Establishment and Characterization of T-ALL Cell Line with KMT2A::Afdn and T/My Mpal Cell Line with ETV6::NCOA2- the First Reports
Authors: Kato Keisuke , Yoshimi Ai , Noda Asami , Ohki Kentaro , Kiyokawa Nobutaka , Panagopoulos Ioannis 
Published: December 2024

&lt;Background&gt;Recent molecular research revealed leukemogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and T-cell and myeloid lineage mixed phenotype acute leukemia (T/My MPAL). However, in vitro model is still an important material. We established a T-ALL cell line that carries KMT2A::AFDN and a T/My MPAL cell line with ETV6::NCOA2. The cell lines harboring these fusion genes have never been reported. We fully characterized and reported the cell lines.
&lt;Cases&gt; The first case is a 12-year-old male who developed T-ALL, characterized by peripheral white blood cell, 239,100/µL, a mediastinal mass, and skin rashes. The bone marrow blast expressed CD2, cyCD3, CD5, CD7, CD33, CD34, and TdT. The karyotype was 46, XY, add(5)(q13), der(11)add(11)(p11)del(11)(q?), add(14)(q11). The patient attained complete remission with AML type induction after induction failure with ALL type induction. Although he received allogeneic hematopoietic cell transplantation (HCT), the patient developed recurrence ten months after the first HCT. The patient died of the obstinate turn-ups of leukemia after the third HCT. The second case was a 16-year-old female who developed acute leukemia with ambiguous lineage. On flow cytometry, the atypical cells were immunoreactive for cytoplasmic CD3, CD7, CD33, CD34, CD56, CD117, HLA-DR, and MPO. The karyotype was 48,XX,+X,t(8;12)(q11.2;p11.2),del(9)(q?),+12. Despite tandem HCT undertaken after recurrence, the patient died of the obstinate disease.
&lt;Method&gt; In both cases, the peripheral blood obtained after the last recurrence was serially cultivated using RPMI1640 containing 20 percent fetal calf serum. We undertook cytogenetic analysis and molecular analysis.
&lt;Result&gt; T-ALL cell line with KMT2A::AFDN: The cells have continuously grown in vitro. The cell line was designated as ICH-TALL-2. The doubling time is approximately 24 hours. The karyotype of the cultured cell was 46,XY,add(6)(q21),add(7)(p11.2),del(11)(q?),der(11)add(11)(p11.2)del(11)(q?),add(12)(q24.1),-18,-20,add(21)(q22.1),+mar1,+mar2. The transcriptome analysis, reverse transcription polymerase chain reaction, and Sanger sequence revealed in-frame fusion of KMT2A exon9 and AFDN exon 2 on the diagnostic sample, recurrent sample and cell line. The exome analysis revealed somatic heterozygous variants of WT1 (c.1264C&gt;T, p.Arg422Cys), NRAS (c.436G&gt;A, p.Ala146Thr), and RUNX2 (c.1282 G&gt;A, p.Gly428Ser) on the diagnostic sample but none of the variants on the recurrent sample and the cell line. The polymerase chain reaction and heteroduplex mobility assay found rearrangement on the TRG locus (TRGV3 and TRGJP2) and TRD locus (TRDV1 and TRDD3) on the recurrent sample and cell line. On the contrary, the diagnostic sample demonstrates different rearrangement patterns (rearrangements between TRGV8 and TRGJP2, TRDV1 and TRDJ1, and TRDD2 and TRDJ1). T/My MPAL cell line with ETV6::NCOA2: The cells have continuously grown in vitro. The cell line was designated as ICH-ALAL-1. The cultured cells are vulnerable to low cell density and requires relative high cell density (1x10^5 - 1x10^6/mL) The doubling time is approximately 24 hours. The karyotype of the cultured cell was 48,XX,+X,del(2)(q?),t(8;12)(q13;p12),del(9)(q?),+12,del(12)(q?),add(12)(q22.1). The cultured cells were immunoreactive for cytoplasmic CD3, CD4, CD7, CD33, CD34, CD56, CD117, and HLA-DR on flow cytometry. Reverse transcription-polymerase chain reaction, and Sanger sequence revealed ETV6::NCOA2 chimeric transcript. The exome analysis revealed somatic heterozygous variants of KRAS (c.C437T, p.A146V) and CREEEB (c.187_188insCA:p.Ser63ThrfsTer26); however, no NOTCH1 variant was evident. There was no rearrangement on TRG, TRD, and TRB. The RT-PCR analysis confirmed MPO expression on the cell line.
&lt;Discussion&gt; This is the first report on novel cell lines derived from T-ALL with KMT2A::AFDN and T/My MPAL with ETV6::NCOA2. It is intriguing to note the oligoclonal nature of KMT2A::AFDN positive T-ALL regarding the variants observed (NRAS, WT1, and RUNX2) and rearrangement on TRG and TRD. Furthermore, no variant of NOTCH1 was evident on T/My MPAL cell line with ETV6::NCOA2. The findings above indicate the strong driver capacity of KMT2A::AFDN and ETV6::NCOA2, respectively. The cell lines are essential for investigating the leukemogenesis of KMT2A::AFDN and ETV6::NCOA2 fusions using ATAC-seq and ChIP-seq.