CEBPA Methylation and Notch1 Mutations Are Common in Immature T-ALLs but Not M0 AML and Cosegregate with TCRG Rearrangement and the TCRgd Lineage.

The CEBPA gene codes for the C/EBPa transcription factor which regulates hematopoietic stem cell homeostasis and orientates towards myeloid differentiation and against a T lymphoid fate. CEBPA silencing by promoter hypermethylation (CEBPA^meth) has recently been identified (Wouters, Blood 2007) in a minor subset of AML, all of which demonstrated T lymphoid features (expression of CD7 and CD3 transcripts, TCRG rearrangement and frequent NOTCH1 mutations (NOTCH1^m)). These AML may be closer to immature T-ALLs, with CEBPa silencing and NOTCH1^m determining lineage orientation. Although expression of CD7 and cytoplasmic CD3 (cCD3) are considered as defining markers in T-ALL, CD7 is commonly expressed in AML and reproducible multicenter detection of low level cCD3, when performed, can be difficult. We have classified CD7+, cCD3+ T-ALLs on the basis of their TCR profiles: immature cases arrested prior to TCRB VDJ rearrangement include TCRgd lineage precursors and can be divided into those with no TCR rearrangement (IM0), TCRd only rearrangement (IMD) or TCRd and TCRg rearrangement (IMG). The CALM-AF10 fusion transcript is found in both AML and T-ALL, where it is restricted to the TCRgd lineage. We initially searched for in CEBPA^meth by methylation specific PCR in 5 AML with downregulated CEBPand confirmed that the 3 cases (FAB-M0/M1) with CEBPA^meth expressed CD3, CD7 and/or TCRg rearrangement. Only 1/49 AML M0, all of which were cCD3 negative, demonstrated CEBPA^meth.We then searched for CEBPA^meth and mutation and NOTCH1^m in 18 IM T-ALL (4 aged <15y; 5 IM0, 5 IMD, 8 IMG) compared to 6 adults with TCRB VDJ+, TCRab lineage T-ALL. No CEBPA mutations were found but 11/24 (56%) demonstrated CEBPA^m and 10/23 (43%) NOTCH1^m. Both correlated with IMG T-ALL, when 6/8 demonstrated CEBPA^meth and NOTCH1^m and a seventh only NOTCH1^m, compared to only 4/10 CEBPA^meth and 1/10 NOTCH1^m in IM0/D cases, with no overlap. In ab-lineage T-ALLs 2/6 demonstrated NOTCH1^m, one of which was also CEBPA^meth, and notably expressed TLX1/HOX11. CEBPA^meth cases were more frequently CALM-AF10+ (5/10), CD2-, CD123/IL3R-, HOXA9 transcript+ and IgH DJ rearranged but did not differ from non-CEBPA^meth T-ALL in CD34, CD5, CD117, CD13/33, CD79a or CD56 expression. As such CEBPA^meth is rare in cCD3- M0 AML but common in cCD3+ immature T-ALL, independently of myeloid antigen expression. CEBPA^meth and NOTCH1^m in IM T-ALL are preferentially associated with cases having undergone TCRG rearrangement but are not found in the majority of more immature cases, demonstrating that CEBPA^meth is not necessary for CD7 and cCD3 expression, within a T-ALL context. It also suggests that CALM-AF10 should be searched for in CEBPA^meth AML, raises issues regarding AML vs. ALL protocol assignement and the use of demethylating agents in IMG T-ALLs and suggests that the potentially misleading “biphenotypic leukaemia” label be reassessed.