ETV6/CBFA2 fusions in childhood B-cell precursor acute lymphoblastic leukemia with myeloid markers.

A translocation resulting in a fusion of ETV6 (TEL) gene at 12p13 and CBFA2 (AML1) gene at 21q22 is variably reported in 16-36% of cases of childhood acute lymphoblastic leukemia (ALL). This t(12;21)(p13;q22) is not detectable by conventional cytogenetic methods and was reported to be associated with B-cell precursor ALL with presumed favorable prognosis. We have examined 18 cases of well characterized childhood B-cell precursor ALL with cytogenetic, immunophenotypic, and clinical data for the presence of the t(12;21) using fluorescence in situ hybridization (FISH). Fourteen of the 18 cases (78%) were positive for fusion ETV6/CBFA2. One of seven adult ALL patients was positive (12% of cells positive in this 21 year old patient). By contrast, no evidence of t(12;21) by FISH was noted in two childhood T-ALL cases and 10 normal bone marrow samples. Twelve of the 14 positive childhood cases had CD13 and/or CD33 expression (myeloid markers) while only one of the four negative cases was CD13 and CD33 positive. Eight of 12 cases positive for t(12;21), and with conventional cytogenetic data, had structural and/or numerical chromosome abnormalities other than the detected t(12;21). One case had relapse with gradual increase in percentage of cells positive for t(12;21) and development of an isochromosome 21 carrying the fusion signals. The data reveal a strong association of t(12;21) with B-cell precursor ALL, especially with myeloid marker expression.     

AML1 amplification in a child with acute lymphoblastic leukemia

We report a case of de novo acute lymphoblastic leukemia with tandem amplification of the AML1 gene located in a chromosome marker that originated from chromosome 21 and a long event-free survival.     

Chromosome 21 abnormalities with AML1 amplification in acute lymphoblastic leukemia

Fluorescence in situ hybridization (FISH) studies were performed in three cases of acute lymphoblastic leukemia (ALL) with marker chromosomes to analyze the contribution of chromosome 21 in these markers. FISH with a chromosome 21 painting probe confirmed that chromosome 21 was involved in all three cases. FISH with YAC probes showed that the number of extra copies varied according to their location on chromosome 21. Attention was focused on the AML1 gene, which was present as five copies in most of the cells exhibiting the marker chromosomes. As controls, 11 cases of childhood ALL were studied with PAC probes covering AML1. The results agreed with the banded karyotypes in 10 patients. FISH uncovered a clone with four copies of AML1 which were only observed by FISH analysis of interphase nuclei in one patient. No point mutation was detected in exons 3-5, encoding the runt domain of AML1, in the three cases, suggesting an oncogenic role of wild-type AML1 amplification.     

Partial chromosome 21 amplification in a child with acute lymphoblastic leukemia

Monosomy 21 and metacentric markers corresponding in size to chromosomes 8 to 12 were found as the only clonal chromosomal changes in a child with acute lymphoblastic leukemia (ALL). Chromosome painting with a whole chromosome 21-specific probe showed that the marker originated from chromosome 21. Fluorescence in situ hybridization with yeast artificial chromosome (YAC) probes to chromosome 21 showed genomic amplification with two, four, or more copies of the probed DNA sequences present on the marker. The most amplified regions of chromosome 21 were centromeric and telomeric to the Down's syndrome region. This observation supports the notion that amplification of only parts of chromosome 21 may be important in the leukemogenic process in spite of the high incidence of complete trisomy 21 in ALL.     

Cytogenetics of long‐term survivors of ETV6‐RUNX1 fusion positive acute lymphoblastic leukemia

This study describes the cytogenetics of 33 children with ETV6‐RUNX1 positive acute lymphoblastic leukemia (ALL) who had been in continuous complete remission for a minimum of 8.8 years [median event‐free survival (EFS) 10.9 years]. The results were compared with a published series of 16 fusion positive patients treated on the same childhood ALL trial, who had relapsed (median EFS, 2.3 years). Interphase fluorescence in situ hybridization (FISH) at diagnosis showed deletion of the second ETV6 signal from all fusion positive cells in 45% of the long‐term survivors but in none of the relapsed patients, whereas patients with mixed populations with retained or lost second signals were more frequent among those who had relapsed (69%) than the long‐term survivors (21%). Interphase populations with two fusion signals in 18% of the long‐term survivors and 31% of relapsed patients were smaller in the long‐term survivors (median, 4% of total cells) than in the relapsed patients (median, 84%). The additional copy of chromosome 21 in 30% of long‐term survivors and in 69% of relapsed patients was a derived chromosome 21 in 20% and 55% of patients, respectively. Metaphase FISH for 26 long‐term survivors and 15 relapsed patients revealed complex karyotypes in both groups. Variant translocations involved different chromosome arms between the long‐term survivors and relapsed patients. It appears that the two groups have some distinguishing cytogenetic features at the time of diagnosis, which may provide pointers to relapse that are worthy of more detailed study. © 2009 Wiley‐Liss, Inc.     

Clonal variation of the immunogenotype in relapsed ETV6/RUNX1-positive acute lymphoblastic leukemia indicates subclone formation during early stages of leukemia development

Recent data suggest that late relapses evolve from an ancestral ETV6/RUNX1-positive (also designated TEL/AML1-positive) clone resulting from secondary changes (ETV6 deletion) that differ from those of the initial leukemia and, as a consequence, may also deviate in their clonotypic immunoglobulin/T-cell receptor (IG/TCR) gene rearrangements. The aim of our study was to compare the immunogenotype and fluorescence in situ hybridization (FISH) patterns of the unrearranged ETV6 allele of matched diagnosis/relapse samples from 12 children with an early or late relapse. We identified varying degrees of differences in the IG/TCR in six of them. A clonal change or evolution of the unrearranged ETV6 allele was also observed in six children but remained unchanged in three. However, these two parameters were not in concordance, nor did the immunogenotype pattern correlate with the duration of the first remission. We therefore propose that the potential of the immunogenotype to diversify depends primarily on the stage of IG/TCR gene configuration of the cell in which the ETV6/RUNX1 gene fusion takes place.     

AML1 amplification in a case of childhood acute lymphoblastic leukemia

We report an 8-year-old girl with B-cell acute lymphoblastic leukemia (ALL). The blast cell karyotype at diagnosis included a marker chromosome revealed by fluorescence in situ hybridization to be a derivative of chromosome 21. A high level amplification of the AML1 gene was identified, but it disappeared upon complete remission. This rare but recurrent abnormality warrants research of B-cell ALL, especially when a marker chromosome is present in the blast cell karyotype.     

The complex genomic profile of ETV6-RUNX1 positive acute lymphoblastic leukemia highlights a recurrent deletion of TBL1XR1

The ETV6-RUNX1 fusion is the molecular consequence of the t(12;21)(p13;q22) seen in approximately 25% of children with acute lymphoblastic leukemia (ALL). Studies have shown that the fusion alone is insufficient for the initiation of leukemia; additional genetic changes are required. Genomic profiling identified copy number alterations at high frequencies in these patients. Focal deletions of TBL1XR1 were observed in 15% of cases; 3 patients exhibited deletions distal to the gene. Fluorescence in situ hybridization confirmed these deletions and quantitative RT-PCR showed that the TBL1XR1 gene was significantly under-expressed. TBL1XR1 is a key component of the SMRT and N-CoR compressor complexes, which control hormone-receptor mediated gene expression. Differential expression of the retinoic acid target genes, RARB, CRABP1, and CRABP2, indicated that deletion of TBL1XR1 compromised the function of SMRT/N-CoR in the appropriate control of gene expression. This study identifies deletions of TBL1XR1 as a recurrent abnormality in ETV6-RUNX1 positive ALL. We provide evidence that implicates this deletion in the inappropriate control of gene expression in these patients. The target of the interaction between TBL1XR1 and the signaling pathways described here may be exploited in cancer therapy.     

Near-tetraploidy in childhood B-cell precursor acute lymphoblastic leukemia is a highly specific feature of ETV6/RUNX1-positive leukemic cases

Near-tetraploidy (82-94 chromosomes) makes up fewer than 1% of childhood acute lymphoblastic leukemia (ALL) cases and has been reportedly associated with a possibly poorer prognosis compared with other ploidy groups. We analyzed 783 patients enrolled in the ALL-BFM-Austria 86, -90, -95, -99/2000 and Interfant-Austria 99 trials in order to assess its incidence, biological characteristics, and prognostic relevance. Twelve of 783 patients (1.5%) had a near-tetraploid ALL. Fluorescence in situ hybridization revealed that eight of the nine B-cell precursor (BCP) cases and none of the three T-cell ALL cases had an ETV6/RUNX1 rearrangement. After a median follow-up of 11.4 years, none of the patients has relapsed or died. Thus, near-tetraploidy appears to be a specific feature of ETV6/RUNX1+ BCP ALL cases that in turn may explain its excellent outcome.     

Coexistence of ETV6/RUNX1 and MLL aberrations in B-cell precursor acute lymphoblastic leukemia discloses a small subclass of BCP-ALL

Out of 76 pediatric cases of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) positive for ETV6/RUNX1 (previously TEL/AML1) resulting from t(12;21), 7 cases revealed coexistence of ETV6/RUNX1 and MLL aberrations. One case of der(21) duplication with ETV6/RUNX1 exhibited a novel MLL translocation variant t(6;11)(p21.1p23;q13q25), with translocation of 3' telomeric MLL and deletion of 5' centromeric MLL. Another case of der(21) duplication with ETV6/RUNX1 showed MLL rearrangement upon Southern blotting. The remaining five ETV6/RUNX1-positive cases had MLL allelic deletion. ETV6/RUNX1 and MLL aberration clone size in these cases was suggestive of ETV6/RUNX1 as an early primary event, originating in the embryonic or infant stage and developing into leukemia by later acquisition of MLL aberration, ETV6 loss, and ETV6/RUNX1 duplication as secondary events. To date, the prognosis has been favorable, which seems to be compatible with ETV6/RUNX1-positive ALL. We conclude that the cases with coexisting ETV6/RUNX1 and MLL aberrations probably exist as a small, hidden group of ETV6/RUNX1-positive BCP-ALL, which invites further investigation, in large series from different populations, to confirm the findings and establish the biological mechanisms and prognostic significance.     

RUNX1 amplification in lineage conversion of childhood B-cell acute lymphoblastic leukemia to acute myelogenous leukemia

Amplification of RUNX1 (alias AML1) is a recurrent karyotypic abnormality in childhood acute lymphoblastic leukemia (ALL) that is generally associated with a poor outcome. It does not occur with other primary chromosomal abnormalities in acute ALL. AML1 amplification in acute myelogenous leukemia (AML) is a rare secondary event described mainly in therapy-related cases. AML1 amplification was found in a 13-year-old patient with AML M4/M5 leukemia that occurred 5 years after she had been diagnosed with common B-cell ALL. Conventional cytogenetic, fluorescent in situ hybridization (FISH), and polymerase chain reaction methods revealed no other chromosomal change expected to occur in a disease that we assumed to be a secondary leukemia. Due to the lack of cytogenetic data from the diagnostic sample, we developed a new approach to analyze the archived bone marrow smear, which had been stained previously with May-Grünwald-Geimsa by the FISH method. This analysis confirmed that in addition to t(12;21), AML1 amplification and overexpression existed already at the time the diagnosis was made. The chromosomal changes, however, were found in different clones of bone marrow cells. While the first course of chemotherapy successfully eradicated the cell line with the t(12;21), the second cell line with AML1 amplification remained latent during the time of complete remission and reappeared with a different immunophenotype.     

Abnormalities of the der(12)t(12;21) in ETV6‐RUNX1 acute lymphoblastic leukemia

ETV6‐RUNX1 fusion [t(12;21)(p13;q22)] occurs in 25% of childhood B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) and is associated with a favorable outcome. Additional abnormalities involving der(21)t(12;21) and nonrearranged chromosome 12 are well characterized but aberrations involving the der(12)t(12;21) have rarely been described. Herein, we describe two novel abnormalities affecting the der(12)t(12;21): a deletion (20/247, 8%) and duplication (10/247, 4%). All 30 patients were under 10 years of age, had a median white blood count of 12.4 × 10⁹/L and 19.2 × 10⁹/L, respectively, with a good outcome. Deletions of der(12)t(12;21) on both sides of the breakpoint were confirmed and mapped: centromeric (12p11.21‐12p13.2) and telomeric (21q22.12‐21q22.3). The size of these deletions extended from 0.4–13.4 to 0.8–2.5 Mb, respectively. The centromeric deletion encompassed the following genes: LRP6, BCL2L14, DUSP16, CREBL2, and CDKN1B. We postulate that this deletion occurs at the same time as the translocation because it was present in all ETV6–RUNX1‐positive cells. A second abnormality representing duplication of the reciprocal RUNX1–ETV6 fusion gene was a secondary event, which we hypothesize arose through mitotic recombination errors. This led to the formation of the following chromosome: der(12)(21qter→21q22.12::12 p13.2‐12 p12.3::12p12.3→12qter). Both abnormalities affect the reciprocal RUNX1–ETV6 fusion product which could either eliminate or amplify its expression and thus contribute to leukemogenesis. However, other consequences such as haploinsufficiency of tumor suppressor genes and amplification of oncogenes could also be driving forces behind these aberrations. In conclusion, this study has defined novel abnormalities in ETV6–RUNX1 BCP‐ALL, which implicate new genes involved in leukemogenesis. © 2012 Wiley Periodicals, Inc.