Advancing a clinically relevant perspective of the clonal nature of cancer Christian Ruiza,b, Elizabeth Lenkiewicza, Lisa Eversa, Tara Holleya, Alex Robesona, Jeffrey Kieferc, Michael J. Demeurea,d, Michael A. Hollingsworthe, Michael Shenf, Donna Prunkardf, Peter S. Rabinovitchf, Tobias Zellwegerg, Spyro Moussesc, Jeffrey M. Trenta,h, John D. Carpteni, Lukas Bubendorfb, Daniel Von Hoffa,d, and Michael T. Barretta,1 aClinical Translational Research Division, Translational Genomics Research Institute, Scottsdale, AZ 85259; bInstitute for Pathology, University Hospital Basel, University of Basel, 4031 Basel, Switzerland; cGenetic Basis of Human Disease, Translational Genomics Research Institute, Phoenix, AZ 85004; dVirginia G. Piper Cancer Center, Scottsdale Healthcare, Scottsdale, AZ 85258; eEppley Institute for Research in Cancer and Allied Diseases, Nebraska Medical Center, Omaha, NE 68198; fDepartment of Pathology, University of Washington, Seattle, WA 98105; gDivision of Urology, St. Claraspital and University of Basel, 4058 Basel, Switzerland; hVan Andel Research Institute, Grand Rapids, MI 49503; and iIntegrated Cancer Genomics Division, Translational Genomics Research Institute, Phoenix, AZ 85004 Edited* by George F. Vande Woude, Van Andel Research Institute, Grand Rapids, MI, and approved June 10, 2011 (received for review March 11, 2011) Cancers frequently arise as a result of an acquired genomic insta- on the basis of morphology alone (8). Thus, the application of bility and the subsequent clonal evolution of neoplastic cells with purification methods such as laser capture microdissection does variable patterns of genetic aberrations. Thus, the presence and not resolve the complexities of many samples. A second approach behaviors of distinct clonal populations in each patient’s tumor is to passage tumor biopsies in tissue culture or in xenografts (4, 9– may underlie multiple clinical phenotypes in cancers. We applied 11). These methods apply selective pressures on the complex DNA content-based flow sorting to identify and isolate the nuclei mixtures of cells and clones present in a patient sample and are of clonal populations from tumor biopsies, which was coupled time-consuming and labor intensive and are not amenable to with array CGH and targeted resequencing. The results produced rapid deployment in most clinical settings. Consequently, the number of xenografts successfully grown varies from site to site, high-definition genomic profiles of clonal populations from 40 and the biological complexity and clinical context of the patient pancreatic adenocarcinomas and a set of prostate adenocarcino- sample may not be reflected in the final processed sample. mas, including serial biopsies from a patient who progressed to Flow cytometry-based cell sorters can select, objectively mea- androgen-independent metastatic disease. The genomes of clonal sure, and sort individual particles such as cells or nuclei using fi populations were found to have patient-speci c aberrations of desired features objectively defined by fluorescent and light- clinical relevance. Furthermore, we identified genomic aberrations scattering parameters in a flow stream. Recent advances in this specific to therapeutically responsive and resistant clones arising technology provide high-throughput flow rates and the detection during the evolution of androgen-independent metastatic pros- of relatively rare events in dilute admixed samples, enabling the tate adenocarcinoma. We also distinguished divergent clonal pop- application of flow cytometry to in vivo high-definition analyses of ulations within single biopsies and mapped aberrations in multiple human cancers (12). The combination of flow sorting and geno- aneuploid populations arising in primary and metastatic pancre- mic analyses has been recently used for the enrichment of pan- atic adenocarcinoma. We propose that our high-definition analy- creas carcinoma cells and to study the clonal composition of ses of the genomes of distinct clonal populations of cancer cells in primary breast tumors (13, 14). However, these studies relied on patients in vivo can help guide diagnoses and tailor approaches to extensive bioinformatic analyses, platform-specific sample prep- personalized treatment. arations, or relatively large amounts of input material to achieve an acceptable signal-to-noise ratio in their genome analyses. To clonal genomics | pancreatic cancer | prostate cancer overcome these limitations, we developed a methodology to adapt genomic DNA isolated from cytometrically purified nuclei for use with array comparative genomic hybridization (aCGH) and next- arge-scale investigations of cancer genomes are expected to generation sequencing. Here we demonstrate the feasibility of Llead to the discovery of common disease elements, including this methodology for efficient high-resolution genomic analysis of recurring genomic aberrations, that can guide the development clonal populations from even minute heavily admixed patient bi- of broadly applicable diagnostics and therapeutics (1–3). For opsies. We profiled patient samples of primary and metastatic example, studies of colorectal and breast carcinomas and glio- pancreatic adenocarcinomas (PAs), a highly lethal tumor type blastoma multiforme (GBM) that surveyed somatic mutations that is difficult to molecularly characterize at the biopsy level due in over 18,000 genes estimated an average of ∼80 gene-specific to complex genomes and heterogeneous cellularity, as cancer mutations in each cancer type (3). Strikingly few highly recurrent cells represent on average only 25% of the cells within the tumor mutations were detected; the majority of mutations occur with (15). In contrast to recent studies of PA, we performed whole- a prevalence of <5% with little overlap between cancers. These genome discovery analyses with distinct clonal populations puri- and similar reports challenge the concept of collective cancer fied directly from patient biopsies (16, 17). Furthermore, we ap- genomes (4–7). Therefore, there is a need for a unique approach (i.e., not reliant on prevalence-based studies) to identify and fi interpret sets of selected aberrations and de ne the clinical de- Author contributions: C.R., M.S., P.S.R., S.M., J.M.T., J.D.C., L.B., D.V.H., and M.T.B. de- pendencies that arise in complex, highly variable carcinoma signed research; C.R., E.L., L.E., T.H., A.R., M.A.H., M.S., D.P., and M.T.B. performed re- genomes in patients in vivo. search; C.R., E.L., J.K., M.J.D., M.A.H., D.P., J.D.C., and M.T.B. analyzed data; T.Z. Cancer genome studies have relied on two main approaches for contributed new reagents/analytic tools; and C.R., S.M., L.B., and M.T.B. wrote the paper. selecting and preparing patient samples for analyses. The first is to The authors declare no conflict of interest. select samples exceeding a threshold for tumor cell content on the *This Direct Submission article had a prearranged editor. basis of histological methods such as evaluation of H&E-stained Freely available online through the PNAS open access option. slides (2). However, many samples fail these criteria; notably, Data deposition: The microarray files reported in this paper have been deposited at the tumors arising in solid tissues exhibit high degrees of tissue het- National Center for Biotechnology Information Gene Expression Omnibus (accession no. erogeneity, with varied admixtures of reactive stroma, inflam- GSE21660). matory cells, and necrosis in immediate contact with tumor cells. 1To whom correspondence should be addressed. E-mail: [email protected]. It is well established that biopsies frequently contain multiple This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. clonal populations of neoplastic cells that cannot be distinguished 1073/pnas.1104009108/-/DCSupplemental. 12054–12059 | PNAS | July 19, 2011 | vol. 108 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1104009108 Downloaded by guest on September 25, 2021 plied this approach to analyze distinct clonal tumor populations would have been obscured by the presence of mixed tumor pop- within clinically annotated prostate cancer specimens and were ulations or normal cells in each biopsy of interest. This is high- able to infer their clonal evolution and their mechanisms of lighted by the detection of distinct homozygous deletions, includ- therapy resistance. The results from this study demonstrate that ing those targeting genes regulating different pathways in PA and our approach can study the clonal diversity and evolution of PC genomes. These included NUMB, DDX10, PARK2, SMAD2, cancer genomes that arise in patients in vivo and reveal clinically SMAD3, JARID2, PARD3, PARDG6, P2RY5, and MAP2K4 in PA relevant contexts at least in the cancer types profiled in this study. and AIM1, FOXO3A, NEDD9, FEZF1, and PTEN in PC. In each case, the deletion was absent in the patient-matched diploid pop- Results ulation, confirming their somatic nature. Significantly, the PA We used DAPI-based DNA content measures to identify and SMAD3 deletion, despite a high tumor cell content (>70% by flow sort distinct diploid and aneuploid tumor populations from each cytometry and H&E) that is widely accepted for genome studies, of 40 PAs and biopsies from patients with varying grades of was not detected in the matched unsorted tumor sample (Fig. S1 C–E prostate adenocarcinoma (PC), including a series from one pa- ) (2). In comparison, amplicons were detected in sorted and tient who progressed to androgen-independent metastatic dis- bulk