935 Genome-wide DNA copy number predictors of lapatinib sensitivity in tumor-derived cell lines Joel Greshock,1 Jie Cheng,2 David Rusnak,1 either EGFR or HER2, lapatinib treatment results in growth Anne Marie Martin,3 Richard Wooster,1 arrest and cell death (1), suggesting that it may be possible Tona Gilmer,1 Kwan Lee,2 Barbara L. Weber,1 to predict clinical response based on a tumor’s genetic in vitro and Tal Zaks3 profile. Although larger screens confirm strong associations between EGFR and HER2amplification with 1Translational Medicine Oncology, GlaxoSmithKline, King of lapatinib responsiveness, some tumor models seem to Prussia, Pennsylvania and 2Department of Biostatistics and Data 3 respond in the absence of these markers (2, 3). This Management and Medicines Development Center, Oncology, observation indicates that additional proteins may cooper- GlaxoSmithKline, Collegeville, Pennsylvania ate with HER2in promoting lapatinib responsiveness, thereby forming the basis of a more inclusive means of Abstract predicting clinical response. Such proteins may include the A common aim of pharmacogenomic studies that use linear cascades that constitute the mitogen-activated genome-wide assays on panels of cancers is the unbiased cascade, the stress-activated protein kinase cascade, protein discovery of genomic alterations that are associated with kinase C, and the Akt pathway (4). clinical outcome and drug response. Previous studies of Altered DNA copy number is a hallmark feature of the lapatinib, a selective dual-kinase inhibitor of epidermal cancer genome. A number of tumor suppressor genes have growth factor receptor (EGFR) and HER2 tyrosine kinases, been mapped to homozygous deletions, including RB1 and have shown predictable relationships between the activity SMAD4/DPC4 (5, 6). Similarly, oncogenic copy number of these target genes and response. Under the hypothesis amplifications have been described in such loci as PDGFRA, EGFR HER2 that additional genes may play a role in drug sensitivity, a , and (7–9). Until recent technolog- predictive model for lapatinib response was constructed ical advances, the assembly of more complete lists of genes from genome-wide DNA copy number data from 24 cancer whose copy number is altered in cancer has been limited by cell lines. An optimal predictive model which consists of nonsystematic techniques (10). However, the development aberrations at nine distinct genetic loci, includes gains of of high-resolution microarray-based genome-wide DNA HER2, EGFR, and loss of CDKN2A. This model achieved copy number has remedied this limitation. Although an area under the receiver operating characteristic curve earlier BAC clone–based platforms provided a resolution of f0.85 (80% confidence interval, 0.70–0.98; of f1 Mb (11), more recent oligonucleotide-based plat- P < 0.01), and correctly classified the sensitivity status forms such as Representational Oligonucleotide Microarray of 8 of 10 head and neck cancer cell lines. This study Analysis decreased probe spacing considerably (12). Single- shows that biomarkers predictive for lapatinib sensitivity, nucleotide polymorphism arrays (SNP chips) represent a including the previously described copy number gains of type of high-density oligonucleotide-based microarrays EGFR and HER2, can be discovered using novel genomic that have been adapted for DNA copy number analyses assays in an unbiased manner. Furthermore, these results (13) and perform comparably with traditional two-channel show the utility of DNA copy number profiles in pharma- arrays (14). cogenomic studies. [Mol Cancer Ther 2008;7(4):935–43] The increasing role of pharmacogenomics in oncology compound development and treatment has closely paral- Introduction leled the advent of high-throughput genomics platforms. A major attraction of using genome-wide assays is the Lapatinib is a potent, orally active dual kinase inhibitor of increased ability to more comprehensively characterize the epidermal growth factor receptor (EGFR) and HER2 genomic mechanisms of drug sensitivity. For example, tyrosine kinase receptors. In tumor cells that overexpress gross patterns in gene expression effectively show that in vitro response to a farnesyltransferase inhibitor of the Ras pathway is directly correlated with Ras pathway activity (15). Translational research focuses on the discovery of Received 9/18/07; revised 12/19/07; accepted 1/10/08. rational predictive markers derived from such genomic The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked assays that could then be used for response enrichment in a advertisement in accordance with 18 U.S.C. Section 1734 solely to clinical setting. A growing number of these studies include indicate this fact. an effective predictor of bortezomib response in myelomas Requests for reprints: Tal Z. Zaks, Translational Medicine Oncology, that was retrospectively calculated and validated from gene GlaxoSmithKline, 1250 South Collegeville Road, UP 4W-4230, Collegeville, PA 19426. Phone: 610-917-5124; Fax: 610-917-4830. expression microarrays run on patient tissue collected from E-mail: [email protected] multiple independent clinical sites (16). Although genome- Copyright C 2008 American Association for Cancer Research. wide expression profiling is currently the most widespread doi:10.1158/1535-7163.MCT-07-2072 platform for pharmacogenomic studies, the mapping of Mol Cancer Ther 2008;7(4). April 2008 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. 936 DNA Copy Number Predictors of Lapatinib Sensitivity specific DNA copy number alterations to multiple path- (ranging between 0.008 and 10 Amol/L). Cells were ways is also effective in delineating markers associated with harvested by trypsinization on day 7 and counted using a the responsiveness to targeted therapy (reviewed in ref. 17). particle counter (Z1, Beckman Coulter, Inc.). Growth Fixed genetic alterations such as DNA copy number gains inhibition was calculated as a percentage of the untreated and losses are appealing for clinical study because (a) they controls. Experiments were carried out twice or thrice in are relatively stable in vivo over time, and (b) they are duplicate for each cell line. The log of the fractional growth faithfully modeled in cell lines in vitro (18, 19). For example, inhibition was plotted against the log of the drug copy number studies have associated specific DNA copy concentration, and the IC50s were interpolated from the number alterations in ovarian carcinomas with carboplatin resulting linear regression curve fit (Calcusyn, Biosoft). IC50 response (20), whereas amplifications of the multidrug values <1.0 Amol/L were considered as sensitive, 1.0 to resistance gene MDR1 are correlated with therapeutic 2.5 Amol/L as intermediate, and values >2.5 Amol/L were resistance (21, 22). considered resistant. Proliferation data for several of these The growing use of targeted therapies in cancer treatment lines have been previously presented in ref. (2). reflects an increasing knowledge of key oncogenic path- SNP Chip/Transcript Analysis ways. Difficulties in predicting response to targeted DNA was extracted from each line using Mini DNeasy kit therapies are likely a consequence of the limited global (Qiagen, Inc.) and subsequently purified. For the 24 cell knowledge of altered genetic pathways in each cancer. lines of mixed tumor type composing the gtrainingh panel, Also, unique genomic profiles observed in each cancer two aliquots (250 ng each) were digested with the suggests the existence of many more contributing path- restriction enzyme XbaIorHindIII (New England Biolabs), ways than previously thought. For example, contrasting whereas a similar quantity of material was digested with sets of recurring alterations found to differentiate between Sty and Nsp enzymes for the 10 head and neck lines in the certain types of lung cancers indicate that common validation set. Digested DNA was subsequently ligated to oncogenic pathways may be subject to unique alterations an adaptor and amplified by PCR using Platinum Pfx DNA (23). Collectively, these results suggest that developing Polymerase (Invitrogen), yielding a product of f250 to genome-wide strategies to discover predictive models of 2,000 bp. For each enzyme digest, PCR was carried out in therapeutic response can be more sensitive than traditional four 100-AL aliquots, pooled, purified, quantified, normal- single-gene approaches. In this study, we present a model ized to 40 Ag/45 AL, and fragmented with DNase I to yield that takes advantage of global characterizations of DNA a size range of f25 to 200 bp. The fragmented products of copy number to accurately predict in vitro responses to the training set and test set were then labeled, denatured, lapatinib. The model, which emphasizes high-level copy and hybridized to the Affymetrix 100K and 500K chip, number alterations such as homozygous deletions and respectively (Affymetrix Inc.). Upon completion of hybrid- oncogenic amplifications, rediscovers the HER2 locus, ization, each assay was washed and stained using which is already a known predictor of clinical activity. Affymetrix fluidics stations. Image data were acquired Furthermore, this model discovers novel markers of using the GeneChip Scanner 3000. For all 34 cell lines, response that are capable of classifying a panel of non– transcript abundance was quantified in triplicate by using HER2-amplified