SHORT REPORT Gene signatures of pulmonary metastases of renal cell carcinoma reflect the disease-free interval and the number of metastases per patient Daniela Wuttig 1 * , Barbara Baier 2 , Susanne Fuessel 1 , Matthias Meinhardt 3 , Alexander Herr 4 , Christian Hoefling 1 , Marieta Toma 3 , Marc-Oliver Grimm 1 , Axel Meye 1 , Axel Rolle 2 and Manfred P. Wirth 1 1 Department of Urology, Dresden University of Technology, Dresden, Germany 2 Department of Thoracic and Vascular Surgery, Center for Pneumology, Thoracic and Vascular Surgery, Coswig Specialized Hospital, Coswig, Germany 3 Institute of Pathology, Dresden University of Technology, Dresden, Germany 4 Institute of Clinical Genetics, Dresden University of Technology, Dresden, Germany Our understanding of metastatic spread is limited and molecular mechanisms causing particular characteristics of metastasis are largely unknown. Herein, transcriptome-wide expression profiles of a unique cohort of 20 laser-resected pulmonary metastases (Mets) of 18 patients with clear-cell renal cell carcinoma (RCC) were analyzed to identify expression patterns associated with two important prognostic factors in RCC: the disease-free interval (DFI) after nephrectomy and the number of Mets per patient. Dif- ferentially expressed genes were identified by comparing early (DFI  9 months) and late (DFI  5 years) Mets, and Mets derived from patients with few (8) and multiple (16) Mets. Early and late Mets could be separated by the expression of genes involved in metastasis-associated processes, such as angiogenesis, cell migration and adhesion (e.g., PECAM1, KDR). Samples from patients with multiple Mets showed an elevated expression of genes associated with cell division and cell cycle (e.g., PBK, BIRC5, PTTG1) which indicates that a high number of Mets might result from an increased growth potential. Minimal sets of genes for the prediction of the DFI and the number of Mets per patient were identified. Microarray results were confirmed by quantitative PCR by including nine further pulmonary Mets of RCC. In sum- mary, we showed that subgroups of Mets are distinguishable based on their expression profiles, which reflect the DFI and the number of Mets of a patient. To what extent the identified molecu- lar factors contribute to the development of these characteristics of metastatic spread needs to be analyzed in further studies. ' 2009 UICC Key words: kidney cancer; lung metastases; oligonucleotide microarrays In numerous tumour types, the development of metastases (Mets) causes the patients’ death. The median survival of renal cell carcinoma (RCC) patients amounts to merely 6 to 12 months after Mets have been diagnosed. 1 RCC is the urological cancer with the highest percentage of tumour-related deaths 2 because me- tastasis occurs in about 60% of the patients. 3 The preferential localization of RCC Mets is the lung. 1 In contrast to the emerging development of molecular-based therapies for RCC in the last few years, 3 molecular prognostic markers are still missing. Despite the knowledge of several molecular factors involved in metastatic spread like angiogenesis, cell adhesion, invasion or migration, 4,5 little is known about specific characteristics of this complex pro- cess. These are, for example, primary-dependent site-specific me- tastasis, 6 varying dormancy periods of Mets originating from the same primary tumour entity causing disease-free intervals (DFI) ranging from several months to many years, or the differing num- ber of Mets in patients with the same primary tumour. Knowing the molecular fundamentals of these phenomena would support the prognosis of patients’ outcome and facilitate the decision for an appropriate therapy regime, particularly in RCC where the DFI and the number of Mets are important predictors of clinical out- come. 7,8 So far, most microarray studies on RCC metastasis com- prise metastatic and nonmetastatic primary RCC. 9,10 Only a few studies include Mets of RCC to identify expression patterns asso- ciated with metastatic spread 11–13 since fresh-frozen metastatic lesions are rare because of their restricted surgical treatment. However, these studies have never addressed the particular char- acteristics of metastatic spread mentioned above. RCC patients early diagnosed or with multiple Mets have an extremely with poor prognosis. 7,8,14 In the present study, we investigated if this aggressive behaviour is reflected at the gene expression profile of these Mets. Therefore, we performed tran- scriptome-wide expression analyses on a homogenous cohort of pulmonary RCC Mets obtained by a new resection technique. 14 Material and methods Tissue specimens Renal cell carcinoma lung Mets were removed by 1,318-nm laser resection. 14 One-half of each tissue was snap-frozen in liquid nitrogen, while the other half was embedded in paraffin. Paraffin- embedded tissues were histologically evaluated to ensure a com- plete resection in the healthy lung tissue. Twenty cryo-preserved pulmonary Mets from 18 patients were analyzed by oligonucleotide microarrays. For validation of the array data by quantitative PCR nine additional samples obtained from nine patients were included. All patients underwent nephrec- tomy for clear-cell RCC, showed clinically no other distant Mets before diagnosis of lung Mets, received no immune- or immune- chemotherapy (except one patient) and had no other primary tumours. Pulmonary Mets were diagnosed by the attending urologist or the general practitioner by CT or MRT. After pulmonary metasta- sectomy, patients were generally monitored every 3 months for 2 years, afterwards semi-annually for 3 years and annually thereafter. Clinical and follow-up data of the patients are shown in Table I. Tissue collection and investigation was approved by the ethics committee of the Dresden University of Technology and informed consent was obtained from each patient. RNA isolation and microarray processing Cryo-sections of Mets (4 lm) were made after apparent nonma- lignant lung tissue had been removed. Representative H&E-stained sections were histologically re-evaluated to ensure a clear-cell his- tology and a tumour cell amount of 70%. Tissue sections were homogenized using QiaShredders (Qiagen, Hilden, Germany). Additional Supporting Information may be found in the online version of this article. Abbreviations: CP, crossing point; DFI, disease-free interval after ne- phrectomy; FDR, false discovery rate; KNN, k-nearest neighbouring; Mets, metastases; RCC, renal cell carcinoma; WV, weighted voting. *Correspondence to: Department of Urology, Faculty of Medicine, Dresden University of Technology, Fetscherstr. 74, 01307 Dresden, Germany. Fax: 149-351-458-5771. E-mail: daniela.wuttig@uniklinikum-dresden.de Received 9 June 2008; Accepted after revision 8 January 2009 DOI 10.1002/ijc.24353 Published online 10 February 2009 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 125, 474–482 (2009) ' 2009 UICC Publication of the International Union Against Cancer