Escalas de detección: Son escalas que contribuyen a facilitar el

The fold change calculation used was applied to the two probe set groups previously identified as “present in at least 1 sample” and

“present and changing” through filtering on flags (Chapter 3, Section 3.3.1a) to remove any inconsistent data. Filtering was applied to both groups of probe sets to assess the robustness and reliability of the analysis.

The group of “present in at least 1 sample” probe sets were filtered on a 2-, 3-, 4- and 5-fold change in expression level between the t(8;21)

patients and non-t(8;2 1) patients and between the t(8;2 1) patients and

the normal healthy donor patients (Table 1). By using a £3 fold change as the threshold for significance, the group of 94 probe sets identified as having a greater expression in the t(8;2 1) patients than in the non-

t(8;21) patients (probe set list 4.1) was combined with the group of 272 probes sets identified as having a greater expression in the t(8;2 1)

patients than in the normal healthy donors (probe set list 4.2) to generate a group of 60 probe sets with a greater level of expression in t(8;21) AML patients when compared to other AML patient sub-groups or normal healthy donors (Probe set list 4.3) (Figure 1; Table 2).

The “present and changing” probe sets were filtered on a 2-, 3-, 4- and 5-fold change in expression level between the t(8;21) patients and non- t(8;2 1) patients and between the t(8;2 1) patients and the normal healthy

donors (Table 1). By using a £3 fold change as the threshold for significance the group of 94 probe sets identified as having greater expression in the t(8;2 1) patients than in the non-t(8;2 1) patients (probe

set list 4.4) was combined with the group of 272 probe sets identified as having a greater expression in t(8;2 1) patients than in the normal

healthy donors (probe set list 4.5) to generate a group of 60 probe sets with a greater level of expression in t(8;21) AML patients when compared to other AML patient sub-groups or normal healthy donor (Probe set list 4.6) (Figure 1; Table 2).

The group of 60 probe sets identified from the “present in at least 1 sample” probe sets (Probe set list 4.3) and the group of 60 probe sets identified from the “present and changing" probe sets (Probe set list 4.6) were identical confirming the robustness of the filtering analysis and the unlikelihood that the probe sets identified as specific to t(8;21) AML were false-positives.

Probe set group Fold

Change t(8;21)>non-t(8;21) t(8;21)>Donors

Present in at least 1 sample 2 318 930

3 94 272

4 31 95

5 23 64

Present and changing 2 313 930

3 94 272

4 31 75

5 23 23

Table 1. The number of probe sets identified as having a greater expression in t(8;21) AML patients than in non-t(8;21) patients or normal healthy donors. Fold change filtering was applied to both sets of probe sets generated for quality control purposes as described in section 3.3.1a (Chapter 3).

The 60 probe sets represented 46 genes. Furthermore amongst the 16 probe sets with the highest fold changes (greater than 8 fold) 7 probe

sets identified the tryptase alpha 1 and beta 1 genes (TPSAB1) and

Probe Set _SymbolGene Gene Title Fold Change

31 203913_s_at HPGD Tydroxyprostaglandin dehydrogenase 15-(NAD) 4.220 32 203065_s_at CAV1 Caveolin 1, caveolae protein, 22kDa 4.439 33 _{204468_s_at} TIE1 Tyrosine kinase with immunoglobulin-like and EGF-like _{domains 1} 4.551

34 219686_at STK32B Serine/threonine kinase 32B 4.716

35 204885_s_at MSLN Mesothelin 5.182

36 209170_s_at GPM6B Glycoprotein M6B 5.236

37 218876_at TPPP3 Tubulin polymerization-promoting protein family member 3 5.312

38 208534_s_at RASA4 RAS p21 protein activator 4 5.353

39 209010_s_at TRIO Triple functional domain (PTPRF interacting) 5.453

40 209167_at GPM6B Glycoprotein M6B 6.328

41 206726_at PGDS Prostaglandin D2 synthase, hematopoietic 6.844

42 206940_s_at POU4F1 POU class 4 homeobox 1 7.522

43 212097_at CAV1 Caveolin 1, caveolae protein, 22kDa 8.016

44 _{213194_at} ROBOI Roundabout, axon guidance receptor, homolog 1 _(Drosophila 8.209

45 207741_x_at TBSAB1 Tryptase alpha/beta 1 8.355

46 204086_at PRAME Preferentially expressed antigen in melanoma 8.990

47 207134_x_at TPSB2 Tryptase beta 2 9.150

48 201655_s_at HSPG2 Heparan sulfate proteoglycan 2 9.649

49 205683_x_at TPSAB1 Tryptase alpha/beta 1 10.01

50 216474_x_at TPSAB1 Tryptase alpha/beta 1 12.06

51 216831_s_at RUNX1T1* Runt-related transcription factor 1 13.09 52 205529_s_at RUNX1T1* Runt-related transcription factor 1 13.25

53 215382 x at TPSAB1 Tryptase alpha/beta 1 13.61

54 217023_x_at TPSB2 Tryptase alpha/beta 1 14.81

55 210744_s_at IL5RA Interleukin 5 receptor, alpha 16.56

56 205528_s_at RUNX1T1* Runt-related transcription factor 1 18.12

57 210084_x_at TPSAB1 Tryptase alpha/beta 1 21.71

58 211517_s_at IL5RA Interleukin 5 receptor, alpha 25.35

59 211341_at POU4F1 POU class 4 homeobox 1 39.08

60 206622_at TRH Thyrotropin-releasing hormone 39.27

Table 2. The top 30 “present in at least 1 sample” and “present and changing "probe sets identified from the 305 sample dataset as upregulated in t(8;21) AML patients samples when compared to non-t(8;21) patients and to healthy donor samples by fold change (FC>3). The fold change value displayed is the mean fold change value of the probe sets across the t(8;21) samples. The numbers assigned to the probe sets correspond to their location in figure 1. For a complete list of the 60 probe sets see appendix \\.*RUNX1T1 is also known as £70, the gene previously mentioned as involved in the t(8;21) (Section 4.2.1a).

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Donor Samples t(8;21) samples non-t(8;21) samples

Figure 1. The relative mean expression levels of the 60 probe sets identified, from probe sets that were classified as present in at least 1 sample and as present and changing, as being more significantly expressed in t(8;21) samples than non-t(8;21) samples and in donor samples by £3 fold. The probe sets are coloured according to their mean expression levels in their respective sub-group relative to their mean expression level in the donor samples. The numbers on each block correspond to the probe set it is representing - see table 2 (only top 20 probe sets numbered).

4.3.1b Identification of genes differentially expressed in t(8;21)