Abstract
Tandem duplications (TDs) in exons of upstream binding transcription factor (UBTF-TD) are a rare recurrent alteration in pediatric and adult acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)/neoplasm. Although recently identified, AML with UBTF-TD is now considered a distinct subtype of AML. To further our understanding of myeloid neoplasms with UBTF-TD, we analyzed clinical, morphologic, and immunophenotypic characteristics of 27 pediatric patients with UBTF-TD-positive myeloid neoplasm, including 21 diagnosed as AML and 6 as MDS. Our data demonstrated that UBTF-TD is frequently associated with cytopenia, hypercellular marrow with erythroid hyperplasia, and trilineage dysplasia. Blasts and maturing myeloid cells show a characteristic dysplastic feature with condensed eosinophilic cytoplasm. Blasts have a myeloid or myelomonocytic immunophenotype with a variably dim expression of CD34 and/or CD117, and except for CD7 expression lack a consistent pattern of aberrant lineage-specific antigen expression. Patients with MDS had a lower blast count in the peripheral blood (P = 0.03) and bone marrow (P <0.001) but otherwise had no significant differences in other hematological parameters. Three patients with MDS rapidly progressed to AML in 33, 39, and 210 days from the initial diagnosis and there was no difference in overall survival between patients with MDS and AML (P = 0.18). Our data suggest that MDS with UBTF-TD is prognostically equivalent to AML with UBTF-TD and thus should be considered as a continuum of the same molecularly defined myeloid neoplasm. These collective data also provide morphologic and immunophenotypic clues that can prompt screening for UBTF-TD in patients with MDS or AML.
Key Words: UBTF-TD, acute myeloid leukemia, pediatric leukemia, MDS
The upstream binding transcription factor (UBTF) gene is located on chromosome 17 and encodes the UBTF/UBF protein, a transcription factor required for ribosomal RNA transcription, processing, and regulating the chromatin structure of ribosomal DNA.1 Recently, in-frame tandem duplications (TDs) in exons of UBTF (UBTF-TD) have been described as recurrent alterations in newly diagnosed or relapsed/persistent (R/P) adult acute myeloid leukemia (AML).2–8 UBTF-TD accounts for 4% and 9% of pediatric AML cases at diagnosis and relapse, respectively. While enriched in pediatric and young adult patients, similar UBTF alterations have been observed in 1.2% to 3% of adult patients aged 18 to 79 years. Although less common, UBTF-TD has also been described in myelodysplastic syndrome (MDS)/neoplasm cases, including both in children and adults.7,8 UBTF-TD in AMLs is mutually exclusive with other class-defining cytogenetic and molecular abnormalities. It is commonly associated with high-risk disease, normal karyotype or isolated trisomy 8, comutations in WT1 and FLT3-ITD, and upregulation of HOX gene clusters. Nevertheless, the immunophenotype and detailed morphologic features of UBTF-TD+ myeloid neoplasms have not been extensively explored.
While molecular studies are widely used to diagnose and risk-stratify patients with myeloid malignancies, challenges exist in detecting UBTF-TD since most targeted next-generation sequencing panels do not cover this gene. Further, most current bioinformatic pipelines may also miss this complex alteration.2,9–11 Thus, a full understanding of the morphologic and immunophenotypic features of UBTF-TD MDS and AML cases is needed to potentially identify clues to trigger appropriate genetic testing for UBTF-TD.
MATERIALS AND METHODS
Group Study
We have previously reported molecular features of UBTF-TD cases2,8 and identified 27 cases collected between 1994 and 2023 from these cohorts with available material for additional morphologic and flow cytometry immunophenotyping examination, which was retrospectively reviewed by 2 hematopathologists (M.K. and J.M.K.). Clinical information and follow-up data were obtained from the electronic medical records, including age, sex, history of therapy, initial pathologic diagnosis (when applicable), immunophenotyping, interval from initial diagnosis of MDS to progression to AML, clinical response, status of last follow-up, and overall survival (OS). This study is approved by the St. Jude Children’s Research Hospital Institutional Review Board.
Morphologic Assessment
Wright–Giemsa-stained peripheral blood (PB) and bone marrow (BM) aspirate smears and hematoxylin-eosin-stained core biopsy (with or without BM clot) sections were reviewed. A total of 200 leukocytes in PB and 500 cells in BM were examined for cell differential count, including blast and erythroid cell percentages. At least 20% myeloid blasts in total cells were required to diagnose AML. These specimens were assessed for morphologic dysplasia of all lineages. Dysplasia was defined by 10% or more of the respective lineage cells with dysplastic features. Evidence of progression to AML in patients with MDS was also evaluated, defined as the occurrence of ≥20% blasts in BM or PB after the initial diagnosis of MDS.
The diagnosis was based on WHO 2022 (fifth edition) and ICC 2022 classifications.9–11 Considering the recent recognition of UBTF-TD as a recurrent alteration with clear outcome associations, these cases were also classified as “acute myeloid leukemia with other defined genetic alterations” in WHO 2022 (fifth edition).9 In the absence of other defining alterations, such as those allowing for classification in myelodysplasia-related gene mutations or cytogenetic abnormalities, these cases were classified as “AML not otherwise specified (NOS)” in ICC 2022.11 Lastly, the AML and MDS cases were also subclassified by the French-American-British (FAB) classification based on morphology and immunophenotype.12–15
Immunophenotyping Studies
Flow cytometry immunophenotyping was performed on BM aspirates and/or PB using standard multicolor analysis, which evolved during the study interval. The antibody panels varied in different institutions (for patients with the initial diagnosis rendered elsewhere) and different time periods. The following antibodies were used in various combinations: CD2, CD3 (surface and cytoplasmic), CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD19, CD20, CD22, CD25, CD33, CD34, CD36, CD38, CD41, CD42, CD45, CD56, CD58, CD61, CD64, CD66c, CD71, CD73, cytoplasmic CD79a, CD86, CD90, CD117, CD123, CD133, CD200, CD235a, human leukocyte antigen-DR (HLA-DR), cytoplasmic myeloperoxidase (MPO), cytoplasmic TDT, surface Ig kappa, surface Ig lambda, NG2, TSLP-R, TCR A/B, TCR G/D (Becton-Dickinson, Biosciences). Data were analyzed using FCS Express (DeNovo Software).
Immunohistochemical studies were performed on BM core biopsy specimens according to standard protocols. Myeloperoxidase was performed by flow cytometry, cytochemistry, or both in the majority of cases. Cases were categorized as showing monocytic or monoblastic differentiation using blast morphology, cytochemical studies (positivity for nonspecific esterase), and/or surface marker expression by flow cytometry (positivity for CD64, CD14, CD11b, and/or CD36). Cases were characterized as myeloid based on blast morphology (presence of Auer rods), positivity for myeloperoxidase, and absence of monocytic marker expression. Myelomonocytic differentiation was characterized by ≥20% blasts with maturing granulocytic and monocytic cells constituting ≥20% of total marrow cells or in some cases blasts expressed both monocytic and myeloid markers simultaneously.
Cytogenetics
Conventional karyotypic analysis was performed on G-banded metaphase cells prepared from unstimulated BM aspirate or PB cultures using standard techniques. Twenty metaphases were analyzed, and the results were reported using the 2016 International System for Human Cytogenetic Nomenclature. A complex karyotype was defined as ≥3 chromosomal abnormalities.
Statistical Analysis
Comparisons among categorical and numerical variables were carried out using the Fisher exact test for categorical variables and unpaired t test or analysis of variance (ANOVA) for numeric variables, respectively. OS was calculated from the date of MDS or AML diagnosis to the date of death or last clinical follow-up. The interval from MDS to AML was calculated as the time from the initial diagnosis of MDS to the time of AML progression. The survival swimmer plot was generated using the “ggplot2” R package (version: 3.5.1). Statistical analysis was performed using SPSS for Windows, version 29 (IBM SPSS Statistics), with significance set at a P <0.05 (2-sided).
RESULTS
Baseline Clinical Characteristics
The clinical characteristics and laboratory features are summarized in Table 1. The cohort included 27 patients with myeloid neoplasm harboring UBTF-TD initially diagnosed as MDS (n = 6) or AML (n = 21); 13 males and 14 females with a median age of 15.3 years (range: 4.9 to 21.2) at initial diagnosis. No differences in sex or age were observed between patients with MDS and AML (median age: 13.6 vs 15.3, P = 0.52). Samples at the time of UBTF-TD detection were either from a R/P (AML: n = 8) or diagnosis/initial (I) timepoint (AML: n = 14/MDS: n = 5). Three patients initially diagnosed with MDS progressed to AML in 33 (#28), 39 (#29), and 210 (#27) days. Patient #27 was initially diagnosed with MDS and progressed to AML 7 months later; UBTF-TD was identified at the time of AML as there was no sample available at the MDS timepoint for testing. The other 3 patients with MDS (#30, 31, 32) underwent hematopoietic stem cell transplantation (HSCT) and did not progress to AML, although #30 and #31 relapsed after HSCT (Supplemental Fig. 1, Supplemental Digital Content 1, http://links.lww.com/PAS/B994).
TABLE 1.
Clinical and Genetic Features of Patients With UBTF-TD+Myeloid Neoplasm
| Demographic data | Overall (n = 27) | MDS (n = 6) | AML (n = 21) | P |
|---|---|---|---|---|
| Age (y), median (range) | 15.3 (4.9-21.2) | 13.6 (4.9-17.9) | 15.3 (5.7-21.2) | 0.52 |
| Sex (F/M) | 14/13 | 3/3 | 11/10 | 1.00 |
| PB, median (range) | ||||
| WBC (×109/L) | 3.0 (0.6-103.6) | 2.6 (0.6-4.3) | 9.9 (1.1-103.6) | 0.13 |
| Hgb (g/dL) | 7.9 (5.0-10.8) | 7.5 (5.5-9.0) | 8.1 (5.0-10.8) | 0.42 |
| PLT (×109/L) | 65 (7-197) | 30 (20-163) | 69 (7-197) | 0.68 |
| Blasts (%) | 16 (0.8-93) | 2 (0.8-16) | 32 (1-93) | 0.03 |
| BM | ||||
| Blasts %, median (range) | 29 (4-92) | 10 (4-15) | 40 (24-92) | <0.001 |
| Dyserythropoiesis, n (%) | 25/25 (100) | 5/5 (100) | 12/12(100) | NA |
| Dysgranulopoiesis, n (%) | 24/24 (100) | 5/5 (100) | 12/12 (100) | NA |
| Dysmegakaryopoiesis, n (%) | 23/23 (100) | 5/5 (100) | 11/11 (100) | NA |
| Multilineage dysplasia, n (%) | 25/25 (100) | 5/5 (100) | 12/12 (100) | NA |
| Erythroid: myeloid ratio, median (range) | 0.70 (0.01-6.10) | 0.60 (0.10-1.00) | 0.70 (0.01-6.10) | 0.29 |
| FAB subtype, n (%) | ||||
| M2 | 13 (41) | — | 13 (50) | NA |
| M4 | 8 (25) | — | 8 (31) | NA |
| M6a | 6 (19) | 2 (33) | 4 (15) | 0.31 |
| M7 | 1 (3) | — | 1 (4) | NA |
| RAEBt | 4 (12) | 4 (66) | — | NA |
| CG, n (%) | ||||
| Diploid | 16 (76) | 5 (100) | 11 (68) | 0.27 |
| Trisomy 8 | 4 (20) | 0 | 4 (25) | 0.53 |
| Del (9q) | 1 (5) | 0 | 1 (6) | 1.00 |
| Complex | 0 | 0 | 0 | NA |
| Molecular data, n (%) | ||||
| FLT3-ITD | 10 (37) | 0 | 10 (45) | 0.06 |
| WT1 | 13 (48) | 3 (60) | 10 (45) | 0.65 |
| NRAS | 6 (22) | 1 (20) | 5 (23) | 1.00 |
| MDS-related gene mutations | 2 (9) | 0 | 2 (9) | 1.00 |
| Therapy | ||||
| Chemotherapy | 24/24 (100) | 5/5* (100) | 19/19 (100) | NA |
| Hematopoietic stem cell transplant | 20/25 (80) | 5/6 (83) | 15/19 (79) | 1.00 |
| Clinical follow-up (mo) | ||||
| Duration, median (range) | 32.7 (3.5-291.0) | 18.7 (3.5-291.0) | 33.3 (5.2-195.6) | 0.53 |
| Patient outcome, n (%) | ||||
| CR rate | 13 (52) | 3 (50) | 10 (53) | 1.00 |
| Death | 14 (56) | 4 (66) | 10 (53) | 1.00 |
Data on chemotherapy in one patient was not available (unknown).
Bold values are statistically significant of P value.
CR indicates complete remission; NA, not applicable; RAEBt, refractory anemia with excess blasts in transformation.
Laboratory Findings
The complete blood count (CBC) data (hemoglobin [Hgb], platelet [PLT], and white blood cell [WBC]) at the time of diagnosis/I, R/P, or both were available in 20, 10, and 5 patients, respectively. At the time of diagnosis/I, the median (range) of CBC was Hgb level, 7.9 (5.0 to 10.8) g/dl; WBC count, 3.0 (0.6 to 103.6)×109/L, and PLT count 65 (7 to 197)×109/L (Supplemental Table 1, Supplemental Digital Content 2, http://links.lww.com/PAS/B995). The median percentage of blasts in the BM at diagnosis/I (n = 22) was 29% (4% to 92%; Table 1, Supplemental Table 2, Supplemental Digital Content 3, http://links.lww.com/PAS/B996). All tested diagnosis/I samples (n = 21) had circulating leukemia cells in the PB revealed by morphologic examination and/or flow cytometry immunophenotypic analysis with a median percentage of 16% (0.8% to 93%). There was no significant difference in Hgb (P = 0.42), WBC (P = 0.13), or PLT numbers (P = 0.68) between patients with MDS and AML. As expected, patients with MDS presented with a lower blast count in the PB (median: 2% vs 32%, P = 0.03) and BM (median: 10% vs 40%, P < 0.001) than those with AML.
Morphologic Findings
Dysplastic myeloid, megakaryocytic, and erythroid precursors exceeding 10% of the lineage elements were detected in all cases with adequate cells for evaluation (myeloid: 24/24 cases, megakaryocytic: 23/23 cases, erythroid: 25/25 cases, Supplemental Table 2, Supplemental Digital Content 3, http://links.lww.com/PAS/B996). A range of reproducible and striking morphologic findings was recorded in myeloblasts, including large forms with condensed eosinophilic cytoplasm (23/24, 96%) and occasionally with voluminous cytoplasm (Figs. 1 and 2). Erythroid cells showed nuclear-cytoplasmic asynchrony, binucleation, karyorrhexis, irregular nuclear borders, and megaloblastic changes. The most frequent dysplastic feature in the megakaryocyte lineage was small megakaryocytes with single lobes. Significant increases in megakaryocytes at diagnosis were detected in 2 cases: #32 had myelodysplastic features with myelofibrosis (Fig. 1F–I) and #9 had features of megakaryoblastic leukemia. One or 2 small Auer rods in occasional blasts were detected in 24/27 (88%).
FIGURE 1.
Morphologic and immunophenotypic features of case #19 and morphologic features of case #32: Case ID #19 was initially diagnosed as AML-M2/AML with maturation (FAB classification), showing hypercellular marrow with increased blasts (A, hematoxylin and eosin). Aspirate smears show characteristic blasts, myeloid precursor cells with eosinophilic granules (B, Wright-Giemsa), dysplastic erythroid cells, and blasts with small Auer rods (C, Wright-Giemsa). Myeloperoxidase stain is brightly expressed in Myeloblasts with eosinophilic granules while dim to negative in other myeloblasts (D). Flow cytometry immunophenotyping shows blasts with high side scatter merging with granulocytes. Blasts are positive for CD7 (dim), CD13 (partial), CD33 (bright), CD34 (dim), CD117 (bright), CD123 (bright), CD133 (dim), and HLA-DR (moderate) (E). Case ID #32 was diagnosed as MDS with EBs, showing hypercellular marrow with a marked increase in megakaryocytes and osteosclerosis, features commonly seen in MDS with myelofibrosis (F, hematoxylin and eosin). Aspirate smears show characteristic myeloid precursor cells with eosinophilic granules (G, Wright-Giemsa) and small megakaryocytes (H, Wright-Giemsa). BM biopsy at post-stem cell transplant in the most recent follow-up is morphologically unremarkable and normocellular (I, hematoxylin and eosin).
FIGURE 2.
Morphologic features of cases #2, 9, and 4: Case ID #2 was initially diagnosed as AML-M6a. BM biopsy is hypercellular, mainly composed of blasts and mature erythroid cells (A, hematoxylin and eosin). BM aspirates show many erythroblasts (blue arrow) and some myeloblasts (black arrow). Dyserythropoiesis and dysmegakaryopoiesis (red arrow) are prominent (B, Wright-Giemsa, and C, Wright-Giemsa). Case ID #9 was diagnosed initially as AML-M0/M7. Initial BM examination (D–F) showed a hypercellular marrow with an increased number of small-sized blasts, partially highlighted by CD61 immunohistochemical stain, consistent with megakaryocytic lineage ([D] hematoxylin and eosin, and [E] CD61 immunohistochemistry). BM aspirates showed morphologically different types of blasts, including some intermediate -large sized blasts with cytoplasmic granules (red arrowhead) and some small-sized blasts with scant agranular cytoplasm (black arrowhead) (F, Wright-Giemsa). During therapy, the BM biopsy showed persistent disease with foci of fibrosis and persistent blasts ([G] hematoxylin and eosin and [H] hematoxylin and eosin). There was an increased number of CD61-positive blasts compared with the initial diagnosis (I, CD61 immunohistochemistry), and the BM aspirate is mainly composed of small-sized blasts with scant agranular cytoplasm most consistent with AML-M7 (J, Wright-Giemsa). Case ID #4 presented with leukocytosis (K, Wright-Giemsa) and an increased number of circulating blasts and was diagnosed as AML-M4. The BM biopsy was hypercellular with increased blasts (L, hematoxylin, and eosin) and scattered small megakaryocytes (yellow arrowheads). BM aspirate smears show blasts with myelomonocytic features and scattered dysplastic erythroid cells (M, Wright-Giemsa).
Blast Immunophenotype
The blasts had a myeloid or myelomonocytic immunophenotype with consistent expression of CD13 (25/25, 100%), CD33 (25/25, 100%), CD34 (21/25, 84%), CD38 (10/12, 83%), CD117 (24/25, 96%), CD123 (10/10, 100%), HLA-DR (25/25, 100%), and MPO (17/23, 74%). Despite the expression of CD34 and CD117 in the majority of cases, 66% (14/21) of CD34+ and 54% (13/24) of CD117+ cases had partial/dim expression. Similarly, MPO was positive in the majority of cases, however, only 2 cases had a bright expression of this marker (2/17, 12%). In contrast, CD123 was moderate to bright in all tested cases (10/10, 100%). A subset of cases had blasts that expressed CD15 (12/24, 50%), and/or monocyte-associated antigens including CD4 (7/25, 28%), CD14 (1/25, 4%), CD36 (14/18, 77%), and CD64 (13/25, 52%). Blasts were negative for lymphoid antigens including CD2 (n = 25), cytoplasmic CD3 (n = 23), CD5 (n = 23), and CD10 (n = 14). Positive staining for CD7, CD56, and CD19 was detected in 52% (13/25), 3% (1/25), and 8% (2/24), respectively. No AML cases in our cohort met the criteria for mixed phenotype acute leukemia (Figs. 1E and 3). No significant differences were identified in the expression of selected markers between UBTF-TD+ myeloid neoplasms with and without FLT3-ITD (Fig. 3).
FIGURE 3.
Heatmap depicting the presence and absence of flow cytometry immunophenotype markers, selected mutations,8 and CG in pediatric patients with UBTF-TD. Absolute numbers are shown in the bars along the right side.
Cytogenetic Findings
Complete karyotype results at diagnosis/I were available in 21 patients (AML: n = 16; MDS: n = 5; Table 1, Fig. 3, and Supplemental Table 3, Supplemental Digital Content 4, http://links.lww.com/PAS/B997). Among 16 AML cases, 11 (68%) had a normal karyotype. All 5 MDS cases with cytogenetics (CG; 100%) had a normal karyotype. Trisomy 8 (n = 4, 20%) was the most common chromosomal aberration in AML. None of the patients at diagnosis/I had a complex karyotype. Nine patients had a karyotype in R/P samples with a normal karyotype in 6 (66%). The karyotype in 4 cases was available at both timelines (diagnosis/I and R/P; cases #5, 13, 28, 29), 3 with a normal karyotype at both time points, and 1 (#5) with a change from normal to acquiring a t(14;22) balanced translocation. Both MDS cases progressing to AML (#28 and 29) with available karyotypes at both time points had normal karyotypes at both MDS and AML samples (Fig. 3 and Supplemental Table 2, Supplemental Digital Content 2, http://links.lww.com/PAS/B995).
Molecular Findings
Molecular profiles, which have been previously reported,8 were available at diagnosis/I (AML: n = 14; MDS: n = 5) or R/P (AML: n = 8; Fig. 3 and Supplemental Table 3, Supplemental Digital Content 4, http://links.lww.com/PAS/B997). As previously observed, FLT3-ITD mutation was detected in 10 of 27 (37%) cases assessed (Table 1, Fig. 3, and Supplemental Table 3, Supplemental Digital Content 4, http://links.lww.com/PAS/B997) and is not observed in MDS cases. Case #27 reportedly tested negative for FLT3 mutations at the MDS timepoint and acquired FLT3-ITD at the AML sample. Although limited in the number of cases, those with M6a morphology did not have FLT3-ITD alterations.
Disease Classification
Although UBTF-TD is now recognized as a recurrent and subtype-defining alteration, it is still not clinically reported at many centers. Thus, we opted to classify these myeloid tumors both using UBTF-TD as a defining event that could potentially meet the criteria for WHO 2022 as “acute myeloid leukemia with other defined genetic alteration”9 and without recognizing UBTF-TD as such a molecular alteration. The classification of the MDS cases (n = 6) using the WHO 2022 and ICC 2022 systems was as follows: childhood MDS with excess blasts (EBs) or increased blasts (IBs) by WHO 2022 (fifth edition) and MDS-EBs by ICC 2022. By FAB, MDS cases were classified as refractory anemia with EBs in transformation (n = 4, 66%) or M6a (n = 2, 33%; Supplemental Table 2, Supplemental Digital Content 2, http://links.lww.com/PAS/B995). Using UBTF-TD as an established genetic alteration allowed for 18 (18/21, 86%) of the AMLs to be classified as “acute myeloid leukemia with other defined genetic alteration (AML with ODGA)” and 3 (3/21, 14%) as “acute myeloid leukemia, myelodysplasia related (AML-MR)” assuming that the myelodysplasia-related alterations take precedence of alterations that allow for the recognition of “other defined genetic alterations.” In the absence of UBTF-TD, these cases would be classified into AML “defined by differentiation” in the WHO 2022 classification. Using ICC 2022, the AMLs would be grouped into the “NOS” category, with or without the recognition of UBTF-TD in the cases lacking myelodysplasia-related mutations or cytogenetic alterations. The discrepancy between WHO 2022 and ICC 2022 resulted from the inclusion of trisomy 8 as a myelodysplasia-related cytogenetic abnormality (AML-MDSk) only in ICC 2022. By FAB, AML samples (n = 26) from 24 patients were classified as follows: M2: 13/26 (50%), M4: 8/26 (31%), M6a: 4/26 (15%), and M7:1/26 (4%). Morphologic evolution was observed in 5 cases (AML cases: n = 2, M6a to M2, #13 and 15; MDS cases: n = 3, refractory anemia with EBs in transformation to M2 in #28 and 29; and M6a to M4 in #27) at R/P compared with diagnosis/I (Table 1 and Supplemental Table 2, Supplemental Digital Content 2, http://links.lww.com/PAS/B996).
Treatment and Prognosis
Twenty-five patients (AML, n = 19; MDS, n = 6) had a follow-up with a median follow-up time of 32.7 months (range: 3.5 to 291.0 mo; Table 1, and Supplemental Fig. 1, Supplemental Digital Content 1, http://links.lww.com/PAS/B994). At the end of induction, 18 (72%) patients had persistent/progressive disease by positive minimal residual disease by flow cytometry immunophenotyping or dysplasia in BM examination (Supplemental Table 1, Supplemental Digital Content 2, http://links.lww.com/PAS/B995). At the last follow-up examination, 11 (44%) patients were alive and 14 (56%; AML, n = 10; MDS, n = 4) patients died (Table 1, and Supplemental Table 1, Supplemental Digital Content 2, http://links.lww.com/PAS/B995). The median OS was 37.7 months. The most common cause of death for these patients (11/14, 78%) was R/P disease. The other 3 patients died while in complete remission due to complications related to pulmonary hemorrhage, sepsis, or graft-versus-host disease. Among 11 patients who were alive at the last follow-up, 10 had complete remission (91%).
Patients with MDS did not show a survival advantage over patients with AML (10.7 vs 45.9 mo, P = 0.25), whereas these data have limitations due to the small number of patients with MDS. Among 24 patients with available treatment history, all were treated with conventional chemotherapy (24 patients, 100%) along with a subset receiving targeted therapies (15 patients, 62%), and HSCT (20 patients, 83%). Eleven patients received an FLT3-inhibitor in addition to chemotherapy and HSCT.
Case Illustration
While there are some unifying morphologic and immunophenotypic features of UBTF-TD myeloid tumors (Fig. 1A–E, case ID #19), including morphologic dysplasia, there is a remarkable variety of possible presentations. Four examples are highlighted below.
MDS (With Myelofibrosis)
Case ID #32 (Fig. 1F–I): A 12-year-old male with pancytopenia and 1% blasts in the blood smear. Initial BM examination showed a cellular marrow (60% cellularity) with IBs (5%), a few with Auer rods, left-shifted myeloid maturation, trilineage dysplasia, and erythroid hyperplasia consistent with the diagnosis of MDS-EB (ICC 2022)/childhood MDS with IB (WHO 2022). Megakaryocytes showed dense clustering and cellular atypia with a wide range of sizes, from small, naked megakaryocytes with hyperchromatic chromatin to large megakaryocytes with bulbous nuclei. Erythroid cells showed dysplastic features, including cytoplasmic/nuclear asynchrony, irregular nuclear borders, karyorrhexis, and megaloblastic changes. Myeloid cells were dysplastic with irregular and folded nuclei, and voluminous cytoplasm with abnormal and occasionally asymmetric eosinophilic granulation. Additional morphologic findings included increased reticulin and collagen fibrosis with osteosclerosis. Molecular tests detected WT1 p.H448R missense mutation and UBTF-TD (exon 13); CG revealed a normal karyotype (Supplemental Table 3, Supplemental Digital Content 4, http://links.lww.com/PAS/B997).
AML, Erythroid Predominant
Case ID #2 (Fig. 2A–C): An 18-year-old male presented with progressive weakness. CBC showed pancytopenia and circulating blasts: WBC: 1.1×109/L, Hgb: 9.7 g/dL, PLT: 12.0×109/L, and 8% blasts. BM examination at the time of admission showed hypercellular marrow (95% cellularity) with erythroid hyperplasia (50% of the marrow cellularity composed of erythroid precursors at all stages of maturation and prominent dysplastic changes). Of the nonerythroid cells, >50% were myeloblasts that contained few Auer rods. Myeloblasts accounted for 26% of total nucleated cells. Maturing myeloid cells decreased in number and consisted predominantly of dysplastic elements. Megakaryocytes were significantly decreased and consisted mainly of small dysplastic forms and micromegakaryocytes. Flow cytometry immunophenotyping of the BM revealed a myeloid blast phenotype with moderate to bright expression of CD13, CD33, CD117, and HLA-DR and partial/dim expression of CD34, MPO, CD7, and CD19. Blasts did not express monocytic markers and CD56. CG demonstrated 47,XY,+8(17)/46,XY(3) and molecular tests detected UBTF-TD (exon 13). The findings were consistent with AML-M6a (FAB)/AML-MDSk (ICC 2022). There is no terminology for such cases in WHO 2022 in the absence of UBTF-TD, and trisomy 8 is not considered a defining cytogenetic abnormality. This case could be classified as “acute myeloid leukemia with other defined genetic alteration” (WHO 2022) with the recognition of UBTF-TD.
AML, Megakaryocytic Predominant
Case ID #9 (Fig. 2D–J): An 11-year-old male with relapsed acute myeloid leukemia and pancytopenia: WBC: 0.7×109/L, Hgb: 9.4 g/dL, and PLT: 41.0×109/L at admission. BM examination showed cellular marrow (70% cellularity) with a marked increase in blasts with round to irregular nuclei, scant to moderate cytoplasm, fine chromatin, and indistinct to prominent nucleoli. Occasional blasts had cytoplasmic eosinophilic granulation. Maturing dysplastic myeloid and erythroid precursor cells were seen, and mature megakaryocytes were virtually absent. Marrow fibrosis was not present. Flow cytometry immunophenotyping of the BM revealed a distinct abnormal population (21% of total events) located in the blast gate (dim to moderate CD45 with slightly increased side scatter) positive for CD7(dim), CD11c, CD13, CD33, CD34, CD36, CD41, CD42b (small subset), CD61(dim), CD71, HLA-DR, CD117(dim), CD133, and negative for cytoMPO, cytoTdT, CD235a, specific B-cell and T-cell associated antigens. Immunohistochemical stains confirmed the expression of CD42b and CD61 in a subset of blasts (<50%), yet this case was still difficult to classify the case as megakaryocytic leukemia and was classified as FAB M0/M7 (Fig. 2D-F). However, CD42+/CD61+ blasts increased in follow-up samples with persistent disease (FAB M7; Fig. 2G–J). Trisomy 8 was detected in the R/P sample: 47,XY,+8(12)/46,XY(8). Molecular tests revealed FLT3-ITD and WT1 p.L361_S364fs frameshift mutation and UBTF-TD (exon 13). With UBTF-TD, this case would be classified as “acute myeloid leukemia with other defined genetic alteration” (WHO 2022). Alternatively, the findings were consistent with AML-M7 (FAB)/AML with minimal or megakaryocytic differentiation (WHO 2022, fifth edition) /AML-MDSk (ICC 2022).
AML With Leukocytosis
Case ID #4 (Fig. 2K–M): An 11-year-old male presented with leukocytosis: WBC: 103.6×109/L, Hgb: 6.3 g/dL, and PLT: 197.0×109/L. BM examination showed a hypercellular marrow (~100% cellularity) with an expansion of intermediate to large blasts with abundant pale blue cytoplasm, round to delicately folded nuclear contours, finely dispersed chromatin, and prominent nucleoli. Maturing myeloid elements showed dysplastic features. Monocytes accounted for ~20% of cellularity and showed dysplastic features. Scattered eosinophils (some with a few basophilic granules) were noted. Maturing erythroid elements were markedly decreased and showed dysplastic features. Megakaryocytes were rare and mostly composed of hypolobated forms. Flow cytometric analysis performed on the PB demonstrated an expanded population of blasts (~46% of total) that were positive for CD33, CD34, CD117 (dim), CD15 (dim, subset), CD13, CD36 (subset), CD64 (dim, subset), CD11b (subset), HLA-DR, CD133, CD71 (dim), CD9 (dim), and cyMPO; and negative for B-cell and T-cell associated markers, CD14, CD16, CD42b, and CD56. Cytogenetic analysis showed 46,XY(20), and molecular findings reported FLT3-ITD and UBTF-TD (exon 13), allowing for the classification as “ acute myeloid leukemia with other defined genetic alteration” (WHO 2022). In the absence of UBTF-TD, these findings would be diagnostic of acute myelomonocytic leukemia (FAB: M4)/AMML (WHO 2022, fifth edition)/AML-NOS (ICC 2022)
DISCUSSION
UBTF-TD is a newly recognized driver of AML and MDS that is enriched in adolescent patients but can occur across the age spectrum.2,16 Previous molecular evaluations have highlighted a strong cooccurrence with FLT3-ITD and WT1 mutations along with either normal karyotype or trisomy 8,8 however, an in-depth morphologic and immunophenotypic evaluation has not been reported. Here we evaluated the morphologic and immunophenotypic features of 27 UBTF-TD-positive myeloid neoplasms (MDS, n = 6; AML, n = 21) from a previously published pediatric cohort8 to better define the clinicopathologic characteristics of this new subtype.
A near-universal morphologic hint for the presence of UBTF-TD is multilineage dysplasia, which is equally present in the MDS and AML cases. While a range of different dysplastic features was observed in erythroid cells, micromegakaryocytes were the most frequent dysplastic feature in megakaryocytes. Granulocytes showed a distinctive dysplastic morphologic feature with the presence of large blasts with abundant basophilic cytoplasm, often containing condensed eosinophilic cytoplasm. With the presence of Auer rods and eosinophilic granulation, many cases may be morphologically suggestive of core binding factor AML. These morphologic features also make an accurate blast count challenging, which is further compounded by the fact that the UBTF-TD alteration is also present in mature myeloid cells in UBTF-TD-positive cases.8 Collectively, these data support the notion that MDS and AML cases with UBTF-TD are part of a disease continuum independent of blast count, like other defined alterations in myeloid diseases, and should be classified together as a single entity. This is further supported by the rapid progression of MDS to AML progression observed in this study.
Erythroid hyperplasia and dysplastic changes have been recognized in UBTF-TD cases in adults,6 which was further confirmed in this study. Notably, the most significant morphologic change during therapy between diagnosis/I and R/P) sample was a decrease in erythroid features during disease progression. This was observed both in relapsed AML cases as well as in MDS cases that progressed to AML. Like MDS cases, those cases with M6a morphologic features also lacked FLT3-ITD mutations. These data collectively suggest that the acquisition of FLT3-ITD as a secondary mutation is associated with more aggressive disease and less erythroid features. Supporting this is our previous observation that FLT3-ITD mutations are associated with higher WBC and blast counts in UBTF-TD cases.8 This was also observed in a previously published adult cohort which found that high VAF (≥20%) FLT3-ITD mutations were associated with AML-M1/2 and significant BM blast infiltrates.6
The timely and accurate diagnosis of UBTF-TD is clinically important since these AMLs are known to be associated with inferior outcomes. Recent studies have importantly shown that cells harboring UBTF-TD are sensitive to Menin inhibitors,17,18 further adding to the clinical need to accurately diagnose these cases. The possibility of UBTF-TD needs to be considered by the presence of dysplasia in an AML in the absence of other known genetic alterations associated with AML and dysplasia and in the presence of normal CG or trisomy 8. This is especially true for the evaluation of adolescent patients with a myeloid abnormality. However, the variable clinicopathologic features of these cases outlined in the case presentations will complicate the identification of these cases, especially since UBTF is not covered currently by most sequencing panels. Ideally, upfront evaluation with unbiased sequencing strategies or updated next-generation sequencing panels is needed for appropriate evaluation of all new AML/MDS cases. While the immunophenotypic findings reported here may also provide some clues, such as variable CD34 and CD117, uniform HLA-DR, CD33 and CD123 expression, and aberrant CD7 expression, these findings are not specific to UBTF-TD myeloid tumors. In fact, a recently published study with fewer patients and more variable patient ages noted less consistent expression of HLA-DR and CD34 compared with our cohort.18 This further underscores the lack of clear and consistent immunophenotypic features of UBTF-TD myeloid tumors and also suggests that it may be difficult to monitor residual disease in UBTF-TD-positive AMLs by flow cytometry and that molecular strategies may be ideal.19 While not necessarily helpful for diagnosis, the relatively uniform expression of CD123, which was observed by both studies, suggests that targeted anti-CD123 therapy may be effective in UBTF-TD myeloid neoplasms.
AML cases with UBTF-TD could be defined as “AML with other defined genetic alterations,” “AML, defined by differentiation,” “AML, NOS, or AML, myelodysplasia-related” in WHO/ICC 2022. The observed trilineage dysplasia present in all cases would have led to a diagnosis of AML with myelodysplasia-related changes using WHO2016 criteria, however, morphologic dysplasia is no longer a defining feature in WHO 2022, nor is it part of the ICC 2022 classification. Importantly, the results presented here and elsewhere6–8 highlighting the prevalence of this mutation in pediatric and adolescent patients with MDS and AML, lack of other defining alterations, consistent molecular features characterized by high HOX gene expression, cooccurring WT1 and FLT3-ITD mutations, and the high mortality rate strongly suggest that UBTF-TD should be a distinct molecular category in future WHO and ICC classifications that encompasses both MDS and AML cases.
Supplementary Material
Footnotes
The work was funded by the American Lebanese and Syrian Associated Charities of St. Jude Children’s Research Hospital, the Jane Coffin Childs Fund (J.M.B.), and funds from the US NIH, including F32 HL154636 (J.M.B.) and R01 CA276079 (J.M.K.).
The content, however, does not necessarily represent the official views of the NIH and is solely the responsibility of the authors.
Conflicts of Interest and Source of Funding: J.M.K. and M.U.: Honorarium, AstraZeneca Japan. J.M.K. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund. For the remaining authors, none were declared.
Supplemental Digital Content is available for this article. Direct URL citations are provided in the HTML and PDF versions of this article on the journal's website, www.ajsp.com.
Contributor Information
Mahsa Khanlari, Email: Mahsa.Khanlari@Stjude.org.
Wei Wang, Email: wwang13@mdanderson.org.
Yonghui Ni, Email: yonghui.ni@stjude.org.
Paul E. Mead, Email: paul.mead@stjude.org.
Masayuki Umeda, Email: masayuki.umeda@stjude.org.
Tami Westover, Email: tamara.westover@stjude.org.
Jing Ma, Email: jing.ma@stjude.org.
Jeffrey E. Rubnitz, Email: rubnitzj@gmail.com.
Juan M. Barajas, Email: juanmartin.barajas@stjude.org.
Stanley Pounds, Email: stanley.pounds@stjude.org.
Jeffery M. Klco, Email: jeffery.klco@stjude.org.
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