A 27-year-old Malay woman presented at our hematology clinic with a fever and sore throat. She had a history of hereditary thrombocytopenia that affected her father and 3 brothers. Preliminary clinical investigation at diagnosis revealed abnormal levels of hematological markers - the patient had a white blood cell count of 1.97 x 109 /L (normal: 3.4-9.6 x 109 /L), hemoglobin levels of 7.0 g/dL (normal: 10.9-15.1 g/dL) and platelet count of 20 x 109 /L (normal: 132-372 x 109/L). On morphological analysis, her bone marrow was moderately cellular, with dysplastic changes as well as increased numbers of myeloblasts and monoblasts/promonocytes, indicative of AML with myelodysplastic changes (Fig. 1a). Initial molecular testing by conventional Sanger sequencing revealed the same novel RUNX1 mutation in the patient, her father and 3 brothers (Fig. 2). A buccal analysis also confirmed that the RUNX1 mutation identified in this FPD/AML patient is germline in nature.
The patient received standard induction chemotherapy that consisted of high-dose anthracycline and cytarabine in a 3 + 7 regimen and subsequently attained complete morphological remission. Post-induction evaluation of her bone marrow revealed a regenerating bone marrow in morphological remission with myelodysplastic changes (Fig. 1b), as well as 0.3% residual disease, measured using Flow Cytometry. Thereafter, she was given consolidation chemotherapy with cytarabine. As no matched sibling donor or matched unrelated donor was available, she received a haploidentical stem cell transplant (haplo-SCT) from her mother, who is RUNX1-mutation negative. While she engrafted on Day 15 of her transplant, her admission was complicated by Klebsiella bacteraemia, acute Graft-versus-Host Disease (GvHD) of her liver and Cytomegalovirus reactivation. Post-haplo-SCT, she was given Tacrolimus, Mycophenolate Mofetil and Methotrexate as GvHD prophylaxis. A post-transplant bone marrow aspirate after count recovery showed a normocellular reactive marrow (Fig. 1c) and 0% residual disease on Flow Cytometry.
With informed consent obtained from the patient, a comprehensive genomic mutational analysis on a targeted next-generation sequencing (NGS) platform was conducted using either peripheral blood (PB) or bone marrow (BM) samples collected at three different stages - at diagnosis (pre-treatment), post-treatment (after induction and consolidation chemotherapy) and post-haplo-SCT. These time points coincided with the bone marrow examinations done for the patient, as mentioned above. In brief, 568 amplicons ('tiles') covering 54 genes that were previously implicated in myeloid neoplasms were assessed for the presence of genomic variants. At each stage, the patient's genomic DNA was extracted and processed using the TruSeq Custom Amplicon (TSCA) assay for the TruSight Myeloid Sequencing Panel, according to a previously described methodology. The 54 genes evaluated in this targeted gene panel were as follows: ABL1, ASXL1, ATRX, BCOR, BCORL1, BRAF, CALR, CBL, CBLB, CBLC, CDKN2A, CEBPA, CSF3R, CUX1, DNMT3A, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, WT1 and ZRSR2. After sequence alignment against the reference GRCh37/hg19 human genome assembly, BAM and VCF files were produced by the TruSeq Amplicon software (V.1.1.0.0). Genome annotation was done on the VCF files using the Illumina VariantStudio (V.2.2). Only variants that met the following criteria were selected: non-synonymous mutation, variant not present in dbSNP, read-depth > 100x, variant-allele frequency (VAF) > 5% and acceptable sequence quality. The comprehensive list of mutations, including the hitherto unreported germline RUNX1 mutation, identified in the pre-treatment, post-treatment and post-haplo-SCT blood samples can be found in (Table 2; Fig. 3). A total of 8 mutations were detected in the pre-treatment sample when she had AML, 5 mutations remained on her post-chemotherapy analysis where there was residual myelodysplasia, and no mutations were present after haplo-SCT. At the time of writing, she is currently 14 months post-transplant, with no evidence of disease relapse, with 100% Donor chimerism.