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Lineage Relationship of Gleason Patterns in Gleason Score 7 Prostate Cancer

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Lineage Relationship of Gleason Patterns in Gleason Score 7 Prostate Cancer Published OnlineFirst May 21, 2013; DOI: 10.1158/0008-5472.CAN-12-2803 Cancer Molecular and Cellular Pathobiology Research Lineage Relationship of Gleason Patterns in Gleason Score 7 Prostate Cancer Irina V. Kovtun1, John C. Cheville2, Stephen J. Murphy3, Sarah H. Johnson3, Shabnam Zarei3, Farhad Kosari3, William R. Sukov2, R. Jeffrey Karnes4, and George Vasmatzis3 Abstract Gleason score 7 (GS7) prostate cancer [tumors with both Gleason patterns 3 (GP3) and 4 (GP4)] portends a significantly more aggressive tumor than Gleason score 6 (GS6). It is, therefore, critical to understand the molecular relationship of adjacent GP3 and GP4 tumor cell populations and relate molecular abnormalities to disease progression. To decipher molecular relatedness, we used laser capture microdissection (LCM) and whole- genome amplification (WGA) to separately collect and amplify DNA from adjacent GP3 and GP4 cell populations from 14 cases of GS7 prostate cancer. We then carried out massively parallel mate-pair next generation sequencing (NGS) to examine the landscape of large chromosomal alterations. We identified four to 115 DNA breakpoints in GP3 and 17 to 480 in GP4. Our findings indicate that while GP3 and GP4 from the same tumor each possess unique breakpoints, they also share identical ones, indicating a common origin. Approximately 300 chromosomal breakpoints were localized to the regions affected in at least two tumors, whereas more than 3,000 were unique within the set of 14 tumors. TMPRSS2–ERG was the most recurrent rearrangement present in eight cases, in both GP3 and GP4. PTEN rearrangements were found in five of eight TMPRSS2–ERG fusion–positive cases in both GP3 and GP4. Hierarchical clustering analysis revealed that GP3 has greater breakpoint similarity to its partner GP4 compared with GP3 from different patients. We show evidence that LCM, WGA, and NGS of adjacent tumor regions provide an important tool in deciphering lineage relationships and discovering chromosomal alterations associated with tumor progression. Cancer Res; 73(11); 1–10. Ó2013 AACR. Introduction molecular changes, such as deregulation of PTEN have also – The elucidation of causal DNA rearrangements is critical to been implicated in prostate cancer (7 12). Despite the discov- the understanding of cancer development and progression, ery of some recurrent DNA rearrangements, association of and clinically relevant in regards to prognosis and treatment. these changes with clinical and pathologic features, particu- Although, the precursor lesions for prostate cancer are not larly with tumor grade, has been limited. completely defined (1, 2), significant progress has been made in Morphologic heterogeneity in prostate cancer, assessed with identifying the molecular events underlying initiation and Gleason pattern (GP) and the summation score (GS; refs. 13, progression using genomic and functional studies. The most 14), is strongly related to prognosis. Gleason score 6 (GS6) frequent chromosomal rearrangement in prostate cancer, the prostate cancer is either cured by treatment or needs no fi TMPRSS2–ERG gene fusion, is considered to be an early event treatment at all (15, 16). In up to 30% of cases, the nding of fi in prostate tumorigenesis. The significance of TMPRSS2–ERG GS6 on needle biopsy specimen is associated with insigni cant fusion protein as a prognostic or predictive marker has been cancer. In contrast, men with Gleason score 7 (GS7) and higher fi controversial (3–5), but recent evidence suggests that ERG have signi cant prostate cancer, and are at increased risk of status defines unique subtypes of prostate cancer (6). Other cancer progression. Despite the close association of GP to tumor behavior, the relationship of morphologic heterogeneity and molecular heterogeneity has not been elucidated. While there is a possibility that GP3 and GP4 regions in GS7 prostate Authors' Affiliations: Departments of 1Molecular Pharmacology and Experimental Therapeutics, 2Laboratory Medicine and Pathology, 3Molec- cancer are of different origin, it is generally accepted that GP4 ular Medicine, and 4Urology, Mayo Clinic, Rochester, Minnesota adjacent to GP3 are related and that GP3 progresses to GP4, Note: Supplementary data for this article are available at Cancer Research which shows more aggressive behavior. However, the transi- Online (http://cancerres.aacrjournals.org/). tion supposition has only been inferred by determining the I.V. Kovtun and J.C. Cheville contributed equally to this work. ERG status using FISH or immunostaining. In addition, there is a poor understanding of genomic differences between GP3 that Corresponding Authors: George Vasmatzis, Department of Molecular Medicine, Mayo Clinic, 200 First Street SE, Rochester, MN 55905. Phone: progresses and GP3 that does not. 507-284-2511; Fax: 507-266-5193; E-mail: [email protected]; Several studies have addressed the issue of tumor hetero- and John C. Cheville, E-mail: [email protected] geneity in prostate cancer by comparing changes in multiple doi: 10.1158/0008-5472.CAN-12-2803 separate tumor nodules within a prostate gland (17–21). Ó2013 American Association for Cancer Research. Assessment of the occurrence of the TMPRSS2–ERG fusion by www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst May 21, 2013; DOI: 10.1158/0008-5472.CAN-12-2803 Kovtun et al. FISH in multifocal prostate cancer show that 40% to 50% of foci variability in chromosomal rearrangements between different are discordant in regards to the presence or absence of the GPs within the same tumor and to determine whether these fusion, but within any one focus the absence or presence of the tumors exhibited a common origin. To investigate lineage fusion is consistent (18–21). These studies support the notion relationships between adjacent GPs, we carried out laser that multifocal primary prostate cancers are composed of capture microdissection (LCM), whole-genome amplification multiple clones. A study of metastatic prostate cancer using (WGA), and massively parallel mate-pair next generation high-resolution genome-wide single polymorphism and copy sequencing (NGS), and compared molecular alterations in GP3 number survey approach has shown the lineage relationship of and GP4 within the same tumor and between tumors from metastases to the primary tumor (22). different patients. Although, it is accepted that multiple separate tumors within the prostate gland arise from different clones, there is Materials and Methods no data regarding a common origin of different GPs within a Isolation of prostate GP3 and GP4 DNA, mate-pair unifocal dominant tumor. Indeed, no study has conclusively library construction and sequencing shown whether cells within a single tumor harbor identical Fourteen cases of fresh-frozen GS7 prostate cancer com- molecular alterations. For instance, the TMPRSS2–ERG fusion posed of GP3 adjacent to GP4 were selected for study. The can occur at a number of breakpoints that indicate a dif- tumors were the dominant tumor nodules within each gland. ferent clonal origin, so that identification of the fusion by In each case, 10 mm unstained sections were cut from fresh- FISH or by immunoperoxidase staining in different GPs within frozen blocks of tissue of patient with prostate cancer collected the same tumor does not conclusively prove a common origin from a radical prostatectomy specimen. LCM (Arcturus) was as these studies are not specific to any breakpoint. Moreover, used to separately isolate cells specific to the GP3 and GP4 molecular changes in the context of morphologic heterogene- populations minimizing contamination of adjacent cells ity within a tumor, specifically related to the differences within (Fig. 1A). Histologically normal glands were additionally col- GPs, have not been analyzed. It is unclear whether molecular lected from same slides at distances minimizing the chances of events characteristic of aggressive GP4 are present in the contaminating with the tumor tissues. The cells were lysed associated GP3 in GS7 cancer. It is also unknown whether the directly on the cap, and whole-genome DNA sequencing was chromosomal changes in the associated GP3 in GS7 cancer are carried out as previously described (23). A mate-pair library more similar to GP3 from other patients or more similar to its was prepared using the Mate-pair Library Prep Kit (Illumina), associated GP4. In this study, we identified patients with a following the manufacturer's recommendations. Briefly, 10 mg unifocal GP7 prostate cancer to determine the extent of of WGA DNA was fragmented using Covaris sonication to 4 and A B GP4 GP3 GP3 GP4 CD GP3 GP4 Figure 1. An example of GS7 prostate cancer specimen used for LCM. A, hematoxylin and eosin staining. B, microdissected areas corresponding to pattern GP3 (C) and GP4 (D), light images. OF2 Cancer Res; 73(11) June 1, 2013 Cancer Research Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst May 21, 2013; DOI: 10.1158/0008-5472.CAN-12-2803 Relationship of Gleason Patterns in Gleason Score 7 Prostate Cancer 5 kb, fragments and mate-pair libraries were prepared as FISH previously described and sequenced at one or 2 libraries per FISH studies were conducted on formalin-fixed, paraffin- lane on the Illumina GAII or HiSeq platforms, respectively (23). embedded tissue using a probe designed to assess PTEN status. Bacterial artificial chromosomes
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