High Frequency of the Ph-like PCB-ALL Subtype in Mexican Pediatric Patients. Experience in Two Institutions
Authors: Perez-Vera Patricia , Moreno Lorenzana Dafne , Juárez-Velázquez María Del Rocío , Martínez-Anaya Daniel Alejandro , Reyes-León Adriana , Hernández-Monterde Adrián , Aguilar Maria Montserrat , Soto Lerma Oscar , Herrera-Almanza Martha , Zapata-Tarres Marta , Juarez-Villegas Luis , Cardenas-Cardos Rocio , Rivera-Luna Roberto , Olaya-Vargas Alberto 
Published: October 2019

Abstract
Background: Philadelphia-like (Ph-like) subtype occurs in 15-20% of children patients diagnosed with precursor B cell (PCB) acute lymphoblastic leukemia (ALL). It is characterized by alterations in kinases and their receptors, affecting signaling pathways such as Jak2-Stat5, class Abl and Ras. It is particularly frequent (35%) in Hispano-Latino high risk PCB-ALL patients, residing in the USA. In Mexico, based on our ethnicity and the poor response to treatment of PCB-ALL pediatric patients, the Ph-like subtype could be frequent. However, this entity is widely variable and the diagnosis is based on genomic methods. In our country the incidence of leukemia is high, 78.1/1,000,000, and 83% of these cases are ALL. Therefore, it is important to detect Ph-like patients using diagnostic methods accessible to Institutions that treat patients with leukemia in our country.
Aims: To develop an algorithm adapted to our local capacity for the diagnosis of Ph-like ALL children in Mexico. To determine the frequency of Ph-like patients in two Pediatric Institutions in our country
Hypothesis: Ph-like ALL frequency in Mexican children will be higher than that reported in Hispano-Latino PCB-ALL patients.
Methods, study design and laboratory studies: Bone marrow samples from 119 PCB-ALL patients (18≤ years old) at diagnosis were studied. Samples were collected at the Instituto Nacional de Pediatría and the Hospital Infantil de México Federico Gómez in Mexico City. Patients with recurrent gene fusions, ETV6-RUNX1, TCF3-PBX1,KMT2A-var and BCR-ABL1, were excluded. The Ph-like status of each patient was determined using molecular and biochemical methods. We analyzed a gene expression signature conformed by the genes: CRLF2, TSPN7, IGJ, PON2, SEMA6A and BMPR1B (q-RT-PCR), and detected the P2RY8-CRLF2 deletion (RT-PCR) in all patients. In those patients with overexpression of CRLF2, but negative to P2RY8-CRLF2, the IGH-CRLF2 rearrangement (FISH) was examined. The positive cases to P2RY8-CRLF2, without overexpression of CRLF2, were analyzed looking for minor subclones (FISH) with the deletion. IKZF1 deletions (nested RT-PCR) were analyzed in all patients. The biochemical studies included CRLF2 protein expression, phosphoflow analysis of Jak2-Stat5, class Abl and Ras targets (flow cytometry). All these assays were performed whenever the cell sample allowed it. Patients with two or more of the following characteristics were considered as Ph-like cases: with overexpression of 50% or more of the expression signature, positive to P2RY8-CRLF2 or IGH-CRLF2, alteration of one or more signaling pathways, with IKZF1 deletions.
Results: In 50% (n=74) of patients CRLF2 overexpression was detected, within them 15% were positive to P2RY8-CRLF2 deletion, 17% to IGH-CRLF2 rearrangement, and 18% had an unidentified CRLF2 alteration that cause the overexpression. Within the 50% of cases without CRLF2 overexpression, 18% were positive to the P2RY8-CRLF2 rearrangement. IKZF1 deletion was evaluated in 55 patients and 72% were positive. Results obtained revealed a high frequency of CRLF2 and IKZF1.
In 68 patients the gene expression profile was evaluated, 60% presented overexpression of 50% or more genes. The biochemical analysis revealed that 67% (n=42) of patients were positive to CRLF2 protein, 29% (n=21) had abnormal Jak2/Stat5 activation, 34% (n=32) presented abnormal Abl activation, and in 43% (n=7) abnormal Ras activation was detected. Not all the CRLF2 protein positive patients presented Jak2-Stat5 pathway activation (2%). Patients with more than one abnormal pathway were detected (18% with Jak2-Stat5/Abl or Abl/Ras, n=21).
Based on our algorithm of analysis, 53% of the analyzed patients present genetic and biochemical characteristics of the Ph-like subtype.
Conclusions: The combined methods contributed to identify Ph-like patients, the results demonstrate that the frequency of PCB-ALL children with Ph-like characteristics in Mexican patients is higher compared to other populations. This study demonstrates high frequency of CRLF2 alterations and IKZF1 deletions in PCB-ALL in Mexican population. In our experience, analysis of the pathway activation assays and the identification of CRLF2 and IKZF1 alterations are required to detect Ph-like patients. These results could be useful to stratify the childhood PCB-ALL patients in our country.
Acknowledgments: CONACYT: PDCPN-2004/248591; Cátedra 2038.

Disclosures
No relevant conflicts of interest to declare.