Abstract
Acute myeloid leukemia with minimal differentiation (AML-M0) is associated with overall poor survival with no clinically useful molecular or phenotypic markers. Recently, two distinct gene expression signatures were observed in AML-M0 based on the presence or absence of RUNX1 mutation in AML-M0. Based on the reported upregulation of TdT expression in AML-M0 with RUNX1 mutation, we characterized the features of AML-M0 divided by presence or absence of TdT expression and determined if TdT expression can be used as a surrogate marker for RUNX1 mutation in AML-M0 using a relatively pure group of AML-M0. TdT expression in AML-M0 is associated with higher peripheral blood and bone marrow blast counts, direct correlation with trisomy 13, inverse correlation with aberrations in chromosomes 5 and 17 and longer overall survival in patient receiving stem cell transplant. Only a partial overlap was observed between the features of AML with RUNX1 mutation and AML-M0 with TdT expression.
Introduction
“Acute myeloid leukemia (AML) not otherwise specified, with minimal differentiation” is an AML with no evidence of myeloid differentiation by morphology and light microscopy cytochemistry (1). It corresponds to AML-M0 in the French-American-British (FAB) classification scheme (2). This group comprises approximately 5% of all AML, most patients being infants or older adults and usually portends a poor prognosis with low remission rate (1, 3–5). The blasts are usually medium sized but can be small, resembling lymphoblasts, and MPO by cytochemistry is by definition <3%. Still their myeloid lineage is evidenced by expression of early myeloid antigens (CD13 and/or CD33, CD117). Mature myeloid/monocytic markers are usually not expressed. Nuclear terminal deoxynucleotidyl transferase (TdT) is reported to be positive in approximately 50% of cases, and CD7 in approximately 40% of cases (1–6). No specific chromosomal abnormality has been associated with M0 (1). Although a higher percentage of complex and unbalanced cytogenetic changes have been reported with M0 (3, 7), many of theses cases will likely be reclassified as AML with myelodysplasia-related changes by the new WHO classification (2008) (1), thus making the category of AML with minimal differentiation more “exclusive”.
So far, no single genetic mechanism explaining leukemogenesis in all AML-M0 has been identified. RUNX1, a transcription factor involved in hematopoietic differentiation, has been shown to be frequently mutated in AML-M0 and, is associated with poor clinical outcome (8–10). Mutations in RUNX1 were originally described in two studies in familial and sporadic myeloid leukemias, followed by identification in AML, predominantly in the M0 subtype, and, more recently in AML with myelodysplasia related changes and in secondary (therapy-related) AML (11). Mutations in RUNX1 are clustered in, but not restricted to the Runt homology domain and, requires sequencing of at least 8 exons for comprehensive mutational analysis (8, 9, 12). Clinical testing for RUNX1 mutations is, therefore, not routinely performed. Recent genome wide analyses showed that TdT expression was up-regulated in AML-M0 with RUNX1 mutation. (10, 13). Three TdT probe sets were among top 4 probe sets in a panel of 8 probe set classifier used to classify AML-M0 by RUNX1 mutation (10). Since TdT expression is routinely tested by flow cytometry for all new acute leukemias, it could be used as a convenient surrogate marker for RUNX1 mutation. The goal of this single institution study was to review features of AML with minimal differentiation subdivided according to TdT expression and to test the hypothesis that TdT expression can be used as surrogate marker for RUNX1 mutation.
Our study provides first detailed clinicopathologic characteristics of a “pure” cohort of AML-M0 per new 2008 WHO classification criteria without myelodysplasia-related changes or therapy-related AML. To the best of our knowledge, this is the only report examining significance of TdT expression in AML-M0, by itself and in comparison with reported features of RUNX1 mutation in AML.
Methods
Patient Selection
The study was approved by the institutional review board. A total of 30 cases of de novo AML with minimal differentiation were identified in the files of our department for the period 2002–2008. We excluded any case with history or concurrent findings of myelodysplasia, myeloproliferative neoplasm or therapy-related changes. Cases with history of prior chemotherapy with or without relapse were also excluded from this study. Clinical histories including stem cell transplant and, outcomes such responses to chemotherapy and survival data were retrieved from the institutional medical information system.
Cytochemistry and Immunophenotyping
Flow cytometric immunophenotyping (FCI) was performed on bone marrow aspirates in all patients and was analyzed using three-color flow cytometric immunophenotyping and a FACScan (BD Biosciences, San Jose, CA) instrument as described previously (14). The panel of antibodies included: CD2, surface CD3, cytoplasmic CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD19, CD20, cytoplasmic CD22, CD33, CD34, CD38, CD45, CD52, CD56, CD64, CD123 and TdT.
Cytogenetics and Molecular Studies
All cases were analyzed by conventional cytogenetics (CG) analysis as described previously (14). Molecular testing for mutations in RAS (KRAS and NRAS) and FLT3 was performed as described previously (15, 16). PCR-based testing was performed for the detection of clonal rearrangements (GRs) in immunoglobulin heavy chain gene (IGH), T-cell receptor beta gene (TCRB) and T-cell receptor gamma gene (TCRG) as described previously (17, 18).
Statistical Analysis
Statistical analysis was performed using Student’s two-tailed t-test and Fisher’s two-tailed exact test as applicable.
Results
TdT expression in AML-M0 is associated with significantly higher blast count at presentation
AML-M0 cases in our study included 19 males and 11 females (M:F ratio=1.7:1) with a median age of 60 years (Range: 16–87, Table 1). Median hemogram values at presentation were as follows (Table 1): WBC count, 2.2 k/μL (range: 0.3–226); hemoglobin 9.7 g/dl (range: 5.6–13.3) and platelets 46 k/μL (range: 11–272). Median peripheral blood and bone marrow blasts were 52% (range: 0–99) and 78% (range: 20–98) respectively.
Table 1.
Demographics and Clinical Presentation of AML-M0
| Characteristic | Total (N=30) |
TdT-Pos (N=10) |
TdT-Neg (N=20) |
p-value |
|---|---|---|---|---|
| Gender | 0.7† | |||
| M:F | 19:11 | 7:3 | 12:8 | |
| Age (years) | 0.37†† | |||
| Average | 57.8 | 62.6 | 55.3 | |
| Median | 60 | 65.5 | 57 | |
| Range | 16–87 | 17–83 | 16–87 | |
| WBC (k/μl) | 0.23†† | |||
| Average | 25.5 | 45.9 | 15.3 | |
| Median | 2.2 | 5.05 | 2.05 | |
| Range | 0.3–226 | 1–226 | 0.3–178 | |
| Hgb (g/dl) | 0.8†† | |||
| Average | 9.7 | 9.8 | 9.6 | |
| Median | 9.7 | 9.5 | 9.8 | |
| Range | 5.6–13.3 | 7.6–12.6 | 5.6–13.3 | |
| Platelet (k/μl) | 0.9†† | |||
| Average | 75 | 77 | 74 | |
| Median | 46 | 60 | 45 | |
| Range | 11–272 | 23–272 | 11–229 | |
| PB Blast (%) | 0.04†† | |||
| Average | 44 | 68 | 34 | |
| Median | 52 | 77 | 14 | |
| Range | 0–99 | 0–99 | 0–92 | |
| BM Blast (%) | 0.01†† | |||
| Average | 70 | 81 | 65 | |
| Median | 78 | 84 | 71 | |
| Range | 20–98 | 58–98 | 20–96 | |
| LDH (U/L) | 0.26†† | |||
| Average | 1358 | 759 | 1657 | |
| Median | 635 | 608 | 673 | |
| Range | 334–15544 | 349–1828 | 334–15544 | |
| LDH | 0.71† | |||
| In normal range | 14 | 4 | 10 | |
| Increased | 16 | 6 | 10 |
, Student’s two-tailed t-test;
, Fisher’s two-tailed exact test;
AML-M0 cases showing expression of TdT in 25% or more blasts by flow cytometry were considered TdT-Positive (TdT-Pos). Using this cut-off, 10/30 (33.3%) cases were classified as TdT-Pos and 20/30 as TdT-Negative (TdT-Neg). TdT-Pos AML-M0 cases (N=10) showed significantly higher peripheral blood blast count and BM blast percentage as compared to TdT-Neg (N=20) cases (For PB, median: 77 vs 14, range: 0–99 vs 0–92, p<0.05; For BM, median: 84 vs 71, range: 58–98 vs 20–96, p<0.05; Table 1). TdT-Pos AML-M0 cases (N=10) showed higher male:female ratio and, higher median age, WBC count and platelet count compared to TdT-Neg cases (N=20), however, the differences were not statistically significant (Table 1).
Clonal IGH and TCR gene rearrangements are frequent in AML-M0 and are independent of TdT expression
Molecular testing showed mutations in RAS (KRAS and NRAS) in 5/28 (18%) and in FLT3 in 3/25 (12%) AML-M0 cases. Overall, TdT-Pos cases showed a slightly higher rate of FLT3 mutation compared to TdT-Neg cases (3/10, 30% vs. 2/18, 11%). No mutations in KRAS or NRAS codon 12, 13 or 61 were detected in 7 TdT-Pos cases. All 3 KRAS/NRAS mutation were limited to TdT-Neg cases (3/18, 17%). No significant differences were observed in the frequency of RAS and FLT3 mutations between TdT-Pos and TdT-Neg cases (Table 2).
Table 2.
Selected Molecular, Cytogenetic and Flow Cytometric Features of AML-M0
| Characteristic | Total (N=30) |
TdT-Pos (N=10) |
TdT-Neg (N=20) |
p-value (Fisher’s two-tailed exact test) |
|---|---|---|---|---|
| Molecular | ||||
| FLT3 | 0.34 | |||
| Mutant | 5/28 (18%) | 3/10 (30%) | 2/18 (11%) | |
| WT | 23/28 (82%) | 7/10 (10%) | 16/18 (89%) | |
| RAS | 0.53 | |||
| Mutant | 3/25 (12%) | 0/7 | 3/18 (17%) | |
| WT | 22/25 (88%) | 7/7 (100%) | 15/18 (83%) | |
| IGH | 0.57 | |||
| Clonal | 4/18 (22%) | 3/9 (33%) | 1/9 (11%) | |
| Non-clonal | 14/18 (78%) | 6/9 (67%) | 8/9 (89%) | |
| TCR-Beta/Gamma | 0.41 | |||
| Clonal | 11/23 (48%) | 6/10 (60%) | 5/13 (38%) | |
| Non-Clonal | 12/23 (52%) | 4/10 (40%) | 8/13 (62%) | |
| IGH/TCRB/TCRG | 0.42 | |||
| Clonal | 12/23 (52%) | 6/10 (60%) | 6/13 (46%) | |
| Non-clonal | 11/23 (48%) | 4/10 (40%) | 7/13 (54%) | |
| Cytogenetics | ||||
| Karyotype | ||||
| Diploid | 8/30 (27%) | 3/10 (30%) | 5/20 (25%) | 1.00 |
| Simple | 7/30 (23%) | 3/10 (30%) | 4/20 (20%) | 0.41 |
| Complex | 15/30 (50%) | 4/10 (40%) | 11/20 (55%) | 0.46 |
| Risk Category | ||||
| Favorable | 0 | 0 | 0 | |
| Intermediate | 15/30 (50%) | 6/10 (60%) | 9/20 (45%) | 0.47 |
| Poor | 15/30 (50%) | 4/10 (40%) | 11/20 (55%) | 0.47 |
| Chromosomes | ||||
| 5 | 8/30 (27%) | 0/10 | 8/20 (40%) | 0.03 |
| 7 | 8/30 (27%) | 1/10 (10%) | 7/20 (35%) | 0.20 |
| 8 | 6/30 (20%) | 2/10 (20%) | 4/20 (20%) | 1.00 |
| 11 | 7/30 (23%) | 3/10 (30%) | 4/20 (20%) | 0.67 |
| +13 | 3/30 (10%) | 3/10 (30%) | 0/20 | 0.03 |
| −13 | 3/30 (10%) | 0/10 | 3/20 (15%) | 0.5 |
| 17 | 7/30 (23%) | 0/10 | 7/20 (35%) | 0.03 |
| 21 | 4/30 (13%) | 1/10 (10%) | 3/20 (15%) | 1.00 |
| Immunophenotype* | ||||
| CD2 | 2/26 (8%) | 1/10 (10%) | 1/16 (6%) | 1.00 |
| CD3 | 0/30 | 0/10 | 0/20 | |
| CD5 | 4/27 (15%) | 0/10 | 4/17 (24%) | 0.13 |
| CD7 | 9/27 (33%) | 3/10 (30%) | 6/17 (35%) | 1.00 |
| CD19 | 4/29 (14%) | 2/10 (20%) | 2/19 (11%) | 0.61 |
| Any T- or B- | 13/30 (43%) | 4/10 (40%) | 9/20 (45%) | 0.72 |
, no significant difference in expression of CD34, CD117, HLA-DR, CD13, CD33 and CD56.
Previous studies have shown frequent clonal gene rearrangements (GRs) in IGH and TCR in AML-M0 (19). We, therefore, tested the bone marrow aspirates for clonal GRs in IGH, TCRB and TCRG. Clonal GRs in at least one of the tested genes were observed in 12/23 (52%) of cases, of which TCRB/TCRG GRs were more frequent (11/23, 48%) than IGH GRs (4/18, 22%). No significant differences were observed in the frequency of these GRs, either individually or combined together, between TdT-Pos and TdT-Neg cases (Table 2).
TdT expression in AML-M0 shows positive relationship with gain of chromosome 13 and inverse relationship with aberrations in chromosomes 5 and 17
Conventional karyotyping analysis of bone marrow aspirates from 31 AML-M0 cases showed diploid karyotype in 8/30 (27%), less than 3 chromosomal abnormalities (non-complex) in 7/30 (23%) and, more than 3 chromosomal abnormalities (complex) in 15/30 (50%) of cases. Based on the number and type of chromosomal aberrations 16/30 (50%) AML-M0 cases belonged to intermediate-prognosis group and the rest (15/30, 50%) were in poor-prognosis group. No significant differences in the cytogenetics risk-groups were observed between TdT-Pos and TdT-Neg AML-M0 (Table 2).
The most frequent chromosomal abnormalities in AML-M0 involved chromosomes 5 (8/30, 27%), 7 (8/30, 27%), 11 (7/30, 23%), 17 (7/30, 23%), 13 (6/30, 20%), 8 (6/30, 20%) and 21 (4/30, 13%) (Table 2). All TdT-Pos cases with chromosome 13 abnormalities (3/10, 30%) showed extra copies of chromosome 13 (p=0.03), two with trisomy 13 and one with tetrasomy 13. All TdT-Neg cases with abnormalities involving chromosome 13 (3/20, 15%) showed loss/deletion (p=0.5). TdT-Pos showed no aberrations in chromosomes 5 and 17, whereas TdT-Neg cases showed abnormalities in 8/20 (40%) and 7/20 (35%) cases respectively (p=0.03 for each). The significant differences were observed in frequency and nature of aberrations for other commonly aberrant chromosomes 7, 8, 11 and 21 between TdT-Pos and TdT-Neg AML-M0 (Table 2).
TdT expression in AMl-M0 is not associated with a distinct immunophenotype
By definition, all cases were negative for myeloperoxidase (MPO) expression (<3% positive blasts) by cytochemistry and flow cytometry. A total of 13/30 (43%) cases showed expression of a T- or B-cell associated antigen (CD2, CD5, CD7 or CD19), but did not meet the criteria for mixed phenotype acute leukemia, NOS by 2008 WHO classification (Table 2). No significant difference was noted for the expression of a T- or B-cell associated antigen expression between TdT-Pos (4/10, 40%) and TdT-Neg (9/20, 45%) AML-M0 (p=0.7). No significant differences were observed for the expression of CD34, CD117, HLA-DR, CD13, CD33 and CD56 between TdT-Pos and TdT-Neg AML-M0 (data not shown). No significant association was observed between % blasts expressing TdT expression and lymphoid marker expression (data not shown).
TdT-Pos AML-M0 show better overall survival with a stem cell transplant compared to TdT-Pos AML-M0 with no transplant and TdT-Neg AML-M0 with transplant
Analysis of available clinical outcome in response to the induction chemotherapy in 26 AML-M0 patient showed complete remission (CR) in 13/26, 50%), complete remission with incomplete marrow recovery (CRi) in 1/26 (4%), partial response (PR) in 5/26 (19%) and refractory AML in 7/26 (%) patients (Table 1). Overall, 17/26 (65%) patients achieved CR and 9/26 (35%) did not. Average and median overall survival of AML-M0 patients were 23.5 and 10.6 months respectively (Range: 2.4 to 96.6). In 24 patients with available survival information, 1-year and 5-year survivals were 11/24 (46%) and 2/24 (8%) respectively. TdT-Pos AML-M0 showed slightly better 5-year survival (2/9, 22%) compared to TdT-Neg AML-M0 (0/15), however, this difference did not reach statistical significance (p=0.13). There was no significant difference in induction responses (CR, CRi, PR, refractory), achievement of complete remission, overall survival and 1-year survival between TdT-Pos and TdT-Neg AML-M0 (Table 2).
Available clinical information showed that 6/26 (3/8 TdT-Pos and 3/18 TdT-Neg) AML-M0 patients received stem cell transplant. Overall survival (p=0.02) was significantly longer in 3 TdT-Pos patients (43.2, 91.3, 97.4 months; average: 76.3 months) compared to 3 TdT-Neg patients (4.7, 10.3 and 11.7 months; average: 8.9 months). Similar beneficial outcome was noted within the TdT-Pos group. Three TdT-Pos patients receiving stem cell transplant showed significantly better (p=0.007) overall survival (43.2, 91.3, 97.4 months; average: 76.3 months) compared to 5 TdT-Pos patients who did not receive a stem cell transplant (5.1, 5.1, 9.5, 20, 40.2 months; average: 16 months). The overall survival is likely to improve in TdT-Pos AML-M0 group, as all 3 patients receiving stem cell transplant are still alive. There was no significant difference in the cytogenetics, response to induction therapy or achievement of complete remission between the groups compared suggesting that TdT-Pos expression can be used an indicator of beneficial outcome with stem cell transplant (data not shown).
Characteristics of AML-M0 with TdT expression in our study are different than reported characteristics of AML with RUNX1 mutation
Recent studies showed a high degree of correlation between RUNX1 mutation and TdT expression in AML (10, 13). We, therefore compared the characteristics of TdT-Pos AML in our study with reported characteristics of AML with RUNX1 mutation (8, 9, 20). We detected TdT expression in 10/30 (33%) AML-M0, comparable to reported frequency of RUNX1 mutation (35% and 41%) in AML-M0 (8, 13). AML with RUNX1 expression are shown to have a higher male:female ratio and higher median age at presentation (20). We did not see significant differences for these parameters between TdT-Pos and TdT-Neg AML. Trisomy 13 is shown to be associated with RUNX1 mutation in AML-M0 (8, 9). We detected Trisomy/Tetrasomy 13 in 3/11 (27%) of cases. Interestingly, 7/8 (87.5%) AML-M0 with Trisomy 13 showed RUNX1 mutation (9). In our study, 3/3 (100%) AML-M0 with Trisomy/Tetrasomy 13 were positive for TdT expression.
RUNX1 mutations are shown to be associated with presence of CD34 and HLA-DR expression and, absence of CD33, CD15, CD19 and CD56 expression (20). We did not see any immunophenotypic differences between TdT-Pos and TdT-Neg AML-M0 (Table 2). AML with RUNX1 mutation are reported to have a significantly higher lactate dehydrogenase (LDH) level compared to AML with wild-type RUNX1 (20). In our study, we did not find statistically significant differences in the LDH levels between TdT-Pos and TdT-Neg AML-M0 (Table 1). RUNX1 mutation in AML is shown to be associated with poor induction response, failure to achieve complete remission, poor overall survival and poor disease-free survival (20). We did not see significant differences in these parameters between TdT-Pos and TdT-Neg AML-M0. On the contrary, TdT-Pos AML-M0 showed a tendency for better overall survival compared to TdT-Neg AML-M0 in patients receiving stem cell transplantation (p=0.056).
Thus, despite reported correlation between RUNX1 mutation and TdT expression, the clinical presentation, immunophenotype and outcomes in AML-M0 with TdT expression appears to be different than AML with RUNX1 mutation except a common association with Trisomy/Tetrasomy 13.
Discussion
RUNX1 (runt-related transcription factor 1; also known as AML1; CBFA2; EVI-1; AMLCR1; PEBP2aB; AML1-EVI-1), a heterodimeric transcription factor, is a member of core binding factor (CBF) family. It encodes the alpha subunit of CBF and is currently thought to be involved in the development of normal hematopoiesis (21). Most of the RUNX1 mutations are clustered in, but not limited to, the Runt domain and result in defective DNA binding but active beta-subunit binding (12). Chromosomal translocations involving RUNX1 gene are well-documented and have been associated with several types of leukemia such as the RUNX1-RUNX1T1 fusion transcript in AML with t(8;21)(q22;q22) (1). RUNX1 has been described to contribute to leukemogenesis both as a tumor suppressor gene (AML-M0) as well as an oncogene in various other hematologic malignancies (22, 23). Sporadic point mutations are frequently found in three leukemia entities: AML-M0 subtype, MDS-AML, and secondary (therapy-related) MDS/AML (11, 12, 24). The incidence of RUNX1 point mutations in therapy-related leukemias appears comparable to the incidence of the AML with t(8;21)(q22;q22) (12). In AML-M0, half of the RUNX1 point mutations are reported to be biallelic, although the frequency varies with ethnicity (12). Gene expression profiling of AML-M0 showed two distinct subgroups, one of which is fully associated with RUNX1 mutations (10, 13) and shows poor overall survival. This suggests potential clinical utility for determining RUNX1 mutation status. Unfortunately, routine clinical testing for RUNX1 is not feasible due to multiple reasons and, therefore, identification of a surrogate marker may provide a more efficient alternative.
Terminal deoxynucleotidyl transferase (TdT), although traditionally considered to be a marker of immature lymphoid cells, is now known to be a marker of hematopoietic immaturity in both lymphoid and myeloid lineages, correlating with CD34 expression and a poorer prognosis (25). A recent gene expression profiling study showed that TdT expression highly correlates with RUNX1 mutation. The authors proposed that TdT can be used as a surrogate for RUNX1 mutation status in AML-M0, however, to our knowledge no such comparative studies have been reported (10). Since TdT expression can be conveniently detected by flow cytometric analysis and is a standard of care for the workup of new acute leukemias, we determined the properties of AML-M0 with TdT expression and tested that the hypothesis that TdT expression can be used as a surrogate marker for RUNX1 mutations.
Firstly, we removed AML-M0 cases with prior myelodysplasia and chemotherapy to obtain a purer group of AML-M0 cases per new 2008 WHO classification. Our study, therefore, represents a more accurate description of AML-M0 compared to previous reported studies that contained cases which will not belong to AML-M0 by new 2008 WHO classification. TdT expression was detected by flow cytometry in 10/30 (33%) of AML-M0 using a cut-off of expression by at least 25% blasts. The reported frequency of TdT expression in AML-M0 varies from 30% to 100% depending on the method and cut-off used to determine TdT expression (26–29). In our cohort, we used a cut-off of TdT expression by 25% blasts to be called TdT-Pos to avoid flow cytometry artifact of cell permiabilization and to ensure TdT expression by a significant number of blasts. TdT-Pos AML-M0 showed higher blast count at presentation, correlation with trisomy/tetrasomy 13, inverse correlation with aberrations in chromosomes 5 and 17 and, longer overall survival with stem cell transplant. To our knowledge, no studies showing features of TdT-Pos AML-M0 are reported. A meta-analysis of features of TdT-Pos cases within reported cohorts of AML-M0 could not be performed due to inclusion of AML with MDS related changes or therapy-related AML in the cohort, inclusion of cases showing higher expression of myeloperoxidase than allowed by the current diagnostic criteria and inconsistencies in immunophenotyping studies used in prior reports (26–29). Absence of chromosomal 5 abnormalities in our study is in contrast to a previous report showing abnormalities in chromosomes 5 and/or 7 in 5/19 (26%) cases (29). The frequency of aberrations affecting chromosome 5 alone is not clear from the report, but is expected to be low (<20%).
TdT encodes a DNA polymerase normally expressed during early stages of pre-B and pre-T lymphocyte development (30). In initial reports, TdT expression was believed to be limited to acute lymphoblastic leukemias (ALL). Soon, TdT expression in AML was discovered and is now routinely tested at diagnosis. Some reports suggest that TdT expression in AML-M0 may reflect biphenotypic acute leukemia with myeloid predominance. Additionally, frequent clonal IGH/TCRB rearrangements have been reported in AML-M0 with TdT expression (19). We detected clonal IGH/TCRB/TCRG rearrangements in 6/10 (60%) TdT-Pos AML-M0 cases, similar to previously reported frequency of 61% (8/13) in TdT-Pos AML-M0 (19). No statistically significant difference was observed in the frequency of clonal gene rearrangements or expression of T/B-cell markers between in TdT-Pos and TdT-Neg AML-M0. This was true even when T/B-cell marker expression was correlated to the amount of TdT expression. These findings suggest that TdT expression does not correlate with a partial lymphoid differentiation in AML-M0 defined using the 2008 WHO criteria. TdT expression in AML-M0 appears to be a correlate of minimally differentiated nature of the blasts rather than representation of a lymphoblastic component.
At best, only a partial overlap was observed between the feature of TdT-Pos AML-M0 in our study and reported features of AML with RUNX1 mutations. The frequency of TdT expression in AML-M0 in our study is comparable to the reported frequency of RUNX1 mutation in AML-M0 (8, 13). Interestingly, all chromosome 13 aberrations in TdT-Pos AML-M0 involved gain of chromosome 13, whereas chromosome 13 aberrations in TdT-Neg AML-M0 involved losses. Similarly, aberrations involving chromosome 5 and 17 were limited to TdT-Neg AML-M0. Association of trisomy 13 with RUNX1 mutation and increased FLT3 expression has been reported before (8, 9). It is possible that in TdT-Pos AML-M0 with trisomy 13, higher levels of FLT3 could contribute to a poor outcome. While we did not test for FLT3 mRNA expression levels, we did not notice any differences in FLT3 mutation status between TdT-Pos and TdT-Neg AML-M0. Interestingly, the gene expression signature in AML-M0 with RUNX1 mutation showed a unique signature of genes, many of which are related to early B-cell development (10). Our study does not show association of TdT expression with expression of lymphoid markers in AML-M0.
Reported association of RUNX1 mutation with male predominance, higher median age, high LDH levels, lower rate of complete remission upon induction chemotherapy and poor outcome was not observed for TdT-Pos AML-M0 in our study (20). In contrast, our study shows improved overall survival in TdT-Pos AML-M0 after stem cell transplant. Several explanations can be provided for partial but not complete overlap between the features of RUNX1-mutated AML and TdT-Pos AML-M0. The studies that reported correlation between RUNX1 mutation and TdT expression also contained cases (5/40, 12.5%) that were positive for either RUNX1 mutation or TdT expression, but not both (13). It is not clear from the manuscript whether or not the discordant cases were retained in the analysis that showed correlation between RUNX1 mutation and TdT expression. This further supports our observation that TdT expression. These findings suggest that the nature of RUNX1 mutation (mono- vs. bi-allelic, point mutation vs. insertion/deletion, domain affected) may influence TdT expression. Also, TdT may be up-regulated pathways other than RUNX1 mutation.
Importantly, significant improvements in overall survival were noted with stem cell transplant for TdT-Pos AML-M0 compared to TdT-Neg AML-M0. TdT-Pos AML-M0 who received a stem cell transplant showed better outcome compared to TdT-Pos AML-M0 who did not. No significant contributing factors could be identified that could explain these differences. The findings suggest that TdT expression may correlate with unique biologic properties that directly or indirectly contribute to the beneficial effect of stem cell transplant. Due to the retrospective nature of the study, the patients were not uniformly treated. The observation that TdT-Pos AML-M0 patients show beneficial effect of stem cell transplant need to be confirmed in a controlled clinical study with uniformly treated patients.
Our study is limited by not having RUNX1 mutation analysis for comparison with TdT expression, however, the whole purpose of the study is to see if cumbersome RUNX1 analysis can be replaced with a more convenient assessment of TdT expression. From previously reported studies it is evident that while there is a correlation between RUNX1 mutation and TdT expression as a whole in the study group, it may not be true for an individual patient. (10, 13). Our study shows limited information on commonly mutated genes in AML. Also, in an attempt to obtain a pure group of AML-M0 the sample size has become smaller. Some of the properties of TdT-Pos AML and TdT-Neg AML-M0 that trended towards being different, but could not reach statistical significance, need to be investigated in a larger cohort of patients.
Our study reports features of a relatively pure group of de novo AML-M0 cases, as any case with either history of or changes related to myelodysplasia, myeloproliferative neoplasm or therapy-related changes were excluded. In summary, our study shows that TdT-Pos AML-M0 show different clinical features than those reported for AML with RUNX1 mutation. However, TdT-Pos AML-M0 shows unique clinicopathologic features and seems to benefit from stem cell transplant in comparison to TdT-Neg AML-M0. Further clinical studies are required to confirm and elaborate the beneficial effects of TdT expression in the setting of stem cell transplant in AML-M0.
Table 3.
Clinical Outcome of AML-M0
| Characteristic | Total (N=30) |
TdT-Pos (N=10) |
TdT-Neg (N=20) |
p-value |
|---|---|---|---|---|
| Induction Response | ||||
| CR | 13 | 4 | 9 | 1.00† |
| CRi | 1 | 0 | 1 | 1.00† |
| PR | 5 | 2 | 3 | 1.00† |
| Refractory | 7 | 2 | 5 | 1.00† |
| NA | 5 | 2 | 2 | |
| Achieved CR | 0.67† | |||
| Y | 17 | 6 | 11 | |
| N | 9 | 2 | 7 | |
| NA | 5 | 2 | 2 | |
| Current Status | 0.03† | |||
| Alive | 3 | 3 | 0 | |
| Deceased | 25 | 6 | 19 | |
| NA | 3 | 1 | 1 | |
| Overall Survival (months) | N=24 | N=9 | N=15 | 0.2†† |
| Average | 23.5 | 34.7 | 16.7 | |
| Median | 10.6 | 20 | 8.4 | |
| Range | 2.4–96.6 | 3.3–96.6 | 2.4–57.9 | |
| 1-year survival rate | 11/24 (46%) | 5/9 (56%) | 6/15 (40%) | 0.68†† |
| 5-year survival rate | 2/24 (8%) | 2/9 (22%) | 0/15 | 0.13†† |
, Student’s two-tailed t-test;
, Fisher’s two-tailed exact test;
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