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Molecular Genetic Characterization of Acute Myeloid Leukemia With Trisomy 4 as the Sole Chromosome Abnormality

Background/Aim: The aim of the study was to determine the genetic and molecular consequences of trisomy 4, a recurrent but rare chromosomal abnormality in acute myeloid leukemia (AML). Materials and Methods: Interphase fluorescence in situ hybridization, reverse transcriptase-quantitative polymerase chain reaction for 28 chromosomal gene translocations/fusion genes, and targeted sequencing analyses were performed on five AMLs with trisomy 4 as the sole chromosomal anomaly. Results: An NPM1 frameshift mutation was found in all leukemic bone marrows, DNMT3A, FLT3, and IDH1 mutations were found in three, KIT and NRAS mutations in two, whereas IDH2 (R140Q), RUNX1, and WT1 mutations were found in only one patient each. The three patients with a DNMT3A (R882H) mutation have died. In contrast, the two patients whose leukemic cells were without this mutation, are alive 55 and 31 months after diagnosis, respectively. Conclusion: The results suggest a possible association between trisomy 4 and additional mutations that may influence prognosis.

Acute myeloid leukemia (AML) is a heterogenous group of malignancies with different clinical phenotypes and responses to therapy (1,2). AMLs can be classified according to distinct cytogenetic and genetic abnormalities of leukemic cells at diagnosis, but also by phenotypic and epigenetic differences that act as a guide to risk assessment and choice of treatment (1,2). AML cases with a single trisomy constitute a heterogenous subgroup with regard to clinical, morphological, and immunophenotypic features, as well as the mutational profiles (3).
Trisomy 4 is a recurrent, but rare chromosomal abnormality in AML. It may be the sole aberration or one of several chromosomal changes (4). Trisomy 4 was first described by Mecucci et al. in 1986, and a year later by Sandberg et al. (5), as the sole chromosomal abnormality in AML (6). In the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, 83 (48 female and 35 male patients) out of 18029 AML-cases had trisomy 4 as the sole chromosomal abnormality (http://cgap.nci.nih.gov/Chromosomes/Mitelman, database last updated on February 20, 2017). Most cases are morphologically classified as M1, M2, or M4 using the French-American-British (FAB) classification.
Because trisomy 4 is rare, few studies have addressed its clinical impact. As a consequence, the prognosis of AML with solitary trisomy 4 remains unclear (7,8). However, a recent study based on 87 AML cases with trisomy 4 concluded that the prognostic significance of the aberration depended on the patient's age, and despite an initial good response to treatment, patients with trisomy 4 were prone to relapse (4).
Recently, we reported the genetic, molecular and clinical features of a pediatric AML case with trisomy 4 and an FLT3-ITD mutation (9). Fluorescence in situ hybridization assay demonstrated that part of the RUNX1 probe had moved to chromosome band 6q25 indicating a cryptic t(6;21)(q25;q22) translocation. Molecular analysis of the translocation showed fusion of RUNX1 with an intergenic sequence from 6q25, resulting in a putative RUNX1 truncated protein that would contain the Runt homology domain responsible for both heterodimerization with CBFB and DNA binding (9). The findings prompted us to investigate additional cases of myeloid malignancies with trisomy 4 as the sole karyotypic anomaly, for submicroscopic or molecular aberrations.

Materials and Methods
Patients and samples. Permission to perform the study was granted by the Regional Committee for Medical and Health Research Ethics of South-East Norway, and written informed consent was obtained from the patients.
Searching our archive of 5700 cytogenetically analyzed leukemia samples (from 2009 until 2017) five additional cases of AML with trisomy 4 as the sole anomaly were found (Table I).

Results
Neither interphase FISH analyses found any aberrations nor HemaVision 28Q RT-qPCR-KIT analysis detected any of the most common AML-specific fusion transcripts.
Targeted sequencing showed that all five examined AML samples carried mutations in addition to trisomy 4 (Table II). Samples from patients 1, 2, 4, and 5 harbored four mutations whereas patient 3 had seven mutations. NPM1 frameshift mutation was found in all patients, whereas KIT and NRAS mutations were found in two patients. DNMT3A, FLT3, and IDH1 mutations were found in three patients, whereas IDH2 (R140Q), RUNX1, CUX1, KRAS, and WT1 mutations were found in one patient each. Patients 1, 2, and 5, whose AMLs showed a DNMT3A (R882H) mutation, have died. In contrast, patients 3 and 4, whose leukemic cells did not carry this mutation, are alive 55 and 31 months after diagnosis, respectively. The leukemic cells from the deceased patients 2 and 5 also harbored the KIT D816V mutation, whereas patients 3 and 4 without this mutation are alive 55 and 31 months after diagnosis. Patient 1, also deceased, did not have the acquired KIT D816V mutation. This patient succumbed suddenly probably due to a cardiac event rather than AML.

Discussion
The genetic and molecular consequences of trisomy 4 are, in general, unknown (3,4,7,8). Possible mechanisms involve global gene expression alterations because of gene dosage effects, including duplication of rearranged or mutated genes on chromosome 4. No studies have as yet specifically examined general expression patterns in AML with trisomy 4. Instead, analyses have focused on KIT at 4q12, which encodes a receptor tyrosine kinase related to FLT3. Ferrari et al. (10) reported overexpression of KIT in one case with trisomy 4 as compared to AML without this abnormality, suggesting a possible pathogenetic role. Beghini et al. (11) showed that trisomy 4 resulted in duplication of a mutated KIT allele in two out of six AML cases with trisomy 4 as the sole change. Similarly, Schnittger et al. (12) reported KIT mutations in two cases with trisomy 4. On the other hand, Bains et al. (7) studied 13 AML cases with isolated trisomy 4 finding a KIT mutation in only one of them, and concluded that trisomy 4 was not generally associated with KIT mutations. Instead, FLT3 and NPM1 mutations were found in 50% and 40% of the cases, respectively.
In the present study, targeted sequencing showed concurrent multiple mutations in all patients (Table II). An NPM1 frameshift mutation was found in all leukemic bone marrows. NPM1 mutations are common in AML and are found in 20-30% of the cases (13). In addition, mutations were found in genes which are involved in the regulation of DNA methylation (DNMT3A, IDH1 and IDH2) and genes involved in cell signaling (FLT3, NRAS and KIT). RUNX1, CUX1, KRAS, and WT1 mutations were found in one patient each.
The three patients with a DNMT3A (R882H) mutation have died. In contrast, the two patients whose leukemic cells did not carry a DNMT3A mutation, are alive 55 and 31 months after diagnosis, respectively.
DNMT3A mutations are the most frequent recurrent gene mutations in AML after NPM1 and FLT3 mutations (14). The prognostic significance of DNMT3A mutations is thought to be adverse. DNMT3A mutations arise early in AML evolution and may persist during remission (1,14). There is, however, no clear information today as to whether any particular relationship exists between trisomy 4 and mutations of DNMT3A, let alone whether any such combination impacts prognosis. It is unclear whether the poor outcome for patients 1, 2, and 5 was influenced by additional mutation(s) in DNMT3A, KIT, IDH1 or a combination of these changes. The results, nevertheless, suggest a possible association between trisomy 4 and additional mutations that may affect prognosis. Novel techniques, such as targeted sequencing used in this study, may help identify the critical genes and understand their role in leukemogenesis and their influence on prognosis. Such investigations in large groups of patients are needed to collect quality data for this purpose.

Conflicts of Interest
The Authors declare that they have no potential conflicts of interest.

Authors' Contributions
ST made hematological evaluations, treated patients, designed the research, and wrote the manuscript. LG interpreted the cytogenetics and FISH data. SH interpreted the cytogenetics and FISH data and wrote the manuscript. GET made hematologic evaluations, treated patients and wrote the manuscript. SS made hematopathologic evaluations and interpreted the targeted sequencing data. BR performed targeted sequencing and interpreted the data. HTTT made hematologic evaluations and treated patients. IP designed and supervised the research, interpreted the data and wrote the manuscript.