Quantification of the Minimal Residual Disease in Pediatric Philadelphia-Positive Acute Lymphoblastic Leukemia
Authors: Ferri Cristian , Riccheri Cecilia , Bietti Julieta , Cedola Alejandra , Bianchini Michele , Aversa Luis Alberto , Pennesi Sandra , Belli Carolina , Milone Gustavo , Larripa Irene B 
Published: October 2019

Abstract
Philadelphia (Ph) chromosome in acute lymphoblastic leukemia (ALL) is found nearly in 20-30% adults and 5% children. Ph chromosome results from the reciprocal translocation t(9;22)(q34;q11) joining the tyrosine kinase c-ABL1 proto-oncogene on chromosome 9 to BCR gene on chromosome 22. The majority of Ph breakpoint in ALL occurs in the minor breakpoint cluster region at first intron of the BCR gene, and results in the fusion of BCR exon 1 to ABL1 exon 2 (e1a2) generating the oncoprotein p190BCR-ABL1. Ph+ ALL patients present an aggressive disease with a poor prognosis. Current treatment includes potent chemotherapy and tyrosine kinase inhibitors, such as imatinib. The aim of this study was to quantify BCR-ABL1 transcripts in ALL Ph+ patients at different stages of treatment in order to evaluate the kinetics of the tumor clone in response to treatment.
We reported a preliminary analysis of the clinical trial GATLA LLA-Ph+2011 (Grupo Argentino de Tratamiento de la Leucemia Aguda LLA-Ph+) that includes patients from 1 to 21 years old newly diagnosed Ph+ ALL. Written informed consent was obtained from all subjects. Diagnosis of Ph+ALL was confirmed by the presence of Ph chromosome and/or positive molecular analysis for detection of BCR-ABL1 fusion transcripts. All patients received induction treatment consisting of vincristine, daunorubicin, prednisolone, asparaginase, and metrotexate. Imatinib was administered 300 mg/m2/day from day 15 of induction until the initiation of conditioning for alloHSCT or to the end of maintenance therapy. The clinical protocol consist in an induction IA (33 days) and IB (28 days), consolidation RA1, RA2 and RA3, re-induction (Protocol II A and B, 36 days each) and maintenance to complete 2 years of treatment. Eligible patients are transplanted after the third block.
Minimal residual disease (MRD) monitoring was performed by one-step quantitative RT-PCR assays in bone marrow and/or peripheral blood, using RotorGene® Qiagen; at the end of induction AI, previous RA1, RA2, RA3 and protocol II Fase IIA, IIB and during maintenance or post-alloHSCT. BCR-ABL1 quantification was expressed relative to the amount of ABL1 mRNA. We studied a control group of pediatrics positive P190 isoform bone marrow samples at diagnosis in order to establish our basal reference to estimate the logarithmic reduction of BCR-ABL1 transcripts for our cohort. The obtained mean value of BCR-ABL1(e1a2)/ABL1 in the control group was 55.5% (range 29% - 92%). The molecular response was defined as optimal (OMR, OMR-undetectable) if the BCR-ABL1/ABL1 ratio was lower than 0.055%.
A total of 25 subjects (male: female = 16: 9) were enrolled into the study from November 2011 to July 2014. The median age was 7 (range 2 - 19) years old. Type of BCR breakpoint was minor (e1a2) in 96% of patients, and only one present a rearrangement corresponding to major BCR (b3a2). During the follow up (median: 15 months), five patients (20%) died: 3 by sepsis, 1 by post alloHCT complications, and 1 due to disease progression. Two patients had to rotate to dasatinib because of disease relapse: one in central nervous system and the other post alloHCT. Our preliminary results showed that the overall survival was 79%±10% and the survival free of event (death, molecular and disease relapse) was 67%±10% at 12 months.
We serially measured BCR-ABL1 at different trial stage (AI, RA1, RA2, RA3, Protocol II) and OMR was 13.3%, 41.2%, 33.3%, 45.5% and 76.9%, respectively. Among 17 evaluable patients on maintenance and post alloHCT, 88% (15/17) obtained OMR values, and among them, 87% (13/15) presented OMR-undetectable values.
We evaluated the kinetics of the tumor clone under treatment regarding prognostic factors. Patients older than 10 years showed a higher rate of OMR at RA1 (83% vs 27%, p=0.05). However, all evaluated younger patients reached an OMR at protocol II stage (100% vs 33%, p=0.045), showing a sustained and deeper molecular response. Other evaluated parameters did not show a relationship with OMR at different stages.
Our preliminary data showed that BCR-ABL1 quantification may be a good marker to evaluate response to treatment in Ph+ positive ALL patients. BCR–ABL1 transcripts dropped to undetectable levels in 76% (13/17) of evaluable cases at the later stage of treatment. Also, 77% of patients achieved an OMR at Protocol II not only might provide a better chance for receiving alloHSCT but also might contribute to durable remissions.

Disclosures
No relevant conflicts of interest to declare.