Background: Traditionally, the diagnosis of hematologic malignancies relied on morphological, immunological along with cytogenetic evaluation of bone marrow and peripheral blood. However, these methods sometimes yield non-diagnosis, diagnostic ambiguity or discordance. The advent of next generation sequencing (NGS) can be of help in such settings. Here, we describe our experience of using NGS data in guidance to diagnose and treat an indeterminate case of hematologic malignancy.

Case: A 77-year old female presented to the clinic with worsening dyspnea on exertion with pancytopenia (white blood cells 1.25×109/L – hemoglobin 7.6g/dL – platelet 22×109/L). Her bone marrow examination showed hypercellular marrow morphologically suggesting acute myeloid leukemia (AML) M4 by French-American British classification, but blast count of only 6.9%. The conventional karyotyping showed 46, XX [20]. Fluorescence in situ panel, including PML-RARA, RUNX1T1-RUNX1, CBFB, KMT2A, MECOM, were all negative, and so were the results of HemaVision®. Direct sequencing and PCR for FLT-3 ITD and TLD and KIT sequencing for exon 9, 11, 13, 17 results were all negative. In order to reconcile the discrepancies between morphological and cytogenetic diagnosis, NGS was performed.

Method: The DNA from bone marrow collected at the time of diagnosis was analyzed using whole exome sequencing (WES). Saliva DNA collected at the time of remission was used matched normal sample. Somatic mutations were determined by MuTect. For structure variation analysis, insertion size was calculated by CollectInsertSizeMetrics module of Picard package and Pindel to call SVs. Gene to gene network analysis was done using STRING database.

Discussion: We identified 13 somatically mutated non-synonymous small nucleotide variants with WES. These included NBPF1/8/9, PBRM1, DDX60L, VPS52, TIMM23B, KIF26A, MYO1E, KRTAP9-8, CRELD2, FOXO4 and TNMD. Through gene to gene networking, we recognized FOXO gene in relation to AKT pathway and JUN signaling. Since FOXO4 amplification appears in 0.5% of AML cases and AKT/FOXO and JNK/c-JUN plays a pivotal role in myeloid leukemia, we recognized this as possible driver mutation. Moreover, fragment analysis with direct sequencing recognized NPM1 gene exon12 c.860_863dupTCTG. NGS data revealed NPM1 1 copy repeat (2 bp insertion). Based on the NGS data, the patient received low dose cytarabine (20mg/m2/day for 14 days), as in elderly AML patients. She responded surprisingly well, and after 3 cycles of chemotherapy, her hemogram was almost normalized (WBC 3.02×109/L – Hb 10.4g/dL – platelet 180×109/L). She is currently being followed up without any further interventions.

Conclusion: Our data highlights the importance of molecular diagnosis based on NGS when confronted with diagnostic challenges in hematology. Efforts to effectively incorporate NGS data in real-world clinical settings should be encouraged to realize the goals of tailored medicine.

Citation Format: Ja Min Byun, Youngil Koh, Sung-Soo Yoon, Daeyoon Kim. Value of next generation sequencing in a diagnostically challenging case of hematologic malignancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3185.