Somatic Mutations Associated with Myeloid Transformation of RUNX1 -Mutated Familial Platelet Disorders with Propensity to Myeloid Malignancies Using Targeted Next-Generation Sequencing

INTRODUCTION: RUNX1 is a master transcription factor associated with hematopoiesis. Germ-line RUNX1 mutations are associated with familial thrombocytopenia and platelet disorders with bleeding phenotype and propensity to myeloid malignancies (FPDMM), such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Although only 40 families with germ-line RUNX1 mutations have been described, its autosomal dominant inheritance pattern and the finding that 50% of the families with more than one first degree relative with MDS and/or AML had RUNX1 mutations underscore the importance of this entity.

Despite the elevated frequency of transformation to AML/MDS the array of somatic mutations involved in this process is not fully characterized. Here we describe three patients from two families who had a diagnosis of FPDMM, progressed to MDS and/or AML and had DNA samples available for Next-Generation Sequencing (NGS).

METHODS: We identified five patients from two different family pedigrees with AML/MDS between 2014 and 2016. We've had access to DNA samples from three of these patients extracted from one of three sources (buccal swab, peripheral blood leukocytes, and bone marrow) throughout their follow-up. Patients agreed with sample collection after informed consent, according to the Declaration of Helsinki.

NGS was performed using a custom-designed 48 gene panel related to myeloid malignancies, using the IonTorrent® sequencing platform as previously described (PMID: 28486043).

RESULTS: The pedigrees of both families are described in Figure 1. Patient I-2 from pedigree 1 was diagnosed with acute lymphoblastic leukemia at age 57 and died shortly after. Her son, patient II-3 came to us initially with thrombocytopenia (80,000-100,000/ μl) and mild bleeding symptoms (bleeding assessment tool score: 3) 12 years prior to his AML diagnosis. He also had a colorectal cancer (treated surgically) diagnosed 6 months prior. At AML diagnosis, he had a normal karyotype and underwent standard induction chemotherapy and achieved a complete response. He received one cycle of consolidation with high-dose cytarabine and died shortly after due to metastatic colorectal cancer. His oral epithelial cell DNA sample from the beginning of follow-up was positive for a RUNX1 p.Arg166Ter mutation. Bone marrow (BM) DNA sample from the AML diagnosis showed an additional p.Arg204Ter RUNX1 mutation and an additional p.Gln139fs NFE2 mutation.

Patients III-4 and III-5 from pedigree 2 belonged to a family with an extensive previous history of thrombocytopenia and AML. Patient III-5 came to us with thrombocytopenia (50,000-70,000/μl) 2 years prior his AML diagnosis. BM DNA obtained at this time was positive for a p.Arg201Ter RUNX1 mutation and for a p.Pro95Arg SRSF2 mutation. At AML diagnosis, his karyotype was normal and BM DNA was positive for a p.Gly12Asp NRAS mutation in addition to the aforementioned mutations. He underwent three cycles of induction and received a matched unrelated donor bone marrow transplant (BMT) while in refractory disease. He achieved a complete remission that lasted a year and is currently under treatment with hypomethylating agents. His oral epithelial cell DNA sample obtained prior his BMT was only positive for the RUNX1 p.Arg201Ter mutation.

Patient III-4 had been followed up for thrombocytopenia for three years and presented to us with pancytopenia and multilineage dysplasia. BM DNA at the onset of pancytopenia showed a p.Arg201Ter RUNX 1 mutation, and emerging TET2 Ser471fs and NRAS Gly12Asp mutations. These findings are summarized in Table 1.

CONCLUSIONS: We report a longitudinal DNA sequencing follow-up of three patients with FPDMM. Our findings highlight the heterogeneity of genetic pathways to myeloid transformation in FPDMM. Some of these, as loss of the functioning RUNX1, have already been described. However, here we report for the first time the association of NFE2 and NRAS mutations with myeloid transformation in FPDMM.

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