Submit articles online
ISMPO
THIEME

Year (Archive)

Year

Issues

Search

Recent Articles

IJMPO—A Journey of a Thousand Miles

Author : Padmaj S. Kulkarni

Coronavirus Disease 2019 Treatment—T-Cells Hold the Key in Severe Cases

Author : Kunal Das, Nitika Agrawal, Mansi Kala, Rakhee Khanduri

Why Is China Importing COVID-19 Vaccine Now?

Author : Purvish M. Parikh

Terminal Deoxynucleotidyl Transferase (TdT) Positive Acute Myeloid Leukemia with C-MYC and BCL2 Expression: A Distinct Biological Entity with Potential Clinical Implications

CC BY 4.0 · Indian J Med Paediatr Oncol 2025; 46(05): 508-516

DOI: DOI: 10.1055/s-0045-1809173

Author : Chinmayee Panigrahi,Biswajit Bhuyan,Prabodha Kumar Das,Somanath Padhi,Gaurav Chhabra,Ashutosh Panigrahi,Arushi Choudhary,Saheeta Sudarsini,Prapti,Acharya,Debasis Jash

Abstract

Terminal deoxynucleotidyl transferase (TdT) is a unique deoxyribonucleic acid polymerase whose overexpression has been used as an important biomarker in the diagnosis of precursor B or T cell acute lymphoblastic leukemia/lymphoma. Over the last few years, TdT has been increasingly implicated in the genesis of mutant Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and NPM1 isoforms, two key events in the myeloid stem cell leukemogenesis. This article studies the diagnostic and prognostic implications of immunoexpression of TdT, C-MYC, and BCL2 in acute myeloid leukemia (AML). We retrospectively reviewed seven cases of TdT positive (+), myeloperoxidase negative AMLs (2022–2024) with C-MYC and/or BCL2 expression by immunohistochemistry (IHC), and correlated these with flow cytometric (FC), molecular, and/or cytogenetics data (wherever available) along with outcome following induction chemotherapy. Morphologically, all these cases resembled French-American-British M0, M1, and M5 AMLs, or even high-grade lymphoproliferative neoplasms with moderate to stronger nuclear positivity for TdT and C-MYC in a higher proportion neoplastic cell along with stronger cytoplasmic BCL2 expression. Two were FLT3-ITD mutated, one was TP53 mutated, one had high-risk cytogenetics, whereas the remainder had no detectable abnormalities on molecular testing with variable degree of therapeutic response. Literature review highlighted the negative impact of overall, event-free, and relapse-free survivals among TdT and C-MYC positive AMLs whereas BCL2 expressors were likely to have increased chemoresistance. We suggest that TdT +/C-MYC +/BCL2+ AMLs may serve as distinct biological subtype; and IHC/FC-based testing should be a part of routine diagnostic workup for better risk stratification in AMLs.

Keywords

immunohistochemistry - TdT - myeloid leukemia - molecular testing - prognosis

Authors' Contributions

C.P. and S.P.: Conceptual design, data curation, diagnosis, writing the original draft, review, and editing. B.B., P.K.D., and A.P.: Management of cases, follow-up data, and critical review of scientific content. G.C., S.P., and D.J.: Flow cytometry evaluation of cases. S.S. and P.A.: Diagnosis and literature review. A.C.: Literature review.


Ethical Approval

The study was part of the retrospective review proposal approved by the Institutional Ethics Committee of AIIMS, Bhubaneswar (T/IM-NF/Patho/24/52).


Patient's Consent

Patient consent is not required.




Publication History

Article published online:
19 May 2025

© 2025. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)

Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India

We recommend

Abstract

Terminal deoxynucleotidyl transferase (TdT) is a unique deoxyribonucleic acid polymerase whose overexpression has been used as an important biomarker in the diagnosis of precursor B or T cell acute lymphoblastic leukemia/lymphoma. Over the last few years, TdT has been increasingly implicated in the genesis of mutant Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and NPM1 isoforms, two key events in the myeloid stem cell leukemogenesis. This article studies the diagnostic and prognostic implications of immunoexpression of TdT, C-MYC, and BCL2 in acute myeloid leukemia (AML). We retrospectively reviewed seven cases of TdT positive (+), myeloperoxidase negative AMLs (2022–2024) with C-MYC and/or BCL2 expression by immunohistochemistry (IHC), and correlated these with flow cytometric (FC), molecular, and/or cytogenetics data (wherever available) along with outcome following induction chemotherapy. Morphologically, all these cases resembled French-American-British M0, M1, and M5 AMLs, or even high-grade lymphoproliferative neoplasms with moderate to stronger nuclear positivity for TdT and C-MYC in a higher proportion neoplastic cell along with stronger cytoplasmic BCL2 expression. Two were FLT3-ITD mutated, one was TP53 mutated, one had high-risk cytogenetics, whereas the remainder had no detectable abnormalities on molecular testing with variable degree of therapeutic response. Literature review highlighted the negative impact of overall, event-free, and relapse-free survivals among TdT and C-MYC positive AMLs whereas BCL2 expressors were likely to have increased chemoresistance. We suggest that TdT +/C-MYC +/BCL2+ AMLs may serve as distinct biological subtype; and IHC/FC-based testing should be a part of routine diagnostic workup for better risk stratification in AMLs.

Keywords

immunohistochemistry - TdT - myeloid leukemia - molecular testing - prognosis

Introduction

Terminal deoxynucleotidyl transferase (TdT) is a nuclear template-independent deoxyribonucleic acid (DNA) polymerase that catalyzes the addition of deoxynucleotides to the 3′-hydroxyl terminus of oligonucleotide primers during V(D)J recombination to enhance antigen receptor diversity. Therefore, it plays a crucial role in the development of immature (precursor) B (pre-B) and T (pre-T) lymphoid cells in the bone marrow (BM) and thymus, respectively.[1] The template-independent insertion of nucleotide is unique for TdT that also makes it a potentially vulnerable target for neoplastic transformation of pre-B or pre-T lymphoid cells.[2] TdT overexpression is considered as an important biomarker in the diagnostic evaluation of pre-B/pre-T acute lymphoblastic leukemia/lymphoma (ALL), lymphoblastic blast crisis in chronic myeloid leukemia (CML-LB), and some non-Hodgkin lymphomas (NHLs).[3] Although TdT expression is primarily limited to lymphoid stem cells to restrict genetic infidelity, discovery of frequent TdT expression among myeloid stem cell lines has potentially increased the risk for development of acute myeloid leukemia (AML).[4] [5] In this article, we aimed to describe TdT expression in a series of AML cases at our center and comprehensively review the literature on TdT expression in AML and their possible interaction with other prognostic biomarkers such as C-MYC and BCl2.

Materials and Methods

We retrospectively reviewed all myeloperoxidase (MPO) negative AML cases (≥ 18 years) diagnosed and managed at our center over the last 2 years (April 2022–April 2024). The study was part of the retrospective record review and analyses cleared by the Institutional Ethics Committee (ref. no. T/IM-NF/Patho/24/52). All cases of AML were diagnosed and differentiated from ALL, early T-precursor ALL (ETP-ALL), and mixed phenotypic acute leukemia based upon constellation of morphologic (blast proportion, French-American-British [FAB] morphology, cytochemistry) and immunophenotypic characteristics supplemented with molecular/cytogenetics data (wherever available) as per the 2017 World Health Organization (WHO) guidelines.[6] MPO negativity was confirmed by cytochemistry and flow cytometric (FC) immunophenotypic analysis performed on either peripheral blood (PB) and/or BM aspirate (BMA) sample as well as on BM trephine biopsy (BMBx) sections using immunohistochemistry (IHC) (mentioned below). Immunohistochemical expression of TdT of any intensity (weak/dim [+], moderate [++], or strong [++ + ]) positivity in at least 10% myeloid blasts was considered as positive; and the same was correlated with FC [removed]wherever available) and other antigen expression profile. Additionally, TdT expression was corroborated with other markers such as CD10, BCL2, C-MYC, and cyclin D1 wherever performed to confirm or rule out B cell NHLs. All cases of acute promyelocytic leukemias, myelodysplastic syndrome-increased blast 2 (MDS-IB2), and CML in myeloid blast crisis (CML-MB) were excluded from the review. The clinicomorphological and immunophenotypic features were correlated with molecular and/or cytogenetics data (wherever available) and therapeutic outcome following induction chemotherapy (CT). The Institutional Review Board of our institute approved the retrospective study protocol.

Immunophenotyping

IHC was performed on paraffin-embedded, 14% ethylenediaminetetraacetic acid decalcified trephine biopsy sections following standard protocol and using following selected antibodies in variable combination on a case-to-case basis as follows: CD34 (Q-band 10, 1:100), CD117 (EP10, 1:100), MPO (EP151, ready to use, RTU), TdT (E266, RTU), BCl2 (EP36, 1:100), C-MYC (EP121, RTU), cyclin D1 (EP12, RTU) (all PathnSitu), and CD10 (DAKO, RTU). FC immunophenotypic analysis was performed on PB and/or BMA sample using FACS Canto II flow cytometer (BD Biosciences, India) using a panel of antibodies and appropriate fluorochromes (presented in [Table 1]). The sample was processed using the stain-lyse-wash procedure. Initially, the sample was stained with an antibody panel consisting of pre-titrated antibodies for surface staining followed by the fixation, permeabilization, and staining for nuclear (Nu) TdT antibody (APC, clone E-17–1519, BD Biosciences, San Jose, California, United States).


Table 1

Panel of antibodies used in the flow cytometric evaluation for acute leukemia[a]

Fluorochromes

V450

V500c

FITC

PE

PerCPCy5.5

PECy7

APC

APCH7

A LOT

cyCD3

CD45

cyMPO

cyCD79a

CD34

CD19

CD7

smCD3

B-tube

CD20

CD45

CD58

CD66c

CD34

CD19

CD10

CD38

AML tube

HLA-DR

CD45

CD64

CD13

CD34

CD117

CD33

CD14

T tube

CD4

CD45

CD1a

–

CD5

CD2

nuTdT

CD8

AML extra tube

CD45

CD15

CD56

CD61

CD34

CD11c

CD41a

CD123

CD71

Abbreviations: AML, acute lymphoblastic leukemia; APC, adenomatous polyposis coli; FITC, fluorescein isothiocyanate; PE, phycoerythrin.


a FACS Canto II flow cytometer (BD Biosciences, India).

Cytogenetics and/or Molecular Studies

Heparinized BMA samples were processed for conventional G-banded karyotyping for any numerical and/or structural abnormalities and reported following the International System for Human Cytogenomic Nomenclature 2020 guidelines. Interphase fluorescent in situ hybridization (FISH) probes were used to detect AML defining fusion genes as per the 2017 WHO classification.[6] Heparinized BMA samples from last four subjects were subjected to targeted next-generation sequencing (NGS) assays to detect the mutant DNA and fusion transcripts as per the Oncomine Myeloid Panel.

Therapy and Monitoring

All patients < 60 years with good performance status were managed with conventional 3 + 7 induction CT regimen comprising of injection daunorubicin (60 mg/m2 from day 1 to day 3 as intravenous infusion in normal saline over 2 hours) and cytosine arabinoside (100 mg/m2 from day 1 to day 7 as continuous infusion over 24 hours daily) along with broad-spectrum antibiotics, transfusion support, and antifungal prophylaxis (tablet voriconazole 200 mg twice daily). Fms-like tyrosine kinase (FLT) tyrosine kinase inhibitor (midostaurin) was used in those patients (cases 1 and 2, [Table 2]) with FLT-internal tandem duplication (ITD) positivity. Those patients ≥ 60 years and unfit for intensive CT regimen were managed with tablet venetoclast and injection azacitidine (Ven-Aza) combination as per protocol. Response to therapy was assessed after hematological recovery post-day 14 of starting CT by assessing BM blast percentage, absolute neutrophil count, platelet count, presence or absence of extramedullary disease, and transfusion dependency.[7] [8]



Table 2

Case series of TdT positive acute myeloid leukemia with C-MYC and BCL2 expression

Sl. No.

Age, y, gender

Presentation

Peripheral blood smear

Bone marrow blasts, FAB morphology

TdT/ intensity[a]

FC intensity

C-MYC (%), intensity[a]

BCL2 (%), intensity[a]

Molecular/cytogenetics

Follow-up

1

21, male

Bone pain, fatigue

Pancytopenia, 9% blast

95%, M0/M1

90%, +++

CD342+ CD1172+

TdTNP

MPO−

CD2+

CD19+

20%, ++

Not done

FLT/ITD; mutated by FISH

Remission, lost to follow-up

2

18, male

Weakness, lethargy

A + T with 70% blast

90%, M1/M5

90%, +++

CD342+ CD1172+ MPO−/+

TdTNP

CD19+

–

30%, ++

FLT/ITD; mutated

NF1; mutated

PHF6; mutated

(by FISH)

Remission

3

61, male

Weakness, fever, k/c/o tuberculosis on ATT

A + L with 3% blast

50% (AML-MR), M0

60%, +++

CD34+ CD117+

HLA-DR+

MPO−

CD13+

TdT2+

CD7 + (aberrant)

Not done

20%, ++

46, XY;

t(6;17) (q15;p13), t(4;8) (q31.3;q24.1)

FISH; negative

Expired

4

32, female

Heavy menstrual bleed

Pancytopenia

50%, M4/M5

60%, ++

CD34+ CD117+ MPO+

TdTNP

CD79a+

Not done

Not done

Del 17q; positive (by NGS)

Partial remission, lost to follow-up

5

73, male

Coronary artery disease

Pancytopenia with 35% blast

70%, M0/M1

10%, ++

CD342+ CD117+

MPO+

TdT+

30%, +++

Not done

No fusion identified (by NGS)

Lost to follow-up

6

57, male

Fatigue

A + L with lymphoplasmacytoid cells, platelet; 300 × 109/L

Rouleaux present; ?LPL/WM

60%, M0/M5/PLL

70%, +++

CD342+ TdT+++ CD1172+ HLA- DR+++

MPO+/−

CD19−

CD20−

25%, ++

70%, +++

No fusion identified (by NGS)

Remission, alive

7

17, male

Breathlessness, lymphadenopathy, pleural effusion

A + T

Marked leukocytosis (500 × 109/L)

> 95% blasts

ETP-ALL

> 90% undifferentiated blasts;

FAB M0 > ETP-ALL

15%, ++

CD34+++

CD117+++

HLA-DR2+

TdT+

MPO−

CD13+++

CD332+

cyCD3−

CD1a−

CD4−CD8−

CD5−

CD7+++

CD19−

CD79a+

60%, ++

80%, +++

No fusion identified (by NGS)

Remission, alive
Abbreviations: -, negative; +, weak positivity; ++, intermediate intensity positivity; ++ + , strong intensity positivity; A + T, anemia with thrombocytopenia; AML, acute myeloid leukemia; ETP-ALL, early T-precursor acute lymphoblastic leukemia; FAB, French-American-British; FC, flow cytometry; FISH, fluorescent in situ hybridization; FLT/ITD, Fms-like tyrosine kinase-internal tandem duplication; LPL/WM, lymphoplasmacytic lymphoma/leukemia/Waldenstrom macroglobulinemia; MPO, myeloperoxidase; NGS, next-generation sequencing; NP, not processed in these cases; PLL, prolymphocytic leukemia; TdT, terminal deoxynucleotidyl transferase.

a On trephine biopsy immunohistochemistry (IHC).

Results

The clinicopathological, immunophenotypic, and molecular data of seven cases of TdT positive AML are presented in [Table 2]. These constituted 11% of 64 adult AMLs diagnosed during the last 2 years. These included six males and one female with a median age of 32 years (range: 17–73) at diagnosis. One of the cases (no. 7) presented with breathlessness, generalized lymphadenopathy, and marked leukocytosis (500 × 109/L), thus simulating clinically a lymphoma/leukemia such as ALL. Peripheral smear and BMA examination revealed mostly undifferentiated blasts that resembled FAB M0, M1, or M5 phenotype or a high-grade lymphoproliferative neoplasm; prominent rouleaux formation with presence of lymphoplasmacytoid lymphoid cells in presence of adequate platelets resembled that of lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia in another (no. 6); and of ETP-ALL phenotype with prominent cytoplasmic blebbing in case no. 7. In case no. 3, the BM morphology was consistent with AML-myelodysplasia-related (AML-MR, WHO 2022) where it resembled that of a prolymphocytic leukemia in another (case no. 6). Trephine biopsy section showed an interstitial to diffuse marrow infiltration pattern (blast percentage; 50–95%) in all with focal, myelofibrosis 1 to 2 reticulin (WHO) fibrosis.

On IHC, intermediate to strong, homogenous, nuclear TdT immunopositivity was noted in all cases with a TdT positive blast proportion ranging from 10 to 90% (median: 60%), whereas FC immunoexpression was variable along with variable intensity aberrant expression of B and T lineage markers. Moreover, C-MYC (nuclear) and BCL2 (cytoplasmic), performed in 4/7 cases, showed variable intensity positivity in 20 to 70% of blasts ([Figs. 1] [2] [3] [4] [5] [6]). Molecular/cytogenetic analyses revealed mutated FLT3/ITD in two, translocation t(6;17) (q15;p13) and t(4;8) (q31.3;q24.1) in one with AML-MR, del17q in one, whereas no abnormalities were identified in either FISH- or NGS-based studies in the remainder of the cases. Mutated NPM1 was not identified in any of these cases. Three achieved complete remissions, one expired, and the remaining two were lost to follow-up after in remission.


  Fig 1: Flowchart 1. The stepwise evaluation of all cases of terminal deoxynucleotidyl transferase (TdT) positive acute myeloid leukemia cases among adults (≥ 18 years) (2022–2024) at our center.


 

Fig 2 : Leishman–Giemsa stained hypercellular bone marrow aspirate smears from iliac crest at two different occasions at baseline (A, B) from a 57-year-old male with acute leukemia with undifferentiated morphology mimicking a lymphoproliferative neoplasm, and trephine biopsy sections showing a packed marrow with total replacement by lesional cells (C, hematoxylin and eosin). On immunohistochemistry (IHC), these cells were positive for CD34 (strong, D), myeloperoxidase (MPO) (weak, E), TdT (strong, F), CD117 (weak, focal, G), HLA-DR (strong, H), C-MYC (strong, I), and BCL2 (strong, J), consistent with TdT + acute myeloid leukemia (AML) (all magnifications ×400).


 

Fig 3 : Flow cytometric immunophenotyping on second bone marrow aspirate sample from the above case ([Fig. 2]) depicting gated population of abnormal cells (dim to negative CD45 vs. low-intermediate side scatter [SSC], blast, A), which were positive for CD117 (heterogeneous) and HLA-DR (bright) (B), CD64 and CD14 (C), negative for T (D) and B (E) cell markers, and positive for nuclear terminal deoxynucleotidyl transferase (nuTdT) (F).


 


 Fig 4 : Bone marrow aspirate smear from a 61-year-old male with worsening pancytopenia showing sheets of highly undifferentiated neoplastic cells resembling high grade lymphoproliferative neoplasm or even, leukemia (A). On trephine biopsy and IHC, these cells were strongly and diffusely positive for CD117 (B) and terminal deoxynucleotidyl transferase (TdT) (C), negative for myeloperoxidase (MPO) (not shown), and CD19 (D). Original magnification, ×400 (A–C) (×200, D). Note the corresponding flow cytometric immunophenotyping panel (E to I) depicting dim expression of nuTdT (G) from the same case [blasts (in red) are positive for CD34 (E), CD117 (F), CD7 (H, aberrant), and CD13 (I, heterogeneous)].


 

Fig 5 : Immunohistochemical expression of terminal deoxynucleotidyl transferase (TdT) (A), C-MYC (B), and BCL2 (C) from an adolescent male (case 7, [Table 1]) with generalized lymphadenopathy and high leukocytosis clinically and morphologically suspected to be that of lymphoma/leukemia (possibly early T-precursor acute lymphoblastic leukemia [ETP-ALL]) (×400).


 

Fig 6 : Flow cytometric (FC) immunophenotypic analysis of peripheral blood sample from the above case ([Fig. 5] (Case 7)) showing gated population of cells (dim to moderate CD45 vs. low side scatter [SSC], A) immunopositive for CD34, HLA-DR (B), CD117 (bright, C), CD7 (E), nuclear terminal deoxynucleotidyl transferase (nuTdT) (F), CD2 (H), and CD10 (I). The lesional cells were negative for cytoCD3 (D, dotted black line representing the control T cell population), CD1a (F), both CD4 and CD8 (G), CD5 (H), and other B cell markers. Although the cyCD3 expression pattern did reach up to the control population (green), (D) the possibility of early T-precursor acute lymphoblastic leukemia (ETP-ALL) was ruled out by negative CD3 expression on immunohistochemistry (IHC) (not shown) as well as on repeat flow cytometry evaluation on fresh marrow aspirate sample.

Discussion

Borrow et al in their 2019 cutting edge work have highlighted the pivotal role of TdT in both pediatric and adult myeloid leukemia stem cell lines.[3] [4] They have tried to shed more light into the underlying molecular events that lead to the generation and accumulation of mutated FLT3-ITD and NPM1; two important driver mutations that facilitate risk-adapted therapy in AML. It was proven in TdT models that illegitimate insertional activity of TdT at the 3′ end was the main factor behind the priming of replication slippage of both FLT3-ITD and NPM1 molecules producing more mutated forms in nearly half of all AMLs.[3] [4] [5] Further reports also suggested significantly higher TdT expression among t(6;9) DEK-NUP214 (with marrow basophilia and myelodysplasia) and t(5;11) NUP98-NSD1 fusion (AML in children with FAB M4/M5 phenotype), but uncommonly with t(8;21) RUNX1/RUNX1T1 and inv16 AMLs, which are more likely to harbor FLT3-ITD mutations and conferring a poor prognosis.[9] [10] Moreover, TdT expression was also reported to be linked to the accumulation of mutated FLT3-ITD in variant acute promyelocytic leukemia (M3v) with aberrant lymphoid antigenic expression than the classical/hypergranular phenotype.[11]

Literature on the diagnostic and prognostic implications of TdT expression in AML is sparse.[12] [13] [14] Shaforostova et al,[12] using FC, reported cytoplasmic TdT (cyTdT) positivity among ≥ 20% blasts among 19 of 324 (6%) adult AMLs (median age: 67 years), which was linked to aberrant CD2 co[removed]p < 0.05), lesser incidence of mutated FLT3-ITD (p = 0.52), but wild-type NPM1 (p = 0.04). However, cyTdT and CD2 coexpression was associated with inferior overall survival (OS) (log rank p = 0.10), relapse-free survival (RFS) (p = 0.037), and 3-year event-free survival (EFS) (p = 0.003).[12] De Bellis et al[13] supported Burrow's models where TdT positivity was reported by FC analysis among 49 of 143 (34.2%) de novo AML subjects (62.5 years, range: 21–86 years) and this was linked to male gender (p = 0.078), mutated FLT3-ITD subgroups (p = 0.035), and an inferior 5-year OS (log rank p = 0.03).[13] Another Japanese group reported a significantly inferior 1-year RFS (hazard ratio: 3.309, 95% confidence interval: 1.334–8.209, p = 0.009) among TdT positive (12/45, 25%) (≥ 10% blasts by FC) de novo AMLs (68 years, range: 16–87) with intermediate-risk karyotype.[14]

Interestingly, we reported significant proportion of myeloid blasts with C-MYC and BCL2 overexpression in some of our TdT positive AML cases. The largest data from MD Anderson Cancer Center[15] tried to evaluate the prognostic impact of C-MYC [removed]moderate to strong nuclear positivity among ≥ 3% myeloid blasts by IHC) among 265 newly diagnosed AMLs (median age: 63 years, range: 22–88, male-to-female: 1.15:1) diagnosed over a period of 7 years. C-MYC expression was reported in a higher proportion (238/265, 90%, de novo > secondary AMLs, p = 0.012) of cases with mean percentage of blast positivity of 32%. A cutoff ≥ 6% blasts with C-MYC positivity was associated with shorter complete response duration (p = 0.007), inferior OS (log rank p = 0.042), EFS (p = 0.048), and RFS (p = 0.024) among older (≥ 55 years) subjects and those with intermediate-high risk cytogenetics.[15] Another study by Gajzer et al[16] demonstrated that de novo MDS subjects with high MYC [removed]in ≥ 5% myeloid blasts by IHC) (N = 16/41, 39%) had significantly shorter OS (19.7 vs. 51.7 months, p = 0.053) and MDS-to-AML progression-free survival (median: 9.3 vs. 17.7 months, respectively, p = 0.013) than low expressors; and high MYC expression was an independent prognostic indicator for MDS-to-AML progression in multivariate regression analysis (hazard ratio = 2.275, p = 0.046).[16]

Overexpression of BCL2 group of antiapoptotic proteins in AMLs has been increasingly reported to be associated with increased chemoresistance and inferior survival.[17] [18] In 2017, discovery of venetoclax, a BCL2 inhibitor, led to a major breakthrough in AML-CT; and this in combination with azacitidine, decitabine, or low-dose cytarabine (LDAC) got Food and Drug Administration approval in 2018 for the use in elderly (≥ 75 year) AMLs as well as those found unfit for intensive induction CT.[18]

To summarize, we described seven adult AML cases with TdT expression using IHC with correlation with flow cytometry, molecular, and follow-up details. IHC is a simple and cost-effective diagnostic tool to evaluate the immunoexpression of TdT, C-MYC, and BCL2 on BMBxs in cases with newly diagnosed AMLs. Available and emerging new evidences point to an increased prognostic impact of such biomarkers in risk stratification and therapy in AMLs, which most likely involve several key players like FLT3-ITD and NPM1. Incorporation of such vital prognostic information into the routine hematopathology is necessary for devising better therapeutic strategies in AML and MDS cases.

Conflict of Interest

None declared.

Acknowledgments

The authors sincerely thank Dr. Pavithra Ayyanar, Associate Professor, Department of Pathology and Laboratory Medicine, All India Institute of Medical Sciences, for her intellectual inputs on the study.

Authors' Contributions

C.P. and S.P.: Conceptual design, data curation, diagnosis, writing the original draft, review, and editing. B.B., P.K.D., and A.P.: Management of cases, follow-up data, and critical review of scientific content. G.C., S.P., and D.J.: Flow cytometry evaluation of cases. S.S. and P.A.: Diagnosis and literature review. A.C.: Literature review.


Ethical Approval

The study was part of the retrospective review proposal approved by the Institutional Ethics Committee of AIIMS, Bhubaneswar (T/IM-NF/Patho/24/52).


Patient's Consent

Patient consent is not required.

References


  1. Motea EA, Berdis AJ. Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase. Biochim Biophys Acta 2010; 1804 (05) 1151-1166
    Crossref PubMed Search in Google Scholar
  2. Patel KP, Khokhar FA, Muzzafar T. et al. TdT expression in acute myeloid leukemia with minimal differentiation is associated with distinctive clinicopathological features and better overall survival following stem cell transplantation. Mod Pathol 2013; 26 (02) 195-203
    Crossref PubMed Search in Google Scholar
  3. Borrow J, Dyer SA, Akiki S, Griffiths MJ. Terminal deoxynucleotidyl transferase promotes acute myeloid leukemia by priming FLT3-ITD replication slippage. Blood 2019; 134 (25) 2281-2290
    Crossref PubMed Search in Google Scholar
  4. Borrow J, Dyer SA, Akiki S, Griffiths MJ. Molecular roulette: nucleophosmin mutations in AML are orchestrated through N-nucleotide addition by TdT. Blood 2019; 134 (25) 2291-2303
    Crossref PubMed Search in Google Scholar
  5. Vassiliou GS. The curious incident of TdT-mediated mutations in AML. Blood 2019; 134 (25) 2229-2231
    Crossref PubMed Search in Google Scholar
  6. Arber DA, Orazi A, Hasserjian R. et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016; 127 (20) 2391-2405
    Crossref PubMed Search in Google Scholar
  7. Pollyea DA, Altman JK, Assi R. et al. Acute myeloid leukemia, version 3.2023, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw 2023; 21 (05) 503-513
    Crossref PubMed Search in Google Scholar
  8. Heuser M, Ofran Y, Boissel N. et al; ESMO Guidelines Committee. Electronic address: clinicalguidelines@esmo.org. Acute myeloid leukaemia in adult patients: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2020; 31 (06) 697-712
    Crossref PubMed Search in Google Scholar
  9. Akiki S, Dyer SA, Grimwade D. et al. NUP98-NSD1 fusion in association with FLT3-ITD mutation identifies a prognostically relevant subgroup of pediatric acute myeloid leukemia patients suitable for monitoring by real time quantitative PCR. Genes Chromosomes Cancer 2013; 52 (11) 1053-1064
    Crossref PubMed Search in Google Scholar
  10. Oyarzo MP, Lin P, Glassman A, Bueso-Ramos CE, Luthra R, Medeiros LJ. Acute myeloid leukemia with t(6;9)(p23;q34) is associated with dysplasia and a high frequency of FLT3 gene mutations. Am J Clin Pathol 2004; 122 (03) 348-358
    Crossref PubMed Search in Google Scholar
  11. Takenokuchi M, Kawano S, Nakamachi Y. et al. FLT3/ITD associated with an immature immunophenotype in PML-RARα leukemia. Hematol Rep 2012; 4 (04) e22
    Crossref PubMed Search in Google Scholar
  12. Shaforostova I, Call S, Evers G. et al. Prevalence and clinical impact of CD56 and T-cell marker expression in acute myeloid leukaemia: a single-centre retrospective analysis. eJHaem 2023; 5 (01) 93-104
    Crossref PubMed Search in Google Scholar
  13. De Bellis E, Ottone T, Mercante L. et al. Terminal deoxynucleotidyl transferase (TdT) expression is associated with FLT3-ITD mutations in acute myeloid leukemia. Leuk Res 2020; 99: 106462
    Crossref PubMed Search in Google Scholar
  14. Saburi M, Ogata M, Satou T. et al. Prognostic implications of TdT expression in acute myeloid leukemia with an intermediate-risk karyotype. Int J Hematol 2020; 112 (01) 17-23
    Crossref PubMed Search in Google Scholar
  15. Ohanian M, Rozovski U, Kanagal-Shamanna R. et al. MYC protein expression is an important prognostic factor in acute myeloid leukemia. Leuk Lymphoma 2019; 60 (01) 37-48
    Crossref PubMed Search in Google Scholar
  16. Gajzer D, Logothetis CN, Sallman DA. et al. MYC overexpression is associated with an early disease progression from MDS to AML. Leuk Res 2021; 111: 106733
    Crossref PubMed Search in Google Scholar
  17. Wei Y, Cao Y, Sun R. et al. Targeting Bcl-2 proteins in acute myeloid leukemia. Front Oncol 2020; 10: 584974
    Crossref PubMed Search in Google Scholar
  18. Tiribelli M, Michelutti A, Cavallin M. et al. BCL2 expression in AML patients over 65 years: impact on outcomes across different therapeutic strategies. J Clin Med 2021; 10 (21) 5096
    Crossref PubMed Search in Google Scholar

Address for correspondence

Somanath Padhi, MD
Department of Pathology and Laboratory Medicine, All India Institute of Medical Sciences
Bhubaneswar 751019, Odisha
India   

Publication History

Article published online:
19 May 2025

© 2025. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)

Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India

We recommend

      Fig 1: Flowchart 1. The stepwise evaluation of all cases of terminal deoxynucleotidyl transferase (TdT) positive acute myeloid leukemia cases among adults (≥ 18 years) (2022–2024) at our center.


      Fig 2 : Leishman–Giemsa stained hypercellular bone marrow aspirate smears from iliac crest at two different occasions at baseline (A, B) from a 57-year-old male with acute leukemia with undifferentiated morphology mimicking a lymphoproliferative neoplasm, and trephine biopsy sections showing a packed marrow with total replacement by lesional cells (C, hematoxylin and eosin). On immunohistochemistry (IHC), these cells were positive for CD34 (strong, D), myeloperoxidase (MPO) (weak, E), TdT (strong, F), CD117 (weak, focal, G), HLA-DR (strong, H), C-MYC (strong, I), and BCL2 (strong, J), consistent with TdT + acute myeloid leukemia (AML) (all magnifications ×400).


      Fig 3 : Flow cytometric immunophenotyping on second bone marrow aspirate sample from the above case ([Fig. 2]) depicting gated population of abnormal cells (dim to negative CD45 vs. low-intermediate side scatter [SSC], blast, A), which were positive for CD117 (heterogeneous) and HLA-DR (bright) (B), CD64 and CD14 (C), negative for T (D) and B (E) cell markers, and positive for nuclear terminal deoxynucleotidyl transferase (nuTdT) (F).


      Fig 4 : Bone marrow aspirate smear from a 61-year-old male with worsening pancytopenia showing sheets of highly undifferentiated neoplastic cells resembling high grade lymphoproliferative neoplasm or even, leukemia (A). On trephine biopsy and IHC, these cells were strongly and diffusely positive for CD117 (B) and terminal deoxynucleotidyl transferase (TdT) (C), negative for myeloperoxidase (MPO) (not shown), and CD19 (D). Original magnification, ×400 (A–C) (×200, D). Note the corresponding flow cytometric immunophenotyping panel (E to I) depicting dim expression of nuTdT (G) from the same case [blasts (in red) are positive for CD34 (E), CD117 (F), CD7 (H, aberrant), and CD13 (I, heterogeneous)].


      Fig 5 : Immunohistochemical expression of terminal deoxynucleotidyl transferase (TdT) (A), C-MYC (B), and BCL2 (C) from an adolescent male (case 7, [Table 1]) with generalized lymphadenopathy and high leukocytosis clinically and morphologically suspected to be that of lymphoma/leukemia (possibly early T-precursor acute lymphoblastic leukemia [ETP-ALL]) (×400).


      Fig 6 : Flow cytometric (FC) immunophenotypic analysis of peripheral blood sample from the above case ([Fig. 5] (Case 7)) showing gated population of cells (dim to moderate CD45 vs. low side scatter [SSC], A) immunopositive for CD34, HLA-DR (B), CD117 (bright, C), CD7 (E), nuclear terminal deoxynucleotidyl transferase (nuTdT) (F), CD2 (H), and CD10 (I). The lesional cells were negative for cytoCD3 (D, dotted black line representing the control T cell population), CD1a (F), both CD4 and CD8 (G), CD5 (H), and other B cell markers. Although the cyCD3 expression pattern did reach up to the control population (green), (D) the possibility of early T-precursor acute lymphoblastic leukemia (ETP-ALL) was ruled out by negative CD3 expression on immunohistochemistry (IHC) (not shown) as well as on repeat flow cytometry evaluation on fresh marrow aspirate sample.

    References


    1. Motea EA, Berdis AJ. Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase. Biochim Biophys Acta 2010; 1804 (05) 1151-1166
      Crossref PubMed Search in Google Scholar
    2. Patel KP, Khokhar FA, Muzzafar T. et al. TdT expression in acute myeloid leukemia with minimal differentiation is associated with distinctive clinicopathological features and better overall survival following stem cell transplantation. Mod Pathol 2013; 26 (02) 195-203
      Crossref PubMed Search in Google Scholar
    3. Borrow J, Dyer SA, Akiki S, Griffiths MJ. Terminal deoxynucleotidyl transferase promotes acute myeloid leukemia by priming FLT3-ITD replication slippage. Blood 2019; 134 (25) 2281-2290
      Crossref PubMed Search in Google Scholar
    4. Borrow J, Dyer SA, Akiki S, Griffiths MJ. Molecular roulette: nucleophosmin mutations in AML are orchestrated through N-nucleotide addition by TdT. Blood 2019; 134 (25) 2291-2303
      Crossref PubMed Search in Google Scholar
    5. Vassiliou GS. The curious incident of TdT-mediated mutations in AML. Blood 2019; 134 (25) 2229-2231
      Crossref PubMed Search in Google Scholar
    6. Arber DA, Orazi A, Hasserjian R. et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016; 127 (20) 2391-2405
      Crossref PubMed Search in Google Scholar
    7. Pollyea DA, Altman JK, Assi R. et al. Acute myeloid leukemia, version 3.2023, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw 2023; 21 (05) 503-513
      Crossref PubMed Search in Google Scholar
    8. Heuser M, Ofran Y, Boissel N. et al; ESMO Guidelines Committee. Electronic address: clinicalguidelines@esmo.org. Acute myeloid leukaemia in adult patients: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2020; 31 (06) 697-712
      Crossref PubMed Search in Google Scholar
    9. Akiki S, Dyer SA, Grimwade D. et al. NUP98-NSD1 fusion in association with FLT3-ITD mutation identifies a prognostically relevant subgroup of pediatric acute myeloid leukemia patients suitable for monitoring by real time quantitative PCR. Genes Chromosomes Cancer 2013; 52 (11) 1053-1064
      Crossref PubMed Search in Google Scholar
    10. Oyarzo MP, Lin P, Glassman A, Bueso-Ramos CE, Luthra R, Medeiros LJ. Acute myeloid leukemia with t(6;9)(p23;q34) is associated with dysplasia and a high frequency of FLT3 gene mutations. Am J Clin Pathol 2004; 122 (03) 348-358
      Crossref PubMed Search in Google Scholar
    11. Takenokuchi M, Kawano S, Nakamachi Y. et al. FLT3/ITD associated with an immature immunophenotype in PML-RARα leukemia. Hematol Rep 2012; 4 (04) e22
      Crossref PubMed Search in Google Scholar
    12. Shaforostova I, Call S, Evers G. et al. Prevalence and clinical impact of CD56 and T-cell marker expression in acute myeloid leukaemia: a single-centre retrospective analysis. eJHaem 2023; 5 (01) 93-104
      Crossref PubMed Search in Google Scholar
    13. De Bellis E, Ottone T, Mercante L. et al. Terminal deoxynucleotidyl transferase (TdT) expression is associated with FLT3-ITD mutations in acute myeloid leukemia. Leuk Res 2020; 99: 106462
      Crossref PubMed Search in Google Scholar
    14. Saburi M, Ogata M, Satou T. et al. Prognostic implications of TdT expression in acute myeloid leukemia with an intermediate-risk karyotype. Int J Hematol 2020; 112 (01) 17-23
      Crossref PubMed Search in Google Scholar
    15. Ohanian M, Rozovski U, Kanagal-Shamanna R. et al. MYC protein expression is an important prognostic factor in acute myeloid leukemia. Leuk Lymphoma 2019; 60 (01) 37-48
      Crossref PubMed Search in Google Scholar
    16. Gajzer D, Logothetis CN, Sallman DA. et al. MYC overexpression is associated with an early disease progression from MDS to AML. Leuk Res 2021; 111: 106733
      Crossref PubMed Search in Google Scholar
    17. Wei Y, Cao Y, Sun R. et al. Targeting Bcl-2 proteins in acute myeloid leukemia. Front Oncol 2020; 10: 584974
      Crossref PubMed Search in Google Scholar
    18. Tiribelli M, Michelutti A, Cavallin M. et al. BCL2 expression in AML patients over 65 years: impact on outcomes across different therapeutic strategies. J Clin Med 2021; 10 (21) 5096
      Crossref PubMed Search in Google Scholar

    CONTACT US

    QUICK LINKS

    NEWSLETTER

    Get all the information, latest updates delivered directly in your inbox

    © Indian Journal of Medical and Paediatric Oncology. Designed and Developed by Stackloom & Minerrals