In-Utero Origin of Childhood Acute Leukaemias.
Authors: Burjanivova Tatiana , Madzo Jozef , Muzikova Katerina , Meyer Claus , Schneider Björn , Stary Jan , Marschalek Rolf , Trka Jan , Zuna Jan 
Published: April 2020

Abstract
While there is enough convincing evidence in childhood ALL, the data on the pre-natal origin in childhood AML are less comprehensive. Our study aimed to screen Guthrie cards (neonatal blood spots) of childhood acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) patients for the presence of their respective leukaemic markers. For the analysis we used PML/RARa, AML1/ETO and CBFb/MYH11 fusion genes, translocations of MLL gene and internal tandem duplication of Flt3 gene (Flt3/ITD) in patients with AML and TEL/AML1 fusion gene and immunoglobulin (Ig) and/or T-cell receptor (TCR) gene rearrangements in patients with ALL. These molecular markers on the DNA level present suitable candidates for backtracking of leukaemic cells in newborn material, because they are clonotypic and altogether their incidence is ~40% and &gt;90% in AML and ALL, respectively. We screened Guthrie cards from 13 AML patients (4x PML/RARa,, 3x CBFb/MYH11, 2x AML1/ETO, 2xFlt3/ITD, 3x MLL rearrangement - MLL/AF6, MLL/AF9 and MLL/AF10) and from 15 ALL patients (13x Ig/TCR rearrangements, 3x TEL/AML1). One AML patient from our group had PML/RARa fusion gene together with Flt3/ITD, in 1 ALL patient we screened the Guthrie card for both Ig/TCR and TEL/AML1. We designed patient-specific PCR primers and we established nested PCR assay for each clonotypic sequence prior to the analysis of the corresponding Guthrie card. Assay specificity was determined using serial dilutions of patient DNA into the DNA of a healthy donor. The sensitivity of PCR was &gt;=10(−4) in all patients. Therefore, this approach allowed us to detect the pre-leukaemic clone provided 1–10 positive cells were present on the Guthrie card. In three patients with ALL we reproducibly detected identical rearrangements both in the presentation sample and on the Guthrie card. The first patient harboured two independent rearrangements: TCR-delta Vd2/Dd3 and IgH VH3/JH5 and both were detectable on Guthrie card. In the second patient two PCR systems were optimised with adequate identical sensitivity: Igkappa Vk1/Kde and TCRgamma VgI/Jg1.3-2.3. However, only the latter detected pre-leukaemic cells on Guthrie card. The third patient had TEL/AML1 and we found this translocation on her Guthrie card. We did not find patient-specific molecular markers in any patient with AML. In the present study we confirmed the prenatal origin in 20 % of ALL patients. The negative results in the AML group can not disprove the theory that some childhood AML cases are also initiated in-utero. The negative results might be caused also by the fact that the age at presentation was higher in our AML group compared to ALL patients (median 7 and 3 years, respectively), the size of pre-leukaemic clone in AML might be below the sensitivity threshold of our approach, and Flt3/ITD could be of post-natal origin as recently suggested. However, our present data based on the largest cohort examined so far show that there is much less evidence for the pre-natal origin of childhood AML. Support: grants #7436 (Czech Ministry of Health) and #0021620813 (Czech Ministry of Education).