Supplemental Table 1. Comparison among Q-RT-PCR, ISH-TMA and IHC for the detection of ErbB-2 in breast cancers. IHC ISH-TMA Q-RT-PCR Q-RT-PCR range Q-RT-PCR score 0 0 0* 0 0 0 0* 0 0 0 1 0 0 0 1 0 0 0 1 0 0 1 2 0 1 1 2 0 0 0 3 0 0 0 3 0 0 0 3 0 0 0 3 0 0 0 3 x<10 0 1 1 3 0 0 0 4 0 0 0 5 0 0 0 5 0 0 0 5 0 1 1 5 0 0 0 6 0 0 0 8 0 0 0 8 0 1 1 8 0 0 1 8 0 1 1 11 1 1 1 11 1 1 1 11 1 1 0 12 1 1 1 13 10<x<20 1 0 1 13 1 1 1 16 1 1 1 17 1 2 1 17 1 2 2 22 2 2 2 43 2 20<x<100 3 2 74 2 3 2 86 2 3 3 213 3 3 3 259 x>100 3 3 3 362 3 Legend to Supplemental Table 1. This experiment was set up to demonstrate that there is good semi-quantitative correlation between the levels of expression detected by ISH-TMA, IHC and Q-RT-PCR. We compared the three methods on levels of ErbB-2 expression in breast cancer, since ErbB-2 is overexpressed in breast cancers, over a wide range of levels. 1 Thirty-nine primary infiltrating ductal carcinomas were divided into two aliquots, one of which was snap-frozen in liquid nitrogen and stored at –80°C for RNA extraction, and the other was fixed in buffered formalin and embedded in paraffin blocks for TMA construction. All frozen sections were reviewed by a pathologist before analysis and selected for cellularity (at least 70% of tumor cells), before RNA extraction. Column “Q-RT-PCR”: The cohort of 39 breast cancer patients was analyzed by Q-RT-PCR. The housekeeping gene ß-actin was used for the normalization. Results were expressed as relative levels of ErbB-2 mRNA, referred to a sample, called calibrator, chosen to represent 1X expression of this gene. The calibrator was a breast cancer cell line (MCF-7) that was analyzed on every assay plate with the unknown samples. The amount of target, normalized to the endogenous reference (ß-actin) and relative to the calibrator, was defined by ∆∆Ct method as described by Livak K (Sequence Detectro User Bulletin 2; Applied Biosystems). Specifically, the formula is applied as follows: Target amount = 2 -∆∆Ct where ∆∆Ct = [Ct (HER-2/neu sample) – Ct (ß-actin sample)] – [Ct (HER-2/neu calibrator) – Ct (ß-actin calibrator)]. The two samples labeled with an asterisk (0*) gave no amplification after 40 cycles. Cores from paraffin blocks, corresponding to these cases, were then arrayed on a TMA (in duplicate) and subjected to ISH with an ErbB-2-specific riboprobe. ISH scores were assigned as described in Methods (column “ISH-TMA”). Parallel sections of the TMA were also evaluated by IHC. Immunohistochemical staining for ErbB-2 was performed using the primary Ab (anti-HER-2/neu, DAKO, Carpinteria, CA, 1:2000 dilution) followed by detection with the Dako EnVision+ system, peroxidase (DAKO). Briefly, formalin-fixed, paraffin-embedded tissue sections were deparaffinized, rehydrated, pretreated in 0.25 mM EDTA pH 8.0 at 95°C and exposed to the primary antibody for 60 min at room temperature. Positive and negative controls were included in each experiment. ErbB-2 overexpression was evaluated according to the scoring system recommended by the DAKO HercepTest: score 0, no staining or membrane staining in <10% of the tumor cells; score 1, barely perceptible membrane staining in >10% of the tumor cells; score 2, weak-to-moderate staining of the entire membrane in >10% of the tumor cells; score 3, strong staining of the entire membrane in >10% of the tumor cells. Scores of 2 and 3 were considered to represent overexpression. Since data from ISH-TMA and IHC analyses are discontinuous, whereas those obtained by Q-RT-PCR analyses are continuous, we transformed the Q-RT-PCR values into discontinuous data (1). The discontinuous Q-RT-PCR scores (column “Q- RT-PCR score”) are shown along with the ranges of actual values used to attribute the score (column “Q-RT-PCR range”). Pearson Correlation was calculated in Excel Software (Microsoft). The Pearson Correlation Coefficients were Q-RT-PCR vs. ISH- TMA, r = 0.906; Q-RT-PCR vs. IHC, r = 0.909; ISH-TMA vs. IHC, r = 0.910. To test if the frequency of the observed values of expression between ISH, IHC and PCR are evenly distributed, a chi square test was performed (ISH vs. IHC, Chi = 2.15 and P=0.54; ISH vs. PCR, Chi = 3.85 and P = 0.27; IHC vs. PCR, Chi = 2.41 and P = 0.49), confirming that the three method used in the analysis are not significantly different. Twenty of the 39 samples displayed in this Table are the same used in a previously published validation of ISH vs. Q-RT-PCR of ErbB-2 expression (2). 2 References to Supplemental Table 1 1. Ginestier, C., Charafe-Jauffret, E., Bertucci, F., et al. Distinct and complementary information provided by use of tissue and DNA microarrays in the study of breast tumor markers. Am J Pathol 2002;161:1223-33. 2. Nicassio, F., Bianchi, F., Capra, M., et al. A cancer-specific transcriptional signature in human neoplasia. J Clin Invest 2005;115:3015-25. 3 Supplemental Table 2. List of screened kinases. Symbol Sugen Aliases Group Name cDNA Acc AAK1 AAK1 KIAA1048 OTHER AP2 associated kinase 1 NM_014911 ACVR1C ALK7 ALK7, ACVRLK7 TKL Activin A receptor, type IC AY127050 ADCK1 ADCK1 ADCK1 ATYPICAL AarF domain containing kinase 1 BC058906 ADRBK2 BARK2 GRK3, BARK2 AGC Adrenergic, beta, receptor kinase 2 NM_005160 ALS2CR2 STLK6 ILPIP STE Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 2 NM_018571 ALS2CR7 PFTAIRE2 CMGC Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 7 BC038807 ANKK1 SgK288 ANKK1, PKK2 TKL Ankyrin repeat and kinase domain containing 1 AF487542 AURKA AurA AIK, STK6 OTHER Aurora kinase A NM_003600 AURKB AurB STK12, AIK2, ARK2 OTHER Aurora kinase B NM_004217 BRSK1 BRSK1 KIAA1811 CAMK BR serine/threonine kinase 1 NM_032430 BRSK2 BRSK2 PEN11B, STK29, SAD1 CAMK BR serine/threonine kinase 2 NM_003957 BUB1 BUB1 BUB1, BUB1L, BUB1A OTHER BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) NM_004336 C9orf96* SgK071 OTHER Chromosome 9 open reading frame 96 NM_153710 CAMK1D CaMK1d CKLIK CAMK Calcium/calmodulin-dependent protein kinase ID AF286366 CAMK2D CaMK2d CAMKD CAMK Calcium/calmodulin-dependent protein kinase (CaM kinase) II delta NM_001221 CAMKK1 CaMKK1 CAMKKA OTHER Calcium/calmodulin-dependent protein kinase kinase 1, alpha AF425301 CAMKK2 CaMKK2 CAMKK, CAMKKB OTHER Calcium/calmodulin-dependent protein kinase kinase 2, beta AF287631 CDKL3 CDKL3 NKIAMRE CMGC Cyclin-dependent kinase-like 3 NM_016508 CDKL4 CDKL4 CMGC Cyclin-dependent kinase-like 4 NM_001009565 CIT CRIK CRIK, STK21 AGC Citron (rho-interacting, serine/threonine kinase 21) NM_007174 CLK2 CLK2 hCLK2 CMGC CDC-like kinase 2 NM_003993 CLK4 CLK4 CMGC CDC-like kinase 4 NM_020666 4 CRK7 CRK7 CRKR, KIAA0904 CMGC CDC2-related protein kinase 7 NM_016507 CSNK1A1L CK1a2 CK1 Casein kinase 1, alpha 1-like BC028723 DCAMKL2 DCAMKL2 DCDC3, DCK2 CAMK Doublecortin and CaM kinase-like 2 NM_152619 DCAMKL3 DCAMKL3 KIAA1765 CAMK Doublecortin and CaM kinase-like 3 AB051552 DYRK3 DYRK3 DYRK5, RED, REDK CMGC Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3 BC015501 EIF2AK1 HRI HRI, KIAA1369 OTHER Eukaryotic translation initiation factor 2-alpha kinase 1 NM_014413 EIF2AK4 GCN2_d2 GCN2, KIAA1338 OTHER Eukaryotic translation initiation factor 2 alpha kinase 4 NM_001013703 ERN2 IRE2 IRE1b OTHER Endoplasmic reticulum to nucleus signalling 2 NM_033266 FLJ23356* SgK196 OTHER Hypothetical protein FLJ23356 NM_032237 FLJ25006 SgK494 AGC Hypothetical protein FLJ25006 NM_144610 HIPK2 HIPK2 CMGC Homeodomain interacting protein kinase 2 NM_022740 HIPK4 HIPK4 CMGC Homeodomain interacting protein kinase 4 BC034501 HUNK HUNK CAMK Hormonally upregulated Neu-associated kinase NM_014586 IRAK3* IRAK3 IRAK-M TKL Interleukin-1 receptor-associated kinase 3 BC057800 KALRN Trad HAPIP, DUO, TRAD CAMK Kalirin, RhoGEF kinase NM_007064 KIAA0999 QSK KIAA0999 CAMK KIAA0999 protein NM_025164 KSR2 KSR2 TKL Kinase suppressor of ras 2 AY345972 LATS2 LATS2 AGC LATS, large tumor suppressor, homolog 2 (Drosophila) NM_014572 LIMK1 LIMK1 LIMK1, LIMK TKL LIM domain kinase 1 NM_002314 LIMK2 LIMK2 TKL LIM domain kinase 2 NM_016733 LOC340156 SgK085 LOC340156 CAMK hypothetical protein LOC340156 NM_001012418 LOC91461 SgK493 OTHER Hypothetical protein BC007901 NM_138370 LRRK2 LRRK2 DARDARIN TKL Leucine-rich repeat kinase 2 XM_058513 LYK5* STLK5 STRAD STE Protein kinase LYK5 NM_001003786 MAP3K15 MAP3K7 FLJ16518 STE Mitogen-activated protein kinase kinase kinase 15 NM_001001671 5 MAP3K6 MAP3K6 MAPKKK6 STE Mitogen-activated protein kinase kinase kinase 6 AF100318 MAP3K9 MLK1 MLK1 TKL Mitogen-activated protein kinase kinase kinase 9 NM_033141 MAP4K1 HPK1 HPK1 STE Mitogen-activated protein kinase kinase kinase kinase 1 NM_007181 MAP4K3 KHS2 MAPKKKK3, GLK, RAB8IPL1 STE Mitogen-activated protein kinase kinase kinase kinase 3 NM_003618 MAP4K4 ZC1/HGK HGK STE Mitogen-activated protein kinase kinase kinase kinase 4 NM_004834 MAPK15 Erk7 ERK8 CMGC Mitogen-activated protein kinase 15 AY065978 MASK MST4 MST4 STE Mst3 and SOK1-related kinase NM_016542 MASTL MASTL FLJ14813 GENE AGC Microtubule associated serine/threonine kinase-like NM_032844 MELK MELK KIAA0175, HPK38 CAMK Maternal embryonic leucine zipper kinase NM_014791 MGC16169* TBCK OTHER Hypothetical protein MGC16169 NM_033115 MGC42105 NIM1 MGC42105 CAMK Hypothetical protein MGC42105 NM_153361 MINK1 ZC3/MINK MINK, B55, MAP4K6 STE Misshapen-like kinase 1 (zebrafish) NM_015716 MLCK caMLCK LOC91807 CAMK Cardiac-MyBP-C associated Ca/CaM kinase NM_182493 MLKL* MLKL FLJ34389 OTHER Mixed lineage kinase domain-like BC028141 MYLK2 skMLCK KMLC, MLCK CAMK Myosin light chain kinase 2, skeletal muscle NM_033118 MYO3B