ONCOGENOMICS Through the Use of Chemotherapeutic Agents

Oncogene (2002) 21, 7749 – 7763 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 ª<a href="/goto?url=http://www.nature.com/onc" target="_blank">www.nature.com/onc </a>Expression profiling of primary non-small cell lung cancer for target identification Jim Heighway*,1, Teresa Knapp1, Lenetta Boyce1, Shelley Brennand1, John K Field1, Daniel C Betticher2, Daniel Ratschiller2, Mathias Gugger2, Michael Donovan3, Amy Lasek3 and Paula Rickert*,3 1Target Identification Group, Roy Castle International Centre for Lung Cancer Research, University of Liverpool, 200 London <ul style="display: flex;"><li style="flex:1">2</li><li style="flex:1">3</li></ul>Road, Liverpool L3 9TA, UK; Institute of Medical Oncology, University of Bern, 3010 Bern, Switzerland; Incyte Genomics Inc., 3160 Porter Drive, Palo Alto, California, CA 94304, USA Using a panel of cDNA microarrays comprising 47 650 transcript elements, we have carried out a dual-channel analysis of gene expression in 39 resected primary human non-small cell lung tumours versus normal lung tissue. Whilst *11 000 elements were scored as differentially expressed at least twofold in at least one sample, 96 transcripts were scored as over-represented fourfold or more in at least seven out of 39 tumours and 30 sequences 16-fold in at least two out of 39 tumours, including 24 transcripts in common. Transcripts (178) were found under-represented fourfold in at least seven out of 39 tumours, 31 of which are underrepresented 16-fold in at least two out of 39 lesions. The relative expression levels of representative genes from these lists were analysed by comparative multiplex RT – PCR and found to be broadly consistent with the microarray data. Two dramatically over-represented genes, previously designated as potential tumour suppressors in breast (maspin) and lung and breast (S100A2) cancers, were analysed more extensively and demonstrate the effectiveness of this approach in identifying potential lung cancer diagnostic or therapeutic targets. Whilst it has been reported that S100A2 is downregulated in NSCLC at an early stage, our microarray, cmRT – PCR, Western and immunohistochemistry data indicate that it is strongly expressed in the majority of tumours. Introduction The lack of generally effective therapies for lung cancer leads to the high mortality rate associated with this common disease. Recorded 5-year survival figures vary, to some extent perhaps reflecting differences in ascertainment methods, but across Europe are approximately 10% (Bray et al., 2002). Whilst surgery is an effective strategy for the treatment of localized lesions, most patients present with overtly or covertly disseminated disease. Unfortunately, despite the many advances in chemotherapeutic regimes, for most lung cancer patients, metastatic disease present at diagnosis will be fatal. We have chosen to use microarray transcript profiling to illuminate potential targets with application in the detection or treatment of this disease. Microarray analysis allows a reversal of the standard cancer genetics paradigm. Instead of attempting to identify areas of the genome which are perturbed in disease and then looking for key cancer-associated genes within these regions, the technology allows us to first identify genes that are differentially expressed in a condition and then to look at the genomic context of that abnormal expression. Perhaps the most powerful prioritization tool in the search for disease significance will be the coupling of gene expression changes to genomic damage events in the tumour DNA. Lung cancer is commonly divided into small cell (SCLC) and non-small cell (NSCLC) disease. SCLC, approximately 20% of cases, is invariably metastatic at presentation and is therefore treated primarily through the use of chemotherapeutic agents. In contrast, NSCLC may appear to be localised or only locally metastatic and therefore amenable to surgical resection. NSCLC is further subdivided into three main classes, squamous cell carcinoma, adenocarcinoma and large cell carcinoma. However, many if not most tumours are composed of multiple histological types (Thurlbeck and Churg, 1995; Fraire et al., 1987). Such variation in a presumably clonal lesion has led to the suggestion that lung cancers arise from a multipotential stem cell component of the normal tissue, a hypothesis supported by the analysis of model systems Oncogene (2002) 21, 7749 – 7763. doi:10.1038/ sj.onc.1205979 Keywords: lung; cancer; microarray; expression; target identification *Correspondence: J Heighway, Gene Function, Roy Castle International Centre for Lung Cancer Research, 200 London Road, Liverpool L3 9TA, UK; E-mail: [email protected] or Paula Rickert, Incyte Genomics, 3160 Porter Drive, Palo Alto, California, CA 94304, USA; E-mail: [email protected] Received 27 May 2002; revised 8 August 2002; accepted 13 August 2002 Lung cancer target identification J Heighway et al 7750 (Sunday et al., 1999). It is very likely that transcript profiling will lead to further subdivisions within these overall classes of disease, some which mirror preexisting pathological classes and some which are novel (Garber et al., 2001; Bhattacharjee et al., 2001). Whilst such classification regimes may be very important in understanding disease biology and may become critical in the future with respect to assessing lung cancer prognosis and directing subsequent therapeutic practice, such uses are perhaps currently secondary to the need for improved diagnostic and therapeutic agents. Given that it is generally easier pharmacologically to inhibit rather than to replace function and given that a lung cancer diagnostic is likely to be most effective where it measures an excess of rather than the lack of a gene product, our attention has been directed primarily towards genes that are over-represented in disease tissue. However, it is likely that the study of underrepresented sequences will considerably enhance our understanding of the biology of the disease process and may lead to the discovery of linked and more conventional targets. We have therefore generated and validated gene lists of both tumour over and under-represented sequences. As additional verification, we independently assayed expression profiles of two of these genes, osteopontin and PRAME, using multiplex quantitative real-time RT – PCR (TaqManTM, Applied Biosystems) with the housekeeping gene b2-microglobulin as an internal reference (Figure 1). Osteopontin was found to be over-represented at least fourfold in 14 out of 14 tumours assayed, with an average CT (cycle threshold of detectable logarithmic phase amplification) of 22 in lung tumours and 32 in normal tissue. PRAME was measured as over-represented in 13 out of 14 tumours, with an average CT of 28 in tumours and 36 in normal tissue. Our microarray approach for identifying overrepresented genes in lung tumours was thus confirmed by two distinct methods using independent control genes. Having validated the basic experimental protocol, a wider analysis was undertaken. Microarray data analysis RNA was extracted from resectable NSCLC tumours from 33 additional patients giving 39 in total, including 13 who had undergone neo-adjuvant chemotherapy (Table 1). A further series of dual channel microarray Results Method validation Dual channel microarray hybridizations were carried out in duplicate for six patients comprising tumour against matched normal tissue across five cDNA microarrays: Human Drug Target (formerly known as Human Genome GEMTM 1) and Human Foundation 1 – 4 (formerly known as Human Genome GEM 2 – 5) (Incyte Genomics, Inc.). Clones representing 14 <a href="/goto?url=http://www.roycastle.org/research/) with" target="_blank">distinct genes (http://www.roycastle.org/research/) with </a>a differential expression (DE) value greater than two in at least five of the six patients were selected. The validity of these predictions was tested by an independent gene expression analysis, comparative multiplex RT – PCR (cmRT – PCR). As many of the standard control genes are known to be variable in expression between different human tissues and states (Lee et al., 2002) we selected new control genes from within the array data; sequences that showed DE values below two in the tumour versus normal hybridizations from the first six patients. Control primer sets were validated against each other in reactions with paired cDNA samples. The most robust of the tested control loci in terms of ease of amplification and wide reaction compatibility was the KIAA0228 (228) gene (Acc: D86981), located on 17q23.2. We used cmRT – PCR and at least one control gene to test the 14 genes identified as differentially expressed (83 individual hybridised elements). The cmRT – PCR data was consistent with the hybridization data predicting a gene expression difference between the tumour and normal tissue, in each case, in the same direction. Figure 1 Illustrates TaqManTM quantitative real-time PCR data for matched normal lung and tumour pairs for osteopontin (a) and PRAME (c), confirming the over-representation of these genes in a majority of non-small cell lung tumours. The housekeeping gene b2-microglobulin was used as an internal reference for normalization of sample concentrations. Relative expression values (y-axis) were determined using normal lung tissue from patient 185 (detectably expressing both genes) as the calibrator. (b and d) show cmRT – PCR (b) and RT – PCR (d) analyses of osteopontin and PRAME in matched samples Oncogene Lung cancer target identification J Heighway et al 7751 Table 1 Lists the patient details and tumour histology <ul style="display: flex;"><li style="flex:1">Patient </li><li style="flex:1">Diagnosis </li><li style="flex:1">Sex </li><li style="flex:1">Age at surgery </li><li style="flex:1">T</li><li style="flex:1">N</li><li style="flex:1">M</li><li style="flex:1">Stage </li></ul>57 61 64 65 67 69 70 74 78 80 83 86 87 89 91 111 122 123 125 128 130 132 134 138 141 142 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B14 <ul style="display: flex;"><li style="flex:1">Squamous </li><li style="flex:1">M</li></ul>MMF 67 73 79 70 68 73 75 71 66 70 73 67 72 68 62 61 66 54 78 50 43 54 60 75 71 53 71 71 50 54 39 57 58 62 53 54 62 54 62 221121212322222122222222212223122222322 010001000110020012001010002222222222222 XXXXXXXXXXXXXXXXXXXXXXXXXX0000000000000 IB IIB IA IA IB IIA IB IA IB IIB IIB IB IB IIIA IB Large cell endocrine Pulmonary carcinoid Adenocarcinoma <ul style="display: flex;"><li style="flex:1">Squamous </li><li style="flex:1">F</li></ul><ul style="display: flex;"><li style="flex:1">Squamous </li><li style="flex:1">M</li></ul>MFMMMMMFMMFMMF Adenocarcinoma Adenocarcinoma Squamous Squamous Squamous Adenocarcinoma Adenocarcinoma Squamous Adenocarcinoma Atypical carcinoid Adenocarcinoma Adenocarcinoma Adenocarcinoma Squamous IA IIB IIIA IB IB IIB IB IIB IB IB IA IIIA IIIA IIIA IIIA IIIA IIIA IIIA IIIA IIIA IIIA IIIA IIIA IIIA <ul style="display: flex;"><li style="flex:1">Squamous </li><li style="flex:1">M</li></ul>FF Large cell endocrine Squamous <ul style="display: flex;"><li style="flex:1">Squamous </li><li style="flex:1">F</li></ul><ul style="display: flex;"><li style="flex:1">Squamous </li><li style="flex:1">M</li></ul>MMMMF Neuroendocrine ca. Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Squamous Adenocarcinoma Adenocarcinoma Large cell Squamous Adenocarcinoma Adenocarcinoma FFMMFMMMMT, N and M refer to the tumour, node and metastasis status according to WHO guidelines hybridizations was carried out for all patients comparing tumour against matched normal tissue (27 patients) or tumour against a pooled normal sample (six patients). For the majority of patient samples, duplicate hybridizations were performed across all five cDNA microarrays. The data presented in this study was obtained from 298 individual array analyses that passed hybridization QC, screening 34 – 37 different patient samples across each of 47 650 clones representing 39 176 unique gene/EST clusters. Approximately 11 000 clones were scored as signifi- cantly differentially expressed (4twofold: Yue et al., 2001) in at least one sample. We were primarily interested in identifying sequences dramatically differentially expressed in a wide range of disease samples, or else sequences that were very dramatically differentially expressed in a smaller number of tumours. Gene lists were constructed of (a) genes that were differentially expressed fourfold or more in seven or more (approximately 20%) tumours and (b) genes that were differentially expressed 16-fold or more in two or more tumours (Tables 2 and 3). Approximately 50% of the elements highlighted in the fourfold tumour overrepresented list were various immunoglobulin transcripts, the relative expression levels of which correlated with each other closely across the tumour series (data not shown). Removing these sequences left a list of 96 genes with Genbank annotation, some of which were represented in the primary list by multiple array elements. These remaining 96 were ranked in order of number of tumours in which the element passed the restriction criteria. Following removal of Igrelated genes, 30 elements were scored as overrepresented 16-fold or more in at least two tumours and of those, 24 were common to both lists. One hundred and seventy-eight non-Ig genes with Genbank annotation were scored as under-represented fourfold or more in seven or more tumours with 30 of these under-represented 16-fold in two or more lesions. One element, representing the calcium binding protein S100A8 (MRP8), was present in both the over and under-represented lists. The expression profile of this gene did not correlate closely with those of immunoglobulin related elements. Oncogene Lung cancer target identification J Heighway et al 7752 Table 2 Lists in ranked order four- and 16-fold tumour over-represented elements that passed the restriction criteria Genbank Acc Chr. loc. 46up in: 166up in: 26up in: Gene description (closest PD match to element sequence) 12224892 30057 3413861 14250681 35147 416114 2088550 790537 6688166 544761 2065174 3150034 393318 35569 31894 13516384 12653176 14043321 14198020 1903383 452483 4481752 13111934 292829 8q23.3 2q32.2 1p36.33 12q13.13 4q22.1 Xq28 6q22.2 11q22.3 2q24.1 10p15.1 1q23.1 7q33 13q14.11 8q22.2 14q23.3 8q22.1 4q26 20q13.12 17p11.2 22q11.22 10p15.1 13q12.11 15q26.1 17q21.2 11p11.2 4p13 6q24.1 11q22.2 4p16.1 6q22.1 Xp11.23 Xq28 10q21.2 17q21.33 17q21.33 1q32.1 19q13.2 17q25.3 12q23.3 2q36.1 15q15.3 3p25.2 3q24 17q21.2 15q14 2q35 22q11.23 2q37.1 20q11.21 11q23.3 5q34 26 22 22 18 18 18 18 18 17 17 17 17 17 16 16 16 16 15 15 15 15 14 14 14 13 13 13 13 13 12 12 12 12 12 12 11 11 11 11 11 11 11 11 10 10 10 10 10 10 10 10 10 10 9 65 33 30 31 20 28 21 26 25 23 20 19 18 24 24 19 28 27 24 23 20 20 18 24 23 23 20 24 22 17 19 21 16 25 30 23 25 14 22 18 22 20 17 23 16 24 15 25 13 21 17 23 27 19 19 17 19 18 10 14 16 13 16 19 18 23 18 15 21 16 Homo sapiens mRNA; cDNA DKFZp667P184. Human mRNA for pro-alpha-1 type 3 collagen. Homo sapiens mRNA for KIAA0450 protein. Homo sapiens, keratin 6A. Human mRNA for osteopontin. Human antigen (MAGE-1) gene. Human hereditary haemochromatosis region, histone 2A-like gene. Human glutamate receptor (GluR4). Homo sapiens partial mRNA for GalNAc-T5 (GALNT5 gene). Chlordecone reductase {clone HAKRa} [human, liver, mRNA]. Homo sapiens S100A2 gene, exon 1-3. Homo sapiens aldose reductase-like peptide. OSF-2p1 Human mRNA for polyA binding protein. Human mRNA for glutathione peroxidase-like protein. Homo sapiens genomic DNA, chromosome 8q23, clone: KB1241F12. Homo sapiens, MAD2 (mitotic arrest deficient, yeast, homolog)-like 1. Homo sapiens, ubiquitin carrier protein E2-C. 11 2432287533<ul style="display: flex;"><li style="flex:1">5</li><li style="flex:1">H.sapiens gene for cytokeratin 17. </li></ul>Human preferentially expressed antigen of melanoma (PRAME). Human dihydrodiol dehydrogenase Human connexin 26. Human, protein regulator of cytokinesis 1. DNA topoisomerase II (EC 5.99.1.3) Human midkine gene, complete cds Homo sapiens, similar to ubiquitin carboxy-terminal hydrolase L1. Homo sapiens mRNA for transmembrane protein (THW gene). Human mRNA for type I interstitial collagenase (MMP1) Homo sapiens, S100 calcium-binding protein P. H.sapiens COL10A1 gene for collagen (alpha-1 type X). Homo sapiens prostate associated PAGE-1. 219928 12653130 11558106 10437218 13905069 30094 3300091 15079897 29840 8919392 1418927 7677071 5834583 339708 3483454 394340 13676499 2367444 5419854 34070 334452 Homo sapiens, melanoma antigen, family A, 3. Human cell cycle control gene CDC2. Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 781354. H.sapiens mRNA for prepro-alpha1(I) collagen. Homo sapiens ubiquitin-conjugating enzyme E2. Human mRNA encoding rat C4.4-like protein Human thymidine kinase mRNA, complete cds Homo sapiens full length insert cDNA clone ZA02A01. H. sapiens (D2S351) DNA segment containing (CA) repeat; AFM294yf5. Macaca fascicularis brain cDNA clone:QflA-13282. Myoblast city Homo sapiens mRNA; cDNA DKFZp566N043. Human mRNA for cytokeratin 15 Human thrombospondin 2 (THBS2). Human, insulin-like growth factor binding protein 2 (36kD). Homo sapiens genomic DNA, chromosome 22q11.2, clone KB1125A3. Human bilirubin UDP-glucuronosyltransferase isozyme 1. Homo sapiens mRNA for fls353. Homo sapiens tripartite motif protein TRIM29 alpha. Homo sapiens pituitary tumor-transforming 1 (PTTG1) gene, exon 4. Human PNG pseudogene, complete sequence Human ARF-activated phosphatidylcholine-specific phospholipase D1a (hPLD1). Homo sapiens pro-alpha 2(I) collagen (COL1A2) gene, complete cds. Homo sapiens:Probable G-protein coupled receptor (GPR87). Homo sapiens, keratin 14. 307505 13279202 5103013 184472 4589928 12275863 6318669 2911809 1185462 179595 14042718 12803708 14602472 14279429 7328949 533527 13516845 12052859 14042497 10440167 914203 22q12.2 3q26.31 7q21.3 3q25.1 17q21.2 1q32.1 Xq28 999999999999888 Human ladinin (LAD) mRNA. Human chondrosarcoma CSAG2b mRNA sequence Human 20-alpha HSD gene for 20 alph-hydroxysteroid dehydrogenase. Homo sapiens, melanoma antigen, family A, 9, mRNA, complete cds. Human hxCT mRNA for cystine/glutamate exchanger, complete cds Human mRNA; cDNA DKFZp564O2071 (from clone DKFZp564O2071); complete cds Human cDNA FLJ14751 fis, similar to R. norvegicus potassium channel regulator 1 Human cDNA: FLJ23468 fis, clone HSI11603 blk=protein tyrosine kinase [Human, B lymphocytes, mRNA, 2608 nt]. Homo sapiens keratin 19 (KRT19) gene. 10p15.1 Xq28 4q28.3 10q24.33 12p11.1 4q35.1 8q23.1 17q21.2 NP 6729680 15012098 340159 Human, similar to small inducible cytokine subfamily B (Cys-X-Cys), member 10 Source: Human pro-urokinase. Human gremlin mRNA 10q22.2 <ul style="display: flex;"><li style="flex:1">15q13.3 </li><li style="flex:1">6274527 </li></ul>Continued Oncogene Lung cancer target identification J Heighway et al 7753 Table 2 (Continued ) Genbank Acc Chr. loc. 46up in: 166up in: 26up in: Gene description (closest PD match to element sequence) 2306914 188691 2546963 30374 14286293 12803652 14043731 929656 10439469 14603393 35322 17p12 1q22 888888888777777777777777777 22 10 16 13 19 19 12 12 22 14 19 9 17 19 21 13 18 21 19 11 10 10 16 17 22 23 17 13 9 Homo sapiens protein kinase mRNA. Human migration inhibitory factor-related protein 8 (MRP8) gene. Human mRNA for diubiquitin Human radiated keratinocyte mRNA for cysteine protease inhibitor. Homo sapiens, similar to RIKEN cDNA 1200008O12 gene. Source: Homo sapiens, keratin 13. Homo sapiens, clone MGC:14128 IMAGE:3610547. H.sapiens mRNA for neutrophil gelatinase associated lipocalin. FLJ22932 fis, highly similar to Human carcinoma-associated antigen GA733-2. Human, CK2 interacting protein 1; HQ0024c protein. H.sapiens mRNA for p cadherin. 6p21.31 3q21.1 10q23.33 17q21.2 8q24.13 9q34.13 2q16.3 1q21.3 16q22.1 19q13.11 5q13.2 17p11.2 14q22.2 17p11.2 18q21.33 11q12.3 17p11.2 18q21.33 1q22 23<ul style="display: flex;"><li style="flex:1">897916 </li><li style="flex:1">Human 11kd protein mRNA. </li></ul>1737211 12653220 414584 14286195 453368 12653112 181915 1172085 338424 6688152 4929718 14286301 5689824 36154 Human basic transcription factor 2 p44 (btf2p44) gene. Human, ubiquitin B, clone MGC:8385 IMAGE:2820408. Human protein tyrosine phosphatase (CIP2). Human, aldehyde dehydrogenase 3 family, member A1. Human maspin mRNA, complete cds. Homo sapiens, flap structure-specific endonuclease 1. Human ubiquitin carrier protein (E2-EPF) mRNA, complete cds. Human squamous cell carcinoma antigen (SCCA1) gene, exon 8. Human small proline rich protein (sprII) mRNA, clone 174N. Homo sapiens mRNA for small proline-rich protein 3 (SPRR3 gene). Human CGI-125 protein. 3<ul style="display: flex;"><li style="flex:1">4</li><li style="flex:1">1q22 </li></ul>17q13.1 1q25.1 15q13.1 Xp11.3 3p21.31 1q22 1q22 1q22 12q23.2 18q21.32 10q11.23 Homo sapiens, lactate dehydrogenase B. Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 295344. H.sapiens RR2 mRNA for small subunit ribonucleotide reductase. <ul style="display: flex;"><li style="flex:1">Human lactoferrin (HLF1). </li><li style="flex:1">187121 </li></ul><ul style="display: flex;"><li style="flex:1">338433 </li><li style="flex:1">5</li></ul>43322 Human small proline-rich protein gene. 3327379 14599490 12804462 13325357 338414 Source: Homo sapiens small proline-rich protein 1 (SPRR1A) gene. Human small proline-rich protein 2F (SPRR2F) gene. Human, similar to achaete-scute complex (Drosophila) homolog-like 1. Homo sapiens, gastrin-releasing peptide. 10 42<ul style="display: flex;"><li style="flex:1">7</li><li style="flex:1">Human prostate secreted seminal plasma protein. </li></ul>Listed is the closest Genbank match to the Incyte cDNA element sequence, the most likely genomic position of the sequence (BLAT similarity), the number of times the element score exceeded the filter threshold, the number of times it was scored as differentially expressed (DE42) and the abbreviated Genbank annotation for the matched sequence Table 3 Lists in ranked order four- and 16-fold tumour under-represented elements that passed the restriction criteria 46 down in: down in: down in: Gene description (closest PD match to element sequence) 166 26 <ul style="display: flex;"><li style="flex:1">Genbank Acc </li><li style="flex:1">Chrom. Loc. </li></ul>

ONCOGENOMICS Through the Use of Chemotherapeutic Agents

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