Unexpectedly High Prevalence of iAMP21 in Children with Acute Lymphoblastic Leukemia in Mexico: A Report from the “Mexico in Alliance with St. Jude” (MAS) Collaborative Group

Keywords: acute lymphoblastic leukemia, intra-amplification of chromosome 21, genetic characterization.

Mexico in Alliance with St. Jude (MAS) has carried out a multi-center collaboration for children with acute lymphoblastic leukemia (ALL) in Mexico since 2019. The "Bridge Project" has granted access to diagnostic tests that allow genetic characterization of children with ALL in participating centers. The Molecular Genetics Laboratory of Hospital Infantil Teleton de Oncologia (HITO) serves as the referral laboratory. 253 newly diagnosed ALL patients from 5 hospitals in Mexico have utilized a comprehensive diagnostic panel that includes cytogenetic and molecular tests to identify relevant abnormalities. iAMP21 is recognized by the 2017 World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues as a characteristic genetic alteration of B-lineage Leukemia/Lymphoma, with a reported incidence of up to 2-5%. It involves amplification of the RUNX1 gene and/or duplication of chromosome 21 dup(21q), and defines a relatively new cytogenetic subgroup in B-lineage ALL with poor prognosis, derived from the genetic instability of chromosome 21.The heterogeneity of iAMP21 contributes to the difficulty in its detection with the sole use of conventional cytogenetic techniques, rendering Fluorescent In Situ Hybridization (FISH) as a better strategy for the distinction between iAMP21 (amplification in tandem) and the sporadic gain of copies of the RUNX1 gene. METHODS: We performed a comprehensive diagnostic panel for 253 patients with ALL; the FISH B-cell lymphoid panel included (KMT2A, ETV6 (TEL)/RUNX1 (AML1), BCR/ ABL, E2A/(TCF3)/(PBX1) and the T-cell lymphoid panel included (MLL, BCR / ABL, E2A / (TCF3) / (PBX1), TLX1, TLX3, CDKN2A and TRA / D). Diagnosis of iAMP21 was made using the interphase FISH technique with the LSI ETV6/RUNX1 (Vysis®) double color double fusion probe (Abbott Molecular®, Ill), and Cytocell probe (OGT Company®, NY). A finding of ≥ 5 RUNX1 gene signals in interphase FISH and/or more than 3 signals within chromosome 21 was considered positive for the identification of iAMP21. Samples were processed according to the specifications to the AGT Cytogenetics Laboratory Manual. FISH analysis was performed by analyzing 200 interphase cells. When ETV6/ RUNX1 probes are used, samples of patients carrying iAMP21 usually show two normal copies of the ETV6 signal and multiple RUNX1 signals (three or more additional signals) and are negative for the ETV6-RUNX1 fusion gene. RESULTS: Among 253 patients with ALL, 233 cases were of lineage B (92%) and 20 (8%) were of lineage T. FISH was reported negative in 57 (23%) and positive in 185 (73%) cases, 11 results were not available (4.3%). We detected iAMP21 in 28 (11%); these cases were classified as high risk. We also report t(12;21) in 41 (16%), MLL gene disruption t(4;11) in 10 (4%), t(1;19) in 14 (6%), and t(9;22) in 7 (3%); 75 (30%) patients had nonspecific genetic gains. The T lineage showed CDKN2A del(9)(p21) in 6 (2.5%) and TRA/D(14)(q11.2) rearrangement in 4 (1.7%) cases. CONCLUSION: Our experience shows that FISH analysis with the ETV6/RUNX1 probe is useful for identifying iAMP21, a chromosomal abnormality with prognostic significance. Genetic risk factors in the Hispanic population that adversely impact survival in Latin America are not yet fully identified. The Molecular genetic analysis and the fluorescence in situ hybridization (FISH) technique are the basic methods used to detect the presence of the most common genetic abnormalities, the presence or absence of which has an impact on the patient's classification into the appropriate risk group. Our findings from this cohort point to an increased prevalence of iAMP21 in ALL patients in Mexico. We will soon incorporate gene sequencing to the diagnostic panel and improve the characterization of these patients.