TO THE EDITOR: NUP214-ABL1 translocation is probably the most common tyrosine kinase inhibitor (TKI)-targetable cytogenetic abnormality observed in T-cell acute lymphoblastic leukemia (T-ALL), accounting for 5–6% of the T-ALL cases [1]. However, there are only few case reports and small series in the literature, possibly indicating the underdiagnosis of this entity. Unlike in B-cell acute lymphoblastic leukemia, testing for gene fusions using reverse transcriptase polymerase chain reaction (RT-PCR) or fluorescent in-situ hybridization (FISH) is often avoided in T-ALL because of the absence of distinct associations with prognosis or targeted therapy, especially in resource-limited settings. However, TKI-targetable abnormalities have also been described in T-ALL, including NUP214-ABL1, BCR-ABL1, and mutations in STAT5b [2, 3]. Among these, FISH testing using BCR-ABL1 dual-color dual-translocation probes can help to identify not only BCR:ABL1 but also NUP214-ABL1 based on the characteristic patterns in FISH testing. This is an illustrative case highlighting an easy and cost-effective approach for routine detection of cytogenetic abnormalities.

A developmentally normal male child, appropriately immunized for his age, presented with continuous moderate-grade fever, progressive pallor, and mild hepatosplenomegaly since 10 days. Peripheral blood examination revealed that the hemoglobin level was 71 g/L; total leukocyte count, 45.3×10⁹/L; and platelet count, 65×10⁹/L. The peripheral blood film revealed 72% blasts, 9% neutrophils, 16% lymphocytes, and 3% monocytes. The blasts were negative for myeloperoxidase. Flow cytometry confirmed T-ALL [positive for cluster of differentiation (CD) 7, CD4, CD5, CD2, cytoCD3, and terminal deoxynucleotidyl transferase (TdT); negative for surface CD3, CD1a, CD8, T-cell receptor (TCR) ab, TCRgd, B cell, and myeloid antigens]. FISH testing using BCR-ABL1 dual-color dual-fusion probe (Metasystems, Germany) showed amplification of ABL1 (3–50 copies) using the protocol as previously described [4]. Further testing using ABL1 (Metasystems, Germany) and NUP214 (Zytolight, Germany) dual-color break-apart probes showed a characteristic signal pattern confirming NUP214-ABL1 translocation (Fig. 1). Details of immunophenotyping and FISH cytogenetics are summarized in Table 1.

Table 1 . Hematological and laboratory parameters at diagnosis and after induction therapy..

	Diagnosis	After induction
Peripheral blood
Total leukocyte count	45.3×109/L	4.9×109/L
Hemoglobin	71 g/L	119 g/L
Platelet count	65×109/L	195×109/L
Peripheral blood blasts	72%	0%
Bone marrow blasts	Not done	2%
Immunophenotyping (flow cytometry)
Gated events	CD45-dim low side scatter events (progenitors) -90% of viable events	CD7-positive low side scatter events -5% of viable events
Positivea,b) markers on gated cells	CD2 (39.2%), CytoCD3 (98%), CD4 (79.5%), CD5 (56%), CD7 (98%), CD10 (70%), CD81 (98%), CD33 (28.7%), CD45 (dim), CD58 (93.4%), CD38 (72%), Tdt (55%)	Surface CD3 (86%), cytoplasmic CD3 (100%), CD4 (42.8%), CD8 (40.9%), CD5 (84%), CD56 (6.8%), CD45 (100%)
Negative markers on gated cells	Surface CD3, CD8, CD13, CD19, CD20, CD34, CytoCD79a, CytoCD22, CD86, CD117, CD56, TCRab, TCRgd, anti-MPO, CD14, CD36, CD64, CD15, CD123, HLA-DR	CD34, CD38
FISH cytogenetics	XL BCR/ABL1 dual color dual fusion probes,	Not done
Probes tested	Cytotest LSI KMT2A dual color break apart rearrangement probe, and Zytologht SPEC NUP214 dual color break-apart probe
Pattern	nuc ish (ABL13–50, BCR×2)[160/200], (5′KMT2A×3,3′KMT2A×3)(5′KMT2A con 3′KMT2A)×3[120/200], (5′NUP214×3-50,3′NUP214×2)(5′NUP214 con 3′NUP214)×1

^a)≥20% positive, ^b)CD2, CD10, CD81, CD13, CD33, CD117, CD34, CD58, and Tdt were not included in post-induction measurable residual disease analysis. Only T-cell tube was studied for measurable residual disease testing..

Abbreviations: CD, cluster of differentiation; FISH, fluorescent in-situ hybridization; HLA-DR, human leukocyte antigen-DR isotype; MPO, myeloperoxidase; TCR, T-cell receptor; Tdt, terminal deoxynucleotidyl transferase..

Figure 1. (A) FISH using BCR-ABL1 dual-color dual-fusion probe showing two green signals (normal BCR gene) and multiple orange signals (3–50 copies), indicating amplification of the ABL1 gene; (B, C) FISH using ABL1 break-apart probe showing one fusion (normal ABL1 gene) and multiple orange signals, indicating amplification of the 3′ region of the ABL1 gene along with deletion of the 5′ region of the ABL1 gene; (D, E) FISH using NUP214 break-apart probe showing one fusion (normal NUP214 gene), one green (normal 3′ region of the other NUP214 allele), and multiple orange signals, indicating amplification of the 5′ region of the NUP214 gene.
Abbreviation: FISH, fluorescent in-situ hybridization.

Bone marrow examination was not performed, as a confirmatory diagnosis could be made from the peripheral blood investigation, and the child was not willing to undergo bone marrow examination. Conventional cytogenetics of the peripheral blood did not show metaphase. Augmented Berlin-Frankfurt-Munich protocol plus imatinib was administered. Post-induction bone marrow was hypocellular with 2% blasts, and no measurable residual disease was detected using 10-color flow-cytometric immuno-phenotyping. The delayed intensification phase 2 was completed uneventfully, and the child is now in the maintenance phase.

Both ABL1 and NUP214 genes are located at 9q34.1, with the latter on the telomeric side. The fusion of these genes results from extrachromosomal episome formation and amplification of both genes. The episome containing the fused gene exists autonomously and freely replicates in the cytoplasm or integrates with the chromosome and replicates with it. This episomal amplification, varying between 5–50 copies/cell, can be visualized using FISH, multiplex ligation-dependent probe amplification, or chromosomal microarray; however, it is undetectable with conventional cytogenetics. Amplification of ABL1 does not appear to be the only mechanism involved in the pathogenesis of T-ALL; there have been reports of associated alterations of other genes, such as CDKN2A, TLX1, TLX3, and NOTCH1. These observations indicate a multigene contribution to the pathogenesis of T-ALL with NUP214-ABL1 fusion. NUP214-ABL1 fusion is found predominantly in men, and these patients usually present with high-risk factors, including elevated leukocyte count, mediastinal mass, or extramedullary involvement, often with early relapse and dismal outcomes [5]. While an occasional patient has survived for more than 194 months, the median overall survival reported in previous series is only 18 months. These patients are reported to benefit from TKI, especially dasatinib; hence, it is imperative to diagnose this entity in the clinics [6]. However, the long-term benefit of adding TKI in the treatment remains unclear owing to the lack of randomized controlled trials. A summary of the cases reported until now is presented in Table 2.

Table 2 . Summary of the cases of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia [1,5,9-15]..

Variable	Value
Total N of cases	65a)
Male:female	52:13
Age, median (range; IQR) in years	18 (1–68; 10–28.5)
Total leukocyte count, median (range; IQR)×109/L	54 (1.8–480; 18–122)
Blast % (peripheral blood/bone marrow), median (range; IQR)	86 (10–98; 78–93)
Conventional cytogenetics
Not available/failed	17 (26.2%)
Normal karyotype	18 (27.7%)
Abnormal karyotype	30 (46.1%)
Method of detection of NUP214-ABL1b)
FISH using BCR/ABL1 probe	45 (69.2%)
FISH using NUP214 break-apart probe	28 (43%)
Sanger sequencing	7 (10.7%)
Reverse transcriptase PCR	24 (36.9%)
Mate pair sequencing	1 (1.5%)
Overall outcome
Complete response	32
Relapse	15c)
Death	8c)
No data	11
N of patients receiving tyrosine kinase inhibitor	4 (6.1%)
Complete response	4 (6.1%)
Relapse	NIL
Death	NIL
Overall survival (N=64), median (range; IQR) in months	17.7 (1 week–194; 7–39.4)
Event free survival (N=46), median (range; IQR) in months	16.8 (1–125; 9.3–36.1)

^a)Including present case; ^b)Some cases were detected by multiple methods; ^c)Includes patients who had complete response but subsequently relapsed/died..

Abbreviations: FISH, fluorescent in-situ hybridization; IQR, interquartile range; NIL, none; PCR, polymerase chain reaction..

The limited number of reports and absence of definite treatment guidelines may indicate underdiagnosis of this entity. Amplification of ABL1 (9q34) is an indirect indicator of NUP214-ABL1 fusion, which can easily be detected by routine testing of BCR-ABL1 using FISH in T-ALL cases. NUP214-ABL1 fusion can be further confirmed using NUP214 break-apart FISH testing or RT-PCR. In addition to BCR-ABL1 translocation, this dual-color probe FISH helps to detect other ABL1-related and possibly TKI-responsive cytogenetic abnormalities associated with T-ALL, such as EML1-ABL1 and ETV6-ABL1 fusions or 9q34 duplication associated with therapeutic resistance [7]. Although BCR-ABL1-positive T-ALL is rare, the frequency of its detection has increased, with approximately 30 cases reported in the literature and pediatric cases accounting for more than 40% of the total cases. In most reported cases, the prognosis was poor with a median survival of only 7 months (range, 0.1–60 mo), and nearly 50% of the patients died by the time of the last follow-up [8-15].

To summarize, this illustrative report highlights the utility of incorporating routine FISH testing using a BCR/ABL1 dual-color probe in the workup for T-ALL in resource-constrained settings, especially in the absence of advanced testing, such as ribonucleic acid -sequencing.

This study was supported by a grant from the National Cancer Grid, Government of India.

No potential conflicts of interest relevant to this article were reported.