Two rare cases of acute myeloid leukemia with t(8;16)(p11.2;p13.3) and 1q duplication: case presentation and literature review

Background Acute myeloid leukemia (AML) is a complex hematological disease characterized by genetic and clinical heterogeneity. The identification and understanding of chromosomal abnormalities are important for the diagnosis and management of AML patients. Compared with recurrent chromosomal translocations in AML, t(8;16)(p11.2;p13.3) can be found in any age group but is very rare and typically associated with poor prognosis. Methods Conventional cytogenetic studies were performed among 1,824 AML patients recorded in our oncology database over the last 20 years. Fluorescence in situ hybridization (FISH) was carried out to detect the translocation fusion. Array comparative genome hybridization (aCGH) was carried out to further characterize the duplication of chromosomes. Results We identified three AML patients with t(8;16)(p11.2;p13.3) by chromosome analysis. Two of the three patients, who harbored an additional 1q duplication, were detected by FISH and aCGH. aCGH characterized a 46.7 Mb and 49.9 Mb gain in chromosome 1 at band q32.1q44 separately in these two patients. One patient achieved complete remission (CR) but relapsed 3 months later. The other patient never experienced CR and died 2 years after diagnosis. Conclusion A 1q duplication was detected in two of three AML patients with t(8;16)(p11.2;p13.3), suggesting that 1q duplication can be a recurrent event in AML patients with t(8;16). In concert with the findings of previous studies on similar patients, our work suggests that 1q duplication may also be an unfavorable prognostic factor of the disease.


Background
Acute myeloid leukemia (AML) is a common disease characterized by immature myeloid cell proliferation and bone marrow failure, which can be subdivided into 9-11 pathogenetically different subtypes [1]. Over the past two decades, the incidence has increased by 30% [2,3]. Furthermore, AML has poor long-term survival with a high relapse rate [4]. Therefore, AML represents a substantial health problem that requires strict monitoring and innovative treatment strategies. The development of newer, effective treatment strategies is necessary for AML patients.

Patients
This study was approved by the Institutional Review Board (IRB) of Oklahoma University (IRB Number: 2250). A total of 1,824 AML patient samples were studied cytogenetically from 2000 to 2019 at the Genetics Laboratory of Oklahoma University Health Sciences Center. Bone marrow samples were obtained from three of the 1,824 patients who had t(8;16)(p11.2;p13.3).

Conventional cytogenetic analysis
Short-term cultures of unstimulated bone marrow samples were established and harvested according to standard laboratory protocols. Karyotype analysis was performed using Giemsa and trypsin techniques for G-banding. The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature (ISCN 2016).

Fluorescence in situ hybridization analysis
Fluorescence in situ hybridization (FISH) assays were performed according to the manufacturer's instructions in combination with our established laboratory protocols. A PML/RARA dual-color, dual-fusion translocation probe (Abbott Molecular Inc., Des Plaines, IL, USA), subtelomere-specific probes for chromosome 3 p-arm and q-arm, and whole chromosome painting (WCP) probes for chromosomes 1, 3 and 14 were purchased from Cytocell Ltd, NY, USA. A spectrum green-labeled probe mapping to the 8p11.21 region and a spectrum orange-labeled probe mapping to the 16p13. 3  . The KAT6A gene located on 8p11.21 and the CREBBP gene located on 16p13.3 were covered by the green-labeled and red-labeled home-brewed probes, respectively. All probes were validated before use. Chromosome spreads were counterstained with 4,6-diamidino-2-phenylindole (DAPI4) in antifade medium (Vector Laboratories Inc., CA, USA). Digital images carrying specific hybridization signals were captured and processed on CytoVision version 7.0 (Applied Spectral Imaging, Carlsbad, CA, USA).

aCGH analysis
Genomic DNA was extracted from each of the three patients' bone marrow pellets according to the standard operating procedure using the phenol and chloroform method with a commercially available DNA extraction kit (Puregene blood kit, Qiagen, Valencia, CA) or Nucleic Acid Isolation System (QuickGene-610L, FUJI-FILM Corporation, Tokyo, Japan). Two aCGH platforms, NimbleGen and Agilent, were used in this study. For the NimbleGen aCGH platform, human reference genomic DNA was purchased from Promega Corporation (Promega Corporation, Madison, WI, USA). The patient's DNA and the reference DNA were labeled with either Cyanine 3 (Cy-3) or Cyanine 5 (Cy-5) by random priming, and then equal quantities of both labeled products were mixed and loaded onto a 720 K oligonucleotide chip (Roche NimbleGen Inc., Madison, WI, USA) to hybridize at 42 °C for 40 h in a MAUI hybridization system (BioMicro Systems, Salt Lake City, UT) according to the manufacturer's protocols with minor modifications. The slides were washed with washing buffers (Roche NimbleGen Inc.) after hybridization and scanned using a Roche Scanner MS 200 Microarray Scanner (Roche NimbleGen Inc.). Images were analyzed using NimbleScan software version 2.6 and SignalMap software version 1.9 (Roche NimbleGen Inc.). The genomic positions were determined using GRCh36/hg18, UCSC Genome Browser. For the Agilent aCGH platform, human reference genomic DNA was purchased from Agilent Corporation (Agilent Corporation, Santa Clara, CA, USA). The patient's DNA and the purchased reference DNA were labeled with either Cyanine 3 (Cy-3) or Cyanine 5 (Cy-5) by random priming (Agilent Corporation). Patient DNA (labeled with Cy-3) was combined with a normal control DNA sample (labeled with Cy-5) of the same sex and hybridized to an Agilent 2 × 400 K oligo microarray chip (Agilent Technologies) by incubating in an Agilent Microarray Hybridization Oven (Agilent Technologies). After 40 h of hybridization at 67 °C, the slides were washed and scanned using the NimbleGen MS 200 Microarray Scanner (Roche NimbleGen Inc.). Agilent's CytoGenomics 2.7 software (Agilent Technologies.) was applied for data analysis. The genomic positions were determined using GRCh37/hg19, UCSC Genome Browser.

Case presentation
Case 1 An 82-year-old male presented with anemia was referred to us for AML evaluation. His subsequent lab results and hospital records were not available in our clinical database.
Case 2 A 28-year-old female presented with disseminated intravascular coagulopathy was referred to rule out APL. Her complete blood examination and bone marrow aspirate smears were not available. Flow cytometry revealed 57% monocytic cells positive for CD4, CD11b (partial), CD13 (bright), CD14 (partial), CD15, CD33 (bright) and HLA-DR (partial) but negative for CD3, CD7, CD34, CD117, MPO and TdT, consistent with a diagnosis of AML with monocytic differentiation (subtype M5). The patient achieved hematological CR on day 15 and cytogenetic CR on day 33 after induction chemotherapy and then relapsed 3 months later.
Case 3 A 69-year-old female with a medical history of breast cancer after lumpectomy, chemotherapy, and radiation presenting with generalized weakness, pancytopenia, and fever was referred to us for disease progression evaluation. A complete blood examination showed a white blood cell count of 216 × 10 9 /L with 53% blasts, a hemoglobin count of 66 g/L and a platelet count of 31 × 10 9 /L. Her bone marrow aspirate smear demonstrated over 90% myeloblasts. Flow cytometry revealed that 69% of the blast cells expressed CD45 (moderate), CD34 (dim), CD38, HLA-DR, CD13, CD15, and CD33 and were negative for CD117, consistent with a diagnosis of AML with monocytic differentiation (subtype M5). The patient started consolidation chemotherapy but had spontaneous regression and died 2 years after AML diagnosis.

Results
In case 1, routine chromosome analysis detected an abnormal karyotype with a translocation between the short arms of chromosomes 8 and 16 (Fig. 1a) [3]. No other consistent karyotypic aberrations were detected. Thus, this male patient was excluded from subsequent FISH and aCGH analyses.
In case 3, t(8;16)(p11.2;p13.3) with a gain of a similar chromosome segment on the long arm of chromosome 3 was detected in 18 of 20 cells by karyotyping analysis (Fig. 1c). FISH confirmed the KAT6A-CREBBP fusion and revealed additional chromosome 1 material (Fig. 2c  and d). Loss of the end portion of the chromosome 3 long arm was not found by FISH (Fig. 3e). aCGH further detected a gain from chromosome 1 at bands 1q32.1q44  (Fig. 3b). The molecular size was 49.9 Mb.

Discussion
AML is one of the most common diseases characterized by the proliferation of blast cells in bone marrow or peripheral blood, which accounts for approximately 30% of adult leukemia cases. As reported previously, common chromosomal translocations such as t(8;21)/RUNX1-RUNX1T1, t(15;17)/PML-RARA , and inv(16)/CBFB-MYH11 are frequently observed, and numerous uncommon chromosomal aberrations also exist in AML [12]. The detection of these fusion transcripts is important for the diagnosis and progression monitoring of AML patients [21].

Fig. 1 continued
Over the past 15 years, cytogenetic and molecular technologies have largely promoted the efficiency of the identification and characterization of this disease [5]. Compared with conventional cytogenetic analysis and FISH methods, aCGH is an attractive method for the investigation of cancer genomes [23]. aCGH has higher resolution, simplicity, high reproducibility, shorter turnaround time and precise mapping of aberrations. Most importantly, it avoids the need for cell culture and dividing cells [24][25][26]. Furthermore, aCGH chromosomal analysis facilitates rapid detection and duplication of cytogenetic abnormalities previously undetectable by conventional cytogenetics [27]. In our investigation, we applied aCGH to characterize the additional chromosome 1 materials in patients 2 and 3 and interestingly found that the two patients revealed the same extra copy of 1q at bands q32.1q44. Patients with 1q duplication have also demonstrated a wide range of multiple malformations, such as intellectual disability, macrocephaly, large fontanels, prominent foreheads, broad flat nasal bridges, high-arched palates, retrognathia, low-set ears, and cardiac defects [28,29]. More recent studies have shown that a 1q gain is also related to a portion of solid tumors. For instance, the gain of 1q is well known as a poor prognostic biomarker of Wilms tumor [30], and it plays an important role in predicting poor clinical outcome in patients with thyroid carcinoma as well [31]. In addition, patients with a 1q duplication showed worse survival and high risk in acute leukemia, Burkitt lymphoma, and myeloproliferative neoplasms [32][33][34][35][36]. The outcomes of 1q duplication in the nine reported AML patients with t(8;16)(p11.2;p13.3) are summarized in Table 1. Seven patients' data were available. These seven patients (two adult and five pediatric) all received induction chemotherapy, and six achieved CR. At the time of last follow-up, two adult patients and three of five pediatric patients had died. Only two pediatric patients were alive. We reported two adult patients here: patient 2 achieved CR but relapsed 3 months later, and patient 3 had spontaneous regression and died 2 years after diagnosis. Taken together, the findings suggest that 1q duplication might be associated with adverse outcomes in AML patients with t(8;16)(p11.2;p13.3). However, the significance of the 1q duplication in AML with t(8;16) needs to be further investigated. Since such changes have been seldom reported, the pathogenic effects of 1q duplication in AML patients with t(8;16) (p11.2;p13.3) require more studies to be delineated.

Conclusion
Three patients were detected with t(8;16)(p11.2;p13.3) from an 1,824 AML patient database. Two female patients were identified with a 1q duplication by FISH and aCGH analyses. Combining our investigation with the findings of published studies, we conclude that 1q duplication is a recurrent finding in AML patients with t(8;16). Our data also suggest that 1q duplication might be associated with unfavorable prognosis in these cases. The understanding of cytogenetic data would contribute to the diagnosis and treatment evaluation of AML.