St. Jude Research/Supplemental Data/ALL2/section D

D. COMMON GENE EXPRESSION CHANGES AFTER ALL TREATMENTS

Fig. 6: Commonly regulated genes after all four initial treatments.

Displayed are the median and the inter-quantile range of the 17 genes that exhibited higher expression after treatment (red) or lower expression after treatment (green), in at least 70% of patients, across all four treatments (Wilcoxon’s rank sum test P<0.01).

Table 4 (a) and (b): Changes in gene expression after single agent chemotherapy versus the same agents in combination

(a) Of the 97 gene probe sets that changed by more than 50% in at least 70% of patients after treatment with HDMTX alone, 38 increased and 59 decreased. As shown in Table D (1) below, only 21.1% of genes (8/38) that were up-regulated after HDMTX alone were also up-regulated when HDMTX was given with MP. Further, only 11.9% of genes (7/59) were down-regulated after HDMTX alone and also down-regulated after HDMTX when given with MP. The genes that concordantly changed after HDMTX alone and after HDMTX+MP are listed below. Negative numbers indicate a decrease and positive an increase in expression.

(a) probe set ID	accession number	gene name	median FC HDMTX	median FC HDMTX+MP
36161_at	M34175	adaptor-related protein complex 2, beta 1 subunit	5.7	3.6
37277_at	U80017	baculoviral IAP repeat-containing 1	2.6	2.4
38819_at	U33635	PTK7 protein tyrosine kinase 7	2.5	2.4
36651_at	X15525	acid phosphatase 2, lysosomal	2.5	1.9
40123_at	D87435	golgi-specific brefeldin A resistance factor 1	2.2	2.0
34279_at	AL050141	hypothetical protein FLJ20719	2.0	2.5
32125_at	AA928996	Tho2	1.9	3.0
38464_at	X87237	glucosidase I	1.9	3.2
36432_at	AL079298	methylcrotonoyl-Coenzyme A carboxylase 2 (beta)	-1.8	-2.0
35074_at	AF004715	jerky homolog-like (mouse)	-1.9	-2.5
36246_at	Z35309	adenylate cyclase 8 (brain)	-2.2	-2.8
32413_at	M13934		-2.8	-2.4
32583_at	J04111	v-jun sarcoma virus 17 oncogene homolog (avian)	-3.5	-2.3
725_i_at	J03071		-4.0	-5.5
1915_s_at	V01512		-5.7	-2.4

b) Of the 197 probe sets that changed by more than 50% in at least 70% of patients after treatment with MP alone 40 increased and 157 decreased. As shown in Table 4 (b) below, only 17.5% of genes (7/40) that were up-regulated after MP alone were also up-regulated when MP was given with HDMTX and only 11.4% of genes (18/157) that were down-regulated after MP alone were also down-regulated after MP when given with HDMTX. The genes that concordantly changed after MP alone and after HDMTX+MP are listed below. Negative numbers indicate a decrease and positive an increase in expression.

(b) probe set ID	accession number	gene name	median FC MP	median FC HDMTX+MP
36161_at	M34175	adaptor-related protein complex 2, beta 1 subunit	4.0	3.6
32125_at	AA928996	Tho2	2.5	3.0
35436_at	L06147	golgi autoantigen, golgin subfamily a, 2	2.1	2.5
34836_at	U18420	RAB5C, member RAS oncogene family	2.0	1.9
36822_at	U51334	TAF15 RNA polymerase II, TATA box binding protein	2.0	1.9
37277_at	U80017	baculoviral IAP repeat-containing 1	2.0	2.4
38915_at	AB011135	KIAA0563 gene product	2.0	1.9
31652_at	AB023217	KIAA1000 protein	-1.9	-2.2
41117_s_at	AB016243	solute carrier family 9 (sodium/hydrogen exchanger)	-2.0	-1.7
38146_at	AB011107	zinc finger protein 387	-2.1	-3.5
940_g_at	D12625	neurofibromin 1	-2.1	-1.7
31785_f_at	U92817	unnamed HERV-H protein	-2.3	-2.5
34702_f_at	M27826	chorionic somatomammotropin hormone 2	-2.3	-2.9
41303_r_at	AI378632	Homo sapiens mRNA; cDNA DKFZp564P233	-2.5	-3.6
450_g_at	U66469	cell growth regulatory with ring finger domain	-2.5	-1.9
40590_at	AA166687	cell division cycle 27	-2.8	-7.7
31529_at	X99141	keratin, hair, basic, 3	-3.0	-2.1
39407_at	M22488	bone morphogenetic protein 1	-3.0	-4.3
33047_at	AI971169	ESTs, Highly similar to BCL2-like 11	-3.2	-2.1
34704_r_at	AA151971	chorionic somatomammotropin hormone 2	-3.5	-3.0
40387_at	U80811	endothelial differentiation,G-protein-coupled receptor	-3.5	-3.7
32583_at	J04111	v-jun sarcoma virus 17 oncogene homolog (avian)	-3.7	-2.3
39586_at	AF097935	desmoglein 1	-4.0	-3.0
1915_s_at	V01512		-4.9	-2.4
725_i_at	J03071		-9.2	-5.5

Table 5 (a) and (b): Human leukemia cell lines differ from primary ALL cells.

(a) When the treatment with HDMTX alone (12 nM x 24 h plus 18 h drug-free media) was recapitulated (i.e., comparable level of cytotoxicity), with two human ALL cell lines in vitro (i.e., B-lineage Nalm6 [N.MTX] and T-lineage CEM [C.MTX]), we found very little overlap in the genes that changed after treatment in the cell lines compared to the primary leukemia cells in patients. Specifically, only seven out of the 97 genes (7.2%) that changed by more than 50% in at least 70% of patients after HDMTX also changed on average by more than 50% in the cell lines. Table 5 (a) lists the genes that concordantly change after HDMTX in cell lines and in patients, negative numbers indicate a decrease and positive an increase in expression.

(a) probe set ID	accession number	gene name	median FC N.MTX	median FC C.MTX	median FC HDMTX
32264_at	L23134	granzyme M (lymphocyte met-ase 1)	1.9	1.6	2.9
36591_at	X06956	tubulin, alpha 1 (testis specific)	1.9	1.2	1.9
33143_s_at	U81800	solute carrier family 16 (monocarboxylic acid transporters),	1.5	1.7	3.6
2067_f_at	L22475	BCL2-associated X protein	7.0	1.0	3.7
2001_g_at	U26455	ataxia telangiectasia mutated	2.1	1.6	2.5
35692_at	AL080235	Ras-induced senescence 1	-2.6	-1.0	-2.5
1916_s_at	V01512	c-fos FBJ murine osteosarcoma viral oncogene homolog	-1.4	-1.9	-11.3

(b) When the treatment with MP alone (10 µM x 24 h) was recapitulated with two human ALL cell lines in vitro (i.e., B-lineage Nalm6 [N.MP] and T-lineage CEM [C.MP]), we found very little overlap in the genes that changed after treatment in the cell lines compared to the primary leukemia cells in patients. Only 27 of the 197 genes (13.7%) changed in a consistent manner by more than 50% after MP treatment of cell lines and primary cells in vivo. Table 5 (b) lists the genes that concordantly changed after MP in cell lines and in patients as negative numbers indicate a decrease and positive an increase in expression.

(b) probe set ID	accession number	gene name	median FC N.MP	median FC C.MP	median FC MP
37881_at	AF100907	growth differentiation factor 11	2.6	3.7	2.1
38547_at	Y00796	integrin, alpha L (antigen CD11A (p180), lymphocyte function	4.9	-1.4	1.7
39286_at	D64109	transducer of ERBB2, 2	1.9	3.0	3.0
40329_at	AL031228	ring finger protein 1	1.3	3.2	1.7
41743_i_at	AF061034	tumor necrosis factor alpha-inducible cellular protein	1.7	1.5	2.3
34335_at	AI765533	ephrin-B2	1.6	1.5	1.7
34818_at	X96381	ets variant gene 5 (ets-related molecule)	2.5	1.5	2.0
40951_at	AL049250		1.7	1.7	1.9
292_s_at	L29219	CDC-like kinase 1	1.1	2.5	1.7
31777_at	AF006464	muscle, skeletal, receptor tyrosine kinase	1.2	-5.7	-2.6
33069_f_at	U06641	UDP glycosyltransferase 2 family, polypeptide B15	-2.5	1.1	-1.7
34068_f_at	X86174	synovial sarcoma, X breakpoint 1	-1.1	-2.5	-2.8
35081_at	D14838	fibroblast growth factor 9 (glia-activating factor)	-1.0	-4.9	-2.3
35109_at	AB018299	KIAA0756 protein	-5.3	-1.0	-2.6
37871_at	X68830	islet amyloid polypeptide	-3.0	-0.9	-2.0
40322_at	D12763	interleukin 1 receptor-like 1	-1.1	-4.6	-2.1
40387_at	U80811	endothelial differentiation, lysophosphatidic acid	-4.3	1.4	-3.5
32083_at	AF027826	transmembrane 7 superfamily member 1 (upregulated in kidney)	-1.0	-10.6	-3.2
35178_at	W27944	Wnt inhibitory factor-1	-6.5	1.1	-5.3
39407_at	M22488	bone morphogenetic protein 1	-1.2	-3.7	-3.0
32834_r_at	AF013591	sudD (suppressor of bimD6, Aspergillus nidulans) homolog	-3.7	-1.1	-3.0
39448_r_at	W27095	B7 protein	-1.9	-1.3	-1.6
41244_f_at	X80910	protein phosphatase 1, catalytic subunit, beta isoform	-2.6	-1.7	-2.1
32531_at	X52947	gap junction protein, alpha 1, 43kD (connexin 43)	-1.2	-5.7	-3.2
32583_at	J04111	v-jun sarcoma virus 17 oncogene homolog (avian)	2.0	-16.0	-3.7
1152_i_at	J00117	chorionic gonadotropin, beta polypeptide	-2.8	-2.3	-6.1
618_at	M26167	platelet factor 4 variant 1	-2.1	-4.3	-4.0