Supplemental material to
1.
Gene expression shift towards normal B cells, decreased proliferative capacity and distinct surface receptors characterize leukemic blasts persisting during induction therapy in childhood acute lymphoblastic leukemia.
Peter Rhein1, Stefanie Scheid2, Richard Ratei1, Christian Hagemeier3, Karl Seeger4, Renate Kirschner-Schwabe4, Anja Moericke5, Martin Schrappe5, Rainer Spang2, Wolf-Dieter Ludwig1 and Leonid Karawajew1
1 Department of Hematology, Oncology, and Tumor Immunology, Robert Rossle Clinic at the HELIOS Klinikum Berlin-Buch, Charité Medical School, Berlin, Germany
2 Department of Computational Molecular Biology, Max Planck Institute for Molecular Genetics; Berlin Center for Genome Based Bioinformatics, Berlin, Germany
3 Department of General Pediatrics, Charité Medical School, Berlin, Germany
4 Department of Pediatric Oncology and Hematology, Charité Medical School, Berlin, Germany
5 Department of Pediatrics, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
1
Contents
Cell sorting accuracy, isolated RNA quality and RT-PCR control measurements
Data preprocessing and statistical analysis
Genome-wide gene expression in matched BM and PB ALL samples
References
Table S1 Patient series analyzed by flow cytometry.
Table S2 List of monoclonal antibodies used for flow cytometric analysis.
Table S3 The set of genes differentially expressed at d8 and d0.
1
Cell sorting accuracy, isolated RNA quality and RT-PCR control measurements
Mean purity of isolated samples assessed by re-analysis of the sorted cells was 98% +/- 0.3 and 95% +/- 0.9 for the d0 and d8 samples, respectively. Normal B cells were isolated with a mean purity of 93% +/- 0.9 (Figure S1A). To further ensure that leukemic samples were free of contaminations with normal B cells, we analysed the microarray data for expression levels of the genes encoding for immunoglobulin (Ig) lambda and Ig kappa light chains. In normal B cells, high expression levels of both, Ig lambda and kappa chains, were observed in accordance with the polyclonal nature of B-cell samples. By contrast, light chains expression in leukemic samples was by more than 10-fold lower. Moreover, it did not significantly differ between d0 and d8 samples (Figure S1B).
Recent technical reports demonstrated that flow sorting does not impair RNA quality, hybridization efficiency, and gene expression of purified small cell subpopulations1,2. In our setting, the integrity of total RNA was assessed by 28S and 18S ratios in 17 samples (2.4 +/- 0.1) (Figure S1C). The values of the GAPDH signal ratio for 3´/M (2.1 +/- 0.1) and 3´/5´ (6.3 +/- 0.4) indicated good quality of microarray hybridizations, given two rounds of RNA amplification.
Given limitations of primary material at d8 (Figure 1), three representative genes were chosen to verify the microarray data by real-time RT-PCR. One of them (TXNIP) showed increased expression, another (TYMS) decreased and a third (UBB) unchanged expression. mRNA levels of these genes, which could be recorded in 17 patient samples, revealed a good correlation between RT-PCR and microarray based measurements (Figure S1D).
Data preprocessing and statistical analysis
Normalization and microarray data quality
The computational preprocessing and statistical analysis was done using the statistical software R which is freely available from and add-on packages distributed by the Bioconductor project which are freely available from The analysis is based on Bioconductor release 1.8. In total, 46 Affymetrix GeneChip HGU133A arrays were submitted to the preprocessing protocol. Starting from the raw scanner data, the intensity data was normalized on probe level by applying the variance-stabilizing method of Huber et al.5 implemented in package vsn. Next, the perfect match probes within a probe set were summarized by the median polish method of Irizarry et al.6, using package affyPLM. Mismatch probes were not taken into account in the summarization step. After these consecutive preprocessing steps, gene expression values were returned on additive scale.
Data quality
The summarization method implemented in package affyPLM fits a linear model to the data. The model includes array effects and probe set effects. Figure S2 displays boxplots of the resulting model residuals per array. The B-cell samples are marked with "B". The consistent behavior of the boxplots indicates good data quality.
Filtering
After preprocessing and exclusion of the 10 B-cell samples, the data set consisted of 22283 probe sets measured for 18 patients on day 0 and day 8 of prednisone treatment. A filtering step was applied to remove non-informative probe sets, that is Affymetrix control probe sets and probe sets connected to hemoglobin were excluded from further analysis. The number of remaining probe sets was 22194.
Differential expression between d0 and d8 blast samples
The 36 paired leukemic samples from day 0 and day 8 were evaluated in a permutation based test on average differences in gene expression. The test procedure is implemented in the Bioconductor package twilight and works as follows: For each patient and each gene the difference between day 8 and day 0 expression was computed. The differences averaged over the patients are called log ratio scores. To assess the significance of the observed log ratio scores, the average differences for 100 random permutations of the given class labels were computed. Here, "permutation" refers to random flipping of day0/day8 labels within a pair of samples. The average differences of all permutations including the observed scores were joint into one set. From this set, the observed log ratio scores were transformed into empirical p-values by computing the percentage of absolute permutation scores exceeding the absolute observed score. To correct for multiple testing, the set of p-values was transformed to q-value7. For a list of genes with q-values up to 0.05, the estimated false discovery rate (FDR) is just 0.05. For further reading, we refer to Scheid and Spang8 and the technical report9.
Principal Component Analysis
For the majority of genes with significant average differences between day 0 and day 8 we expected that the averages on day 0 and day 8 either increase or decrease towards the average of the corresponding gene expression in B-cells. To examine this hypothesis, Principal Component Analysis (PCA) was applied first. Via singular value decomposition, PCA transforms a set of correlated variables into a set of uncorrelated variables while preserving as much of the data variance as possible. Thus, the gene expression of the d0, d8 and B-cell samples was mapped from the space spanned by the set of significant genes to a lower-dimensional space. After PCA transformation, the 523-dimensional data space of the set of significant probes is reduced to 46 dimensions. These new dimensions preserve decreasing amounts of the original data variance. Based on the data mapped onto the new PCA dimensions, equality of sample averages was tested by applying the Jonckheere-Terpstra (JT) test10,11. The JT test is a non-parametric test against the alternative hypothesis that the sample averages increase or decrease monotonically. The test works as follows: for each pair of conditions d0/d8, d0/B and d8/B, the Mann-Whitney U-statistic is computed. The sum over these three scores is the JT score.
Genome-wide gene expression in matched BM and PB ALL samples
Differences between bone marrow (BM) and peripheral blood (PB) samples were investigated in an independent series of 20 matched samples collected at diagnosis from ten PBC-ALL patients. Blast cells were extracted from BM and PB samples by flow-cytometric isolation. We refer to the original paper for details on cell sorting and hybridization protocol. The independent data set was then preprocessed and analysed in the same way as the main data set. The significance of the observed log ratio scores was assessed on the set of all possible 512 permutations of the 10 pairs of BM/PB-sample labels. Among the 22194 probe sets, only a single gene (RRM2) was found to be significantly differentially expressed between BM and PB samples (FDR < 0.05). Hence we conclude that differences between PB and BM samples are negligible.
References
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2. Szaniszlo P, Wang N, Sinha M, Reece LM, Van Hook JW, Luxon BA, et al. Getting the right cells to the array: Gene expression microarray analysis of cell mixtures and sorted cells. Cytometry. 2004;59a:191-202
3. R_Development_Core_Team. R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing. Vienna, Austria, ISBN 3-900051-07-0. 2005
4. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004;5:R80
5. Huber W, von Heydebreck A, Sultmann H, Poustka A, Vingron M. Variance stabilization applied to microarray data calibration and to the quantification of differential expression. Bioinformatics. 2002;18 Suppl 1:S96-104
6. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31:e15
7. Storey JD. The positive false discovery rate: A Bayesian interpretation and the q-value. Annals of Statistics. 2003;31:2013-2035
8. Scheid S, Spang R. twilight; a Bioconductor package for estimating the local false discovery rate. Bioinformatics. 2005;21:2921-2922
9. Scheid S, Spang R. Estimation of local false discovery Rate - User's guide to the Bioconductor package twilight. CompDiag Technical Report Nr. 2004/01. Nov. 2004
10. Odeh RE. On Jonckheere's k-sample test against ordered alternatives. Technometrics. 1971;13:912-918
11. Skillings JH. On the null distribution of Jonckheere's statistic used in two-way models for ordered alternatives. Technometrics. 1980;22:431-436
Table S1 Patient series analyzed by flow cytometry.
N / Median age,in years
(range) / Sex / Diagnosis1 / Median WBC2
109/l
(range) / DNA ploidy / Molecular
translocation / Prednisone
response
CD10,
CD20,
CD34 / 109 / 3.6
(1.2-17.4) / male: 62
female: 47 / pro: 3
cALL: 87
pre: 14
other: 5 / 13.5
(1.0-196.9) / hyperdiploid: 24
diploid: 72
hypo: 1
NA: 12 / MLL: 1
TEL-AML: 28
BCR-ABL: 2
ND: 68
NA: 10 / good: 105
poor: 4
CD45 / 42 / 3.3
(1.6-17.4) / male: 27
female: 15 / pro: 1
cALL: 36
pre: 3
other: 2 / 9.9
(2.0-136.0) / hyperdiploid: 11
diploid: 27
NA: 4 / MLL: 0
TEL-AML: 12
BCR-ABL: 0
ND: 27
NA: 3 / good: 39
poor: 3
TdT / 21 / 3.0
(1.7-6.6) / male: 8
female: 13 / pro: 0
cALL: 17
pre: 3
other: 1 / 10.7
(1.2-73.4) / hyperdiploid: 5
diploid: 12
NA: 4 / MLL: 0
TEL-AML: 5
BCR-ABL: 0
ND: 12
NA: 4 / good: 21
poor: 0
CD11b,
IFNGR1 / 29 / 4.4
(1.2-16.6) / male: 17
female: 12 / pro: 0
cALL: 25
pre: 3
other: 1 / 13.3
(1.9-232.5) / hyperdiploid: 10
diploid: 18
NA: 1 / MLL: 0
TEL-AML: 11
BCR-ABL: 3
ND: 12
NA: 3 / good: 28
poor: 1
Cell cycle / 13 / 3.6
(1.5-7.3) / male: 8
female: 5 / pro: 1
cALL: 11
pre: 0
other: 1 / 18.3
(5.7-104.0) / hyperdiploid: 6
diploid: 6
NA: 1 / MLL: 0
TEL-AML: 2
BCR-ABL: 0
ND: 10
NA: 1 / good: 12
poor: 1
1 Diagnosis: cALL = common ALL, pre-B = pre-B ALL, pro-B = pro-B ALL, other = non-specified or bi-phenotypic
2 WBC = white blood cell count at diagnosis
NA = not available
ND = none detected
Table S2 List of monoclonal antibodies used for flow cytometric analysis.
Antibodies against / Clone / Fluorochrome1 / Source2CD10 / SS2/36 / PE / Dako
CD11b / Bear1 / PC5 / Coulter
CD19 / J4.119 / PC7 / Coulter
CD20 / L27 / FITC / BD
CD34 / 581 (class III) / PC5 / Coulter
CD45 / J.33 / PC5 / Coulter
CD119 / GIR-208 / PE / BD
TdT / HT-6 / FITC / Dako
1 FITC, fluorescein isothiocyanate; PE, phycoerythrin; PC5, phycoerythrin-cyanin 5; PC7, phycoerythrin-cyanin 7
2 Dako, Dako, Glostrup, Denmark; Coulter, Coulter/Immunotech, Miami, FL; BD, Becton Dickinson, San Jose, CA
Table S3 The set of genes differentially expressed at d8 and d0.
Affymetrix ID / Gene / GO1 / B-cell2 / d8/d03 / good/poor4 / Affymetrix ID / Gene / GO1 / B-cell2 / d8/d03 / good/poor4203504_s_at / ABCA1 / 1.61 / 1.66 / 202713_s_at / KIAA0391 / 0.64 / 0.64°
202003_s_at / ACAA2 / 0.62 / 0.68° / 212311_at / KIAA0746 / 1.76 / 1.96
202502_at / ACADM / 0.52 / 0.29° / 212314_at / KIAA0746 / 1.65 / 2.09
205412_at / ACAT1 / 0.62 / 0.83 / 222139_at / KIAA1466 / 1.83 / 0.82
216705_s_at / ADA / 2,3 / 0.61 / 0.73 / 204444_at / KIF11 / 1 / 0.39 / 0.53°
204639_at / ADA / 2,3 / 0.64 / 0.72 / 221841_s_at / KLF4 / 2 / 0.45 / 0.79
208268_at / ADAM28 / 2 / 1.99 / 1.3 / 203542_s_at / KLF9 / 2 / 1.88 / 0.7
202144_s_at / ADSL / 2 / 0.62 / 0.55° / 203543_s_at / KLF9 / 2 / 1.55 / 0.61
200903_s_at / AHCY / 0.55 / 0.59° / 219157_at / KLHL2 / 1.52 / 1.45
200850_s_at / AHCYL1 / 0.65 / 0.53° / 219306_at / KNSL7 / 1 / 0.55 / 0.81
211986_at / AHNAK / 1.56 / 0.44° / 214039_s_at / LAPTM4B / 0.66 / 0.74
212543_at / AIM1 / 1.68 / 1.08 / 221581_s_at / LAT2 / 3 / + / 0.63 / 0.49°
212175_s_at / AK2 / 2 / 0.64 / 0.41° / 200650_s_at / LDHA / 2 / 0.65 / 0.47°
210517_s_at / AKAP12 / 3 / 0.59 / 1.8 / 213564_x_at / LDHB / 2 / 0.55 / 0.52°
201900_s_at / AKR1A1 / 2 / 0.6 / 0.56° / 201030_x_at / LDHB / 2 / 0.51 / 0.51°
211560_s_at / ALAS2 / 1.73 / 1.14 / 211596_s_at / LRIG1 / 0.53 / 0.79
203608_at / ALDH5A1 / 3 / 0.57 / 0.42° / 211615_s_at / LRPPRC / 0.59 / 0.68°
221589_s_at / ALDH6A1 / 2 / 0.64 / 0.50° / 202737_s_at / LSM4 / 2 / 0.65 / 0.51°
219437_s_at / ANKRD11 / 1.61 / 1.76 / 202904_s_at / LSM5 / 2 / 0.66 / 0.63
201051_at / ANP32A / 3 / 0.66 / 0.47° / 208771_s_at / LTA4H / 2,3 / 0.64 / 0.54°
201306_s_at / ANP32B / 0.64 / 0.64° / 202018_s_at / LTF / 3 / 1.58 / 0.76
221505_at / ANP32E / 0.62 / 0.50° / 218729_at / LXN / 0.63 / 0.65
210027_s_at / APEX1 / 2,3 / 0.48 / 0.51° / 205859_at / LY86 / 3,4 / 1.54 / 0.86
204205_at / APOBEC3G / 2 / 0.54 / 0.49° / 206584_at / LY96 / 3 / 1.62 / 0.69
205239_at / AREG / 3 / 1.92 / 0.27° / 213975_s_at / LYZ / 3 / 2.29 / 0.48
218694_at / ARMCX1 / 0.51 / 0.73 / 203362_s_at / MAD2L1 / 1 / 0.39 / 0.48°
220966_x_at / ARPC5L / 2 / 0.63 / 2.18° / 209014_at / MAGED1 / 0.62 / 0.92
213902_at / ASAH1 / 1.55 / 0.91 / 205027_s_at / MAP3K8 / 2 / 1.66 / 1.02
208758_at / ATIC / 2 / 0.51 / 0.54° / 200644_at / MARCKSL1 / 0.65 / 0.96
213366_x_at / ATP5C1 / 2 / 0.52 / 0.45° / 214108_at / MAX / 2 / 1.54 / 1.69°
205711_x_at / ATP5C1 / 2 / 0.55 / 0.44° / 202107_s_at / MCM2 / 1,2 / 0.56 / 0.64°
208870_x_at / ATP5C1 / 2 / 0.56 / 0.49° / 222037_at / MCM4 / 2 / 0.53 / 0.69
207508_at / ATP5G3 / 2 / 0.61 / 0.62° / 222036_s_at / MCM4 / 2 / 0.62 / 0.77
207507_s_at / ATP5G3 / 2 / 0.64 / 0.68° / 216237_s_at / MCM5 / 1,2 / 0.53 / 0.57
202325_s_at / ATP5J / 2 / 0.64 / 0.74 / 201755_at / MCM5 / 1,2 / 0.63 / 0.72
208745_at / ATP5L / 2 / 0.63 / 0.54° / 201930_at / MCM6 / 1,2 / 0.51 / 0.67
212062_at / ATP9A / 0.64 / 0.76 / 218163_at / MCTS1 / 1 / 0.61 / 0.59°
208860_s_at / ATRX / 2,3 / 0.58 / 0.5 / 200978_at / MDH1 / 2 / 0.6 / 0.53°
208833_s_at / ATXN10 / 0.53 / 0.39° / 218288_s_at / MDS025 / 0.65 / 0.68
205345_at / BARD1 / 2 / 0.65 / 0.46° / 209397_at / ME2 / 2 / 0.62 / 0.36°
212384_at / BAT1 / 2 / 1.58 / 2.04° / 209200_at / MEF2C / 2 / 0.61 / 0.39°
200837_at / BCAP31 / 4 / 0.66 / 0.79 / 204825_at / MELK / 2 / 0.43 / 0.68
218264_at / BCCIP / 1 / 0.64 / 0.64° / 202016_at / MEST / 0.61 / 0.46°
203685_at / BCL2 / 1,3,4 / 0.63 / 0.37° / 209861_s_at / METAP2 / 2 / 0.65 / 0.8
201170_s_at / BHLHB2 / 2 / 0.63 / 0.99 / 221570_s_at / METTL5 / 2 / 0.62 / 0.47°
210538_s_at / BIRC3 / 2,3,4 / 1.68 / 1.08 / 207098_s_at / MFN1 / 1.63 / 1.07
202095_s_at / BIRC5 / 1,4 / 0.64 / 0.76 / 214696_at / MGC14376 / 2.69 / 2.00°
202179_at / BLMH / 2 / 0.58 / 0.64° / 217871_s_at / MIF / 3,4 / 0.56 / 0.82
207655_s_at / BLNK / 3 / + / 0.49 / 0.33° / 218883_s_at / MLF1IP / 0.43 / 0.58
203755_at / BUB1B / 1,2 / 0.53 / 0.54° / 211071_s_at / MLLT11 / 0.57 / 0.53°
217809_at / BZW2 / 2 / 0.61 / 0.63 / 204959_at / MNDA / 2,3 / 1.79 / 0.68°
212460_at / C14orf147 / 0.57 / 0.71 / 202974_at / MPP1 / 3 / 0.66 / 0.74
218383_at / C14orf94 / 0.62 / 0.67° / 204438_at / MRC1 / 0.57 / 0.75
215087_at / C15orf39 / 1.5 / 1.1 / 208787_at / MRPL3 / 2 / 0.66 / 0.48°
219555_s_at / C16orf60 / 0.53 / 0.85 / 218654_s_at / MRPS33 / 2 / 0.66 / 0.68°
212055_at / C18orf10 / 0.54 / 0.47° / 217942_at / MRPS35 / 0.64 / 0.69°
219506_at / C1orf54 / 0.62 / 1.07 / 213164_at / MRPS6 / 2 / 0.63 / 0.74
208910_s_at / C1QBP / 0.49 / 0.65° / 202309_at / MTHFD1 / 2 / 0.6 / 0.48°
214214_s_at / C1QBP / 0.5 / 0.59° / 204798_at / MYB / 2 / 0.63 / 0.66
219004_s_at / C21orf45 / 0.66 / 0.8 / 212372_at / MYH10 / 0.58 / 0.45°
218123_at / C21orf59 / 0.63 / 0.59 / 218966_at / MYO5C / 0.58 / 0.63
201310_s_at / C5orf13 / 0.59 / 0.36° / 214349_at / NA / 0.54 / 0.97
215380_s_at / C7orf24 / 0.46 / 0.36° / 222079_at / NA / 0.59 / 0.76
218905_at / C8orf52 / 0.66 / 0.54° / 221973_at / NA / 0.63 / 0.94
205950_s_at / CA1 / 2.22 / 0.97 / 218764_at / NA / 0.66 / 0.99
211761_s_at / CACYBP / 2 / 0.58 / 0.47° / 208754_s_at / NAP1L1 / 2 / 0.52 / 0.57°
211985_s_at / CALM1 / 0.54 / 0.91 / 208753_s_at / NAP1L1 / 2 / 0.6 / 0.73
211984_at / CALM1 / 0.64 / 1.12 / 208752_x_at / NAP1L1 / 2 / 0.64 / 0.70°
200068_s_at / CANX / 2 / 0.52 / 0.47° / 212967_x_at / NAP1L1 / 2 / 0.66 / 0.70°
208852_s_at / CANX / 2 / 0.66 / 0.55° / 201969_at / NASP / 2 / 0.57 / 0.66°
201432_at / CAT / 3 / 0.62 / 0.37° / 201970_s_at / NASP / 2 / 0.58 / 0.66
202705_at / CCNB2 / 1 / 0.56 / 0.72 / 217860_at / NDUFA10 / 2 / 0.65 / 0.55°
201700_at / CCND3 / 1 / 0.58 / 0.47° / 218160_at / NDUFA8 / 0.57 / 0.55°
208796_s_at / CCNG1 / 1 / 0.66 / 0.79 / 203621_at / NDUFB5 / 0.54 / 0.43°
200910_at / CCT3 / 2 / 0.5 / 0.55° / 204634_at / NEK4 / 1,2 / 0.61 / 0.56°
201326_at / CCT6A / 2 / 0.56 / 0.49° / 218334_at / NIF3L1BP1 / 0.66 / 0.59°
201327_s_at / CCT6A / 2 / 0.59 / 0.49° / 201577_at / NME1 / 1,2 / 0.52 / 0.54°
200812_at / CCT7 / 1,2 / 0.5 / 0.54° / 209104_s_at / NOLA2 / 0.62 / 0.65
200873_s_at / CCT8 / 2 / 0.62 / 0.57° / 212377_s_at / NOTCH2 / 1,2,3,4 / + / 1.8 / 0.93
203435_s_at / CD10 / 2,3 / + / 0.55 / 0.82 / 202443_x_at / NOTCH2 / 1,2,3,4 / + / 1.79 / 0.9
203434_s_at / CD10 / 2,3 / + / 0.48 / 1.23 / 221691_x_at / NPM1 / 1,2,3,4 / 0.54 / 0.73
205786_s_at / CD11b / 3 / 2.31 / 0.47° / 204621_s_at / NR4A2 / 2,3 / 1.59 / 1.91
210356_x_at / CD20 / 3 / + / 3.04 / 2.24 / 204622_x_at / NR4A2 / 2,3 / 1.52 / 1.81
217418_x_at / CD20 / 3 / + / 2.94 / 2.29 / 203675_at / NUCB2 / 0.47 / 0.34°
209543_s_at / CD34 / 3 / + / 0.63 / 0.55° / 204766_s_at / NUDT1 / 2,3 / 0.64 / 0.76
212587_s_at / CD45 / 2,3 / + / 1.8 / 0.99 / 213311_s_at / NULP1 / 1.56 / 2.24°
212588_at / CD45 / 2,3 / + / 1.58 / 0.74 / 218622_at / NUP37 / 0.64 / 0.49°
201925_s_at / CD55 / 3 / 1.54 / 1.60° / 218039_at / NUSAP1 / 0.44 / 0.76
201005_at / CD9 / 2,3 / + / 0.54 / 2.39 / 212320_at / OK/SW-cl.56 / 2,4 / 0.51 / 0.49°
201028_s_at / CD99 / 3 / + / 0.63 / 0.40° / 202780_at / OXCT1 / 2 / 0.62 / 0.56°
203213_at / CDC2 / 1,2 / 0.62 / 0.64° / 210448_s_at / P2RX5 / 1.92 / 0.42°
204695_at / CDC25A / 1,2 / 0.59 / 0.86 / 214615_at / P2RY10 / 3 / 1.73 / 1.99
210473_s_at / CDC2L2 / 1,2,4 / 0.63 / 0.96 / 201014_s_at / PAICS / 2 / 0.4 / 0.44°
202246_s_at / CDK4 / 1,2 / 0.64 / 0.53° / 201013_s_at / PAICS / 2 / 0.48 / 0.46°
212501_at / CEBPB / 2,3 / 1.51 / 1.34 / 208877_at / PAK2 / 2,3 / 0.65 / 0.52°
218542_at / CEP55 / 0.53 / 0.61° / 200006_at / PARK7 / 2,3 / 0.62 / 0.51°
203493_s_at / CEP57 / 3 / 0.63 / 0.52° / 203860_at / PCCA / 0.65 / 0.64°
212675_s_at / CEP68 / 0.61 / 0.56 / 212694_s_at / PCCB / 0.61 / 0.62°
209194_at / CETN2 / 1 / 0.65 / 0.52° / 205202_at / PCMT1 / 2 / 0.65 / 0.42°
203166_at / CFDP1 / 0.6 / 0.55° / 201202_at / PCNA / 1,2,3 / 0.57 / 0.65
206932_at / CH25H / 1.76 / 0.32° / 204025_s_at / PDCD2 / 4 / 0.61 / 0.58°
217972_at / CHCHD3 / 0.56 / 0.51° / 215635_at / PDE8A / 2,3 / 1.51 / 1.35
205394_at / CHEK1 / 1,2,3 / 0.54 / 0.83 / 216640_s_at / PDIA6 / 2 / 0.59 / 0.60°
221675_s_at / CHPT1 / 1.63 / 1.1 / 207668_x_at / PDIA6 / 2 / 0.63 / 0.62°
209357_at / CITED2 / 2 / 0.67 / 0.92 / 208639_x_at / PDIA6 / 2 / 0.64 / 0.59°
201897_s_at / CKS1B / 1 / 0.63 / 0.62° / 208690_s_at / PDLIM1 / 3 / 0.6 / 0.61°
204170_s_at / CKS2 / 1 / 0.5 / 1.14 / 218025_s_at / PECI / 0.55 / 0.48°
209143_s_at / CLNS1A / 0.52 / 0.34° / 218319_at / PELI1 / 1.69 / 0.78
201405_s_at / COPS6 / 0.65 / 0.57° / 210976_s_at / PFKM / 2 / 0.61 / 0.62°
211727_s_at / COX11 / 2 / 0.48 / 0.40° / 221521_s_at / Pfs2 / 2 / 0.6 / 0.81
202119_s_at / CPNE3 / 0.62 / 0.67° / 201118_at / PGD / 2 / 0.62 / 0.62°
204314_s_at / CREB1 / 2,3 / 0.65 / 0.50° / 217356_s_at / PGK1 / 2 / 0.62 / 0.66
209967_s_at / CREM / 2,3 / 2.21 / 1.42 / 204866_at / PHF16 / 2 / 0.65 / 0.62°
207630_s_at / CREM / 2,3 / 1.71 / 1.37 / 203335_at / PHYH / 0.57 / 0.55
208669_s_at / CRI1 / 0.65 / 0.42° / 221689_s_at / PIGP / 0.54 / 0.86
201390_s_at / CSNK2B / 3 / 0.6 / 0.59° / 214224_s_at / PIN4 / 2 / 0.52 / 0.43°
207030_s_at / CSRP2 / 0.67 / 0.32° / 204571_x_at / PIN4 / 2 / 0.61 / 0.54°
204971_at / CSTA / 1.66 / 0.67 / 204146_at / PIR51 / 2,3 / 0.38 / 0.43°
203392_s_at / CTBP1 / 2 / 0.58 / 0.46° / 203688_at / PKD2 / 3 / 0.65 / 0.52°
215726_s_at / CYB5A / 0.65 / 0.51° / 205909_at / POLE2 / 2,3 / 0.64 / 0.68°
208923_at / CYFIP1 / 0.66 / 0.49° / 217848_s_at / PPA1 / 0.42 / 0.48°
219837_s_at / CYTL1 / 3 / 0.53 / 0.77 / 211765_x_at / PPIA / 2 / 0.66 / 0.67°
200046_at / DAD1 / 4 / 0.53 / 0.42° / 201293_x_at / PPIA / 2 / 0.66 / 0.70°
201623_s_at / DARS / 2 / 0.64 / 0.46° / 218009_s_at / PRC1 / 1 / 0.66 / 0.73
201624_at / DARS / 2 / 0.67 / 0.58° / 208680_at / PRDX1 / 0.43 / 0.8
218325_s_at / DATF1 / 2,4 / 1.5 / 0.82 / 201619_at / PRDX3 / 0.63 / 0.43°
201572_x_at / DCTD / 2 / 0.65 / 0.60° / 201923_at / PRDX4 / 2,3 / 0.5 / 0.9
210137_s_at / DCTD / 2 / 0.65 / 0.66° / 200845_s_at / PRDX6 / 3 / 0.57 / 0.41°
206414_s_at / DDEF2 / 0.63 / 0.37° / 205053_at / PRIM1 / 2 / 0.48 / 0.54°
213998_s_at / DDX17 / 2 / 1.52 / 0.71 / 208694_at / PRKDC / 2,3 / 0.66 / 0.58°
205033_s_at / DEFA1 / 3 / 1.95 / 0.83 / 206445_s_at / PRMT1 / 2,3 / 0.66 / 0.70°
207269_at / DEFA4 / 3 / 1.51 / 0.67 / 203401_at / PRPS2 / 2 / 0.58 / 0.48°
202534_x_at / DHFR / 2 / 0.54 / 0.59 / 202529_at / PRPSAP1 / 2 / 0.65 / 0.58°
202532_s_at / DHFR / 2 / 0.56 / 0.62° / 209606_at / PSCDBP / 1.5 / 0.92
48808_at / DHFR / 2 / 0.6 / 0.67° / 209337_at / PSIP1 / 0.62 / 0.43°
219590_x_at / DPH5 / 2 / 0.65 / 0.72° / 208805_at / PSMA6 / 2 / 0.42 / 0.54°
200762_at / DPYSL2 / 2,3 / 0.47 / 0.28° / 200039_s_at / PSMB2 / 2 / 0.63 / 0.68°
208370_s_at / DSCR1 / 3 / 0.4 / 0.34° / 209040_s_at / PSMB8 / 2 / 0.63 / 0.54°
203405_at / DSCR2 / 0.6 / 0.68° / 204279_at / PSMB9 / 2 / 0.58 / 0.32°
218585_s_at / DTL / 0.43 / 0.73 / 219485_s_at / PSMD10 / 0.65 / 0.55
221419_s_at / DUSP23 / 2 / 1.61 / 1.05 / 212296_at / PSMD14 / 2 / 0.6 / 0.54°
204014_at / DUSP4 / 1,2,3 / 1.59 / 3.72° / 219293_s_at / PTD004 / 0.5 / 0.53°
209457_at / DUSP5 / 2 / 1.83 / 0.65 / 215894_at / PTGDR / 3 / 0.65 / 0.6
208891_at / DUSP6 / 1,2,3 / 0.58 / 1.05 / 219654_at / PTPLA / 2,3 / 0.38 / 0.52°
208892_s_at / DUSP6 / 1,2,3 / 0.58 / 1.13 / 203554_x_at / PTTG1 / 1,2,3 / 0.66 / 0.73
208956_x_at / DUT / 2 / 0.61 / 0.73 / 212013_at / PXDN / 0.61 / 1.07
208955_at / DUT / 2 / 0.62 / 0.62° / 212012_at / PXDN / 0.66 / 1.16
209932_s_at / DUT / 2 / 0.66 / 0.72 / 202252_at / RAB13 / 3 / 0.6 / 0.62
200703_at / DYNLL1 / 0.47 / 0.63 / 222077_s_at / RACGAP1 / 3 / 0.52 / 0.52°
202971_s_at / DYRK2 / 2 / 0.65 / 0.74 / 205024_s_at / RAD51 / 1,2,3 / 0.57 / 0.79
219990_at / E2F8 / 1,2 / 0.58 / 0.74° / 210371_s_at / RBBP4 / 1,2,3 / 0.63 / 0.72
205419_at / EBI2 / 3 / 3 / 0.89 / 214113_s_at / RBM8A / 2 / 0.66 / 0.54°
219787_s_at / ECT2 / 0.56 / 0.47° / 213762_x_at / RBMX / 0.51 / 0.59°
201694_s_at / EGR1 / 2 / 0.27 / 1.45 / 209219_at / RDBP / 2 / 0.65 / 0.62°
201693_s_at / EGR1 / 2 / 0.52 / 1.3 / 204023_at / RFC4 / 2 / 0.62 / 0.54°
206115_at / EGR3 / 2 / 0.43 / 1.68 / 218723_s_at / RGC32 / 1 / 0.59 / 0.81
212653_s_at / EHBP1 / 0.62 / 0.42° / 210258_at / RGS13 / 3 / 1.57 / 1.78
211074_at / EIF1 / 2,3 / 3.84 / 0.98 / 202388_at / RGS2 / 1,3 / 1.75 / 2.01
201726_at / ELAVL1 / 2 / 0.59 / 0.55° / 200059_s_at / RHOA / 3 / 0.59 / 0.61°
201324_at / EMP1 / 4 / 0.64 / 1.26 / 201528_at / RPA1 / 2,3 / 0.63 / 0.33°
201231_s_at / ENO1 / 2 / 0.54 / 0.45° / 214042_s_at / RPL22 / 2 / 0.66 / 0.81
200843_s_at / EPRS / 2 / 0.6 / 0.52° / 221726_at / RPL22 / 2 / 0.61 / 0.68
213541_s_at / ERG / 2,3 / 0.42 / 0.59 / 200888_s_at / RPL23 / 2 / 0.53 / 0.95
211626_x_at / ERG / 2,3 / 0.66 / 0.66° / 211666_x_at / RPL3 / 2 / 0.62 / 0.88
209009_at / ESD / 0.61 / 0.52° / 200962_at / RPL31 / 2 / 0.66 / 0.47°
201931_at / ETFA / 0.65 / 0.42° / 214167_s_at / RPLP0 / 2 / 0.61 / 0.81
202345_s_at / FABP5 / 0.62 / 1.06 / 213427_at / RPP40 / 2 / 0.63 / 0.59
218397_at / FANCL / 2,3 / 0.61 / 0.48° / 201477_s_at / RRM1 / 2 / 0.64 / 0.78
211623_s_at / FBL / 2 / 0.57 / 0.56° / 201890_at / RRM2 / 2 / 0.26 / 0.31°
213145_at / FBXL14 / 2 / 0.67 / 0.75 / 209773_s_at / RRM2 / 2 / 0.49 / 0.60°
210299_s_at / FHL1 / 0.46 / 0.69 / 213750_at / RSL1D1 / 0.66 / 0.66°
201540_at / FHL1 / 0.6 / 0.81 / 201980_s_at / RSU1 / 3 / 0.6 / 0.48°
204236_at / FLI1 / 2 / 0.63 / 0.43° / 203594_at / RTCD1 / 2 / 0.62 / 0.58°
205511_at / FLJ10038 / 1.57 / 0.86 / 219549_s_at / RTN3 / 0.63 / 0.50°
201307_at / FLJ10849 / 1 / 0.57 / 0.48° / 202917_s_at / S100A8 / 3 / 7.97 / 1
222378_at / FLJ43663 / 1.96 / 1.77 / 208740_at / SAP18 / 2 / 0.61 / 0.78
215330_at / FLJ43663 / 1.54 / 2.04 / 201826_s_at / SCCPDH / 0.58 / 0.48°
209189_at / FOS / 2,3 / 0.5 / 1 / 201825_s_at / SCCPDH / 0.59 / 0.50°
202768_at / FOSB / 1,2 / 0.61 / 1.2 / 204030_s_at / SCHIP1 / 0.66 / 1.09
218880_at / FOSL2 / 2,4 / 1.73 / 1.49 / 211733_x_at / SCP2 / 0.59 / 0.37°
213056_at / FRMD4B / 0.48 / 0.77 / 201339_s_at / SCP2 / 0.61 / 0.41°
201637_s_at / FXR1 / 4 / 0.63 / 0.68 / 215088_s_at / SDHC / 2 / 0.58 / 0.38°
213524_s_at / G0S2 / 1 / 1.71 / 1.32 / 217724_at / SERBP1 / 0.6 / 0.49°
208869_s_at / GABARAPL1 / 3 / 1.85 / 1.37 / 202628_s_at / SERPINE1 / 3 / 0.66 / 0.94
211458_s_at / GABARAPL3 / 2.74 / 1.6 / 213047_x_at / SET / 2 / 0.54 / 0.37°
213453_x_at / GAPD / 2 / 0.43 / 0.65 / 201312_s_at / SH3BGRL / 0.66 / 0.36°
217398_x_at / GAPDH / 2 / 0.45 / 0.68 / 219493_at / SHCBP1 / 0.54 / 0.81
212581_x_at / GAPDH / 2 / 0.46 / 0.64 / 214096_s_at / SHMT2 / 0.6 / 0.72°
203282_at / GBE1 / 2 / 0.57 / 0.46° / 218988_at / SLC35E3 / 0.64 / 0.61
213129_s_at / GCSH / 0.46 / 0.24° / 201195_s_at / SLC7A5 / 1.51 / 2.38°
200009_at / GDI2 / 0.65 / 0.60° / 201589_at / SMC1L1 / 1,2,3 / 0.62 / 0.6
203560_at / GGH / 0.52 / 0.49° / 204240_s_at / SMC2L1 / 1 / 0.61 / 0.61°
219777_at / GIMAP6 / 0.65 / 0.49 / 202043_s_at / SMS / 0.44 / 0.58°
200681_at / GLO1 / 2 / 0.64 / 0.72 / 200826_at / SNRPD2 / 2 / 0.65 / 0.97
215001_s_at / GLUL / 3 / 1.73 / 1.05 / 202567_at / SNRPD3 / 2 / 0.63 / 0.64
218350_s_at / GMNN / 1,2 / 0.64 / 0.92 / 203316_s_at / SNRPE / 2 / 0.66 / 0.79
214431_at / GMPS / 2 / 0.64 / 0.68° / 201563_at / SORD / 2 / 0.61 / 0.59°
209576_at / GNAI1 / 3 / 0.6 / 1.03 / 200665_s_at / SPARC / 0.66 / 0.8
200708_at / GOT2 / 0.65 / 0.69° / 201240_s_at / SPCS2 / 2 / 0.53 / 0.38°
208308_s_at / GPI / 2,3 / 0.66 / 0.68 / 202591_s_at / SSBP1 / 2 / 0.52 / 0.47°
209170_s_at / GPM6B / 0.51 / 0.77 / 203787_at / SSBP2 / 2 / 0.66 / 0.42°
209167_at / GPM6B / 0.56 / 0.63 / 200890_s_at / SSR1 / 0.65 / 0.63°
210279_at / GPR18 / 3 / 1.92 / 1.41 / 200957_s_at / SSRP1 / 2 / 0.61 / 0.51°
214467_at / GPR65 / 3,4 / 1.9 / 1.02 / 208666_s_at / ST13 / 2 / 0.47 / 0.44°
213170_at / GPX7 / 3 / 0.65 / 0.85 / 208667_s_at / ST13 / 2 / 0.49 / 0.45°
204396_s_at / GRK5 / 2,3 / 1.74 / 2.73 / 207040_s_at / ST13 / 2 / 0.61 / 0.71
219357_at / GTBP1 / 1.52 / 1.68 / 219686_at / STK32B / 0.55 / 1.02
201036_s_at / HADHSC / 0.49 / 0.35° / 200783_s_at / STMN1 / 3 / 0.48 / 0.68
211569_s_at / HADHSC / 0.61 / 0.51° / 215416_s_at / STOML2 / 1,2,3 / 0.63 / 0.58°
218662_s_at / HCAP-G / 1 / 0.55 / 0.79 / 209306_s_at / SWAP70 / + / 1.66 / 1.18
203259_s_at / HDDC2 / 0.64 / 0.49° / 201463_s_at / TALDO1 / 2 / 0.61 / 0.33°
218450_at / HEBP1 / 0.64 / 0.52° / 203386_at / TBC1D4 / 0.63 / 0.86
214472_at / HIST1H3D / 2 / 0.65 / 1.22 / 203667_at / TBCA / 2 / 0.58 / 0.71
206110_at / HIST1H3H / 0.6 / 1.11 / 202371_at / TCEAL4 / 0.64 / 0.96
206074_s_at / HMGA1 / 2 / 0.66 / 0.93 / 208986_at / TCF12 / 2 / + / 0.64 / 0.53°
203744_at / HMGB3 / 2 / 0.61 / 0.98 / 204849_at / TCFL5 / 2 / 0.59 / 0.48°
208668_x_at / HMGN2 / 2 / 0.66 / 0.47° / 210487_at / TdT / 2,3 / + / 0.18 / 0.38
209786_at / HMGN4 / 0.65 / 0.49° / 202039_at / TIAF1 / 3,4 / 0.65 / 0.68
207165_at / HMMR / 0.6 / 0.73° / 208700_s_at / TKT / 0.6 / 0.7
201054_at / HNRPA0 / 2 / 0.61 / 0.56 / 208699_x_at / TKT / 0.63 / 0.64
211933_s_at / HNRPA3 / 0.58 / 0.82 / 215802_at / TLE1 / 2,3 / 1.72 / 1.86°
211932_at / HNRPA3P1 / 0.54 / 0.62 / 215269_at / TMEM1 / 2 / 1.55 / 1.41
206809_s_at / HNRPA3P1 / 0.65 / 0.65° / 222209_s_at / TMEM135 / 0.65 / 0.46°
214737_x_at / HNRPC / 2 / 0.59 / 0.61° / 218477_at / TMEM14A / 0.66 / 0.50°
212626_x_at / HNRPC / 2 / 0.64 / 0.62° / 202857_at / TMEM4 / 0.59 / 0.45°
202854_at / HPRT1 / 2 / 0.54 / 0.61 / 212281_s_at / TMEM97 / 0.43 / 0.61
218402_s_at / HPS4 / 0.56 / 0.72 / 212282_at / TMEM97 / 0.56 / 0.69°
54037_at / HPS4 / 2 / 0.6 / 0.79 / 201291_s_at / TOP2A / 2 / 0.54 / 0.78
214085_x_at / HRB2 / 1.52 / 0.5 / 213011_s_at / TPI1 / 2 / 0.63 / 0.49°
204405_x_at / HSA9761 / 2 / 0.66 / 0.74 / 221952_x_at / TRMT5 / 0.64 / 0.50°
214359_s_at / HSP90AB1 / 2,3 / 0.57 / 0.65 / 204822_at / TTK / 1,2 / 0.66 / 0.89
200064_at / HSP90AB1 / 2,3 / 0.62 / 0.69 / 209251_x_at / TUBA6 / 2 / 0.63 / 0.56°
208687_x_at / HSPA8 / 2,3 / 0.63 / 0.45° / 211750_x_at / TUBA6 / 2 / 0.64 / 0.60°
201841_s_at / HSPB1 / 2,3 / 0.51 / 1.07 / 211714_x_at / TUBB / 2,4 / 0.57 / 0.73
217900_at / IARS2 / 2 / 0.66 / 0.41° / 209026_x_at / TUBB / 2,4 / 0.58 / 0.74
210046_s_at / IDH2 / 2 / 0.6 / 0.58° / 201113_at / TUFM / 2 / 0.65 / 0.63°
206342_x_at / IDS / 2 / 1.8 / 1.57 / 201008_s_at / TXNIP / 2 / 1.1
202727_s_at / IFNGR1 / 3 / 1.74 / 1.02 / 201009_s_at / TXNIP / 1.98 / 0.76
201163_s_at / IGFBP7 / 0.6 / 0.3 / 201010_s_at / TXNIP / 1.89 / 0.76
201887_at / IL13RA1 / 3 / 1.86 / 0.58 / 202589_at / TYMS / 2 / 0.25 / 0.44
201888_s_at / IL13RA1 / 3 / 1.54 / 0.72 / 201649_at / UBE2L6 / 2 / 0.6 / 0.43°
205403_at / IL1R2 / 1.52 / 0.81 / 205480_s_at / UGP2 / 2 / 0.64 / 0.28°
202859_x_at / IL8 / 1,3 / 2.5 / 1.38 / 202330_s_at / UNG / 2,3 / 0.62 / 0.53°
201892_s_at / IMPDH2 / 2 / 0.39 / 0.57 / 200883_at / UQCRC2 / 2 / 0.56 / 0.51°
205981_s_at / ING2 / 2,3 / 0.66 / 0.91 / 220370_s_at / USP36 / 2 / 1.77 / 3.97°
200992_at / IPO7 / 3 / 0.67 / 0.60° / 212038_s_at / VDAC1 / 4 / 0.62 / 0.66
203906_at / IQSEC1 / 1.66 / 0.87 / 217234_s_at / VIL2 / 1.52 / 1.88
203907_s_at / IQSEC1 / 1.55 / 1.35 / 205922_at / VNN2 / 1.53 / 0.85
218170_at / ISOC1 / 0.62 / 0.79 / 221349_at / VPREB1 / + / 0.58 / 1.39
205176_s_at / ITGB3BP / 2,3,4 / 0.66 / 0.46° / 206660_at / VPREB2 / + / 0.5 / 0.61
201189_s_at / ITPR3 / 3 / 1.72 / 1.94° / 203856_at / VRK1 / 2 / 0.56 / 0.63
206245_s_at / IVNS1ABP / 2,3 / 0.65 / 0.93 / 204165_at / WASF1 / 2 / 0.46 / 0.44°
201466_s_at / JUN / 2 / 0.51 / 0.73 / 221532_s_at / WDR61 / 0.63 / 0.40°
201464_x_at / JUN / 2 / 0.62 / 1.01 / 221531_at / WDR61 / 0.65 / 0.45°
213646_x_at / K-ALPHA-1 / 2 / 0.59 / 0.56° / 209053_s_at / WHSC1 / 2 / 0.65 / 0.69°
212639_x_at / K-ALPHA-1 / 2 / 0.6 / 0.56° / 218919_at / ZFAND1 / 0.54 / 0.54°
211072_x_at / K-ALPHA-1 / 2 / 0.6 / 0.57° / 206683_at / ZNF165 / 2 / 2.11 / 1.39
201090_x_at / K-ALPHA-1 / 2 / 0.61 / 0.56° / 219228_at / ZNF331 / 2 / 2.06 / 1.89
211058_x_at / K-ALPHA-1 / 2 / 0.64 / 0.61° / 206648_at / ZNF571 / 2 / 1.99 / 2.28°
217894_at / KCTD3 / 0.64 / 0.59° / 206158_s_at / ZNF9 / 2 / 0.62 / 0.49°
200700_s_at / KDELR2 / 0.66 / 0.49° / 204026_s_at / ZWINT / 0.32 / 0.50°
202503_s_at / KIAA0101 / 0.18 / 0.4
1 GO annotated groups: 1-cell cycle (GO node 7049), 2-metabolism of proteins, nucleic acids, carbohydrates (GO nodes 19538, 6139, 5975), 3-cell communication, response to stress (GO nodes 7154, 6950), 4-cell death (GO 8219)
2 Genes involved in B-cell development / differentiation.
3 Mean fold changes of gene expression at d8 and d0 found to be significant (FDR < 0.05) within the whole gene expression dataset.
4 Comparison of gene expression in prednisone good vs poor responders at d8: mean ratios (shown as numbers) and t-test statistics (not shown but indicated by ° if FDR < 0.05).
NA = gene symbol not available
Figure Legends: Figure S1. Cell sorting accuracy, isolated RNA quality and RT-PCR control measurements. A: Leukemic cells at days 0 and 8 of induction therapy were stained with CD19, CD34 and CD10 antibodies and isolated by flow sorting. Dot plots of cell samples from one patient (patient 3) prior to and after the sorting and the corresponding percentages of leukemic cells are shown. B: Box plots of expression levels (in arbitrary fluorescence units) of genes encoding for Ig kappa (IGKC) and Ig lambda (IGL@) light chains indicate the absence of significant contaminations with normal B cells in the sorted blast samples. C: Electropherogram of total RNA isolated from the sorted leukemic cells (d0 sample, patient 3). The ratio of the areas under the 28S and 18S ribosomal RNA peaks was 1.7, thus indicating integrity of the total RNA isolated from the sorted cells. D: mRNA expression assessed by DNA microarray and real-time RT-PCR techniques. Primers specific to TYMS, TXNIP and UBB were obtained from Applied Biosystems (Foster City, CA). Real-time PCR was performed using ABI PRISM 5700 (Applied Biosystems). rs: Spearman correlation coefficient. Figure S2. Boxplots of residuals per array. Shown are boxplots of model residuals of all perfect match probes on the array. A residual is defined as the difference between the observed value and the expected value derived from the fitted model.
Figure S1
Figure S2
1