Gene expression profiling in ALL

U95Av2 microarray data on four subtypes of adult ALL

Firstly, U95Av2 gene expression profiles were analyzed to accurately identify the known prognostically important adult leukemia subtypes, i.e., B-lineage leukemias that contain t(9;22), t(11q23)/MLL (all t(4;11)-positive), or t(8;14), as

well as T-lineage leukemias, respectively. In total, ten comparisons within the four groups were performed (pairwise and OVA). Using leave-one-out cross- validation, all samples were correctly classified on the basis of 17 genes represented by 19 probe sets. The corresponding cluster dendrogram is given as Figure 26.

Figure 26. Analysis of adult ALL samples using U95Av2 microarrays. Hierarchical clustering based on U95Av2 expression data of 17 ALL samples (columns) from the subgroups t(11q23)/MLL

(n=4), t(9;22) (n=7), t(8;14) (n=3), and precursor T-ALL (n=3) vs. a minimal set of 17 informative genes represented by 19 probe sets (rows). The normalized expression value for each gene is coded by color. Red cells represent high expression and green cells represent low expression.

To further demonstrate that the presented genes were characteristic for the respective leukemia subtype, also the analyses of ALL subtypes were extended to the U133A microarray. Thus, all previously used 17 hybridization cocktails and two additional samples for each subgroup, respectively, were rehybridized to the U133A microarray. Again, a stringent search determined for the presented U95Av2 probe sets their best corresponding U133A counterparts. As shown in Figure 27, now based on U133A microarray expression data and including two new samples of each of the distinct subgroups, all adult ALL cases were repeatedly separated according to their underlying chromosomal aberration or immunophenotype. Taken together, this section successfully demonstrates a subclassification of ALL samples based on gene expression profiling. It could be shown that only a small set of differentially expressed genes was necessary to correctly discriminate the different ALL subtypes. New differentially expressed genes were identified and defined as potential diagnostic markers. 60 70 80 90 100 t(8;14) t(11q23) BCR/ABL T-ALL HELO1 TRB CD3D ANXA5 SIT LYN CCL3 VLDLR FLJ12443 CTNNA1 CTNNA1 CTGF NR3C1 ADA MYB MYB MME MONDOA KIAA0870

Figure 27. Analysis of adult ALL samples using U133A microarrays. Hierarchical clustering based on U133A expression data of 25 ALL

samples (columns) comprising the subgroups t(11q23)/MLL (n=6),

t(9;22) (n=9), t(8;14) (n=5), and precursor T- ALL (n=5) vs. 17 informative genes (rows). New patient samples which were not previously hybridized to U95Av2 arrays are accordingly marked (asterisks). The normalized expression value is coded by color.

Analysis of heterogeneous precursor B-ALL cases

After obtaining these results that specific signatures are also observed in adult ALL subtypes the analyses were extended to expression profiles of heterogeneous precursor B-ALL cases not positive for t(9;22) or t(11q23)/MLL.

Therefore, in addition 7 precursor B-ALL patients (c-ALL and Pre-B-ALL) were hybridized to obtain new insights into the molecular features of these cases. This additional cohort included patients who showed a normal karyotype (n=2) or a variety of different karyotype abnormalities (n=5).

Firstly, an unsupervised analysis, i.e., hierarchical clustering and PCA of the complete data set was performed. However, this analysis did not reveal informative structures. Therefore, a supervised analysis by use of the SAM software was performed to identify differentially expressed genes correlated with T-ALL, BCR/ABL, and t(11q23)/MLL cases. A selection of the top 510

genes accurately separated the latter three ALL subtypes (Figure 28A). Next, the 7 precursor B-ALL samples without BCR/ABL or t(11q23)/MLL

chromosomal aberrations were added to the data set and all cases were projected into the space of the 510 ALL subtype relevant genes. As shown in Figure 28B, the other precursor B-ALL samples (yellow spheres) intercalate with BCR/ABL-positive samples (red spheres).

75 80 85 90 95 100 MME MONDOA KIAA0870 CTNNA1 CTGF FLJ12443 VLDLR ADA MYB NR3C1 HELO1 CD3D ANXA5 SIT TRB LYN CCL3 t(8;14) T-ALL t(11q23) BCR/ABL * * * * * * * *

T-ALL _t(11q23)/MLL BCR/ABL T-ALL _t(11q23)/MLL BCR/ABL heterogeneous precursor B-ALL

A

B

Figure 28. PCA including heterogeneous precursor B-ALL cases. The PCA is based on 510 differentially expressed genes that were identified by use of the SAM software to separate T- ALL (n=9), t(11q23)/MLL (n=10), and BCR/ABL (n=15) cases (U133A). (A) T-ALL, t(11q23)/MLL

and BCR/ABL samples can accurately be separated using the three components capturing

most of the variance in the data set. (B) When added to the data set, profiles from 7 heterogeneous precursor B-lineage ALL samples (yellow spheres) intercalate with BCR/ABL-

positive samples (red spheres).

Thus, the other precursor B-ALL share similar characteristics with BCR/ABL-

positive ALLs. This is in line with the definition and subclassification of precursor B-ALL according to EGIL, which is based on the immunophenotype and distinguishes Pro-B-ALL, common ALL, and Pre-B-ALL (Bene et al., 1995). Most importantly, both other ALL cases and BCR/ABL cases belong to the

common ALL and Pre-B-ALL groups and are thus anticipated to have common gene expression profiles. This finding can also be visualized by use of the hierarchical clustering technique.

As shown in Figure 29, due to inherent similarities in their expression profiles three major branches of the top dendrogram can be observed. The t(11q23)/MLL and T-ALL samples are accurately grouped. The more

heterogeneous precursor B-ALL cases are exclusively distributed in the branch containing all BCR/ABL patient samples. Several subtrees in the left

dendrogram indicate co-expression of genes for the distinct ALL subtypes.

Figure 29. Hierarchical clustering including hetero- geneous precursor B-ALL cases. Hierarchical cluster analysis of T-ALL (n=9), t(11q23)/MLL (n=10), BCR/ABL (n=15), and heterogeneous precursor B- ALL (n=7) patients. This analysis is based on 510 differentially expressed genes (rows) which have been identified in a supervised approach to separate the three entities BCR/ABL, T-

ALL, and t(11q23)/MLL,

(U133A). When analyzed with this set of genes, profiles from 7 heterogeneous precursor B- ALL samples intercalate with

BCR/ABL-positive samples.

The normalized expression value for each gene is coded by color. Red cells indicate high expression and green cells indicate low expression. Five subtrees with interesting differentially expressed genes are marked on the right.

Subtree 1 contains genes overexpressed in T-ALL: TRB, CD3D, CD3E, CD2, CD6, MAL, LCK, ITM2A, and SH2D1A. A large number of these genes and

additional candidates like transmembrane adapters (LAT, TRIM), further CD3

complex signal transducing members (CD3G, CD3Z), CD8A coreceptor, and ZAP70 tyrosine kinase can be correlated with a functional role in the class I

MHC-restricted T cell receptor signalosome (Leo et al., 2002). Subtrees 2 and 4 group genes with high expression in MLL gene rearranged ALL cases: ADAM10, BLK, CD72, CD79A, CSPG4, HOXA9, HOXA10, IGHM, LGALS1, LMO2, MBNL, MEF2A, PPP2R5C, PTPRC, and VLDLR. Subtree 3 contains

96 97 98 99 100

heterogeneous precursor B-ALL

BCR/ABL T-ALL t(11q23)/MLL 1 4 5 2 3

mainly genes with functional role in immune response. BLNK, BRDG1, CD24, MHC2TA, CD74, HLA-DMA, HLA-DMB, HLA-DPA1, HLA-DRA, HLA-DPB1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4, and TNFRSF14 demonstrate

similar patterns for BCR/ABL, t(11q23)/MLL and the more heterogeneous

precursor B-ALL cases.

An interesting cluster of genes is organized in subtree 5, which is enlarged in Figure 30. Twenty-six probe sets demonstrate similar expression signatures for both BCR/ABL-positive and the more heterogeneous precursor

B-ALL cases. All candidate genes are consistently overexpressed in these cases compared to T-ALL and t(11q23)/MLL samples. The probe sets represent

for example LGMN, also called asparaginyl endopeptidase (AEP), a receptor

tyrosine kinase activated by collagen (DDR1), CD52, an excellent target for

complement-mediated lysis and antibody-dependent cellular cytotoxicity, a cytokine-like protein (C17), a retinoic acid induced gene (RAI14), or the

hypothetical protein LOC54103.

98 98. 5 99 99 .5 10 0 98 98. 5 99 99 .5 10 0

heterogeneous precursor B-ALL

BCR/ABL T-ALL t(11q23)/MLL ABL1 DDEF1 TRIO KIAA0582 HYA22 C17 RAI14 HYA22 HYA22 CDW52 CDW52 LOC54103 LOC54103 APP EST LGMN DDR1 DDR1 D2S448 EST P164RHOGEF MYO1B TNFRSF14 SCHIP1 FXYD5 EMP2 KIAA0870 TCF4 EST EST EST EST LARGE ALOX5 DKFZP564A2416 HSA250839

Figure 30. Zoomed image of subtree 5 out of Figure 29. Heterogeneous precursor B-ALL share similar expression patterns with BCR/ABL-positive ALLs. In this subtree, 26 probe sets

were consistently overexpressed compared to both T-ALL and t(11q23)/MLL. The normalized

expression value for each gene is coded by color. Red cells indicate high expression and green cells indicate low expression.

A similar distribution of the adult ALL samples can be observed when these cases were projected into the gene expression space of markers previously reported from Yeoh et al. to discriminate six distinct pediatric ALL subtypes, i.e., T-ALL, E2A/PBX1, BCR/ABL, TEL/AML1, t(11q23)/MLL, and hyperdiploid

leukemias (Yeoh et al., 2002). As anticipated, genetically heterogeneous precursor B-ALL samples again cluster together with BCR/ABL cases

confirming the previous observation. They do not show up as an independent fourth distinct cluster separated from adult T-ALL, t(11q23)/MLL, and BCR/ABL-

positive leukemias. Figure 31 visualizes the observed dendrogram structure of a hierarchical cluster analysis as well as a three-dimensional plot from a PCA. Thus, signatures, previously reported to correlate with E2A/PBX1, TEL/AML1,

and hyperdiploid childhood leukemias could not separate these two groups. This is not unexpected as none of the heterogeneous precursor B-ALL showed one of these genetic characteristics.

t(11q23)/MLL t(11q23)/MLL t(11q23)/MLL t(11q23)/MLL t(11q23)/MLL t(11q23)/MLL t(11q23)/MLL t(11q23)/MLL t(11q23)/MLL t(11q23)/MLLL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL BCR/ABL BCR/ABL precursor B-ALL BCR/ABL BCR/ABL BCR/ABL BCR/ABL BCR/ABL precursor B-ALL BCR/ABL BCR/ABL precursor B-ALL precursor B-ALL precursor B-ALL BCR/ABL BCR/ABL BCR/ABL BCR/ABL precursor B-ALL precursor B-ALL BCR/ABL BCR/ABL BCR/ABL heterogeneous precursor B-ALL T-ALL t(11q23)/MLL

A

B

Figure 31. Heterogeneous precursor B-ALL in the context of pediatric markers. The analyses are based on reported differentially expressed genes to distinguish pediatric T-ALL,

E2A/PBX1, BCR/ABL, TEL/AML1, t(11q23)/MLL and hyperdiploid leukemias (Yeoh et al.,

2002). (A) Hierarchical clustering and (B) PCA of adult T-ALL (n=9), t(11q23)/MLL (n=10), BCR/ABL (n=15), and heterogeneous precursor B-lineage ALL (n=7) patients (U133A). Each

patient sample is represented by a color-coded sphere. Heterogeneous precursor B-lineage ALL samples (yellow spheres) cluster together with BCR/ABL-positive samples (red spheres)