Un cuento mexicano: experiencia con los RDP

The cohort included in the first part of the study were those treated using ALL97/99 protocol, while the second part, which investigated the third aim, involved patients who were registered in both trials ALL97/99 and UKALL2003 (see section 2.2). A total of 49 patients with suitable material were included in this Chapter. Most of the data included in this Chapter was recently published (Al-Shehhi et al., 2013).

4.3.2 Methods

4.3.2.1 FISH

All FISH studies were performed as described in section 2.8. Different probe sets were applied to accomplish the following objectives:

A. Mapping der(12)t(12;21) deletion

Deletion mapping of der(12)t(12;21) was performed using two different probe sets, depending on the region of the deletion and comprised differentially labelled target and control probes (Appendix C).

Using the ETV6 BA probe, a signal pattern of 1R 0G 1F was suggestive of a deletion of 3’ETV6 sequences on der(12)t(12;21) (Figure 4.1 E.). Therefore, these cases were screened using a series of target probes mapping along 12p (12p11.21- 12p13.2). For each FISH test, the 5’RUNX1 probe (21q22.12) was used as the control probe (labelled

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red) combined with a target probe (labelled green). In a cell with a standard t(12;21) translocation, one would observe a signal pattern of 1R 1G 1F indicating a normal chromosome 21, normal chromosome 12 and intact der(12)t(12;21), respectively (Figure 4.2 B). Thus in each test, a signal patterns of 2R 1G 0F or 2R 0G 0F would suggest the 3’ETV6 sequence deletions at der(12)t(12;21) with or without intact non- rearranged ETV6, respectively (Figure 4.2 C).

On the other hand, those with suggestive deleted 5’RUNX1 sequences (21q sequences) of der(12)t(12;21) indicated by the unexpected signal pattern of 0R 1G 1F using RUNX1 BA probe (Figure 4.1 F.), target probes mapping along 21q from 21q22.12-21q22.3 were serially applied. For each FISH test, 3’ETV6 probe (12p13.2) was used as the control probe (labelled green) combined with a target probe (labelled red). In a cell with a standard t(12;21) translocation, a signal pattern of 1R 1G 1F is expected indicating a normal chromosome 21, normal chromosome 12 and intact der(12)t(12;21), respectively (Figure 4.2 B). Thus in case of cells with deleted 5’RUNX1 sequences at der(12)t(12;21) with associated intact or deleted non-rearranged ETV6, one would observe a signal pattern of 1R 2G 0F or 1R 1G 0F, respectively (Figure 4.2 D).

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Figure 4.2 Ideograms show the signal patterns generated from the mapping deletion probes targeting either 3’ETV6 or 5’RUNX1 sequences of der(12)t(12;21): in a normal [2R 2G 0F] (A), a

standard t(12;21) [1R 1G 1F] (B), a deleted 3’ETV6 region [2R 1G 0F] (C) and a deleted 5’RUNX1 region of der(12)t(12;21) cells [1R 2G 0F] (D).

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B. Confirmation of the origin of deleted 12p sequences

In order to confirm the origin of deleted 12p sequences, three colour FISH was carried out using home-grown probes which comprised of the control probe (5’RUNX1 probe) (labelled red), a known intact 3’ETV6 probe (labelled green) and a known deleted 3’ETV6 target probe (labelled gold). In a standard translocation, a signal pattern of 1R 1F (green- gold) 1F (green-red-gold) would be observed representing the normal chromosome 21, normal chromosome 12 and intact der(12)t(12;21), respectively (Figure 4.3 B). Thus, a signal pattern of 1R 1F (green-gold) 1F (red-green) would indicate the presence of a normal chromosome 21, normal chromosome 12 and deletion of the gold probe from the der(12)t(12;21), respectively (Figure 4.3 C).

Figure 4.3 Ideograms of the home grown probes used in the three colour FISH: A. a normal

signal pattern [2R 0G 2F (green-gold)], B. a standard t(12;21) with signal pattern of [1R 1F (green- gold) 1F (green-gold-red)] and C. deleted 3’ETV6 sequences (gold probe) from der(12)t(12;21) indicated by the signal pattern of [1R 1F (green-gold) 1F (green-red)].

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C. Investigations of a possible duplicated der(12)t(12;21)

In order to investigate those patients with the unexpected signal patterns of 1R 2G 0F and 1G 2R 1F, using ETV6 and RUNX1 BA probes, respectively, (Figure 4.1 G-H), suggestive of duplicated RUNX1-ETV6 fusion gene on der(12)t(12;21), a home grown dual colour single fusion (DC SF) probe was applied. The DC SF probe was comprised of the 5’RUNX1 probe (labelled red) and 3’ETV6 probe (labelled green). In a cell with a standard t(12;21) translocation, a signal pattern of 1R 1G 1F would be observed indicating a normal chromosome 21, a normal chromosome 12 and an intact der(12)t(12;21), respectively (Figure 4.2 B). Thus, in each FISH test, a signal pattern of 1R 0G 2F, showing an extra fusion signal with associated loss of the green signal was sought (Figure 4.4).

Figure 4.4 The expected signal pattern of the suggested duplicated RUNX1-ETV6 fusion gene using 5’RUNX1 (labelled red)/3’ETV6 (labelled green) DS SF probe set. A signal pattern of 1R

0G 2F is shown.

To confirm the number of chromosomes 12 and to assess the genetic stability by obtaining the relative number of chromosome 12 centromeres and 12p subtelomeres in those cases with duplicated RUNX1-ETV6 fusion gene, FISH using a commercial probe set was carried out. The probe set consisted of centromeric 12 (CEP 12) probe (labelled red, Cytocell) and subtelomeric 12p (subtel 12p) probe (labelled green, Vysis). This probe

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set would give a signal pattern of 2R 2G 0F in either normal or a standard t(12;21) cells. Thus, the number of the centromeres and the subtelomeres was investigated.

D. Investigation of cases with isolated ETV6 exon 8 deletions

In order to identify further cases with deleted 3’ETV6 sequences of der(12)t(12;21), cases with isolated deletions of ETV6 exon 8 (3’ETV6 region) by MLPA (see section 4.3.2.2), whether mono or biallelic losses, were further investigated by FISH studies. Firstly, application of ETV6 and RUNX1 DC BA probes was carried out to uncover the possible abnormalities responsible for the isolated ETV6 exon 8 losses (e.g. deletions of either the non-rearranged ETV6 or 3’ETV6 sequences of der(12)t(2;21) (Figure 4.1). If the observed signal patterns suggested that both ETV6 alleles were intact using ETV6 DC BA probe, a home grown fosmid probe targeting ETV6 exon 8 (labelled green) was used as a target probe to identify smaller deletions. This fosmid probe was combined with the 5’ETV6 probe (labelled red) (Figure 4.5), thus, this probe set was considered similar to ETV6 DC BA, but it was able to identify smaller deletions undetectable by the ordinary ETV6 DC BA probe.

In order to define the deletion sizes in those new cases with 3’ETV6 sequences of der(12)t(12;21), FISH mapping was done (see section 4.3.2.1 A. ).

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Figure 4.5 The locations of ETV6 exon 8 [3’ ETV6] probes with either 5’ETV6 or 5’RUNX1 probes.

A. ETV6 gene at 12p13.2, composed of 8 exons in which the t(12;21) breakpoint occurred within intron 5; Locations of the green and red probes used in the ETV6 DC BA probe (RP11-525I3 or

ETV6 exon 8 - RP11-434C1), B. The breakpoint at der(12)t(12;21); locations of the green and red

probes used in the 3’ETV6-5’RUNX1 SF DC probe (RP11-525I3 or ETV6 exon 8 - RP11-396G11).

E. Investigations of cases with isolated ETV6 exons 1-5 deletions

To identify further cases with duplicated RUNX1-ETV6 fusion gene, cases with MLPA ETV6 profile of isolated exons 1-5 losses were further investigated by FISH using both BA probes targeting ETV6 and RUNX1 loci (Figure 4.1). In case of suspected duplication, further application of 5’RUNX1/3’ETV6 DC SF probe was carried out (see section 4.3.2.1. C. ).

4.3.2.2 MLPA

Genomic DNA from nine diagnostic bone marrow samples was extracted using standard procedures. In one case, the DNA was extracted from the same fixed cell pellet as used for FISH (see section 2.6). DNA was analysed using the SALSA MLPA kit P335 (MRC Holland, Amsterdam, The Netherlands). Data was analysed using GeneMarker V1.85 analysis software (SoftGenetics) (see section 2.9).

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4.3.2.3 SNP6

Five DNA samples were hybridized to Affymetrix Genome-Wide Human SNP Array 6.0 at AROS Applied Biotechnology (Aarhus, Denmark) according to the manufacturer’s instructions (Affymetrix) (see section 2.10).

4.4 Results