Comprehensive Characterization of Cytogenetic and Mutational Analysis of Acute Promyelocytic Leukemia: Is PML-Rara Everything?
Authors: Awada Hassan , Durrani Jibran , Kewan Tariq Zuheir , Kishtagari Ashwin , Visconte Valeria , Mahfouz Reda Z. 
Published: November 2019

Acute promyelocytic leukemia (APL) is characterized by PML-RARA fusion caused by the t(15;17)(q24;q21) translocation. Although PML-RARA fusion explains the dedifferentiation in most of APL patients, it still does not entirely represent the unique cause of all the clinical manifestations of the disease failing to determine the full leukemic phenotype. Up to 40 % of APL patients have an additional chromosomal abnormality other than PML-RARA. Murine studies have reported that additional cytogenetic abnormalities and secondary somatic mutations (for instance FLT3-ITD) might contribute to leukemia progression. Indeed, mice expressing mutant PML-RARA develop definitive leukemia after one year, suggesting that additional hits are required for transformation. Moreover, no distinct genetic signature has been characterized by next generation sequencing (NGS). This confirms that no gene has been reproducibly identified. APL respond to all-trans retinoic acid (ATRA) in the great majority of patients. However, one quarter of APL develop ATRA resistance suggesting that additional secondary chromosomal abnormalities might be evolving resistance. Combination of low dose arsenic, modify certain epigenetics, with ATRA decreased resistance potential and improved response. In the line with other possible factors involved in ATRA resistance, is the broad nature of the targets of ATRA. A molecular core network of ATRA's targets has been clustered in differentiation, growth factors and nuclear receptors possibly cooperating with PML-RARA and additional chromosomal abnormalities. Herein, we aimed to characterize the gene mutations and chromosomal abnormalities playing key roles in cellular differentiation and epigenetic regulation and to correlate the occurrence of these alterations with treatment response and survival outcomes in APL.
We took advantage of a large cohort of APL patients (n=145). Median age of the cohort was 50 yrs (19-85); equal gender distribution; median blood counts were: [WBC 6.2 x 109/L (0.4-155); 37% had leukopenia], hemoglobin [9.8 g/dL (2.7-16.2); 32% had anemia] and platelets [29 x 109/L (range of 0-228); 93% had thrombocytopenia]. In terms of karyotype, 15% of the patients carried +8, 7% had complex karyotyping (≥3 cytogenetic abnormalities), 2% had -7/del (7q) or del (12p), 1% had -17/del(17p), and 1 patient had -5. Mutational analysis of 30 genes panel, identified 141 mutations carried by 65% (94/145) of APL patients. The most frequent mutations were observed in FLT3-ITD (61/143; 43%), WT1 (26/139; 23%), and ASXL1 (7/136; 5%) genes. Less frequent mutations were found in 3.7% of CEBPA, KRAS, and NRAS genes as well as in CBL, EZH2, TET2 (3% each) genes. Additionally, we noted that all mutations were recurrent in specific functional pathways and patients carried mutations in more than 1 gene of the same pathway. Of note, cell signaling and proliferation genes (CBL, NRAS, KRAS, KIT, FLT3) were the most frequently mutated (77/141, 55%) and impacted OS (HR: 1.7, P=0.02). Moreover, transcriptional factors which are often mutated in AML (e.g. CEBPA, TP53, NPM1, RUNX1, WT1) as well as major determinants of cell's fate were markedly mutated (38/141, 27%) suggesting that genetic impairment of signaling and transcription might contribute to the lack of differentiation observed in APL phenotypes. Mutations in epigenetic genes and histone methyltransferases (ASXL1, BCORs, DNMT3A, EZH2, IDH1/2, TET2) were also found in 18/141 (13%) while genes regulating cell proliferation and RAS family (CBL, NRAS, KRAS, NPM1) were enriched in 16/132 (12%) of APL cohort. We then analyzed the genetic picture of remission (APLRm, n=131, 90%) and relapsed (APLR, 1sr relapsed to ATRA, n=14, 10%) patients. Acknowledging the low number of APLR, we observed that molecular mutations did not make a key difference in APLRvs. APLRm [except for a complete lack of mutations in epigenetic pathways (0% vs. 13%)]. Contrarily, specific cytogenetic abnormalities were more common in APLR compared to APLRm as the case of +8 (36% vs. 11%; P= .02) and -17/del(17p) (2/14 vs. 0/131; P= .008).
In sum, our study demonstrates that PML-RARA might be accompanied by additional acquired chromosomal change with a variety of genetic mutations in key pathways driving cellular differentiation. These molecular/ cytogenetic associations could determine resistance to ATRA and overall APL patients' survival.

Disclosures
No relevant conflicts of interest to declare.