In all 1635 patients with acute leukemia admitted to our center over a period of more than five years, there were 39 patients (2.4%) satisfied the criteria of EGIL, and 30 (1.8%) patients were diagnosed as AUL or MPAL according to the WHO standards. As shown in Table 1, there was no significant difference regarding to the baseline and clinical characteristics between the patients included (WHO 2016, n = 30) and excluded (EGIL-WHO, n = 9) from EGIL criteria, except that patients excluded by WHO system had a lower level of platelet count (p = 0.039) and did not express MPO/cMPO (p = 0.001).
Table 1
Clinical and laboratory features of patients in this study
Characteristics | EGIL | WHO 2016 | EGIL-WHO | p value༊ |
(n = 39) | (n = 30) | (n = 9) |
Sex, m/f | 23/16 | 18/12 | 5/4 | 0.812 |
Age, y | 39 ± 16.6 | 39 ± 14.9 | 38 ± 22.4 | 0.935 |
WBC, ×109/L | 11 (3–40) | 10 (3–39) | 37 (2-107) | 0.588 |
HB, g/L | 80 (62–106) | 86 (64–105) | 68 (42–124) | 0.501 |
PLT, ×109/L | 71 (35–131) | 79 (44–145) | 29 (7–80) | 0.039 |
BM blast (%) | 80 (55–92) | 76 (51–90) | 92 (75–97) | 0.069 |
Karyotype abnormality, N (%) | | | | |
Abnormal | 17 (53.1) | 15 (60.0) | 2 (28.6) | 0.141 |
Ph+ | 7 (21.9) | 7 (28.0) | 0 (0) | 0.113 |
MLL rearrangment | 1 (3.1) | 0 (0) | 1 (14.3) | 0.055 |
Complex | 9 (28.1) | 8 (32.0) | 1 (14.3) | 0.357 |
Immunophenotype profile, N/tested | | | | |
CD34+ | 36/39 | 27/30 | 9/9 | 0.323 |
MPO+ | 19/39 | 19/30 | 0/9 | 0.001 |
CD13+ | 30/38 | 22/29 | 8/9 | 0.402 |
CD11c+ | 9/17 | 7/13 | 2/4 | 0.893 |
CD14+ | 5/29 | 3/21 | 2/8 | 0.495 |
CD64 | 6/25 | 5/17 | 1/8 | 0.356 |
CD33+ | 24/34 | 17/25 | 7/9 | 0.581 |
CD117+ | 24/36 | 16/27 | 8/9 | 0.102 |
CD19+ | 17/38 | 15/29 | 2/9 | 0.120 |
CD20+ | 4/23 | 4/19 | 0/4 | 0.313 |
CD22+ | 10/13 | 8/10 | 2/3 | 0.631 |
CD10+ | 18/32 | 15/24 | 3/8 | 0.217 |
CD79a+ | 17/36 | 14/27 | 3/9 | 0.335 |
CD3+/cCD3+ | 20/37 | 14/28 | 6/9 | 0.383 |
CD2+ | 9/23 | 6/16 | 3/7 | 0.809 |
CD5+ | 11/24 | 9/16 | 2/8 | 0.148 |
CD7+ | 25/38 | 18/29 | 7/9 | 0.386 |
Patients with mutations, N/tested | 17/19 | 15/17 | 2/2 | 0.608 |
EGIL: European Group for the Classification of Acute Leukemia; WHO: World Health Organization; BM: bone marrow; |
* p values were evaluated by comparing EGIL-WHO with WHO 2016 |
For all 39 patients, mean age was 39 years and 23 (59%) of them were male. The median value of WBC count on presentation was 11 × 109, hemoglobin 80 g/L, platelet 71 × 109, and bone marrow blasts 80%. According to the FAB criteria, 14 (35.9%) patients were classified as ALL, and 5 (12.8%) patients displayed an AML morphology, and others were classified as AUL (7.7%), mix phenotype leukemia (2.6%), or inclusive (41%) (Supplementary Table 2).
Four AUL cases (10%) expressed no lineage specific markers but CD34, HLA-DR with or without TdT. Among the remaining cases, 16 (41%) cases had a myeloid and B-lymphoid phenotype (B-M), 16 (41%) for a myeloid and T-lymphoid phenotype (T-M), 2 (5.1%) for a B-T-lymphoid phenotype (B-T), and 1 cases (2.6%) with evidence of tri-lineage concomitant expression (myeloid, B, and T lymphoid [B-T-M]; Supplementary Table 2).
Among all, 32 patients underwent karyotyping, and 17 (53.1%) of them were abnormal. Complex karyotypes were observed in 9 (28.1%) patients with a high degree of heterogeneity as shown in Table 2. The most frequent abnormal karyotype is t (9; 22) (q34; q11.2), resulting in the BCR-ABL1 fusion gene, appeared in 7 patients. Interestingly, 6 patients were B-M MPAL while one of them was T-M MPAL. MLL rearrangement was observed in a patients diagnosed as T-M MPAL (Table 2). In addition to BCR-ABL and SET/CAN, which was previously reported, we also firstly detected E2A-PBX1 fusion genes in a B-M MPAL patient (Table 2).
Table 2
Detailed information of the patients with cytogenetic abnormalities
Patient No. | Gender | Age | EGIL | 2016 WHO | Gene fusion | Chromosome karyotype / FISH | CR | OS (day) |
(year) | classification | classification |
1 | Male | 24 | BAL | MPAL, B-M, NOS | E2A/PBX1 | 46,XY,[9]/43༌XY,b (1q), 6q-, -9, -10,11q-,-15,-17,-18,19p-,-21 [1] | Yes | 478+ |
2 | Female | 42 | BAL | MPAL, BCR/ABL+ | BCR/ABL (P210) | 46, XX, t(9;22) (q34;q11) [20] | Yes | 115+ |
3 | Female | 24 | BAL | MPAL, B-M, NOS | Neg | 46, XX, t (2:5) (p21:p15), del (6) (q16-q22), t (9:x) (p24:q21) | Yes | 578 |
13 | Female | 56 | BAL | MPAL, T-B, NOS | Neg | 46, XX, dic(11:22)(q24;q12),del(12)(q22)[7]/46,XX [3] | No | 381 |
15 | Male | 20 | BAL | MPAL,BCR/ABL+ | BCR/ABL (P190) | 46,XY, r (5) (p15q35), t (9; 22) (q34, q11) [14] / 46, XY, t (9; 22) (q34; q11) [6] | Yes | 530+ |
16 | Female | 58 | BAL | MPAL, T-M, NOS | SET/CAN | Neg | No | 360 |
19 | Male | 26 | BAL | MPAL, T-B, NOS | Neg | Subtriploid karyotype | Yes | 602+ |
21 | Female | 29 | BAL | MPAL,BCR/ABL+ | BCR/ABL | 46, XX [6] / [CP] 46, XX, 2q+, t (9; 22) (q34; q11), 14p+ [8] | Yes | 579+ |
22 | Male | 25 | BAL | T-ALL | / | 43–47, XY, t (4; 11) (q21; p15), + 6, del (6) (q21), del (7), (q21), del (7) (q14), add (9) (p24), add (11) (p15), add (14) (q32), -17, del (17), (p13), +ace [cp6] / 46, XY [14] FISH: Isolation of MLL gene P53/CSP17(17p13) deletion | No | 83 |
25 | Female | 43 | BAL | MPAL, T-M, NOS | / | 47, XX, + 21 [4] / 46, XX [2] | Yes | 545+ |
26 | Female | 41 | BAL | MPAL,BCR/ABL+ | BCR/ABL (P210) | / | Yes | 2293+ |
29 | Female | 60 | BAL | B-ALL | Neg | 47, XX, t(8; 13), (q10; p10), + 22 [10] | No | 51 |
31 | Male | 50 | BAL | MPAL,BCR/ABL+ | BCR/ABL (P210) | 47, XX,+11[9] / 46, XX, [1] | Yes | 850+ |
32 | Female | 36 | AUL | AUL | Neg | 47, XX, + 8 [3] | No | 382 |
36 | Male | 18 | BAL | MPAL,BCR/ABL+ | BCR/ABL (P190) | Neg | Yes | 565+ |
37 | Male | 42 | BAL | MPAL,BCR/ABL+ | BCR/ABL (P190) | 49, XY, t (9; 22) (q34; q11), + 10, +21, +der (22) t (9; 22) (q34; q11) [1] / 53, idem, M31 + x, + 1, +5, + 8 [1] / 46, XY [18] | No | 731 |
44 | Male | 44 | BAL | MPAL, T-M, NOS | Neg | 46, XY [6] /47, XY, + 10 [14] | Yes | 351+ |
45 | Male | 23 | AUL | AUL | Neg | 47,XY, add (2) (q37), + 4, t (10;11) (p12;q21), -17, +mar [4]/47, idem, add (2) (q37) [1] /46, XY, [15] | Yes | 141 |
Genetic testing was conducted in 27 cases. Nineteen out of them were tested by 173-gene panel in next-generation deep sequencing platform, and 8 of them, who admitted in hospital in earlier time, were tested by Sanger sequencing. 17 cases (89.5%) were detected at least one somatic mutation by high-throughput sequencing system, while only 2 patients were positive mutated by screening limited hot spot mutation of AML by Sanger sequencing. The two mutated genes detected by first-generation sequencing were the CEBPA in No.36 patient and the FLT3-ITD repetitive tandem sequence in No. 42 patient, respectively.
To avoid bias, subsequent mutation analysis only included 19 patients who had been sequenced in next-generation platform. Based on the sequencing data, 53 high-confidence somatic mutations were detected in 33 genes in 17 patients. The two patients with no detectable mutations were both B-M phenotype. The median number of gene mutation was 3 (0–8) per sample. The most frequently mutated genes were NRAS (4, 21%), CEBPA (4, 21%), JAK3 (3, 16%), RUNX1 (3, 16%). Mutations in genes such as DNMT3A, ETV6, IDH2, KMT2D, KRAS, NOTCH1, PHF6, TP53, and WT1 were detected in two different patients. CEBPA mutations were detected in 3 patients with M-T phenotype and one with B-T phenotype. Notably, the mutations in NRAS gene (reoccurring in 4 cases) were concentrated at its 12th/13th amino acid site, which suggested that this region of NRAS gene may play an important role in the pathogenesis of ALAL.
We divided genes into the following categories according to gene attribute: transcription factor, chromatin regulation, epigenetics, cell apoptosis, signaling pathway (RTK-RAS, NOTCH, MAPK-ERK, PI3K/AKT, JAK-STAT), and others. The results showed that mutations detected in BAL cases enriched in transcription factor, chromatin regulation and epigenetic, all related to genomic stability and transcriptional regulation; while AUL cases frequently mutated in genes in signal pathway regulation such as RTK-RAS, NOTCH, and PI3K/AKT (Fig. 1A).There was no significant difference in the distribution of B/M and T/M gene mutations, except for the particularly high frequency of transcription factor mutations in T/M cases (71.4%) (Fig. 1B). The detailed information profile was shown in Fig. 1C.
Survival analyses were used to compare the prognosis of clinical subsets and types of treatment. Up to January 16, 2019 (mean follow up time 428 days), 34 patients received chemotherapy, while others, according to patients' wishes or limitation of general condition, only received symptomatic support treatment. Among the patients who received chemotherapy, 7 cases received ALL-like therapy, 6 cases received AML-like therapy, and 21 cases received AML + ALL combined therapy. Decitabine was included in the therapy of 9 patients, 5 patients received tyrosine kinase inhibitors (TKI), and 4 patients received allogeneic hematopoietic stem cell transplantation (HSCT).
Among all the patients, 21 patients (53.8%) achieved complete remission (CR) at least once. The CR rate was not affected by immunophenotype (p = 0.167), complex karyotype (p = 0.761), whether mutated (p = 0.156), or chemotherapy regimen (p = 0.326); while showed a slight association with no. of mutation/person (p = 0.096) (Table 3). During the follow up, 27 cases (69.2%) progressed or died. Of all factors considered, mutation complexity, defined as no. of mutation/person was prognostically significant in PFS and OS (Log rank p = 0.009 and Log rank p = 0.047 respectively) (Table 3 and Fig. 2A-B). When the WHO diagnostic criteria were applied, we obtained the similar results in the 30 patients included. In specific, mutation complexity was the only clinical risk factor that remarkably associated with PFS (Log rank p < 0.001), and OS (Log rank p < 0.001), manifested as patients with more complex mutation profiles had much worse clinical outcomes (Supplementary Table 3 and Fig. 2C-D). Of note, 9 patients were excluded according to WHO 2016 classification system. Eight of them were diagnosed as acute lymphoblastic leukemia, and one was determined as unclassified. We found the patients excluded by WHO criteria had a even worse prognosis than those patients included, characterized as shorter PFS (Log rank p = 0.023) and OS (Log rank p = 0.031) (Table 3 and Fig. 2E-F). Whether the clinical diagnosis and treatment of these subsets of patients is the optimal strategy may need to be further explored.
Table 3
Clinical outcomes of patients according to different parameters for all the patients
Parameter | CR | p-value* | PFS, day | p-value# | OS, day | p-value# |
patients (%) | median | median |
Immunophenotype | | 0.167 | | 0.184 | | 0.217 |
AUL | 1 (25.0) | | 133 | | 141 | |
B/M | 11 (73.3) | | 578 | | 731 | |
T/M | 8 (50.0) | | 264 | | 299 | |
B/T + B/T/M | 1 (25.0) | | 205 | | 205 | |
Complex karyotype | | 0.761 | | 0.936 | | 0.741 |
Yes | 6 (66.7) | | 578 | | 578 | |
No | 14 (60.9) | | 338 | | 360 | |
Gene mutation | | 0.156 | | 0.058 | | 0.176 |
Yes | 8 (47.1) | | 252 | | 382 | |
No | 2 (100) | | / | | / | |
Mutation complexity& | | 0.096 | | 0.009 | | 0.047 |
Low (n = 0) | 2 (100) | | / | | / | |
Middle(1 ≤ n ≤ 3) | 7 (53.8) | | 338 | | 731 | |
High (n ≥ 4) | 1 (25.0) | | 77 | | 83 | |
Treatment | | 0.326 | | 0.907 | | 0.145 |
ALL-like | 6 (85.7) | | 422 | | 478 | |
AML-like | 3 (50.0) | | 424 | | 1181 | |
AML + ALL-like | 12 (57.1) | | 338 | | 381 | |
Classification system | | 0.907 | | 0.023 | | 0.031 |
WHO | 16 (53.3) | | 422 | | 578 | |
EGIL-WHO | 5 (55.6) | | 207 | | 211 | |
CR: complete remission; PFS: progression-free survival; OS: overall survival; * evaluated by chi square test |
WHO: World Health Organization; EGIL: European Group for the Classification of Acute Leukemia;# evaluated by Log-rank test |
& defined as number of pathogenic mutations carried by each patient |