Haematology
Contributions
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:30 to 13.06.2015 12:45
Location: Room Stolz 1
Background
Runt-related transcription factor 1 (Runx1)/ AML1 plays a crucial role in haematopoiesis and its dysfunction may contribute to leukaemogenesis. RUNX1 is frequently involved in chromosomal translocations including t(8;21), t(3;21), and t(16;21). Normal RUNX1 function is repressed by all truncated variants of RUNX1 that retain the entire Runt domain and lack the C-terminal domain. RUNX1 with various point mutations also has a dominant-negative effect on wild type and exhibits decreased transactivation activity; these have been found in AML, myelodysplastic syndromes and accelerated phase/blast crisis (AP/BC) of CML.
Aims
To explore the role of RUNX1 lesions in disease evolution of CML, we investigated RUNX1 splice variants and point mutations in CML chronic phase (CP) and BC.
Methods
The cases studied included 54 cases of Philadelphia chromosome positive (Ph+) acute leukaemia (22 cases of myeloid, 21 lymphoid, 3 bi-phenotypic and 8 unknown lineage of leukaemia). Of these 54 cases, 35 evolved from previous CP CML and 19 were de novo Ph+ acute leukaemia. Twenty cases of newly diagnosed CP CML were also studied. RNA was extracted and cDNA synthesised from whole blood white cells. First round PCR primers were designed to amplify the full length cDNA of the longest RUNX1 isoform 1c. Then two overlapping fragments were amplified by nested PCR primers. For several cases, it was found that there were PCR products of different size in addition to the PCR product of wild type RUNX1 1c. All PCR products of various sizes for each sample were gel-purified and subject to Sanger sequencing.
Results
A total of 18 types of transcript variant were found. Of these, 2 types (39 bp spliced out from exon3 and 99 bp spliced out from the end of exon10) were universally present and were excluded from this analysis. The 16 other types each had either all or the majority of the RUNT domain spliced out (named as Group 1 variants, 8 types) or had apparent aberrant C-terminal alteration (named as Group2 variants, 8 types). Group 2 variants were detected predominantly in Ph+ acute leukaemia cases, i.e. 8 of 54 Ph+ acute leukaemia cases. In contrast, Group 2 variants were only found in 1 of 20 CP cases and this one case was the only case that could not be controlled by imatinib treatment. The presence of Group1 variants was not significantly different between Ph+ acute leukaemia cases and CP cases.
There were 35 types of nonsynonymous mutation and 14 types of synonymous mutation detected. All nonsynonymous mutations have been aligned to various single nucleotide polymorphism databases to exclude known polymorphisms. Nonsynonymous RUNX1 mutations were detected in of 20 of 54 cases of Ph+ acute leukaemia (37%). In contrast only 2/20 (10%) cases of CP CML had a nonsynonymous RUNX1 point mutation (p=0.025). Combining RUNX1 splicing variant Group 2 and point mutation together, 23/54 (43%) Ph+ acute leukaemia cases had either a Group 2 splice variant or point mutations, which were only present in 3/20 (15%) CML CP (p=0.03). 5/8 cases of CML who had transformed had point mutations at the BC stage. In 4 of these 5 cases new point mutations were only detected at the BC stage; 1 case had mutations that were present in the earlier CP sample. 6/8 cases of CML who had transformed had variants detected, and 3 of these had Group 2 splice variants that were not present in CP.
Summary
RUNX1 splicing variants with disrupted C-terminal domain and RUNX1 point mutations are predominantly found in Ph+ acute leukaemia and at the transformation of CML, but are uncommon in chronic phase; these lesions may therefore be crucial transformation events, and candidate biomarkers of transformation.
Keyword(s): Blast crisis, Chronic myeloid leukemia, Mutation, RUNX1
Session topic: Novel actors in chronic myeloid leukemia biology
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:30 to 13.06.2015 12:45
Location: Room Stolz 1
Background
Runt-related transcription factor 1 (Runx1)/ AML1 plays a crucial role in haematopoiesis and its dysfunction may contribute to leukaemogenesis. RUNX1 is frequently involved in chromosomal translocations including t(8;21), t(3;21), and t(16;21). Normal RUNX1 function is repressed by all truncated variants of RUNX1 that retain the entire Runt domain and lack the C-terminal domain. RUNX1 with various point mutations also has a dominant-negative effect on wild type and exhibits decreased transactivation activity; these have been found in AML, myelodysplastic syndromes and accelerated phase/blast crisis (AP/BC) of CML.
Aims
To explore the role of RUNX1 lesions in disease evolution of CML, we investigated RUNX1 splice variants and point mutations in CML chronic phase (CP) and BC.
Methods
The cases studied included 54 cases of Philadelphia chromosome positive (Ph+) acute leukaemia (22 cases of myeloid, 21 lymphoid, 3 bi-phenotypic and 8 unknown lineage of leukaemia). Of these 54 cases, 35 evolved from previous CP CML and 19 were de novo Ph+ acute leukaemia. Twenty cases of newly diagnosed CP CML were also studied. RNA was extracted and cDNA synthesised from whole blood white cells. First round PCR primers were designed to amplify the full length cDNA of the longest RUNX1 isoform 1c. Then two overlapping fragments were amplified by nested PCR primers. For several cases, it was found that there were PCR products of different size in addition to the PCR product of wild type RUNX1 1c. All PCR products of various sizes for each sample were gel-purified and subject to Sanger sequencing.
Results
A total of 18 types of transcript variant were found. Of these, 2 types (39 bp spliced out from exon3 and 99 bp spliced out from the end of exon10) were universally present and were excluded from this analysis. The 16 other types each had either all or the majority of the RUNT domain spliced out (named as Group 1 variants, 8 types) or had apparent aberrant C-terminal alteration (named as Group2 variants, 8 types). Group 2 variants were detected predominantly in Ph+ acute leukaemia cases, i.e. 8 of 54 Ph+ acute leukaemia cases. In contrast, Group 2 variants were only found in 1 of 20 CP cases and this one case was the only case that could not be controlled by imatinib treatment. The presence of Group1 variants was not significantly different between Ph+ acute leukaemia cases and CP cases.
There were 35 types of nonsynonymous mutation and 14 types of synonymous mutation detected. All nonsynonymous mutations have been aligned to various single nucleotide polymorphism databases to exclude known polymorphisms. Nonsynonymous RUNX1 mutations were detected in of 20 of 54 cases of Ph+ acute leukaemia (37%). In contrast only 2/20 (10%) cases of CP CML had a nonsynonymous RUNX1 point mutation (p=0.025). Combining RUNX1 splicing variant Group 2 and point mutation together, 23/54 (43%) Ph+ acute leukaemia cases had either a Group 2 splice variant or point mutations, which were only present in 3/20 (15%) CML CP (p=0.03). 5/8 cases of CML who had transformed had point mutations at the BC stage. In 4 of these 5 cases new point mutations were only detected at the BC stage; 1 case had mutations that were present in the earlier CP sample. 6/8 cases of CML who had transformed had variants detected, and 3 of these had Group 2 splice variants that were not present in CP.
Summary
RUNX1 splicing variants with disrupted C-terminal domain and RUNX1 point mutations are predominantly found in Ph+ acute leukaemia and at the transformation of CML, but are uncommon in chronic phase; these lesions may therefore be crucial transformation events, and candidate biomarkers of transformation.
Keyword(s): Blast crisis, Chronic myeloid leukemia, Mutation, RUNX1
Session topic: Novel actors in chronic myeloid leukemia biology