Coexistence of t(15;17) and t(15;16;17) detected by fluorescence in situ hybridization in a patient with acute promyelocytic leukemia: A case report and literature review

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), which results in the fusion of the promyelocytic leukemia (PML) gene at 15q22 with the retinoic acid α-receptor (RARA) gene at 17q21. The current study presents the case of a 54-year-old female with APL carrying the atypical PML/RARA fusion signal due to a novel complex variant translocation t(15;16;17)(q22;q24;q21), as well as the classical PML/RARA fusion signal. Subsequent array comparative genomic hybridization revealed somatic, cryptic deletions on 3p25.3, 8q23.1 and 12p13.2-p13.1, and a duplication on 8q11.2; however, no genetic material loss or gain was observed in the breakpoint regions of chromosomes 15, 16 or 17. To the best of our knowledge, this is the first report of the coexistence of two abnormal clones, one classical and one variant, presenting simultaneously in addition to cryptic chromosome segmental imbalances in an adult APL patient.


Introduction
Acute promyelocytic leukemia (APL) is characterized by particular clinical features that are important in differentiating it from other acute myeloid leukemias (AMLs) and determining an accurate diagnosis. These clinical features include unique hemorrhagic syndrome, disseminated intravascular coagulation (DIC) and association with the translocation between chromosomes 15 and 17, resulting in the formation of two reciprocal fusion genes; promyelocytic leukemia (PML)/retinoic acid α-receptor (RARA) on chromosome 15 and RARA/PML on chromosome 17. These fusion genes are sensitive to retinoid differentiating agents, such as all-trans retinoic acid (ATRA), and novel antiapoptotic agents, including arsenic trioxide (1,2).
Although t(15;17) has been found in ~90% of APL patients, variant translocations have been reported in a few APL patients, which are described as simple translocations involving chromosome 15 or 17 with any other chromosomes [t (15;v) or t(17;v)] or complex translocations characterized by the involvement of additional chromosome(s) in addition to chromosomes 15 and 17 [ct(15;17;v)] (3)(4)(5). In the last few years, a number of studies have focused on the alternate translocation in APL, for example t(5;17)(q35;q21) forming NPM/RARA, t(11;17)(q23;q21) producing PLF/RARA fusion and t(11;17) (q13;q21) generating NUMA/RARA, providing advanced insights into the pathogenesis of APL (3,6,7). However, little is known concerning the complex variant translocations in APL. The current study reports a patient who presented with the classical t(15;17) and complex variant t(15;16;17)(q22;q24;q21), which were demonstrated by traditional cytogenetic analysis, including G-banding karyotype and fluorescence in situ hybridization (FISH). In addition, cryptic losses on 3p25.3, 8q23.1 and 12p13.2-p13.1, and a gain of chromosome 8q11.2, were revealed in the level of array comparative genomic hybridization (CGH). This study was approved by the Institutional Review Board (IRB) at the University of Oklahoma Health Sciences Center (IRB no. 13100; Oklahoma City, OK, USA). The patient provided consent.

Case report
Clinical presentation. The current study presents a 54-year-old female with APL who was admitted to the University of Oklahoma Health Sciences Center due to fever and bleeding gums. The peripheral blood examination showed a hemoglobin count of 7.1 g/dl (normal range, 12-15.5 g/dl) and platelet count of 35x10 3 /µl (normal range, 150-450x10 3 /µl), as well as a white blood cell count of 11.95x10 3 /µl (normal range, 3.5-10.5x10 3 /µl) with 86% blasts, characterized by small to large cells with irregular, lobulated and bilobed nuclei with prominent nucleoli. The blast cells in the peripheral blood exhibited scant to moderate rare blue granulated cytoplasm and the bone marrow aspirate revealed no cellular particles. The touch preparations showed suboptimal cellular morphology, but revealed numerous blasts that were more frequently granulated than those present in the peripheral smear. The core biopsy of the bone marrow revealed a cellularity of >95% consisting of sheets of immature cells. A diagnosis of APL, M3 variant was determined according to the French-American-British Cooperative Group criteria (8).
Cytogenetics, FISH and array CGH analyses. The karyotype analysis at diagnosis revealed that the 20 metaphases    (23,29). However, the breakpoint of chromosome 16 in the current case (16q24) was different from those identified in the two previously reported cases. The breakpoint 16q24 has been described in AML with t(16;21) (q24;q22), which leads to the fusion gene RUNX1/MTG16 and is predominantly associated with therapy-related AML (41). Whether the MTG16 gene is located at the breakpoint and forms a fusion gene in the present case requires clarification. In addition, the association between classical t(15;17) and ct (15;17;v) clones remains unclear. Several previous studies have assumed that the complicated rearrangements originate from the single standard translocation and then quickly outgrow this stem line (16,26,33). The current study is the first to report the presentation of two abnormal clones, classical t(15;17) and ct (15;17;v), simultaneously in an adult APL patient, providing indirect evidence that the variant translocation possibly evolves from the classical t (15;17).
It is well known that chromosomal imbalances, including the deletion(s) or amplification(s) of key driver gene(s), may promote the malignant transformation of leukemia (42)(43)(44). In the reported 45 APL cases of ct (15;17;v), eight cases presented with trisomy 8 or partial trisomy, including 8p+, 4p+, 9p+, +10 and +X. However, deletions, particularly small or cryptic deletions, have not yet been reported. In the present study, array CGH revealed the cryptic chromosome aberrations, including the deletions of ATP2B2, ANGPT1 and ETV6, and a gain of RB1CC1 genes, but no imbalances of the breakpoints of chromosomes 15, 16 or 17. FISH analysis not only confirmed the deletions of ATP2B2, ANGPT1 and ETV6, but also demonstrated that these deletions were somatically acquired events since normal cells coexist with abnormal cells, which may potentially be involved in the leukemogenesis. However, the gain of 8q11.23 has not been confirmed due to the one size of duplication, which is too small to confirm by FISH. Furthermore, the possibility of a constitutional gain rather than disease-related gain cannot be ruled out. A divergence of ratio has also been identified between the deletions of ATP2B2 (97%), ANGPT1 (52%), ETV6 (90%) and the PML/RARA fusion (classical and variant combined; 90%) by FISH, suggesting that these unbalanced genetic events may occur asynchronously in leukemogenesis.
In conclusion, the current study was the first to identify the classical t(15;17) and complex variant t (15;16;17) in an adult patient with APL using FISH. Furthermore, cryptic genomic alterations involving leukemia-related genes, such as ATP2B2, ANGPT1, ETV6 and RB1CC1, were inferred in the level of array CGH and confirmed by FISH. It may be proposed that the malignant transformation of APL with complex variant translocation presents the following multi-step progression: i) Classical translocation; ii) formation of complex variant translocation by third chromosome involvement; and iii) further genomic changes. The further genomic changes, with the exception of the fusion of PML/ RARA in APL, may be implicated in the heterogenicity of therapy outcome. Advanced study on these genes is likely to aid the elucidation of the oncogene or tumor suppressor gene candidates that potentially affect the prognosis of APL.