Epidemiology of Philadelphia Cromosome like (Ph-like) in Acute Lymphoblastic Leukemia (ALL) in Ecuadorian Population
Authors: Arias Calvachi Claudia Patricia , Larrea Esteban David , Luna Beatriz , Silva Sofia Stefany , Ramos Oscar Enrico , Di Stefano Marcos 
Published: December 2024

Philadelphia chromosome-like (Ph-like) is a high-risk subtype of Acute Lymphoblastic Leukemia (ALL) that lacks the expression of the BCR-ABL1 (Breakpoint cluster region-Abelson oncogene) fusion gene, typically found in Ph-positive ALL, yet exhibits similar clinical behavior. The Ph-like subtype accounts for 18-25% of worldwide cases in young adults, with males from 21 to 29 age group being the most affected. There is no official registry of the incidence of Ph-like translocation but there's documentation of positive cases among White, African-American and Asian populations, especially in children. Particularly, Ph-like subtype which is also present in Hispanic adults is characterized by chemo-resistance to therapy leading to higher relapse rates and overall poorer survival compared to other ALL diagnoses. Ph-like ALL is characterized by molecular alterations affecting B cell proliferation, differentiation and cell cycle regulation. These include fusion translocations involving the ABL1 gene with a variety of partner genes such as RANBP2-ABL1. The resulting chimeric proteins exhibit enhanced tyrosine kinase activity, disrupting normal signaling pathways by up regulating the mitogen-activate protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K) pathways. Additionally, CRLF2 rearrangements and alterations in JAK2, EPOR, FLTS, NTRK3 and RAS genes are commonly found in Ph-like ALL. This case describes a Ph-like ALL patient in Ecuador, the first reported instance in the country.

Identifying Ph-like ALL is challenging due to varying diagnostic method sensitivities. Thus, we employed next generation sequencing (NGS) to analyze DNA and RNA extracted from the patient's bone marrow. DNA and RNA quantification were performed by spectrophotometry (nanodrop) and fluorometric methods (Qubit). Then, using the Ampliseq Myeloid panel, we assessed the presence of hotspot genes of higher oncological risk (n=23), complete exonic genes sequencing (n=17) and RNA fusion initiator genes (n=29) of clinical relevance. This process began with the retrotranscription of the RNA sample to obtain cDNA, followed by amplification of cDNA and DNA targets included in the panel. Then the amplicons were partially digested to allow the index ligation (i7 and i5). Afterwards, the libraries were amplified to achieve an optimal concentration. As a quality control step, a fragment analysis was performed using capillary electrophoresis. We obtained DNA library fragments between 200 and 550 bp and cDNA library fragments between 181 and 373 bp as expected, according to the panel instructive. Finally, the libraries were quantified, denatured, diluted and pooled to reach a final concentration of 8pM to analyze in Miseq Illumina sequencer. The bioinformatic analysis was performed in Franklin platform by Geenox revealing the presence of the RANBP2/ABL1 fusion transcript as a result of translocation at exon 18 of RANBP2 and exon 2 of ABL1 t(2;9)(q12;q34). This finding was classified as a TIER 1 in a 21 male patient with ALL, which event is concordant with the diagnosis and relapse situation of the patient (central nervous system and testicular compromised). These types of fusions only involving the ABL1 gene without BCR are kinase activators, typically distinctive of Ph-like ALL. ABL1 is considered as the key partner that defines the functional/oncogenic effects of the fusion protein. Our findings reveal the first Ph-Like ALL case documented in Ecuador, highlighting the importance of molecular analysis with vanguard technology as NGS to define a true diagnosis in order to stratify risk and apply targeted therapy in the management of Ph-like ALL patients.