. 2017 Mar 16;15(3):e04691. doi: 10.2903/j.efsa.2017.4691

Table B.3.

Biological plausibility of the KERs; WoE analysis

1 Support for biological plausibility of KERs	Defining question	High (Strong)	Moderate	Low(Weak)
MIE → KE1 In utero exposure to DNA topoisomerase II poison leads to In utero MLL chromosomal translocation	Strong	Rationale: Although type II topoisomerases are essential to cell proliferation and survival, they have a significant genotoxic potential consequent to the resulting (double) strand breaks. Mis‐repair of accumulated of DNA double strand breaks can result in chromosomal translocations which can persist in survived cells (Mc Clendon et al. 2009). Studies on identical twins and neonatal blood samples strongly implicate an in utero occurrence of the KER (Sanjuan‐Pla et al., 2015). Furthermore, a study in pregnant mice demonstrates that in utero exposure of the fetus to etoposide causes the MLL chromosomal translocation analogous to the human translocation except the principal fusion partner (Nanya et al., 2015). Indirect evidence from human prehaematopoietic/mesenchymal stem cells and fetal liver haematopoietic progenitor and stem cells strengthen the plausibility. Experimental evidence in these cell lines has demonstrated that etoposide as a TopII poison causes DSBs in MLL and partner genes, which leads to the formation of fusion genes and their products (Sanjuan‐Pla et al., 2015) MLL translocation sites (breakpoint sequences) in the therapy‐related leukaemia fall within a few base pairs of etoposide‐induced enzyme‐mediated DNA cleavage site. Although rearrangements associated with infant leukaemias are often more complex than those observed in treatment‐related leukaemias, many are nevertheless associated with stable TopII‐mediated DNA cut sites (Cowell and Austin, 2012; Pendleton et al., 2014)
KE1 → AO In utero MLL chromosomal translocation leads to Infant leukaemia	Strong	Rationale: The basic processes underlying overt leukaemia development are well understood and accepted. There is a general understanding of the molecular and epigenetic mechanisms leading to differentiation blockage and clonal expansion and there is evidence that the principal MLL‐fusion genes and proteins harbour the necessary properties to execute the pathways associated with differentiation blockage and clonal expansion (Benito et al., 2015; Chen and Armstrong, 2015; Chen et al., 2015)

1 Support for biological plausibility of KERs

Defining question

High (Strong)

Moderate

Low(Weak)

Is there a mechanistic (i.e. structural or functional) relationship between KE_up and KE_down consistent with established biological knowledge?

Extensive understanding of the KER based on extensive previous documentation and broad acceptance

The KER is plausible based on analogy to accepted biological relationships, but scientific understanding is not completely established

There is empirical support for a statistical association between KEs but the structural or functional relationship between them is not understood

MIE → KE1

In utero exposure to DNA topoisomerase II poison leads to In utero MLL chromosomal translocation

Strong

Rationale: Although type II topoisomerases are essential to cell proliferation and survival, they have a significant genotoxic potential consequent to the resulting (double) strand breaks. Mis‐repair of accumulated of DNA double strand breaks can result in chromosomal translocations which can persist in survived cells (Mc Clendon et al. 2009).

Studies on identical twins and neonatal blood samples strongly implicate an in utero occurrence of the KER (Sanjuan‐Pla et al., 2015). Furthermore, a study in pregnant mice demonstrates that in utero exposure of the fetus to etoposide causes the MLL chromosomal translocation analogous to the human translocation except the principal fusion partner (Nanya et al., 2015). Indirect evidence from human prehaematopoietic/mesenchymal stem cells and fetal liver haematopoietic progenitor and stem cells strengthen the plausibility. Experimental evidence in these cell lines has demonstrated that etoposide as a TopII poison causes DSBs in MLL and partner genes, which leads to the formation of fusion genes and their products (Sanjuan‐Pla et al., 2015)

MLL translocation sites (breakpoint sequences) in the therapy‐related leukaemia fall within a few base pairs of etoposide‐induced enzyme‐mediated DNA cleavage site. Although rearrangements associated with infant leukaemias are often more complex than those observed in treatment‐related leukaemias, many are nevertheless associated with stable TopII‐mediated DNA cut sites (Cowell and Austin, 2012; Pendleton et al., 2014)

KE1 → AO

In utero MLL chromosomal translocation leads to Infant leukaemia

Strong

Rationale: The basic processes underlying overt leukaemia development are well understood and accepted. There is a general understanding of the molecular and epigenetic mechanisms leading to differentiation blockage and clonal expansion and there is evidence that the principal MLL‐fusion genes and proteins harbour the necessary properties to execute the pathways associated with differentiation blockage and clonal expansion (Benito et al., 2015; Chen and Armstrong, 2015; Chen et al., 2015)