% each, with trisomy 9 and trisomy 21, respectively. There were no cases with a complex or monosomal karyotype. NGS studies have identified recurrent gene mutations involving; ASXL1, TET2, SETBP1, EZH2, CBL, JARID2, IDH2, CEBPA, KRAS, NRAS, JAK2V617F, SUZ12, RUNX1, ETNK1 and EED in aCML. [6–8, 13] Amongst these, commonly mutated genes include ASXL1 (~30%), TET2 (~40%) and SETBP1 (~25%).[7, 8] ASXL1 mutations are seen in a variety of myeloid neoplasms and have an independent negative prognostic impact in CMML, CNL and PMF, with a less clear impact in aCML.[10, 11, 14–16] In our series, 28% had nonsense and frameshift ASXL1 mutations, the most common being, c.1888_1907del p.H630Gfs*21(43%), with no impact on survival. TET2 mutations alter DNA methylation/hydroxy-methylation and are common in myeloid malignancies, especially CMML(~60%), with an unclear prognostic impact.[11, 17] Thus far in aCML, their exact prevalence and impact remains to be elucidated. In our series, 16% had TET2 mutations, with the presence of these mutations independently and adversely impacting survival. With the exception for a higher frequency of SH2B3, NRAS and PTPN11 mutations in TET2 mutated patients, there were no other clinical or laboratory differences with their wild type counterparts. Similar to CMML, 2 of 4 (50%) patients had >1 TET2 mutations, with a strong correlation with increasing age (p=0.03).[17] SETBP1 mutations are seen in ~25% of aCML patients and are generally associated with leukocytosis and inferior survival.[4, 7] In our series 12% (n=3) had SETBP1 mutations, all demonstrating the c.2062G>A; p. D868N change, without a significant association with leukocytosis or impact on survival. The set binding protein (SETBP1) interacts with SET, a negative regulator of the tumor suppressor protein phosphatase 2A (PP2A), protecting SET from cleavage; thus repressing the activity of PP2A. SETBP1 mutations are seen in ~15% of CMML and ~11% of CNL patients.[10, 18]

ETNK1 encodes an ethanolamine kinase, which catalyzes the first step of the de novo phosphatidylethanolamine biosynthesis pathway, critical for regulating membrane architecture and the topology of transmembrane domains of membrane biding proteins. Lasho et al. first described 10 ETNK1 mutations (N244S=6, N244T=1, N244K=1, G245A=2) in 290 patients with myeloid neoplasms and idiopathic hypereosinophilia.[13] The distribution of mutations was as follows; systemic mastocytosis (SM) 6% (n=82), CMML 14% (n=29), idiopathic hypereosinophilia <1% (n=139) and PMF 0% (n=32).[13] In SM, these mutations were often associated with concomitant eosinophilia (~20%). Subsequently in a larger European data set (n=515) these mutations were documented in ~8% (n=68) of aCML and ~3% of CMML (n=77) patients, with an unclear prognostic impact.[6] In our series there were 2 (8%) patients with ETNK1 mutations, both of whom demonstrated the c.731A>G; p.N244S change, with no associated eosinophilia and without an impact on survival. The 2016, WHO revision recommendations for aCML urge caution while establishing a diagnosis in a patient with a previous history of MPN, or in the presence of MPN-associated driver mutations such as JAK2, CALR and MPL.[3] While there were no CALR or MPL mutations seen, two (8%) patients did have JAK2V617F mutations, with variant allele frequencies of 42% and 46%, respectively, with WHO grade 1 BM fibrosis; who otherwise met all requisite criteria for a diagnosis of aCML.[3]

CSF3R mutations are common in CNL (~80%) and fall into two classes, non-sense or frameshift mutations that lead to premature truncation of the cytoplasmic tail of the receptor (truncation mutations) and point mutations in the extracellular domain of CSF3R (membrane proximal mutations); with the membrane proximal T618I mutation being the most common.[18] These mutations are uncommon in aCML and in our prior study were documented in 83% of patients with CNL (n=12) and 0% of aCML (n=9) patients.[19] In our current series of 25 aCML patients, we identified CSF3RT618I mutations in 2 (8%) patients, with variant allele frequency burdens of 33% and 48%, respectively. Both these patients had marked neutrophilia with prominent dysgranulopoiesis, excluding a diagnosis of CNL. Five patients (20%) received therapy with HMA, with no (0%) complete or partial responses, with 2 of 5(40%) demonstrating stable disease.

Prognostic factors reported to impact survival in aCML include; age >65 years, female gender, leukocyte count >50 × 10(9)/L, hemoglobin <10 gm/dl, circulating IMC and SETBP1 mutations. [2, 5, 7] In 2002, Onida et al. proposed a prognostic model for BCR-ABL1 negative CML, which stratified patients into low (median OS 38 months) and high risk (median OS 9 months) categories based on age (< or >65 years), hemoglobin level (< or >10 gm/dl) and leukocyte count (< or >50 × 10(9)/L).[5] This model however was not specific for aCML and included patients with non-classical ABL translocations. Breccia et al. analyzed prognosis in 55 patients with aCML and identified age > 65 years, female sex, leukocyte count >50 × 10(9)/L and the presence of IMC as negative prognosticators.[2] In this study hemoglobin levels did not impact survival. [2] In 2013, Piazza et al. identified SETBP1 mutations in 17 of 70 (25%) aCML patients and found them to be associated with leukocytosis (p=0.008) and inferior survival (median OS 22 versus 77 months, p=0.01, HR-2.27).[7] In our series of 25 patients we analyzed multiple clinical and laboratory variables, along with myeloid relevant gene mutations and on a multivariable analysis identified the following to be independently prognostic; increasing age (p=0.003) [age >67: HR 10.1, 95% CI 1.3–119], low hemoglobin (p=0.008) [HB< 10gm/dl: HR 8.2, 95% CI 1.6–23.2] and TET2 mutations (p=0.01) [HR 8.8, 95% CI 1.6–47.7]. Interestingly, ASXL1, SETBP1, and ETNK1 mutations had no prognostic impact. Fifteen patients (60%) had >1 mutation, while 9 (36%) had ≥3, and 3 (12%) had ≥4 mutations. Unlike in MDS, and similar to CMML, the number of concurrent mutations did not independently impact survival.[11, 20] Based on our findings, we created a hazards ratio weighted prognostic model; effectively stratifying patients into two risk categories; low (0–1 risk factor), and high (≥2 risk factors), with median OS of 18 and 7 months respectively.

In summary, aCML is an aggressive myeloid neoplasm with limited treatment options and a median survival of <12 months. In comparison to other myeloid neoplasms, mutations in SETBP1 and ETNK1 are relatively more common; however, in the present series they did not impact survival. Increasing age, progressive anemia and the presence of TET2 mutations adversely impacted survival, providing us with an effective risk stratification system for affected patients. Response to conventional therapies, including HMA is dismal, indicating the urgent and unmet need to rationally develop newer therapies.